## Markers of genuine (species-specific) sensitization

Examples: Fel d 1 (cat), Api m 1, Api m 3, Api m 4, Api m 10 (honey bee venom), Ves v 1, Ves v 5 (Vespula species), Bet v 1 (fagales), Ole e 1 (olive tree, plantane), Phl p 1, Phl p 5 (grass) Art v 1 (mugwort), Amb a 1 (ragweed), Par j 2 (pellitory).

In summary, after taking the history and performing extract-based sensitization tests a diagnostic work-up including specific IgE to allergenic molecules is useful to increase assay sensitivity for single allergens of low abundance (in extracts) or weak stability. An increased analytical specificity will help to identify risk- or severity-associated allergens, indicators for cross-reactivity and marker allergens of genuine (primary) sensitization.

Interpretation is an integral part of each sensitization test: Positive results are only clinically relevant in case of corresponding symptoms; negative results can ideally rule out an allergic sensitization and subsequent clinical reaction to the tested allergen.

## 4 – From molecules to symptoms: the “bottom-up approach”

Instead of performing symptom-oriented focused molecular allergy diagnostics (“top-down approach,” see above), one can simply turn this approach around and start from the bottom, i.e., with the molecules.⁷⁹ In an ideal scenario, diagnostic tools would allow us to characterize the entire IgE repertoire to all potential allergens that

a patient has been exposed to. It would then be conceivable that we first analyze the entire IgE repertoire and then start talking to the patient to find out which of the detected IgE sensitizations are clinically relevant. This would be a “broad bottom-up approach,” i.e., turning the diagnostic pyramid upside down. However, a number of reasons suggest that in real life this is not appropriate: (a) We are far from being able to characterize the entire IgE repertoire, i.e., the individual IgE response to the entire allergome—currently only approx. 200 of the 3000 known allergens are available for diagnostic purposes. (b) An entire IgE repertoire characterization would be exceedingly expensive and yield enormous amounts of information that require processing and interpretation. (c) At present molecular allergy research attempts and multiplex technologies still depend on the availability of allergens for diagnosis. Many research projects have so far focused on certain molecules, i.e., Bet v 1-homologous proteins in various sources, leading to a broad spectrum of available proteins. However, this does not mean that this group is more relevant than other allergens to which less attention was paid in the past or which are more difficult to be produced as recombinant allergens. (d) Finally, the number of positive IgE results to a certain allergen source usually exceeds by far the number of clinically relevant allergies. Screening the IgE response to the entire allergome thus would most likely result in generation of large proportions of positive test results that have no clinical relevance (as it would be the case of performing extensive extract-based skin prick tests without the guidance of a previous thorough clinical history).

<figure><img src="image_15.png" /><figcaption>FIGURE 4 Diagnostic work-up with broad (multiplex) molecular-based IgE testing: In complex cases and/or inconclusive diagnostic outcomes after previous testing (A) a panel of molecular allergens might be applied for subsequent (multiplex) IgE testing (B2). After final interpretation (B3) with an optional challenge (C) this approach, coined “bottom-up,” might facilitate improved decisions on therapeutic consequences.</figcaption></figure>
In conclusion, screening of IgE sensitization profiles to large panels of allergens irrespective of the clinical history ("broad bottom-up approach") is of limited value for the management of the allergy patient. However, there are a number of situations, in which a "targeted bottom-up approach," i.e., using molecular information and asking for corresponding symptoms can be helpful for patient management and consultation in clinical practice as outlined below:

**Definition:** Diagnostic work-up from (cross-reactive) molecules to clinical implications ("targeted bottom-up approach" Figure 4) aims for more detailed characterization of the IgE-repertoire or clinical reaction pattern unfolding important hints from a panel of potential molecular IgE-mediated cross-reactions. This diagnostic algorithm "targeted bottom-up approach" (Figure 4) is designated for molecular work-up in case of positive IgE to a certain, potentially clinically relevant cross-reactive molecule (protein family). The finding might explain broad cross-reactivities to other allergen sources if the candidate belongs to a protein family with many members of similar structure and a high degree of cross-reactive IgE recognition.

In clinical practice, the diagnostic work up in such cases (IgE to potentially clinically relevant cross-reactive molecules) can be based purely on clinical assessment to determine the relevance of potential symptom driving cross-reactivities, extending the clinical history or applying optional challenge tests with the allergen source in question.

Alternatively, or in addition, molecular IgE serology using the "targeted bottom up approach" i.e., screening for sIgE to corresponding cross-reactive allergens, may provide useful information on the presence or absence of cross-sensitizations.

The decision on which allergenic molecules have to be tested in this context should be based on the known degree of cross-reactivity. Here, two different patterns emerge:

1. Protein families with highly cross-reactive allergens do not require further IgE testing, but thorough clinical work-up to identify relevant clinical cross-reactions.

* A single IgE test is sufficient to demonstrate cross-reactivity to a prominent (most IgE-binding) member of an allergen family with broad cross-reactivity.

* Additional tests would only demonstrate more cross-reactions (Figure 5B,C) without addressing the clinical consequences (i.e., symptoms, clinical reactions).

* Subsequently, detailed clinical work-up is required to clarify potential clinically relevant cross-reactivities.

**Reasoning:** Further IgE tests would potentially create many (more) positive results with questionable clinical relevance. Therefore, the physician should sort out potential clinically relevant cross-reactions to related allergen sources in question containing a cross-reactive member of the same allergen family. In conclusion, it

<figure><img src="image_16.png" /></figure>

**FIGURE 5** Allergen-specific IgE concentrations to various allergen molecules depending on structural similarity within one allergen family. A. Variable, limited cross-reactions (illustrated by circles with limited overlap) between different 2S-albumins (stable seed storage proteins in nuts, legumes and seeds) and corresponding IgE values (bars of various sizes) B. Cross-reactions of moderately limited variability (illustrated by circles with moderately limited overlap) between different Bet v 1-homologous food allergens C. High degree of cross-reactivity (illustrated by largely overlapping circles) between highly conserved, similar structures and epitopes of profilins (in pollen, foods and natural rubber latex) with corresponding IgE concentrations (bars of almost identical size).

is commonly sufficient to test only one member of a highly cross-reactive allergen family

Examples: Bet v 1 and its homologues, profilins, polcalcins (Ca++-binding proteins), serum albumins, grass pollen major group 1 and 5 alergens, parvalbumins, tropomyosins.

2. Protein families with allergens of limited cross-reactivity are an option for further IgE testing, if an IgE test to a member of the same allergen family has been positive. In general, the highest IgE concentration to a member of the same family might indicate the primary sensitizer (Figure 5A). A negative result would generally exclude an IgE sensitization and make subsequent clinical reactions highly unlikely. However, in the case of a positive IgE result, only a thorough clinical work-up would be able to clarify potential clinically relevant cross-reactivities and subsequent reactions. If the case
In conclusion, screening of IgE sensitization profiles to large panels of allergens irrespective of the clinical history ("broad bottom-up approach") is of limited value for the management of the allergy patient. However, there are a number of situations, in which a "targeted bottom-up approach," i.e., using molecular information and asking for corresponding symptoms can be helpful for patient management and consultation in clinical practice as outlined below:

**Definition:** Diagnostic work-up from (cross-reactive) molecules to clinical implications ("targeted bottom-up approach" Figure 4) aims for more detailed characterization of the IgE-repertoire or clinical reaction pattern unfolding important hints from a panel of potential molecular IgE-mediated cross-reactions. This diagnostic algorithm "targeted bottom-up approach" (Figure 4) is designated for molecular work-up in case of positive IgE to a certain, potentially clinically relevant cross-reactive molecule (protein family). The finding might explain broad cross-reactivities to other allergen sources if the candidate belongs to a protein family with many members of similar structure and a high degree of cross-reactive IgE recognition.

In clinical practice, the diagnostic work up in such cases (IgE to potentially clinically relevant cross-reactive molecules) can be based purely on clinical assessment to determine the relevance of potential symptom driving cross-reactivities, extending the clinical history or applying optional challenge tests with the allergen source in question.

Alternatively, or in addition, molecular IgE serology using the "targeted bottom up approach" i.e., screening for sIgE to corresponding cross-reactive allergens, may provide useful information on the presence or absence of cross-sensitizations.

The decision on which allergenic molecules have to be tested in this context should be based on the known degree of cross-reactivity. Here, two different patterns emerge:

1. Protein families with highly cross-reactive allergens do not require further IgE testing, but thorough clinical work-up to identify relevant clinical cross-reactions.

* A single IgE test is sufficient to demonstrate cross-reactivity to a prominent (most IgE-binding) member of an allergen family with broad cross-reactivity.

* Additional tests would only demonstrate more cross-reactions (Figure 5B,C) without addressing the clinical consequences (i.e., symptoms, clinical reactions).

* Subsequently, detailed clinical work-up is required to clarify potential clinically relevant cross-reactivities.

**Reasoning:** Further IgE tests would potentially create many (more) positive results with questionable clinical relevance. Therefore, the physician should sort out potential clinically relevant cross-reactions to related allergen sources in question containing a cross-reactive member of the same allergen family. In conclusion, it

<figure><img src="image_16.png" /></figure>

**FIGURE 5** Allergen-specific IgE concentrations to various allergen molecules depending on structural similarity within one allergen family. A. Variable, limited cross-reactions (illustrated by circles with limited overlap) between different 2S-albumins (stable seed storage proteins in nuts, legumes and seeds) and corresponding IgE values (bars of various sizes) B. Cross-reactions of moderately limited variability (illustrated by circles with moderately limited overlap) between different Bet v 1-homologous food allergens C. High degree of cross-reactivity (illustrated by largely overlapping circles) between highly conserved, similar structures and epitopes of profilins (in pollen, foods and natural rubber latex) with corresponding IgE concentrations (bars of almost identical size).

is commonly sufficient to test only one member of a highly cross-reactive allergen family

Examples: Bet v 1 and its homologues, profilins, polcalcins (Ca++-binding proteins), serum albumins, grass pollen major group 1 and 5 alergens, parvalbumins, tropomyosins.

2. Protein families with allergens of limited cross-reactivity are an option for further IgE testing, if an IgE test to a member of the same allergen family has been positive. In general, the highest IgE concentration to a member of the same family might indicate the primary sensitizer (Figure 5A). A negative result would generally exclude an IgE sensitization and make subsequent clinical reactions highly unlikely. However, in the case of a positive IgE result, only a thorough clinical work-up would be able to clarify potential clinically relevant cross-reactivities and subsequent reactions. If the case
history is not informative, a challenge test with the allergen source in question has to be applied to ultimately address the question of potential clinical relevance.

**Reasoning:** In case of allergens of limited cross-reactivity (see Figure 5A) an appropriate panel of related allergens (from the same protein family) could be used to demonstrate or exclude subsequent (serological) cross-reactivities. Therefore, additional IgE testing with related allergen molecules of the same family might establish a hierarchy of allergen-specific IgE values (Figure 5): Ideally the one with the highest IgE antibody level will represent the primary sensitizer. A negative result could exclude serological (and subsequently clinical) cross-reactivity. A positive result, however, would indicate serological cross-reactivity, which should be addressed with the patient according to her/his individual symptoms. Only in the case of corresponding symptoms, sometimes backed by an oral challenge, these cross-reactivities have to be considered in terms of present clinical relevance.

*Examples:* Seed storage proteins like 2S-albumins, 7S-globulins (vici-lins), 11S-globulins (legumins); lipocalin subfamilies, nsLTPs

In conclusion, dissecting the relevance of a panel of related, cross-reactive allergens can be obtained by (a) a purely clinical work-up and/or (b) a further introduction of related, cross-reactive molecules. In case of negative IgE tests, serological and clinical cross-reactions can be ruled out with certainty. Positive IgE results would confirm serological cross-reactivity, the clinical relevance that needs to be addressed with the patient according to her/his

individual symptoms. This approach is only recommended for protein families with a low or limited degree of cross-reactivity (i.e., seed storage proteins, nsLTP), where the individual's IgE repertoire is highly variable and its binding to related molecules cannot be predicted.

## 5 – “U-shaped” molecular diagnostics in IgE-mediated diseases

**Definition:** A previous diagnostic work-up from symptoms to molecules ("top-down approach," (Figure 2)) is combined with a subsequent diagnostic sequence from molecules to clinical implications ("targeted bottom-up approach," (Figure 4)), coined "U-shaped molecular diagnosis" (Figure 6), dissecting the relevance of potential molecular IgE-mediated cross-reactions.

Satisfactory diagnostic conclusions after applying selected molecules (top-down approach) for IgE testing would result in appropriate advice to the patient without the need of further work-up (i.e., bottom-up approach).

However, if open questions remain, regarding the implications of potentially cross-reactive allergens after identification of one key allergen, the U-shaped molecular diagnosis (Figure 6) might help to solve the diagnostic problem. Subsequently, criteria for further molecular work-up after a previous "top-down approach" with the "bottom-up approach" have to be applied on an individual basis depending on the diagnostic outcome after the initial diagnostic steps.

<figure><img src="image_17.png" /><figcaption>**FIGURE 6** Combined diagnostic work-up with “top-down” and “bottom-up” approach. The diagnostic flow chart starts with the history, extract diagnostics, molecular diagnostics and subsequent application of extended molecular panels for further differentiation of the allergen-specific IgE repertoire. The approach, coined “U-shape,” has been proposed for complex cases.</figcaption></figure>
history is not informative, a challenge test with the allergen source in question has to be applied to ultimately address the question of potential clinical relevance.

**Reasoning:** In case of allergens of limited cross-reactivity (see Figure 5A) an appropriate panel of related allergens (from the same protein family) could be used to demonstrate or exclude subsequent (serological) cross-reactivities. Therefore, additional IgE testing with related allergen molecules of the same family might establish a hierarchy of allergen-specific IgE values (Figure 5): Ideally the one with the highest IgE antibody level will represent the primary sensitizer. A negative result could exclude serological (and subsequently clinical) cross-reactivity. A positive result, however, would indicate serological cross-reactivity, which should be addressed with the patient according to her/his individual symptoms. Only in the case of corresponding symptoms, sometimes backed by an oral challenge, these cross-reactivities have to be considered in terms of present clinical relevance.

*Examples:* Seed storage proteins like 2S-albumins, 7S-globulins (vici-lins), 11S-globulins (legumins); lipocalin subfamilies, nsLTPs

In conclusion, dissecting the relevance of a panel of related, cross-reactive allergens can be obtained by (a) a purely clinical work-up and/or (b) a further introduction of related, cross-reactive molecules. In case of negative IgE tests, serological and clinical cross-reactions can be ruled out with certainty. Positive IgE results would confirm serological cross-reactivity, the clinical relevance that needs to be addressed with the patient according to her/his

individual symptoms. This approach is only recommended for protein families with a low or limited degree of cross-reactivity (i.e., seed storage proteins, nsLTP), where the individual's IgE repertoire is highly variable and its binding to related molecules cannot be predicted.

## 5 – “U-shaped” molecular diagnostics in IgE-mediated diseases

**Definition:** A previous diagnostic work-up from symptoms to molecules ("top-down approach," (Figure 2)) is combined with a subsequent diagnostic sequence from molecules to clinical implications ("targeted bottom-up approach," (Figure 4)), coined "U-shaped molecular diagnosis" (Figure 6), dissecting the relevance of potential molecular IgE-mediated cross-reactions.

Satisfactory diagnostic conclusions after applying selected molecules (top-down approach) for IgE testing would result in appropriate advice to the patient without the need of further work-up (i.e., bottom-up approach).

However, if open questions remain, regarding the implications of potentially cross-reactive allergens after identification of one key allergen, the U-shaped molecular diagnosis (Figure 6) might help to solve the diagnostic problem. Subsequently, criteria for further molecular work-up after a previous "top-down approach" with the "bottom-up approach" have to be applied on an individual basis depending on the diagnostic outcome after the initial diagnostic steps.

<figure><img src="image_17.png" /><figcaption>**FIGURE 6** Combined diagnostic work-up with “top-down” and “bottom-up” approach. The diagnostic flow chart starts with the history, extract diagnostics, molecular diagnostics and subsequent application of extended molecular panels for further differentiation of the allergen-specific IgE repertoire. The approach, coined “U-shape,” has been proposed for complex cases.</figcaption></figure>
The question remains if singleplex or multiplex testing should be applied:

* if possibly a large number of allergens from one family is involved, multiplex testing might carry some advantages.

* singleplex testing, however, offers an enhanced assay sensitivity, allowing the ultimate exclusion of IgE-mediated sensitization to the allergen in question if the allergen-specific IgE does not exceed the cut-off of 0.1 kU/l.

* therefore, the decision of singleplex or multiplex testing should consider the number of allergens to be tested and the preferred test sensitivity (very low or not as low).

Examples: seed storage proteins like 2S-albumins, 7S-globulins (vi-cilins), 11S-globulins (legumins). Note of caution: The number of storage proteins of different nuts, legumes and seeds that are available for diag-nostics is still limited. This does not allow one to check for the presence of allergen-specific IgE to a full panel of these stable and risk-associated al-lergens. Therefore, allergen extracts are still needed, to indirectly get infor-mation on the potential relevance of the risk-associated storage proteins.

In conclusion, certain scenarios require a complete molecular di-agnostic work-up after taking the history and performing focused extract- and molecule-based sensitization tests. This approach ex-plores the degree and potential clinical relevance of further cross-reactivities to related molecules of a protein family. Singleplex assays would guarantee maximum assay sensitivity; multiplex assays would rather provide a broad panel of related, cross-reactive molecules for further definition of the IgE repertoire. The clinical relevance must be determined by the physician and not by the test, based on pa-tient's history and outcome of challenge tests if needed.

# 6 - Molecular IgE sensitization profiles as biomarkers

Biomarkers are usually molecules that indicate physiological or pathologic phenomena. They reflect an objectively quantifiable measure of disease expression, severity and/or response to therapy. They can be beneficial in many different settings, especially in diagnostic processes and disease staging, identifying patients who will benefit from the treatment, monitoring disease trends, treatment efficacy and its side effects, predicting long-lasting protection, and thus im-proving acceptance and compliance. Along these lines, detection of molecular IgE sensitization profiles may function as biomarker in several settings as outlined below.

## 6.1 Sensitization to species-specific and cross-reactive molecules as diagnostic biomarkers

Grass pollen allergy represents a typical example for the use of allergen-specific IgE profiles as a biomarker, not only for an etiologi-cal confirmation of the presumptive diagnosis but also for the subse-quent prescription of AIT. Patients with symptoms of allergic rhinitis during the grass pollen season and a positive SPT/IgE response to

grass pollen extracts are further investigated in order to detect serum IgE antibodies to Phl p 1, Phl p 2, Phl p 5, Phl p 7, Phl p 11, and Phl p 12. The identification of IgE antibodies towards one or more of the molecules Phl p 1, Phl p 2, Phl p 5, and/or Phl p 11 is then followed by the prescription of grass pollen-AIT. The presence of IgE to Phl p 12 (profilin), however, is followed by an investigation of a potential Oral Allergy Syndrome (OAS) including SPT/IgE as-says with other pollen, fruit, or vegetable extracts. Furthermore, the identification of IgE to Phl p 7 may be indicative and alerts the doc-tor of a greater severity of disease including a higher risk of asthma (21). In addition, the presence of IgE to pan allergens such as Phl p 12 (profilin) and/or Phl p 7 (polcalcin) may provide an explanation for broad sensitization profiles obtained by extract-based skin test or sIgE testing.

Hymenoptera venom allergy is another excellent example in which sIgE to individual allergens serve as biomarker for genuine honey bee (Api m 1, Api m 3, Api m 4, Api m 10) or yellow jacket sen-sitization (Ves v 1, Ves v 5), while IgE to homologous allergens (such as the hyaluronidases Api m 2 and Ves v 2; the dipeptidyl peptidase IV Api m 5 and Ves v 3 and the vitellogenins Api m 12 and Ves v 6) indicate positive extract-based test results based on cross-reactivity (see chapters B20 and B21).

## 6.2 The impact of molecular assays on doctors' decisions

Providing a clear distinction between co-sensitization and cross-sensitization is an explicit advantage of CRD in allergology.⁸⁰,⁸¹ A study with 651 Italian children suffering from moderate-to-severe pollen-related allergic rhinitis showed interesting results. No IgE to the respective major allergens was detected in significant pro-portions of patients with supposed clinically relevant sensitiza-tion (based on SPT) to mugwort (69%), Betulaceae (60%), pellitory (30%), olive (28%), cypress (15%), and grass (10%). IgE to profilins, polcalcins, or both could justify 37% of these SPT reactions. The SPT-based decision of prescribing specific immunotherapy and/or its composition was adapted in 277 (42%) or 315 (48%) of children according to the⁸² European or American approach, respectively, when taking into account the CRD results. This study reiterates the high and valuable role of CRD on the prescription and composition of AIT, particularly in geographical regions where polysensitization to airborne allergens is frequently observed. Another study, com-prising 1263 Spanish patients and their sensitization patterns to the allergen molecules of grass and olive pollen had an analogous out-come. The entire study population had seasonal allergic rhinitis, with positive SPT results to grass and olive pollens. Of these patients, 922 (73%) would have been prescribed AIT with both grass and olive pollen, if following the traditional diagnostic approach. Incidentally, the AIT composition was modified for 56.8% of the patients after considering additional IgE results obtained by CRD.⁸³

## 6.3 Molecular IgE sensitization profiles as biomarkers predicting efficacy and safety

The heterogeneity of molecular sensitization profiles—A cross-sectional study with 176 Italian grass pollen allergic children
The question remains if singleplex or multiplex testing should be applied:

* if possibly a large number of allergens from one family is involved, multiplex testing might carry some advantages.

