Isolation and Characterization of Antigen-Specific Plasmablasts Using a Novel Flow Cytometry–Based Ig Capture Assay

Abstract

We report the development of a novel flow cytometry-based Ig capture assay (ICA) for the identification and sorting of individual Ab-secreting cells based on their Ag reactivity. The ICA represents a fast and versatile tool for single-cell sorting of peripheral plasmablasts, streamlining subsequent Ab analysis, and cloning. We demonstrate the utility of the assay by isolating Ag-reactive plasmablasts from cryopreserved PBMC obtained from volunteers vaccinated with a recombinant HIV envelope protein. To show the specificity of the ICA, we produced Ag-specific Abs from these cells and subsequently verified their Ag reactivity via ELISA. Furthermore, we used the ICA to track Ag-specific plasmablast responses in HIV-vaccine recipients over a period of 42 d and performed a head-to-head comparison with a conventional B cell ELISpot. Results were highly comparable, highlighting that this assay is a viable alternative for monitoring Ag-specific plasmablast responses at early time points after infection or vaccination. The ICA provides important added benefits in that phenotypic information can be obtained from the identified Ag-specific cells that can then be captured for downstream applications such as B cell sequencing and/or Ab cloning. We envisage the ICA as being a useful tool in Ab repertoire analysis for future clinical trials.

Cross-Reactive Bactericidal Antimeningococcal Antibodies Can Be Isolated From Convalescing Invasive Meningococcal Disease Patients Using Reverse Vaccinology 2.0

“…Larger patient cohorts will be required to investigate this theory. Optimization of the IgG-cloning technique to increase the number of antimeningococcal plasmablasts using a recently published Ig capture-based assay ( 40 ) should improve productivity of the approach.…”

Techniques to Study Antigen-Specific B Cell Responses

“…With technologic advancements increasing the number of simultaneously measurable parameters, antigen-specific B cells can be further characterized by cell surface markers and intracellular staining. Additionally, the immunoglobulin capture assay is a flow cytometry-based adaptation of the ELISPOT assay in which a streptavidin-conjugated anti-CD45 antibody carrying four biotinylated anti-IgG antibodies is used to simultaneously bind plasmablasts and capture secreted antibody followed by fluorescent-labeled antigen to detect antigenspecific plasmablasts (61). The mean fluorescence intensity measured by flow cytometry and normalized to the level of BCR expression also provides a measure of the relative amount of antigen binding to a B cell and can be used as a rough surrogate for binding affinity (79,81,82).…”

Screening of Antigen-Specific Antibody-Secreting Cells

“…Streptavidin can link both anti-CD45 and biotinylated anti-IgG antibodies that can simultaneously bind to the plasmablasts and secreted mAbs (Fig. 1d) (Pinder et al 2017). However, the aforementioned techniques increase the complexity of the secretion assays.…”