Chapter 11
Monitoring Memory B Cells by Next-Generation
ImmunoSpot® Provides Insights into Humoral Immunity that
Measurements of Circulating Antibodies Do Not Reveal
Paul V. Lehmann, Zhigang Liu, Noémi Becza, Alexis V. Valente,
Junbo Wang, and Greg A. Kirchenbaum
Abstract
Memory B cells (Bmem) provide the second wall of adaptive humoral host defense upon specific antigen
rechallenge when the first wall, consisting of preformed antibodies originating from a preceding antibody
response, fails. This is the case, as recently experienced with SARS-CoV-2 infections and previously withseasonal influenza, when levels of neutralizing antibodies decline or when variant viruses arise that evadesuch. While in these instances, reinfection can occur, in both scenarios, the rapid engagement of preexisting
Bmem into the recall response can still confer immune protection. Bmem are known to play a critical role inhost defense, yet their assessment has not become part of the standard immune monitoring repertoire. Here
we describe a new generation of B cell ELISPOT/FluoroSpot (collectively ImmunoSpot®) approaches
suited to dissect, at single-cell resolution, the Bmem repertoire ex vivo, revealing its immunoglobulin class/subclass utilization, and its affinity distribution for the original, and for variant viruses/antigens. Becausesuch comprehensive B cell ImmunoSpot® tests can be performed with minimal cell material, are scalable,
and robust, they promise to be well-suited for routine immune monitoring.
Key words ELISPOT, FluoroSpot, B cells, Immune monitoring, Antibodies, Antibody titers,
Immune memory, Affinity

1

Introduction
Clinical immune diagnostics are currently confined to the detectionof specific antibodies in serum and other bodily fluids. Diagnostic
decisions on whether a person has been infected by, and subsequently developed immunity, e.g., to a particular virus or vaccination, and the magnitude of the ensuing immune response arepresently deduced from measurements of serum antibody titers.
In this chapter, we will describe why it is necessary to include
memory B cell (Bmem) measurements into such considerations,and how this can be reliably accomplished with minimal cell

Alexander E. Kalyuzhny (ed.), Handbook of ELISPOT: Methods and Protocols, Methods in Molecular Biology, vol. 2768,
https://doi.org/10.1007/978-1-0716-3690-9_11, © The Author(s) 2024

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material and labor by performing B cell ELISPOT/FluoroSpot,collectively “ImmunoSpot®” assays (see Note 1).
There are conceptual and practical reasons why, thus far, mainstream efforts toward monitoring antigen-specific Bmem have notbeen sufficiently undertaken. Namely, it has largely been acceptedthat antibody titer measurements directly reflect Bmem frequencies.
This notion has been based on the antiquated assumption thatduring the B cell response to antigen, long-lived Bmem and longlived plasma cells (PC) arise in a fixed ratio to each other. Thus, thefeeling had been that cumbersome assays to detect Bmem utilizing
fragile live cell material are unnecessary because simple measurements of stable proteins in serum reliably provide the sought-after
information. However, recent advances in the understanding of
B cell lineage differentiation pathways imply the requirement for adifferent approach. This topic has been the subject of several excellent reviews recently [1–3] and is only outlined briefly in thefollowing section.
1.1 Differential Fate
Decisions for B Cell
Differentiation into theMemory vs. Plasma
Cell Lineages

During the antigen-driven B cell response, within a germinal center(GC) present in the draining lymph node(s), Bmem and PC arise
along differential, affinity-driven maturation pathways (Fig. 1).
Briefly, when germinal center B cells (GCB) undergo proliferation
and acquire somatic hypermutations (SHM) (see Note 2), daughter
cells arise with mutated antigen-binding sites of their B cell antigen
receptors (BCR) (see Note 3). Because SHM occurs somewhat
randomly, a subset of these mutated BCR can acquire an increased
affinity for the antigen, although the affinity of most BCR is either
unaltered or attenuated as a result of these mutations. In
subsequent steps, the progeny of GCB that gained an increased
affinity for the antigen undergoes additional cycles of proliferation,
SHM, and affinity-based positive selection. Multiple repetitions of
this process eventually lead to the differentiation of PC, whichconstitutes the cellular basis of the affinity-matured antibody
response. Progeny of GCB that do not meet the increasingly stringent antigen-driven affinity selection criterion (see Note 4) exit the
GC and become long-lived Bmem.
Bmem and PC, therefore, emerge along alternative routes driven
by different selection criteria, and, subsequently, their frequencies
are not necessarily linked. Consequently, one cannot expect antigen-specific serum antibody levels and Bmem frequencies to be
proportional to each other. From the above understanding of
antigen-driven B cell differentiation, it also follows that, whileboth PC and Bmem are antigen-specific (i.e., their BCR exceeds aminimal binding constant for antigen), PC (and hence secreted
antibodies) constitute primarily the high affinity end of this affinity
spectrum, while Bmem also encompass a lower affinity fraction.
Recent interpretation of these findings even implies that PC and
Bmem play fundamentally different roles in humoral immune
defense [1].

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Fig. 1 Affinity-based differential differentiation of plasma- and memory B cells. In a secondary lymphoidtissue, e.g., a lymph node, a naive B cell (Bnaive) encounters the homotypic antigen presented by a folliculardendritic cell (FDC) for which its B cell receptor (BCR) has sufficient affinity to trigger activation. The B cell thenpresents BCR-acquired antigen-derived peptides to cognate T follicular helper (TFH) cells, resulting in theactivation/polarization and proliferation of both cell types. The B cell then enters a germinal center within thelymph node, where it undergoes additional rounds of proliferation and somatic hypermutation (SHM) of its
BCR. Because the latter is random, the daughter cells display BCR with a broad spectrum of affinities for thehomotypic antigen. Cells endowed with high-affinity BCR for the homotypic antigen are positively selected andundergo additional cycles of proliferation, acquisition of further somatic hypermutations, and affinity-based
positive selection, eventually differentiating into antibody-secreting plasma cells (PC). Daughter cells that
express lower affinity BCR variants for the homotypic antigen exit the lymph node as memory B cells (Bmem).
By chance cross-reactivity, however, some of these Bmem will have high affinity for heterotypic antigen
variants

1.2 Serum
Antibodies Reflect theFirst Wall of Adaptive
Humoral Defense,
Bmem the Second

While PC elicited during the primary immune response can secrete
large amounts of antibodies, their lifespans are heterogenous and,contrary to the previous assumption that PC are long-lived, likely
fall on a continuum with possibly only a fraction of them surviving
long-term [4, 5]. The antibody molecules they secrete are also
relatively short-lived in the body and possess half-lives of several
weeks, at best (see Note 5). Thus, serum antibodies that are170Paul V. Lehmann et al.

detected at any one time have been recently produced and, therefore, the maintenance of serum antibody levels depends on constant active replenishment by PC. Such antibodies in bodily fluids
and their cell-bound variants (see Notes 5–7) constitute the firstwall of the adaptive humoral immune defense since they are alreadypresent prior to antigen reencounter. Such antibodies (IgA andIgM) are transported across the mucosa and can confer instanthost protection by preventing entry of the pathogen/antigen into
the body. If the offending pathogen/antigen succeeds in crossing
this interface and gains access into the body, preformed antibodies(IgG and IgM) already present in serum can still confer protection
through direct neutralization. Serum antibodies also possess precipitating, opsonizing, and complement activating activity to further combat the dissemination of the pathogen/antigen.
Preformed antibodies can additionally initiate inflammatory reactions through their interactions with Ig-binding receptors
expressed on various immune cell populations (IgE, IgG, or IgM).
Many times, however, the first wall of adaptive humoral
immune defense fails to prevent reinfection. Such is the case whenantibody levels either decline to sub-protective levels, or whenantigenic viral variants are encountered that evade neutralization,
as evidenced, e.g., in the recent COVID pandemic. Boosting thelevels of specific antibodies that were already established throughinfection or vaccination against the previously circulating virus
strain, the “homotype,” will not confer sterilizing immunity against
a newly emerging antigenic variant strain, the “heterotype.” In thiscase, Bmem provide the second wall of adaptive humoral immune
defense. Within the Bmem pool established during a preceding
immune response, BCR specificities possessing a reduced affinity
for the homotypic virus/antigen will be present that, by chance,
have an increased affinity for the variant, heterotypic virus/antigen.
As such, heterotypic antigen-reactive Bmem occur at higher frequencies than would occur within a B cell repertoire that is naive
to the homotypic virus/antigen; moreover, as such Bmem havealready undergone immunoglobulin (Ig) class switch recombination (CSR), they can readily engage into faster and more efficient
recall responses, including potential reentry into a GC for acquisition of further affinity-enhancing SHM. While this Bmem-based
second wall of adaptive humoral immunity is not able to prevent
infection with a heterotypic virus, it still mediates immune protection against it by enabling the host to rapidly mount a secondarytype immune response at the first encounter with a heterotypicvirus.
Studying antigen-specific serum antibodies vs. Bmem, therefore,
provides fundamentally different information on adaptive humoral
immunity. The former reflects only on the past remnants ofpreviously established, still deployable, but passive, and fading

