Production of single-chain Fv antibodies against melamine from a rabbit phage display library for the development of a melamine immunoassay

A rabbit phage display library of single chain Fv (scFv) antibodies was constructed to screen specific scFv antibodies against melamine. Rabbits were immunized with melamine immunogens prepared via glutaraldehyde method. Two recombinant scFv antibodies with high specificity for melamine, termed A9 and B4, were isolated via library panning. SDS-PAGE analysis showed that the soluble A9-scFv and B4-scFv antibodies were expressed successfully and the scFvs showed good sensitivity with an IC₅₀ of 12.64 and 10.32 μM to melamine, respectively. The cross-reactivity of 2 scFv antibodies with cyanuric acid and chloromycetin were below 1%. The high similarity of 2 scFv nucleotide sequences indicated that they may originate from 1 B cell clone that had undergone somatic diversification. As a consequence, the conjugation synthesis was successful and the selected scFvs could be used for sensitive detection of melamine in foods and feeds by immunoassay.