CONSTRUCTION OF RECOMBINANT EXTENDED SINGLE-CHAIN ANTIBODY PEPTIDE CONJUGATES FOR USE IN THE DIAGNOSIS OF HIV-1 AND HIV-2

The construction, expression and evaluation of recombinant scFv based HIV diagnostic reagents are described, In a whole-blood, erythrocyte a gglutination assay format, recombinant scFv antibodies (expressed in E scherichia coli), linked to a spacer domain and HIV-gp36 or -gp41 pept ides, were shown to be able to detect efficiently natural antibodies a gainst HIV in human serum, Performance in trials suggests that these s ingle chain reagents have potential as alternatives to existing Fab-pe ptide chemical conjugates. We also report the construction of an induc ible expression vector, pGC, which can be used both in laboratory expe riments and in large-scale fed-batch fermentations. It was found that while the base scFv reagent (lacking a spacer) functioned as well as t he Fab peptide conjugate in assays where whole (negative) blood was sp iked with mouse monoclonal anti-HIV antibodies (IgG or IgM), clinical assays using human sera showed lower sensitivities and increased false negatives, This deficiency was overcome by inclusion of the natural 1 C3 kappa (light) chain domain as a spacer arm between the scFv and HIV peptide tags. This spacer was thought to overcome steric constraints which would otherwise prevent efficient interaction between the reagen t (once bound to the surface of red blood cells) and the various serum antibodies against the respective C-terminal peptide epitopes. As a r esult of this important modification, performance of the extended scFv reagent (for both HIV-1 and HlV-2) equalled that of the current comme rcial technology in limited trials.