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immunological memory. In contrast, studies of Bmem provide
insights into the ability of an individual to actively mount secondary
immune responses against homo- and/or heterotypic antigens.
Thus, studying Bmem permits one to take a glance into the future.
1.3 Emerging
Evidence for Serum
Antibody Levels andMemory Cell
Frequencies Providing
Divergent Informationon the Magnitude of
B Cell-MediatedImmunity

Recently, we concluded a systematic study (to the best of ourknowledge the first on this subject) in which we measured circulating antibody levels to SARS-CoV-2, EBV, and different seasonal
influenza virus strains and compared them with the frequencies ofBmem reactive with the same antigens [6]. These ImmunoSpot®based Bmem detections were enabled by our newly acquired ability
to achieve high-density antigen coating, thus reliable detection of
Bmem specific for essentially any antigen ([11] and see Note 8).
Representative results for the SARS-CoV-2 Spike protein are
shown in Fig. 2. Notably, elevated frequencies of Spike-specific,
Bmem-derived antibody-secreting cells (ASC) were detected in all ofthese convalescent COVID-19 donors despite variable levels of IgG
antibody reactivity in the plasma at the time of sample collection. In

Fig. 2 Discordance between circulating antibody levels and Bmem frequencies in
PBMC against SARS-CoV-2 Spike antigen. Correlation between SARS-CoV2 Spike (FL = full length)-specific IgG+ antibody-secreting cell (ASC)frequencies (x-axis) and plasma IgG levels (y-axis) in convalescent COVID-19
donors (n = 8). Pearson correlation analysis was performed using GraphPad
Prism software on log-transformed antigen-specific ASC data and thecorresponding antigen-specific IgG titers (expressed as μg/mL of IgGequivalents). Regression analysis was also performed using GraphPad Prism
and the 95% confidence bands are plotted. These data are representative ofsimilar findings reported previously for this and other viral antigens [6]172Paul V. Lehmann et al.

this same study [6], discordance between antibody levels and Bmemderived ASC frequencies was also noted for the SARS-CoV2 NCAP antigen and multiple antigenically unrelated seasonal
influenza strains, all of which represent respiratory viruses thatcause acute, short-term infections. Furthermore, discordance
between antibody levels and Bmem-derived ASC frequencies was
also evidenced against the EBNA1 antigen of Epstein–Barr virus(EBV), a latent herpesvirus that occasionally reactivates [7].
1.4 Emerging
Evidence for Bmem
Detection Being More
Reliable for Revealing
Past Antigen Exposure
than Serum Antibody
Measurements

It is not uncommon to find individuals who exhibit little if any
seropositivity for an antigen, yet possess significant, often high,frequencies of antigen-specific, Bmem-derived ASC (Fig. 2, [6],and Kirchenbaum et al., manuscript in preparation). Among theantigens tested, SARS-CoV-2 proteins are the most informative inthis regard because SARS-CoV-2 Spike and NCAP antigen-specificBmem-derived ASC are not detected in samples from individuals
cryopreserved prior to the onset of the COVID-19 pandemic(which, therefore, are verifiably immunologically naive to thisvirus owing to the time period in which these samples were collected). In contrast, each of the seronegative, yet Bmem-positive,
donors in our study cohort had undergone PCR-verified SARSCoV-2 infections (see Note 9). In these instances, serum antibody
measurements provided clearly false-negative results on the infection history of these individuals, while the presence of Bmemderived ASC reliably revealed it.
We have also previously noted clearly false-negative serologicalassessments for human cytomegalovirus (HCMV) exposure[8]. Obtaining PBMC (peripheral blood mononuclear cells) fromdifferent FDA-approved blood banks, all of which screen theirdonors for HCMV seropositivity, we found that PBMC frommany such HCMV-seronegative donors possessed HCMV
antigen-reactive, Bmem-derived ASC in ImmunoSpot® assays following in vitro polyclonal stimulation. Such subjects also exhibited
CD4+ and CD8+ T cell memory to HCMV. Thus, the presence of
Bmem-derived ASC reactivity (and T cell memory) more reliably
revealed the HCMV-exposed status of these donors than did
serum antibody levels.
In yet another independent line of investigation, studying
patients with multiple sclerosis (MS), an autoimmune disease of
the central nervous system (CNS), we detected neuroantigenspecific Bmem, in the absence of detectable serum antibody levels
in most patients, whereas such Bmem were absent in healthy controls[9]. Collectively these findings suggest that detecting Bmem-derived
ASC might be a more reliable way to diagnose past infections andpossibly also autoimmune diseases than afforded presently byserum antibody measurements.

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1.5 Assessing Bmem
Might Be Important,but how, and why?

Presently there are two major techniques that permit the detectionof antigen-specific Bmem in blood, lymphoid tissues, and otherprimary cell material. One approach is based on labeling B cellswith fluorescently tagged antigen(s) followed by their detection
using flow cytometry; the other is by ImmunoSpot®. While thestrength of the former approach is that it allows for the segregationof antigen probe-binding B cells into phenotypically distinct subsets based on their surface marker expression, it has several disadvantages compared to ImmunoSpot®. One crucial difference is thelower limit of detection of the former. While ImmunoSpot®
enables measurement of a single ASC within a bulk population ofcells with essentially no intrinsic lower detection limit (see Note10), flow cytometry falls short of detecting antigen-specific B cells
when they are present as low-frequency events. Bmem, however,frequently occurs in very low frequencies in PBMC (Fig. 3). Additionally, the number of PBMC required for flow cytometric detection of antigen-specific B cells is considerably higher compared to
ImmunoSpot® (see Note 11). Notably, a surface staining-only
approach for the identification of antigen-specific B cells does notreliably reveal the Ig class/subclass usage of ASC, while four-color
ImmunoSpot® assays provide this key information with ease as partof routine Bmem frequency measurements (see Note 12). Lastly,
unlike for flow cytometry, the actual wet lab implementation of B
cell ImmunoSpot® assays is scalable for high-throughput analysis(see Note 13) and multi-color ImmunoSpot® analysis can be fully
automated (see Chap. 5 by Karulin et al. in this volume [10]).

1.6 Affinity Coating:
Enabling B Cell
ImmunoSpot® Assays“to See”

With so many potential advantages in favor of ImmunoSpot® assays
for the detection of Bmem, the question arises as to why this technique has not become more widely used for immune monitoring
purposes. The answer is simple and practical: the original protocol(that involves direct coating of the antigen to the membrane) only
works well for a rather limited set of antigens (see Note 14).
Consequently, many investigators likely gave up on this assay after
not being successful in their initial attempts to establish such assays
for the antigen(s) of interest. Our recent introduction of affinity
capture coating [11] represents a breakthrough to this end, as itprovides a universal strategy for successful assay development foressentially any antigen: the membrane is first coated with an anti(His- or other) affinity tag-specific antibody followed by the addition of recombinant (His- or other) affinity-tagged recombinantantigen. In this way, low-affinity absorption of the antigen to themembrane via weak, non-specific binding forces (primarily hydrophobicity) is replaced by specific, high-affinity binding. The assay
principle is depicted in Fig. 4b, and the protocol is described indetail below. This version of the B cell ImmunoSpot® assay is

174Paul V. Lehmann et al.

Fig. 3 Antigen-specific Bmem occur over a wide frequency range in blood. PBMC
from healthy human subjects – each represented by a dot – were tested via
ImmunoSpot® assays to establish the frequency of IgG+ Bmem-derived ASC
specific for the panel of antigens shown: Tetanus toxoid heavy chain (TTHc),
Epstein–Barr nuclear antigen (EBNA1) of EBV, gH pentamer complex (consistingof gH, gL, UL128, UL130, and UL131A proteins) of HCMV, recombinanthemagglutinin (rHA) proteins representative of A/California/2009 (H1N1),
A/Texas/2013 (H3N2), and B/Phuket/2013 (Yamagata lineage) seasonal
influenza vaccine strains, as well as SARS-CoV-2 Spike (full-length) andNucleocapsid (NCAP) proteins. Serving as specificity controls for SARS-CoV2 antigens, donors whose PBMC were cryopreserved prior to widespread COVID
infection and vaccination (before June 2021) are depicted as red dots, andsubjects whose PBMC were frozen after June 2021 are denoted as black dots.
Note, the data shown are results of testing PBMC without serial dilution at a fixed
input of 3 × 105 PBMC per well, and because quantification in wells with >100
antigen-specific spot-forming units (SFU) is no longer precise, we defined theupper limit for accurate counts at 330 SFU per 106 PBMC. Values exceeding thisthreshold, denoted by the gray shading in the figure, require lower cell inputs for
accurate quantification of antigen-specific ASC frequencies. Likewise, the lower
gray-shaded region denotes low-frequency responses in which Poisson noise
necessitates seeding higher PBMC numbers per well and larger numbers of
replicate wells to be evaluated for accurate determination of antigen-specificASC frequencies (see Note 10)particularly well-suited for detecting and characterizing rare antigen-specific, Bmem-derived ASC in a test sample, whereas an alternative variant of the B cell ImmunoSpot® assay approach, whichenables assessment of ASC functional affinity and is described inanother chapter of this volume [12], is better suited for samples inwhich antigen-specific ASC are present at an elevated frequency
among all ASC (see Note 15).

