Chapter 15
Four-Color ImmunoSpot® Assays Requiring Only 1–3 mL
of Blood Permit Precise Frequency Measurementsof Antigen-Specific B Cells-Secreting Immunoglobulinsof All Four Classes and Subclasses
Lingling Yao, Noémi Becza, Andrea Maul-Pavicic, Jack Chepke,
Greg A. Kirchenbaum, and Paul V. Lehmann
Abstract
The B lymphocyte response can encompass four immunoglobulin (Ig) classes and four IgG subclasses, eachcontributing fundamentally different effector functions. Production of the appropriate Ig class/subclass iscritical for both successful host defense and avoidance of immunopathology. The assessment of an antigenspecific B cell response, including its magnitude and Ig class/subclass composition, is most often confinedto the antibodies present in serum and other biological fluids and neglects monitoring of the memory B cell
(Bmem) compartment capable of mounting a faster and more efficient antibody response following antigen
reencounter. Here, we describe how the frequency and Ig class and IgG subclass use of an antigen-specific
Bmem repertoire can be determined with relatively little labor and cost, requiring only 8 × 105 freshly
isolated peripheral blood mononuclear cells (PBMC), or if additional cryopreservation and polyclonal
stimulation is necessary, 3 × 106 PBMC per antigen. To experimentally validate such cell saving assays,
we have documented that frequency measurements of antibody-secreting cells (ASC) yield results indistinguishable from those of enzymatic (ELISPOT) or fluorescent (FluoroSpot) versions of the ImmunoSpot®
assay, including when the latter are detected in alternative fluorescent channels. Moreover, we have shownthat frequency calculations that are based on linear regression analysis of serial PBMC dilutions using asingle well per dilution step are as accurate as those performed using replicate wells. Collectively, our data
highlight the capacity of multiplexed B cell FluoroSpot assays in conjunction with serial dilutions tosignificantly reduce the PBMC requirement for detailed assessment of antigen-specific B cells. The protocols presented here allow GLP-compliant high-throughput measurements which should help to introduce
high-dimensional Bmem characterization into the standard immune monitoring repertoire.
Key words ELISPOT, FluoroSpot, B cells, Immune monitoring, Immune memory, Ig class, IgG
subclass, High throughput, Antibody-secreting 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_15, © The Author(s) 2024

251

252

1

Lingling Yao et al.

Introduction
One might ask why bother with detecting antigen-specific memory
B cells (Bmem), fragile live cells that need to be processed within ashort time window after they have been collected from the bodywhen simple serum antibody measurements provide the soughtafter information? The answer is simple: Bmem measurements canprovide insights into immune responsiveness that serum antibodies
cannot. Because another chapter in this volume [1] is dedicated tothis issue, here we will just touch on the major points. Antigenspecific plasma cells (PC) and Bmem both arise during an immune
response triggered by antigen encounter, but the generation ofthese two daughter cell lineages follows different fate-decision
pathways. Precursors of both cell types, germinal center B cells(GCB), undergo somatic hypermutations (SHM) that result in
the generation of subclones with slightly modified B cell antigen
receptors (BCR). From this repertoire of daughter cells, subclones
that have an increased affinity for the antigen are positively selected
to undergo further rounds of proliferation and SHM, and eventually differentiate into PC. Contrary to the previously held notion,
PC are not necessarily long-lived (see Note 1) and neither are the Ig
molecules they secrete (see Note 2). In contrast, GCB progeny
endowed with lower affinity BCR for the antigen can still join thelong-lived Bmem compartment. Hence, PC and Bmem fulfill different roles in maintaining host immune defense.
The antibodies produced by PC constitute the first wall of
acquired humoral immune defense [2]. They provide immediate
protection by preventing the re-entry of the antigen, or, if it enters,by neutralizing it and/or facilitating its elimination by phagocytes
via immune complex formation (precipitation), opsonization, andcomplement fixation. As evidenced during the recent COVID-19
pandemic, and previously with seasonal influenza, the first wall ofadaptive humoral defense may fail to prevent (re)-infection whenantibody titers drop below protective levels, or upon emergence ofviral escape mutants capable of evading the neutralizing activity ofantibodies elicited by the original (homotype) virus strain. In suchcases, Bmem provide the second wall of adaptive humoral hostdefense [2]. Owing to their increased frequencies compared toantigen-specific naive B cells, and having already switched fromIgM to the expression of specialized Ig classes and subclasses (seeNote 3), Bmem not only mediate a faster and more efficient “secondary” antibody response against the same (homotypic) virus but
also against antigenically-related viral escape mutants (heterotypes).
This is because, within the antigen-specific B cell repertoire that wasclonally expanded by the homotypic virus during the primary
response, there will be Bmem that joined the memory compartment
with mutated BCR that have affinity for the heterotype as well.

