New sensitive and specific assay for human immunodeficiency virus antibodies using labeled recombinant fusion protein and time-resolved fluoroimmunoassay.
A new, rapid method for the detection of human immunodeficiency virus type 1 (HIV-1) antibody by time-resolved fluoroimmunoassay (TR-FIA) was developed. In this assay format, microtitration strips were coated with a recombinant fusion protein, and the same protein was labeled with europium and added into the wells simultaneously with the test specimens. The recombinant fusion protein contained the HIV-1 p24 gag protein sequence that carried an insertion, near the carboxyl terminus, of a 23-amino-acid sequence from a highly conserved region of the HIV-1 gp41 envelope protein. This recombinant antigen enabled the detection of antibodies to both gag and env gene products. When this assay was compared with a commercially available recombinant enzyme-linked immunoabsorbent assay (ELISA) by using four quality-control panels, the TR-FIA detected all 20 positive specimens, while the recombinant ELISA detected only 16 of them. This increased sensitivity could be demonstrated directly by the assay of dilution series of HIV-1-positive sera. The analysis of two seroconversion panels by TR-FIA and six ELISAs showed that TR-FIA allowed detection of antibody in infected individuals 16 days earlier than the other assays did. In addition to being highly sensitive, the assay was highly specific; of the 57 samples shown to be repeatedly positive by ELISA but known to be HIV-1 negative by Western immunoblot analysis, only 1 sample reacted positively in this assay. The specificity of the assay was 99.9% when 1.054 random serum specimens were tested.
Siitari
H
H
Wallac Molecular Biology Laboratory, Turku, Finland.
Turunen
P
P
Schrimsher
J
J
Nunn
M
M
D000595:Amino Acid Sequence; D015153:Blotting, Western; D004797:Enzyme-Linked Immunosorbent Assay; D005069:Evaluation Studies as Topic; D015200:Fluoroimmunoassay; D015683:Gene Products, gag / Q000276:immunology; D015483:HIV Antibodies / Q000032:analysis*; D015488:HIV Antigens; D016655:HIV Core Protein p24; D015497:HIV-1 / Q000276:immunology*; D006801:Humans; D008969:Molecular Sequence Data; D011993:Recombinant Fusion Proteins / Q000276:immunology; D014758:Viral Core Proteins / Q000276:immunology
D015683:Gene Products, gag; D015483:HIV Antibodies; D015488:HIV Antigens; D016655:HIV Core Protein p24; D011993:Recombinant Fusion Proteins; D014758:Viral Core Proteins