Protein microarrays for antibody profiling: Specificity and affinity determination on a chip

Abstract

Protein microarray technology facilitates the detection and quantification of hundreds of binding reactions in one reaction from a minute amount of sample. Proof-of-concept studies have shown that the set-up of sensitive assay systems based on protein arrays is possible, however, the lack of specific capture reagents limits their use. Therefore, the generation and characterisation of capture molecules is one of the key topics for the development of protein array based systems. Recombinant antibody technologies, such as HuCAL (human combinatorial antibody library; MorphoSys, Munich, Germany), allow the fast generation of highly specific binders to nearly any given target molecule. Although antibody libraries comprise billions of members, it is not the selection process, but the detailed characterisation of the pre-selected monoclonal antibodies that presents the bottleneck for the production of high numbers of specific binders. In order to obtain detailed information on the properties of such antibodies, a microarray-based method has been developed. We show that it is possible to define the specificity of recombinant Fab fragments by protein and peptide microarrays and that antibodies can be classified by binding patterns. Since the assay uses a miniaturised system for the detection of antibody-antigen interactions, the observed binding occurs under ambient analyte conditions as defined by Ekins (J. Pharm. Biomed. Anal. 1989, 7, 155-168). This allows the determination of a relative affinity value for each binding event, and a ranking according to affinity is possible. The new microarray based approach has an extraordinary potential to speed up the screening process for the generation of recombinant antibodies with pre-defined selection criteria, since it is intrinsically a high-throughput technology.

ProteomeBinders: planning a European resource of affinity reagents for analysis of the human proteome

“…Although different binder types may have superior characteristics for defined applications, certain criteria (affinity, specificity 8 and cross-reactivity, native or denatured target, stability in vivo and in vitro, association and dissociation rate constants 26,27 ) are applicable across all formats. The consortium will establish reference criteria for binder quality control and validation, which could eventually become a 'gold standard' in the research and commercial areas.…”

“…ProteomeBinders aims to integrate existing, but presently fragmented, bioinformatics tools into distributed and/or virtual facilities that specifically address target-site selection within proteins. In the context of constructing a large-scale binder resource, an important goal will be to reduce the amount of target required (and hence also the cost), for example, by using microarrays for selection or using micro-or nanotechnology for specificity and affinity assays 8 .…”

Multiplexed Immunoassays

“…To solve this problem, several attempts have been made to optimize antibodies characterization and selection by using high-throughput methods based on multiplexed immobilized proteins or peptides. For example, Poetz et al 107 reported the use of a protein microarray to simultaneously analyze epitope recognition and binding affinity of antibodies to determine their specificity and affinity. 114 And Schwenk et al 108 also reported the use of Luminex platform to determine antibody specificity towards up to 100 antigens.…”

Porous Silicon Integrated Photonic Devices for Biochemical Optical Sensing

“…The microarray technology has demonstrated a great potential in drug discovery, genomics, proteomics research, and medical diagnostics (Pregibon et al, 2007;Poetz et al, 2005; Fig. 18.…”