# Development and evaluation of ELISA serological immunoassays for influenza and respiratory syncytial viruses

Alexandra Claudet, Noémi Alcover, Elise Lebigot, Hana-Sofia Bouhelal, Fairly Warnakulasuriya, Marie-Pierre Soutiere, Marie Galloux, Jean-François Éléouët, Marie-Anne Rameix-Welti, Etienne Bizot, et al.

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## To cite this version:

Alexandra Claudet, Noémi Alcover, Elise Lebigot, Hana-Sofia Bouhelal, Fairly Warnakulasuriya, et al. Development and evaluation of ELISA serological immunoassays for influenza and respiratory syncytial viruses. Diagnostic Microbiology and Infectious Disease, 2025, 112 (3), pp.116835. 10.1016/j.Diagmicrobio.2025.116835. hal-05050932

HAL Id: hal-05050932

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Submitted on 29 Apr 2025

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Original Article

# Development and evaluation of ELISA serological immunoassays for influenza and respiratory syncytial viruses

Alexandra Claudet<sup>a</sup>, Noémi Alcover<sup>a</sup>, Elise Lebigot<sup>b</sup>, Hana-Sofia Bouhelal<sup>a</sup>,

Fairly Warnakulasuriya<sup>a</sup>, Marie-Pierre Soutiere<sup>a</sup>, Marie Galloux<sup>c</sup>, Jean-François Eleouet<sup>c</sup>,

Marie-Anne Rameix-Welti<sup>d</sup>, Etienne Bizot<sup>e,f</sup>, Christelle Vauloup-Fellous<sup>a,f</sup>,

Vincent Portet-Sulla<sup>a,f,*</sup>

ARTICLE INFO

Key words:

RSV
Influenza

Serology
IgG

Transfer

Neonatal

Placental

ABSTRACT

**Introduction:** Respiratory syncytial virus (RSV) and influenza are major causes of respiratory infections globally. Although vaccines are available, serological tools to assess population-level immunity and maternal antibody transfer remain limited. This study aimed to develop and evaluate ELISA assays for detecting anti-RSV and anti-influenza antibodies as a basis for future maternal-fetal immunity research.

**Materials and Methods:** In this study, we designed ELISA immunoassays using various influenza and RSV antigens.

Sensitivity and specificity were calculated using sera from presumed seronegative infants and seropositive adults.

**Results:** The influenza vaccine-based ELISA achieved 98.3 % sensitivity and 100 % specificity. The pre-fusion F protein of RSV (Arexvy®) showed 97.5 % sensitivity and 97.4 % specificity. Other antigen combinations performed less optimally.

**Discussion:** These ELISA assays are scalable tools for seroepidemiological and maternal transfer studies. Future work will include correlation with neutralizing antibodies and paired maternal-infant analyses.

# 1. Introduction

Influenza virus and respiratory syncytial virus (RSV) are the primary viral pathogens responsible for pediatric hospitalizations due to respiratory infections [1,2]. *Influenza viruses (Orthomyxoviridae)* possess two key glycoproteins, haemagglutinin (HA) and neuraminidase (NA), which facilitate viral entry and release and induce neutralizing antibody responses [3]. Globally, influenza leads to up to 650,000 deaths annually, mainly among infants, pregnant women and the elderly [4]. RSV belongs to the *Orthopneumovirus* family and has two major glycoproteins, F and G. The F protein, responsible for membrane fusion,

undergoes a structural transition from a pre-fusion to a post-fusion conformation. The pre-fusion F form exposes epitopes such as site Ø, which are highly immunogenic and targeted by neutralizing antibodies [5]. The G protein, by contrast, is highly variable and poorly immunogenic for serological purposes [6]. In addition to these surface glycoproteins, RSV also has internal proteins, notably the nucleoprotein, which has already been targeted for the development of serological assays [7]. The nucleoprotein N which encapsidates the viral genome, forming the nucleocapsid NC (serving as a matrix for the viral replication complex) is highly conserved and highly immunogenic and has already been tested as a target protein in serology [8,9]. RSV is the main

