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. 2010 Mar;17(3):335-41.
doi: 10.1128/CVI.00283-09. Epub 2010 Jan 20.

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

Sheikh M Talha  1 , Teppo Salminen, Deepti A Chugh, Sathyamangalam Swaminathan, Tero Soukka, Kim Pettersson, Navin Khanna
Affiliations

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

Sheikh M Talha et al. Clin Vaccine Immunol. 2010 Mar.

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 third-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.

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Figures

FIG. 1.
FIG. 1.
The r-HIV-MEP antigen designed for this study. (A) Computer-generated graphic visualization (http://www.sbg.bio.ic.ac.uk/∼3dpssm) of r-HIV-MEP. (B) Complete nucleotide (lowercase letters) and predicted amino acid (capital letters) sequences of the r-HIV-MEP gene showing four epitopes (aa 1 to 51, p24 of HIV-1; aa 56 to 92, gp41 of HIV-1 group O; aa 97 to 124, gp36 of HIV-2; and aa 129 to 174, gp41 of HIV-1 group M) linked together with flexible tetraglycyl linkers (underlined). The asterisk indicates the engineered stop codon.
FIG. 2.
FIG. 2.
Multiple sequence alignment of the four r-HIV-MEP epitopes with the corresponding epitopes of different HIV types, groups, and subtypes (http://bioinfo.genotoul.fr/multalin/multalin.html). The black dots indicate identical residues. Variants are indicated by the standard single-letter amino acid code. Letters in the virus names indicate subtypes. In the case of HIV-1 group O gp41, the alignment has been done with different isolates within the group.
See this image and copyright information in PMC

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