Abstract
BackgroundAntibodies 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.ResultsHere, 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.ConclusionThis 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.
Highlights
Antibodies constitute a powerful tool to study protein function, protein localization and proteinprotein interactions, as well as for diagnostic and therapeutic purposes
Production of human single-chain Fv (scFv) against cell-free expressed antigens The cDNAs-encoding green fluorescent protein (GFP) and Trx were cloned into pIVEX and pET32b plasmids, respectively, and used for the in vitro transcription/translation reactions
The purity and homogeneity of the expressed proteins were confirmed by SDSPAGE and western blot using 10 μl of Rapid Translation System (RTS) reaction (Figure 1A, B)
Summary
Antibodies constitute a powerful tool to study protein function, protein localization and proteinprotein interactions, as well as for diagnostic and therapeutic purposes. Several high-throughput alternatives have been developed to generate antibodies to the entire proteome [3,4,5]. The production of mAbs and/or rabbit antibodies requires large amounts of antigens, it is time-consuming due to the immunization step of the animals and, in the case of mAbs, the screening and clone selection can take from 6 months to 1 year [10].The development of recombinant antibodies in single-chain Fv (scFv) formats is a good alternative to obtain high-affinity antibodies against any target without time-consuming immunization [11,12,13,14]. In vitro phage display pipelines have been setup to generate antibodies to the complete human proteome, but the selections are still carried out manually [8,9,22]. Screening of phage display antibody libraries is constrained by the necessity of having considerable amounts of antigen, at least 0.10.5 mg of protein for the whole procedure (selection, screening and validation)
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