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New sensitive and specific assay for human immunodeficiency virus antibodies using labeled recombinant fusion protein
H Siitari1, P Turunen, J Schrimsher
1Wallac Molecular Biology Laboratory, Turku, Finland.
Insights
A novel time-resolved fluoroimmunoassay (TR-FIA) offers a rapid, sensitive, and specific method for detecting human immunodeficiency virus type 1 (HIV-1) antibodies, enabling earlier diagnosis than current ELISA methods.
Area of Science:
- Immunology
- Virology
- Biochemistry
Background:
- Accurate and timely detection of human immunodeficiency virus type 1 (HIV-1) antibodies is crucial for patient management and public health.
- Existing enzyme-linked immunosorbent assays (ELISAs) have limitations in sensitivity and early detection of seroconversion.
Purpose of the Study:
- To develop and evaluate a new, rapid time-resolved fluoroimmunoassay (TR-FIA) for the detection of HIV-1 antibodies.
- To compare the sensitivity, specificity, and early detection capabilities of TR-FIA against commercially available recombinant ELISAs.
Main Methods:
- Development of a TR-FIA using a recombinant fusion protein containing HIV-1 p24 gag and gp41 env sequences.
- Coating microtitration strips with the recombinant antigen and simultaneous addition of europium-labeled antigen and test specimens.
- Comparative analysis using quality-control panels, dilution series of positive sera, seroconversion panels, and random serum specimens.
Main Results:
- The TR-FIA detected all 20 positive specimens in a quality-control panel, outperforming a commercial ELISA which detected 16 out of 20.
- TR-FIA identified HIV-1 antibodies 16 days earlier in seroconversion panels compared to six ELISAs.
- The assay demonstrated high specificity (99.9%) with only 1 false positive out of 57 repeatedly ELISA-positive, Western blot-negative samples.
Conclusions:
- The developed TR-FIA is a highly sensitive and specific method for HIV-1 antibody detection.
- TR-FIA offers significant advantages in early HIV-1 diagnosis, potentially improving patient outcomes.
- This assay represents a valuable advancement in HIV-1 diagnostic technologies.
Abstract:
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.