Monitoring Memory B Cells

253

Because such cross-reactive Bmem occur in increased frequencies
compared to naive B cells and have already undergone Ig classswitching, this population is poised to engage in a quasi-secondary
B cell response if infection with a variant virus occurs.
From the above, it follows that measurements of serum antibodies provide insights only about the fading first wall of immune
protection. Measurements of Bmem, in contrast, provide insights
into the cellular basis of long-term immunity. Through measuring
the frequency of antigen-specific Bmem within all peripheral bloodmononuclear cells (PBMC) (see Note 4), the magnitude of theexisting memory compartment within an individual can be directly
quantified. Such information sheds light on the vigor of futuresecondary antibody responses upon antigen encounter. Moreover,by establishing the Ig class/subclass utilization of the Bmem compartment, one can also predict the type of antibody that will be
produced upon antigen encounter (see Note 5).
There are few techniques capable of detecting rare antigenspecific Bmem while also providing information regarding theirrelative abundance, Ig class/subclass usage, and functional affinity(see Note 6). B cell ImmunoSpot® assays are ideally suited for thispurpose as they enable the detection of Ig molecules secreted byindividual antibody-secreting cells (ASC). While resting Bmem do
not secrete antibodies, such cells can readily be differentiated into
ASC following a simple in vitro stimulation protocol (see Note 7).
The ImmunoSpot® assay principle for detecting ASC, irrespectiveof their antigen specificity, is described in Fig. 1a, and its variant for
detecting antigen-specific ASC in Fig. 1b.
In this chapter, we share our expertise on how to best establish
the frequency of antigen-specific, Bmem-derived ASC in human
PBMC, including their Ig class and subclass usage, and how to do
so with the least labor, and the lowest number of PBMC possible(see Note 4). The type of testing described here is also essential for
determining the so-called “Goldilocks” number of PBMC to beseeded into subsequent ImmunoSpot® assays aimed at evaluating
the affinity distribution present among antigen-specific ASC (seethe chapter by Bezca et al. in this volume, [3]), or the crossreactivity of homotype antigen-primed ASC with heterotypic antigens (see the chapter by Lehmann et al., also in this volume [1]).
Owing to the requirement to detect individual ASC-derived
secretory footprints to accurately determine the frequency of antigen-specific B cells, an ImmunoSpot® assay-related challenge toovercome is that Bmem-derived ASC producing different classes
and subclasses of Ig span orders of magnitude [4]. Importantly,
this problem is readily overcome by seeding PBMC (or othersingle-cell suspensions) in serial dilutions. For establishing thefrequencies of antigen-specific ASC following in vitro polyclonal
stimulation of PBMC, we recommend starting at 2 × 105 PBMC
per well and progressing in a 1 + 1 (two-fold dilution series) down

254

Lingling Yao et al.

Fig. 1 Schematic representation of (a) the pan (total), or (b) the antigen-specific, direct ImmunoSpot® assays(the latter, using the affinity capture coating variant). For (a), the PVDF membrane on the bottom of a 96-wellplate is densely coated with a pan anti-Ig capture antibody that will bind the ASC-secreted Ig(G) with highaffinity irrespective of the ASC’s antigen specificity. In (b), the membrane is coated first with an anti-affinity
tag-specific antibody (in this example anti-His) that captures the (His)-tagged antigen with high affinity. In thisway, a dense coating of the membrane with the antigen is accomplished. As the next step in both assay
variants, the PBMC containing the ASC are plated. In (a), an ASC-secreted Ig(G) antibody is captured aroundeach ASC that is secreting Ig(G), and results in the formation of individual secretory footprints. In (b), theantibody produced by antigen-specific ASC is captured only on the lawn of the antigen. The subsequent steps
are similar for both assay variants. After removal of the cells, the membrane-associated antibody is visualizedusing biotinylated anti-human Ig class/subclass-specific detection antibody reagents that subsequently are
revealed by the addition of a fluorescently conjugated streptavidin (FluoroSpot, as shown) or via an enzymatic
reaction (ELISPOT, not shown). Counting the spot-forming units (SFU) per well reveals the number of (a) total Ig(G) or (b) antigen-specific, Ig(G)-producing ASC within the PBMC plated. The spot morphologies in (b) 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 [3]the 96-well plate to generate 8 additional data points. Similarly, forestablishing the frequency of all ASC-producing IgM, IgG, or IgA,
irrespective of their antigen-specificity, we recommend starting at
2 × 104 and performing a similar two-fold dilution series down the
96-well plate for 8 points of cell titration (see Note 10).
Four-color ImmunoSpot® assays are suited to generate maximal data while saving on cells (see Note 11). Figures 2 and 3 showthat such fluorescence-based tests detect the secretory footprints ofindividual Bmem with the same efficacy as single-color enzymatic
assays. As can be seen for higher cell numbers in Fig. 3, the confluence of secretory footprints and the resulting ELISA effect interferewith the accurate recognition and counting of individual spotforming units (SFU). At SFU counts lower than 100 SFU perwell, however, a close to perfect linear relationship exists between
the number of PBMC seeded per well and the number of SFU perwell, from which, by linear regression, the frequency of SFU within
all PBMC plated can be accurately extrapolated. A fully automatedsoftware module built into the ImmunoSpot® software permits theidentification of the linear range of SFU counts, the calculation ofmeans of replicates, and the frequency extrapolation (see the chapter by Karulin et al. on this issue in this volume [5]).

Monitoring Memory B Cells

255

Fig. 2 Single-color ELISPOT and FluoroSpot assays have similar sensitivities for detecting ASC-derived
secretory footprints. Peripheral blood mononuclear cells (PBMC) were polyclonally stimulated with B-Poly-S(or B-Poly-SE for detection of IgE+ ASC in panel D) for 5 days in vitro (see Notes 7 and 10), washed, andseeded into single-color ELISPOT or FluoroSpot assays in parallel, detecting the immunoglobulin (Ig) class/subclass specified in panels A–J, respectively; the type of assay shown in Fig. 1a was performed. Since
antibody-secreting cells (ASC) producing the different Ig classes or subclasses occur in vastly different
frequencies in PBMC following in vitro polyclonal stimulation (as detailed in the text), the assays were
performed by plating PBMC in a 1 + 1 serial dilution series, starting at 2 × 105, in 4 replicates per cell
dilution. Images of representative wells are shown for the dilution step in which the individual secretory
footprints were clearly discernable; between 50 and 70 SFU/well. The cell inputs for panels A-J were:781, 391, 3125, 200,000, 781, 6250, 6250, 25,000, 3125, and 6250 per well, respectively. The number ofsecretory footprints (spot forming units, SFU) measured using ImmunoSpot® software is reported in the left
upper corner of the corresponding well images, and means ± SD of the four replicate wells are specified undereach pair of wells. Statistical analysis (unpaired Student’s t-test) indicated no significant difference (“ns”)
between the ELISPOT and corresponding FluoroSpot counts between the replicates for each condition. The
presented results are representative of 4 independent experiments using different PBMC donors, leading to
the same conclusion

