ASSAY AND PURIFICATION OF FV FRAGMENTS IN FERMENTER CULTURES - DESIGNAND EVALUATION OF GENERIC BINDING REAGENTS

Fv fragments whose genes have been cloned using common PCR primers car ry identical peptide motifs at their termini. We have raised antibodie s against the C-terminal motif of the VH chain GQGTTVTVSS and evaluate d their utility as reagents for the assay and purification of Fvs in f ermenter culture. Three different Fvs were included in the investigati on. We found that the motif was exposed and available for capture when Fv fragments were blotted onto nitrocellulose paper or adsorbed direc tly onto microtiter plates. In contrast, the motif was either partiall y or totally obscured when the Fv was complexed with immobilised antig en or when free in solution. This reactivity profile enabled us to dev elop a general-purpose assay for Fv protein, but not a general-purpose assay for monitoring active Fv. The apparent inaccessibilty of the C- terminus of VH conflicts with currently held views on the three-dimens ional structure of these molecules.