Inexpensive designer antigen for anti-HIV antibody detection with high sensitivity and specificity.
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.
Talha
Sheikh M
SM
ecombinant Gene Products Group, International Centre for Genetic Engineering & Biotechnology, Aruna Asaf Ali Marg, New Delhi 110067, India.
Salminen
Teppo
T
Chugh
Deepti A
DA
Swaminathan
Sathyamangalam
S
Soukka
Tero
T
Pettersson
Kim
K
Khanna
Navin
N
D000595:Amino Acid Sequence; D000956:Antigens, Viral / Q000235:genetics / Q000276:immunology*; D001483:Base Sequence; D015483:HIV Antibodies / Q000097:blood* / Q000276:immunology; D015658:HIV Infections / Q000175:diagnosis* / Q000276:immunology; D015497:HIV-1 / Q000235:genetics; D015498:HIV-2 / Q000235:genetics / Q000276:immunology; D006801:Humans; D007118:Immunoassay / Q000191:economics / Q000379:methods*; D016056:Immunodominant Epitopes / Q000276:immunology; D008969:Molecular Sequence Data; D015202:Protein Engineering; D011933:Reagent Kits, Diagnostic / Q000191:economics*; D011994:Recombinant Proteins / Q000235:genetics / Q000276:immunology; D012680:Sensitivity and Specificity
D000956:Antigens, Viral; D015483:HIV Antibodies; D016056:Immunodominant Epitopes; D011933:Reagent Kits, Diagnostic; D011994:Recombinant Proteins