Our in-depth studies of such regression analysis-based frequency calculations showed that performing serial dilution experiments involving replicates offers only a negligible advantage inprecision over performing the assay with single wells (Fig. 4 andBecza et al., manuscript in preparation). Doing such serial dilutionbased frequency measurements in four-color reduces the required
number of PBMC by four-fold compared to performing 4 independent single-color assays, and doing so using a single well serial
dilution approach permits another four-fold reduction in cell material compared to testing each cell input in quadruplicate (as wasdone in the data presented in Figs. 2 and 3).
Figure 5 depicts the results obtained from a typical serial
dilution-based four-color ImmunoSpot® test detecting all four
antibody classes (IgM, IgG, IgA, and IgE) of SARS-CoV-2 Spikespecific ASC in a convalescent individual with PCR-verified infection. Similarly, the IgG subclass usage of Spike-specific ASC wasdetermined in parallel using an IgG1/IgG2/IgG3/IgG4 fourcolor assay. The abundance of all ASC in the test sample producing
each Ig class or IgG subclass was also established to permit

256

Lingling Yao et al.

Fig. 3 Single-color ELISPOT and multi-color FluoroSpot assays have similar sensitivities for detecting
ASC-derived secretory footprints. Peripheral blood mononuclear cells (PBMC) were polyclonally stimulatedwith B-Poly-S for 5 days in vitro, washed, and then seeded into single-color enzymatic (SCE) ELISPOT, singlecolor FluoroSpot (SCF), three-color FluoroSpot (TCF), or four- (quadruple) color FluoroSpot (QCF) assays inparallel, diluting the cell inputs two-fold per well, detecting pan IgG+ (a, b) or IgM+ ASC (c, d) in four replicate
wells, irrespective of antigen specificity; the type of assay illustrated in Fig. 1a was performed. In panels (a)and (c), representative images (one of the four replicates tested) depicting secretory footprints visualized using
anti-IgG or anti-IgM detection reagents are shown, respectively, with the corresponding cell input specified.
ELISA effects are evident at high cell inputs. In panels (b) and (d), the spot-forming unit (SFU) counts for thefour replicate wells for each condition are shown as means ± SD (y-axis) at the corresponding cell inputs(x-axis). The detection modalities are color-coded, as specified, including the matching trend lines calculated
through linear regression analysis also denoting R2 values

calculation of the frequency of antigen-specific ASC among all
ASC, for each Ig class and subclass (see Notes 12 and 13). As
seen for this individual, and consistent with the majority of convalescent COVID-19 donors we tested thus far (Kirchenbaum,
unpublished observation), the SARS-CoV-2 Spike-specific Bmem
primarily secrete IgG, and IgG1 in particular. Notably, although
our IgA class-specific detection system works perfectly well for thedetection of total IgA+ ASC activity (see Fig. 2c), very few Spikespecific IgA+ Bmem-derived ASC were detectable in this individual.
Moreover, a small population of Spike-specific IgG3+ Bmem-derived
ASC was also detectable in this donor. Importantly, for the precise
enumeration of the latter very rare antigen-specific ASC, higher cell

Monitoring Memory B Cells

257

Fig. 4 Linear regression analysis performed using single wells in serial dilution experiments yields similar
accuracy for determining the frequency of ASC compared to that calculated from the mean of 4 replicate wells
at each cell input. Peripheral blood mononuclear cells (PBMC) were polyclonally stimulated with B-Poly-S for
5 days in vitro, washed, and then serially diluted two-fold, with four replicate wells for each cell input, in afour-color FluoroSpot assay to determine the frequency of antibody-secreting cells (ASC) producing IgA (a),IgM (b), or IgG (c). The type of assay shown in Fig. 1a was performed, detecting ASC irrespective of theirantigen specificity. Red symbols denote means ± SD of SFU counts in the four replicate wells, at each of thecell inputs, respectively; the solid red trend line was calculated by linear regression analysis of these means
with the R2 value specified. The respective frequencies as calculated by extrapolating the linear regression
line to a 106 PBMC input are given in parentheses. The color-coded “Cell titration 1-4” data with thecorresponding dashed trend lines, R2 values, and frequencies were obtained by independent single-wellanalysis of the four replicates. The results are representative of 5 independent experiments using different
PBMC donors leading to the same conclusion

inputs would be required, inputs that would be too high for determining the frequency of antigen-specific ASC producing
IgG/IgG1 (highlighting the importance of testing samples acrossmultiple cell inputs through serial dilutions and the value of multicolor analysis). Of note, the large number of IgM+ SFU detected in
Spike antigen-coated wells does not appear to be “specific” sincecomparable numbers of SFU were also present in negative control
wells. Such IgM+ SFU likely originate from naive B cells possessing
broadly reactive BCR specificities, and which differentiated into
ASC following in vitro polyclonal stimulation. This lack of “specificity” exhibited by IgM+ ASC following polyclonal stimulation ofhuman PBMC has also been reported previously [6] and reiterates
the importance of including negative controls in such B cell ImmunoSpot® assays. In contrast, while IgG+ Spike- or NCAP-antigenspecific ASC were absent in all PBMC collected in the pre-COVID
era, abundant such IgG+ ASC were detected in individuals with
PCR-verified SARS-CoV-2 infection [4]. Therefore, unlike IgM,
detecting antigen-specific IgG (and IgG subclass)-producing ASC
in PBMC following short-term in vitro polyclonal stimulation signifies in vivo primed and class-switched memory B cells. Antigenspecific, IgM+ ASC can be detected, however, when PBMC are
studied acutely (5–9 days) after induction of a primary B cell
response.
Performing the above test required 8 × 105 PBMC to be seeded
into the antigen-specific assay, plus we added an optional pan Ig
class/subclass assay requiring an additional 8 × 104 PBMC (see258Lingling Yao et al.

