Cloning, expression, purification, and characterization of a novel single-chain variable fragment antibody against the 2-nitrobenzaldehyde derivative of a furaltadone metabolite in Escherichia coli

Abstract

Furaltadone is an illicit veterinary drug that shows toxic, carcinogenic, and mutagenic effects, as does its metabolite 3-amino-5-morpholinomethyl-2-oxazolidone (AMOZ)1Abbreviations used: scFv, single-chain variable fragment; AMOZ, 3-amino-5-morpholinomethyl-2- oxazolidone; NPAMOZ, 2-nitrobenzaldehyde derivative of furaltadone metabolite 3-amino-5-morpholinomethyl-2-oxazolidone; VH, heavy chain variable region; VL, light chain variable region; SDS-PAGE, sodium dodecyl sulfate-polyacrylamide gel electrophoresis; Ni-NTA, nickel nitrilotriacetic acid; icCLEIA, indirect competitive chemiluminescence enzyme immunoassay; PAb, polyclonal antibody; MAb, monoclonal antibody; RAb, recombinant antibody; PCR, polymerase chain reaction; IPTG, isopropyl β-d-1-thiogalactopyranoside; RLU, relative light units.1. Recombinant antibodies with desirable affinity and specificity that can replace polyclonal or monoclonal antibodies are important factors for effective AMOZ immunoassays. In the present study, a novel single-chain variable fragment (scFv) antibody against the 2-nitrobenzaldehyde derivative of AMOZ (NPAMOZ) was prepared and characterized. The scFv gene was cloned into the pET-22b(+) expression vector, and 6His-tagged scFv antibodies expressed as inclusion bodies in Escherichia coli BL21 (DE3), which were then purified by nickel nitrilotriacetic acid column chromatography. Characterization of the target protein by sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS–PAGE), western blotting, and a novel indirect competitive chemiluminescence enzyme immunoassay (icCLEIA) showed that the scFv antibody was ∼27kDa and exhibited HRP-anti-His-tag antibody-recognized activity. The final purity, yield and mg of this scFv antibody after ultrafiltration concentration were 97%, 20% and 29.1mg, respectively. The icCLEIA indicated that the antibody competitively combined with NPAMOZ, exhibiting an IC50 value of 1.46±0.01ng/ml (n=6). Cross-reactivity studies revealed that the antibody showed desirable specificity to NPAMOZ and little reactivity to analogs except the parent furaltadone. In summary, these findings suggested that the prepared recombinant scFv antibody can be used for future immunoassay screening for AMOZ.