<footnote><sup>a</sup> Division of Virology, WHO Rubella National Reference Laboratory, Dept of Biology Genetics, Paul Brousse Hospital, Paris Saclay University Hospital, APHP, Villejuif, France</footnote>

<footnote><sup>b</sup> Division of Biochemistry, Kremlin-Bicêtre Hospital, APHP, Le Kremlin Bicêtre, France</footnote>

<footnote><sup>c</sup> Unité de Virologie et Immunologie Moléculaires (VIM), Université Paris-Saclay, INRAE, Jouy-en-Josas, France</footnote>

<footnote><sup>d</sup> National Reference Center for Respiratory Infection Viruses (CNR VIR), M3P, Institut Pasteur, Université Paris-Saclay, Université de Versailles St. Quentin, Université Paris Cité, UMR 1173 (2I), INSERM, Assistance Publique - Hôpitaux de Paris, 75015 Paris, France</footnote>

<footnote><sup>e</sup> General Pediatrics and Pediatric Emergency Department, Antoine-Béclère Hospital, AP-HP, Clamart</footnote>

<footnote><sup>f</sup> Paris Saclay University, INSERM U1184, CEA, Center for Immunology of Viral, Auto-immune, Hematological and Bacterial diseases (IMVA-HB/IDMIT), Fontenay-aux-Roses, France</footnote>

<footnote>* Corresponding author at: Laboratoire de virologie du Groupe Hospitalier Paris-Saclay, Hôpital Paul Brousse, 12-14 avenue Paul Vaillant Couturier, 94804 Villejuif, France.</footnote>

<footnote>E-mail address: vincent.portetsulla@aphp.fr (V. Portet-Sulla).</footnote>

<footnote>https://doi.org/10.1016/j.diagmicrobio.2025.116835</footnote>

<footnote>Received 16 February 2025; Received in revised form 27 March 2025; Accepted 5 April 2025</footnote>

<footnote>Available online 7 April 2025</footnote>

<footnote>0732-8893/© 2025 The Author(s). Published by Elsevier Inc. This is an open access article under the CC BY license (http://creativecommons.org/licenses/by/4.0/).</footnote>
cause of bronchiolitis in infants and represents a significant global health concern. Worldwide, it accounts for 33 million cases a year among children under the age of 5, including 3.6 million hospitalizations and 101,400 deaths. It is the cause of 1 in 28 deaths under 6 months of age, and 1 in deaths 50 among children under 5 years old [9–11]. In France, RSV is responsible for 50,000 hospitalizations each year, 69 % of which involve infants under 12 months 9,12. There is also a significant morbidity and mortality rate in elderly or frail individuals. Globally, RSV causes approximately 1.5 million cases of acute respiratory infections annually among adults aged 65 years and above, resulting in an estimated 336,000 hospitalizations and 14,000 in-hospital deaths [13]. In populations at risk of severe influenza, annual vaccine prophylaxis is recommended, and in the event of severe infection, antiviral treatment with a neuraminidase inhibitor (Oseltamivir or Zanamivir) may be offered [14]. However, despite recurrent vaccination campaigns, France remains a country where vaccination coverage is low even in the most at-risk populations. For example, only 7 % pregnant women were vaccinated in 2016 [15]. Concerning RSV, vaccines are available since few months and have shown to be effective for adults above 60 years old (Arexyv®) and in neonates born to mothers vaccinated during pregnancy (Abrysvo®) [16,17]. Moreover, monoclonal antibodies (Palivizumab or Nirsevimab) is another way to protect neonates (born to mothers unvaccinated) [18].