Fig. 5 Four-color ImmunoSpot® assays permit assessment of SARS-CoV-2 Spike-specific ASC frequency, plus
the frequency of all ASC producing different Ig classes and IgG subclasses using 8.8 × 105 PBMC (see Note 4).
Cryopreserved peripheral blood mononuclear cells (PBMC) from a convalescent donor with PCR-verified
SARS-CoV-2 infection were polyclonally stimulated with B-Poly-S for 5 days in vitro, washed, and thenevaluated in four-color ImmunoSpot® assays. (a) Recommended plate layout for determining ASC frequencies
for each Ig class and IgG subclass using a serial dilution approach: columns 1 and 2 measure pan (total) Ig
class and subclass usage are illustrated in Fig. 1a; columns 3 and 4 for measuring the antigen-specific ASC.
(b) Spike-specific SFU counts were measured using the four-color Ig class detection system over the entire
PBMC range plated, and (d) for the IgG subclass system with Ig class/subclass specified by color. Notedeviation from linearity at high cell inputs/SFU counts. (c) Trend line for Spike-specific IgG+ ASC calculated bylinear regression analysis of the wells containing SFU < 100/well with the R2 value; in parenthesis theextrapolated frequency per 106 PBMC. (d) The results for IgG1+ ASC are represented in (e)Fig. 5a and Note 4). As PBMC can be cryopreserved without lossof B cell functionality [7], samples can be run in batches instead of
testing them one by one as soon as the blood is drawn (see Note13). During freeze-thawing up to 30% of the cells may be lost, but
the functionality of the recovered B cells will be unaltered compared to freshly isolated PBMC ([7] and Becza et al., manuscript in
preparation). If an additional polyclonal stimulation is needed prior
to the actual ImmunoSpot® test to convert resting Bmem into ASC,approximately 50% of the frozen PBMC will be recovered afterthawing and performing the 5 day in vitro polyclonal stimulation
protocol (N. Becza, manuscript in preparation). Therefore, only
8.8 × 105 PBMC are needed for the type of test shown in Fig. 5,
and allowing for a safety margin in cell recovery, we suggest that
2–3 × 106 PBMC should be cryopreserved per aliquot to perform
such a test. Furthermore, we recommend freezing several aliquotsof PBMC (or other single-cell suspensions) to permit subsequent
tests using higher cell inputs and/or additional replicates in scenarios where antigen-specific ASC frequencies are very low (see Note

Monitoring Memory B Cells

259

15). Furthermore, the availability of additional aliquots of cellmaterial permits further in-depth characterization of affinity distributions or heterotypic cross-reactivity within the Bmem-derived
ASC repertoire (see Notes 16 and 17) and references [1, 3].
In the following, we provide detailed protocols for cryopreservation of PBMC to maintain their full functionality, subsequentpolyclonal stimulation of these PBMC to differentiate resting Bmem
into ASC, and four-color ImmunoSpot® assays for defining the Ig
class and IgG subclass usage of antigen-specific ASC.

2

Materials

2.1 Isolation and
Cryopreservation of
PBMC from Whole
Blood

1. Class II biosafety cabinet (BSC)
2. Green vacutainer tubes containing sodium heparin
3. Lymphoprep™
4. 15 or 50 mL conical tubes
5. Centrifuge capable of spinning tubes at 800 × g (temperature
set to 25 °C)
6. Sterile transfer pipette
7. Ca2+, Mg2+-free phosphate-buffered saline (PBS), pH 7.2(room temperature)
8. Parafilm
9. CTL-LDC™ counting kit
10. CTL-Cryo™ ABC media kit
11. ImmunoSpot® S6 Ultimate 4 LED analyzer, or a suitable
instrument equipped with the appropriate detection channels,
running CTL’s live/dead cell counting suite software
12. DMSO
13. Bead bath (set to 37 °C)
14. 1.8 mL Cryovials (with internal thread and silicone
washer seal)
15. Mr. Frosty™ freezing container, or controlled rate freezer.
16. Isopropyl alcohol
17. -80 °C freezer
18. Liquid nitrogen tank

2.2 Thawing of
Cryopreserved PBMC

1. Cryopreserved PBMC sample(s) (see Note 14)
2. 70% EtOH
3. DNase-containing washing medium (pre-warmed to 37 °C)(see Note 18)
4. Complete B cell medium (BCM) (pre-warmed to 37 °C) (seeNote 19)

260

Lingling Yao et al.

