Chapter 13
Assessing the Affinity Spectrum of the Antigen-Specific
B Cell Repertoire via ImmunoSpot®
Noémi Becza, Zhigang Liu, Jack Chepke, Xing-Huang Gao,Paul V. Lehmann, and Greg A. Kirchenbaum
Abstract
The affinity distribution of the antigen-specific memory B cell (Bmem) repertoire in the body is a criticalvariable that defines an individual’s ability to rapidly generate high-affinity protective antibody specificities.
Detailed measurement of antibody affinity so far has largely been confined to studies of monoclonal
antibodies (mAbs) and are laborious since each individual mAb needs to be evaluated in isolation. Here,
we introduce two variants of the B cell ImmunoSpot® assay that are suitable for simultaneously assessing theaffinity distribution of hundreds of individual B cells within a test sample at single-cell resolution using
relatively little labor and with high-throughput capacity. First, we experimentally validated that both
ImmunoSpot® assay variants are suitable for establishing functional affinity hierarchies using B cell hybridoma lines as model antibody-secreting cells (ASC), each producing mAb with known affinity for a defined
antigen. We then leveraged both ImmunoSpot® variants for characterizing the affinity distribution of
SARS-CoV-2 Spike-specific ASC in PBMC following COVID-19 mRNA vaccination. Such ImmunoSpot®
assays promise to offer tremendous value for future B cell immune monitoring efforts, owing to their easeof implementation, applicability to essentially any antigenic system, economy of PBMC utilization, highthroughput capacity, and suitability for regulated testing.
Key words ELISPOT, FluoroSpot, B lymphocytes, Immune monitoring, Antibodies, Antibody titers,
Immune memory, Functional affinity

1

Introduction

1.1 Definition of
Affinity, Avidity, andFunctional Affinity

Antibodies, also called immunoglobulins (Ig), can specifically bindto nearly any type of molecule (collectively termed an antigen),whereby the antigen-binding domain located in the hypervariable
region of the antibody molecule (called the paratope) associateswith a defined region of the antigen referred to as an epitope. The
specificity and strength of antigen-antibody interactions depends

Paul V. Lehmann and Greg A. Kirchenbaum contributed equally and share senior authorship.
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_13, © The Author(s) 2024

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on the presence of complementary structures on both surfaces inthree-dimensional space (“lock and key principle”) with the summation of attractive/repulsive forces contributing to the net binding strength. The binding forces involved in antibody-antigen
interactions include (a) pairs of oppositely charged moleculargroups, (b) hydrophobic regions capable of attracting each other,(c) coordinated hydrogen bonds, and (d) van der Walls-type interactions. As all four binding forces are reversible, the binding ofantibodies to antigens is reversible too. Consequently, there is aconstant “on-off” flickering of the paratope-epitope associationthat follows a second-order biochemical reaction. The association(Kon) and dissociation- (Koff) constants of the paratope-epitope
interaction jointly determine the equilibrium constant (Kd) thatdefines “affinity” (see Note 1).
The affinity of antibodies elicited during the course of an
immune response can range from nearly undetectable (e.g.,
Kd < 10-4 M), to extremely high (e.g., Kd > 10-10) [1–8]. Consequently, a difference of greater than six orders of magnitude in
antibody concentration can be required for low vs. high-affinity
antibodies to attain the same level of epitope coverage. Thus, while
all such “specific” antibodies can result in “specific” antigenbinding if they are present in sufficiently high concentration, it
would take one million times more antibody molecules for a
low-affinity antibody compared to a high-affinity antibody toachieve the same density of antigen binding! Therefore, the affinityof an antibody is a critical factor determining its capacity to contribute towards protective immune reactions.
Affinity will suffice for defining the binding strength of an
antibody to an antigen when only a single arm (so-called Fabfragment) of the antibody can interact with the antigen. This is
the typical scenario when antibodies (immobilized or not) interact
with individual antigen molecules in solution. This is because, on
most protein antigens, the epitopes are singular unique structures,i.e., there are no other epitopes present on the same antigen molecule for the other arm of the antibody to associate with. Avidity
defines the net binding strength when a single antibody molecule(that possesses 2–10 Fab fragments or arms) can attach to multiple
epitopes that are structurally connected. This is the case whenrepetitive epitopes occur on the same antigen molecule and arewithin the reach of the antibody’s arms, or when separate, butphysically linked, antigen molecules are sufficiently close in proximity that the antibody can associate with more than one antigen
molecule simultaneously (e.g., when two membrane-anchoredantigen molecules are in close proximity to each other). The net
binding strength attained from such multivalent binding (avidity) is
exponentially higher than that resulting from monovalent binding(affinity). This is because, as mentioned above, the interaction of
the antibody’s paratope with the antigen’s epitope is a reversible

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213“on” and “off” flickering; during the “off” state, in case of amonovalent interaction, the reaction partners can diffuse away. If,
however, more than one arm of the same antibody molecule issimultaneously involved in unsynchronized “on/off” flickering,
the time window in which two or more arms are dissociated isgreatly reduced, and the ligand(s) could separate and diffuse away.
To define affinity, single arms of antibodies (so-called Fab fragments) need to be studied; however, these do not occur naturally.
As all naturally occurring antibodies in humans (and other commonly used animal models) have at least two arms (IgG, IgE, andmonomeric IgA have two; IgM and dimeric IgA have 10 and4, respectively), the occurrence of monovalent or multivalent binding (i.e., whether affinity or avidity applies), depends on the special
circumstances of the antigen encounter. In terms of immunobiological consequences, however, the overall attachment strength of
the antibody molecule with the antigen is fundamental for hostdefense. The latter is pragmatically called functional affinity. Since,in this chapter, we will also be analyzing the binding of bivalent
antibodies to antigens, we adopt the term functional affinity, whenappropriate, hereafter.
1.2 Biological
Benefits of Antibodies
with High Functional
Affinity

A critical effector function of antibodies is their ability to bind andneutralize antigens (e.g., toxins or viruses) by preventing theirassociation with cells of the host. Importantly, for this process tooccur effectively, these antibodies need to possess a functional
affinity for the antigen that surpasses that of the toxin/virus fortheir endogenous host receptor(s). Thus, the higher the functional
affinity of these antibodies (e.g., the lower the Kd), the lower theconcentration required for them to exert their neutralizing effector
function. Moreover, another effector function of antibodies withhigh functional affinity is their ability to cross-link soluble antigens
and generate immune complexes facilitating the effective elimination of the antigen by phagocytes. Activation of the complement
system, another effector function initiated by antibodies, depends
on the cross-linking of at least two of the six “arms” of the C1q
molecule [9–11]; which is the first component that initiates theclassical complement cascade. For this to occur, two antibody
molecules need to be bound simultaneously, in close vicinity, so
C1q can bind to both at once. As antibodies with high functional
affinity bind more stably, even when they occur at low concentrations, they outperform antibodies with lower functional affinity intheir capacity to initiate complement activation. The same applies
when antibodies bind to antigen and label the latter for
FcR-mediated elimination via phagocytosis (opsonization) or
destruction (antibody-dependent cellular cytotoxicity, ADCC).
For all these effector functions, the concentration of “specific”
antibodies in bodily fluids is critical for their efficacy. As antibody

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levels tend to decline with time after an immune response, only
those antibodies at the high-level end of the functional affinity
spectrum will effectively contribute to host defense.
1.3 Affinity
Maturation of the
Antibody Response