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Fig. 4 Principle of antigen-specific, (a) inverted vs. (b) direct B cell ImmunoSpot® assays (the latter, using theaffinity capture coating approach). In (a), the PVDF membrane on the bottom of a 96-well plate is densely
coated with a pan anti-Ig(G) class-specific (in this example IgG) capture antibody that will bind theASC-secreted Ig(G) with high affinity irrespective of the ASC’s antigen specificity. In (b), the membrane iscoated first with an anti-affinity tag-specific antibody (in this example anti-His) that captures the (His)-tagged
antigen with high affinity. In this way, dense coating of the membrane with the antigen is accomplished. As thenext step in both assay variants, the PBMC containing the ASC are plated. In (a), ASC-produced IgG is captured
around each ASC that is secreting IgG and results in the formation of individual secretory footprints. In (b), only
the antibody produced by antigen-specific ASC is captured on the lawn of antigen. After removal of the cells,(a) the affinity-tagged (in this example His) antigen is added at a sufficient concentration to be retained byantigen-specific secretory footprints generated by ASC producing low- or high-affinity antibody. Alternatively,
after removal of cells, (b) antigen-bound antibody is visualized by biotinylated human Ig class/subclass(in this example IgG) specific detection antibodies. In (a), antigen-specific secretory footprints are thenvisualized using a biotinylated anti-affinity tag detection reagent, which is revealed by the addition of afluorescently conjugated streptavidin (FluoroSpot, as shown) or via an enzymatic reaction (ELISPOT, notshown). In (b), antigen-specific secretory footprints are revealed by the addition of a fluorescently conjugatedstreptavidin (FluoroSpot, as shown) or via an enzymatic reaction (ELISPOT, not shown). In both B cell
ImmunoSpot® assays, counting the spot-forming units (SFU) per well reveals the number of antigen-specific
ASC within the PBMC plated. Furthermore, spot morphologies in such antigen-specific ImmunoSpot® assays
also provide insights into the functional affinities of the antibody secreted by the individual ASC for the antigen,a topic covered in detail in the chapter by Becza et al., in this issue [12]1.7 High-Content
Information Provided
by Assessing
Individual ASC via
ImmunoSpot® vs.
Serum Antibody
Measurements

Owing to the single-cell resolution afforded by ImmunoSpot®
assays, they are ideally suited to study individual ASC that comprisean antigen-specific B cell response. While traditional serum antibody measurement techniques readily perceive large increases inantigen-specific antibody titers, they fail to appreciate more subtle
changes in antibody levels, especially when the abundance of antibodies is very small, e.g., in the context of allergen-specific IgE[13], or when an elevated level of preexisting antibody reactivity isalready present, e.g., in the context of studying seasonal influenza
vaccine responses [14, 15]. In contrast, B cell ImmunoSpot® assays
circumvent ambiguity in both cases through quantifying the precise
number of cells that are actively secreting antigen-specific antibody
and thus offer increased sensitivity and resolution.
When optimal antigen-coating conditions exist in the direct
B cell ImmunoSpot® assay, each secretory footprint will be composed of antibody originating from a single ASC and thus permits
assessment of antibody affinity similar to that achieved throughstudying an individual monoclonal antibody (mAb). In this setup,176

Paul V. Lehmann et al.

the morphology of an ASC-derived secretory footprint is primarily
defined by the affinity of the secreted antibody for the membranebound antigen. In agreement with prior computational modeling[16], we routinely observe variable spot morphologies in suchantigen-specific B cell ImmunoSpot® assays [6, 11]. Importantly,
beyond enumeration of such antigen-specific secretory footprints, amultitude of morphological features including metrics of intensity
and size are also captured for each spot-forming unit (SFU) and arereadily exported as flow cytometry standard (FCS) files by the
ImmunoSpot® software. Such FCS files can be leveraged to visualize variable spot morphology, as was done using scatter plots in ourrecent publications [6, 11]. Here, we direct the reader to Chap. 5
by Karulin et al. in this volume that is dedicated to high-content
analysis of spot morphologies [10]. Additionally, in the context of
an inverted B cell ImmunoSpot® assay (see Note 16) in which
ASC-derived secretory footprints are efficiently captured irrespective of antigen specificity, secretory footprints originating fromindividual antigen-specific ASC are revealed by their ability to retain
an antigen probe. While also briefly introduced below, Chap. 13 inthis volume by Becza et al. [12] describes in detail how the inverted
ImmunoSpot® assay variant and high-content data analysis enableassessment of the functional affinity distribution present in a polyclonal population of ASC, such as the ASC response elicited following COVID-19 vaccination.
1.8 Frequency of
Antigen-Specific,
Bmem-Derived ASC
Reflect Memory
Potential

Because ImmunoSpot® assays detect the secretory footprints ofindividual ASC, frequency information is revealed by counting thenumbers of antigen-specific SFU, either per cells plated per well, or,better, as the frequency of antigen-specific ASC secreting a given Ig
class/subclass among all ASC producing that Ig class/subclass (seeNote 16). In either case, however, the crowding of secretory
footprints along with the ELISA effect (see Note 17) can lead toundercounting of secretory footprints [6]. Systematically studying
this phenomenon previously, we found that there exists a range inwhich cell numbers plated per well are directly proportional to thenumber of SFU detected. Depending on the nature of the assay andmorphology of the resulting SFU, however, the corresponding
SFU counts measured at higher cell inputs may break down at
approximately 100–200 SFU per well (see Note 18). This is amajor concern for establishing accurate frequencies of antigenspecific, Bmem-derived ASC, especially in light of our previous
data demonstrating that frequencies may span orders of magnitude
for the same antigen in different individuals (Fig. 2 and [6]). Also,
within any single individual, the frequency of Bmem-derived ASC
specific for different antigens can span a similarly wide distribution[6]. Furthermore, even the number of ASC producing a given Ig
class/subclass, irrespective of antigen specificity, exhibits considerable interindividual variation. ELISA effects in B cell

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ImmunoSpot® assays can also jeopardize high-content analysis ofspot morphologies. A simple and efficient solution to this problem
is to serially dilute the cell input to establish the linear range of SFU
counts from which the accurate frequency of ASC can be extrapolated by linear regression ([6] and see Note 19). Karulin et al.,
Chap. 5 in this volume [10], introduce software for automaticallyestablishing frequencies from such serial dilution experiments
making this approach suitable for high-throughput workflows.
1.9 Defining the Ig
Class/Subclass Usageof Antigen-SpecificBmem

The different Ig classes and subclasses are endowed with distinct
effector functions and each contributes nonredundant roles toward
maintaining host defense (see above and reviewed in greater detailin [17]). During the primary immune response, B cells can transition from IgM-expressing naive B cells into effector cells (PC) and
Bmem that have undergone class switch recombination (CSR)[1]. CSR is an irreversible process involving the excision of DNA
encompassing the exons of the Igμ heavy chain required for expression of IgM and juxtaposition of the upstream variable region genes(VDJ; which jointly define the antigen specificity of the BCR or
secreted antibody) with downstream exons encoding alternative Ig
classes or IgG subclasses [18]. Class switching of the BCR to
downstream Ig classes or IgG subclasses is an instructed process
and can be influenced by the cytokine milieu and co-stimulation
provided by CD4+ T helper cells. For the latter, the differentiationof naive CD4+ T cells into different T helper cell classes (Th1, Th2,
Th17, etc.) is defined by the circumstances of antigen encounter, inparticular by Toll-like receptor (TLR)-derived signals [19, 20]. Triggering the “appropriate” type of T helper cells capable of stimulating the optimal Ig class usage during an infection or following
vaccination is vital to successful host defense and the avoidance ofcollateral immune-mediated pathology (reviewed in [21]). The
same applies to reinfection with the same pathogen.
Upon antigen reencounter and subsequent reactivation, Bmem
rapidly differentiates into PC that secrete the same Ig class/subclassexpressed by the parental Bmem. Therefore, detecting the Ig class/subclass that Bmem-derived ASC produced in B cell ImmunoSpot®
assays permits the prediction of the specific types of antibodies thatwill be produced following the next antigen encounter (see Notes
20–22). Learning about the full spectrum of Ig classes/subclasses
that the antigen-specific Bmem repertoire will produce when theantigen is reencountered is thus essential for predicting the protective efficacy of future antibody responses to an antigen. Moreover,such B cell ImmunoSpot® assays may also predict the likelihood of
antibody-mediated complications occurring upon antigen
re-exposure (as illustrated by the example of antibody-dependentenhancement leading to exacerbation of clinical disease following
secondary dengue infection [22]). Importantly, the frequency ofantigen-specific Bmem capable of secreting different Ig classes or178

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IgG subclasses can be orders of magnitude apart [6] (and see alsoYao et al., Chap. 15 in this volume [23]). Thus, it is not only
recommended to assess all antigen-specific Ig classes/subclasses,
but also to do so over a wide frequency range for each. The lattercan be readily accomplished by combining the serial dilution strategy described above with four-color ImmunoSpot® analysis (fordetails, see the Chap. 15 of Yao et al. in this volume, [23]).
1.10 Assessing theAffinity Distribution of
the Antigen-Specific
Bmem Repertoire