2.3 In Vitro Polyclonal Stimulation of B
Cells in PBMC
2.4 Single-Color
ELISPOT Assay

1. B-Poly-S
2. Tissue culture plate (48 or 24-well) 25 cm2 sterile culture flask
3. Humidified incubator set at 37 °C, 5% CO2
1. Commercially available, single-color human Ig class (IgA, IgE,
IgG, or IgM) or subclass (IgA1, IgA2, IgG1, IgG2, IgG3, or
IgG4) ELISPOT kit (see Note 20)
2. 190 proof (95%) EtOH
3. Cell culture-grade water
4. 96-well, round bottom dilution plate
5. 0.05% Tween-PBS wash solution
6. 0.1 μm low-protein binding syringe filter
7. Plate washer
8. ImmunoSpot® S6 Ultimate 4 LED analyzer, or a suitable
instrument equipped with the appropriate detection channels,
running CTL’s ImmunoSpot® UV

2.5 Single-, Three- or
Four-Color FluoroSpot
AssaysCommercially available, single-color human Ig class (IgA, IgE,
IgG, or IgM) or subclass (IgA1, IgA2, IgG1, IgG2, IgG3, or
IgG4) FluoroSpot kit (see Note 21).
1. Commercially available, three-color human Ig class (IgA, IgG,
and IgM) FluoroSpot kit (see Note 21)
2. Commercially available, four-color human Ig class (IgA, IgE,
IgG, and IgM) FluoroSpot kit (see Note 21)
3. Vacuum manifold

2.6 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 Note22)
2. Commercially available, four-color human IgG subclass affinity
capture (His) FluoroSpot kit (see Note 22)
3. The His-tagged recombinant protein (see Notes 23 and 24)

3

Methods

3.1 Isolation and
Cryopreservation of
PBMC from Whole
Blood (Sterile
Conditions)

1. Obtain blood samples according to IRB-approved protocol.
Keep blood at ambient temperature, do not refrigerate.
2. Keeping donors’ material separate, inside a class II BSC, pooleach donor’s blood into labeled conical tubes. Rinse eachvacutainer tube with PBS at ambient temperature and combine
it with whole blood.

Monitoring Memory B Cells

261

3. Measure blood volume and dilute 1:1 with PBS. If the volumeof whole blood to be processed is ≥20 mL, transfer half of thevolume into another labeled 50 mL tube prior to diluting
with PBS.
4. Layer diluted blood at ambient temperature slowly over Lymphoprep™, also at ambient temperature taking care not to
disrupt the interface.
5. Centrifuge balanced tubes at 800 × g for 20 min with thecentrifuge brake off, non-refrigerated.
6. Identify the buffy coat at the interface between Lymphoprep™
and diluted plasma layers. Carefully remove the cells at thisinterface and transfer them to a fresh conical tube (see Note25).
7. Wash the harvested PBMC by adding additional PBS at RT andpellet cells by spinning at 330 × g for 10 min with centrifuge
brake on, non-refrigerated.
8. Decant supernatant and resuspend cell pellet(s) using PBS at
RT. If appropriate, pool cell pellets from a single donor into
one conical tube. Centrifuge balanced tubes at 300 × g for
15 min with centrifuge brake on, non-refrigerated.
9. Decant supernatant and resuspend the cell pellet(s) using PBS
to achieve a cell density of ~2–5 × 106 cells/mL.
10. Pipet 15 μL of live/dead cell counting dye onto a piece ofparafilm to form a droplet.
11. 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.
12. Transfer 15 μL of the cell and dye suspension into each chamber of a hemacytometer.
13. Determine live cell count and viability using CTL’s live/deadcell counting suite.
14. Increase volume of cell suspension(s) with additional PBS andcentrifuge balanced tubes at 330× g for 10 min with centrifuge
brake on, non-refrigerated.
15. Decant supernatant and gently resuspend the cell pellet(s) using pre-warmed CTL-Cryo™ medium at a cell densityof 6–10 × 106 cells/mL to generate aliquots containing
3–5 × 106 PBMC/vial, respectively.
16. Double the volume of the sample(s) by dropwise addition of
pre-warmed CTL-Cryo™ medium containing 20% v/v
DMSO while gently swirling the tube to ensure adequate
mixing.

262

Lingling Yao et al.

17. Immediately transfer sample(s) into labeled 1.2 mL cryovials
and place into Mr. Frosty™ freezing containers and transfer
into -80 °C freezer (see Note 26), or use a controlled ratefreezer.
3.2 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).
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.

Monitoring Memory B Cells

263

14. Decant supernatant and gently resuspend the cell pellet(s) using pre-warmed BCM at a cell density of ~2–4 × 106
cells/mL.
3.3 Polyclonal In
Vitro 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).

3.4 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 solution into each well (or designated wells, Note 28) of the low
autofluorescence PVDF-membrane plate provided with the 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 (see Note 24).
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 (see Note 29).
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 pre-warmed BCM to block the plate(≥1 h at RT).
9. If using PBMC following in vitro polyclonal activation, collectthe cell suspension(s) and transfer it into labeled conical tube(s). Keep the cells warm during processing. Wash the culture
vessel’s interior with sterile warm PBS to collect residual
PBMC and transfer it into the corresponding conical tube(s).
Increase the volume to fill the tube with additional warm PBS

264

Lingling Yao et al.

and then centrifuge balanced tubes at 330 × g for 10 min
non-refrigerated, centrifuge with the 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 antigenspecific 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 ~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 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
warm PBS and centrifuge balanced tubes at 330 × g for
10 min with centrifuge brake on, non-refrigerated (see Note30).
16. Decant supernatant and resuspend the cell pellet(s) using
pre-warmed BCM at 2 × 106 PBMC/mL (see Note 31).
17. Decant the BCM used for blocking the ImmunoSpot® assay
plate and replace it with 100 μL/well of pre-warmed BCM.
18. Prepare PBMC serial dilution series in a round bottom 96-wellpolystyrene plate to match the plate layout shown in Fig. 5a (seeNote 32). For this, we recommend the following procedure.
Into the round bottom 96 well dilution plate, add 10 μL of
pre-warmed BCM into all wells, except for row A of columns
1 and 2 for the pan-Ig assay and columns 3 and 4 for theantigen-specific assay. Into wells A3 and A4 add 200 μL/welleach of the 2 × 106 PBMC stock (for the pan-Ig assays), dilute
40 μL of the 2 × 106 PBMC stock with 360 μL of warm BCM
to obtain 400 μL of cell suspension at 2 × 105 PBMC/mL, ofwhich plate 200 μL each into wells A1 and A2. Using a multichannel pipettor, perform a two-fold dilution series of the
PBMC by transferring 100 μL from each row to the next,
diluting the cells by gently aspirating and ejecting twice ateach dilution step. Once the cell dilution in the round bottom
plate is complete, using a multichannel pipettor and fresh tips,
transfer 100 μL of the serially diluted cells from the dilution
plate into the actual ImmunoSpot® test plate.