* singleplex testing, however, offers an enhanced assay sensitivity, allowing the ultimate exclusion of IgE-mediated sensitization to the allergen in question if the allergen-specific IgE does not exceed the cut-off of 0.1 kU/l.

* therefore, the decision of singleplex or multiplex testing should consider the number of allergens to be tested and the preferred test sensitivity (very low or not as low).

Examples: seed storage proteins like 2S-albumins, 7S-globulins (vi-cilins), 11S-globulins (legumins). Note of caution: The number of storage proteins of different nuts, legumes and seeds that are available for diag-nostics is still limited. This does not allow one to check for the presence of allergen-specific IgE to a full panel of these stable and risk-associated al-lergens. Therefore, allergen extracts are still needed, to indirectly get infor-mation on the potential relevance of the risk-associated storage proteins.

In conclusion, certain scenarios require a complete molecular di-agnostic work-up after taking the history and performing focused extract- and molecule-based sensitization tests. This approach ex-plores the degree and potential clinical relevance of further cross-reactivities to related molecules of a protein family. Singleplex assays would guarantee maximum assay sensitivity; multiplex assays would rather provide a broad panel of related, cross-reactive molecules for further definition of the IgE repertoire. The clinical relevance must be determined by the physician and not by the test, based on pa-tient's history and outcome of challenge tests if needed.

# 6 – Molecular IgE sensitization profiles as biomarkers

Biomarkers are usually molecules that indicate physiological or path-ologic phenomena. They reflect an objectively quantifiable measure of disease expression, severity and/or response to therapy. They can be beneficial in many different settings, especially in diagnostic processes and disease staging, identifying patients who will benefit from the treatment, monitoring disease trends, treatment efficacy and its side effects, predicting long-lasting protection, and thus im-proving acceptance and compliance. Along these lines, detection of molecular IgE sensitization profiles may function as biomarker in several settings as outlined below.

## 6.1 Sensitization to species-specific and cross-reactive molecules as diagnostic biomarkers

Grass pollen allergy represents a typical example for the use of allergen-specific IgE profiles as a biomarker, not only for an etiologi-cal confirmation of the presumptive diagnosis but also for the subse-quent prescription of AIT. Patients with symptoms of allergic rhinitis during the grass pollen season and a positive SPT/IgE response to

grass pollen extracts are further investigated in order to detect serum IgE antibodies to Phl p 1, Phl p 2, Phl p 5, Phl p 7, Phl p 11, and Phl p 12. The identification of IgE antibodies towards one or more of the molecules Phl p 1, Phl p 2, Phl p 5, and/or Phl p 11 is then followed by the prescription of grass pollen-AIT. The presence of IgE to Phl p 12 (profilin), however, is followed by an investigation of a potential Oral Allergy Syndrome (OAS) including SPT/IgE as-says with other pollen, fruit, or vegetable extracts. Furthermore, the identification of IgE to Phl p 7 may be indicative and alerts the doc-tor of a greater severity of disease including a higher risk of asthma (21). In addition, the presence of IgE to pan allergens such as Phl p 12 (profilin) and/or Phl p 7 (polcalcin) may provide an explanation for broad sensitization profiles obtained by extract-based skin test or sIgE testing.

Hymenoptera venom allergy is another excellent example in which sIgE to individual allergens serve as biomarker for genuine honey bee (Api m 1, Api m 3, Api m 4, Api m 10) or yellow jacket sen-sitization (Ves v 1, Ves v 5), while IgE to homologous allergens (such as the hyaluronidases Api m 2 and Ves v 2; the dipeptidyl peptidase IV Api m 5 and Ves v 3 and the vitellogenins Api m 12 and Ves v 6) indicate positive extract-based test results based on cross-reactivity (see chapters B20 and B21).

## 6.2 The impact of molecular assays on doctors' decisions

Providing a clear distinction between co-sensitization and cross-sensitization is an explicit advantage of CRD in allergology.⁸⁰,⁸¹ A study with 651 Italian children suffering from moderate-to-severe pollen-related allergic rhinitis showed interesting results. No IgE to the respective major allergens was detected in significant pro- portions of patients with supposed clinically relevant sensitiza-tion (based on SPT) to mugwort (69%), Betulaceae (60%), pellitory (30%), olive (28%), cypress (15%), and grass (10%). IgE to profilins, polcalcins, or both could justify 37% of these SPT reactions. The SPT-based decision of prescribing specific immunotherapy and/or its composition was adapted in 277 (42%) or 315 (48%) of children according to the⁸² European or American approach, respectively, when taking into account the CRD results. This study reiterates the high and valuable role of CRD on the prescription and composition of AIT, particularly in geographical regions where polysensitization to airborne allergens is frequently observed. Another study, com-prising 1263 Spanish patients and their sensitization patterns to the allergen molecules of grass and olive pollen had an analogous out-come. The entire study population had seasonal allergic rhinitis, with positive SPT results to grass and olive pollens. Of these patients, 922 (73%) would have been prescribed AIT with both grass and olive pollen, if following the traditional diagnostic approach. Incidentally, the AIT composition was modified for 56.8% of the patients after considering additional IgE results obtained by CRD.⁸³

## 6.3 Molecular IgE sensitization profiles as biomarkers predicting efficacy and safety

The heterogeneity of molecular sensitization profiles—A cross-sectional study with 176 Italian grass pollen allergic children
illustrated the vast amount of additional information that can be
obtained with molecular diagnostic tests when compared to extract-
based ones. All patients presented similar profiles when testing
them for the allergenic extract of Timothy grass with conventional
ELISA. They were all positive for Timothy grass, with the concen-
tration ranging in amplitude. This homogeneity disappeared upon
using a molecular assay, which uncovered a noteworthy diversity
of responses. Overall, 39 different IgE sensitization profiles to grass
pollen molecules were detected.⁸⁴ In another cross-sectional study,
82 different profiles were found among 1120 children. Both studies
included monosensitized patients (in most cases to Phl p 1), as well
as those sensitized to 5 of the examined 8 allergenic molecules. A
correlation between the clinical phenotype and molecular sensitiza-
tion profiles was observed. Specifically, Phl p 7 served as a reliable
biomarker for asthma and possibly increased severity of seasonal al-
lergic rhinitis, while Phl p 12 served as a biomarker of oral allergy
syndrome (OAS).⁸⁵ In Germany, IgE results to Phleum pratense mol-
ecules were compared with nasal and conjunctival provocation tests
in 101 adult patients with pollinosis. A significant heterogeneity of
sensitization profiles, as well as a positive correlation between the
number of recognized molecules and the likelihood of a positive
provocation test result was observed. Interestingly, no match was
observed between these IgE profiles and the composition of a previ-
ously published component-resolved specific AIT-containing Phl p
1, Phl p 2, Phl p 5a/b, and Phl p 6.⁸⁶ In a similar fashion, the indi-
vidual sensitization profiles of 119 house dust mite allergic patients
at 20 years of age from the German Multicenter Allergy Study cohort
were extremely heterogenous. Of these, 27 subjects had a monomo-
lecular profile, 50 subjects had an oligomolecular profile (responding
to 2 to 4 molecules), and 42 subjects had positive IgE to ≥5 of the 12
tested molecules.²⁸ A similar picture emerged, when individual mo-
lecular sensitization profiles of 144 patients with honey bee venom
allergy were analyzed. The characterization of IgE reactivity to Api
m 1, Api m 2, Api m 3, Api m 4, Api m 5 and Api m 10 demonstrated
39 of 64 possible sensitization profiles, the ten most frequent pro-
files covering two thirds of the study population.⁸⁷ The heterogene-
ity of the AIT preparations—The standardization of allergic extracts
requires consistent composition combined with stable potency re-
lated to clinical efficacy. Nevertheless, the Monograph on Allergen
Products, a European regulation, allows a wide range of variation.
Through measurement by IgE inhibition tests, it was uncovered that
the percentage of individual allergenic molecules within an extract
varies from 50% to 200%. A fairly simple allergen extract composed
of only 3 major allergenic proteins, can therefore originate differ-
ent batches with different allergen loads that range between very
low and very high, including all intermediate possibilities. As an af-
termath of these regulations, different manufacturers may provide
allergen extracts of the same allergen source that differ greatly in
their molecular composition and potency. The use of molecular as-
says that allow the comparative analysis of single allergens hetero-
geneity of AIT preparation has conclusively been demonstrated for
a number of different allergen sources including birch and grass pol-
len, house dust mite, and insect venom preparations.⁸⁸⁻⁹⁰ Given this
lack of standardization, occurrence of divergent SPT wheal reactions

for the same allergen species in the same patient elicited by different
allergen extracts are not surprising.⁸⁹,⁹¹ As for the *in vitro* settings,
differences in IgE test results to extracts of the same allergen source
may similarly be attributed to the composition of the extract.⁹²

Molecular sensitization profiles as biomarkers predicting AIT efficacy and safety—The use of allergenic molecules in various clinical studies aimed at monitoring changes in the specific antibody repertoire of patients receiving AIT has shown good outcomes.⁹³,⁹⁴ For instance, IgE sensitization profile before the start of AIT was proven to directly impact the efficacy of SLIT in patients with mite allergy. No efficacy of a house dust mite (HDM)-SLIT was observed among the total HDM-allergic population, including all IgE variabilities. Interestingly, a positive outcome is seen when analyzing only patients with IgE to Der p 1 or Der p 2. These results lead to believe that patients with stronger molecular spreading have a lower efficacy of AIT.⁸⁴,⁹⁵ However, specific studies designed to address the efficacy of molecular diagnosis driven AIT need to be performed since to date only post-hoc analysis have been performed with non-uniform results. While the results of Chen KW et al. suggest that the use of molecular assays is a promising approach for predicting and monitoring AIT efficacy, Arroabarren et al.⁹⁶ could not find a significant association between AIT efficacy and the HDM sensitization profile. A recent study on 24 HDM allergic patients who had received 1 year of treatment with Alutard SQ 510 concluded that the stratification of patients with HDM allergy according to molecular sensitization profiles and molecular monitoring of AIT-induced IgG responses may enhance the success of AIT.⁹⁷

The potential use of molecular sensitization profiles as biomark-
ers predicting AIT efficacy and safety has also been suggested in
the field of Hymenoptera venom allergy. In patients with honey
bee venom allergy sensitization to Api m 4 has been reported to
be associated with an increased risk of systemic side effects during
the induction phase of venom IT.⁹⁸ Furthermore, dominant IgE
sensitization to the low abundance allergen Api m 10, which has
been reported to be absent or underrepresented in certain venom
preparations,⁹⁹,¹⁰⁰ has been associated with an increased risk of
treatment failure in honey bee venom AIT.¹⁰¹ These recent studies
emphasizes that the use of molecular assays for the prediction and
monitoring of AIT efficacy is a promising approach. However, more
investigations in particular prospective studies are needed to con-
firm molecular IgE sensitization profiles as a predictive biomarker
of efficacy.

7 – Allergenic molecules sorted by allergen
sources and their relevance for clinical diagnostics

The use of molecular allergy diagnostics can only be successfully
implemented in our daily routine when we have all the available
information on the clinical relevance of each and every allergen at
our fingertips. In an attempt to provide information on the clinically
most relevant allergens, the authors of MAUG 2.0 provide a compre-
hensive list of allergenic molecules at the end of the e-book version
at www.eaaci.org
illustrated the vast amount of additional information that can be
obtained with molecular diagnostic tests when compared to extract-
based ones. All patients presented similar profiles when testing
them for the allergenic extract of Timothy grass with conventional
ELISA. They were all positive for Timothy grass, with the concen-
tration ranging in amplitude. This homogeneity disappeared upon
using a molecular assay, which uncovered a noteworthy diversity
of responses. Overall, 39 different IgE sensitization profiles to grass
pollen molecules were detected.⁸⁴ In another cross-sectional study,
82 different profiles were found among 1120 children. Both studies
included monosensitized patients (in most cases to Phl p 1), as well
as those sensitized to 5 of the examined 8 allergenic molecules. A
correlation between the clinical phenotype and molecular sensitiza-
tion profiles was observed. Specifically, Phl p 7 served as a reliable
biomarker for asthma and possibly increased severity of seasonal al-
lergic rhinitis, while Phl p 12 served as a biomarker of oral allergy
syndrome (OAS).⁸⁵ In Germany, IgE results to Phleum pratense mol-
ecules were compared with nasal and conjunctival provocation tests
in 101 adult patients with pollinosis. A significant heterogeneity of
sensitization profiles, as well as a positive correlation between the
number of recognized molecules and the likelihood of a positive
provocation test result was observed. Interestingly, no match was
observed between these IgE profiles and the composition of a previ-
ously published component-resolved specific AIT-containing Phl p
1, Phl p 2, Phl p 5a/b, and Phl p 6.⁸⁶ In a similar fashion, the indi-
vidual sensitization profiles of 119 house dust mite allergic patients
at 20 years of age from the German Multicenter Allergy Study cohort
were extremely heterogenous. Of these, 27 subjects had a monomo-
lecular profile, 50 subjects had an oligomolecular profile (responding
to 2 to 4 molecules), and 42 subjects had positive IgE to ≥5 of the 12
tested molecules.²⁸ A similar picture emerged, when individual mo-
lecular sensitization profiles of 144 patients with honey bee venom
allergy were analyzed. The characterization of IgE reactivity to Api
m 1, Api m 2, Api m 3, Api m 4, Api m 5 and Api m 10 demonstrated
39 of 64 possible sensitization profiles, the ten most frequent pro-
files covering two thirds of the study population.⁸⁷ The heterogene-
ity of the AIT preparations—The standardization of allergic extracts
requires consistent composition combined with stable potency re-
lated to clinical efficacy. Nevertheless, the Monograph on Allergen
Products, a European regulation, allows a wide range of variation.
Through measurement by IgE inhibition tests, it was uncovered that
the percentage of individual allergenic molecules within an extract
varies from 50% to 200%. A fairly simple allergen extract composed
of only 3 major allergenic proteins, can therefore originate differ-
ent batches with different allergen loads that range between very
low and very high, including all intermediate possibilities. As an af-
termath of these regulations, different manufacturers may provide
allergen extracts of the same allergen source that differ greatly in
their molecular composition and potency. The use of molecular as-
says that allow the comparative analysis of single allergens hetero-
geneity of AIT preparation has conclusively been demonstrated for
a number of different allergen sources including birch and grass pol-
len, house dust mite, and insect venom preparations.⁸⁸⁻⁹⁰ Given this
lack of standardization, occurrence of divergent SPT wheal reactions

for the same allergen species in the same patient elicited by different
allergen extracts are not surprising.⁸⁹,⁹¹ As for the *in vitro* settings,
differences in IgE test results to extracts of the same allergen source
may similarly be attributed to the composition of the extract.⁹²

Molecular sensitization profiles as biomarkers predicting AIT efficacy and safety—The use of allergenic molecules in various clinical studies aimed at monitoring changes in the specific antibody repertoire of patients receiving AIT has shown good outcomes.⁹³,⁹⁴ For instance, IgE sensitization profile before the start of AIT was proven to directly impact the efficacy of SLIT in patients with mite allergy. No efficacy of a house dust mite (HDM)-SLIT was observed among the total HDM-allergic population, including all IgE variabilities. Interestingly, a positive outcome is seen when analyzing only patients with IgE to Der p 1 or Der p 2. These results lead to believe that patients with stronger molecular spreading have a lower efficacy of AIT.⁸⁴,⁹⁵ However, specific studies designed to address the efficacy of molecular diagnosis driven AIT need to be performed since to date only post-hoc analysis have been performed with non-uniform results. While the results of Chen KW et al. suggest that the use of molecular assays is a promising approach for predicting and monitoring AIT efficacy, Arroabarren et al.⁹⁶ could not find a significant association between AIT efficacy and the HDM sensitization profile. A recent study on 24 HDM allergic patients who had received 1 year of treatment with Alutard SQ 510 concluded that the stratification of patients with HDM allergy according to molecular sensitization profiles and molecular monitoring of AIT-induced IgG responses may enhance the success of AIT.⁹⁷

The potential use of molecular sensitization profiles as biomark-
ers predicting AIT efficacy and safety has also been suggested in
the field of Hymenoptera venom allergy. In patients with honey
bee venom allergy sensitization to Api m 4 has been reported to
be associated with an increased risk of systemic side effects during
the induction phase of venom IT.⁹⁸ Furthermore, dominant IgE
sensitization to the low abundance allergen Api m 10, which has
been reported to be absent or underrepresented in certain venom
preparations,⁹⁹,¹⁰⁰ has been associated with an increased risk of
treatment failure in honey bee venom AIT.¹⁰¹ These recent studies
emphasizes that the use of molecular assays for the prediction and
monitoring of AIT efficacy is a promising approach. However, more
investigations in particular prospective studies are needed to con-
firm molecular IgE sensitization profiles as a predictive biomarker
of efficacy.

7 – Allergenic molecules sorted by allergen
sources and their relevance for clinical diagnostics

The use of molecular allergy diagnostics can only be successfully
implemented in our daily routine when we have all the available
information on the clinical relevance of each and every allergen at
our fingertips. In an attempt to provide information on the clinically
most relevant allergens, the authors of MAUG 2.0 provide a compre-
hensive list of allergenic molecules at the end of the e-book version
at www.eaaci.org
# A04 - Singleplex and multiplex Immunoassays

Robert G. Hamilton, Jörg Kleine-Tebbe

## Highlights

* IgE antibody tests are run as singleplex (one), multi-allergen (<100) and multiplex (>100 allergen specificities) assays, all with particular design and performance features

* Allergen extracts remain the principal reagents for IgE assays; allergenic molecules supplement labile or missing allergens in extracts or are analyzed individually

* Allergenic molecules enhance the IgE assay's analytical sensitivity, and improve its analytical specificity by separating serological cross-reactivity from (genuine) sensitization to an allergen source.

* The relevance of positive allergen-specific IgE responses to extracts or molecules can only be determined by the physician based on the clinical context and not by the test itself.

## 1 - Introduction

The serological measurement of allergen-specific IgE antibodies provides the clinician with a measure of a patient's allergic sensitization profile. Two fundamental types of serological IgE antibody assays are performed in the clinical immunology laboratory. "Singleplex" or "monoplex" assays refer to laboratory methods in which one analyte is measured per analysis. "Multiplex" assays permit more than one analyte to be detected and quantified in a single assay analysis.¹⁰²

This report examines the technology, performance, and application of singleplex and multiplex IgE antibody assays that utilize allergen extracts, allergenic molecules (components) and select epitopes on allergenic molecules in the diagnosis and subsequent management of human allergic disease.

### ImmunoChemistry Design Considerations

Two fundamental immunoassay chemistries that have been referred to as "classical" or "reverse" assay formats have been used to detect IgE antibody (Table 1). The noncompetitive, heterogeneous (separation of free and bound), immunometric (labeled antibody) immunoassay¹⁰³ that employs allergen immobilized on a solid phase "allergosorbent" to bind specific antibodies of all isotypes from serum is the design that has endured in both singleplex and multiplex assays that are used in clinical laboratories (Figure 7). Following a buffer wash to separate free and bound human antibody, radionuclide-, enzyme- or fluorescence-labeled anti-human IgE is added to detect IgE antibodies that have bound to immobilized allergen. The magnitude of the response (counts per minute-radioactivity, optical density, chemiluminescence, or fluorescence) after the final buffer wash is proportional to the quantity of allergen-specific IgE antibody in the original test serum.

## 2 - Reverse IgE format: basis of certain singleplex assays

A reverse or capture anti-IgE assay design uses a second step liquid-phase allergen to detect allergen-specific IgE antibody. In this assay (Figure 7 - bottom right panel), all IgE (in theory) is initially captured from serum by a paramagnetic particle solid-phase anti-IgE

<table><caption>TABLE 1 Different formats and features of allergen-specific IgE-assays. "Classical" IgE assay format: Basis of most current singleplex and multiplex assays</caption><thead><tr><th></th><th>"classical" IgE assay format</th><th>"reverse" IgE assay format</th></tr></thead><tbody><tr><td>1st assay step</td><td>allergen-specific IgE and other antibody isotypes (e.g., IgG) bind immobilized allergen reagent</td><td>Immobilized Anti-IgE ideally binds entire (total) IgE in the reaction vessel</td></tr><tr><td>2nd assay step</td><td>Labeled anti-IgE binds only allergen-specific IgE antibody</td><td>Labeled allergen reagent binds only allergen-specific IgE</td></tr><tr><td>Advantages (pro)</td><td>Detection of the entire allergen-specific IgE repertoire (low and high affinity)</td><td>detection of allergen-specific IgE principally of higher affinity no competitive inhibition through allergen-specific immunoglobulins other than IgE, (i.e. IgG)</td></tr><tr><td>Limitations (con)</td><td>competitive inhibition by allergen-specific immunoglobulins other than IgE, (i.e. IgG antibody after subcutaneous immunotherapy or natural exposure)</td><td>large amounts of anti-IgE are needed for sera with high total IgE levels biased results (lower values) in samples with a low specific to total IgE-ratio.¹⁰⁶</td></tr><tr><td>Potential use in multiplex assay formats</td><td>micronization of binding chemistry and limited amounts of allergen reagents required</td><td>less useful due to the need of multiple labeled allergens (to be put into one reaction vessel)</td></tr><tr><td>References</td><td>Ekins¹⁰³</td><td>Ricci et al.¹⁰⁴; Petersen et al.¹⁰⁵</td></tr></tbody></table>
# A04 - Singleplex and multiplex Immunoassays

Robert G. Hamilton, Jörg Kleine-Tebbe

## Highlights

* IgE antibody tests are run as singleplex (one), multi-allergen (<100) and multiplex (>100 allergen specificities) assays, all with particular design and performance features

* Allergen extracts remain the principal reagents for IgE assays; allergenic molecules supplement labile or missing allergens in extracts or are analyzed individually

* Allergenic molecules enhance the IgE assay's analytical sensitivity, and improve its analytical specificity by separating serological cross-reactivity from (genuine) sensitization to an allergen source.