Owing to the fundamental role that antibody functional affinity
plays in immune protection, the B cell system has evolved means tomaximize the affinity of antibodies deployed in the course of an
immune response. This starts with the recombination of V(D)J
gene segments within the IgH/IgL loci during early B cell ontogeny, a process capable of generating an estimated 1014 unique B cell
receptors (BCR) [12] (see Note 2). As B cells are limited to theexpression of a single BCR (with rare exceptions), each B cell isendowed with a unique antigen-binding specificity. Importantly,
any given specificity occurs at a very low frequency among naive B(and T cells) and such cells continuously recirculate through secondary lymphoid tissues where they may encounter their cognate
antigen (see Note 3). Both naive B and T cells are efficiently
retained in secondary lymphoid tissues during the onset of a primary immune response when they first encounter “their” antigen,and, driven by (affinity-based) antigen receptor triggering, thesecells are induced to proliferate. In this way, clonal expansions occur
resulting in an increase in the frequency of antigen-specific B and T
cells among all lymphocytes in the body. The activated T and B cells
also acquire specialized effector functions, i.e., T cells become
Th1/Th2/Th17/Tfh21 cells, each of which is capable of secretinga distinct cytokine signature, and B cells undergo Ig class switchingthat is characteristic of antigen-experienced B cells: they switchfrom IgM-expressing naive B cells to downstream Ig classes suchas IgG or IgA, and within the IgG producers, to subclasses IgG1/
IgG2/IgG3/IgG4. Of note, the process of Ig class switching isgoverned by instructive signals provided primarily by T helper cells(see Note 4).
During the primary immune response, the proliferating
antigen-stimulated B cells also undergo an additional fundamental
process aimed at improving the efficacy of the ensuing antibody
response: the acquisition of somatic hypermutations (SHM) in the
IgH and IgL encoding the antigen-binding variable regions of their
BCR. Initially, naive B cells with adequate affinity for the antigen
become stimulated; these cells can then acquire SHM as they
undergo additional rounds of proliferation within the germinal
center reaction. Their progeny, therefore, will consist of subclones
that have BCR with a spectrum of affinities for the eliciting antigen:some with higher, and others with lower affinity. Of these daughter
cells, only those with the highest affinity for the antigen continue toparticipate in the germinal center reaction and undergo further
rounds of proliferation and acquisition of additional SHM. This
process of positive selection for high-affinity subclones (and negligence of daughter cells with lower affinity) continues throughout

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the primary immune response and becomes more and more stringent as the antigen becomes gradually eliminated as a consequenceof the ensuing successful immune response. This process of affinity
maturation through the acquisition of SHM can also be re-initiatedat a later time point if the antigen is reencountered, providing thecellular basis for why booster immunizations can progressively raise
the affinity of the elicited antibody response. Whether in the context of a primary or secondary (recall) immune response, the B cellprogeny endowed with the highest affinity BCR for the eliciting
antigen will differentiate into plasma cells (PC) that secrete antibodies with identical specificity of the BCR expressed at the time of
their disengagement from the germinal center reaction. If suchantibody-secreting cells (ASC) settle into the bone marrow orother suitable niches, they can become long-lived PC (see Note 5).
Relevant to this chapter, the affinity distribution of B cells forany given antigen is expected to be variable among human subjects,
dependent on the dose and duration of antigen persistence during
the primary immune response (that dictates the initial affinity-based
positive selection process) and on the timing, dose, and duration of
secondary, and possibly subsequent, antigen encounters, thus triggering further rounds of affinity-based selection of the antigenspecific B cell repertoire. Based on past efforts generating monoclonal antibody (mAb)-secreting B cell hybridomas, experience
supports that the more booster shots that are given, the higher
the chance of isolating a clone that secretes antibodies with very
high affinity.
1.4 Measuring
Antibody Functional
Affinity in the Serumvs. the B Cells
Themselves

It is important to understand the fundamental differences between
immune protection mediated by the B cell system via the firstvs. second walls of adaptive humoral defense. Antibodies alreadypresent in serum and other bodily fluids can instantly bind to
antigens as soon as they attempt to enter the body. This first wallof defense can prevent reinfections and it can be readily assessed byserum antibody measurements. Serum antibodies, however, arerelatively short-lived molecules (see Note 6) and their continued
presence depends on constant replenishment by PC. While PC are
potentially long-lived, their lifespans are heterogenous and likely
fall on a continuum [13, 14]. During the recent COVID-19 pandemic, we were reminded how rapidly following natural infection,
or after vaccination, the induced specific serum antibody levels candecline [15, 16]. We also frequently learned that the detection ofmemory B cells (Bmem) is far more reliable for revealing whether an
infection has occurred than measuring serum antibodies [17].
The second wall of B cell-mediated protection is conferredthrough the reactivation of Bmem. If the first wall of adaptive
humoral defense fails, and a (re-)infection occurs, antigen-specific
Bmem (and memory T cells) can rapidly engage in secondary
immune responses. These lymphocytes can mount a stronger and216

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faster counterattack against the offending pathogen because they
are present in greatly increased numbers in the body compared to
the numbers of naive B (and T) cells in the pre-immune repertoire.
Moreover, many of these pre-existing memory cells have alreadyundergone differentiation into effector lineages (Th1/Th2/Th17,
etc. for T cells, IgG subclass, or IgA-switched B cells). Additionally,
the B cell affinity maturation process re-engages during secondary
immune responses, starting, however, from the elevated levels
established following the prior antigen encounter(s).
Studying the first wall (existing antigen-specific serum antibodies) and the second wall (the antigen-specific Bmem repertoire in
the blood, see Note 7) of adaptive humoral immunity therefore
provides fundamentally different information. The former provides
a low-resolution and fading image of the past as it still applies to theintegrity of the first wall, and the latter permits to gain a highresolution image of the second wall, thus assessing the immune
potential present in case of a subsequent antigen encounter. Highresolution in this context means being able to assess individual B
cells within the entire antigen-specific repertoire, the type andquantity of antibody they produce, and, relevant for this chapter,the functional affinity distributions of the individual B cells.
1.5 Measuring
Functional Affinity by
B Cell ImmunoSpot®

Being able to assess the functional affinity distribution of the antigen-specific Bmem compartment in an individual requires defining
the functional affinity for hundreds (the more the better) of individual B cells to obtain a representative picture. Doing so by thestandard approaches, either through surface plasmon resonance(SPR) or biolayer interferometry (BLI) measurements of mAbs,
would be an effort too involved for immune monitoring purposes(see Note 8). We introduce here modifications of the ImmunoSpot® assay that promise to fill this gap.
The principle of one of the underlying ImmunoSpot® variants
suited for this purpose, the inverted assay, is shown in Fig. 1a. In
brief, the membrane is coated with an antibody suited for capturing
the Ig produced by ASC irrespective of their antigen specificity oraffinity: in case of testing human PBMC, the capture reagentstypically would be xenogeneic anti-human IgG, A, or E-specificantibodies, or a pan Ig-specific (anti-kappa + anti-lambda lightchain) antibody (see Notes 9 and 10). Onto this lawn of Ig capture
reagent, the cells are plated (see Note 11). During the time in which
the ASC resides on the membrane, their secreted antibodies will be
captured in close proximity to the respective ASC in the form of asecretory footprint (often also referred to as a “spot forming unit,”
SFU) (see Note 12). After a brief period of cell culture during
which the secreted Ig analyte is captured, the cells are removed
from the plate (see Note 13), and the antigen probe is added toreplicate wells containing the same number of ASC, but decreasing