Monitoring Memory B Cells

265

19. Incubate cells in the ImmunoSpot® 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 the plate two times with warm
PBS (200 μL/well), followed by two additional washing steps
with 0.05% Tween-PBS wash solution (see Note 33).
21. Prepare anti-Ig class/subclass-specific detection antibody solution(s) according to kit protocol and pass through a 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 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.
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 34).
28. Allow the plate to dry completely, protected from light (seeNote 38).
29. Scan and count plate(s) with a suitable analyzer equipped withthe appropriate detection channels (see Note 35).

4

Notes
1. While plasma cells (PC) elicited during the primary immune
response can secrete large amounts of antibodies, their lifespans
are heterogeneous and likely fall on a continuum [8, 9].
2. The half-life of different Ig classes, and IgG subclasses, isvariable 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 [10]. For IgA and IgM, their half-lives are evenshorter (3–7 days) [11, 12] and IgE has the shortest half-life inserum, ~2–3 days [13].

266

Lingling Yao et al.

3. A hallmark of immunological memory is the rapid increase in
the level of class-switched, antigen-specific IgG and IgA.
4. 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 8.8 × 105 PBMC to be seeded into the assay – see
the plate layout shown in Fig. 5a. Therefore, if freshly isolated
PBMC are to be tested on day 5–9 post onset of the B cell
response when spontaneously Ig-secreting, antigen-specificplasmablasts are present in the blood, 1 mL of blood should
suffice to complete such a test. If the PBMC from such blood is
to be cryopreserved before testing, 1.5–2 × 106 PBMC shouldbe frozen to obtain, with a safety margin, 8.8 × 105 viable andfully functional PBMC after thawing [7]. If resting memory B
cells in PBMC are to be assessed, however, the PBMC need firstto be subject to a 5 day in vitro stimulation culture [15], after
which about 50% of the (fresh or thawed) PBMC are recovered(Becza et al., manuscript in preparation). Thus, for working
with thawed PBMC and after polyclonal stimulation, freezing
3 (with added safety, 4) × 106 PBMC is required to end up with
8.8 × 105 PBMC on the day of the test. Any number of freshly
isolated PBMC between 1 and 10 × 106 per vial can be cryopreserved recovering the proportional number of fully functional PBMC after thawing (Becza et al., manuscript in
preparation).
5. Stimulating optimal Ig class usage during an infection or following vaccination is vital to successful host defense and the
avoidance of collateral immune-mediated pathology (reviewed
in [14]).
6. Flow cytometry does not reliably reveal the class/subclass of Ig
produced by the individual B cell because surface BCR expression can be highly variable and this is an underappreciatedcomplexity of probe staining. In particular, in the case of
IgG+ ASC, they express little if any surface BCR and thisundermines the assessment of their antigen specificity and subclass usage by traditional surface staining approaches. Consequently, fixation and intracellular staining are required to define
the IgG subclass usage of these cells (a procedure that results insubstantial cell loss in the sample).
7. Memory B cells exist in a quiescent state in the absence ofrecent antigen encounter and do not secrete their individual
BCR as soluble antibodies. To overcome this obstacle fordetecting them in ImmunoSpot® assays, in vitro polyclonal
stimulation protocols can be used to trigger the antigenindependent activation of resting memory B cells into
ASC [15].

Monitoring Memory B Cells

267

8. Spontaneous ASC activity can also be evaluated directly ex vivo
(e.g. plasmablasts that occur in PBMC 5–9 days after onset of a
B cell response, or PC residing in the bone marrow).
9. If testing PBMC in the absence of prior stimulation (e.g. tomeasure plasmablasts), or other samples for spontaneous ASC
activity directly ex vivo, the optimal cell inputs for establishing
the frequency of all ASC-producing IgM, IgG, and IgA will bemuch higher.
10. IgE+ ASC are quite rare and consequently, ImmunoSpot®
assays that aim to determine their relative frequency require
high initial starting cell inputs. Despite testing numerous
human PBMC samples (>50) of healthy, non-allergic individuals following in vitro stimulation with CTL’s B-Poly-S, wehave not detected IgE+ ASC activity in any of these samples
thus far. IgE+ ASC can be detected in PBMC of such individuals, however, following polyclonal stimulation with IL-21 inconjunction with anti-CD40 and IL-4 [15] that mimics T cell
help (suggesting class switching to IgE during the in vitro cell
culture period using this particular polyclonal stimulation
protocol).
11. Cell material is most often the limiting component for immune
monitoring.
12. B cell ImmunoSpot® data can be expressed as SFU per cell
input per well to determine the frequency of antigen-specificcells. However, owing to the variable abundance of pan IgG+
ASC in test samples following polyclonal stimulation, some
prefer to report data as the frequency of antigen-specific B
cells secreting a given Ig class/subclass among all B cells secreting that Ig class/subclass.
13. Each investigator in our laboratory can routinely test, following the protocol outlined in this chapter, in a single experiment, 10–20 PBMC samples for reactivity against a panel ofantigens, assessing the frequency of ASC producing each of the
Ig classes and IgG subclasses. With additional logistical refinements, this throughput is readily upward scalable.
14. If a special protocol is followed, PBMC can be frozen without
impairing the B cells’ functionality ([7] 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 [4], or extending those studies. Of note, whenplanning the numbers of PBMC to be frozen per cryovial, asa rule of thumb, one can anticipate recovery of ~50% of PBMC
initially frozen after these cells are thawed and have undergone
5 days of polyclonal stimulation to promote terminal differentiation of resting Bmem into ASC (see also Note 4). It is also