* The relevance of positive allergen-specific IgE responses to extracts or molecules can only be determined by the physician based on the clinical context and not by the test itself.

## 1 - Introduction

The serological measurement of allergen-specific IgE antibodies provides the clinician with a measure of a patient's allergic sensitization profile. Two fundamental types of serological IgE antibody assays are performed in the clinical immunology laboratory. "Singleplex" or "monoplex" assays refer to laboratory methods in which one analyte is measured per analysis. "Multiplex" assays permit more than one analyte to be detected and quantified in a single assay analysis.¹⁰²

This report examines the technology, performance, and application of singleplex and multiplex IgE antibody assays that utilize allergen extracts, allergenic molecules (components) and select epitopes on allergenic molecules in the diagnosis and subsequent management of human allergic disease.

### ImmunoChemistry Design Considerations

Two fundamental immunoassay chemistries that have been referred to as "classical" or "reverse" assay formats have been used to detect IgE antibody (Table 1). The noncompetitive, heterogeneous (separation of free and bound), immunometric (labeled antibody) immunoassay¹⁰³ that employs allergen immobilized on a solid phase "allergosorbent" to bind specific antibodies of all isotypes from serum is the design that has endured in both singleplex and multiplex assays that are used in clinical laboratories (Figure 7). Following a buffer wash to separate free and bound human antibody, radionuclide-, enzyme- or fluorescence-labeled anti-human IgE is added to detect IgE antibodies that have bound to immobilized allergen. The magnitude of the response (counts per minute-radioactivity, optical density, chemiluminescence, or fluorescence) after the final buffer wash is proportional to the quantity of allergen-specific IgE antibody in the original test serum.

## 2 - Reverse IgE format: basis of certain singleplex assays

A reverse or capture anti-IgE assay design uses a second step liquid-phase allergen to detect allergen-specific IgE antibody. In this assay (Figure 7 - bottom right panel), all IgE (in theory) is initially captured from serum by a paramagnetic particle solid-phase anti-IgE

<table><caption>TABLE 1 Different formats and features of allergen-specific IgE-assays. "Classical" IgE assay format: Basis of most current singleplex and multiplex assays</caption><thead><tr><th></th><th>"classical" IgE assay format</th><th>"reverse" IgE assay format</th></tr></thead><tbody><tr><td>1st assay step</td><td>allergen-specific IgE and other antibody isotypes (e.g., IgG)<br>bind immobilized allergen reagent</td><td>Immobilized Anti-IgE ideally binds entire (total) IgE in the reaction vessel</td></tr><tr><td>2nd assay step</td><td>Labeled anti-IgE binds only allergen-specific IgE antibody</td><td>Labeled allergen reagent binds only allergen-specific IgE</td></tr><tr><td>Advantages (pro)</td><td>Detection of the entire allergen-specific IgE repertoire (low and high affinity)</td><td>detection of allergen-specific IgE principally of higher affinity no competitive inhibition through allergen-specific immunoglobulins other than IgE, (i.e. IgG)</td></tr><tr><td>Limitations (con)</td><td>competitive inhibition by allergen-specific immunoglobulins other than IgE, (i.e. IgG antibody after subcutaneous immunotherapy or natural exposure)</td><td>large amounts of anti-IgE are needed for sera with high total IgE levels biased results (lower values) in samples with a low specific to total IgE-ratio.¹⁰⁶</td></tr><tr><td>Potential use in multiplex assay formats</td><td>micronization of binding chemistry and limited amounts of allergen reagents required</td><td>less useful due to the need of multiple labeled allergens (to be put into one reaction vessel)</td></tr><tr><td>References</td><td>Ekins¹⁰³</td><td>Ricci et al.¹⁰⁴; Petersen et al.¹⁰⁵</td></tr></tbody></table>
<figure><img src="image_18.png" /><figcaption>FIGURE 7 Top panel: General principle of “classical” solid phase IgE assay formats for total and allergen-specific IgE quantification; Bottom panel: General principle of the fluid phase “classical” (left) and “reverse” IgE assay format (right) for allergen-specific IgE quantification</figcaption></figure>

in molar excess to the amount of IgE in most test sera. Following the capture of human IgE, allergen-specific IgE antibody is detected with limited quantities of labeled allergen. The reverse phase assay format has been used for IgE antibody quantitation in the ADVIA Centaur.<sup>104,105</sup> The principal advantage of the reverse phase assay over the classical allergosorbent-based singleplex assay is its tendency to measure principally high affinity IgE antibody that is assumed to be “more” clinically relevant (Table 1). By contrast, assays that use molar excess amounts of allergen that have been immobilized on an allergosorbent tend to more broadly detect both low- and high-affinity IgE antibody. The reverse assay format also addresses the concern of competitive inhibition caused by allergen-specific antibodies of non-IgE isotypes origin such as IgG anti-allergen that can achieve microgram per ml levels in sera from individuals receiving immunotherapy. The reverse assay format is, however, less amenable to use in multiplex assays where multiple labeled allergens would have to be added to the same reaction vessel. It suffers from a requirement for large amounts of anti-IgE capture antibody to insure the binding of all IgE molecules from the test serum. The reverse assay design can also show a major bias because its performance depends on the fraction of the total IgE that is specific for the allergen

of interest. These assay design constraints have resulted in the disappearance of the reverse assay format from use in clinical laboratories in the USA and elsewhere.

### 3 – Heterologous calibration based on total IgE for singleplex allergen-specific IgE systems

Consensus has been established that a single generic total serum IgE calibration system is the only workable calibration strategy for use in clinical IgE antibody assays.<sup>102</sup> It allows interpolation of IgE antibody results from any of the hundreds of allergen specificities as long as the total serum IgE and allergen-specific IgE portions of the assay dilute out in parallel with each other. The total serum IgE “heterologous” calibration system that is used in all regulatory cleared singleplex assays is traceable to the World Health Organization's recently depleted 75/502 and currently used third 11/234 human IgE Reference Preparations.<sup>106</sup> This calibration system allows interpolation of IgE antibody results from a limit of quantitation of 0.1 kU/L to 100 kU/L levels of IgE antibody. While rarely performed in clinical testing, serum levels of IgE antibody greater than 100 kU/L can be accurately determined by re-analysis of the serum at a dilution and subsequent mathematical correction for the dilution factor. The alternative to the total serum IgE-based heterologous interpolation scheme is the use of individual allergen-specific calibrations, one for each allergen specificity. Early attempts involving the use of this approach exposed its major limitation, which involved a demand for liter quantities of IgE-positive sera for each specificity.<sup>107</sup> This made the multiple specific IgE antibody calibration strategy impractical, especially since there are no internationally recognized polyclonal human IgE antibody reference preparations.

### 4 – Multi-allergen versus multiplex assays

A true multiplex antibody assay allows many specificities of a single antibody isotype (e.g., IgE) to be individually detected and semi-quantified in a single analysis.<sup>102</sup> This assay design can be distinguished from a “multi-allergen” screening assay in which many allergen specificities from a common group (aeroallergens or food allergens) are mixed and immobilized together as extracts or components on a single solid phase. This multi-allergen reagent is typically used in a singleplex assay format to simultaneously detect specific IgE to multiple antibody specificities in a single reaction. A single qualitative (positive or negative) result is generated for each specimen based on a positive/negative cutpoint. However, the actual allergen specificities that produce a positive IgE antibody response in the multi-allergen screen cannot be definitively identified by the requesting physician without further analysis using additional singleplex assay analyses, one for each of the individual allergen specificities on the multi-allergosorbent. One widely used multi-aeroallergen screening assay measures IgE antibody to 10 or more aeroallergen extracts.<sup>108</sup> The allergen specificities immobilized on
<figure><img src="image_18.png" /><figcaption>FIGURE 7 Top panel: General principle of “classical” solid phase IgE assay formats for total and allergen-specific IgE quantification; Bottom panel: General principle of the fluid phase “classical” (left) and “reverse” IgE assay format (right) for allergen-specific IgE quantification</figcaption></figure>

in molar excess to the amount of IgE in most test sera. Following the capture of human IgE, allergen-specific IgE antibody is detected with limited quantities of labeled allergen. The reverse phase assay format has been used for IgE antibody quantitation in the ADVIA Centaur.<sup>104,105</sup> The principal advantage of the reverse phase assay over the classical allergosorbent-based singleplex assay is its tendency to measure principally high affinity IgE antibody that is assumed to be "more" clinically relevant (Table 1). By contrast, assays that use molar excess amounts of allergen that have been immobilized on an allergosorbent tend to more broadly detect both low- and high-affinity IgE antibody. The reverse assay format also addresses the concern of competitive inhibition caused by allergen-specific antibodies of non-IgE isotypes origin such as IgG anti-allergen that can achieve microgram per ml levels in sera from individuals receiving immunotherapy. The reverse assay format is, however, less amenable to use in multiplex assays where multiple labeled allergens would have to be added to the same reaction vessel. It suffers from a requirement for large amounts of anti-IgE capture antibody to insure the binding of all IgE molecules from the test serum. The reverse assay design can also show a major bias because its performance depends on the fraction of the total IgE that is specific for the allergen

of interest. These assay design constraints have resulted in the disappearance of the reverse assay format from use in clinical laboratories in the USA and elsewhere.

### 3 – Heterologous calibration based on total IgE for singleplex allergen-specific IgE systems

Consensus has been established that a single generic total serum IgE calibration system is the only workable calibration strategy for use in clinical IgE antibody assays.<sup>102</sup> It allows interpolation of IgE antibody results from any of the hundreds of allergen specificities as long as the total serum IgE and allergen-specific IgE portions of the assay dilute out in parallel with each other. The total serum IgE "heterologous" calibration system that is used in all regulatory cleared singleplex assays is traceable to the World Health Organization's recently depleted 75/502 and currently used third 11/234 human IgE Reference Preparations.<sup>106</sup> This calibration system allows interpolation of IgE antibody results from a limit of quantitation of 0.1 kU/L to 100 kU/L levels of IgE antibody. While rarely performed in clinical testing, serum levels of IgE antibody greater than 100 kU/L can be accurately determined by re-analysis of the serum at a dilution and subsequent mathematical correction for the dilution factor. The alternative to the total serum IgE-based heterologous interpolation scheme is the use of individual allergen-specific calibrations, one for each allergen specificity. Early attempts involving the use of this approach exposed its major limitation, which involved a demand for liter quantities of IgE-positive sera for each specificity.<sup>107</sup> This made the multiple specific IgE antibody calibration strategy impractical, especially since there are no internationally recognized polyclonal human IgE antibody reference preparations.

### 4 – Multi-allergen versus multiplex assays

A true multiplex antibody assay allows many specificities of a single antibody isotype (e.g., IgE) to be individually detected and semi-quantified in a single analysis.<sup>102</sup> This assay design can be distinguished from a “multi-allergen” screening assay in which many allergen specificities from a common group (aeroallergens or food allergens) are mixed and immobilized together as extracts or components on a single solid phase. This multi-allergen reagent is typically used in a singleplex assay format to simultaneously detect specific IgE to multiple antibody specificities in a single reaction. A single qualitative (positive or negative) result is generated for each specimen based on a positive/negative cutpoint. However, the actual allergen specificities that produce a positive IgE antibody response in the multi-allergen screen cannot be definitively identified by the requesting physician without further analysis using additional singleplex assay analyses, one for each of the individual allergen specificities on the multi-allergosorbent. One widely used multi-aeroallergen screening assay measures IgE antibody to 10 or more aeroallergen extracts.<sup>108</sup> The allergen specificities immobilized on
a single solid phase are carefully selected because they are known to be unique or cross-reactive with the major specificities that induce the majority of aeroallergen-related allergic symptoms. Due to its high negative predictive value, this particular multi-aeroallergen assay serves as a cost-effective screen to rule out allergic sensitization in an individual with a questionable respiratory allergy history and to define the atopic status of individuals enrolling in asthma studies.¹⁰⁹

## 5 – Heterogeneity in IgE antibody measurements from different assay types and manufacturers

In most multiplex assays, the small quantities of the individual allergens bound to a solid phase contrast with the higher IgE antibody-binding capacity present on individual hydrophilic polymer and bead-based allergosorbents that are used clinically. Law of Mass Action constraints cause these assays to detect different distributions of allergen-specific IgE antibody in any given serum. The amount of antibody detected in the assay is dependent on multiple factors including the IgE antibody's concentration, affinity, epitope specificity, IgE-specific activity (specific to total IgE ratio)¹¹⁰ and level of non-IgE antibody specific for the allergen.¹⁰² The more antigen-limiting multiplex allergosorbents tend to bind more allergen-specific IgE antibody when it is higher in concentration, has a higher affinity, the serum has a higher specific IgE antibody to total IgE ratio and a lower concentration of competing allergen-specific non-IgE (typically IgG) antibody. These mass action considerations have important assay performance consequences, especially when analyzing sera with nanogram quantities of IgE antibody that are present with high microgram/ml levels of allergen-specific IgG antibody.¹¹¹ Such high IgG anti-allergen levels can result from inadvertent natural exposure to high levels of allergen or hyper-immunization through allergen immunotherapy. IgG antibody competes with the lower nanogram/ml levels of IgE antibody for limited allergen binding sites on the multiplex chip allergosorbent.¹¹¹,¹¹² This constraint has been cited as an advantage of the multiplex assay format in that its lower level of detected allergen-specific IgE antibody in the presence of high allergen-specific IgG may more closely reflect the true biological consequence of IgG interference with allergen binding to IgE attached to effector cells.

## 6 – Advantages and limitations of multiplex assays

Table 2 summarizes commonly cited performance and assay design advantages and limitations of a multiplex assay in comparison with the singleplex assay. Multiplex assays are attractive because they tend to have a shorter turn-around time for result generation. They tend to use less specimen volume by simultaneously testing multiple IgE antibody specificities in a small surface area on the solid phase. Their assay design tends to be simpler, with fewer reagents and less technician time that reduces overall costs. Multiplex assays,

especially in a hand-held cassette format, are attractive for use as point of care tests.¹¹³,¹¹⁴ These advantages are offset by the multiplex assays' potentially higher limit of quantitation, reduced ability to provide quantitative levels of antibody for each respective IgE specificity, and an increased challenge in optimizing the assay, which involves simultaneous quality control of many immobilized allergens. There is the potential for greater inter-lot variability as a result of the need to balance multiple reagents in different spots on a single allergosorbent. The fixed allergen menus of the multiplex assay encourages the testing of IgE antibody for unwanted or unnecessary specificities. Finally, there can be additional expense associated with the need to purchase new equipment to perform a multiplex assay.

## 7 – Current Assay Technology

Common IgE assay systems based on singleplex technology

Many versions of the “classical” IgE assay format have been cleared by governmental regulators over the years. Worldwide, three singleplex autoanalyzers that use the “classical” allergosorbent design dominate the current clinical laboratory market. These are the ImmunoCAP Specific IgE test (Thermo Fisher Scientific/Phadia); Immulite (Siemens Healthcare Diagnostics) and the Noveos (Hycor Biomedical). In Europe, there are additional assays with the EU mark that use a similar assay design but that are not available worldwide for use in clinical laboratories. The performance characteristics of the three predominant singleplex autoanalyzers have been assessed using masked patient specimens and inter-laboratory proficiency data. All three singleplex autoanalyzers use an analogous total IgE calibration curve. They display good precision, reproducibility and they report down to the same 0.1 kU/L limit of quantitation. Multiple studies have confirmed, however, that they report different levels of IgE antibody for any given specificity, which indicates that they detect different distributions of allergen-specific IgE antibody most probably due to the use of different allergen-containing reagents and possibly a result of slightly different procedures for assay calibration and data computation.

## 8 – Introduction of single molecules (components) into singleplex and multiplex assays

The single most important scientific advance to impact on the use of multiplex assays in the diagnostic allergy laboratory has been the identification since 2000 and purification of allergenic components from principal aero-, food and venom allergens as discussed extensively throughout this book. Molecular biology techniques have been employed to generate recombinant forms of many of the allergens and others are isolated from extracted native sources using various purification procedures. Allergen libraries have been created as illustrated by the food allergen library from the EuroPrevall project that has established rigorous verification and purity requirements for allergenic molecules.¹¹⁵ Well-characterized allergenic
a single solid phase are carefully selected because they are known to be unique or cross-reactive with the major specificities that induce the majority of aeroallergen-related allergic symptoms. Due to its high negative predictive value, this particular multi-aeroallergen assay serves as a cost-effective screen to rule out allergic sensitization in an individual with a questionable respiratory allergy history and to define the atopic status of individuals enrolling in asthma studies.¹⁰⁹

## 5 – Heterogeneity in IgE antibody measurements from different assay types and manufacturers

In most multiplex assays, the small quantities of the individual allergens bound to a solid phase contrast with the higher IgE antibody-binding capacity present on individual hydrophilic polymer and bead-based allergosorbents that are used clinically. Law of Mass Action constraints cause these assays to detect different distributions of allergen-specific IgE antibody in any given serum. The amount of antibody detected in the assay is dependent on multiple factors including the IgE antibody's concentration, affinity, epitope specificity, IgE-specific activity (specific to total IgE ratio)¹¹⁰ and level of non-IgE antibody specific for the allergen.¹⁰² The more antigen-limiting multiplex allergosorbents tend to bind more allergen-specific IgE antibody when it is higher in concentration, has a higher affinity, the serum has a higher specific IgE antibody to total IgE ratio and a lower concentration of competing allergen-specific non-IgE (typically IgG) antibody. These mass action considerations have important assay performance consequences, especially when analyzing sera with nanogram quantities of IgE antibody that are present with high microgram/ml levels of allergen-specific IgG antibody.¹¹¹ Such high IgG anti-allergen levels can result from inadvertent natural exposure to high levels of allergen or hyper-immunization through allergen immunotherapy. IgG antibody competes with the lower nanogram/ml levels of IgE antibody for limited allergen binding sites on the multiplex chip allergosorbent.¹¹¹,¹¹² This constraint has been cited as an advantage of the multiplex assay format in that its lower level of detected allergen-specific IgE antibody in the presence of high allergen-specific IgG may more closely reflect the true biological consequence of IgG interference with allergen binding to IgE attached to effector cells.

## 6 – Advantages and limitations of multiplex assays

Table 2 summarizes commonly cited performance and assay design advantages and limitations of a multiplex assay in comparison with the singleplex assay. Multiplex assays are attractive because they tend to have a shorter turn-around time for result generation. They tend to use less specimen volume by simultaneously testing multiple IgE antibody specificities in a small surface area on the solid phase. Their assay design tends to be simpler, with fewer reagents and less technician time that reduces overall costs. Multiplex assays,

especially in a hand-held cassette format, are attractive for use as point of care tests.¹¹³,¹¹⁴ These advantages are offset by the multiplex assays' potentially higher limit of quantitation, reduced ability to provide quantitative levels of antibody for each respective IgE specificity, and an increased challenge in optimizing the assay, which involves simultaneous quality control of many immobilized allergens. There is the potential for greater inter-lot variability as a result of the need to balance multiple reagents in different spots on a single allergosorbent. The fixed allergen menus of the multiplex assay encourages the testing of IgE antibody for unwanted or unnecessary specificities. Finally, there can be additional expense associated with the need to purchase new equipment to perform a multiplex assay.

## 7 – Current Assay Technology

Common IgE assay systems based on singleplex technology

Many versions of the “classical” IgE assay format have been cleared by governmental regulators over the years. Worldwide, three singleplex autoanalyzers that use the “classical” allergosorbent design dominate the current clinical laboratory market. These are the ImmunoCAP Specific IgE test (Thermo Fisher Scientific/Phadia); Immulite (Siemens Healthcare Diagnostics) and the Noveos (Hycor Biomedical). In Europe, there are additional assays with the EU mark that use a similar assay design but that are not available worldwide for use in clinical laboratories. The performance characteristics of the three predominant singleplex autoanalyzers have been assessed using masked patient specimens and inter-laboratory proficiency data. All three singleplex autoanalyzers use an analogous total IgE calibration curve. They display good precision, reproducibility and they report down to the same 0.1 kU/L limit of quantitation. Multiple studies have confirmed, however, that they report different levels of IgE antibody for any given specificity, which indicates that they detect different distributions of allergen-specific IgE antibody most probably due to the use of different allergen-containing reagents and possibly a result of slightly different procedures for assay calibration and data computation.