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Fig. 1 Schematic representation of antigen-specific inverted or direct ImmunoSpot® assays. The PVDF
membrane on the bottom of a 96-well plate is densely coated (a) with a pan anti-Ig(G) class-specific(in this example IgG) capture antibody that binds with high-affinity to secreted Ig(G) irrespective of antigen
specificity or (b) with an antigen. The PBMC are plated on the resulting lawn of (a) pan anti-Ig(G) class-specificcapture antibody, and individual ASC producing the particular Ig class will generate a secretory footprint or (b)antigen and individual ASC-producing antibody with sufficient affinity to the antigen will generate a secretory
footprint. After removal of the cells, (A) the affinity-tagged (in this example His) antigen is added at a sufficientconcentration to be retained by antigen-specific secretory footprints generated by ASC producing low- or highaffinity antibody or (b) antigen-bound antibody is visualized by biotinylated human Ig class/subclass (in thisexample IgG) specific detection antibodies. (a) Antigen-specific secretory footprints are then visualized using a
biotinylated anti-affinity tag detection reagent; which is revealed by the addition of a fluorescently-conjugatedstreptavidin (FluoroSpot, as shown) or via an enzymatic reaction (ELISPOT, not shown). (b) Antigen-specificsecretory footprints are revealed by the addition of a fluorescently-conjugated streptavidin (FluoroSpot, asshown) or via an enzymatic reaction (ELISPOT, not shown). (a, b) Counting the spot-forming units (SFU) per
well reveals the number of antigen-specific ASC within the PBMC plated(graded) concentrations of antigen. When the antigen probe isadded in excess, all secretory footprints retain the antigen probe:
ASC with low- and high-affinity will be revealed alike. As theconcentration of antigen probe becomes limiting, however, increasingly only the high-affinity secretory footprints (SFUs) will capturea sufficient quantity of the antigen probe for their detection.
For such affinity measurements, the cells need to be plated at apredetermined “Goldilocks” number that, being assay dependent,
is between 50 and 200 SFUs/well (being at the upper end of thelinear range between cell numbers plated and SFU counted, so thatas many individual ASC can be assessed per well as possible, yet
without ASC crowding interfering with the image analysis, see also
Note 14). Testing a fixed number of replicate wells for each antigen
concentration thus permits to assessment of the affinity spectrum
within the same number (ideally ≥300) of antigen-specific ASC foreach antigen concentration. The percentage of SFU lost with eachsuccessive reduction in antigen probe concentration reveals theaffinity thresholds for the ASC subpopulations lost, respectively. A
detailed protocol for such B cell affinity measurements is provided
below.

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Fig. 2 Assessing the affinity of Granzyme B (GzB)-specific mAb by inverted ImmunoSpot® assay vs. surface
plasmon resonance testing. In columns 1–4, the results of an inverted ImmunoSpot® assay are shown. Onto alawn of anti-murine Igκ capture antibody, B cell hybridoma clones 12.4 and 30.1, each of which secretes
GzB-specific mAb, and a third hybridoma clone that secretes a SARS-CoV-2 Spike-specific mAb, were plated
at 200 cells per well, in the rows specified. His-tagged recombinant human GzB (rhGzB) was added indecreasing concentrations, as shown, and the captured rhGzB probe was detected via the His-tag. In thecolumn on the right, replicate wells seeded with the B cell hybridomas, respectively, were detected using ananti-murine IgG detection capture antibody to verify the presence of IgG+ secretory footprints. KD values of the
GzB-specific hybridomas for rhGzB, as established by surface plasmon resonance testing, are indicated on theright. ND not determined
1.6 B Cell Hybridoma
Studies Validating
ImmunoSpot® Affinity
Measurements

While performing B cell affinity measurements using the inverted
ImmunoSpot® approach outlined above seems simple and intuitive, this strategy has not yet been introduced. To experimentally
validate this approach, we tested B cell hybridomas with establishedaffinities for defined antigens. An example is shown in Fig. 2 using apair of anti-human Granzyme B (GzB) hybridomas we generated
during our efforts to raise mAbs against this protein. Among 7 such
hybridomas, two clones secreted mAb at opposing ends of theantigen-retention spectrum when tested using an inverted ImmunoSpot® approach: GzB12.4 and GzB30.1. These two clones,
along with an additional SARS-CoV-2 Spike-specific control
hybridoma (generously provided by Giuseppe A. Sautto), were allplated at ~200 cells per well. Visualizing the secretory footprints
with an anti-murine IgG detection antibody showed that most cells
within each of the three hybridoma clones were capable of generating a secretory footprint and that the per-cell IgG productivity rates
were comparable across the three (the SFU sizes and fluorescenceintensities were similar between individual ASC within each hybridoma line and between the three B cell hybridoma clones (Fig. 2).
When recombinant His-tagged human GzB (rhGzB) was added at
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GzB12.4 and GzB30.1 bound the antigen revealing ~200 SFU,
whereas the footprints of the control hybridoma did not capture
rhGzB. When the concentration of rhGzB was decreased in a 1:5
dilution series, footprints of GzB12.4 continued to capture thisantigen down to a concentration of 4 ng/mL, whereas the secretory footprints of clone GzB30.1 were no longer discernable when
the rhGzB probe was added at concentrations lower than 100 ng/
mL. By ImmunoSpot®, clone GzB12.4 was identified as secretingmAb with high affinity for rhGzB, whereas clone GzB30.1 secreted
mAb that appeared to possess a substantially lower affinity for thismolecule. To verify by an independent method that this was indeed
the case, we purified mAb from GzB12.4 and GzB30.1 and established their functional affinity for rhGzB using surface plasmon
resonance (Biacore): a KD value of 7.0 × 10-11 was calculated
for mAb GzB12.4, while the KD value of mAb GzB30.1 was
2.3 × 10-8. Thus, both techniques concurred that clone GzB12.4
secreted mAb with a substantially higher functional affinity for
rhGzB than the mAb produced by clone GzB30.1. Supplementary
Figures 1 and 2 (see Note 15) provide further examples in which
inverted ImmunoSpot® assays provided confirmatory results forhybridoma pairs secreting mAb with differences in functional affinity for alternative antigens; influenza hemagglutinin (H1) or SARSCoV-2 Spike, respectively.
Another semiquantitative approach for assessing the affinity of
individual ASC via ImmunoSpot® is based on the direct assay(Fig. 1b), studying the SFU morphologies on antigen-coatedwells, in particular when coated with graded antigen densities. In
direct B cell ImmunoSpot® assays, the membrane is either coatedwith the antigen itself, or adapter molecules are used for attaching
the antigen to the membrane (see Note 16). As such, only ASC
which produce an antibody with sufficient functional affinity for themembrane-associated antigen will be capable of generating a secretory footprint. Next to the per cell quantity of secreted antibody,the antibodies’ functional affinity for the antigen defines the sizeand density characteristics of the resulting secretory footprint: ASC
that produce high-affinity antibodies will create crisp and more
intense spots, whereas ASC-producing antibodies with reduced
functional affinity yield more diffuse and fainter/sparse footprints[18]. The difference between secretory footprints generated byhigh- or low-affinity ASC will be further accentuated when thebonus effect of multivalent binding is negated by reducing theantigen-coating density. At low antigen-coating densities, antibodies can only bind using a single “arm,” i.e., the binding is definedby affinity alone; if the antigen is coated densely onto the assay
membrane, however, the antibody can attach with both arms toneighboring antigen molecules and now avidity can amplify the
antibody’s functional affinity. Figure 3 illustrates the spot morphologies observed when murine B cell hybridomas secreting mAb with

