ELISpot Assay for Quantifying Antigen-Specific and Total Antibody-Secreting Cells in Blood and Bone Marrow of Immunized Rhesus Macaques

Summary

B-Cell ELISpot is a validated method for quantifying the magnitude and durability of antigen-specific IgG, IgM, and IgA long-lived plasma cells, as well as total antibody-secreting cells (ASCs), in both blood (reflecting short-lived plasmablasts following immunization) and more durable bone marrow ASCs.

Abstract

Evaluation of novel vaccine antigens and adjuvants traditionally relies on measuring antibody titers, which require time to develop following immunization. Quantification of antibody-secreting cells (ASCs) after primary and booster immunizations provides an earlier, mechanistically informative measure of vaccine-induced humoral immunity and can facilitate the optimization of vaccine formulations and immunization strategies. Here, a protocol for enumerating ASCs from peripheral blood mononuclear cells (PBMCs) and bone marrow (BM) cells of rhesus macaques is described; rhesus macaques are an important preclinical model for HIV vaccine research. This enzyme-linked immunospot (ELISpot) assay enables the quantification of both antigen-specific and total IgG, IgM, and IgA ASCs by measuring their absolute and relative frequencies. In peripheral blood, the assay captures the peak plasmablast response following immunization, whereas in bone marrow it detects the accumulation of antigen-specific plasma cells of different isotypes, thereby assessing the durability of long-lived ASCs. This protocol offers a robust and reproducible approach for evaluating immunogen and adjuvant combinations in preclinical vaccine studies and provides valuable data to support the translation of promising vaccine candidates into clinical development.

Introduction

Antibody-secreting cells (ASCs) are key components of adaptive immunity and include both short-lived plasmablasts, which are generated rapidly following immunization, and long-lived plasma cells, which are responsible for sustained antibody production. Plasmablasts are transiently detectable in peripheral blood and secondary lymphoid organs, reaching peak frequencies at defined time points following immunization that vary according to the vaccine formulation and host species (typically days 4–5 in rhesus macaques and approximately day 7 in humans). In contrast, mature plasma cells are long-lived and primarily reside within secondary lymphoid tissues and the bone....

Protocol

The rhesus macaques used in this protocol were sourced from the specific pathogen-free (SPF) colonies of the New Iberia Research Center and housed at the Michale E. Keeling Center for Comparative Medicine and Research, the University of Texas MD Anderson Cancer Center. Animal care and all experimental procedures complied with the Guide for the Care and Use of Laboratory Animals and were approved by the University of Texas MD Anderson Cancer Center Institutional Animal Care and Use Committee (Protocol #2616RN00).

This protocol represents an optimization of the technique described in previous immunization pre-clinical studies

Results

In this B-cell ELISpot assay, antibody-secreting cells (ASCs) were detected using CH505 SOSIP, a soluble HIV-1 envelope glycoprotein (Env) trimer, as the coating antigen in a vaccine study aimed at eliciting broadly neutralizing antibodies (bnAbs) against HIV-1. Figure 1 illustrates the ELISpot plate layout used to quantify total and antigen-specific ASCs on separate plates. The layout identifies individual animals and the well positions assigned for IgG, IgM, and IgA analyses. Plate labels .......

Discussion

B-cell ELISpot is a sensitive method for detecting total immunoglobulin and antigen-specific antibody-secreting cell (ASC) responses in blood, plasma, and bone marrow cells^8,9,10,11. Several steps are critical to ensuring the protocol's reproducibility. First, membranes should be precisely coated with the optimal concentration of the capture antibody. Membrane blocking is essential to preve.......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
ACK Lysis	Quality Biological	118-156-101	500 ml
70µm strainer	Fisher	22363548	Sterile cell strainer, fit for 50 mL conical tubes
AEC working Substrate	BD Biosciences	551951	Reagents for 10 plate(s)
Analysis: Excel	Microsoft office	Version 2508 Build 16.0.19127.20678	Microsoft Excel for Microsoft 365
Analysis: GraphPad Prism	GraphPad Software, LLC	Version 10.6.1 (892)	GraphPad Prism 10
Anti Rhesus IgA biotin	NHPRR	AB-3086851	lyophilized
Anti-monkey IgG biotin	Rockland	P/N 617-106-012	lyophilized
Anti-monkey IgM biotin	Rockland	P/N 617-106-007	lyophilized
Blocking media	Lab prepared reagents	formulated in-house	RPMI-10
Buffer for diluting the secondary antibody and HRP	Lab prepared reagents	formulated in-house	1X DPBS with 1%FBS
Centrifuge	Thermo Scientific	75004521	Sorvall Legend XTR, Make: 2013, TX-750 rotor and 75003608 (Round bucket)
Coating buffer	Lab prepared reagents	formulated in-house	1X DPBS
DPBS	Gibco	14190-136	1X
ELISpot plates	Millipore Sigma	MSHAN4B50	50 plates pack
Fetal Bovine Serum (FBS)	Omega Scientific	FB-01	1X
Ficoll	STEMCELL Technologies	18061	500 ml
GNL Unconjugated	Vector Laboratories	L-1240-5	lyophilized
Horseradish Peroxidase (HRP) - Avidin	Vector Laboratories	A-2004	5mg/ml ready to use
Mabtech IRIS Fluorospot/ELISpot reader	Mabtech AB	P/N: 2000	Automated imaging and analysis for ELISpot and Flurospot assay
Penicillin/Streptomycin	Gibco	15140-122	100X
Plate washer	BioTek Instruments, Inc.	REF# 405TSRS	405 TS Microplate washer
RPMI-10	Lab prepared reagents	formulated in-house	RPMI-1640 + 10% Fetal Bovine Serum and 1% Penicillin/Streptomycin
RPMI-1640	Gibco	22400-071	1X
Tween20	Sigma Life sciences	P7949	500ml
Unconjugated Polyclonal Goat anti monkey IgG+IgA+IgM Antibody IgG/IgM/IgA	LS-Bio	C347340	lyophilized
Wash buffer (Antigen Specific Plates)	Lab prepared reagents	formulated in-house	(DPBS 1X only)
Wash buffer (Total Ig Plates)	Lab prepared reagents	formulated in-house	( DPBS 1X + 0.05% Tween 20)