## 8 – Introduction of single molecules (components) into singleplex and multiplex assays

The single most important scientific advance to impact on the use of multiplex assays in the diagnostic allergy laboratory has been the identification since 2000 and purification of allergenic components from principal aero-, food and venom allergens as discussed extensively throughout this book. Molecular biology techniques have been employed to generate recombinant forms of many of the allergens and others are isolated from extracted native sources using various purification procedures. Allergen libraries have been created as illustrated by the food allergen library from the EuroPrevall project that has established rigorous verification and purity requirements for allergenic molecules.¹¹⁵ Well-characterized allergenic
<table><caption>TABLE 2 Advantages and limitations of singleplex and multiplex assay technology for allergen-specific IgE testing that utilizes allergenic molecules (components)</caption><thead><tr><th></th><th>Singleplex IgE assay format</th><th>Multiplex IgE assay format</th></tr></thead><tbody><tr><td>Performance Related<br>Advantages (pro)</td><td><ul><li>Increased assay analytical sensitivity (lower Limit of Quantitation, LoQ)</li><li>Potentially more precise quantification and precision, facilitating comparisons between different allergen reagents (extracts versus molecules)</li><li>More established internal and external quality control measures (proficiency testing)</li></ul></td><td><ul><li>Increased speed of analysis and reduced result turn-around time</li><li>Conservation of sample volume facilitating pediatric testing</li></ul></td></tr><tr><td>Assay Design and Cost<br>Related Advantages (pro)</td><td><ul><li>Traceable of allergen-specific IgE values to a total human IgE International Reference Preparation</li><li>Similar units for total IgE and allergen-specific IgE due to heterologous calibration (permits calculation of allergen-specific IgE/total IgE-ratio)</li><li>Global availability in many countries</li><li>In case of limited number of samples more cost efficient</li><li>Minimizes unneeded testing</li></ul></td><td><ul><li>Greater simplicity</li><li>Reduced cost due to fewer required reagents</li><li>Reduced technician intervention</li><li>Optimal design applications for point of care tests</li></ul></td></tr><tr><td>Performance Limitations<br>(con)</td><td>More costly due to increased need for reagents to evaluate polysensitized patients<br>More technical intervention<br>Limited answers in case of few samples per subject<br>Expensive in case of large scale screening (i.e. multi-sensitised subjects)</td><td><ul><li>Potentially lower analytical sensitivity for each analyte specificity measured (higher limit of detection, LoD)</li><li>Reduced ability to accurately quantify each IgE antibody</li><li>Encouragement of abusive testing which involves the measurement of unwanted or unneeded IgE antibody specificities</li></ul></td></tr><tr><td>Assay Design and Cost<br>Related Limitations (con)</td><td><ul><li>More serum required, particularly in case of many samples</li><li>Potentially slower analysis</li><li>Likely more sophisticated assay format</li></ul></td><td><ul><li>Less global availability</li><li>Cost of the new instrumentation and reagents</li><li>Greater challenge in managing different levels of non-specific binding</li><li>Enhanced challenges in optimizing, balancing and standardizing assay reagents and assay quality control</li><li>Potential greater inter-lot variability</li></ul></td></tr></tbody></table>

components from cow's and goat's milk, chicken egg, fish, shrimp, hazelnut, peanut, celery and fruits from the Rosaceae family (apple and peach) have been produced. Documentation of these allergenic components has involved extensive analytical, immunochemical and 3-dimensional structural analyses.

The availability of unlimited quantities of the molecular allergens has allowed multiplex chip microarray based assay methods to be used for rapid simultaneous evaluation of human sera for IgE antibodies to multiple allergen specificities. The most important illustration of technology transition from singleplex to multiplex assays has involved the chip-based multiplex IgE antibody assay initially reported by Hiller et al.¹¹⁶ The original chip-based microarray utilized 49 purified allergen molecules, which were covalently immobilized in fixed microdot arrays on a preactivated glass slide. IgE antibody profiles of allergic individuals were evaluated to disease-causing

allergens in a single multiplex analysis using 30 microliters of undiluted serum. With this report, serious clinical application of both allergenic components and multiplex assay methods became available to evaluate individuals for allergic disease. From this initial proof of concept, the repertoire of allergens has increased and the assays' lower limit of quantitation and reproducibility have continued to improve. The commercially available version of this assay is the immune solid phase allergen chip or ImmunoCAP ISAC (Thermo Fisher Scientific/Phadia), which requires 30 microliters of serum to detect IgE antibody to 112 individual allergenic molecules that are in a static or planar array in triplicate on a glass slide.¹¹⁷⁻¹¹⁹

The ImmunoCAP ISAC reports IgE antibody levels in ISU units, which are considered semi-quantitative¹¹⁸ A good correlation exists between the summed IgE anti-cow's milk components levels (Bos d 4,5,6,8 and lactoferrin, r² = 0.66) as measured in 44 sera from
<table><caption>TABLE 2 Advantages and limitations of singleplex and multiplex assay technology for allergen-specific IgE testing that utilizes allergenic molecules (components)</caption><thead><tr><th></th><th>Singleplex IgE assay format</th><th>Multiplex IgE assay format</th></tr></thead><tbody><tr><td>Performance Related<br>Advantages (pro)</td><td><ul><li>Increased assay analytical sensitivity (lower Limit of Quantitation, LoQ)</li><li>Potentially more precise quantification and precision, facilitating comparisons between different allergen reagents (extracts versus molecules)</li><li>More established internal and external quality control measures (proficiency testing)</li></ul></td><td><ul><li>Increased speed of analysis and reduced result turn-around time</li><li>Conservation of sample volume facilitating pediatric testing</li></ul></td></tr><tr><td>Assay Design and Cost<br>Related Advantages (pro)</td><td><ul><li>Traceable of allergen-specific IgE values to a total human IgE International Reference Preparation</li><li>Similar units for total IgE and allergen-specific IgE due to heterologous calibration (permits calculation of allergen-specific IgE/total IgE-ratio)</li><li>Global availability in many countries</li><li>In case of limited number of samples more cost efficient</li><li>Minimizes unneeded testing</li></ul></td><td><ul><li>Greater simplicity</li><li>Reduced cost due to fewer required reagents</li><li>Reduced technician intervention</li><li>Optimal design applications for point of care tests</li></ul></td></tr><tr><td>Performance Limitations<br>(con)</td><td>More costly due to increased need for reagents to evaluate polysensitized patients<br>More technical intervention<br>Limited answers in case of few samples per subject<br>Expensive in case of large scale screening (i.e. multi-sensitised subjects)</td><td><ul><li>Potentially lower analytical sensitivity for each analyte specificity measured (higher limit of detection, LoD)</li><li>Reduced ability to accurately quantify each IgE antibody</li><li>Encouragement of abusive testing which involves the measurement of unwanted or unneeded IgE antibody specificities</li></ul></td></tr><tr><td>Assay Design and Cost<br>Related Limitations (con)</td><td><ul><li>More serum required, particularly in case of many samples</li><li>Potentially slower analysis</li><li>Likely more sophisticated assay format</li></ul></td><td><ul><li>Less global availability</li><li>Cost of the new instrumentation and reagents</li><li>Greater challenge in managing different levels of non-specific binding</li><li>Enhanced challenges in optimizing, balancing and standardizing assay reagents and assay quality control</li><li>Potential greater inter-lot variability</li></ul></td></tr></tbody></table>

components from cow's and goat's milk, chicken egg, fish, shrimp, hazelnut, peanut, celery and fruits from the *Rosaceae* family (apple and peach) have been produced. Documentation of these allergenic components has involved extensive analytical, immunochemical and 3-dimensional structural analyses.

The availability of unlimited quantities of the molecular allergens has allowed multiplex chip microarray based assay methods to be used for rapid simultaneous evaluation of human sera for IgE antibodies to multiple allergen specificities. The most important illustration of technology transition from singleplex to multiplex assays has involved the chip-based multiplex IgE antibody assay initially reported by Hiller et al.¹¹⁶ The original chip-based microarray utilized 49 purified allergen molecules, which were covalently immobilized in fixed microdot arrays on a preactivated glass slide. IgE antibody profiles of allergic individuals were evaluated to disease-causing

allergens in a single multiplex analysis using 30 microliters of undiluted serum. With this report, serious clinical application of both allergenic components and multiplex assay methods became available to evaluate individuals for allergic disease. From this initial proof of concept, the repertoire of allergens has increased and the assays' lower limit of quantitation and reproducibility have continued to improve. The commercially available version of this assay is the immune solid phase allergen chip or ImmunoCAP ISAC (Thermo Fisher Scientific/Phadia), which requires 30 microliters of serum to detect IgE antibody to 112 individual allergenic molecules that are in a static or planar array in triplicate on a glass slide.¹¹⁷⁻¹¹⁹

The ImmunoCAP ISAC reports IgE antibody levels in ISU units, which are considered semi-quantitative¹¹⁸ A good correlation exists between the summed IgE anti-cow's milk components levels (Bos d 4,5,6,8 and lactoferrin, r² = 0.66) as measured in 44 sera from
clinically milk allergic individuals by the singleplex ImmunoCAP Specific IgE test (x-axis) and multiplex ImmunoCAP ISAC (y axis).¹²⁰ (Figure 8 - top panel). The correlation remains impressive when one compares the individual IgE anti-cow's milk components (Bos d 4,5,6,8 and lactoferrin, r² = 0.77) as measured in the same sera by ImmunoCAP Specific IgE and ImmunoCAP ISAC tests (Figure 8 - bottom panel). The lower analytical sensitivity of the ImmunoCAP ISAC test, however, depends on the allergen in question¹¹⁸⁻¹²⁰ and is evident with some strongly positive IgE antibody levels as detected in the ImmunoCAP Specific IgE test that are undetectable in the same sera when analyzed in the ImmunoCAP ISAC test.

In 2014, a research version of the ImmunoCAP ISAC called the “Mechanisms for the Development of ALLergy” or MeDALL allergen chip was produced with 170 allergen molecules to more broadly study IgE and IgG antibody development in children.¹¹² Using defined concentrations of chimeric IgE and IgG antibodies specific for Bet v 1, the study demonstrated that the simultaneous presence of IgG blocking antibodies can effectively inhibit IgE antibody binding to Bet v 1 allergen that has been immobilized onto the multiplex chip. By contrast, the same levels of IgG anti-Bet v1 produce minimal competitive interference in the more antigen laden singleplex ImmunoCAP Specific IgE test. The authors suggest that the inhibition of IgE binding by IgG antibodies of the same specificity to limited allergen immobilized on the chip may more closely reflect biological responses under conditions of natural allergen exposure. However, the clinical relevance of this inhibition needs further investigation. The smaller amount of allergen on the chip also reduces the working range of the IgE antibody assay in comparison to the singleplex ImmunoCAP Specific IgE test, whose allergosorbent has 10,000,000 times more allergen coupled.¹¹²

The Allergy Explorer (ALEX²) (Macro-Array Diagnostics, Wien, Austria) employs nearly 300 allergen extracts (n=117) and recombinant or purified native molecules (n=178) that are spotted on a solid phase by the use of nanoparticles. Quan et al.¹²¹ evaluated the clinical and technical performance of the ALEX² against the ImmunoCAP ISAC-112 microarray and the ImmunoCAP Specific IgE singleplex assay. Repeatability and inter-assay, inter-batch, and inter-laboratory reproducibility were evaluated using sera from clinically allergic and nonatopic patients.

EUROLINE (Euroimmun, Lübeck, Germany) produces a multiplex IgE antibody test that is immunoblot-based and widely used in Europe. Di Fraia et al.¹²² studied the multi-parameter semi-quantitative immunoblot molecular “Pollen Test” produced by EUROLINE that is designed to detect IgE antibodies to pollen extracts and molecules, which are clinically relevant to patients in Southern Europe. The test strip consists of nine membrane chips with different allergen extracts or components that are immobilized in parallel lines, which are mounted on a carrier foil. Allergen extracts and allergenic molecules from birch, olive tree, cypressus, Bermuda grass, Timothy grass, mugwort, alternaria and cross-reactive carbohydrate determinant are immobilized. The semi-quantitative response data (range 1–6) are measured as a band intensity and they provide an estimate of IgE antibody concentration.

<figure><img src="image_19.png" /></figure>

<figure><img src="image_20.png" /></figure>

**FIGURE 8** Left panel: correlation between the summed IgE anticow's milk (Bos domesticus [Bos d ]) components as measured in the ImmunoCAP and ISAC (Bos d 4: alpha lactalbumin; Bos d 5: beta lactoglobulin; Bos d 6 bovine serum albumin; Bos d 8: casein; Bos d lactoferrin). Right panel: Correlation between the individual IgE anti-cow's milk (Bos domesticus [Bos d ]) components as measured in the ImmunoCAP and ISAC (Bos d 4: alpha lactalbumin; Bos d 5: beta lactoglobulin; Bos d 6 bovine serum albumin; Bos d 8: casein; Bos d lactoferrin) components. The dashed lines indicate the positive/ negative cut-off for each assay: ImmunoCAP 0.1kU/L; ImmunoCAP ISAC: 0.3 ISU. Reproduced with permission from [120]

The ImmunoCAP ISAC, ALEX,² and Euoline are examples of diagnostic assays that require the physician to compromise between a targeted molecular singleplex IgE antibody assay strategy where individual allergen specificities are selected based on the patient's history, and use of a rigid allergen microarray panels, which contain a prescribed number of allergen specificities, some of which will not be relevant to a patient. The testing of unnecessary allergen specificities in a fixed menu based multiplex assay reduces the test's benefit to cost ratio. A recent literature review-based examination of these and other pro/con issues related to IgE antibody microarray assays is provided by Keshavarz et al.¹²³ As outlined in (Table 3), the authors emphasize the strengths of the microarray
clinically milk allergic individuals by the singleplex ImmunoCAP Specific IgE test (x-axis) and multiplex ImmunoCAP ISAC (y axis).¹²⁰ (Figure 8 - top panel). The correlation remains impressive when one compares the individual IgE anti-cow's milk components (Bos d 4,5,6,8 and lactoferrin, r² = 0.77) as measured in the same sera by ImmunoCAP Specific IgE and ImmunoCAP ISAC tests (Figure 8 - bottom panel). The lower analytical sensitivity of the ImmunoCAP ISAC test, however, depends on the allergen in question¹¹⁸⁻¹²⁰ and is evident with some strongly positive IgE antibody levels as detected in the ImmunoCAP Specific IgE test that are undetectable in the same sera when analyzed in the ImmunoCAP ISAC test.

In 2014, a research version of the ImmunoCAP ISAC called the “Mechanisms for the Development of ALLergy” or MeDALL allergen chip was produced with 170 allergen molecules to more broadly study IgE and IgG antibody development in children.¹¹² Using defined concentrations of chimeric IgE and IgG antibodies specific for Bet v 1, the study demonstrated that the simultaneous presence of IgG blocking antibodies can effectively inhibit IgE antibody binding to Bet v 1 allergen that has been immobilized onto the multiplex chip. By contrast, the same levels of IgG anti-Bet v1 produce minimal competitive interference in the more antigen laden singleplex ImmunoCAP Specific IgE test. The authors suggest that the inhibition of IgE binding by IgG antibodies of the same specificity to limited allergen immobilized on the chip may more closely reflect biological responses under conditions of natural allergen exposure. However, the clinical relevance of this inhibition needs further investigation. The smaller amount of allergen on the chip also reduces the working range of the IgE antibody assay in comparison to the singleplex ImmunoCAP Specific IgE test, whose allergosorbent has 10,000,000 times more allergen coupled.¹¹²

The Allergy Explorer (ALEX²) (Macro-Array Diagnostics, Wien, Austria) employs nearly 300 allergen extracts (n=117) and recombinant or purified native molecules (n=178) that are spotted on a solid phase by the use of nanoparticles. Quan et al.¹²¹ evaluated the clinical and technical performance of the ALEX² against the ImmunoCAP ISAC-112 microarray and the ImmunoCAP Specific IgE singleplex assay. Repeatability and inter-assay, inter-batch, and inter-laboratory reproducibility were evaluated using sera from clinically allergic and nonatopic patients.

EUROLINE (Euroimmun, Lübeck, Germany) produces a multiplex IgE antibody test that is immunoblot-based and widely used in Europe. Di Fraia et al.¹²² studied the multi-parameter semi-quantitative immunoblot molecular “Pollen Test” produced by EUROLINE that is designed to detect IgE antibodies to pollen extracts and molecules, which are clinically relevant to patients in Southern Europe. The test strip consists of nine membrane chips with different allergen extracts or components that are immobilized in parallel lines, which are mounted on a carrier foil. Allergen extracts and allergenic molecules from birch, olive tree, cypressus, Bermuda grass, Timothy grass, mugwort, alternaria and cross-reactive carbohydrate determinant are immobilized. The semi-quantitative response data (range 1–6) are measured as a band intensity and they provide an estimate of IgE antibody concentration.

<figure><img src="image_19.png" /></figure>

<figure><img src="image_20.png" /></figure>

**FIGURE 8** Left panel: correlation between the summed IgE anticow's milk (Bos domesticus [Bos d ]) components as measured in the ImmunoCAP and ISAC (Bos d 4: alpha lactalbumin; Bos d 5: beta lactoglobulin; Bos d 6 bovine serum albumin; Bos d 8: casein; Bos d lactoferrin). Right panel: Correlation between the individual IgE anti-cow's milk (Bos domesticus [Bos d ]) components as measured in the ImmunoCAP and ISAC (Bos d 4: alpha lactalbumin; Bos d 5: beta lactoglobulin; Bos d 6 bovine serum albumin; Bos d 8: casein; Bos d lactoferrin) components. The dashed lines indicate the positive/ negative cut-off for each assay: ImmunoCAP 0.1kU/L; ImmunoCAP ISAC: 0.3 ISU. Reproduced with permission from [120]

The ImmunoCAP ISAC, ALEX,² and Euoline are examples of diagnostic assays that require the physician to compromise between a targeted molecular singleplex IgE antibody assay strategy where individual allergen specificities are selected based on the patient's history, and use of a rigid allergen microarray panels, which contain a prescribed number of allergen specificities, some of which will not be relevant to a patient. The testing of unnecessary allergen specificities in a fixed menu based multiplex assay reduces the test's benefit to cost ratio. A recent literature review-based examination of these and other pro/con issues related to IgE antibody microarray assays is provided by Keshavarz et al.¹²³ As outlined in (Table 3), the authors emphasize the strengths of the microarray
<table><caption>TABLE 3 Potential criteria for assessing assay improvement of sensitization tests following the replacement of allergen extracts with allergenic molecules</caption><thead><tr><th>Exemples (allergen source, allergen carrier)</th><th>Increased analytic sensitivity</th><th>Increased analytical specificity/selectivity</th><th>Cross-reactive allergens</th><th>Species/family-specific major allergens</th></tr></thead><tbody><tr><td>cat</td><td></td><td>Fel d 2</td><td>Fel d 2</td><td>Fel d 1</td></tr><tr><td>hazelnut</td><td>Cor a 1 (Bet v 1-homologue)</td><td>Cor a 14 (2S albumin),<br>Cor a 9 (11S globulin)<br>Cor a 8 (LTP, mediterranean)</td><td></td><td></td></tr><tr><td>kiwi</td><td>Act d 8 (Bet v 1-homologue)</td><td></td><td>Act d 8 (Bet v 1-homologue)</td><td></td></tr><tr><td>peach</td><td>Pru p 1 (Bet v 1-homologue)</td><td>Pru p 3<br>(LTP, marker, mediterranean)</td><td>Pru p 1 (Bet v 1-homologue),<br>Pru p 4 (Profilin)</td><td></td></tr><tr><td>peanut</td><td>Ara h 10, Ara h 11 (oleosins)</td><td>Ara h 1 (7S globulin)<br>Ara h 2 (2S albumin)<br>Ara h 3 (11S globulin)<br>Ara h 6/7 (2S albumin)<br>Ara h 9 (LTP, mediterranean)</td><td>Ara h 8 (Bet v 1-homologue),<br>Ara h 5*</td><td></td></tr><tr><td>soy</td><td>Gly m 4 (Bet v 1-homologue)</td><td>Gly m 5, Gly m 6</td><td></td><td></td></tr><tr><td>wheat</td><td>Tri a 19 (omega-5-gliadin)</td><td></td><td></td><td></td></tr><tr><td>meat</td><td>alpha-GAL</td><td>alpha-GAL</td><td></td><td></td></tr><tr><td>honeybee venom</td><td>Api m 3, Api m 4, Api m 10</td><td>Api m 1, Api m 3, Api m 4, Api m 10</td><td></td><td>Api m 1, Api m 3,<br>Api m 4, Api m 10</td></tr><tr><td>yellow jacket venom</td><td>Ves v 5</td><td>Ves v 1, Ves v 5</td><td>Bet v 2*, Bet v 4**</td><td>Ves v 1, Ves v 5</td></tr><tr><td>birch (hazel, alder, birch pollen) and beech trees (beech, oak pollen)</td><td></td><td>Bet v 1</td><td></td><td>Bet v 1</td></tr><tr><td>oleaceae (ash, olive pollen)</td><td></td><td>Ole e 1</td><td>Ole e 2*, Ole e 3**, Ole e 7</td><td>Ole e 1</td></tr><tr><td>poaceae (pollen from moderate climate grasses)</td><td></td><td>Phl p 1, Phl p 5</td><td>Phl p 12*, Phl p 7**</td><td>Phl p 1, Phl p 5</td></tr><tr><td>mugwort pollen</td><td></td><td>Art v 1</td><td>Art v 4*, Art v 5**</td><td>Art v 1</td></tr><tr><td>ragweed pollen</td><td></td><td>Amb a 1</td><td>Amb a 8*, Amb a 10**</td><td>Amb a 1</td></tr></tbody></table>

technology that reside in its ability to detect IgE to a large number of allergens, simultaneously in a single test, using a small amount of patient serum. This is counter-balanced against a higher relative cost per allergen specificity, a generally lower analytical sensitivity than single-plex assays, semi-quantitative results, and difficulties with data interpretation and managing simultaneous results involving 100s of allergen specificities. They conclude that IgE microarray assays are currently invaluable research tools and increasingly used in the clinical practice of allergy, particularly in Europe. Artificial intelligence algorithms will be increasingly used by clinicians to aid them in digesting the complex inter-relationship of allergen families, cross-reactivities, and unique signature specificities that are provided by the multi-allergen IgE antibody-based arrays.