Numerous serological techniques have been developed to detect antibodies against RSV and influenza. The most widely used are virus neutralization assays (microneutralization or plaque reduction), hemagglutination inhibition (HAI) tests for influenza, and ELISAs targeting the nucleoprotein (NP) [19–25]. For RSV, microneutralization assays are widely used in vaccine trials and correlate with protection, but they are technically complex, time-consuming and require viral culture under high biosafety conditions [20,26]. HAI assays, specific to influenza, suffer from limited standardization and depend on subtype-specific red blood cell interactions. ELISAs based on NP antigens are easier to implement lack to target hemagglutinin, which is a key immunodominant antigen in the antibody response to influenza infection. In contrast, ELISAs using surface antigens such as hemagglutinin for influenza and F protein or N protein for RSV offer better scalability. Furthermore, regarding RSV, the recent success in the stabilization of the F protein in its pre-fusion conformation enables the development of more RSV-specific ELISA assays, as this conformation exposes high immunogenic site Ø [27,28]. These influenza and RSV antigens also align closely with vaccine targets and are less prone to variability or structural homology with unrelated viruses. These advantages make them particularly suitable for population-level studies, including investigations of maternal-fetal antibody transfer. However, no fully clinically validated commercial assays targeting these proteins are currently available for routine clinical use. In this study we focused on the development and validation of standardized ELISA assays using vaccine-grade and recombinant antigens for both influenza and RSV. These tools represent a necessary methodological foundation for future maternal-infant serological studies, including the TransAc cohort (Clinical Trial number: NCT05821764).

# 2. Material and methods

## 2.1. Serum samples

Sera from individuals presumed to be "seronegative" and "seropositive" for influenza and RSV were collected from patients attending our university hospital (usually for biochemistry purposes) from April 2022 and October 2022. Subjects who had received blood-derived products within the previous 3 months were excluded. As part of routine management in our hospital, patients/parents are requested to indicate that if they don't formally object, their biological samples may be used for research purposes. No patient/parent has objected and additional investigations on the same samples were performed in accordance with

the Declaration of Helsinki and French law that allows additional tests on samples collected more than one year before. Serum samples were stored at -20°C until analysis.

To account for the high seroprevalence of RSV and influenza in the study population (> 90 %), we aimed to use a 2:1 ratio of seropositive to seronegative sera (i.e. approximatively 200 seropositive and 100 seronegative). This approach ensures sufficient statistical power to evaluate sensitivity and specificity while optimizing the availability of rare seronegative samples [29,30].

Presumed seronegative sera were collected from children between 2- and 12-months old attending hospital for non-infectious cause. Indeed, at this age, we considered they have already started to lose maternal antibodies and probably might have not encountered RSV or influenza yet [2,31].

Presumed seropositive sera were collected from adults for whom we presume they had already contracted RSV or influenza infection in their life. When it was available in medical report, we collected the anti-influenza vaccination status.

## 2.2. Antigens

For influenza virus, two antigens were tested:

- The hemagglutinin of Influenza A virus H3N2 (A/Darwin/6/2021) (SinoBiological®, Germany) (Darwin HA). Influenza A virus H3N2 was responsible for the 2021/2022 and 2022/2023 epidemics [14]

- The 2022 influenza vaccine Vaxigrip® (Sanofi®, France), including 4 different strains: 2 influenza A strains (H1N1 A/Victoria/2570/2019 and H3N2 A/Darwin/9/2021) and 2 influenza B strains (B/Austria/1359417/2021 and B/Phuket/3073/2013) [14].

For RSV, four antigens were tested:

- F protein (SinoBiological®, Germany), post-fusion conformation, derived from an RSV-A2 strain

- F protein from Arexyv® vaccine, pre-fusion conformation "RSVPreF3" [32]

- N nucleoprotein (SinoBiological®, Germany)

- N nucleoprotein in the form of RNA-N nanorings provided by INRAe (National Research Institute for Agriculture, Food and Environment) [8,33]

## 2.3. Coating

Antigens were diluted in phosphate-buffered solution (PBS, pH 7.4) and distributed in 96-well plates (Maxisorp ThermoScientific®, Denmark): Darwin HA and vaccine (1µg/mL) at a rate of 50 µL per well [25], and F and N proteins (2 or 4 µg/mL) at a rate of 100 µL per well [34]. Arexyv® vaccine contains an additive: AS01E adjuvant (extract of Quillaja saponaria Molina (QS-21) and 3-O-desacyl-4'-monophosphoryl lipid A (MPL) derived from Salmonella minnesota). We hypothesized that optical density could be overestimated because of this antigen, so we decided to dilute the RSVPreF3 powder into PBS directly and not into the adjuvant.