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Fig. 3 Direct ImmunoSpot® assay leveraging limiting antigen coating reveals
differential mAb functional affinity for influenza hemagglutinin. The specified
murine B cell hybridomas, each secreting monoclonal antibody (mAb) reactivewith recombinant hemagglutinin (rHA) protein (COBRA P1), however, with previously defined differential affinity [19], were plated at the Goldilocks numbers(100 cells per well) into wells that had been pre-coated with decreasing
concentrations of rHA protein, as specified. The captured secretory footprints
were visualized using anti-murine IgG1-specific detection antibodies. Secretory
footprints from each of the B cell hybridoma lines were readily apparent in wells
coated with a high concentration of rHA. However, the number of secretory
footprints and their density declined at different rates for the two clones whenhybridoma cells were seeded into wells coated with decreasing concentrationsof rHA. Based on the results of this experiment, the mAb secreted by the 1-B7.2
B cell hybridoma line has an increased functional affinity for the coated rHA
protein compared to the mAb secreted by 2-A10.4, confirming the previously
published affinity hierarchy established by Sautto et al. [19]differential functional affinities for an influenza hemagglutinin(H1) were evaluated for secretory footprint formation on membranes in which the antigen-coating density was progressively
limited.
1.7 B Cell Affinity
Distribution
Measurements in
PBMC with Inverted
ImmunoSpot® Tests

After having experimentally validated the suitability of both ImmunoSpot® assay variants for B cell affinity measurements using hybridomas as model ASC, we set out to translate these tests for studiesof antigen-specific ASC in human PBMC. As described above, B
cells endowed with a wide affinity spectrum for the eliciting antigen
participate in the primary immune response. However, progressively the engaged BCR repertoire is selected for those with increasingly high affinity, particularly in cases of prolonged or repetitive
antigen exposure. Every individual’s Bmem repertoire at a given
time point is therefore a variable defining that person’s capacity toengage in an effective anamnestic, adaptive humoral defense

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reaction. One critical variable for evaluation is the clonal size of theantigen-specific Bmem compartment, as revealed by their frequency
in PBMC. A second critical variable for assessment is the antibody
class/subclass usage of antigen-specific Bmem since this will offer
insight into what types of antibodies will be secreted acutely following antigen reencounter. Third, but not least, the frequency ofhigh-affinity Bmem capable of producing the desired Ig class/subclass will define the efficacy of the anamnestic antibody response
elicited in case of antigen reencounter. Importantly, all three ofthese parameters can readily be assessed using tailored B cell ImmunoSpot® assays.
As PBMC can be frozen without losing B cell functionality (seeNote 11), we recommend undertaking high-resolution B cell
ImmunoSpot® testing using aliquots of the same sample and atiered approach. In the first experiment, the frequency of antigenspecific Bmem-derived ASC is established by performing serial dilutions of the cell input to define the Goldilocks number by linear
regression (see Notes 14 and 17). Of note, this first experiment
only requires ~2 × 106 cells per antigen to define the frequency ofantigen-specific ASC secreting each Ig class/IgG subclass, respectively (see Note 18 and see the chapter by Yao et al. in this volume[20] that is dedicated to such measurements). In a subsequent
experiment, thawing an additional aliquot(s) of the PBMC sample,
the affinity distribution of the antigen-specific ASC is established at
the Goldilocks number, testing each antigen concentration in thesame number of replicate wells. The number of PBMC needed forthis second experiment depends on the Goldilocks number, thenumber of replicate wells needed to obtain a total of minimally
300 SFU, and the concentration range of antigen being evaluated.
It can be calculated with high accuracy once the Goldilocks number
has been established. In the example provided in Fig. 4, as detailed
below, the number of PBMC needed to perform an inverted
ImmunoSpot® assay encompassing 4 concentrations of SARSCoV-2 Spike (receptor-binding domain, RBD) antigen probe,
each evaluated in 10 replicate wells with ~50 SFU/well (thusassessing the affinity distribution within ~500 IgG+ Bmem of thisspecificity) was 4 × 106 PBMC.
In Fig. 4, we present typical results obtained using the inverted
ImmunoSpot® assay to assess the affinity distribution of antigenspecific ASC present within a human PBMC sample using limiting
quantities of the antigen probe for their detection. In this case,cryopreserved PBMC collected from a healthy human donor 7 days
following a second COVID-19 mRNA vaccination were tested. In
the first experiment, the Goldilocks number for the SARS-CoV2 RBD-specific IgG+ ASC was determined for this PBMC sample(as described above) at 50 SFU/1 × 105 PBMC per well. In theinitial test, a saturating concentration of the RBD probe wasused and enabled the detection of all antigen-specific IgG+ ASC;

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Fig. 4 Measuring B cell affinity distribution in PBMC with inverted ImmunoSpot® assays using limiting
quantities of the antigen probe for detection. Cryopreserved PBMC collected from a healthy human donor
7 days following a second COVID-19 mRNA vaccination were tested. The PBMC were seeded into an inverted
assay at 1 × 105 PBMC per well, which in a previous experiment was determined to result in ~50 SARS-CoV2 Spike (RBD)-specific IgG+ spot-forming units (SFU), a number suitable for studying individual secretory
footprints. The His-tagged RBD probe was added to 10 replicate wells at the specified concentrations,followed by the detection of the His-tagged, plate-bound antigen. (a–d) Representative well images areshown for each RBD probe concentration. (e) Standard bar graph representation of cumulative SFU counts ateach probe concentration. (f) Composite bar representation of the same data with color code specifying spot
numbers detected at each probe concentration. (g) Intensity of spots detected at each RBD probe concentration was stratified into ten intensity groupings (denoted using grayscale) and the percentage of SFU residing ineach “bin” is reflected by the width of the corresponding section. (h) Flow cytometry standard (FCS)-type
intensity by size scatter plot analysis of the cumulative spots detected using 100 ng/mL or 12.5 ng/mL of the
RBD probe, respectively

low- and high-affinity alike. Thawing a second aliquot of thesecryopreserved cells, the PBMC were plated into 10 replicate wells
for each of the four RBD probe concentrations tested at the Goldilocks number. In Fig. 4a–d, representative well images are shownfor each RBD probe concentration. A reduction in SFU numbers isreadily seen visually as the RBD probe concentration decreases. The
corresponding SFU numbers are shown in a conventional bar
diagram in Fig. 4e; most of the expected cumulative ~500 SFU
were detected at 100 ng RBD/mL. Figure 4f shows these results in
a composite bar diagram format that is better suited for comparing
affinity distributions between different PBMC samples, e.g., tovisualize affinity maturation for a subject’s B cell response withrepeat immunizations, or for representing such data for cohorts.