## 9 – Additional multiplex IgE assays used in research or in development

While the ImmunoCAP ISAC test, ALEX² and Euroline have been highlighted for their use with purified recombinant and native allergenic molecules, other assays use a combination of allergen extracts

and molecules immobilized in chip microarrays using different multiplex assay configurations.

A In 2015, Williams et al.¹²⁴ reported comparative testing of ImmunoCAP ISAC test, ImmunoCAP Specific IgE test and puncture skin testing with a chip-based multiplex autoanalyzer called the MicrotestDx. In contrast to the ImmunoCAP ISAC test, it uses 100 microliters of serum and employs a limited number of 19 allergen extracts and 16 allergenic molecules covering a total of 26 aero- and food-allergen specificities that are covalently immobilized onto a precoated chip in triplicate. This is a scaled down version of a proof of concept assay that used 95 allergen extracts and 8 recombinant proteins, which were immobilized on aldehyde-activated glass microscope slides. These initial IgE antibody comparative data while initially encouraging have not yielded a viable assay to date to compete with the ImmunoCAP ISAC test and ALEX.²

B Wiltshire et al. spotted a small number of allergen extracts on activated microarray slides and used an interesting rolling DNA circle amplification strategy to detect IgE antibody bound to immobilized allergen.¹²⁵ Feyzkhanova et al.¹²⁶ photo-induced
<table><caption>TABLE 3 Potential criteria for assessing assay improvement of sensitization tests following the replacement of allergen extracts with allergenic molecules</caption><thead><tr><th>Exemples (allergen source, allergen carrier)</th><th>Increased analytic sensitivity</th><th>Increased analytical specificity/selectivity</th><th>Cross-reactive allergens</th><th>Species/family-specific major allergens</th></tr></thead><tbody><tr><td>cat</td><td></td><td>Fel d 2</td><td>Fel d 2</td><td>Fel d 1</td></tr><tr><td>hazelnut</td><td>Cor a 1 (Bet v 1-homologue)</td><td>Cor a 14 (2S albumin),<br>Cor a 9 (11S globulin)<br>Cor a 8 (LTP, mediterranean)</td><td></td><td></td></tr><tr><td>kiwi</td><td>Act d 8 (Bet v 1-homologue)</td><td></td><td>Act d 8 (Bet v 1-homologue)</td><td></td></tr><tr><td>peach</td><td>Pru p 1 (Bet v 1-homologue)</td><td>Pru p 3<br>(LTP, marker, mediterranean)</td><td>Pru p 1 (Bet v 1-homologue),<br>Pru p 4 (Profilin)</td><td></td></tr><tr><td>peanut</td><td>Ara h 10, Ara h 11 (oleosins)</td><td>Ara h 1 (7S globulin)<br>Ara h 2 (2S albumin)<br>Ara h 3 (11S globulin)<br>Ara h 6/7 (2S albumin)<br>Ara h 9 (LTP, mediterranean)</td><td>Ara h 8 (Bet v 1-homologue),<br>Ara h 5*</td><td></td></tr><tr><td>soy</td><td>Gly m 4 (Bet v 1-homologue)</td><td>Gly m 5, Gly m 6</td><td></td><td></td></tr><tr><td>wheat</td><td>Tri a 19 (omega-5-gliadin)</td><td></td><td></td><td></td></tr><tr><td>meat</td><td>alpha-GAL</td><td>alpha-GAL</td><td></td><td></td></tr><tr><td>honeybee venom</td><td>Api m 3, Api m 4, Api m 10</td><td>Api m 1, Api m 3, Api m 4, Api m 10</td><td></td><td>Api m 1, Api m 3,<br>Api m 4, Api m 10</td></tr><tr><td>yellow jacket venom</td><td>Ves v 5</td><td>Ves v 1, Ves v 5</td><td>Bet v 2*, Bet v 4**</td><td>Ves v 1, Ves v 5</td></tr><tr><td>birch (hazel, alder, birch pollen) and beech trees (beech, oak pollen)</td><td></td><td>Bet v 1</td><td></td><td>Bet v 1</td></tr><tr><td>oleaceae (ash, olive pollen)</td><td></td><td>Ole e 1</td><td>Ole e 2*, Ole e 3**, Ole e 7</td><td>Ole e 1</td></tr><tr><td>poaceae (pollen from moderate climate grasses)</td><td></td><td>Phl p 1, Phl p 5</td><td>Phl p 12*, Phl p 7**</td><td>Phl p 1, Phl p 5</td></tr><tr><td>mugwort pollen</td><td></td><td>Art v 1</td><td>Art v 4*, Art v 5**</td><td>Art v 1</td></tr><tr><td>ragweed pollen</td><td></td><td>Amb a 1</td><td>Amb a 8*, Amb a 10**</td><td>Amb a 1</td></tr></tbody></table>

technology that reside in its ability to detect IgE to a large number of allergens, simultaneously in a single test, using a small amount of patient serum. This is counter-balanced against a higher relative cost per allergen specificity, a generally lower analytical sensitivity than single-plex assays, semi-quantitative results, and difficulties with data interpretation and managing simultaneous results involving 100s of allergen specificities. They conclude that IgE microarray assays are currently invaluable research tools and increasingly used in the clinical practice of allergy, particularly in Europe. Artificial intelligence algorithms will be increasingly used by clinicians to aid them in digesting the complex inter-relationship of allergen families, cross-reactivities, and unique signature specificities that are provided by the multi-allergen IgE antibody-based arrays.

## 9 – Additional multiplex IgE assays used in research or in development

While the ImmunoCAP ISAC test, ALEX² and Euroline have been highlighted for their use with purified recombinant and native allergenic molecules, other assays use a combination of allergen extracts

and molecules immobilized in chip microarrays using different multiplex assay configurations.

A In 2015, Williams et al.¹²⁴ reported comparative testing of ImmunoCAP ISAC test, ImmunoCAP Specific IgE test and puncture skin testing with a chip-based multiplex autoanalyzer called the MicrotestDx. In contrast to the ImmunoCAP ISAC test, it uses 100 microliters of serum and employs a limited number of 19 allergen extracts and 16 allergenic molecules covering a total of 26 aero- and food-allergen specificities that are covalently immobilized onto a precoated chip in triplicate. This is a scaled down version of a proof of concept assay that used 95 allergen extracts and 8 recombinant proteins, which were immobilized on aldehyde-activated glass microscope slides. These initial IgE antibody comparative data while initially encouraging have not yielded a viable assay to date to compete with the ImmunoCAP ISAC test and ALEX.²

B Wiltshire et al. spotted a small number of allergen extracts on activated microarray slides and used an interesting rolling DNA circle amplification strategy to detect IgE antibody bound to immobilized allergen.¹²⁵ Feyzkhanova et al.¹²⁶ photo-induced
copolymerization of 21 allergens (15 extracts and 6 molecular allergens) into a hydrogel covered chip and used 60 microliters of serum to perform IgE antibody microarray analyses. Renault et al.¹²⁷ reported a microarray assay in which 350 defatted and extracted foods were imprinted on slides (4800 dots per slide) and human IgG, IgA, IgM and IgE antibodies were simultaneously detected in serum using a 4 laser scanner. Joshi et al.¹²⁸ reported on an ultrasensitive carbohydrate-peptide surface plasmon resonance imaging microarray in which they immobilized peptide and xylosyl glycoside of Ara h 2 onto carboxylated gold slides and amplified the response with 1 micron diameter magnetic beads coated with ~60,000 polyclonal anti-IgE molecules. None of these proof-of-concept assays have been commercialized into viable assays for research and clinical use. The use of novel imaging systems and allergen extracts bound to chips raise theoretical concerns about the analytical sensitivity and specificity of the assays and whether the limited binding capacity of microdot surface on an activated glass or silicon chip can immobilize sufficient molar concentrations and all relevant allergen in an extracted protein mixture to quantitatively bind IgE antibody in the presence of other antibody isotypes.

C Alternative multiplex technologies are capable of detecting IgE antibody in human serum. The Luminex bead based suspension array assay uses fluorescent microspheres that are coupled with allergen, one specificity per bead type. Each bead type emits a different internal fluorescence that allows them to be distinguished from each other in a flow cytometer when they are mixed together. Each well of a microtiter plate is loaded with a mixture of bead types (50 microliters; 2000 beads) and serum (50 microliters at 1:4). Following an incubation and wash, bound IgE antibody is detected with biotinylated anti-IgE and avidin-phycoerythrin. The fluorescence intensity on the surface of the individual bead types is quantified and interpolated from a (fluorescent intensity vs total serum IgE) calibration curve. This assay is provided as a commercial laboratory developed test service by Indoor Biotechnologies (Charlottesville, VA, USA) to measure IgE antibody specific for a panel of 6 molecular aeroallergens from dust mites (Der p 1, Der p 2), cat dander (Fel d 1), dog dander (Can f 1), birch tree pollen (Bet v 1) and Timothy grass pollen (Phl p 5¹²⁹).

D A different multi-array approach has been employed by Meso-Scale Discovery (MSD).¹³⁰Initially, α-lactalbumin, β-lactoglobulin A/B, α-β-κ-casein, lactoferrin and BSA proteins were individually biodotted onto separate spots in NPT 9-spot plates. Each spot within the same reaction well permitted a separate antibody specificity to bind. Following reaction with milk allergic sera, bound IgE antibody was detected with Sulfo-Tag-labeled anti-human IgE antibody. Bound labeled antibody when exposed to an electrical pulse generated chemiluminescence through an oxidation-reduction reaction that was measured in an automated reader. Response levels were interpolated from a calibration curve into IgE antibody units. In 2021, Millen et al.¹³¹ validated a MSD multiplex immunoassay against the skin prick test and ImmunoCAP assay for respiratory allergens

(Dpt, Cat, dog; rye timothy grass; mugwort and birch pollen, Cladosporium, Aspergillus, Alternaria, and a mixture of animals, fungi, grasses, weeds, trees, house dust mites and mixed nuts). They used sera from adult patients with allergic rhinitis. Pearson correlations and Bland-Altman analysis showed high comparability of the MSD multiplex immunoassay with the prick skin test and the ImmunoCAP Specific IgE assay, except for house dust mite. The reproducibility of the MSD multiplex immunoassay as assessed for intra- and interassay reproducibility and biological variability between different sampling periods, showed significantly high correlations. This study shows proof of concept; however, MSD presently offers only a total serum IgE assay and does not commercially produce allergen-containing reagents for specific IgE antibody quantification.

E Chinnasamy et al. investigated a vertical flow allergen microarray assay with 10 purified allergenic molecules at 3 concentrations that were immobilized on 0.1 µm pore size nitrocellulose membranes.¹³² Bound IgE antibodies from human sera were detected with gold nanoparticle bound anti-IgE using a colorimetric read-out. Its precision and relative concordance with the singleplex ImmunoCAP Specific IgE were encouraging. However, to apply this vertical flow strategy, additional verification analyses were needed to validate the technique. Unfortunately, further direct comparison studies with clinical specimens that had been analyzed in parallel with established single and multiplex IgE assays were not performed and this assay format was therefore not pursued further

F A novel nanotechnology biosensor point of care test has been developed by Abionic (Epalinges, Switzerland) in which serum is mixed with fluorescently labeled anti-IgE and the mixture added to a capsule containing 10 allergenic molecules coupled to a biosensor surface. Capillary action drives allergen-specific IgE to bind to immobilized allergen and fluorescent molecular complexes are then optically measured by the abioSCOPE reading unit. The fluorescent response is finally translated to an IgE antibody dose. This is graphically overviewed by Chapman et al.⁷¹ The assay remains available but needs regulatory clearance for clinical use.

G The PROTIA™ Allergy-Q 64 Atopy (Proteometech, Seoul, Korea) has been evaluated in comparison to the ImmunoCAP Specific IgE test using the sera of 125 Korean allergic patients. The agreement for the 10 allergen components tested was > 88% for group 1 house dust mite (HDM) allergen, 100%; group 2 HDM allergen, 94.6%; Bet v 1, 97.4%; Fel d 1, 90.5%; Que a 1, 89.2%; α-lactalbumin, 96%; β-lactoglobulin, 88%; casein, 88%; ω-5 gliadin, 96%; and 100% for α-Gal.¹³³ More extensive cross-validation is needed to elevate this procedure to clinical use.

H McKenzie et al¹³⁴ has developed a novel flow cytometric assay called the CytoBas that uses fluorescent protein tetramers for direct staining of IgE antibodies on blood basophils that are then detected by flow cytometry. In a proof of concept study, recombinant forms of grass Lol p 1 and Lol p 5 and honeybee venom Api m 1 were produced, biotinylated, and tetramerized with
copolymerization of 21 allergens (15 extracts and 6 molecular allergens) into a hydrogel covered chip and used 60 microliters of serum to perform IgE antibody microarray analyses. Renault et al.<sup>127</sup> reported a microarray assay in which 350 defatted and extracted foods were imprinted on slides (4800 dots per slide) and human IgG, IgA, IgM and IgE antibodies were simultaneously detected in serum using a 4 laser scanner. Joshi et al.<sup>128</sup> reported on an ultrasensitive carbohydrate-peptide surface plasmon resonance imaging microarray in which they immobilized peptide and xylosyl glycoside of Ara h 2 onto carboxylated gold slides and amplified the response with 1 micron diameter magnetic beads coated with ~60,000 polyclonal anti-IgE molecules. None of these proof-of-concept assays have been commercialized into viable assays for research and clinical use. The use of novel imaging systems and allergen extracts bound to chips raise theoretical concerns about the analytical sensitivity and specificity of the assays and whether the limited binding capacity of microdot surface on an activated glass or silicon chip can immobilize sufficient molar concentrations and all relevant allergen in an extracted protein mixture to quantitatively bind IgE antibody in the presence of other antibody isotypes.

C Alternative multiplex technologies are capable of detecting IgE antibody in human serum. The Luminex bead based suspension array assay uses fluorescent microspheres that are coupled with allergen, one specificity per bead type. Each bead type emits a different internal fluorescence that allows them to be distinguished from each other in a flow cytometer when they are mixed together. Each well of a microtiter plate is loaded with a mixture of bead types (50 microliters; 2000 beads) and serum (50 microliters at 1:4). Following an incubation and wash, bound IgE antibody is detected with biotinylated anti-IgE and avidin-phycoerythrin. The fluorescence intensity on the surface of the individual bead types is quantified and interpolated from a (fluorescent intensity vs total serum IgE) calibration curve. This assay is provided as a commercial laboratory developed test service by Indoor Biotechnologies (Charlottesville, VA, USA) to measure IgE antibody specific for a panel of 6 molecular aeroallergens from dust mites (Der p 1, Der p 2), cat dander (Fel d 1), dog dander (Can f 1), birch tree pollen (Bet v 1) and Timothy grass pollen (Phl p 5<sup>129</sup>).

D A different multi-array approach has been employed by Meso-Scale Discovery (MSD).<sup>130</sup> Initially, α-lactalbumin, β-lactoglobulin A/B, α-β-κ-casein, lactoferrin and BSA proteins were individually biodotted onto separate spots in NPT 9-spot plates. Each spot within the same reaction well permitted a separate antibody specificity to bind. Following reaction with milk allergic sera, bound IgE antibody was detected with Sulfo-Tag-labeled anti-human IgE antibody. Bound labeled antibody when exposed to an electrical pulse generated chemiluminescence through an oxidation-reduction reaction that was measured in an automated reader. Response levels were interpolated from a calibration curve into IgE antibody units. In 2021, Millen et al.<sup>131</sup> validated a MSD multiplex immunoassay against the skin prick test and ImmunoCAP assay for respiratory allergens

(Dpt, Cat, dog; rye timothy grass; mugwort and birch pollen, Cladosporium, Aspergillus, Alternaria, and a mixture of animals, fungi, grasses, weeds, trees, house dust mites and mixed nuts). They used sera from adult patients with allergic rhinitis. Pearson correlations and Bland-Altman analysis showed high comparability of the MSD multiplex immunoassay with the prick skin test and the ImmunoCAP Specific IgE assay, except for house dust mite. The reproducibility of the MSD multiplex immunoassay as assessed for intra- and interassay reproducibility and biological variability between different sampling periods, showed significantly high correlations. This study shows proof of concept; however, MSD presently offers only a total serum IgE assay and does not commercially produce allergen-containing reagents for specific IgE antibody quantification.

E Chinnasamy et al. investigated a vertical flow allergen microarray assay with 10 purified allergenic molecules at 3 concentrations that were immobilized on 0.1 µm pore size nitrocellulose membranes.<sup>132</sup> Bound IgE antibodies from human sera were detected with gold nanoparticle bound anti-IgE using a colorimetric read-out. Its precision and relative concordance with the singleplex ImmunoCAP Specific IgE were encouraging. However, to apply this vertical flow strategy, additional verification analyses were needed to validate the technique. Unfortunately, further direct comparison studies with clinical specimens that had been analyzed in parallel with established single and multiplex IgE assays were not performed and this assay format was therefore not pursued further

F A novel nanotechnology biosensor point of care test has been developed by Abionic (Epalinges, Switzerland) in which serum is mixed with fluorescently labeled anti-IgE and the mixture added to a capsule containing 10 allergenic molecules coupled to a biosensor surface. Capillary action drives allergen-specific IgE to bind to immobilized allergen and fluorescent molecular complexes are then optically measured by the abioSCOPE reading unit. The fluorescent response is finally translated to an IgE antibody dose. This is graphically overviewed by Chapman et al.<sup>71</sup> The assay remains available but needs regulatory clearance for clinical use.

G The PROTIA™ Allergy-Q 64 Atopy (Proteometech, Seoul, Korea) has been evaluated in comparison to the ImmunoCAP Specific IgE test using the sera of 125 Korean allergic patients. The agreement for the 10 allergen components tested was > 88% for group 1 house dust mite (HDM) allergen, 100%; group 2 HDM allergen, 94.6%; Bet v 1, 97.4%; Fel d 1, 90.5%; Que a 1, 89.2%; α-lactalbumin, 96%; β-lactoglobulin, 88%; casein, 88%; ω-5 gliadin, 96%; and 100% for α-Gal.<sup>133</sup> More extensive cross-validation is needed to elevate this procedure to clinical use.

H McKenzie et al<sup>134</sup> has developed a novel flow cytometric assay called the CytoBas that uses fluorescent protein tetramers for direct staining of IgE antibodies on blood basophils that are then detected by flow cytometry. In a proof of concept study, recombinant forms of grass Lol p 1 and Lol p 5 and honeybee venom Api m 1 were produced, biotinylated, and tetramerized with
streptavidin. These-fluorochrome conjugates were then incubated with whole blood samples from grass and bee venom allergic patients and analyzed by flow cytometry for basophil binding and activation. Direct fluorescence staining of Ap1 m 1 and Lol p 1 tetramers had greater positive predictive values than basophil activation and the staining intensities of allergen tetramers correlated with allergen-specific IgE levels in serum.

