DETERMINATION OF ANTIBODY SPECIFICITY USING AFM

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Description (provided by applicant): This research will use atomic force microscopy (AFM) and peptide combinatorial libraries to quantify and determine directly the specificity of an antibody. The antibody under investigation (mouse anti-beta-endorphin (clone 3-E7) and mouse anti-Escherichia coli beta-galactosidase (clone D19-2F3-2)) is attached to the AFM cantilever tip and used to probe surfaces with covalently attached peptides. Force/distance curves are generated from which the adhesion force of the tip to the surface is calculated. The use of AFM to assess antibody binding has the advantage over conventional techniques involving enzyme-linked immunosorbant assays (ELISA) in that strong and weak binding antigens will be identified rather than just the strongest binding antigen. For the anti-beta-endorphin antibody the peptides PGGFL and YGGFL are attached to a glass cover slip surface. Preliminary results indicate that the adhesion force for the antibody is greater with the cognate peptide (YGGFL) than for the non-cognate peptide (PGGFL). Additional studies will be performed involving alterations to buffer conditions to thoroughly characterize the binding of anti-beta-endorphin antibody. Peptide combinatorial libraries will be used as the surfaces for probing with the anti-E. coli beta-galactosidase derivatized AFM tip as a means to determine the specificity of this antibody. The libraries will be constructed on a silicon substrate using a novel ink-jet printer technology. This technology produces spatially defined peptide combinatorial libraries. The libraries can be synthesized in a short period of time, tailored for the antibody being studied, scanned relatively quickly with AFM, and they are reusable. The utilization of the AFM to probe libraries made by the ink-jet printer technology will provide for a faster, more reliable and reproducible methodology for uncovering the binding characteristics of an antibody.