Protein microarrays for antibody profiling: specificity and affinity determination on a chip.
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.
Poetz
Oliver
O
Natural and Medical Sciences Institute at the University of Tübingen, Reutlingen, Germany.
Ostendorp
Ralf
R
Brocks
Bodo
B
Schwenk
Jochen M
JM
Stoll
Dieter
D
Joos
Thomas O
TO
Templin
Markus F
MF
D000911:Antibodies, Monoclonal / Q000737:chemistry*; D000918:Antibody Specificity; D018604:Epitope Mapping; D006801:Humans; D007128:Immunoglobulin Fragments / Q000737:chemistry*; D010455:Peptides / Q000737:chemistry*; D040081:Protein Array Analysis
D000911:Antibodies, Monoclonal; D007128:Immunoglobulin Fragments; D010455:Peptides