After overnight incubation at +4°C and 3 washes with PBS Tween®, 200 µL of blocking solution was added to each well and the plates incubated for 1 h at room temperature. After 5 washes with PBS Tween®, the plates were sealed and stored at +4°C.

## 2.4. Immunoassay

Sera were diluted and distributed on the coated well plates for 2 h at 37°C. Plates were then washed 3 times with PBS Tween® before revocation by 1 h incubation with goat anti-human IgG coupled to peroxidase (Sigma-Aldricht®, USA), followed by o-Phenylenediamine dihydrochloride substrate (Sigma-Aldricht®, USA). Reaction was stopped with 1
M sulfuric acid solution (BioRad®, USA). Optical densities were read at 490 nm and 620 nm (spectrophotometer MultiSkan GO, Thermofisher Scientific®, USA).

## 2.5. Statistics

GraphPad Prism® software, version 9.5.1 (528) was used to compare optical densities for the different populations tested, to establish ROC curves. A ROC curve is used to determine the sensitivity and specificity of a technique; sensitivity being the probability that the test will be positive if the patient is immunized in our case (or percentage of true positives) and specificity being the probability that the test will be negative if the patient is non-immunized (or percentage of true negatives). ROC curve allows therefore evaluating performances of serology assays in discriminating between immune and non-immune individuals. Positive predictive value (PPV) and negative predictive value (NPV) were also calculated. Student and Mann-Whitney statistical tests were used to compare the studied populations. The alpha risk for our statistics was set at 0.05.

# 3. Results

## 3.1. Study population characteristics

### 3.1.1. Presumed seronegative samples: infants with documented infections or respiratory infection signs excluded

During the period of the study, we were able to collect 114 serum samples from children between 2 and 12 months old. We excluded 12 serums from infants that had respiratory infection signs or documented respiratory infection by RSV, influenza, Rhinovirus, SARS-CoV-2 (Flowcharts in Figs. 1 and 2). At last, 102 samples were available for our study with sufficient volume. Median age was 4 months (IQR [1-7]).

### 3.1.2. Presumed seropositive samples: adults from various backgrounds with partial vaccination data

We collected 180 serum samples including 103 from pregnant women, 56 from prison inmates and 9 from adults hospitalized for an RSV infection. Median age was 31.5. We did not get the information about anti-influenza vaccination for 109 adults (60.5 %), 28 (15.6 %) were vaccinated and 43 (23.9 %) were not vaccinated. (Flowcharts in Figs. 1 and 2).

### 3.1.3. Influenza Immunoassays: high sensitivity and 100 % specificity of ELISA using influenza vaccine antigen

Results of influenza serology with Vaccine Vaxigrip® coating for presumed seronegative samples are shown in Fig. 3. Out of the 102 seronegative serum samples collected, we observed that probably only children aged between 4 months and 12 months old were truly seronegative (56 samples) as OD results were higher in 2- and 3-months old children (probable remaining maternal antibodies). Out of the 180 seropositive serum samples collected, we were able to perform the serology tests on 179 (Fig. 1). Results for these selected 56 presumed negative and the 179 presumed positive samples are shown in Fig. 4. For the best threshold identified by ROC curve (1.05) sensitivity was 98.33 % (IC95 % 0.95-0.99) and specificity was 100 %, (IC95 % 0.94-1.00) (p < 0,0001); Fig. 4. Detailed results regarding vaccination status are shown in Fig. 5.

Results with Darwin HA coating were not conclusive (Fig. 6). Indeed, for the best threshold identified by ROC curve (0.295) sensitivity was 65 % and specificity was 66 % (p < 0,001). All performance results are summarized in Table 1.