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Figure 4 also illustrates that, next to the decrease in SFU, thefluorescence intensity of the individual secretory footprints changes
as the RBD probe concentration decreases. Footprints of highaffinity ASC continue to stain bright as the RBD probe concentration declines, but ASC whose threshold of binding capability isapproximated at the given probe concentration become fainter.
Such transitions can be graphically captured. In Fig. 4g, the intensity of spots detected at each RBD probe concentration is stratified
into ten intensity groupings, and the percentage of SFU residing ineach “bin” is reflected by the width of the corresponding section.
Flow cytometry standard (FCS)-type representation of ImmunoSpot® data also permits such further high-content data analysis.
Figure 4h shows an FCS-type scatter plot representation (fluorescence intensity vs. size) of spot morphologies, and the difference
seen between the highest and lowest RBD probe concentrations.
Inverted ImmunoSpot® assays are particularly well-suited formeasuring B cell affinity distributions when the frequency of antigen-specific ASC producing the Ig class/subclass to be captured on
the membrane (e.g., IgG) is relatively high within all ASC present
in the sample producing that particular Ig class/subclass (see Note19). This is often the case when studying plasmablasts that transiently circulate in the blood acutely (5–9 days) after initiation of a
B cell response (see Note 20). If the frequency of antigen-specific
ASC vs. total ASC is low in a given sample, direct ImmunoSpot®
assays offer an alternative approach for measuring the affinity distribution of Bmem-derived ASC.
1.8 B Cell Affinity
Distribution
Measurements in
PBMC with Direct
ImmunoSpot® Tests

Direct ImmunoSpot® assays inherently reveal information about
ASC affinity. Following basic rules of antigen–antibody binding,ASC that produce high-affinity antibodies will leave dense andsharp secretory footprints on membrane-bound antigens, while
ASC-producing antibodies with lower functional affinity for themembrane-associated antigen will form faint and diffuse spots[18]. Performing direct ImmunoSpot® assays under conditions
when the membrane is coated with graded antigen densities helps
to further enhance such affinity studies. Typical results for thesetypes of tests done on human PBMC are shown in Fig. 5, in thiscase using PBMC from a COVID-19 mRNA vaccine recipient. Likefor the inverted ImmunoSpot® assay, here too the cells were plated
into all replicate wells at an assay-specific, Goldilocks number(3 × 104 PBMC/well) needed to achieve 50 Spike-specific SFU/well. A direct ImmunoSpot® assay was performed in which, however, the Spike protein coating density was graded. In Fig. 5a–d,
representative well images are shown for each coating concentration of Spike protein. Here too, a reduction in SFU numbers is seenas the coating density was decreased. The corresponding SFU
numbers are shown in a conventional bar diagram in Fig. 5e, anda composite bar diagram format in Fig. 5f.

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Fig. 5 ImmunoSpot® enables the evaluation of ASC affinity through limiting antigen coating. Cryopreserved
PBMC collected from a healthy human following a second COVID-19 mRNA vaccination were tested. The
PBMC were seeded into the assay at 3 × 104 PBMC per well, which in a previous experiment was determined
to result in ~50 SARS-CoV-2 Spike-specific IgG+ spot-forming units (SFU), a number suitable for studying
individual secretory footprints. Assay wells were initially coated with anti-His capture antibody, followed byaffinity capture of Spike protein at 10, 5, 2.5, or 1 μg/mL, in 20 replicate wells, respectively. (a–d)
Representative well images are shown for each Spike coating concentration. (e) Standard bar graphrepresentation of cumulative SFU counts at each Spike coating concentration. (f) Composite bar representation of the same data with color code specifying spot numbers detected at each Spike coating concentration.
(g) Flow cytometry standard (FCS)-type intensity by size scatter plot analysis of the cumulative spots detected
in wells coated with either 10 or 2.5 μg/mL of Spike protein, respectively

In this chapter, we present data demonstrating that B cell
ImmunoSpot® assays are capable of distinguishing between
model ASC (murine B cell hybridomas) known to secrete mAb
with variable functional affinity for defined antigens. Further,through characterizing the B cell response elicited in COVID-19
mRNA vaccine recipients, we highlight the utility of antigen-specific, inverted, and direct ImmunoSpot® assays for characterizing
the functional affinity distribution of the antigen-specific B cells
present in PBMC. In the following Materials and Methods section,
we provide detailed protocols for performing such inverted anddirect ImmunoSpot® assays. Here we also refer to a chapter by Yao
et al. [21] in this volume that describes defining of Goldilocks
number in detail, and to a chapter by Karulin et al. [29] on highcontent image analysis of B cell-derived SFU. The underlying B cellbiology is elaborated in greater detail in a chapter by Lehmann et al.
[18], also in this volume.

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2

225

Materials

2.1 Thawing of
Cryopreserved PBMC

1. Class II biosafety cabinet (BSC)
2. Cryopreserved PBMC sample(s) (see Note 11).
3. 70% (v/v) ethanol (EtOH)
4. DNase-containing washing medium (pre-warmed to 37 °C)(see Note 21).
5. Complete B cell medium (BCM) (pre-warmed to 37 °C) (seeNote 22).

2.2 In Vitro
Polyclonal Stimulationof B Cells in PBMC
2.3 Antigen-Specific,
Single-Color Inverted
FluoroSpot Assay

1. B-Poly-S
2. 25 cm2 sterile culture flask
3. Humidified incubator set at 37 °C, 5% CO2
1. Commercially available, single-color human IgG Inverted(His) B cell kit (see Note 23).
2. His-tagged recombinant protein (see Note 24).
3. 190 proof (95%) EtOH
4. Cell culture-grade water.
5. Reagent reservoir(s) (sterile).
6. 0.05% Tween-PBS wash solution
7. 0.1 μm low-protein binding syringe filter
8. Plate washer.
9. Vacuum manifold.
10. ImmunoSpot® S6 Ultimate 4 LED Analyzer, or a suitable
instrument equipped with the appropriate detection channels,
running CTL’s ImmunoSpot® UV.

2.4 Antigen-SpecificFluoroSpot Assay(Affinity Capture
Coating)3

1. Commercially available, single-color human IgG affinity capture (His) B cell kit (see Note 25).
2. His-tagged recombinant protein (see Note 26).

Methods

3.1 Thawing of
Cryopreserved PBMC
(Sterile Conditions)

1. Place cryovial(s) into a 37 °C bead bath, or better, glass beadbath, for 8 min to thaw.
2. Remove cryovial(s) and wipe with 70% EtOH inside the BSC
before unscrewing the cap(s).
3. Using a sterile pipette, transfer contents of cryovial(s) into alabeled conical tube (if applicable, up to 5 vials of the same
donor’s cell material can be pooled in one conical tube).

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4. Rinse each of the cryovials with 1 mL of warm anti-aggregate
solution. Add the warm rinse solution to the conical tube
dropwise while swirling the tube to ensure its adequate mixing
with the cells in the 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.
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
ten-fold. 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, non-frigerated.
8. Decant supernatant and resuspend the cell pellet(s) using
pre-warmed B cell medium (BCM) to achieve a cell density of~2–5 × 106 cells/mL. You may estimate this number assuming
a typical recovery of 70–80% of the frozen PBMC.
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 a droplet oflive/dead cell counting dye. Pipet up and down three to fivetimes to mix 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
with a centrifuge, non-refrigerated, brake on.
14. Decant supernatant and gently resuspend the cell pellet(s) using
pre-warmed BCM at a cell density of ~2–4 × 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 vessels, add 50% vol of BCM containing 2X concentration of B-Poly-S.
2. Add the same volume of cell suspension at ~2–4 × 106 cells/
mL to achieve a final culture at ~1–2 × 106 cells/mL with 1X
potency of CTL’s B-Poly-S polyclonal stimulation reagent (seeNote 27).
3. Transfer culture vessels (flasks or plates) into a humidified
incubator set at 37 °C, 5% CO2 for 4–6 days (96–144 h).