As described above in this chapter, and briefly recapitulated here,
SHM leads to the diversification of Bmem cells that previously
participated in a GC reaction [24]. Upon reencounter with thesame (homo-) or a modified (heterotypic) antigen, previously generated Bmem endowed with high-affinity BCR for the rechallenging
antigen will readily differentiate into PC that are capable of rapidly
increasing antibody titers [3]. Moreover, Bmem can also be
re-recruited into a GC following antigen reencounter, where they
will undergo further rounds of proliferation and acquire additional
SHM necessary to refine and improve their BCR affinity for theoffending antigen. Again, the GCB cell progeny endowed with thehighest affinity BCR for the eliciting antigen will be selected for PC
differentiation and ideally will enter the potentially long-lived fraction of the PC compartment. Thus, a Bmem repertoire that entails ahigher frequency of high-affinity B cells specific for the (homo- or
heterotypic) antigen can generate a faster and more robust anamnestic antibody response upon antigen (re)encounter.
Thus far, the study of BCR/antibody affinity for a particularantigen has largely been confined to the generation of B cell hybridomas and/or paired IgH/IgL sequencing followed by expression
and purification of individual mAb in order to establish their specificities and affinity using surface plasmon resonance or biolayerinterferometry. While these approaches are the gold standard forstudying individual mAbs (i.e., single B cells), it would require
generation and subsequent characterization of hundreds of suchmAbs to appreciate the underlying affinity distribution of antigenspecific Bmem repertoire in just one donor, at a single timepoint, andagainst only a single antigen of interest. While such an exercise is, intheory at least, conceptually possible for a very limited number ofindividuals, it would be inconceivable both in magnitude and costto attempt such an objective for a larger donor cohort, e.g., as partof a clinical trial. Consequently, this traditional method for characterizing the affinity distribution of the antigen-specific B cell
repertoire is not realizable for high-throughput immune monitoring efforts.
The Chap. 13 in this volume by Becza et al. [12] demonstrates
that the affinity distribution information of the antigen-specific
B cell repertoire can be established with ease, and in a readily
scalable manner, using two B cell ImmunoSpot® assay variants.
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elicited IgG+ ASC, a first experiment would need to be performedthat establishes the frequency of such antigen-specific IgG+ ASC
using the serial dilution approach (see Note 23). This information isessential for being able to seed in a follow-up test the PBMC at a“Goldilocks” cell density in which the individual secretory footprints can be studied without interfering with each other. In thissecond experiment, using an independent aliquot of cryopreserved
cell material, the donor PBMC are seeded at the so-called “Goldilocks number,” which is assay dependent and between 50 and
100 SFU per well. As each SFU represents the secretory footprintof an individual ASC, at 50 SFU per well, the secretory footprints of
50 distinct ASC would be assessed. Seeding additional replicate
wells at this “Goldilocks number” would increase the number ofantigen-specific IgG+ ASC being characterized. We recommend to
study a fixed number of ≥300 ASC for each antigen concentration
as this number gives a considerable (and representative) sample size
for assessment of the antigen-specific Bmem repertoire elicited in aparticular person, against an individual antigen, at the time ofsample collection.
Leveraging the inverted ImmunoSpot® approach for B cellaffinity distribution measurements, the soluble antigen probe isadded in decreasing concentrations to the same number of replicate
wells for each antigen concentration, whereby each well was previously seeded with a “Goldilocks number” of cells. At the highest
antigen probe concentration, the secretory footprints of all antigen-specific ASC, high- and low-affinity alike, will be detected as
SFU when the membrane-retained antigen probe is visualized. Inwells containing decreasing concentrations of the antigen probe,
however, only the secretory footprints originating from ASC that
produced an antibody with high affinity for the antigen probe willretain an adequate amount for their eventual detection as SFU; B
cells with an affinity lower than dictated by the actual antigen
concentration go undetected, and the SFU counts will decreaseby their number. Therefore, establishing the SFU counts across a
range of antigen probe concentrations provides insights into theaffinity distribution of the antigen-specific B cell repertoire.
The second B cell ImmunoSpot® assay variant suited for assessment of the affinity distribution present in a polyclonal antigenspecific ASC compartment relies on a direct assay in which theantigen-coating density is graded. Such assays (Fig. 4) inherently
provide information on the affinity of the “monoclonal antibody”
that each antigen-specific ASC secretes. Following the basic rules ofantibody–antigen binding, ASC that produce high-affinity antibodies generate dense, bright, and tight (sharp and small) secretory
footprints (“spots”), while ASC that produce antibodies with lower
affinity yield secretory footprints that are fainter and more diffuse[16]. Coating the membrane with decreasing densities of antigen
and monitoring the resulting changes in SFU number and180

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morphology, therefore, provides information regarding the affinity
distribution of an antigen-specific ASC repertoire (see Note 24).
The Chap. 13 by Becza et al. in this volume [12] lays out in detailboth assay variants for B cell affinity measurements.
1.11 Measuring
Cross-Reactivities ofIndividual BmemDerived ASC

As described above, Bmem also provides a “second wall of adaptive
humoral immunity” against mutated antigens/viruses (heterotypic
antigens) that evolved to evade neutralizing antibodies induced by
the original (homotypic) virus/antigen. While the primary
response leads to fine tailoring of the B cell repertoire directedagainst the homotypic antigen, including affinity maturation,
among the variants created by random SHM, Bmem endowed with
BCR possessing affinity for the heterotypic antigen will be present.
Even if such Bmem would be rare, and even if their initial affinity
would be modest, they still would occur in much higher frequencies compared to the naive B cell repertoire. Moreover, most ofthese Bmem have already undergone Ig class switching. Therefore,
when such clonally expanded, semi-affinity matured, and classswitched, cross-reactive Bmem engage in a primary immune
response against the heterotypic antigen, they can generate amore rapid and efficient antibody response compared to individuals
who have not been previously immunized/infected (see Note 25).
Measuring existing serum antibodies does not detect such crossreactive Bmem, and thus does not provide predictive information
about cross-reactive protection, whereas ImmunoSpot®-based
assessments at the level of individual Bmem-derived ASC do.
There are two ways to measure Bmem-derived ASC crossreactivity by ImmunoSpot®. For both, the appropriate cell input
per well first needs to be defined, which will provide the “Goldilocks” SFU count for the homotypic antigen (between 50 and
100 SFU per well, see above). In the simple version of B cellcross-reactivity studies, a second experiment is performed, an
inverted B cell assay, in which this Goldilocks input of PBMC for
the particular donor is seeded into all wells. Setting up replicate
wells permits to assess ≥300 individual ASC-derived secretory
footprints in independent single-color ImmunoSpot® assays, comparing the numbers of SFU detected in all replicates for the homotypic antigen, vs. the cumulative SFU number in the same numberof replicate wells detected using the heterotypic antigen probe. The
ratio of these two numbers reveals the percentage of cross-reactive
ASC at equimolar concentrations of the respective antigen probes(see Note 26 and Fig. 5). Raising/lowering the concentration ofheterotypic antigen probe enables assessment of the affinity spectrum of Bmem-derived ASC that cross-react with the heterotypic
antigen.
In a second, somewhat more complex but perhaps more elegant, approach, Bmem-derived ASC cross-reactivity is evaluatedusing alternatively labeled homotypic and heterotypic antigens. As

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Fig. 5 Inverted ImmunoSpot® enables assessment of Bmem cross-reactivity against the receptor binding
domain (RBD) of the homotypic prototype strain of SARS-CoV-2, and heterotypic antigenic variants of concernthat emerged later in the COVID-19 pandemic. PBMC from nine convalescent COVID-19 donors with
PCR-verified infection during the initial wave of SARS-CoV-2 infections caused by the prototype Wuhan-Hu1 strain (collected prior to November 1, 2021, i.e., before the onset of the Delta and Omicron waves), were
cryopreserved. After thawing, the PBMC were stimulated for 5 days in vitro to transition resting Bmem into
antibody-secreting cells (ASC). These in vitro stimulated PBMC were then seeded into an inverted ImmunoSpot® assay, leveraging IgG capture, to detect IgG+ secretory footprints that captured RBD protein representative of the homotypic Wuhan-Hu-1, or heterotypic B.1.617.2 (Delta) or B.1.1.529 (Omicron) strains of SARSCoV-2. Each of these RBD-variant specific secretory footprints was detected in single-color assays, first by theaddition of the respective soluble His-tagged RBD antigen probe, followed by the addition of biotinylated antiHis mAb, and finally by the addition of SA-CTL-Red™. The individual secretory footprints, or spot-forming
units (SFU), with reactivity against the His-tagged RBD probes were subsequently enumerated using ImmunoSpot® software. Representative well images for donor A are shown in panels a–c for the three RBD probes, as
specified. (d) The percentage of IgG+ ASC, following in vitro differentiation, exhibiting reactivity with thehomotypic Wuhan-Hu-1, or heterotypic Delta or Omicron RBD probes. (e) The percentage of IgG+ ASC reactivewith the heterotypic Delta or Omicron RBD probes relative to the homotypic Wuhan-Hu-1 strain (denoted as100%). Data are plotted as mean ± SD. Statistical significance was determined using an analysis of variance(ANOVA) and Bonferroni’s multiple comparisons post-hoc test. ***p < 0.001

before, the PBMC are plated at the Goldilocks input number into
replicate wells of an inverted B cell ImmunoSpot® assay. The “tag1” labeled homotypic antigen probe (e.g., His tag) and the “tag 2”labeled heterotypic antigen probe (e.g., FLAG tag) are added
simultaneously, at equimolar concentrations, followed by dualcolor detection of SFU that bound an antigen probe. The ratio of“tag 1” single-color positive SFU and of “tag 1 + tag 2” doublecolor positive SFU is established, revealing the frequency of cross-182