### 3.1.4. RSV immunoassays: ELISA with RSV pre-F antigen achieves high diagnostic accuracy; N protein less effective

Results of RSV F serology for presumed seronegative are shown in Fig. 7. Out of the 102 seronegative samples, we observed that probably only children aged between 4 months and 8 months old were truly seronegative (39 samples) as OD results were higher in 2-3 months (probable remaining maternal antibodies) and 9-12 months (RSV infection early in life) aged children. Out of the 180 seropositive serum samples collected, we were able to perform the serology tests on 161, primarily due to insufficient sample volume in the remaining cases (Fig. 2). Results for these 39 presumed negative and the 161 presumed positive samples are shown in Figs. 8 and 9. For Arexvy® vaccine coating (Fig. 8), the best threshold identified by ROC curve (1.17) allowed to reach 97.5 % sensitivity (IC 95 % 0.93-0.99) and 97.4 % specificity (IC 95 % 0.86-0.99.87) (p < 0,0001). For SinoBiological F coating (Fig. 9), the best threshold identified by ROC curve (0.36) allowed only a 95.3 % (IC 95 % 0.84-0.93) sensitivity and 92.3 % specificity (IC 95 % 0.71-0.93, p < 0,0001).

Performance values for N protein (either SinoBiological® or the one provided by INRAe) coating were disappointing as best sensitivity and specificity were around ~86 % for N SinoBiological and ~50 % for N

<figure><img src="image_3.png" /><figcaption>Fig. 1. Flowchart of the serum used for performances calculation of influenza immunoassays.</figcaption></figure>

Out of the 102 seronegative serum samples tested, we observed that probably only children aged between 4 months and 12 months old were truly seronegative (56 samples) as OD results were higher in 2- and 3-months old children (N = 46, probable remaining maternal antibodies). Out of the 180 seropositive serum samples collected, we were able to perform the serology tests on 179 (N = 1 insufficient volume).
<figure><img src="image_4.png" /><figcaption>**Fig. 2.** Flowchart of the serum used for performances calculation of RSV immunoassays. Out of the 102 seronegative samples tested, we observed that probably only children aged between 4 months and 8 months old were truly seronegative (39 samples) as OD results were higher in 2-3 months (probable remaining maternal antibodies) and 9-12 months (RSV infection early in life) aged children. Out of the 180 seropositive serum samples collected, we were able to perform the serology tests on 161, primarily due to insufficient sample volume in the remaining cases (N = 19).</figcaption></figure>

<figure><img src="image_5.png" /></figure>

<figure><img src="image_6.png" /><figcaption>**Fig. 3.** Influenza immunoassay results in the presumed seronegative samples (Vaccine Vaxigrip®) Optical density distribution in the seronegative population (N = 102) (Vaxigrip® coating) from 2 months after birth (M2) to 12 months after birth (M12). Patients presumed seronegative were taken from M4 to M12 (blue arrows; N = 56/102). Patients from M2 and M3 were excluded from the ROC performance calculations because the titers of maternal antibodies were too high.</figcaption></figure>

INRAe (Supplemental data). All performance results are shown in Table 1.

In order to improve performances of RSV immunoassay we attempted to coat microplates with a mix of the two RSV proteins F and N (SinoBiological®) to coat our plates. Results for 35 presumed negative (children aged between 4 months and 8 months) and 173 presumed positive samples are shown in Fig. 10. For the best threshold identified by ROC curve (0.56) sensitivity was 88.4 % (IC95 % 0.82-092) and specificity was 88.5 % (IC95 % 0.74-0.95) (p < 0.0001).

## 4. Discussion

We report in this article the development and evaluation of influenza and RSV serology immunoassays (for research purposes) as no