Measuring Memory B-Cell Affinity

3.3 Antigen-Specific,
Single-Color HumanIgG Inverted
FluoroSpot Assay(Limiting Antigen
Probe) (See Notes 19,
28, and 29)

227

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
plate. Immediately after the addition of the 70% EtOH solution to the entire plate (or designated wells), add 180 μL/wellof PBS (see Note 30). Decant and wash wells again with180 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 (see Note31).
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 pre-warmed BCM to block the plate (≥1 h
at RT).
6. If using PBMC following polyclonal activation in vitro, collectthe cell suspension(s) and transfer it into labeled conicaltube(s). Wash culture vessel(s) interior to recover remaining
cells with sterile PBS and transfer to the corresponding conicaltube(s). Increase the volume to fill the conical tube with additional PBS and then centrifuge balanced tubes at 330 × g for
10 min with the centrifuge brake on. Alternatively, thaw previously cryopreserved PBMC as detailed above if prior in vitro
stimulation is not required to elicit antigen-specific ASC activity in the sample(s).
7. Decant supernatant and resuspend the cell pellet(s) using
pre-warmed BCM to achieve a cell density of approximately
2–5 × 106 cells/mL.
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 a droplet oflive/dead cell counting dye. Pipet up and down three to fivetimes to mix 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
PBS and centrifuge balanced tubes at 330 × g for 10 min
with centrifuge brake on (see Note 32).

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13. Decant supernatant and resuspend the cell pellet(s) using
pre-warmed BCM at the pre-determined cell density to achieve
the so-called “Goldilocks” cell input of 50 SFU/well (seeNotes 19, 28, and 29).
14. Decant the BCM used for blocking the assay plate and replace
it with 100 μL/well of pre-warmed BCM to block the plate.
15. Into a reagent reservoir, add cell suspension adjusted to adensity of 500 SFU/mL (see Note 14). Using a multichannel
pipet, transfer 100 μL of the cell suspension(s) into designatedwells of the assay plate. Gently tap the edges of the ImmunoSpot® assay plate to ensure equal distribution of the cell input.
16. Incubate cells in the assay plate for 16–18 h at 37 °C, 5% CO2(see Note 33).
17. After completion of the assay incubation period, remove theplate and decant cells. Wash the plate two times with PBS(200 μL/well), followed by two additional washing steps
with 0.05% Tween-PBS wash solution (see Note 34).
18. Prepare His-tagged antigen probe solution at limiting concentrations (see Note 35) according to kit protocol and passthrough a 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
the various His-tagged antigen probe solutions (containing
limiting/graded quantities of antigen probe) into designatedwells, and incubate 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 a 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 a 0.1 μm low-protein binding syringe filter to remove
any aggregates.
25. Wash plates(s) twice with distilled water.
26. Remove the protective underdrain and place the plate facedown on the vacuum manifold. Completely fill the backsideof the plate with distilled water and apply a vacuum to draw
water through the membrane (“back to front”) (see Note 36).

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27. Allow the plate to dry completely, protected from light (seeNote 37).
28. Scan and count plate(s) with a suitable analyzer equipped withthe appropriate detection channel (see Note 38).
3.4 Antigen-Specific(Affinity Capture
Coating) FluoroSpot
Assay (Limiting
Antigen-Coating
Concentrations)

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 (see Note 30). Decant and wash wells again with180 μL/well of PBS.
3. Decant the assay plate, replace underdrain, and immediately
add 80 μL/well of the anti-His affinity capture antibody solution into each well (or designated wells) of the low autofluorescence PVDF-membrane plate provided with the kit (seeNote 39).
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 B (provided with the kit) at pre-determined
limiting/graded concentrations (see Note 26).
6. Decant the assay plate and wash wells with 180 μL/well of PBS.
Immediately, add 80 μL/well of the corresponding His-tagged
protein coating solutions into the designated wells.
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 PBS. Next, decant the plate and add
150 μL/well of pre-warmed BCM to block the plate (≥1 h
at RT).
9. If using PBMC following polyclonal activation in vitro, collectthe cell suspension(s) and transfer it into labeled conical tube(s) (see Note 27). Wash culture vessel(s) interior with sterile
PBMC and also transfer into the corresponding conical tube(s). Increase volu vitrome to fill up tube(s) with additional PBS
and then centrifuge balanced tubes at 330× g for 10 min withcentrifuge brake on. Alternatively, follow the procedures
detailed above to obtain freshly isolated PBMC, or to thaw
PBMC that were previously cryopreserved, if prior in 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
pre-warmed BCM to achieve a cell density of approximately
2–5 × 106 cells/mL.

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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 a droplet oflive/dead cell counting dye. Pipet up and down three to fivetimes to mix 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
PBS and centrifuge balanced tubes at 330× g for 10 min withcentrifuge brake on (see Note 32).
16. Decant supernatant and resuspend the cell pellet(s) using
pre-warmed BCM at the pre-determined cell density to achieve
the so-called “Goldilocks” cell input of 50 SFU/well.
17. Decant the BCM used for blocking the assay plate and replace
it with 100 μL/well of pre-warmed BCM to block the plate.
18. Into a reagent reservoir, add cell suspension adjusted to adensity of 500 SFU/mL (see Note 18). Using a multichannel
pipet, transfer 100 μL of the cell suspension(s) into designatedwells of the assay plate. Gently tap the edges of the ImmunoSpot® assay plate to ensure equal distribution of the cell input.
19. Incubate cells in the assay plate for 16–18 h at 37 °C, 5% CO2(see Note 33).
20. After completion of the assay incubation period, remove theplate and decant cells. Wash the plate two times with PBS(200 μL/well), followed by two additional washing steps
with 0.05% Tween-PBS wash solution (see Note 34).
21. Prepare detection antibody solution by following kit protocol
and pass through a 0.1 μm low protein binding syringe filter toremove any aggregates.
22. Decant 0.05% Tween-PBS wash solution, add 80 μL/well ofdetection antibody solution into designated wells, and incubate for 2 h at RT (protected from light).
23. Wash plate(s) two times with 0.05% Tween-PBS wash solution.
24. Prepare a tertiary solution by following kit protocol and passthrough a 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.

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27. Remove the protective underdrain and place the plate facedown on the vacuum manifold. Completely fill the backsideof the plate with distilled water and apply a vacuum to draw
water through the membrane (“back to front”) (see Note 36).
28. Allow the plate to dry completely, protected from light (seeNote 37).
29. Scan and count plate(s) with a suitable analyzer equipped withthe appropriate detection channels (see Note 38).