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reactive ASC under equimolar conditions. Raising and lowering theconcentration of the heterotypic antigen probe provides insights
into the affinity of the individual homotype-primed Bmem for theheterotypic antigen.
1.12 Concluding
Remarks

The aim of this chapter was to draw attention to the tremendous,
thus far largely unrealized, potential of B cell ImmunoSpot® assays,
and their potential to revolutionize immune diagnostics. Although
the ELISPOT assay was originally introduced 40 years ago for
detecting antigen-specific ASC [25, 26], it was T cell ELISPOT
assays (introduced 5 years later [27]) that took the limelight. For
the latter to occur, however, we needed to introduce fundamental
modifications to the originally described protocol so that the assay
lived up to its potential and was capable of reliably revealing secretory footprints of individual T cells responding to antigen ([28] andsee Note 27). Our introduction of automated, objective, and scientifically validated software-assisted machine reading of T cellderived secretory footprints [29] also largely contributed to thesuccess of T cell ImmunoSpot® assays (see Note 28).
Despite being introduced earlier into the literature, B cell
ImmunoSpot® assays have, thus far, not become a mainstay in
B cell immune monitoring efforts. One likely reason is that formost antigens the classic protocol of directly absorbing the antigen
to the assay membrane simply does not work (see above); only ourrecent introduction of the affinity coating approach [11] enables
one to rapidly develop ImmunoSpot® assays for detecting ASC
with specificity for essentially any affinity-tagged antigen of interest.
But perhaps the primary reason why B cell ImmunoSpot® assays
have not been sufficiently pursued until now is the widely held, andflawed, assumption that serum antibody measurements yield sufficient insight on underlying B cell-mediated adaptive humoral
immunity. As outlined above, from the basic science perspective,
this standpoint is no longer tenable. Importantly, a major component of the critical information pertaining to Bmem-mediatedimmunity is now easily attainable through the implementation of
B cell ImmunoSpot® assays, as outlined above, and is neglected bystudying antigen-specific serum reactivity/titers alone.
Ease of implementation, low cost, and moderate labor investment are major requirements for the success of any assay. The
efficiency of B cell ImmunoSpot® in PBMC utilization has been
repeatedly highlighted in this chapter (see also see Note 23) and iscritical in clinical trial settings. Cryopreserved PBMC can be usedwithout impairing B cell function [30], permitting batch testing ofdozens of samples by a single investigator in a single experiment(and hundreds of samples with a well-trained team). Fully automated image analysis of the assay results is now available, along withretention of audit trails [10]. The reproducibility of B cell ImmunoSpot® is quite remarkable for a cellular assay [6], and the serial

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dilution strategy detailed above permits to extend the upper andlower detection limits of the assay over orders of magnitude[6]. Moreover, B cell ImmunoSpot® assays have been shown tobe suitable for regulated testing [31]. For all of the above reasons,
we believe B cell ImmunoSpot® testing will soon become an indispensable component of the immune monitoring repertoire.
Detailed methodology for the affinity capture-based B cell
ImmunoSpot® assay is described in another Chap. 15 contributed
by Yao et al. in this volume [23]. This is the method of choice forestablishing frequencies of antigen-specific ASC via the serial dilution strategy; including for all Ig classes, or IgG subclasses, simultaneously. Detailed methodology for the single-color, antigenspecific inverted B cell ImmunoSpot® assay described in Fig. 5 isdetailed below. Both the direct and inverted ImmunoSpot® assay
formats are also well-suited for assessment of ASC cross-reactivity.
However, while direct ImmunoSpot® assays provide insights into
the affinity distributions through studying spot morphologies, theinverted assay described in detail by Becza et al. [12] is the methodof choice for such high-content analysis of antigen-specific ASC
repertoires. Software solutions for B cell ImmunoSpot® analysis aredetailed in another Chap. 5 of this volume by Karulin et al. [10].

2Materials

2.1 Thawing of
Cryopreserved PBMC

1. Class II biosafety cabinet (BSC).
2. Cryopreserved PBMC sample(s) (see Note 23).
3. 70% (v/v) ethanol (EtOH)
4. Conical tubes.
5. Sterile transfer pipette.
6. DNase-containing washing medium (prewarmed to 37 °C) (seeNote 29).
7. Centrifuge capable of spinning 50 mL conical tubes at 330 × g(temperature set to 25 °C).
8. Complete B cell medium (BCM) (prewarmed to 37 °C) (seeNote 30).
9. Ca2+, Mg2+-free phosphate-buffered saline (PBS), pH 7.2(room temperature).
10. Parafilm.
11. CTL-LDC™ live/dead cell counting kit.
12. ImmunoSpot® S6 Ultimate 4 LED Analyzer, or suitable
instrument equipped with the appropriate detection channels,
running CTL’s live/dead cell counting suite software.

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2.2 In Vitro Polyclonal Stimulation of B
Cells in PBMC

1. B-Poly-S.
2. Volume-dependent, 48- or 24-well plate, or sterile culture flask(see Note 31).
3. Humidified incubator set at 37 °C, 5% CO2.

2.3 Four-Color,
Antigen-SpecificFluoroSpot Assay(Affinity Capture
Coating)

1. Commercially available, four-color Human Ig class (IgA, IgE,
IgG, and IgM) affinity capture (His) FluoroSpot kit (see Note32).
2. Commercially available, four-color Human IgG subclass affinity capture (His) FluoroSpot kit (see Note 32).
3. His-tagged recombinant protein (see Notes 33 and 34).
4. 190 proof (95% v/v) EtOH
5. Cell culture-grade water.
6. 96-well, round bottom dilution plate.
7. 0.05% Tween-PBS wash solution
8. 0.1 μm low-protein binding syringe filter
9. Plate washer.
10. Vacuum manifold.
11. ImmunoSpot® S6 Ultimate 4 LED Analyzer, or suitable
instrument equipped with the appropriate detection channels,
running CTL’s ImmunoSpot® UV.

2.4 Single-Color,
Antigen-Specific
Inverted ImmunoSpot®
Assay

3

1. Commercially available, single-color inverted (His) human
B cell ImmunoSpot® kit (see Note 35).
2. His-tagged recombinant protein (see Note 36).

Methods

3.1 Thawing of
Cryopreserved PBMC
(Sterile Conditions)

1. Place cryovial(s) into a 37 °C bead bath (or water bath, we
prefer the former for sterility reasons) for 8 min to thaw.
2. Remove cryovial(s) and wipe with 70% EtOH inside the biosafety cabinet (BSC) before unscrewing the cap(s).
3. Using a sterile pipette, transfer contents of cryovial(s) into alabeled conical tube.
4. Rinse each of the cryovials with 1 mL of prewarmed antiaggregate solution. Transfer the rinse solution(s) to the conicaltube(s) dropwise while swirling the tube to ensure adequate
mixing of the cells and thawing medium.
5. Double the volume of the cell suspension by dropwise additionof warm anti-aggregate solution while swirling the tube toensure adequate mixing of the cells and thawing medium.

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6. Continue doubling the volume of the cell suspension by dropwise addition of warm anti-aggregate solution while swirling
the tube until the cryopreserved cell material has been diluted
tenfold. If multiple cryovials are pooled, calculate using 1 mLof cryopreserved cell suspension + 9 mL of anti-aggregate
solution to determine the necessary final resuspension volume.
7. Centrifuge balanced tubes at 330 × g for 10 min with thecentrifuge brake on, at room temperature (RT).
8. Decant supernatant and resuspend the cell pellet(s) using prewarmed B cell medium (BCM) to achieve a cell density of
approximately 2–5 × 106 cells/mL. The number of PBMC
recovered can be estimated at this point being 80% of thenumber frozen.
9. Pipet 15 μL of live/dead cell counting dye onto a piece ofparafilm to form a droplet.
10. Remove 15 μL of cell suspension and combine with droplet oflive/dead cell counting dye. Pipet up and down 3–5 times tomix the sample while avoiding the formation of bubbles.
11. Transfer 15 μL of the cell and dye suspension into each chamber of a hemacytometer.
12. Determine live cell count and viability using CTL’s live/deadcell counting suite.
13. Increase volume of cell suspension(s) with additional sterile
PBS and centrifuge balanced tubes at 330× g for 10 min withcentrifuge brake on, unrefrigerated.
14. Decant supernatant and gently resuspend the cell pellet(s) using prewarmed BCM at a cell density to 2 × 106 cells/mL.
3.2 In Vitro Polyclonal Stimulation of B
Cells in PBMC (Sterile
Conditions)

1. Dilute CTL’s B-Poly-S polyclonal stimulation reagent 1:500
into prewarmed BCM to achieve a final concentration of 2X.
Into labeled sterile culture vessel, add the same volume of
PBMC at 2–4 × 106 cells/mL (see Note 31).
2. Transfer culture vessels (flasks or plates) into humidified incubator set at 37 °C, 5% CO2 for 4–6 days (96–144 h).

