Affiliation: Department of Molecular Biology and Genetics, Democritus University of Thrace, University Campus-Dragana, 68100 Alexandroupolis, Greece.
The development of antibodies for diagnostic and therapeutic applications in inflammatory diseases is a major focus for biotechnology and pharmaceutical companies. Production of monoclonal antibodies requires the development of fast, high-throughput methodologies for screening and selecting appropriate candidate antibodies for development. Capture (sandwich) enzyme linked immunosorbent assay (ELISA) provides a quick and reliable method that could be used for hybridoma screening of potential candidates accompanied with surface plasmon resonance (SPR) biosensor technology for identifying high affinity biomolecular interactions. A sensitive, cost-effective, robust and accurate capture ELISA for detection of murine monoclonal antibodies in culture supernatants was developed. This assay was optimized for high sensitivity and specificity with a capture anti-mouse polyclonal antibody. Using serial dilutions of a defined murine IgG antibody, a linear dose-response was observed between 2 and 1200 ng/ml antibody with a coefficient of determination r2 of 0.98. The detection limit of the assay was established as 2ng/ml (12.5pM). A similar concentration-dependent doseresponse was also observed using serial dilutions of antibody-containing supernatants from anti-alpha glycophorinproducing hybridomas (detection limit 1:2000). Specific capture of antibodies from supernatants in a similar setting was also confirmed using SPR biosensor technology and correlated well with the immunoassay results. The latter technology can be performed in order to provide quick screening results and kinetic analysis of antibody binding interactions aiming at identifying candidates with high affinity and specificity.