Affinity Tag Coating Enables Reliable Detection of Antigen-Specific B Cells in Immunospot Assays

Abstract

Assessment of humoral immunity to SARS-CoV-2 and other infectious agents is typically restricted to detecting antigen-specific antibodies in the serum. Rarely does immune monitoring entail assessment of the memory B-cell compartment itself, although it is these cells that engage in secondary antibody responses capable of mediating immune protection when pre-existing antibodies fail to prevent re-infection. There are few techniques that are capable of detecting rare antigen-specific B cells while also providing information regarding their relative abundance, class/subclass usage and functional affinity. In theory, the ELISPOT/FluoroSpot (collectively ImmunoSpot) assay platform is ideally suited for antigen-specific B-cell assessments since it provides this information at single-cell resolution for individual antibody-secreting cells (ASC). Here, we tested the hypothesis that antigen-coating efficiency could be universally improved across a diverse set of viral antigens if the standard direct (non-specific, low affinity) antigen absorption to the membrane was substituted by high-affinity capture. Specifically, we report an enhancement in assay sensitivity and a reduction in required protein concentrations through the capture of recombinant proteins via their encoded hexahistidine (6XHis) affinity tag. Affinity tag antigen coating enabled detection of SARS-CoV-2 Spike receptor binding domain (RBD)-reactive ASC, and also significantly improved assay performance using additional control antigens. Collectively, establishment of a universal antigen-coating approach streamlines characterization of the memory B-cell compartment after SARS-CoV-2 infection or COVID-19 vaccinations, and facilitates high-throughput immune-monitoring efforts of large donor cohorts in general.

Antibody Levels Poorly Reflect on the Frequency of Memory B Cells Generated following SARS-CoV-2, Seasonal Influenza, or EBV Infection

“…While the methodology for the quantification of antigen-reactive antibodies in plasma by ELISA is well-established, we first needed to develop such for the objective and accurate counting of antigen-reactive B mem cells in the blood. Empirically, antigen-reactive B cell ImmunoSpot ® assays result in diverse spot morphologies [ 47 ] (see also Figure 1 B,C). This outcome is expected as the secretory footprints of individual ASCs are defined by a multitude of parameters [ 61 ] including (a) the net amount of antibodies produced by the individual B cells during the assay’s duration, (b) the kinetics of antibody production by the ASCs, and (c) the functional affinity of each ASC’s antibody for the antigen (that can encompass a broad spectrum among an antigen-reactive B cell repertoire, defining the capture and dissociation rates of antibodies binding to antigens).…”

“…For the enumeration of antigen-reactive IgG + ASCs, ImmunoSpot ® assays were performed with ACC as previously described [ 47 ] and illustrated by Figure S1 . Briefly, assay plates were first preconditioned with 70% ( v / v ) EtOH followed by two washing steps with PBS.…”

“…Successful coating therefore depends on the physical properties of the membrane chosen versus the corresponding properties of the antigen itself. To overcome this limitation in the development of B cell ImmunoSpot ® assays for “any antigen of interest”, we recently introduced an approach termed “affinity capture coating” (ACC) in which improved antigen absorption to the membrane is achieved through preconditioning the assay membrane with an antibody specific for the genetically encoded hexahistidine (6XHis) affinity tag of the recombinant proteins [ 47 ], enabling the subsequent high-affinity capture of any 6XHis-tagged protein. Using this universal ACC approach, we developed ImmunoSpot ® assays that enable the detection of memory B cells reactive with antigens representing SARS-CoV-2, seasonal influenza, and Epstein–Barr virus (EBV) [ 47 ].…”

Monitoring memory B cells by next generation ImmunoSpot® provides insights into humoral immunity that measurements of circulating antibodies do not reveal

“…Recently, we concluded a systematic study (to the best of our knowledge the rst on this subject) in which we measured circulating antibody levels to SARS-CoV-2, EBV, and different seasonal in uenza virus strains and compared them with the frequencies of B mem reactive with the same antigens [6]. These ImmunoSpot®-based B mem detections were enabled by our newly-acquired ability to achieve high density antigen coating, thus reliable detection of B mem speci c for essentially any antigen ( [11] and Note 8).…”

“…Consequently, many investigators likely gave up on this assay after not being successful in their initial attempts to establish such assays for the antigen(s) of interest. Our recent introduction of a nity capture coating [11] represents a breakthrough to this end, as it provides a universal strategy for successful assay development for essentially any antigen: the membrane is rst coated with an anti-(His-or other) a nity tag-speci c antibody followed by the addition of recombinant (His-or other) a nity-tagged recombinant antigen. In this way, low-a nity absorption of the antigen to the membrane via weak, non-speci c binding forces (primarily hydrophobicity) is replaced by speci c, high-a nity binding.…”