Recombinant fusion proteins for haemagglutination-based rapid detection of antibodies to HIV in whole blood

Recombinant fusion proteins, consisting of a monovalent anti-human RBC monoclonal antibody B6, and conserved immunodominant peptide of HIV-1 envelope glycoprotein gp41 or HIV-2 envelope glycoprotein gp36, have been designed and purified after over-expression in E. coli. These fusion proteins are Fab-based and were obtained by assembling the light chain with Fd (variable domain and the first constant domain of the heavy chain) or Fd fusions containing HIV-derived peptide, and following a protocol of in vitro denaturation of inclusion bodies and subsequent renaturation to assemble functional Fab. Using a multistep column chromatographic procedure, monomeric Fab and Fab fusion proteins containing HIV-derived peptide were purified to high degree, free of aggregates. The yield of various proteins on the laboratory scale (1-2 l of shake flask culture) was in the range of tens of milligram. Purified anti-human RBC Fab fusion proteins containing sequences derived from HIV-1 gp41 and HIV-2 gp36 were highly specific for detection of antibodies to HIV-1 and HIV-2, respectively. The described design, expression and purification protocols will make it possible to produce specific recombinant reagents in large quantities for agglutination-based rapid detection of antibodies to HIV in whole blood.