4

Notes
1. The unit in which antibody affinity is measured is molarity (M).
It defines the concentration of soluble antibody required toattain 50% of the maximal antigen binding capacity, e.g., bysurface plasmon resonance (SPR) or biolayer interferometry(BLI).
2. There are 129 functional V (variable), 27 D (diversity), and 9 J(joining) gene segments available within the human IgH locus
located on chromosome 14 [21]. In concert with the large
number of V and J gene segments present within the Igκ(chromosome 2) and Igλ (chromosome 22) loci, respectively,
plus the addition and deletion of nucleotides at the junctional
borders of the recombined gene segments, and the pairing of
IgH and IgL proteins to generate a functional B cell receptor(BCR), it is estimated that up 1014 different BCR specificities
can be created through combinatorial diversity. As the adulthuman body has ~1012 lymphocytes, of which 1–10% in the
blood are B cells, one can estimate that when an antigen is firstencountered, there are very few naive B cells in the entire bodythat are endowed with a specific BCR.
3. An immune response requires that B and T cells encounter
antigens in a secondary lymphoid tissue, such as a draining
lymph node (LN) or the spleen. Naive B and T cells continuously recirculate among secondary lymphoid tissues, arriving
there via the bloodstream and entering via the “lymph node
homing receptors” they express. After searching the secondary
lymphoid tissue for the presence of “their” antigen, if such isabsent, they return to the blood via the afferent lymph vessels
to continue their surveillance of other secondary lymphoidtissues, as well. Antigens are transported to the regional LN(s) that drain these areas of the body. The antigens arrive in that
LN(s) either with the efferent lymph fluid or via dendritic cells
actively transporting them. In this way, the three essential
components of an efficient immune response (antigen, antigen-specific B cells, and antigen-specific T helper cells) are all

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brought together in the LN(s). The architecture of the LN isalso specialized in that it provides the ideal microenvironment
for the ensuing immune response to evolve.
4. Naive T cells undergoing a primary immune response in secondary lymphoid tissues are sensitive to the type of cytokine/
chemokine microenvironment created by cells of the innate
immune system, as the latter become activated by pattern
receptor-perception of the antigen. In this way, through different local microenvironments, Th1, Th2, Th17, and other typeof T cell lineages arise. The process of Ig class switch recombination, in turn, is dictated by the type of cytokines these“helper” T cells secrete in response to recognizing cognate
peptides presented by antigen-specific B cells. Engaging the“right” class/subclass of T and B cell responses is critical for
mounting an effective antigen clearance/control without inciting collateral immunopathology.
5. While still not fully elucidated, plasma cells require specificstimuli provided by stromal and other accessory cells for theirlongevity and long-term residence in specialized survival niches[13, 22].
6. The half-life of IgG1, IgG2, and IgG4 in humans is
21–28 days, and it is 7 days for IgG3 [23]. The half-life of
IgA and IgM is even shorter (3–7 days) [24, 25]. IgE has a halflife in serum of 2–3 days [26].
7. While antibody-secreting plasma cells are tissue-resident in thebone marrow or other specialized survival niches, memory B
cells (Bmem) recirculates among secondary lymphoid tissues,
just as naive B cells do (see also Note 3). Returning to thebloodstream via efferent lymph vessels, the Bmem are carried
with the bloodstream where they egress into remote secondary
lymphoid tissues and survey for their cognate antigen. By
sampling blood, one can therefore gain insights into the Bmem
compartment present in the body.
8. Defining the affinity distribution of, e.g., 300 antigen-specific
B cells in an individual by SPR (or BLI) would mean having toeither establish 300 antigen-specific B cell hybridoma clones,
or generate 300 paired IgH/IgL sequences and suitable expression constructs, to express and purify these 300 monoclonal
antibodies (mAb), and then to test the affinity of each of the
300 mAbs, one by one. Compare this effort and cost to the easewith which information on the affinity distribution of antigenspecific Bmem/ASC can be obtained via the ImmunoSpot®
approach introduced here.
9. To the choice of capture antibody: when plasmablast populations elicited acutely (days 5–9) following induction of a B cell
response are being studied, most of the spontaneouslyMeasuring Memory B-Cell Affinity

233

producing ASC in the blood will be antigen-specific. In such acase, the use of a pan Ig-specific (anti-kappa + anti-lambda lightchain) capture antibody is justified and enables assessment of allclasses and subclasses of antigen-specific antibodies, including
IgM. In this case, IgM+ secretory footprints are inferred tooriginate from antigen-specific B cells engaging in a primary
immune response. Furthermore, such inverted assays performed using pan Ig-specific capture conditions can be multiplexed with detection reagents which enable resolving the Ig
class/subclass usage of distinct antigen-specific secretory
footprints.
10. When inverted ImmunoSpot® assays are performed within vitro polyclonally stimulated PBMC, however, third-party
IgA and IgM-producing B cells are also induced in high numbers and readily compete for “real estate” on membranes
coated with pan Ig capture antibodies. Moreover, the presenceof IgM-derived secretory footprints generated by naive B cells
possessing broad or highly cross-reactive specificities also hinders the specificity of such inverted assays. To overcome theseobstacles, we recommend the usage of Ig class-specific capture
antibodies; such as the anti-human IgG capture antibody
shown in Fig. 1. In this way, only in vivo class-switched B
memory cells are being interrogated in the ImmunoSpot® test.
11. Peripheral blood mononuclear cells (PBMC) can be cryopreserved without loss of spontaneous, or polyclonal stimulationinduced ASC activity ([27] and Becza et al., manuscript in
preparation). This permits “direct ex vivo” batch testing ofsamples irrespective of the time-point of their actual collection.
By freezing the cells in aliquots, assay results can be reproducedwith high accuracy [17], and/or follow-up experiments can be
performed building on the previous test result that establishes
the “Goldilocks number” of antigen-specific ASC in a given
PBMC sample, as described in Note 14. It is important forplanning cryopreservation of PBMC to know that any number
of PBMC can be frozen per vial between 1 and 10 million
PBMC, in each case recovering at thawing about 90% viable
and fully functional B cells, and that after 5 days of in vitro
polyclonal B cell stimulation about 50% of these cells will berecovered as viable cells for testing (Becza et al., manuscript in
preparation).
12. In the inverted assay, as shown in Fig. 1a, the capture ofantibodies produced by ASCs occurs at a fixed high affinity,that of the anti-IgG capture antibody for the ASC-derived IgG.
Only the net amount of IgG produced per ASC defines the sizeand density of the resulting secretory footprint. The affinity of
the antibody captured in the footprint will be revealed, however, when the antigen is added at various concentrations.