commercial assays were available. Our work is, to our knowledge, the first to develop and clinically evaluate a standardized and rapid serological ELISA assay for both RSV and influenza. First, when developing a serology test, it is required to have a population of seropositive patients and a population of seronegative patients. Regarding influenza virus and RSV, well-characterized population of seronegative patients are hardly available as these infections are quite frequent. Indeed, 82 % of patients between 0 and 12 years old are seropositive against influenza virus A, and 100 % after 15 years old [2]. In infants, protective levels of antibodies against influenza wanes below protective level within 2 months after birth, the seroprevalence of children under 3 years old is approximately 14 % [35,36]. For RSV, it is known that 100 % of children are infected with RSV before the age of 2 [17]. As we performed our serology tests on infants between 2 and 12 months, we observed the waning of RSV and influenza virus IgG titers. Based on this, we decided to select the seronegative population in age groups that are the most unlikely to have remaining IgG from the mother and the less likely to have an RSV or Influenza virus infection. As the epidemiology of RSV and influenza is different, the two populations of the seronegative patients were not completely overlapping. Furthermore, it is possible that in our selection of presumably negative sera, some children still had maternal antibodies or had already been infected with RSV or influenza virus, even though we selected sera between April 2022 and October 2022 (which corresponds to a period of low virus circulation). Consequently, we decided to exclude seronegative sera with the highest optical density, since these infants were probably infected during their first year of life or they could still have had maternal antibodies. Although we carefully excluded infants with high optical densities and/or known infections, it is still possible that some presumed-seronegative infants retained low levels of maternal antibodies, leading to potential false positives. Conversely, within the presumed-seropositive adult group, some individuals may have had low or undetectable antibody levels at the time of sampling, resulting in false negatives. We did not exclude adults with low optical density from the presumed seropositive group, as our objective was to reflect the natural heterogeneity of immune responses in the general population. These misclassifications would tend to underestimate the actual sensitivity and specificity of the assays, suggesting that their true diagnostic performance may in fact be even higher. Among the highest optical density for RSV ELISA, we excluded optical densities of 12 children that were diagnosed at the time of sampling with a respiratory infection (SARS-CoV-2, rhinovirus, RSV)
<figure><img src="image_7.png" /><figcaption>**Fig. 4.** (A): Optical density distribution according to presumed immune status (Vaxigrip® vaccine). Red line: best threshold identified by ROC curve at 1.05. Presumed seropositive *N* = 179. Presumed seronegative *N* = 56. (B): ROC curve.</figcaption></figure>

<figure><img src="image_8.png" /><figcaption>**Fig. 5.** Optical density according to flu vaccination status. Vaccinated (N = 27); unvaccinated (N = 43); unknown status (N = 109) and seronegative N = 56.</figcaption></figure>

and 63 children aged between 2- and 3-months old and 9-12-months old (Fig. 2). Moreover, according to a study published in December 2022, RSV seroprevalence, attributed to maternally transferred IgG, falls sharply during infants' first 7 months of life, as they lose these antibodies to develop their own immunity: it drops from 60% at 2 months to almost 10% by the age of 7 months [31]. Seroprevalence then rises again as children are exposed to RSV.

For each of the techniques developed in this report, there was no marketed kit whose performance had been evaluated in literature. In fact, tests that are used in most of influenza and RSV seroprevalence studies, are based on neutralization assays, in particular micro-neutralization for RSV and hemagglutination for influenza [25,37]. Serum neutralization techniques have the advantage of detecting specific neutralizing antibodies after infection or vaccination, either by incubating serum with virus-infected cells (microneutralization) or with virus and red blood cells (hemagglutination test). Recently, these neutralization techniques have been standardized by the WHO through the introduction of international standards [38]. However, these serum

neutralization techniques remain semi-quantitative (rendered in reverse of the minimum dilution of serum required to neutralize viral infection or hemagglutination of red blood cells) and their performances (sensitivity/specificity/PPV/NPV) are rarely reported. Seroneutralization remains a cumbersome and challenging technique to implement, given the laboratory infrastructure it requires (maintenance of cell lines and viral strains) [37]. These are fastidious procedures with a turnaround time of between 4 and 8 days, whereas our ELISA serology technique delivers a result in <7 h. In addition, ELISA serology has the advantage of being inexpensive, quantitative, and standardizable.