3.3 Four-Color,
Antigen-SpecificFluoroSpot Assay(Affinity Capture
Coating)

1. Two days before plating cells (Day 2), prepare 70% EtOH andanti-His affinity capture antibody solutions.
2. Remove underdrain and pipet 15 μL of 70% EtOH solution
into the center of each well (or designated wells) of the assay
plate. Immediately after the addition of the 70% EtOH solution to the entire plate (or designated wells), add 180 μL/wellof PBS. Decant and wash wells again with 180 μL/well of PBS.
3. Decant the assay plate, replace underdrain, and immediately
add 80 μL/well of the anti-His affinity capture antibody

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solution into each well (or designated wells, see Note 37) of the
low autofluorescence PVDF-membrane plate provided withthe kit.
4. Incubate the plate overnight at 4 °C in a humified chamber.
5. The following day (Day 1), dilute the His-tagged protein(s) into Diluent A (provided with the kit) to the previously
determined optimal concentration.
6. Decant the assay plate and wash wells with 180 μL/well ofwarm PBS. Immediately, add 80 μL/well of the corresponding
His-tagged protein coating solution(s) into the designatedwells.
7. Incubate the plate overnight at 4 °C in a humidified chamber.
8. On the day of the assay (Day 0), decant the assay plate and wash
wells with 180 μL/well of warm PBS. Next, decant the plate
and add 150 μL/well of prewarmed BCM to block the plate(≥1 h at RT).
9. If using PBMC following in vitro polyclonal stimulation, collect the cell suspension(s) and transfer into labeled conical tube(s). Keep the cells warm during processing. Wash culture vessel’s interior with sterile warm PBS to collect residual PBMC
and transfer into the corresponding conical tube(s). Increase
volume to fill the tube with additional warm PBS and thencentrifuge balanced tubes at 330 × g for 10 min nonrefrigerated, centrifuge with brake on. Alternatively, follow the procedures detailed above to obtain freshly isolated PBMC, or tothaw PBMC that were previously cryopreserved, if priorin vitro stimulation is not required to elicit antigen-specific
ASC activity in the sample(s).
10. Decant supernatant and resuspend the cell pellet(s) using prewarmed BCM to achieve a cell density of ~2–5 × 106 cells/mL(the cell number recovered at this point can be estimated to be
50% of the number of cells frozen).
11. Pipet 15 μL of live/dead cell counting dye onto a piece ofparafilm to form a droplet.
12. Remove 15 μL of cell suspension and combine with droplet oflive/dead cell counting dye. Pipet up and down 3–5 times tomix the sample while avoiding the formation of bubbles.
13. Transfer 15 μL of the cell and dye suspension into each chamber of a hemacytometer.
14. Determine live cell count and viability using CTL’s live/deadcell counting suite.
15. Increase volume of cell suspension(s) with additional sterile
warm PBS and centrifuge balanced tubes at 330× g for
10 min with centrifuge brake on, unrefrigerated.

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16. Decant supernatant and resuspend the cell pellet(s) using prewarmed BCM at 2 × 106 PBMC/mL.
17. Decant the BCM used for blocking the assay plate and replacewith 100 μL/well of prewarmed BCM.
18. If applicable, e.g., for Goldilocks number determinations or
accurate frequency measurements, prepare PBMC serial dilution series in a round-bottom 96-well polystyrene plate tomatch the plate layout shown in Fig. 5a of the chapter by Yao
et al. in this volume [23] before transferring the PBMC into
the ImmunoSpot® assay.
19. Incubate cells in the assay plate for 16–18 h at 37 °C, 5% CO2.
20. After completion of the assay incubation period, decant(or reutilize) cells and wash plate two times with warm PBS(200 μL/well), followed by two additional washing steps with
0.05% Tween-PBS wash solution (see Note 38).
21. Prepare anti-Ig class/subclass-specific detection antibody solution(s) according to kit protocol and pass through 0.1 μm
low-protein binding syringe filter to remove any protein
aggregates.
22. Decant 0.05% Tween-PBS wash solution, add 80 μL/well of
the anti-Ig class/subclass-specific detection antibody solution
into designated wells, and incubate for 2 h at RT (protectedfrom light).
23. Wash plate(s) two times with 0.05% Tween-PBS wash solution.
24. Prepare tertiary solution by following kit protocol and passthrough 0.1 μm low-protein binding syringe filter to remove
any aggregates.
25. Decant 0.05% Tween-PBS wash solution, add 80 μL/well of
tertiary solution into designated wells, and incubate for 1 h at
RT (protected from light).
26. Wash plates(s) twice with distilled water.
27. Remove protective underdrain and place plate face down on
vacuum manifold. Completely fill the backside of the plate with
distilled water and apply vacuum to draw water through themembrane (“back to front”) (see Note 39).
28. Allow plate to dry completely, protected from light (see Note40).
29. Scan and count plate(s) with suitable analyzer equipped withthe appropriate detection channels (see Note 18).
3.4 Single-Color,
Antigen-Specific
Human IgG-Inverted
ImmunoSpot® Assay

1. One day before plating cells (Day 1), prepare 70% EtOH andanti-human IgG capture antibody solutions.
2. Remove underdrain and pipet 15 μL of 70% EtOH solution
into the center of each well (or designated wells) of the assay

188Paul V. Lehmann et al.

plate. Immediately after addition of the 70% EtOH solution to
the entire plate (or designated wells), add 180 μL/well of PBS.
Decant and wash wells again with 180 μL/well of PBS.
3. Decant the assay plate, replace underdrain, and immediately
add 80 μL/well of the anti-human IgG capture antibody solution into each well (or designated wells) of the low autofluorescence PVDF-membrane plate provided with the kit.
4. Incubate the plate overnight at 4 °C in a humified chamber.
5. On the day of the assay (Day 0), decant the assay plate and wash
wells with 180 μL/well of PBS. Next, decant the plate and add
150 μL/well of prewarmed BCM to block the plate (≥1 h
at RT).
6. If using PBMC following polyclonal stimulation, collect thecell suspension(s) and transfer into labeled conical tube(s).
Rinse interior of the culture vessel to recover all cells andtransfer into the corresponding conical tube(s). Increase volume to fill up tube with additional PBS and then centrifuge
balanced tubes at 330× g for 10 min with centrifuge brake on,
at RT. Alternatively, thaw previously cryopreserved PBMC as
detailed above if prior in vitro stimulation is not required toelicit antigen-specific ASC activity in the sample(s).
7. Decant supernatant and resuspend the cell pellet(s) using prewarmed BCM to achieve a cell density of approximately
2–5 × 106 cells/mL (this number can be estimated to be 50%of that prior to the stimulation culture).
8. Pipet 15 μL of live/dead cell counting dye onto a piece ofparafilm to form a droplet.
9. Remove 15 μL of cell suspension and combine with droplet oflive/dead cell counting dye. Pipet up and down 3–5 times tomix the sample while avoiding the formation of bubbles.
10. Transfer 15 μL of the cell and dye suspension into each chamber of a hemacytometer.
11. Determine live cell count and viability using CTL’s live/deadcell counting suite.
12. Increase volume of cell suspension(s) with additional sterile
warm PBS and centrifuge balanced tubes at 330× g for
10 min with centrifuge brake on, at RT.
13. Decant supernatant and resuspend the cell pellet(s) using prewarmed BCM at approximately 1 × 106 PBMC/mL.
14. Decant the BCM used for blocking the assay plate and replacewith 100 μL/well of prewarmed BCM to block the plate.
15. If the Goldilocks number has already been established for the
PBMC(s), plate cells at that number into the ImmunoSpot®

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assay. If that number is unknown, prepare serial dilution of
PBMC (or other single-cell suspension) accordingly, and thentransfer into the ImmunoSpot® assay plate.
16. Incubate cells in the assay plate for 16–18 h at 37 °C, 5% CO2.
17. After completion of the assay incubation period, remove plateand decant cells. Wash plate two times with PBS (200 μL/well), followed by two additional washing steps with 0.05%
Tween-PBS wash solution (see Note 38).
18. Prepare His-tagged antigen probe solution at optimized concentration and pass through 0.1 μm low-protein binding
syringe filter to remove any protein aggregates.
19. Decant 0.05% Tween-PBS wash solution, add 80 μL/well of
His-tagged antigen probe solution into designated wells, andincubate for 2 h at RT (protected from light).
20. Wash plate(s) two times with 0.05% Tween-PBS wash solution.
21. Prepare anti-His detection antibody solution according to kit
protocol and pass through 0.1 μm low-protein binding syringe
filter to remove any aggregates.
22. Decant 0.05% Tween-PBS wash solution, add 80 μL/well ofanti-His detection antibody solution into designated wells, andincubate for 1 h at RT (protected from light).
23. Wash plate(s) two times with 0.05% Tween-PBS wash solution.
24. Prepare tertiary solution according to kit protocol and passthrough 0.1 μm low protein binding syringe filter to remove
any aggregates.
25. Wash plates(s) twice with distilled water.
26. Remove protective underdrain and place plate face down on
vacuum manifold. Completely fill the backside of the plate with
distilled water and apply vacuum to draw water through themembrane (“back to front”) (see Note 39).
27. Allow plate to dry completely, protected from light (see Note40).
28. Scan and count plate(s) with suitable analyzer equipped withthe appropriate detection channel (see Note 18).