268

Lingling Yao et al.

important to know that any number of PBMC between 1 and
10 × 106 can be frozen per cryovial permitting the optimization of PBMC utilization when planning experiments(N. Becza, manuscript in preparation).
15. In ImmunoSpot® assays, there is no inherent lower limit ofdetection. If, e.g., 3 × 106 PBMC are plated at 3 × 105 PBMC
across 10 replicate wells, 1 in 3 × 106 is the detection limit, etc.
Importantly, however, owing to increased Poisson noise occurring with such low-frequency measurements, the number of
replicate wells evaluated needs to be increased accordingly toobtain accurate low-frequency measurements.
16. The so-called “Goldilocks” number is defined as the maximal
number of cells that can be plated in an assay well while still
being able to discern clearly individual secretory footprints
derived from antigen-specific ASC. As it is assay-dependent, itneeds to be experimentally established, but 50 SFU/well is asafe estimate.
17. Once the so-called “Goldilocks” number has been established,using a serial dilution approach as illustrated in Fig. 5, in asecond experiment, the cell number(s) can be chosen accordingly for generating replicates containing ≥300 individual
ASC-derived footprints for studying the functional affinity
distribution (see also the chapter in this volume by Beczaet al. [3]). Obviously, the higher the assay-specific Goldilocks
number, the fewer replicates are needed to obtain ≥300 cumulative secretory footprints.
18. 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.
19. 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.
20. Kit is suited for detecting all antibody-secreting cells (ASC)producing a given Ig class or Ig subclass, irrespective ofantigen-specificity, that differentiated in vivo, or following an
in vitro polyclonal stimulation protocol to promote their transition to ASC. Each kit contains pan anti-Ig capture antibody,
Ig class or Ig subclass-specific detection reagents, diluent buffers, PVDF-membrane plates, development substrate solutions, and polyclonal B cell activator (B-Poly-S or B-Poly-Monitoring Memory B Cells

269

SE). Of note, B-Poly-SE is capable of stimulating ASC that
produce one of the distinct Ig classes/subclasses; including IgE(see also Note 10).
21. Kit is suited for detecting all antibody-secreting cells (ASC)producing a given Ig class or Ig subclass, irrespective ofantigen-specificity, that differentiated in vivo, or following an
in vitro polyclonal stimulation protocol to promote their transition to ASC. Each kit contains pan anti-Ig capture antibody,
Ig class or Ig subclass-specific detection reagents, diluent buffers, low autofluorescence PVDF-membrane plates, and polyclonal B cell activator (B-Poly-S or B-Poly-SE). Importantly, Ig
class-specific and/or Ig subclass-specific detection reagents canbe combined to generate multiplexed detection systems
enabling two-, three, or four-color B cell ImmunoSpot® assays.
22. 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 antibody, Ig class-specific (IgA, IgE, IgG, and IgM)
detection reagents, diluent buffers, low autofluorescence
PVDF-membrane plates, and polyclonal B cell activator(B-Poly-S). Alternatively, IgG class-specific (IgG1, IgG2,
IgG3, and IgG4) detection reagents can be substituted in thecontext of such four-color B cell ImmunoSpot® assays.
23. 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 [16].
24. 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 [16].
25. Take care to collect as little Lymphoprep™ as possible. At thispoint, the interphase of two conical tubes can be combined
into one tube. If the proportion of Lymphoprep™ is too high(≥ 10% v/v), significant cell loss may occur.
26. A cooling rate of 1 °C/min is optimal for cell cryopreservation.
Be sure to fill the lower compartment of the Mr. Frosty™
Freezing container with 100% isopropyl alcohol and replace itafter five cryopreservation cycles. After approximately 20 h,

270Lingling Yao et al.

and no more than 2 days, transfer cryopreserved PBMC fromthe -80 °C freezer into liquid nitrogen for long-term storage.
27. 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. If larger
numbers of in vitro stimulated PBMC are required for downstream ImmunoSpot® assays, tissue culture flasks should be
used; store flat or standing such the height of the cell suspension in the flask is between 0.5 and 1 cm. Smaller in vitro
stimulation cultures can be initiated in 48- or 24-well plateswith a final volume of 1–2 mL, respectively. Be sure to fill
empty wells in tissue culture plates with sterile PBS to avoid
dehydration of cell cultures.
28. If the entire plate will not be coated with the anti-His affinity
capture antibody solution, the remainder of the EtOH pre-wet
wells should receive 80 μL/well of PBS.
29. If performing both an antigen-specific affinity capture and total
ASC assay on the same plate, we recommend adding the pananti-Ig capture antibody at this stage.
30. If the cells are not washed thoroughly, contaminating antibodies in the cell suspension(s) can compete for binding of theaffinity captured antigen and may also result in elevated membrane staining that interferes with accurate enumeration ofindividual antigen-specific ASC.
31. This starting cell input was used to generate the data presented
in Figs. 2, 3, 4 and 5 and to highlight the vastly different
frequencies of ASC that produce each Ig class or subclassfollowing in vitro polyclonal stimulation.
32. Do NOT do serial dilutions in the actual ImmunoSpot® plates
because the pipet tips can easily damage the membrane. For
multiple PBMC, these cell transfers can readily be done simultaneously using a multichannel pipettor.
33. 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.
34. Optimal removal of background staining, fibers, and otherdebris, along with reduction of “hot spots” in the center of
the assay wells, is achieved through performing the “back tofront” water filtration technique.
35. The chapter by Karulin et al. in this volume [5] introduces
artificial intelligence-based SFU analysis that can partially compensate for ELISA effects and SFU crowding, thus extending
the linear range of accurate quantification for cell numbers
plated per well and SFU detected.

