Inexpensive designer antigen for anti-HIV antibody detection with high sensitivity and specificity

Abstract

A novel recombinant Multiepitope Protein (MEP) has been designed that consists of four linear, immunodominant and phylogenetically conserved epitopes, taken from Human Immunodeficiency Virus (HIV)-encoded antigens that are used in many t hird-generation immunoassay kits. This HIV-MEP has been evaluated for its diagnostic potential in the detection of anti-HIV antibodies in human sera. A synthetic MEP gene encoding these epitopes, joined by flexible peptide linkers in a single open reading frame, was designed and overexpressed in Escherichia coli. The recombinant HIV-MEP was purified using a single affinity step, yielding >20 mg pure protein/liter culture and used as the coating antigen in an in-house immunoassay. Bound anti-HIV antibodies were detected by highly sensitive time-resolved fluorometry, using europium (III) chelate-labeled anti-human antibody. The sensitivity and specificity of the HIV-MEP were evaluated using Boston Biomedica worldwide HIV performance, HIV seroconversion and viral coinfection panels and were found to be comparable with those of commercially available anti-HIV Enzyme Immunoassay (EIA) kits. The careful choice of epitopes, high epitope density and an E. coli-based expression system, coupled with a simple purification protocol and the use of europium (III) chelate-labeled tracer, provide the capability for the development of an inexpensive diagnostic test with high degrees of sensitivity and specificity.