A high-throughput, bead-based, antigen-specific assay to assess the ability of antibodies to induce complement activation

Stephanie Fischinger,Jonathan K Fallon,Ashlin R Michell,Thomas Broge,Todd J Suscovich,Hendrik Streeck,Galit Alter

doi:10.1016/j.jim.2019.07.002

Abstract

The complement system plays a critical role in innate immune defense against pathogens, both via non-specific direct pathogen recognition and killing or via antigen-specific indirect recruitment by complement fixing antibodies. While various assays for measuring complement activation have been developed, few provide a high-throughput, sample-sparing approach to interrogate the qualitative differences in the ability of antibodies to drive complement activation. Here we present a high-throughput, sample-sparing, bead-based assay to evaluate antigen-specific antibody-dependent complement activation against nearly any antigen. Optimization of buffer composition, kinetics of immune complex formation, as well as complement source all contribute critically to the development of a robust, highly flexible and high-throughput approach to analyze antibody-dependent complement deposition (ADCD). Thus, the optimized bead-based, antigen-specific assay represents a simple, highly adaptable platform to profile antibody-dependent complement activation across pathogens and diseases.

Highlights

Antibodies represent the primary correlate of protection following most clinically approved vaccines and infections (Haynes et al, 2012; Sicca et al, 2018; Teo et al, 2016)
To initially determine whether complement deposition could be selectively and observed on antigen-coupled beads in the presence of sero-positive pools of antibodies, an Amnis ImageStreamX imaging flow cytometer was used to visualize the binding of the detection antibody to C3 complement following incubation with pools of HIV-positive pools of polyclonal IgG (HIVIG) or HIV-negative pools of polyclonal IgG (IVIG)
The x-axis represents the differences in C3-FITC fluorescence detected by the secondary antibody, with higher positivity in the presence of the HIVIG compared to the IVIG (Fig. 2A and B)

Summary

Introduction

Antibodies represent the primary correlate of protection following most clinically approved vaccines and infections (Haynes et al, 2012; Sicca et al, 2018; Teo et al, 2016). While preventing pathogen entry is one potential mechanism by which antibodies may confer protection (i.e., neutralization), antibodies can control and help clear infections through non-neutralizing immune effector functions (DiLillo et al, 2016; Lelièvre and Lévy, 2016). After antigen-binding, antibodies mediate these effector functions via interactions between their Fc domains and either Fc receptors (found on all innate immune cells) or components of the complement system. With our emerging appreciation for the role of non-neutralizing antibodies in protection from infection, assays to selectively and profile antibody-mediated immune activation have emerged. While several assays have been described for the analysis of antibodymediated innate immune cellular activation (Ackerman et al, 2011), fewer high-throughput assays and selectively probe the ability of antibodies to trigger the complement cascade

Methods

Results

Conclusion