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13. The B cells survive the ImmunoSpot® assay unharmed and canpotentially be re-used in subsequent functional assays, forcloning, or analysis via means of molecular biology.
14. In the initial test, it is suggested to plate PBMC (or othersingle-cell suspensions) in serial dilution over a wide range ofcell numbers per well because the frequencies of antigen-specific ASC can span orders of magnitudes between individuals[17] and ASC-producing different classes and subclasses ofantigen-specific (and total) Ig also spans a wide range within
an individual, and in the human population. In this way, notonly the frequency of ASC can be reliably established by linear
regression analysis [28] within the entire cell material tested
but the Goldilocks number for secretory footprints can be
defined as the maximal number of cells that can be plated
while still being able to discern clearly individual secretory
footprints. This number for an optimized, antigen-specific(SARS-CoV-2 Spike RBD) inverted assay is 50 (maximally100) SFU/well using anti-IgG specific capture antibodies
and Bmem-derived ASC following in vitro polyclonal stimulation of PBMC [17]. Once the Goldilocks number has been
established, in a second experiment, the cell number(s) can bechosen accordingly for creating replicates containing ≥300
individual ASC-derived footprints per antigen concentration
for studying the functional affinity distribution.
15. Due to spacing limitations precluding their inclusion, two
additional supplementary figures for this chapter can befound at https://immunospot.com/supplfigs-affinity
16. Absorption of biomolecules to the hydrophobic PVDF membrane can occur with variable efficiency and the coating concentration required for reliable detection of antigen-specific
ASC may be prohibitive; and even when raising the coating
antigen concentration “indefinitely,” no, or no satisfactory
antigen coating may be achieved. Affinity coating readily circumvents this problem: it is illustrated in Fig. 4 of the chapter
by Lehmann et al. in this volume [29].
17. A similar linear regression approach can also be used for defining the Goldilocks number of in vivo-differentiated ASC suchas circulating plasmablasts or bone marrow/tissue-resident
plasma cells.
18. To establish the frequency, and Ig class or IgG subclass usage,of Bmem-derived ASC for one antigen using a direct ImmunoSpot® assay approach, a typical serial dilution experimentrequires (with a safety margin) 2 × 106 PBMC that can beobtained from 2 mL of blood, as follows: one starts the
PBMC dilution series with 2 × 105 PBMC per well to permit
the detection of relatively low frequency Bmem-derived ASC as
well, and progresses with a 1 + 1 (two-fold) serial dilution ofMeasuring Memory B-Cell Affinity

235

the cells. Importantly, frequency assessments done with single
wells per cell dilution provide frequency assessments withessentially the same accuracy as serial dilutions done with 4 replicate wells (see Yao et al. in this volume [21]). Inclusion ofnegative control wells seeded with 2 × 105 and 5 × 104 PBMC
is also recommended but not required; cell material permitting. Thus, ~6.5 × 105 PBMC will be needed on day 5 fromin vitro polyclonal stimulation cultures, for which 1.3 million(for safety margin 2 × 106) freshly isolated PBMC need to be
cryopreserved accommodating the 50% cell recovery afterin vitro stimulation. The cell numbers recovered on day
5 from such in vitro cultures even permit to establish thefrequency of all IgA-, IgG- and IgM-producing B cells in thatsample; for such measurements, the serial dilution typicallystarts with an input of 2 × 104 cells per well. Note the efficiency
of PBMC utilization for such B cell ImmunoSpot® assays.
19. Owing to polyclonal stimulation of Bmem to trigger their terminal differentiation, a large majority of IgG+ ASC will not be
antigen-specific yet will compete for “real estate” on the lawnof anti-IgG capture reagent used for coating. Consequently,
inverted assays aimed at studying lower frequency ASC specificities are directly limited by the maximal number of total IgG+
ASC that can be input into a single well while still maintaining
the ability to resolve individual antigen-specific secretory
footprints.
20. The plasmablasts that can be detected circulating in the blood
5–9 days after onset of a B cell response are trafficking from thedraining lymph nodes to the bone marrow where they may
become resident as plasma cells.
21. Thawing of cryopreserved cells causes a fraction of the cells(up to 20%) 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.
22. 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.
23. 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 an anti-human IgG-specific capture
antibody, anti-His detection reagents, diluent buffers, low
autofluorescence PVDF-membrane plates, and polyclonal
B cell activator (B-Poly-S).

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24. 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. Likewise, prior optimization of limiting
antigen probe concentrations used to resolve ASC with differential functional affinity is highly recommended.
25. Kit is suited for detecting either antigen-specific antibodysecreting cells (ASC) that differentiated in vivo, or antigenspecific Bmem that have been polyclonally stimulated in vitro
to promote their transition to ASC. Each kit contains anti-His
capture antibodies, detection reagents, diluent buffers, low
autofluorescence PVDF-membrane plates, and polyclonal
B cell activator (B-Poly-S).
26. For most His-tagged proteins, high-density coating of theassay membrane can be achieved via affinity capture of a
10 μg/mL solution. Lower protein coating concentrations
reduce the density of membrane-associated antigens and concomitantly increase the minimal functional affinity of an antigen-specific ASC to generate a detectable secretory footprint.
27. The volume of in vitro stimulation cultures can be scaled up or
down but we recommend keeping the cell density of PBMC at~1–2 × 106 cells/mL. If larger numbers of in vitro stimulated
PBMC are required for downstream ImmunoSpot® assays, two
or more 25 cm2 flasks can be set up per donor or larger 75 cm2
culture flasks can be used with a final volume of 20–25 mL.
Likewise, smaller in vitro stimulation cultures can be initiatedin 24-well plates with a final volume of 2 mL. Be sure to fill
empty wells in tissue culture plates with sterile PBS to avoid
dehydration of cell cultures.
28. Prior to performing an inverted ImmunoSpot® assay using
limiting quantities of antigen detection probe, it is recommended to first determine the Goldilocks cell input to achieve~50 SFU/well using an aliquot of cryopreserved cell material(see also Note 11).
29. In such instances when the population of antigen-specific ASC
is low among all ASC, we recommend increasing the numberof replicate wells and seeding at lower cell inputs. Moreover, toconserve on cell material required, increasing the fold dilutionof the antigen probe and/or testing only at pre-determined
concentrations are both valid options.
30. Activation of the PVDF membrane with 70% EtOH is instantaneous and can be seen visually as a graying of the membrane.
It is important to be sure that the EtOH solution has spread
across the entire membrane before adding the first wash of
PBS. If needed, tapping the plate can promote contact of theMeasuring Memory B-Cell Affinity

237

EtOH solution with the PVDF membrane. We recommend
only pre-wetting one plate at a time with 70% EtOH to ensurethat the contact time is ≤1 min; longer contact times may
promote leaking of the membrane and result in suboptimal
assay performance.
31. If the entire plate will not be coated with the anti-human IgG
capture antibody solution, the remainder of the EtOH pre-wet
wells should receive 80 μL/well of PBS.
32. Contaminating antibody in the cell suspension(s) is efficiently
captured by the anti-Ig capture antibody and may also result inelevated membrane staining that interferes with accurate enumeration of individual antigen-specific ASC in inverted ImmunoSpot® assays.
33. Shorter B cell ImmunoSpot® assay incubation times are suggested if using an enzymatic-based detection approach to avoid
merging of spots and/or elevated membrane backgroundstaining.
34. 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.
35. Antigen probe concentrations can be generated through serial
dilution (two-fold, three-fold, or five-fold) or by directly adding fixed concentrations of antigen probe into the diluent
provided with the ImmunoSpot® assay kit.
36. Optimal removal of fibers and other debris, along with the
reduction of “hot spots” in the center of the assay wells, isachieved through performing the “back to front” water filtration technique.
37. 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 them
face down 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.
38. The chapter by Karulin et al. in this volume [28] 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.

238

Noémi Becza et al.

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, in particular
acknowledging Dr. Alexey Karulin’s long-term contributions tothis field. We also thank Dr. Graham Pawelec and Diana Roen for
carefully proofreading the manuscript and providing constructive
feedback. Lastly, we thank Gregory Kovacs for his support in thegeneration 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®. N.B., Z.L., J.C., and G.A.K. are employees of CTL. The
data X.G. contributed to this chapter (Fig. 2) were generated
during his employment at CTL.

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