## 10 – Evolution from allergen molecules to allergen epitopes.

Diagnostically relevant allergenic epitopes have been identified by epitope mapping using sera from sensitized and (in some cases) clinically allergic individuals. These sera contain IgE antibody to restricted regions on particular allergens. Immunodominant peptides have been identified from a library of overlapping continuous short peptides by IgE binding to synthetically produced allergen peptide fragments spotted on membranes, or plated in microarray chip or bead-based immunoassays. While these methods have been successful in identifying immunodominant peptides that can be associated with a greater likelihood of a persistent allergy or a severe reaction, the process of their identification is laborious and expensive. Monaco et al.<sup>135</sup> have developed a programmable phage display based procedure that can evaluate the binding of allergen-specific IgE and IgG antibodies to a library of ~2000 allergenic proteins plated as overlapping 56 amino acid peptides using a single multiplex reaction. This procedure reduces cost and provides high-throughput in the identification of novel allergenic epitopes that have potential predictive clinical utility. They use of an oligonucleotide library synthesis to encode a database of allergenic peptide sequences for display on T7 bacteriophages. This AllerScan library permits high throughput DNA sequencing and is used to identify thousands of IgE and IgG antibodies that bind to hundreds of distinct peptides. In their proof of concept study, they identified IgE antibodies that bind to wheat specific linear peptides using sera from wheat sensitized and allergic (or nonallergic control) subjects. From their work, a particular allergenic protein (purothionin) was identified that has promise in distinguishing sensitized allergic subjects who manifest objective clinical reactions from sensitized but nonallergic subjects who can readily consume wheat products.

In the diagnosis of food allergies, allergen epitope-based assays have been developed with the goal of identifying sensitization patterns that could reduce the need for definitive placebo-controlled food challenges that require significant resources, time and risk. These assays are based on early work involving cluster analysis of cow's milk and peanut linear and conformational allergen epitopes. Using microarray peptide immunoassays, IgE and IgG4 antibody patterns were identified that discriminated between food-sensitized individuals who were able to pass from those who failed an oral food challenge. Interpatient heterogeneity provided the promise of enhancing the diagnostic predictability of food allergen-specific IgE antibody analyses.<sup>136-138</sup> In a 2018 study of milk allergic patients

receiving oral milk immunotherapy with and without omalizumab (Anti-IgE) treatment, IgE and IgG4 antibodies to 66 sequential epitopes on 5 cow's milk proteins using a bead-based Luminex assay showed that certain baseline antibody profiles to 6 IgE-binding epitopes appeared more predictive of sustained unresponsiveness to milk exposure. than comparable antibody responses to their associated allergenic cows' milk components. Subsequently, the presumed protective effects of epitope-specific IgG1/4, IgA, and IgD immune responses in relation to epitope-specific IgE were investigated in individuals with a history of chicken egg allergy. Collectively higher ovamucoid epitope-specific IgE and IgD together with lower IgA and IgG antibody levels as measured with a bead-based epitope Luminex assay compared with atopic controls were shown to be important contributors to the pathogenesis of egg allergy<sup>139,140</sup> (49-50). In the most definitive study to date, predictive performance of a peanut bead-based epitope Luminex assay was evaluated using sera from subjects in the noninterventional arm of the LEAP trial, CoFAR2, and POISED clinical studies that used a double-blind placebo-controlled food challenge to document peanut allergy status. Diagnostic performance of IgE antibody measurements to two Ara h 2 sequential linear epitopes were superior to skin prick testing and peanut extract and component specific IgE test results in correctly diagnosing the allergic versus not-allergic status of the sensitized study subjects (92% diagnostic sensitivity and 94% diagnostic specificity)<sup>141</sup> These studies confirm that for select food allergen specificities, IgE antibody measurements at the allergenic epitope level are becoming increasingly diagnostically important in the management of food allergic patients.

## 11 – Rationale for the introduction of allergenic molecules and epitopes into clinical IgE antibody assays

The use of single allergens (molecules/components) that have been prepared by purification from native sources or molecular recombinant methods can enhance the clinical performance of serological IgE assays in different ways (Figures 8 and 9). Recombinant allergens can be generated with or without cross-reactive carbohydrate determinants while allergenic molecules purified from native sources will have a mixture of isoforms and carbohydrate determinants. All available allergenic molecules of one allergen source can be used as a mixture in place of a complex natural allergen extract mixture (Figure 9A). While this approach is theoretically feasible,<sup>142</sup> so far it has not been considered as a serious option since it is considered cumbersome, possibly not all inclusive of relevant allergens, expensive and thus of questionable benefit. A second approach has been to use allergenic molecules individually as single reagents in a singleplex or as individual replicate spots in multiplex microarray assays for targeted allergen-specific IgE detection (Figure 9B – 1 component). This is at present the most common use of molecular allergens. The most extensively used components are those from peanut (Ara h 1,2,3,6,8,9) and hazelnut (Cor a 1,8,9,14) that are used to clarify specific versus cross-reactive sensitivities in the assessment of
streptavidin. These-fluorochrome conjugates were then incubated with whole blood samples from grass and bee venom allergic patients and analyzed by flow cytometry for basophil binding and activation. Direct fluorescence staining of Apι m 1 and Lol p 1 tetramers had greater positive predictive values than basophil activation and the staining intensities of allergen tetramers correlated with allergen-specific IgE levels in serum.

## 10 – Evolution from allergen molecules to allergen epitopes.

Diagnostically relevant allergenic epitopes have been identified by epitope mapping using sera from sensitized and (in some cases) clinically allergic individuals. These sera contain IgE antibody to restricted regions on particular allergens. Immunodominant peptides have been identified from a library of overlapping continuous short peptides by IgE binding to synthetically produced allergen peptide fragments spotted on membranes, or plated in microarray chip or bead-based immunoassays. While these methods have been successful in identifying immunodominant peptides that can be associated with a greater likelihood of a persistent allergy or a severe reaction, the process of their identification is laborious and expensive. Monaco et al.<sup>135</sup> have developed a programmable phage display based procedure that can evaluate the binding of allergen-specific IgE and IgG antibodies to a library of ~2000 allergenic proteins plated as overlapping 56 amino acid peptides using a single multiplex reaction. This procedure reduces cost and provides high-throughput in the identification of novel allergenic epitopes that have potential predictive clinical utility. They use of an oligonucleotide library synthesis to encode a database of allergenic peptide sequences for display on T7 bacteriophages. This AllerScan library permits high throughput DNA sequencing and is used to identify thousands of IgE and IgG antibodies that bind to hundreds of distinct peptides. In their proof of concept study, they identified IgE antibodies that bind to wheat specific linear peptides using sera from wheat sensitized and allergic (or nonallergic control) subjects. From their work, a particular allergenic protein (purothionin) was identified that has promise in distinguishing sensitized allergic subjects who manifest objective clinical reactions from sensitized but nonallergic subjects who can readily consume wheat products.

In the diagnosis of food allergies, allergen epitope-based assays have been developed with the goal of identifying sensitization patterns that could reduce the need for definitive placebo-controlled food challenges that require significant resources, time and risk. These assays are based on early work involving cluster analysis of cow's milk and peanut linear and conformational allergen epitopes. Using microarray peptide immunoassays, IgE and IgG4 antibody patterns were identified that discriminated between food-sensitized individuals who were able to pass from those who failed an oral food challenge. Interpatient heterogeneity provided the promise of enhancing the diagnostic predictability of food allergen-specific IgE antibody analyses.<sup>136-138</sup> In a 2018 study of milk allergic patients

receiving oral milk immunotherapy with and without omalizumab (Anti-IgE) treatment, IgE and IgG4 antibodies to 66 sequential epitopes on 5 cow's milk proteins using a bead-based Luminex assay showed that certain baseline antibody profiles to 6 IgE-binding epitopes appeared more predictive of sustained unresponsiveness to milk exposure. than comparable antibody responses to their associated allergenic cows' milk components. Subsequently, the presumed protective effects of epitope-specific IgG1/4, IgA, and IgD immune responses in relation to epitope-specific IgE were investigated in individuals with a history of chicken egg allergy. Collectively higher ovamucoid epitope-specific IgE and IgD together with lower IgA and IgG antibody levels as measured with a bead-based epitope Luminex assay compared with atopic controls were shown to be important contributors to the pathogenesis of egg allergy<sup>139,140</sup> (49-50). In the most definitive study to date, predictive performance of a peanut bead-based epitope Luminex assay was evaluated using sera from subjects in the noninterventional arm of the LEAP trial, CoFAR2, and POISED clinical studies that used a double-blind placebo-controlled food challenge to document peanut allergy status. Diagnostic performance of IgE antibody measurements to two Ara h 2 sequential linear epitopes were superior to skin prick testing and peanut extract and component specific IgE test results in correctly diagnosing the allergic versus not-allergic status of the sensitized study subjects (92% diagnostic sensitivity and 94% diagnostic specificity)<sup>141</sup> These studies confirm that for select food allergen specificities, IgE antibody measurements at the allergenic epitope level are becoming increasingly diagnostically important in the management of food allergic patients.

## 11 – Rationale for the introduction of allergenic molecules and epitopes into clinical IgE antibody assays

The use of single allergens (molecules/components) that have been prepared by purification from native sources or molecular recombinant methods can enhance the clinical performance of serological IgE assays in different ways (Figures 8 and 9). Recombinant allergens can be generated with or without cross-reactive carbohydrate determinants while allergenic molecules purified from native sources will have a mixture of isoforms and carbohydrate determinants. All available allergenic molecules of one allergen source can be used as a mixture in place of a complex natural allergen extract mixture (Figure 9A). While this approach is theoretically feasible,<sup>142</sup> so far it has not been considered as a serious option since it is considered cumbersome, possibly not all inclusive of relevant allergens, expensive and thus of questionable benefit. A second approach has been to use allergenic molecules individually as single reagents in a singleplex or as individual replicate spots in multiplex microarray assays for targeted allergen-specific IgE detection (Figure 9B – 1 component). This is at present the most common use of molecular allergens. The most extensively used components are those from peanut (Ara h 1,2,3,6,8,9) and hazelnut (Cor a 1,8,9,14) that are used to clarify specific versus cross-reactive sensitivities in the assessment of
<figure><img src="image_21.png" /></figure>

FIGURE 9 Use of recombinant (with and without CCD) and purified natural allergenic molecules. Single and mixed isoforms are immobilized in single solid phase (singleplex) or in multiple spots on multiplex microarray solid phases for use in various IgE antibody assays. Reproduced with permission from [151], recombinant production, purification, 1 isoform without CCD, 1 isoform with CCD, mixed isoforms (±CCD), reagents: recombinant allergen molecules, natural allergen molecules (incl. Isoforms), microarray multiple selected molecules, all available components (instead of using allergen extracts), 1 component, 2 components, “spiked”: extract plus one component, multiplex (“screening”), singleplex methods (“single testing”)

FIGURE 10 Reagent patterns based on allergen sources/extracts (upper row), with typical reasons why it is valuable to use allergen molecules as reagents in allergen-specific singleplex IgE assays (middle row) and how IgE anti-allergenic molecule results can enhance the final analytical sensitivity and specificity of the generated assay results. Table 3 provides specific examples that correspond with the conditions depicted in Figure 10.

<figure><img src="image_22.png" /></figure>

individuals with positive peanut and/or hazelnut extract specific IgE responses. Third, selected single molecular allergens of a given allergen specificity can be combined and used as single molecular mixtures for allergen-specific IgE detection (Figure 9B – 2 components). To illustrate this approach, an equal molar mixture of the unique marker allergens Phl p 1 and Phl p 5 for Timothy grass sensitization are excellent representatives of sweet vernal grasses. Alternatively, a combination of highly cross-reactive allergenic molecules like Phl p 7 and Phl p 12, which are the polcalcin and profilin representatives of Timothy grass pollen can be used to identify a patient's

sensitization to other cross-reactive polcalcin and/or profilin pan-allergen specificities. Fourth, single components can be added to allergenic extracts (“spiked”) to increase assay sensitivity. This has been particularly useful for Hevea brasiliensis latex where Hev b 5 is underrepresented as a result of being in low abundance or missing from certain extracts (Figures 9C and 10A). While, spiking physiological allergen extracts can enhance the assay’s limit of quantitation and increase its analytical sensitivity, it can also lead to problems. For instance, supplementation of the hazelnut ImmunoCAP Specific IgE test (F17) with recombinant Cor a 1 caused Bet v 1-specific IgE to
<figure><img src="image_21.png" /></figure>

**FIGURE 9** Use of recombinant (with and without CCD) and purified natural allergenic molecules. Single and mixed isoforms are immobilized in single solid phase (singleplex) or in multiple spots on multiplex microarray solid phases for use in various IgE antibody assays. Reproduced with permission from [151], recombinant production, purification, 1 isoform without CCD, 1 isoform with CCD, mixed isoforms (±CCD), reagents: recombinant allergen molecules, natural allergen molecules (incl. Isoforms), microarray multiple selected molecules, all available components (instead of using allergen extracts), 1 component, 2 components, “spiked”: extract plus one component, multiplex (“screening”), singleplex methods (“single testing”)

**FIGURE 10** Reagent patterns based on allergen sources/extracts (upper row), with typical reasons why it is valuable to use allergen molecules as reagents in allergen-specific singleplex IgE assays (middle row) and how IgE anti-allergenic molecule results can enhance the final analytical sensitivity and specificity of the generated assay results. Table 3 provides specific examples that correspond with the conditions depicted in Figure 10.

<figure><img src="image_22.png" /></figure>

individuals with positive peanut and/or hazelnut extract specific IgE responses. Third, selected single molecular allergens of a given allergen specificity can be combined and used as single molecular mixtures for allergen-specific IgE detection (Figure 9B – 2 components). To illustrate this approach, an equal molar mixture of the unique marker allergens Phl p 1 and Phl p 5 for Timothy grass sensitization are excellent representatives of sweet vernal grasses. Alternatively, a combination of highly cross-reactive allergenic molecules like Phl p 7 and Phl p 12, which are the polcalcin and profilin representatives of Timothy grass pollen can be used to identify a patient's

sensitization to other cross-reactive polcalcin and/or profilin pan-allergen specificities. Fourth, single components can be added to allergenic extracts (“spiked”) to increase assay sensitivity. This has been particularly useful for Hevea brasiliensis latex where Hev b 5 is underrepresented as a result of being in low abundance or missing from certain extracts (Figures 9C and 10A). While, spiking physiological allergen extracts can enhance the assay’s limit of quantitation and increase its analytical sensitivity, it can also lead to problems. For instance, supplementation of the hazelnut ImmunoCAP Specific IgE test (F17) with recombinant Cora 1 caused Bet v 1-specific IgE to
be increasingly detected. This led unsuspecting clinicians to puzzle
over the elevated values of IgE anti-hazelnut in the serum of their
patients who subsequently were confirmed to have birch pollen al-
lergy.¹⁴³ The use of all available components (Figure 10A) in a micro-
array format can allow targeted and more precise differentiation of
the individual's sensitization profile from their allergen-specific IgE
response. This approach has been coined "Component-Resolved-
Diagnostics" (CRD)¹⁴⁴ and it represents the most important option
in molecular allergy diagnosis.

The utility of single allergenic molecules can be justified by four conditions (Figure 10 and Table 3).⁶⁹,¹⁴⁵ First, the assay's sensitivity can be improved by lowering its limit of quantification (LoQ) while increasing its analytical specificity. Second, if allergen molecules are in low abundance or missing in the extract such as Cor a 1 in hazelnut or Gly m 4 in soy, supplementation improves the assay's sensitivity (LoQ). Third, if allergen molecules are unique to a specificity such as Fel d 1 for cat or Bet v 1 for birch, their use can improve the analytical specificity ("selectivity") of the assay. This allows additional clinical assumption(s) such as assessing increased risk for severe symptoms. Finally, certain allergenic molecules such as Ara h 8 (Bet v 1 homologue) for peanut and Phl p 7 and 12, the polcalcin and profilin representatives in Timothy grass, can serve as indicators for serological cross-sensitizations through the binding of cross-reactive IgE. In case of a positive result, they can demonstrate the lack of analytical specificity of an IgE test with allergen extracts in affected subjects with potential cross-reactions.

**Diagnostic Allergen DataBase (DADB)** Despite the availability of multiple established¹⁴⁶ databases that provide nomenclature, structure, and reference referrals for molecular allergens,¹⁴⁶,¹⁴⁷ none of these databases provide a comprehensive listing of the internationally-accepted nomenclature codes and Linnean system descriptors of the ~1000 extract-based and molecular allergens used world-wide in diagnostic single- and multiplex IgE antibody assays. An international scientific committee of the Clinical and Laboratory Standards Institute has prepared the Diagnostic Allergen DataBase (DADB)¹⁴⁸,¹⁴⁹ to specifically provide these data in a readily-searchable Excel database. The DADB lists the allergen's unique assay code (f13), general category (food), principal IgE antibody assay method(s) where the allergen specificity is available, common name (peanut), taxonomical name (Arachis hypogaea), and the NCBI, NPU, LOINC, Allergome, and IUIS/WHO descriptor codes for allergens and their assays. The DADB is intended for use by manufacturers of allergen-specific IgE assays, allergen extract manufacturers, government regulators, International IgE antibody assay proficiency testing programs, clinicians, and the allergic patient. Its goal is to bring unambiguous clarity to the allergenic specificity being reported by single and multiplex IgE antibody assays being performed world-wide.

**Assay performance evaluation: Assay sensitivity and analytical specificity (selectivity).**

The analytical performance characteristics of laboratory tests
and their predictive value in defining the presence and severity clin-
ical disease have been internationally defined by variables such as

**Textbox 8 Common definitions to describe performance characteristics of a laboratory test (i.e., allergen-specific IgE assay)**

**Analytical sensitivity** is equivalent to the slope of the calibration curve of an (immune)assay. By contrast, assay sensitivity in real terms (=lowest test "cut-off") is currently calculated and provided with following, internationally harmonized variables:

* limit of blank, LoB (i.e., signal of a serum sample without allergen-specific IgE)

* limit of detection, LoD (i.e., signal of a serum sample with the lowest detectable allergen-specific IgE)

* limit of quantitation, LoQ (i.e., signal of a serum sample with the lowest allergen-specific IgE at a predefined assay precision)

**Analytical specificity** of an allergen-specific IgE assay can, first, be related to the specificity of the detected immunoglobulin class, meaning the test will indeed measure IgE and not immunoglobulins of other isotypes and subclasses (IgA 1/2, IgD, IgG1-4 or IgM)

A second definition relates analytical specificity to a tar-
geted, more selective IgE detection against single al-
lergenic molecules. While an allergen extract consists of
complex protein mixtures, ideally the assay binds the entire
IgE reportoire to a specific extracted allergen source. The
use of single allergen molecules will only detect a part of
the IgE reportoire. Thus, the analytical specificity (selectiv-
ity) will be increased.

sensitivity and specificity (Textbox 8).⁶⁹,¹⁴⁵ Two pairs of definitions separate the IgE antibody test's analytical sensitivity and specificity (Table 4, left column, 1-4) from its diagnostic sensitivity and specificity (Table 4, right column, I-IV) that discriminate among the various clinical allergy phenotypes. These definitions have been adopted as part of international guidelines for IgE antibody assays through the Clinical and Laboratory Standards Institute (CLSI). They are particularly important to consider when allergenic molecules are substituted into an IgE antibody assay.¹⁴² Their importance stems from the observation that introducing single allergenic molecules into an IgE antibody assays frequently improves the analytical variables in the left column of Table 3 and this has a direct effect on changing the diagnostic clinical discrimination of disease as defined by the parameters in the right column of (Table 3).

The extent to which an assay performance improvement trans-
lates into improved diagnostic clinical discrimination of disease de-
pends on (a) the cohort of individuals being evaluated in terms of
their age, disease spectrum and severity, (b) the availability and se-
lection of the specific allergenic molecules used in the IgE assay, and
be increasingly detected. This led unsuspecting clinicians to puzzle
over the elevated values of IgE anti-hazelnut in the serum of their
patients who subsequently were confirmed to have birch pollen al-
lergy.¹⁴³ The use of all available components (Figure 10A) in a micro-
array format can allow targeted and more precise differentiation of
the individual's sensitization profile from their allergen-specific IgE
response. This approach has been coined "Component-Resolved-
Diagnostics" (CRD)¹⁴⁴ and it represents the most important option
in molecular allergy diagnosis.

The utility of single allergenic molecules can be justified by four conditions (Figure 10 and Table 3).⁶⁹,¹⁴⁵ First, the assay's sensitivity can be improved by lowering its limit of quantification (LoQ) while increasing its analytical specificity. Second, if allergen molecules are in low abundance or missing in the extract such as Cor a 1 in hazelnut or Gly m 4 in soy, supplementation improves the assay's sensitivity (LoQ). Third, if allergen molecules are unique to a specificity such as Fel d 1 for cat or Bet v 1 for birch, their use can improve the analytical specificity ("selectivity") of the assay. This allows additional clinical assumption(s) such as assessing increased risk for severe symptoms. Finally, certain allergenic molecules such as Ara h 8 (Bet v 1 homologue) for peanut and Phl p 7 and 12, the polcalcin and profilin representatives in Timothy grass, can serve as indicators for serological cross-sensitizations through the binding of cross-reactive IgE. In case of a positive result, they can demonstrate the lack of analytical specificity of an IgE test with allergen extracts in affected subjects with potential cross-reactions.

