Comparison of Phage ELISA and RISE Detection Methods for Analysis of Phage Displayed Antibody Fragment

摘要

Selection for leading candidates is unavoidable during discovery of new antibodies. Since the first reports on application of filamentous phage fusions for protein display and selection the phage display method became the most popular way for discovering new antibodies. During this procedure mRNA isolated from either B-cells or splenocytes serves as a template for amplification of cDNA of antibody variable fragments. The recombinant fragments are usually cloned as single chain variable fragments (scFv) which can be expressed as a fusion with the phage coat protein on the surface of the phage. Naturally, the abilities of the recombinant scFv to bind the antigens of interest need to be confirmed. Many protocols recommend ELISA with antibody-displaying-phages preparation for such assessment (here reported as standard Phage ELISA), however a quicker solution, named RISE detection, has been also described [Ref.1]. The cDNA sequence encoding scFv fragment from monoclonal anti-H5 HA antibody (mAb) was cloned into phagemid pSEX81 vector and analysed by standard Phage ELISA and RISE detection. In both methods, phages exposing scFv on gpIII capsid protein were incubated with BSA or one of the H5 hemagglutinin (HA) from the following influenza strains: "Vietnam" (A/Vietnam/1203/2004(H5N1)), "Qinghai" (A/Bar headed goose/Qinghai/12/05 (H5N1)) or "Poland" (A/swan/Poland/305-135V08/2006(H5N1)). The first two HA variants were full-length, while the third was truncated and contain only the H1 region (17-340) of HA. RISE detection method resulted in the higher OD450 values than the standard Phage ELISA method. Those values were: 3.34±0.01 (mean±SD) for short form of "Poland" HA (17-340), 4.02±0.03 for "Qinghai" and 0.58±0.05 for "Vietnam", while the background OD450 (for BSA) was 0.31±0.04. Subsequent experiments indicated that the results of RISE detection are quite variable and that both, the concentration of phages exposing scFv and the time of incubation have impact on these results. Advantages and disadvantages of both methods (RISE detetion and standard Phage ELISA) are discussed.