In our study, influenza ELISA serology based on vaccine antigens proved to be the most effective with sensitivity and specificity close to 100%. Compared with HA alone, the influenza vaccine contains 4 different strains of influenza (2 strains of influenza A and 2 strains of influenza B), offering a greater number of antigenic targets. Moreover, this coating can be adapted to the annual flu vaccine and offers the opportunity of changing antigens to match as closely as possible to the influenza viruses circulating during each epidemic period. However, this approach also has a limitation: it does not allow for the identification of which specific influenza virus strain the detected antibodies are targeting. As well as Darwin HA, RSV F and N proteins offer only restricted antigenic targets. As a result, their diagnostic performance may be less optimal than coating with a fragmented of the whole virus in the wells. The Sinobiological® F and N proteins did not give completely satisfactory results. Regarding the N protein, some authors have pointed out that its handling conditions could influence assay performance. Specifically, it has been noted that PBS, with a pH close to the isoelectric point of the protein, may cause precipitation, whereas a carbonate-bicarbonate buffer at pH 9.5 has been found to be more suitable [39].

However, the pre-fusion F protein from the Arexyv® vaccine gave 97.5 % sensibility and 97.4 % specificity. In the case of the F protein, these results were expected as antibodies are preferentially produced against the pre-fusion form of the protein whereas the SinoBiological® F protein is a post-fusion form. Indeed, the pre-fusion conformation of the F-protein unveils more epitopes than the post fusion form, especially the site ø that generates potent antibodies that neutralize the virus [40]. In this respect, site ø is also the target of Nirsevimab. Arexyv® vaccine contains an adjuvant to dilute the RSVPreF3 powder but for our experiments, we chose to dilute the RSVPreF3 in PBS to get rid of the false antibody reactivity that could be generated by the presence of the adjuvant. We also compared two N proteins, but results were
<figure><img src="image_9.png" /><figcaption>**Fig. 6.** (A): Optical density distribution according to presumed immune status (Darwin Hemagglutinin). Red line: best threshold identified by ROC curve at 0,295. Presumed seropositive *N* = 179. Presumed seronegative *N* = 56. (B): ROC curve.</figcaption></figure>

<table><caption>**Table 1**
Performances of the immunoassays developed in the study.</caption><thead><tr><td>Pathogen</td><td>Antigens used for coating</td><td>Sensitivity</td><td>Specificity</td></tr></thead><tbody><tr><td>Influenza</td><td>Vaxigrip® Vaccine</td><td>98.3 %</td><td>100 %</td></tr><tr><td>Influenza</td><td>Darwin Hemagglutinin</td><td>65 %</td><td>66 %</td></tr><tr><td>RSV</td><td>Arexyv® vaccine (Pre-fusion F protein)</td><td>97.5 %</td><td>97.4 %</td></tr><tr><td>RSV</td><td>Post-fusion F protein (Sinobiological)</td><td>95.3 %</td><td>92.3 %</td></tr><tr><td>RSV</td><td>N protein (Sinobiological)</td><td>86.5 %</td><td>86.1 %</td></tr><tr><td>RSV</td><td>Post-fusion F protein and N protein (Sinobiological)</td><td>88.4 %</td><td>88.5 %</td></tr></tbody></table>

unsatisfying. We tried coating with a mix of SinoBiological® F protein and SinoBiological® N protein to increase the number of potential antigenic sites. Performances were less satisfactory than with dissociated coatings (sensitivity 81.67 %, specificity 77.78 %). Although we did not perform heterologous cross-reactivity assays using RSV antigens in influenza ELISA or vice versa, the antigens used in our study were selected based on their structural and antigenic specificity. For the

influenza assay, we used the seasonal quadrivalent influenza vaccine as the antigen source, which contains predominantly hemagglutinin proteins from four viral strains (two influenza A and two B) or purified hemagglutinin. For the RSV assay, we used recombinant F proteins (in both pre-fusion and post-fusion conformations) and N protein, which are structurally unrelated to influenza proteins and contain distinct immunodominant epitopes. The pre-fusion RSV F protein in particular presents unique conformational epitopes, such as site Ø, that are highly immunogenic and not shared with other viral proteins [27]. For influenza, hemagglutinin contains both conserved and subtype-specific epitopes, and site-specific serological mapping has demonstrated that highly selective detection is achievable [41]. Given the absence of structural or sequence homology between these antigens, significant cross-reactivity between RSV and influenza is unlikely. Furthermore, in the general adult population, it is extremely difficult to identify individuals who are seropositive for only one of these viruses, as exposure to both RSV and influenza is widespread throughout life. This makes cross-coating experiments challenging to interpret, as dual seropositivity could confound the results. These considerations, together with

