Recombinant single-chain Fv antibody fragment-alkaline phosphatase conjugate for one-step immunodetection in molecular hybridization

Using phage-display technology, a recombinant single-chain Fv antibody frag ment (scFv) was rapidly generated from the K16-16 hybridoma secreting mouse monoclonal antibody (MAb) that binds to acetylaminofluorene-labeled DNA (A AF-DNA). The selected A4 phage-scFv specifically bound to AAF-DNA. The anti -AAF scFv gene was then recloned into a fusion vector for the production of a hybrid protein comprising the antibody fragment fused to a potent bacter ial alkaline phosphatase variant (PhoAv), The anti-AAF scFv-PhoAv hybrid pr otein was bifunctional and possessed both antigen binding capacity and PhoA activity. The recombinant conjugate was directly used, without further pur ification, for one-step immunodetection in dot-blot hybridization. The dete ction limit was identical and the test was quicker than the conventional tw o-step procedure with the purified anti-AAF MAb revealed with a secondary e nzyme-labeled antibody. To assess the value of this new reagent for the imm unodetection of genomic nucleic acids, genomic DNAs of Campylobacter jejuni and Campylobacter coli were then one-step immunodetected with non-purified recombinant scFv-PhoAv conjugate in a Southern-blot hybridization experime nt. The present study shows that the genetic fusion with PhoAv provides a n ew tool for immunodetection which presents easier and quicker production an d use with the same sensitivity and specificity as classical reagents. The recombinant anti-AAF scFv-PhoAv conjugate is a promising alternative reagen t for applications involving the immunodetection of specific DNA or RNA seq uences, such as the detection and characterization of microorganisms. (C) 1 999 Elsevier Science B.V. All rights reserved.