**Diagnostic Allergen DataBase (DADB)** Despite the availability of multiple established¹⁴⁶ databases that provide nomenclature, structure, and reference referrals for molecular allergens,¹⁴⁶,¹⁴⁷ none of these databases provide a comprehensive listing of the internationally-accepted nomenclature codes and Linnean system descriptors of the ~1000 extract-based and molecular allergens used world-wide in diagnostic single- and multiplex IgE antibody assays. An international scientific committee of the Clinical and Laboratory Standards Institute has prepared the Diagnostic Allergen DataBase (DADB)¹⁴⁸,¹⁴⁹ to specifically provide these data in a readily-searchable Excel database. The DADB lists the allergen's unique assay code (f13), general category (food), principal IgE antibody assay method(s) where the allergen specificity is available, common name (peanut), taxonomical name (Arachis hypogaea), and the NCBI, NPU, LOINC, Allergome, and IUIS/WHO descriptor codes for allergens and their assays. The DADB is intended for use by manufacturers of allergen-specific IgE assays, allergen extract manufacturers, government regulators, International IgE antibody assay proficiency testing programs, clinicians, and the allergic patient. Its goal is to bring unambiguous clarity to the allergenic specificity being reported by single and multiplex IgE antibody assays being performed world-wide.

**Assay performance evaluation: Assay sensitivity and analytical specificity (selectivity).**

The analytical performance characteristics of laboratory tests
and their predictive value in defining the presence and severity clin-
ical disease have been internationally defined by variables such as

Textbox 8 Common definitions to describe
performance characteristics of a laboratory test
(i.e., allergen-specific IgE assay)

**Analytical sensitivity** is equivalent to the slope of the
calibration curve of an (immune)assay. By contrast, assay
sensitivity in real terms (=lowest test "cut-off") is currently
calculated and provided with following, internationally har-
monized variables:

* limit of blank, LoB (i.e., signal of a serum sample without allergen-specific IgE)

* limit of detection, LoD (i.e., signal of a serum sample with the lowest detectable allergen-specific IgE)

* limit of quantitation, LoQ (i.e., signal of a serum sample with the lowest allergen-specific IgE at a predefined assay precision)

**Analytical specificity** of an allergen-specific IgE assay can,
first, be related to the specificity of the detected immu-
noglobulin class, meaning the test will indeed measure IgE
and not immunoglobulins of other isotypes and subclasses
(IgA 1/2, IgD, IgG1-4 or IgM)

A second definition relates analytical specificity to a tar-
geted, more selective IgE detection against single al-
lergenic molecules. While an allergen extract consists of
complex protein mixtures, ideally the assay binds the entire
IgE reportoire to a specific extracted allergen source. The
use of single allergen molecules will only detect a part of
the IgE reportoire. Thus, the analytical specificity (selectiv-
ity) will be increased.

sensitivity and specificity (Textbox 8).⁶⁹,¹⁴⁵ Two pairs of definitions separate the IgE antibody test's analytical sensitivity and specificity (Table 4, left column, 1-4) from its diagnostic sensitivity and specificity (Table 4, right column, I-IV) that discriminate among the various clinical allergy phenotypes. These definitions have been adopted as part of international guidelines for IgE antibody assays through the Clinical and Laboratory Standards Institute (CLSI). They are particularly important to consider when allergenic molecules are substituted into an IgE antibody assay.¹⁴² Their importance stems from the observation that introducing single allergenic molecules into an IgE antibody assays frequently improves the analytical variables in the left column of Table 3 and this has a direct effect on changing the diagnostic clinical discrimination of disease as defined by the parameters in the right column of (Table 3).

The extent to which an assay performance improvement trans-
lates into improved diagnostic clinical discrimination of disease de-
pends on (a) the cohort of individuals being evaluated in terms of
their age, disease spectrum and severity, (b) the availability and se-
lection of the specific allergenic molecules used in the IgE assay, and
<table><caption>TABLE 4 Potential criteria for assessing assay improvement of sensitization tests following the replacement of allergen extracts with allergenic molecules</caption><thead><tr><th></th><th>analytical criteria (potential assay improvement)</th><th>diagnostic-clinical criteria (potential clinical advantages)</th></tr></thead><tbody><tr><td>1</td><td>&gt; "analytical" sensitivity &lt; limit of quantitation (LoQ)</td><td>I. &gt; diagnostic sensitivity (proportion of positive IgE antibody tests in patients with allergic disease)</td></tr><tr><td>2</td><td>&gt; analytical specificity</td><td>II. &gt; diagnostic specificity (proportion negative IgE antibody tests in healthy individuals)</td></tr><tr><td>3</td><td>indicator of serological cross reactivity</td><td>III indicator of clinical crossreactivity (allergic symptoms to allergenic sources that did not elicit the primary sensitisation)</td></tr><tr><td>4</td><td>marker of primary/genuine sensitisation</td><td>IV. prediction of clinical reactions (PPV, NPV, thresholds, likelihood ratio etc.)</td></tr></tbody></table>

(c) the preselected study endpoints defined by the clinician.¹⁴⁸ This means that the diagnostic-clinical criteria (right column, I-IV, Table 4) need a thorough individual interpretation based on each IgE antibody test result using the patient's previous history and if needed, additional proof of reproducible and objective symptoms in the affected allergic subject upon allergen exposure (i.e., challenge test). As a consequence, these clinical criteria extend beyond the essential "raw" allergen-specific IgE antibody assay result (e.g., IgE sensitization in question: yes or no). Together these facts support the conclusion that it can be misleading to use sensitization test results alone to define the diagnostic clinical criteria of an IgE antibody test.¹⁴²,¹⁴⁸

One example is enhancement of the analytical sensitivity of an IgE antibody assay by supplementing an extract with a labile allergen molecule (Hev b 5 into the Hevea brasiliensis extract) prior to use in preparing the allergosorbent. The additional Hev b 5 improves the analytical sensitivity by lowering the assay's limit of quantitation (LoQ), and thus increasing the test's diagnostic sensitivity without compromising the analytical specificity of the test. Alternatively, in patients with wheat-dependent, exercise-induced anaphylaxis (WDEIA), only 20-30% of the subjects have IgE-mediated sensitization to wheat flour extract. However, 80-90% of these cases demonstrate allergen-specific IgE to Tri a 19 (Omega-5-gliadin). This gliadin is often responsible for WDEIA; however, it suffers from a poor aqueous solubility and is therefore not well represented in wheat extracts. By using recombinant Tri a 19 as a reagent in the IgE antibody assay, the assay's sensitivity (lowered LoQ) is immediately improved.

Another asset of the use of defined allergenic molecules is the restriction that it provides to the assay. This is especially important when IgE immune responses need to be detected to allergenic specificities that are highly stable or in relatively high abundance (i.e., Ara h 2 or Cor a 14). Their use makes the measurement of IgE antibody more targeted or analytically specific. Identification of IgE immune response patterns to the 2S albumins, Ara h 2 and Cor a 14 (Table 4), have been repeatedly associated with an increased risk for severe reactions to foods and they can facilitate decisions about

the possible elimination of an oral food challenge Alternatively, in a nonselected population study, more than 10% of German children and adolescents demonstrate allergen-specific IgE to peanut extract that is predominantly linked to serological pollen-associated cross-reactions.¹⁵⁰ Moreover, diagnostic tests with the stable and risk-associated peanut storage allergen (Ara h 2) show elevated allergen-specific IgE in only a small proportion of the general children/adolescent population (approximately 0.2% to 0.4%) (Kirsten Beyer, personal communication). Thus IgE anti-Ara h 2 provides a much higher analytical specificity ("selectivity") than the use of a peanut extract based allergosorbent.

## 12 – Clinical evaluation: diagnostic sensitivity and specificity

Diagnostic sensitivity and specificity are related to a clinical history and physical examination based assessment of affected and nonaffected subjects. Requirements for proper calculation and interpretation of the diagnostic sensitivity and specificity of IgE antibody tests of sensitization require sound clinical data from the subject's case history and in some cases additional challenge tests to back up the clinical diagnosis (Table 4, right column). However, the presence of allergen-specific IgE is strictly a marker for allergic sensitization (risk for allergy) and it alone cannot predict the probability of a clinical reaction per se. Thus, concordant results (case history and allergen-specific IgE with a positive clinical or challenge outcome) are effectively considered as clinically relevant (rather than being labeled as true positive). The same applies for concordant negative results, which are used to exclude a clinical state of allergy and an underlying state of allergic sensitization. In case of positive allergen-specific IgE results and a negative case history or provocation test, however, consideration should be given to labeling these discordant results as clinically irrelevant (rather than false positive diagnostic tests). Labeling clinically irrelevant as false positive test results actually
<table><caption>TABLE 4 Potential criteria for assessing assay improvement of sensitization tests following the replacement of allergen extracts with allergenic molecules</caption><thead><tr><th></th><th>analytical criteria (potential assay improvement)</th><th>diagnostic-clinical criteria (potential clinical advantages)</th></tr></thead><tbody><tr><td>1</td><td>&gt; "analytical" sensitivity &lt; limit of quantitation (LoQ)</td><td>I. &gt; diagnostic sensitivity (proportion of positive IgE antibody tests in patients with allergic disease)</td></tr><tr><td>2</td><td>&gt; analytical specificity</td><td>II. &gt; diagnostic specificity (proportion negative IgE antibody tests in healthy individuals)</td></tr><tr><td>3</td><td>indicator of serological cross reactivity</td><td>III indicator of clinical crossreactivity (allergic symptoms to allergenic sources that did not elicit the primary sensitisation)</td></tr><tr><td>4</td><td>marker of primary/genuine sensitisation</td><td>IV. prediction of clinical reactions (PPV, NPV, thresholds, likelihood ratio etc.)</td></tr></tbody></table>

(c) the preselected study endpoints defined by the clinician.¹⁴⁸ This means that the diagnostic-clinical criteria (right column, I-IV, Table 4) need a thorough individual interpretation based on each IgE antibody test result using the patient's previous history and if needed, additional proof of reproducible and objective symptoms in the affected allergic subject upon allergen exposure (i.e., challenge test). As a consequence, these clinical criteria extend beyond the essential “raw” allergen-specific IgE antibody assay result (e.g., IgE sensitization in question: yes or no). Together these facts support the conclusion that it can be misleading to use sensitization test results alone to define the diagnostic clinical criteria of an IgE antibody test.¹⁴²,¹⁴⁸

One example is enhancement of the analytical sensitivity of an IgE antibody assay by supplementing an extract with a labile allergen molecule (Hev b 5 into the Hevea brasiliensis extract) prior to use in preparing the allergosorbent. The additional Hev b 5 improves the analytical sensitivity by lowering the assay's limit of quantitation (LoQ), and thus increasing the test's diagnostic sensitivity without compromising the analytical specificity of the test. Alternatively, in patients with wheat-dependent, exercise-induced anaphylaxis (WDEIA), only 20-30% of the subjects have IgE-mediated sensitization to wheat flour extract. However, 80-90% of these cases demonstrate allergen-specific IgE to Tri a 19 (Omega-5-gliadin). This gliadin is often responsible for WDEIA; however, it suffers from a poor aqueous solubility and is therefore not well represented in wheat extracts. By using recombinant Tri a 19 as a reagent in the IgE antibody assay, the assay's sensitivity (lowered LoQ) is immediately improved.

Another asset of the use of defined allergenic molecules is the restriction that it provides to the assay. This is especially important when IgE immune responses need to be detected to allergenic specificities that are highly stable or in relatively high abundance (i.e., Ara h 2 or Cor a 14). Their use makes the measurement of IgE antibody more targeted or analytically specific. Identification of IgE immune response patterns to the 2S albumins, Ara h 2 and Cor a 14 (Table 4), have been repeatedly associated with an increased risk for severe reactions to foods and they can facilitate decisions about

the possible elimination of an oral food challenge Alternatively, in a nonselected population study, more than 10% of German children and adolescents demonstrate allergen-specific IgE to peanut extract that is predominantly linked to serological pollen-associated cross-reactions.¹⁵⁰ Moreover, diagnostic tests with the stable and risk-associated peanut storage allergen (Ara h 2) show elevated allergen-specific IgE in only a small proportion of the general children/adolescent population (approximately 0.2% to 0.4%) (Kirsten Beyer, personal communication). Thus IgE anti-Ara h 2 provides a much higher analytical specificity ("selectivity") than the use of a peanut extract based allergosorbent.

## 12 – Clinical evaluation: diagnostic sensitivity and specificity

Diagnostic sensitivity and specificity are related to a clinical history and physical examination based assessment of affected and nonaffected subjects. Requirements for proper calculation and interpretation of the diagnostic sensitivity and specificity of IgE antibody tests of sensitization require sound clinical data from the subject's case history and in some cases additional challenge tests to back up the clinical diagnosis (Table 4, right column). However, the presence of allergen-specific IgE is strictly a marker for allergic sensitization (risk for allergy) and it alone cannot predict the probability of a clinical reaction per se. Thus, concordant results (case history and allergen-specific IgE with a positive clinical or challenge outcome) are effectively considered as clinically relevant (rather than being labeled as true positive). The same applies for concordant negative results, which are used to exclude a clinical state of allergy and an underlying state of allergic sensitization. In case of positive allergen-specific IgE results and a negative case history or provocation test, however, consideration should be given to labeling these discordant results as clinically irrelevant (rather than false positive diagnostic tests). Labeling clinically irrelevant as false positive test results actually
<figure><img src="image_23.png" /><figcaption>FIGURE 11 Typical data analyses of IgE antibody laboratory data with single allergenic molecules</figcaption></figure>

misses the key point of the analysis, since the presence of allergen-
specific IgE itself should not be disputed, but rather considered
valid in its own right as a marker for atopy and IgE sensitization.¹⁴⁸
A number of clinical studies have explored diagnostic sensitivity and
specificity of IgE tests with single allergens from a particular allergen
source. The use of previously missing or less represented allergens
in IgE antibody assays was able to increase its diagnostic sensitivity
through the improvement of assay sensitivity, usually by lowering
the assay's limit of assay quantitation (Tables 3 and 4). As a conse-
quence, higher rates of sensitization were found, in general, even
among subjects without clinically relevant reactions or disease.

The reciprocity of diagnostic sensitivity and specificity is
a general feature of diagnostic tests. It is usually depicted as
Receiver Operating Characteristics (ROC) curves (general example
see Figure 11B). Some single allergens like Ara h 2 or other risk-
associated allergens belonging to the seed storage protein family
of 2S-albumins have been found to increase diagnostic sensitivity
and specificity of a patient's evaluation by assessing the risk of se-
vere clinical reactions. Using risk-associated 2S-albumins, predictive
allergen-specific IgE thresholds ("decision points") have been de-
fined to forecast a positive or negative oral challenge in children with
peanut or hazelnut allergy¹⁵⁰ diagnostic sensitivity and specificity,
predictive values and likelihood ratio. (Table 3, right column), the an-
alytical performance of IgE assays can be substantially improved in
many cases by allergenic molecules that are used in parallel with or
in place of allergen extracts. This conceptual view has already been
adopted by international guidelines on allergen-specific IgE assays.
It should further facilitate and possibly accelerate the evalution and

clinical acceptance of allergenic molecules into the diagnostic algo-
rithm for human allergic disease.¹⁵¹

A: Association of log distributed allergen-specific IgE concentra-
tions to a natural (x axis) versus recombinant (y-axis)allergen mol-
ecules. B: Diagnostic efficacy (receiver-operating characteristics,
ROC) displaying performance of IgE antibody assays with allergen
molecules as compared to extracts; C. Single values of allergen
molecule-specific IgE antibody with the median, 25% and 75% per-
centiles that are presented to compare groups. D: Threshold levels
of IgE antibody that are used to predict the probability of a clini-
cal reaction (i.e., 95% probability of a positive oral food challenge)
Despite the performance of elaborate and elegant clinical studies,
they have produced rather variable outcomes with group effects,
displaying considerable overlap between, i.e., clinically affected
and nonaffected (tolerant) individuals (Figure 11C). Presenting data
with smoothed probability plots (Figure 11D) suggests a close rela-
tionship, suitable for individual prediction with the collected data.
However, results of sensitization tests like the allergen-specific IgE
assays cannot yet (and presumably never will) reliably predict clinical
reactions (or their nonappearance).¹⁴⁸,¹⁵² Therefore, future studies
on the diagnostic value of allergenic molecules should establish as
their primarily goal, the improvement of well-defined methodolog-
ical variables (Table 3, left column) that are linked to the analytical
performance characteristics of the assay. This should be done even
without a complete clinical evaluation of the assay, which includes
the assessment of the assay's.

13 – Determination of the clinical relevance of an IgE
antibody assay

The ultimate and essential question with diagnostic allergy test-
ing is "what is the clinical relevance of an allergen-specific IgE
measurement"? Even in this era with the availability of molecular
allergens, the basic rule still applies. Namely, a positive allergen-
specific IgE result represents a state of allergic sensitization (risk
for allergic disease) but not proof of allergic disease.⁶⁹,¹⁴⁵,¹⁴⁸,¹⁵²
A positive IgE antibody response is only clinically relevant in the
case that there are objectively defined corresponding allergic
symptoms that are temporally associated with a known allergen
exposure. A negative allergen-specific IgE result against one re-
combinant allergen molecule or a mixture of natural isoforms of
one single allergen can generally exclude an allergic sensitization
or risk of allergy to that allergen specificity in question. This is,
however, only possible if the total IgE is high enough (i.e., >20
kU/L), the allergen reagent is in sufficient abundance, fully intact,
and presenting all its epitopes and the analytical performance of
the IgE antibody assay has been optimized for a low limit of quan-
titation (i.e., 0.1 kU/L, 0.24 ng/ml).

In conclusion, the clinical relevance of an allergic sensitiza-
tion (i.e., presence of allergen-specific IgE) independent of the
use of allergen extracts or molecules for diagnostic purposes can
<figure><img src="image_23.png" /><figcaption>FIGURE 11 Typical data analyses of IgE antibody laboratory data with single allergenic molecules</figcaption></figure>

misses the key point of the analysis, since the presence of allergen-
specific IgE itself should not be disputed, but rather considered
valid in its own right as a marker for atopy and IgE sensitization.¹⁴⁸
A number of clinical studies have explored diagnostic sensitivity and
specificity of IgE tests with single allergens from a particular allergen
source. The use of previously missing or less represented allergens
in IgE antibody assays was able to increase its diagnostic sensitivity
through the improvement of assay sensitivity, usually by lowering
the assay's limit of assay quantitation (Tables 3 and 4). As a conse-
quence, higher rates of sensitization were found, in general, even
among subjects without clinically relevant reactions or disease.

The reciprocity of diagnostic sensitivity and specificity is
a general feature of diagnostic tests. It is usually depicted as
Receiver Operating Characteristics (ROC) curves (general example
see Figure 11B). Some single allergens like Ara h 2 or other risk-
associated allergens belonging to the seed storage protein family
of 2S-albumins have been found to increase diagnostic sensitivity
and specificity of a patient's evaluation by assessing the risk of se-
vere clinical reactions. Using risk-associated 2S-albumins, predictive
allergen-specific IgE thresholds ("decision points") have been de-
fined to forecast a positive or negative oral challenge in children with
peanut or hazelnut allergy¹⁵⁰ diagnostic sensitivity and specificity,
predictive values and likelihood ratio. (Table 3, right column), the an-
alytical performance of IgE assays can be substantially improved in
many cases by allergenic molecules that are used in parallel with or
in place of allergen extracts. This conceptual view has already been
adopted by international guidelines on allergen-specific IgE assays.
It should further facilitate and possibly accelerate the evalution and

clinical acceptance of allergenic molecules into the diagnostic algo-
rithm for human allergic disease.¹⁵¹

A: Association of log distributed allergen-specific IgE concentra-
tions to a natural (x axis) versus recombinant (y-axis)allergen mol-
ecules. B: Diagnostic efficacy (receiver-operating characteristics,
ROC) displaying performance of IgE antibody assays with allergen
molecules as compared to extracts; C. Single values of allergen
molecule-specific IgE antibody with the median, 25% and 75% per-
centiles that are presented to compare groups. D: Threshold levels
of IgE antibody that are used to predict the probability of a clini-
cal reaction (i.e., 95% probability of a positive oral food challenge)
Despite the performance of elaborate and elegant clinical studies,
they have produced rather variable outcomes with group effects,
displaying considerable overlap between, i.e., clinically affected
and nonaffected (tolerant) individuals (Figure 11C). Presenting data
with smoothed probability plots (Figure 11D) suggests a close rela-
tionship, suitable for individual prediction with the collected data.
However, results of sensitization tests like the allergen-specific IgE
assays cannot yet (and presumably never will) reliably predict clinical
reactions (or their nonappearance).¹⁴⁸,¹⁵² Therefore, future studies
on the diagnostic value of allergenic molecules should establish as
their primarily goal, the improvement of well-defined methodolog-
ical variables (Table 3, left column) that are linked to the analytical
performance characteristics of the assay. This should be done even
without a complete clinical evaluation of the assay, which includes
the assessment of the assay's.

13 – Determination of the clinical relevance of an IgE
antibody assay

The ultimate and essential question with diagnostic allergy test-
ing is "what is the clinical relevance of an allergen-specific IgE
measurement"? Even in this era with the availability of molecular
allergens, the basic rule still applies. Namely, a positive allergen-
specific IgE result represents a state of allergic sensitization (risk
for allergic disease) but not proof of allergic disease.⁶⁹,¹⁴⁵,¹⁴⁸,¹⁵²
A positive IgE antibody response is only clinically relevant in the
case that there are objectively defined corresponding allergic
symptoms that are temporally associated with a known allergen
exposure. A negative allergen-specific IgE result against one re-
combinant allergen molecule or a mixture of natural isoforms of
one single allergen can generally exclude an allergic sensitization
or risk of allergy to that allergen specificity in question. This is,
however, only possible if the total IgE is high enough (i.e., >20
kU/L), the allergen reagent is in sufficient abundance, fully intact,
and presenting all its epitopes and the analytical performance of
the IgE antibody assay has been optimized for a low limit of quan-
titation (i.e., 0.1 kU/L, 0.24 ng/ml).