<figure><img src="image_10.png" /><figcaption>**Fig. 7.** RSV immunoassay results in the presumed seronegative samples (pre-fusion F protein from Arexyv® vaccine (A) and post fusion F protein from Sinobiological (B).</figcaption></figure>

Optical density distribution in the seronegative population from 2 months after birth (M2) to 12 months after birth (M12). Patients presumed seronegative were taken from M4 to M8 (blue arrows; *N* = 39/102). Patients from M2,M3, M9 to M12 were excluded from the ROC performance calculations.
<figure><img src="image_11.png" /><figcaption>**Fig. 8.** (A): Optical density distribution according to presumed immune status (pre-fusion F protein, Arexyvy®): presumed seronegative (N = 39) and presumed seropositive (N = 161). Red horizontal line: best threshold identified by ROC curve at 1.17. (B): ROC curve.</figcaption></figure>

<figure><img src="image_12.png" /><figcaption>**Fig. 9.** (A): Optical density distribution according to presumed immune status (post fusion F protein Sinobiological®): presumed seronegative (N = 39) and presumed seropositive (N = 161). Red horizontal line: best threshold identified by ROC curve at 0.36 (B): ROC curve.</figcaption></figure>

the high specificity values observed in our ELISAs (>97 %), support the robustness of our antigen selection. Nevertheless, we acknowledge that additional cross-reactivity experiments could complement our findings and are worth exploring in future investigations. Finally, it is important to acknowledge that ELISA-based detection reflects total binding IgG and may not directly correlate with neutralizing capacity. Although our assays are not intended to replace microneutralization or hemagglutination inhibition tests, they provide a practical and scalable alternative, particularly for large-scale epidemiological surveillance. Future studies will include correlation analyses with neutralization assays and maternal-infant serum pairs to assess transfer ratios and protective thresholds.

## 5. Informed consent statement

This work was a non-interventional study. Reclassification of biological remnants into research material was approved by the

Institutional Review Board of the Assistance Publique-Hopitaux de Paris University Hospitals participating to the study. According to the French Public Health Code (CSPArtL.1121-1.1), such protocols are exempted from individual informed consent due to the retrospective chart review design and absence of identifying images or personal/clinical details that could compromise anonymity.

Data Availability Statement: Data can be provided by the authors on demand.

Funding sources: none.

## CRediT authorship contribution statement

**Alexandra Claudet:** Writing – original draft, Methodology, Investigation, Formal analysis. **Noémi Alcover:** Conceptualization. **Elise Lebigot:** Resources. **Hana-Sofia Bouhelal:** Investigation. **Fairly War-nakulasuriya:** Writing – review & editing, Resources. **Marie-Pierre Soutiere:** Resources, Methodology, Investigation. **Marie Galloux:**
<figure><img src="image_13.png" /><figcaption>Fig. 10. (A): Optical density distribution according to presumed immune status (mix F & N protein Sinobiological®): presumed seronegative (N = 39) and presumed seropositive (N = 161). Red horizontal line: best threshold identified by ROC curve at 0.56. (B) ROC curve.</figcaption></figure>

Resources, Methodology. **Jean-François Eleouet:** Resources, Methodology. **Marie-Anne Rameix-Welti:** Resources, Methodology. **Etienne Bizot:** Writing – review & editing. **Christelle Vauloup-Fellous:** Writing – review & editing, Supervision, Methodology, Conceptualization. **Vincent Portet-Sulla:** Writing – review & editing, Methodology, Investigation, Formal analysis, Data curation, Conceptualization.

Declaration of competing interest

The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this manuscript.

Supplementary materials

Supplementary material associated with this article can be found, in
the online version, at doi:10.1016/j.diagmicrobio.2025.116835.

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