Development of a sensitive ELISA for HIV-1 p24 antigen using a fluorogenic substrate for monitoring HIV-1 replication in vitro.
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.
Chargelegue
D
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Department of Medical Microbiology, London Hospital Medical College, UK.
O'Toole
C M
CM
D000163:Acquired Immunodeficiency Syndrome / Q000175:diagnosis; D000911:Antibodies, Monoclonal; D004306:Dose-Response Relationship, Immunologic; D004797:Enzyme-Linked Immunosorbent Assay / Q000379:methods*; D016655:HIV Core Protein p24 / Q000032:analysis*; D015497:HIV-1 / Q000502:physiology*; D006801:Humans; D066298:In Vitro Techniques; D012680:Sensitivity and Specificity; D013997:Time Factors; D014779:Virus Replication
D000911:Antibodies, Monoclonal; D016655:HIV Core Protein p24