Measuring affinity constants of 1450 monoclonal antibodies to peptide targets with a microarray-based label-free assay platform

Abstract

Monoclonal antibodies (mAbs) are major reagents for research and clinical diagnosis. For their inherently high specificities to intended antigen targets and thus low toxicity in general, they are pursued as one of the major classes of new drugs. Yet binding properties of most monoclonal antibodies are not well characterized in terms of affinity constants and how they vary with presentations and/or conformational isomers of antigens, buffer compositions, and temperature. We here report a microarray-based label-free assay platform for high-throughput measurements of monoclonal antibody affinity constants to antigens immobilized on solid surfaces. Using this platform we measured affinity constants of over 1,410 rabbit monoclonal antibodies and 46 mouse monoclonal antibodies to peptide targets that are immobilized through a terminal cysteine residue to a glass surface. The experimentally measured affinity constants vary from 10 pM to 200 pM with the median value at 66 pM. We compare results of the microarray-based platform with those of a benchmarking surface-plasmon-resonance-based (SPR) sensor (Biacore 3000).

A microarray-based reaction rate analysis platform. Is there evidence of validity?

“…Furthermore, the affinities of a set of 863 rabbit monoclonal antibodies have, as determined using the present microarray approach, elsewhere been suggested to be about ten to one hundredfold higher than those of mouse monoclonal antibodies (affinity constants that in turn were determined by a range of methodologies) (Abcam, 2015b). However, such difference in affinity between rabbit and mouse antibodies could not be reproduced when antibodies of both origins were analysed using the present technology (Landry et al, 2015). Although these differences might be associated to the way these sets of antibodies were developed using a variety of immunization and screening procedures (Brown et al, 2011), they may also be a result of procedural differences affecting the observed apparent affinity constant of the antigen-antibody interaction itself.…”

“…It was with great interest we approached the recent paper by Landry et al (2015). Antibodies are certainly the main probe type of many clinical and bioscience laboratories but the quality of the reagents used in many applications may be questioned.…”

“…It is in this context that Landry et al (2015) developed a microarraybased label-free platform to assess multiple peptide-antibody interactions in parallel. Although the assay is not intended to create a comprehensive assessment of antibody performance, it is designed to measure factors, reaction rate and affinity constants, important to the performance characteristics of many assays.…”

“…The investigated monoclonal antibodies were originally raised by immunization of rabbits (or in a few cases mice) with peptides conjugated to keyhole limpet hemocyanin (KLH) using a maleimide coupling scheme employing Sulfo-SMCC as the cross-linker. Hybridomas producing specific antibodies were identified by ELISA using either peptideconjugated albumin or peptides as such coated directly onto the surface of microtitre plate wells (Landry et al, 2015). Purified monoclonal antibodies were subsequently used to determine the affinity constant.…”

“…The assay is built on peptides immobilised on epoxy-functionalised glass slides. One hundred monoclonal antibodies isolated against unrelated peptides were allowed to bind their peptide antigens and the binding and dissociation events were followed in real time using a scanning ellipsometry sensor (Landry et al, 2015). The procedural outline as such is straightforward but some of the interpretations made based on the experimental outcomes may not be as straightforward as presented, a fact that may make use of this high throughput platform problematic.…”