Monitoring Memory B Cells

271

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, Diana Roen for carefully proofreading the manuscript, and Gregory Kovacs for his support in the generation ofgraphic illustrations. All efforts were funded from CTL’s researchbudget.
Conflicts of Interest P.V.L. is Founder, President, and CEO of
CTL, a company that specializes in immune monitoring by ImmunoSpot®. L.Y., N.B., A.M.P., J.C., and G.A.K. are employeesof CTL.

References
1. Lehmann PV, Becza N, Liu Z et al (2023)
Monitoring memory B cells by next generation
ImmunoSpot® provides insights into humoral
immunity that measurements of circulating
antibodies do not reveal. Methods Mol Biol
2. Akkaya M, Kwak K, Pierce SK (2020) B cell
memory: building two walls of protection
against pathogens. Nat Rev Immunol 20(4):229–238. https://doi.org/10.1038/s41577019-0244-2
3. Becza N, Liu Z, Chepke J et al (2023) Assessing the affinity spectrum of the antigenspecific B cell repertoire in freshly isolated cellmaterial via ImmunoSpot®. Methods Mol Biol
4. Wolf C, Koppert S, Becza N et al (2022) Antibody levels poorly reflect on the frequency ofmemory B cells generated following SARSCoV-2, seasonal influenza, or EBV infection.
Cell 11(22). https://doi.org/10.3390/
cells11223662
5. Karulin AY, Megyesi Z, Kirchenbaum GA et al(2023) Artificial intelligence-based counting
algorithm enables accurate and detailed analysis of the broad spectrum of spot morphologies
observed in antigen-specific B cell EliSpot andFluoroSpot assays. Methods Mol Biol
6. Bisceglia H, Barrier J, Ruiz J et al (2023) A
FluoroSpot B assay for the detection of IgA
and IgG SARS-CoV-2 spike-specific memory
B cells: Optimization and qualification for use
in COVID-19 vaccine trials. J Immunol Methods 515:113457. https://doi.org/10.1016/j.
jim.2023.113457

7. Fecher P, Caspell R, Naeem V et al (2018) B
cells and B cell blasts withstand cryopreservation while retaining their functionality for producing antibody. Cell 7(6). https://doi.org/
10.3390/cells7060050
8. Lightman SM, Utley A, Lee KP (2019) Survival of long-lived plasma cells (LLPC): piecing
together the puzzle. Front Immunol 10:965.
https://doi.org/10.3389/fimmu.2019.
00965
9. Robinson MJ, Ding Z, Dowling MR et al(2023) Intrinsically determined turnoverunderlies broad heterogeneity in plasma-cell
lifespan. Immunity 56(7):1596–1612. e1594.
https://doi.org/10.1016/j.immuni.2023.
04.015
10. Morell A, Terry WD, Waldmann TA (1970)
Metabolic properties of IgG subclasses inman. J Clin Invest 49(4):673–680. https://doi.org/10.1172/JCI106279
11. Blandino
R,
BaumgarthN(2019)
Secreted IgM: New tricks for an old molecule.
J Leukoc Biol 106(5):1021–1034. https://doi.
org/10.1002/JLB.3RI0519-161R
12. van Tetering G, Evers M, Chan C et al (2020)
Fc engineering strategies to advance IgA antibodies as therapeutic agents. Antibodies 9(4).
https://doi.org/10.3390/antib9040070
13. Normansell R, Walker S, Milan SJ et al (2014)
Omalizumab for asthma in adults and children.
Cochrane Database Syst Rev 1:CD003559.
h t t p s : // d o i . o r g / 1 0 . 1 0 0 2 / 1 4 6 5 1 8 5 8 .
CD003559.pub4

272

Lingling Yao et al.

14. Webb NE, Bernshtein B, Alter G (2021) Tissues: the unexplored frontier of antibody
mediated immunity. Curr Opin Virol 47:52–
67. https://doi.org/10.1016/j.coviro.2021.
01.001
15. Franke F, Kirchenbaum GA, Kuerten S et al(2020) IL-21 in conjunction with anti-CD40
and IL-4 constitutes a potent polyclonal B cellstimulator for monitoring antigen-specificmemory B cells. Cell 9(2). https://doi.org/
10.3390/cells9020433
16. Koppert S, Wolf C, Becza N et al (2021) Affinity tag coating enables reliable detection of
antigen-specific B cells in Immunospot assays.
Cell 10(8). https://doi.org/10.3390/
cells10081843

Open Access This chapter is licensed under the terms of the Creative Commons Attribution 4.0 International
License (http://creativecommons.org/licenses/by/4.0/), which permits use, sharing, adaptation, distribution
and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and thesource, provide a link to the Creative Commons license and indicate if changes were made.
The images or other third party material in this chapter are included in the chapter’s Creative Commons license,
unless indicated otherwise in a credit line to the material. If material is not included in the chapter’s Creative
Commons license and your intended use is not permitted by statutory regulation or exceeds the permitted use,
you will need to obtain permission directly from the copyright holder.