Deep phenotypic characterization of immunization-induced antibacterial IgG repertoires in mice using a single-antibody bioassay

Millie Heo,Jean Baudry,Jérôme Bibette,Andrew D Griffiths,Guilhem Chenon,Carlos Castrillon,Pierre Bruhns,Klaus Eyer

doi:10.1038/s42003-020-01296-3

Abstract

Antibodies with antibacterial activity need to bind to the bacterial surface with affinity, specificity, and sufficient density to induce efficient elimination. To characterize the anti-bacterial antibody repertoire, we developed an in-droplet bioassay with single-antibody resolution. The assay not only allowed us to identify whether the secreted antibodies recognized a bacterial surface antigen, but also to estimate the apparent dissociation constant (KD app) of the interaction and the density of the recognized epitope on the bacteria. Herein, we found substantial differences within the KD app/epitope density profiles in mice immunized with various species of heat-killed bacteria. The experiments further revealed a high cross-reactivity of the secreted IgG repertoires, binding to even unrelated bacteria with high affinity. This application confirmed the ability to quantify the anti-bacterial antibody repertoire and the utility of the developed bioassay to study the interplay between bacteria and the humoral response.

Highlights

Antibodies with antibacterial activity need to bind to the bacterial surface with affinity, specificity, and sufficient density to induce efficient elimination
To comprehensibly address the diversity of surface bacterial antigens, we directly covered the nanoparticles with heat-killed bacteria (HKB) (Figs. 1, 2a; “Methods”)
The bacterial cells were immobilized on 300 nm streptavidin paramagnetic nanoparticles via a biotinylated version of a hydrophobic cholesterol linker[32], in which the cholesterol moiety interacted with the hydrophobic domains on the bacterial cell surface and the biotin with the streptavidin present on the nanoparticle surface

Summary

Introduction

Antibodies with antibacterial activity need to bind to the bacterial surface with affinity, specificity, and sufficient density to induce efficient elimination. Cellbased technologies such as flow cytometry[26], enzyme-linked immunospot assays (ELISPOT)[27] and droplet-microfluidics[28,29,30] have been used to study anti-bacterial antibody repertoires All of these technologies allow to screen the secreted antibody repertoire for surface-binding antibodies with high-throughput on the single-antibody and cell level; they offer low analytical resolution in terms of affinity and epitope density. Our bioassay allowed the classification and comparison of specific and cross-reactive anti-bacterial antibodies according to their apparent dissociation constant (KD app) and epitope availability, and revealed highly cross-reactive IgG repertoires following immunization with full heat-killed bacteria, binding to even unrelated bacteria with high affinity

Objectives

Methods

Results

Conclusion