4

Notes
1. As enzyme-linked immunospot (ELISPOT) and FluoroSpot
assays differ only in the modality of detecting secretory footprints of cells on membranes, we collectively refer to both as
ImmunoSpot® assays. In the former, the detection antibody istagged to enable the engagement of an enzymatic reaction that
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Paul V. Lehmann et al.

white light. In the latter, the plate-bound detection antibodies
are visualized via fluorescent tags using appropriate excitationand emission wavelengths. Data provided in the chapter in thisvolume by Yao et al. [23] establish that ELISPOT and FluoroSpot assays have equal sensitivity for detecting antibodysecreting cell (ASC)-derived secretory footprints.
2. In proliferating germinal center B cells (GCB), somatic hypermutation (SHM) introduces random mutations into the variable regions of the B cell receptors (BCR) responsible for
antibody binding. As a result, subclones endowed withimproved, or decreased, BCR binding affinity for the antigen
are generated. GCB subclones that possess an increased BCR
affinity for antigen are positively selected to proliferate andundergo further rounds of SHM to further improve BCR
affinity. Collectively, the positive selection of GCB subclones
with improved binding for antigen underlies the progressive
affinity maturation of the ensuing antibody response.
3. B cell antigen receptors (BCR) are the membrane-anchoredforms of secreted immunoglobulin (Ig) molecules (commonlyreferred to as antibodies). Surface BCR are encoded by thesame Ig class/subclass and possess an identical antigen specificity to the antibody that a B cell will secrete once it differentiates
into an ASC, such as a plasma cell (PC).
4. As the concentration of antigen declines with time, only thosegerminal center B (GCB) cells with the highest affinity willcontinue to successfully compete for antigen binding, andonly the latter will continue to proliferate and undergo progressive SHM to eventually differentiate along the PC lineage.
5. The half-life of different Ig classes, and IgG subclasses, inserum is variable and relatively short in vivo. The half-life of
IgG1, IgG2, and IgG4 in humans is 21–28 days, whereas for
IgG3 it is ~1 week [32]. For IgA and IgM, their half-lives areeven shorter (3–7 days) [33, 34] and IgE has the shortest halflife in serum, ~2–3 days [35].
6. The extended half-life of serum IgG in vivo is dependent on its
interactions with FcRn [36, 37] which protects it from catabolism and enables IgG recycling. Likewise, the in vivo half-life ofother antibody classes such as IgA, IgE, and IgM are also
greatly extended when in association with Ig-binding surface
receptors [35, 38].
7. IgE secreted by B cells can bind to high-affinity Fc receptors(FcεRI) expressed on mast cells and basophils and endow thesecells with antigen-recognition properties. IgA antibodies are
secreted to mucosal surfaces.
8. Traditional B cell ELISPOT assays have been performed bydirect coating of the assay membrane with the antigen of

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interest. However, many (in fact, most) antigens do not adsorb
sufficiently to the membrane to enable reliable detection ofASC-derived secretory footprints. We have overcome this limitation by introducing an affinity coating approach for achieving
high-density antigen absorption to the assay membrane [11].
9. These samples were collected early in the COVID-19 pandemic
when the prototype Wuhan-Hu-1 strain of the SARS-CoV2 virus was circulating, and the testing was done with Spikeand Nucleocapsid (NCAP) antigens that corresponded to theoriginal Wuhan-Hu-1 strain. Thus, homotypic B cell memory
was studied.
10. While ImmunoSpot® enables measurement of a single ASC
within a bulk population of cells, flow cytometry falls short ofdetecting antigen-specific cells when they are present as very
low-frequency events. In ImmunoSpot® assays, there is noinherent lower limit of detection. If, e.g., 3 million PBMC
are plated at 3 × 105 PBMC across 10 replicate wells, 1 in
3 million is the detection limit, etc. Importantly, owing toincreased Poisson noise occurring with such low-frequency
measurements, the number of replicate wells evaluated needs
to be increased accordingly to obtain accurate low-frequency
measurements. As shown in Fig. 3, antigen-specific Bmem quite
frequently occur in low frequencies.
11. To establish the frequency of antigen-specific ASC among all
ASC-producing IgM/IgG/IgA/IgE or all four IgG subclasses(IgG1-IgG4) requires ~2 million PBMC (that can be isolated
from 2 to 3 mL of blood) when done according to our protocols (see also Note 23). To obtain the same information by flow
cytometry, up to ten-fold more cells would be required[39, 40].
12. For details of four-color B cell ImmunoSpot® detection, see
the chapter by Yao et al. in this volume [23]. Flow cytometry
does not reliably reveal the class/subclass of Ig produced by theindividual B cell because surface BCR expression can be highlyvariable and this is an underappreciated complexity of probe
staining. In particular, in the case of IgG+ ASC, they express
little if any surface BCR and this undermines assessment of
their antigen specificity and subclass usage using traditionalsurface staining approaches. Consequently, fixation and intracellular staining are required to define the IgG subclass usage ofthese cells (a procedure that results in substantial cell loss in thesample).
13. Each investigator in our laboratory can routinely test, in asingle experiment, 10–20 PBMC samples for reactivity against
a panel of antigens, assessing the frequency of ASC producing

192Paul V. Lehmann et al.

each of the Ig classes and IgG subclasses (see also the chapter
by Yao et al. in this volume [23]). With additional logisticalrefinements, this throughput is readily scalable upward.
14. For most antigens we have investigated thus far (SARS-CoV2 NCAP and receptor binding domain (RBD) proteins, theHCMV gH pentamer complex, EBV EBNA1, and OVA), we
have been unsuccessful in establishing reliable B cell ImmunoSpot® assays when following the classic protocol in which theantigen is directly absorbed to the membrane irrespective of
how high an antigen concentration was used for coating. In
contrast, our recent introduction of the affinity coating strategy [11] generated pristine results with each of these antigens.
We succeeded with direct coating only with recombinant hemagglutinin proteins of seasonal influenza viruses or the fulllength trimeric Spike protein of SARS-CoV-2, albeit having
to use very high antigen coating concentrations (exceeding
20 μg/mL) to achieve high-quality secretory footprint
formation.
15. In addition to detecting antigen-specific, B cell-derived secretory footprints on affinity-coated plates, as described here, it isalso possible to detect such cells via the inverted ImmunoSpot®
approach. In the latter, the Ig secretory footprint is captured
irrespective of the ASC’s specificity (e.g., by coating the membrane with an anti-IgG capture antibody) and the antigenspecific B cells’ secretory footprints among all those retainedon the membrane are detected by the capture of labeled antigen. Such inverted assays work perfectly when the frequency ofantigen-specific B cell footprints is high among all secretory
footprints captured, but are challenging for the detection of
low-frequency events. Inverted assays are described in detail inthe chapter by Becza et al. in this volume [12]. While direct
assays permit the capture of all Ig classes or subclasses produced
by antigen-specific ASC via four-color ImmunoSpot® detection (see the chapter by Yao et al. in this volume [23]), theinverted assay detects only the selected Ig class, e.g., IgG+ ASC.
16. B cell ImmunoSpot® data can be expressed as spot-forming
units (SFU) per cell input per well to determine the frequencyof antigen-specific cells. However, owing to considerable interindividual variations in the abundance of total Ig class/subclassproducing ASC in test samples following polyclonal stimulation, and the prevalence of IgG [6], it is better to report data as
the frequency of ASC producing a given Ig class/subclassamong all ASC producing that Ig class/subclass.
17. ASC secrete Ig in an undirected fashion into 3D space. The
antibody released toward the membrane will be captured as asecretory footprint; however, the remainder of the secreted

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antibody will diffuse away from the cell and is diluted into theculture supernatant. As the concentration of diffused antibodies increases in the culture medium, these antibodies arecaptured on the membrane distantly from the source ASC,
increasing the background signal in the assay and undermining
the resolution of individual secretory footprints. Such an elevated background in an ImmunoSpot® assay is termed an
ELISA effect that can obscure the accurate counting, or evendetection of secretory footprints [6].
18. The chapter by Karulin et al. in this volume [10] introduces
machine learning-based spot-forming unit (SFU) analysis thatcan partially compensate for ELISA effects and SFU crowding,thus extending the linear range of accurate quantification forcell numbers plated per well and SFU detected.
19. When performing such serial dilution experiments, using 4 replicate wells per cell input plated, and linear regression of the
SFU frequency through extrapolation from the mean of these4 replicates, that calculated frequency was very close to thefrequencies established by relying on just one replicate well
per cell input dilution (see the chapter by Yao et al. in thisvolume [23], and N. Becza, manuscript in preparation). Thus,
serial dilutions involving single wells for each cell dilution,
progressing in a 1 + 1 (two-fold) dilution series is a valid option
for establishing accurate SFU frequencies and greatly reduces
the cell numbers and reagents required.
20. We failed to detect SARS-CoV-2 Spike or NCAP antigenspecific, class-switched (IgG or IgA) ASC in individuals collected prior to the COVID-19 pandemic that by definition
would be immunologically naive to the SARS-CoV-2 virus[6]. However, IgM+ ASC reactivity against these antigens in
the pre-COVID-19 donor cohort population was ambiguous
because the current polyclonal stimulation approach also
results in the differentiation of naive IgM+ B cells, which may
express broadly reactive specificities and can generate secretory
footprints in negative control wells. In contrast, all subjects
with PCR-verified infection, harbored S and N antigen-specific
IgG+ ASC. These findings are consistent with the notion thatclonal expansions to detectable frequency levels, classswitching, and affinity maturation (all features of Bmem) areneeded for detecting antigen-specific B cells in ImmunoSpot®
assays. Therefore, it appears that detection of class-switched,antigen-specific ASC in ImmunoSpot® assays implies priorin vivo priming of Bmem with (cognate or cross-reactive) specificity for the test antigen.

