antibodies on demand a fast method for the production of human scfvs with minimal amounts of antigen抗体需求快速方法生产的人类scfvs用最小数量的抗原.pdfVIP

Babel et al. BMC Biotechnology 2011, 11:61 /1472-6750/11/61 RESEARCH ARTICLE Open Access Antibodies on demand: a fast method for the production of human scFvs with minimal amounts of antigen † † * Ingrid Babel , Rodrigo Barderas , Alberto Peláez-García and J Ignacio Casal Abstract Background: Antibodies constitute a powerful tool to study protein function, protein localization and protein- protein interactions, as well as for diagnostic and therapeutic purposes. High-throughput antibody development requires faster methodologies with lower antigen consumption. Results: Here, we describe a novel methodology to select human monoclonal recombinant antibodies by combining in vitro protein expression, phage display antibody libraries and antibody microarrays. The application of this combination of methodologies permitted us to generate human single-chain variable fragments (scFvs) against two proteins: green fluorescent protein (GFP) and thioredoxin (Trx) in a short time, using as low as 5 μg of purified protein. These scFvs showed specific reactivity against their respective targets and worked well by ELISA and western blot. The scFvs were able to recognise as low as 31 ng of protein of their respective targets by western blot. Conclusion: This work describes a novel and miniaturized methodology to obtain human monoclonal recombinant antibodies against any target in a shorter time than other methodologies using only 5 μg of protein. The protocol could be easily adapted to a high-throughput procedure for antibody production. Keywords: scFv antibodies, in vitro protein expression, phage display, antibody microarrays Background [7-9]; ) are all good A crucial challe