Construction and preliminary identification of genetically engineered tetravalent antibodies against parathion

Abstract:

Abstract: [Objective]The objective is to improve the affinity of antibody against parathion with an aim to enhance the sensitivity of ELISA assay. [Methods] We recombined anti-parathion single-chain variable fragment gene (scfv) and the core streptavidin gene(sa) to produce an anti-parathion single-chain variable fragment-core of streptavidin-biotin fusion gene (scfv-sa), and inserted the fusion gene (scfv-sa) into the vector pET28a (+) in E.coli BL21 for transformation by prokaryotic expression. The expression was analyzed with SDS-PAGE and Western blot. the affinity of the recombinant protein was measured by ELISA after purified by metal affinity chromatography using Ni-NTA. [Results] The recombinant gene could express a fusion protein about 46 kDa, which formed a tetravalent domain, tetravalent antibody. ELISA results showed that the tetravalent antibody could bound to parathion specifically with the Affinity constant of 4.25 × 107 L / mol and titer of over 1:1×106. [Conclusion] The recombining anti-parathion tetravalent antibody could react with parathion specifically with significantly more binding sites with the monoclonal antibody, based on which the detection sensitivity of ELISA would be improved.

Key words:Keywords: parathion;core streptavidin ;tetravalent antibodies; prokaryotic expression