In conclusion, the clinical relevance of an allergic sensitiza-
tion (i.e., presence of allergen-specific IgE) independent of the
use of allergen extracts or molecules for diagnostic purposes can
ultimately only be determined by the physician and not by the test.<sup>69,145,148,152</sup> Therefore, the complete diagnostic results of sensitizations tests, including allergen-specific IgE assays that have been improved by the use of allergenic molecules, will always have to be interpreted within the individual's clinical context and on the basis of their case history.

## 14 – Summary

For the foreseeable future, clinically validated singleplex assays that use allergen extract-based reagents will remain the principal assays and reagents employed worldwide by clinical immunology laboratories to serologically document sensitization (IgE antibody) in individuals with a positive history of allergic symptoms. Allergen extract-based reagents are being judiciously supplemented with an increasing number of allergenic molecule-based reagents. Their routine use in singleplex assays enhances diagnostic accuracy, predictability of risk for severe reactions and documentation of cross-reactivity. In Europe, novel molecular allergen-based multiplex assays have become more common in diagnostic allergy testing after demonstrating compliance with new IVD-R quality standards. However, in the rest of the world, they will remain invaluable research assays,

until such time as their government regulatory agencies have an opportunity to review multiplex assay performance data. The molecular allergens from peanut and hazelnut provide a pathway for other molecular allergens to be increasingly considered for use in routine diagnostic allergy testing. They have been shown to (a) improve analytical sensitivity by providing molar excess of missing or low abundant allergens (e.g., Ara h 8 in the peanut extract; Cor a 1 in the hazelnut extract), (b) enhance the assay's analytical specificity by defining a clinical risk for systemic reactions (Ara h 1,2,3 [severe] versus Ara h 8 [more mild]) and (c) distinguish cross-reactivity versus (d) genuine (primary) sensitization (Cor a 9,14 [genuine] versus Cor a 1 [Bet v 1 cross-reactive] sensitization to hazelnut). The clinical relevance of allergen-specific IgE detection in a patient's serum is strictly as a marker for allergic sensitization (risk for allergy) and it alone cannot predict the probability of an allergic reaction. The determination of the diagnostic sensitivity and specificity of IgE antibody assays will thus remain difficult to definitively determine because of the lack of an absolute (gold standard) method of defining the presence of allergic disease. This means that the clinical relevance of an allergic sensitization (i.e., presence of allergen-specific IgE) independent of the use of allergen extracts or molecules for diagnostic purposes will ultimately be determined only by the physician and not by the test.
ultimately only be determined by the physician and not by the test.<sup>69,145,148,152</sup> Therefore, the complete diagnostic results of sensitizations tests, including allergen-specific IgE assays that have been improved by the use of allergenic molecules, will always have to be interpreted within the individual's clinical context and on the basis of their case history.

## 14 – Summary

For the foreseeable future, clinically validated singleplex assays that use allergen extract-based reagents will remain the principal assays and reagents employed worldwide by clinical immunology laboratories to serologically document sensitization (IgE antibody) in individuals with a positive history of allergic symptoms. Allergen extract-based reagents are being judiciously supplemented with an increasing number of allergenic molecule-based reagents. Their routine use in singleplex assays enhances diagnostic accuracy, predictability of risk for severe reactions and documentation of cross-reactivity. In Europe, novel molecular allergen-based multiplex assays have become more common in diagnostic allergy testing after demonstrating compliance with new IVD-R quality standards. However, in the rest of the world, they will remain invaluable research assays,

until such time as their government regulatory agencies have an opportunity to review multiplex assay performance data. The molecular allergens from peanut and hazelnut provide a pathway for other molecular allergens to be increasingly considered for use in routine diagnostic allergy testing. They have been shown to (a) improve analytical sensitivity by providing molar excess of missing or low abundant allergens (e.g., Ara h 8 in the peanut extract; Cor a 1 in the hazelnut extract), (b) enhance the assay's analytical specificity by defining a clinical risk for systemic reactions (Ara h 1,2,3 [severe] versus Ara h 8 [more mild]) and (c) distinguish cross-reactivity versus (d) genuine (primary) sensitization (Cor a 9,14 [genuine] versus Cor a 1 [Bet v 1 cross-reactive] sensitization to hazelnut). The clinical relevance of allergen-specific IgE detection in a patient's serum is strictly as a marker for allergic sensitization (risk for allergy) and it alone cannot predict the probability of an allergic reaction. The determination of the diagnostic sensitivity and specificity of IgE antibody assays will thus remain difficult to definitively determine because of the lack of an absolute (gold standard) method of defining the presence of allergic disease. This means that the clinical relevance of an allergic sensitization (i.e., presence of allergen-specific IgE) independent of the use of allergen extracts or molecules for diagnostic purposes will ultimately be determined only by the physician and not by the test.
# A05 - Basophil activation test

Alexandra F. Santos, Bernadette Eberlein, Peter Korosec, Hans-Jürgen Hoffmann, Edward F. Knol

## Highlights

* The basophil activation test (BAT) is a functional test that goes beyond detecting the presence of IgE to measure whether IgE is able to induce cellular activation and degranulation.

* The BAT can be useful to confirm the diagnosis of food, venom and respiratory allergies.

* In the BAT, both allergen extracts and individual molecules can be used to stimulate the basophils and the latter can provide higher specificity in the case of some allergen sources.

## 1 - Introduction

In allergy diagnostics is important not only to test for IgE binding to allergens in a serological assay but also to test the functional interactions of allergens with IgE on effector cells.¹⁵³ This can be tested *in vivo* using skin prick test (SPT); however, SPT requires the appropriate clinical set up with the resources and expertise required to treat acute allergic reactions and does not allow for testing of recombinant allergen components, or even purified components. The Basophil Activation Test (BAT) is an *in vitro* diagnostic test that demonstrates the function of IgE in its ability to induce effector cell activation following stimulation with the allergen. Activation of basophils can be analysed by measuring the mediators released, such as histamine, or the change in plasma membrane markers, such as CD63, that happen during basophil degranulation.¹⁵⁴ CD63 is of particular interest because it is expressed on the membrane of the histamine-containing granules of basophils and is exposed on the plasma membrane after degranulation as the granules fuse with the plasma membrane.¹⁵⁵ The BAT has developed into a robust and straightforward assay that can be implemented in many laboratories using flow cytometry. In addition to supporting the diagnosis of food, insect venom and drug allergies, this test also allows monitoring the acquisition of tolerance in allergic patients. This can be spontaneous tolerance, e.g., in cow's milk allergy in infants who outgrow their allergy, or induced tolerance after Allergen Immunotherapy (AIT) for food, venom, or aero-allergens. Moreover, BAT has been instrumental in the characterization of the potency of allergen components.⁵⁴,¹⁵⁶

This chapter will provide an overview of the methodology for BAT, its potential role in the diagnosis and management of food allergy, insect venom allergy and respiratory allergy. In addition, the advantages and the limitations of the BAT will be discussed.

## 2 - Methodology

For basophil testing, allergen and fluorochrome-conjugated antibodies are required.¹⁵⁶ Heparinized or EDTA-stabilized blood can be used.¹⁵⁷ Normally, 50 – 100 µl of blood are used per assay. In contrast to skin prick testing, anti-histamines do not affect the outcome of BAT.¹⁵⁸ However, treatment with ibrutinib¹⁵⁹ reduces basophil activation and oral steroids can induce¹⁶⁰ basopenia.¹⁶¹ An advantage of the BAT is that it is flexible with regards to the allergen added. Commonly used allergen concentrations are listed in the online supplement of the EAACI position paper on basophil activation.¹⁵⁶ Allergens are added in either 1/10 of the volume of blood used, or in an equal volume depending on the BAT method adopted. Protein allergens like birch, pollen grass pollen or house dust mite allergens are added in concentrations ranging from picogram to microgram per millilitre. Often, basophils in a blood sample will react at four to six of the nine allergen concentrations used, so the response can be extremely dynamic. Sensitivity of blood basophils of an individual successfully treated with allergen immunotherapy may change by 2-3 orders of magnitude and be associated with clear clinical improvement¹⁶² that persists for years.¹⁶³,¹⁶⁴ To improve determination of the sensitivity, half log spacing of the samples may be used. In clinical practice, one may restrict analysis to the range of concentrations at which the basophil response often is dynamic.

Drugs in the BAT are used at higher concentrations than protein allergens—often in the microgram to milligram per millilitre range, and are often tested in five-fold dilutions. Drugs may interact with endogenous proteins to form noncovalent tertiary structures or covalent adducts that can be recognized by IgE on basophils—the so-called haptenization.¹⁶⁵ The response induced by these structures is thus less dramatic than that of protein allergens.

Basophils are identified by fluorochrome conjugated antibodies directed to CD193, CD203c, IgE, or CD123/HLA-DR, with greater precision being achieved with the combination of two or more antibodies. Activation is measured using antibodies directed against CD63 (Figure 12) or, CD203c. There are other markers that are up- and downregulated on activated basophils, such as CD107a and diaminoxidase DAO, and can be used as alternative measures of basophil activation.¹⁶⁶,¹⁶⁷ Antibodies should always be titrated before use; first the antibodies used to identify basophils and afterwards the activation markers, using anti-IgE or allergen-activated basophils.

Blood, allergen and antibodies can be combined and warmed to 37°C for between 15 and 45 minutes in a water bath or incubator. After that, the sample should be hemolysed and analysed by flow cytometry.

For analysis of flow cytometry data, doublets are excluded on a forward scatter area versus height plot, followed by a forward scatter versus side scatter plot in which the region containing basophils
# A05 - Basophil activation test

Alexandra F. Santos, Bernadette Eberlein, Peter Korosec, Hans-Jürgen Hoffmann, Edward F. Knol

## Highlights

* The basophil activation test (BAT) is a functional test that goes beyond detecting the presence of IgE to measure whether IgE is able to induce cellular activation and degranulation.

* The BAT can be useful to confirm the diagnosis of food, venom and respiratory allergies.

* In the BAT, both allergen extracts and individual molecules can be used to stimulate the basophils and the latter can provide higher specificity in the case of some allergen sources.

## 1 - Introduction

In allergy diagnostics is important not only to test for IgE binding to allergens in a serological assay but also to test the functional interactions of allergens with IgE on effector cells.¹⁵³ This can be tested *in vivo* using skin prick test (SPT); however, SPT requires the appropriate clinical set up with the resources and expertise required to treat acute allergic reactions and does not allow for testing of recombinant allergen components, or even purified components. The Basophil Activation Test (BAT) is an *in vitro* diagnostic test that demonstrates the function of IgE in its ability to induce effector cell activation following stimulation with the allergen. Activation of basophils can be analysed by measuring the mediators released, such as histamine, or the change in plasma membrane markers, such as CD63, that happen during basophil degranulation.¹⁵⁴ CD63 is of particular interest because it is expressed on the membrane of the histamine-containing granules of basophils and is exposed on the plasma membrane after degranulation as the granules fuse with the plasma membrane.¹⁵⁵ The BAT has developed into a robust and straightforward assay that can be implemented in many laboratories using flow cytometry. In addition to supporting the diagnosis of food, insect venom and drug allergies, this test also allows monitoring the acquisition of tolerance in allergic patients. This can be spontaneous tolerance, e.g., in cow's milk allergy in infants who outgrow their allergy, or induced tolerance after Allergen Immunotherapy (AIT) for food, venom, or aero-allergens. Moreover, BAT has been instrumental in the characterization of the potency of allergen components.⁵⁴,¹⁵⁶

This chapter will provide an overview of the methodology for BAT, its potential role in the diagnosis and management of food allergy, insect venom allergy and respiratory allergy. In addition, the advantages and the limitations of the BAT will be discussed.

## 2 - Methodology

For basophil testing, allergen and fluorochrome-conjugated antibodies are required.¹⁵⁶ Heparinized or EDTA-stabilized blood can be used.¹⁵⁷ Normally, 50 – 100 µl of blood are used per assay. In contrast to skin prick testing, anti-histamines do not affect the outcome of BAT.¹⁵⁸ However, treatment with ibrutinib¹⁵⁹ reduces basophil activation and oral steroids can induce¹⁶⁰ basopenia.¹⁶¹ An advantage of the BAT is that it is flexible with regards to the allergen added. Commonly used allergen concentrations are listed in the online supplement of the EAACI position paper on basophil activation.¹⁵⁶ Allergens are added in either 1/10 of the volume of blood used, or in an equal volume depending on the BAT method adopted. Protein allergens like birch, pollen grass pollen or house dust mite allergens are added in concentrations ranging from picogram to microgram per millilitre. Often, basophils in a blood sample will react at four to six of the nine allergen concentrations used, so the response can be extremely dynamic. Sensitivity of blood basophils of an individual successfully treated with allergen immunotherapy may change by 2-3 orders of magnitude and be associated with clear clinical improvement¹⁶² that persists for years.¹⁶³,¹⁶⁴ To improve determination of the sensitivity, half log spacing of the samples may be used. In clinical practice, one may restrict analysis to the range of concentrations at which the basophil response often is dynamic.

Drugs in the BAT are used at higher concentrations than protein allergens—often in the microgram to milligram per millilitre range, and are often tested in five-fold dilutions. Drugs may interact with endogenous proteins to form noncovalent tertiary structures or covalent adducts that can be recognized by IgE on basophils—the so-called haptenization.¹⁶⁵ The response induced by these structures is thus less dramatic than that of protein allergens.

Basophils are identified by fluorochrome conjugated antibodies directed to CD193, CD203c, IgE, or CD123/ HLA-DR, with greater precision being achieved with the combination of two or more antibodies. Activation is measured using antibodies directed against CD63 (Figure 12) or, CD203c. There are other markers that are up- and downregulated on activated basophils, such as CD107a and diaminoxidase DAO, and can be used as alternative measures of basophil activation.¹⁶⁶,¹⁶⁷ Antibodies should always be titrated before use; first the antibodies used to identify basophils and afterwards the activation markers, using anti-IgE or allergen-activated basophils.

Blood, allergen and antibodies can be combined and warmed to 37°C for between 15 and 45 minutes in a water bath or incubator. After that, the sample should be hemolysed and analysed by flow cytometry.

For analysis of flow cytometry data, doublets are excluded on a forward scatter area versus height plot, followed by a forward scatter versus side scatter plot in which the region containing basophils
<figure><img src="image_24.png" /><figcaption>FIGURE 12 Example of a BAT analysis (Hoffmann Lab). A FSC-A vs FSC-H plot to select single events, B. FSC-SSC plot to identify basophils be scatter parameters, C CD193 vs CD123 Plot to identify basophils as double positive events, D Histogram for CD63 expression, where the threshold is set to include 2% of the negative population. 77% of activated cells express CD63.</figcaption></figure>

between lymphocytes and monocytes is gated upon. In this region, single- or double-positive cells expressing selective antigens are identified. A threshold should be set on a population of non-stimulated basophils to measure activation in stimulated conditions—a threshold of ca. 2% of CD63 expression is desirable. A donor is a nonresponder if there is no activation through IgE/FceRI pathway, but there is response to non-IgE-mediated stimulants.¹⁶⁸ Patients that are nonresponders have uninterpretable results for BAT.

## 3 - Food allergy

The BAT can be a powerful tool to support the diagnosis of food allergy.¹⁶⁹ As the presence of allergen-specific IgE is not enough to confirm food allergy, the BAT can help to assess the function of allergen-specific IgE and determine whether allergen-specific IgE is able to induce effector cell activation following exposure to the allergen, which can help to establish the clinical relevance of a given IgE sensitization.¹⁷⁰,¹⁷¹ This is particularly useful in the absence of a clear history of an allergic reaction to a specific food or when there is discrepancy between the history and the results of IgE testing. Both food allergen extracts and individual allergen components can be used for basophil stimulation in the BAT, alongside the positive controls, IgE (anti-IgE or anti-FceRI) and non-IgE-mediated (e.g., fMLP).⁵⁴ Table 5 summarizes some of the published studies using allergen components in the BAT. Generally, using individual allergens can be more specific than using allergen extracts; however,

the broader sensitization profile is lost and could potentially lead to false-negative results for patients sensitized to allergens not included in the BAT.

The BAT has been used to distinguish primary from secondary food allergies and to establish the relevance of sensitization to fresh plant foods, such as apple. Primary apple allergy is associated with a shift in the dose-response towards lower concentrations of apple extract.¹⁷² Primary apple allergy can also be confirmed using molecular allergens, such as Mal d 3, the apple LTP, in the BAT, similar to what was previously shown for peach allergy with BAT to Pru p 3.¹⁷³ BAT is also useful to assess and compare allergenicity of individual components and their isoforms, which is not only important for food safety but also to guide guide development of hypoallergenic cultivars.¹⁷⁴,¹⁷⁵

Given BAT's high specificity, it is very useful to confirm the diagnosis of food allergy and therefore can preclude the need for oral food challenges (OFC) in individuals that would otherwise develop an allergic reaction following exposure to the suspected allergen. In peanut allergy studies, BAT using peanut extracts reduced the number of OFC by 67% to 70% and even to 80% when also including specific IgE in the diagnostic algorithm.¹⁷⁶,¹⁷⁷ This approach has been validated in another study of peanut, sesame, and tree nut allergies¹⁷⁸ and gave rise to the diagnostic approach proposed in Figure 13.⁵⁴,¹⁷⁹

For differentiation of patients with a clinically relevant alpha-gal syndrome and alpha-gal-sensitized subjects, commercially available alpha-gal-carrying proteins and pork kidney extracts using adequate
<figure><img src="image_24.png" /><figcaption>FIGURE 12 Example of a BAT analysis (Hoffmann Lab). A FSC-A vs FSC-H plot to select single events, B. FSC-SSC plot to identify basophils be scatter parameters, C CD193 vs CD123 Plot to identify basophils as double positive events, D Histogram for CD63 expression, where the threshold is set to include 2% of the negative population. 77% of activated cells express CD63.</figcaption></figure>

between lymphocytes and monocytes is gated upon. In this region, single- or double-positive cells expressing selective antigens are identified. A threshold should be set on a population of non-stimulated basophils to measure activation in stimulated conditions—a threshold of ca. 2% of CD63 expression is desirable. A donor is a nonresponder if there is no activation through IgE/FceRI pathway, but there is response to non-IgE-mediated stimulants.¹⁶⁸ Patients that are nonresponders have uninterpretable results for BAT.

## 3 - Food allergy

The BAT can be a powerful tool to support the diagnosis of food allergy.¹⁶⁹ As the presence of allergen-specific IgE is not enough to confirm food allergy, the BAT can help to assess the function of allergen-specific IgE and determine whether allergen-specific IgE is able to induce effector cell activation following exposure to the allergen, which can help to establish the clinical relevance of a given IgE sensitization.¹⁷⁰,¹⁷¹ This is particularly useful in the absence of a clear history of an allergic reaction to a specific food or when there is discrepancy between the history and the results of IgE testing. Both food allergen extracts and individual allergen components can be used for basophil stimulation in the BAT, alongside the positive controls, IgE (anti-IgE or anti-FceRI) and non-IgE-mediated (e.g., fMLP).⁵⁴ Table 5 summarizes some of the published studies using allergen components in the BAT. Generally, using individual allergens can be more specific than using allergen extracts; however,

the broader sensitization profile is lost and could potentially lead to false-negative results for patients sensitized to allergens not included in the BAT.

The BAT has been used to distinguish primary from secondary food allergies and to establish the relevance of sensitization to fresh plant foods, such as apple. Primary apple allergy is associated with a shift in the dose-response towards lower concentrations of apple extract.¹⁷² Primary apple allergy can also be confirmed using molecular allergens, such as Mal d 3, the apple LTP, in the BAT, similar to what was previously shown for peach allergy with BAT to Pru p 3.¹⁷³ BAT is also useful to assess and compare allergenicity of individual components and their isoforms, which is not only important for food safety but also to guide guide development of hypoallergenic cultivars.¹⁷⁴,¹⁷⁵

Given BAT's high specificity, it is very useful to confirm the diagnosis of food allergy and therefore can preclude the need for oral food challenges (OFC) in individuals that would otherwise develop an allergic reaction following exposure to the suspected allergen. In peanut allergy studies, BAT using peanut extracts reduced the number of OFC by 67% to 70% and even to 80% when also including specific IgE in the diagnostic algorithm.¹⁷⁶,¹⁷⁷ This approach has been validated in another study of peanut, sesame, and tree nut allergies¹⁷⁸ and gave rise to the diagnostic approach proposed in Figure 13.⁵⁴,¹⁷⁹

For differentiation of patients with a clinically relevant alpha-gal syndrome and alpha-gal-sensitized subjects, commercially available alpha-gal-carrying proteins and pork kidney extracts using adequate