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

Area of Science:

• Immunology
• Cell Biology
• Biotechnology

Background:

• Monitoring antigen-specific antibody responses is crucial for vaccine development and infectious disease research.
• Existing methods for isolating antibody-secreting cells can be time-consuming and may not provide comprehensive phenotypic data.

Purpose of the Study:

• To develop and validate a novel flow cytometry-based Ig capture assay (ICA) for identifying and sorting antigen-reactive antibody-secreting cells.
• To assess the utility of ICA for analyzing plasmablast responses in individuals vaccinated with an HIV envelope protein.
• To compare the performance of ICA with a conventional B cell ELISpot assay.

Main Methods:

• Development of a flow cytometry-based Ig capture assay (ICA) utilizing antigen-specific binding.
• Isolation and sorting of antigen-reactive plasmablasts from peripheral blood mononuclear cells (PBMCs) of HIV-vaccine recipients.
• Production and validation of antigen-specific antibodies via ELISA.
• Head-to-head comparison of ICA with B cell ELISpot assay for monitoring plasmablast responses over 42 days.

Main Results:

• The ICA successfully identified and sorted antigen-reactive plasmablasts from PBMCs of vaccinated volunteers.
• Antigen-specific antibodies produced from ICA-sorted cells demonstrated verified antigen reactivity via ELISA.
• ICA performance was comparable to the conventional B cell ELISpot assay in tracking antigen-specific plasmablast responses.
• ICA facilitated the acquisition of phenotypic information from identified antigen-specific cells.

Conclusions:

• The Ig capture assay (ICA) is a fast, versatile, and effective tool for single-cell sorting of antigen-reactive antibody-secreting cells.
• ICA serves as a viable alternative to ELISpot for monitoring early antigen-specific plasmablast responses post-vaccination or infection.
• The assay's ability to capture phenotypic data and enable downstream applications like B cell sequencing and antibody cloning enhances its utility in antibody repertoire analysis for clinical trials.