Development of a sensitive ELISA for HIV-1 p24 antigen using a fluorogenic substrate for monitoring HIV-1 replication in vitro

• Published in: Journal of virological methods Vol. 38; no. 3; p. 323
• Main Authors: Chargelegue, D, O'Toole, C M
• Format: Journal Article
• Language: English
• Published: Netherlands 01-08-1992
• Subjects: Acquired Immunodeficiency Syndrome - diagnosis; Antibodies, Monoclonal; Dose-Response Relationship, Immunologic; Enzyme-Linked Immunosorbent Assay - methods; HIV Core Protein p24 - analysis; HIV-1 - physiology; Humans; In Vitro Techniques; Sensitivity and Specificity; Time Factors; Virus Replication
• ISSN: 0166-0934
• DOI: 10.1016/0166-0934(92)90077-Q

A two-site sandwich fluorescent-ELISA was optimized for the detection of HIV-1 p24 antigen produced by lymphoid cells infected with HIV-1 in vitro. To improve the sensitivity of the ELISA, a combination of streptavidin-beta-galactosidase and a fluorogenic substrate (4-methylumbelliferyl-beta-D-galactopyranoside) was employed for the enzymatic detection stage. Using recombinant p24 as standard antigen, a two-step assay detected as little as 0.7 pg/ml (3.10(-14) M) with an upper limit of 10,000 pg/ml. This detection range (approx. 50-70-times greater than ELISAs using a chromogenic detection) permitted an accurate and straightforward quantitation of p24 in culture supernatants. Overall, the fluorescent-ELISA had increased detectability, sensitivity and efficiency over existing ELISAs for HIV-1 p24.