194Paul V. Lehmann et al.

21. Thus far, we have not seen evidence for class-switching of Bmemduring short-term in vitro polyclonal stimulation using R848
plus IL-2.
22. So far, having studied “healthy volunteers” exclusively, we only
detect IgE+ ASC following in vitro stimulation with agonistic
anti-CD40 mAb plus IL-4 and IL-21, but not after stimulation
with R848 plus IL-2 [41]. Thus, our detection system enables
identification of IgE+ ASC, but whether IgE+ Bmem exist
in vivo, or whether they switch de novo to IgE in vivo orin vitro is controversial [42, 43].
23. If special protocols are followed, PBMC can be frozen without
impairing the B cells’ functionality ([30] and N. Becza manuscript in preparation). Thus, by freezing B cells of a sample inseveral aliquots, the same PBMC can be tested repeatedly,
reproducing the results of the previous experiment with highaccuracy [6], or for extending those studies, e.g., for assessing
the affinity distribution of the antigen-specific Bmem repertoire.
Of note, when planning the numbers of PBMC to be frozen
per cryovial, as a rule of thumb, one can anticipate recovery of~50% of PBMC initially frozen after these cells are thawed andhave undergone 5 days of in vitro polyclonal stimulation topromote terminal differentiation of resting Bmem into ASC. It
is also important to note that any number of PBMC between
1 and 10 million can be frozen per cryovial permitting the
optimization of PBMC utilization when planning experiments(N. Becza, manuscript in preparation).
24. While the potential of B cell ImmunoSpot® assays to establish
affinity distributions is embedded in the nature of the assay, thispotential has not been realized yet. The chapter by Becza et al.
in this volume [12] is the first step in this direction. To progressalong these lines is one of the major focuses in our laboratory.
25. It can be assumed that after vaccination with the prototype
Wuhan-Hu-1 strain Spike antigen, in addition to T-cell immunity directed against shared antigenic determinants of Spikevariants, heterotype-specific, cross-reactive B cell memory forthe receptor binding domain (RBD) mediates the partial protection seen toward newly emerging antigenic variants of thisvirus. Such SARS-CoV-2 variants evade the first wall (reflectedby serum antibodies) and are thus capable of causing infections, but the engagement of cross-reactive Bmem (the secondwall) can still convey protection from severe disease throughrapidly generating a quasi-secondary neutralizing antibody
response against the mutated RBD of the new variant.
26. In our ongoing studies, we find that ~40% Wuhan-Hu-1
infection-elicited Bmem are cross-reactive with the Omicron(BA.1) variant (Fig. 5 and Liu et al., work in progress).

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27. Our introduction of the PVDF membrane to T-cell ELISPOT
assays [28], with its by far superior capture antibody adsorption
properties [44], has been key for improving our ability todetect secretory footprints to the point needed for transforming ImmunoSpot® into the robust T-cell monitoring platform
it has become for detecting rare – even very rare – antigenspecific T cells ex vivo, in freshly isolated PBMC or otherlymphoid cell material. We refer to Figure 1 in [45] to appreciate the difference in assay performance using the PVDF
membrane vs. the previously used mixed cellulose ester
membrane.
28. The shape of secretory footprints (spot morphologies) produced by T cells follows simple rules since the capture antibody’s (i.e., an anti-cytokine-specific mAb) affinity for the
analyte to be detected is high and fixed. Consequently, only
the quantity of analyte (cytokine) produced by the T cell willdefine the morphology of the resulting secretory footprint[16]. Predictable (log normal, [46]) spot sizes permit objective
automated size gating [47]. However, ASC-derived secretory
footprints in antigen-coated wells are primarily defined by theaffinity of the secreted antibody for the membrane-boundantigen. Thus, the formation of ASC-derived secretory footprints follows a different set of rules, and hence requires afundamentally different analytical approach. The chapter contributed by Karulin et al. in this volume [10] introduces asoftware solution suited for accurate, high-throughput, andhigh-content B cell ImmunoSpot® analysis.
29. Thawing of cryopreserved cells causes a fraction of the cells(up to 30%) to die, and the DNA released from such cells cancause clumping of the thawed cell material. This cell clumping
can be reduced, if not completely eliminated, by including an
immunologically neutral endonuclease, Benzonase. Ready-touse Benzonase-containing serum-free wash solutions are available: CTL anti-aggregate Wash™ 20X solution.
30. A suitable assay medium for use in B cell ImmunoSpot® is
RPMI 1640 with 10% FCS, 2 mM L-glutamine, 100 U/mL
penicillin, 100 μg/mL streptomycin, 8 mM HEPES, and50 μM 2-mercaptoethanol.
31. The volume of in vitro stimulation cultures can be scaled up or
down accordingly, but we recommend keeping the cell density
of PBMC at approximately 1–2 × 106 cells/mL. Smaller in vitro
stimulation cultures can be initiated in 48- or 24-well plateswith a final volume of 1 or 2 mL, respectively. Be sure to fill
empty wells in tissue culture plates with sterile PBS to avoid
dehydration of cell cultures. For larger volumes, use appropriate tissue culture flasks, upright or laying, with the culture
medium between 0.5 and 1 cm in height.

196

Paul V. Lehmann et al.

32. Kit is suited for detecting either antigen-specific ASC thatdifferentiated in vivo, or antigen-specific Bmem that have beenpolyclonally stimulated in vitro to promote their transition toASC. Each kit contains anti-His capture antibody, Ig classspecific (IgA, IgE, IgG, and IgM) detection reagents, diluent
buffers, low autofluorescence PVDF-membrane plates, andpolyclonal B cell activator (B-Poly-S). Alternatively, IgG classspecific (IgG1, IgG2, IgG3, and IgG4) detection reagents canbe substituted in the context of such four-color B cell ImmunoSpot® assays.
33. Traditional B cell ELISPOT assays have been performed bydirect coating of the assay membrane with the antigen ofinterest. However, many (in fact, most) antigens do not adsorb
sufficiently to the membrane to enable reliable detection ofASC-derived secretory footprints. We have overcome this limitation by introducing an affinity coating approach for achieving
high-density antigen absorption to the assay membrane [11].
34. We recommend optimizing the concentration of His-tagged
protein(s) used for affinity capture coating. A concentration of
10 μg/mL His-tagged protein has yielded well-formed secretory footprints for most antigens, but increased concentrationsof the anti-His affinity capture antibody and/or His-tagged
protein may be required to achieve optimal assay
performance [11].
35. Kit is suited for detecting either antigen-specific ASC thatdifferentiated in vivo, or antigen-specific Bmem that have beenpolyclonally stimulated in vitro to promote their transition toASC. Each kit contains pan anti-human IgG capture antibody,anti-His detection reagents, diluent buffers, low autofluorescence PVDF-membrane plates, and polyclonal B cell activator(B-Poly-S).
36. The optimal concentration of affinity (His)-tagged antigenprobe used for detection of all antigen-specific secretory footprints (e.g., SFU), low- or high-affinity alike, should be determined empirically.
37. If the entire plate will not be coated with the anti-His affinity
capture antibody solution, the remainder of the EtOH prewet
wells should receive 80 μL/well of PBS.
38. Plate washes may also be performed manually. For automatedwashing, the pin height and flow rate should be customized to
avoid damaging the assay membranes, which is the case for the
CTL 405LSR plate washer.
39. Lowering nonspecific background staining, and reduction of“hot spots” in the center of the assay wells, is achieved throughperforming the “back to front” water filtration technique.

Monitoring Memory B Cells

197

40. To completely dry plates, blot assay plate(s) on paper towels to
remove residual water before either placing them in a runninglaminar flow hood at a 45° angle for >20 min or placing facedown on paper towels for >2 h in a dark drawer/cabinet. Do
not dry assay plates at temperatures exceeding 37 °C as thismay cause the membrane to warp or crack. Fluorescent spots
may not be readily visible while the membrane is still wet andthe background fluorescence may be elevated. Scan and countplates only after membranes have dried completely.

Acknowledgments
We wish to thank the R&D and the Software Development teams at
CTL for their continued support and technological innovation thatmade our B cell ImmunoSpot® endeavor possible. We thank Drs.
Alexey Y. Karulin and Graham Pawelec for in-depth discussions of
the subject matter, and Diana Roen for carefully proofreading themanuscript, and also Gregory Kovacs for his support in the generation of graphic illustrations. All efforts were funded from CTL’s
research budget.
Conflicts of Interest P.V.L. is Founder, President, and CEO of
CTL, a company that specializes in immune monitoring by ImmunoSpot®. Z.L., N.B., A.V.V., and G.A.K are employees of CTL.
J.B. was a summer intern at CTL when he developed the tetanus
toxoid heavy chain (TTHc)-specific B cell ImmunoSpot® assay andcontributed the frequency measurements of TTHc-specific Bmemcells incorporated in Fig. 3.

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