Measurement of the anti-HIV agent 2′,3′-didehydro-2′,3′-dideoxythymidine (D4T) by competitive ELISA

• Published in: Journal of immunological methods Vol. 176; no. 1; pp. 103 - 110
• Main Authors: Ferrua, Bernard, Tran, Thanh Thu, Quaranta, Jean François, Kubar, Joanna, Roptin, Clotilde, Condom, Roger, Durant, Jacques, Guedj, Roger
• Format: Journal Article
• Language: English
• Published: Amsterdam Elsevier B.V 10.11.1994; Elsevier
• Subjects: 2′,3′-Didehydro-2′,3′-dideoxythymidine; AIDS; AIDS/HIV; Animals; Antibiotics. Antiinfectious agents. Antiparasitic agents; Antiviral agents; Binding, Competitive - immunology; Biological and medical sciences; Cell Line; Cells, Cultured; Chromatography, high pressure liquid; Cross Reactions; ELISA; Enzyme-Linked Immunosorbent Assay - methods; Female; Fundamental and applied biological sciences. Psychology; Fundamental immunology; HIV; HIV Infections - drug therapy; HIV-1 - drug effects; human immunodeficiency virus; Humans; Immunodeficiencies; Immunodeficiencies. Immunoglobulinopathies; Immunopathology; Medical sciences; Molecular immunology; Pharmacology. Drug treatments; Rabbits; Reproducibility of Results; Sensitivity and Specificity; Stavudine - analysis; Stavudine - therapeutic use; T-Lymphocytes - cytology; T-Lymphocytes - drug effects; Techniques; PBS; D4T-MP; D4T; HRP; AIDS; Chromatography, high pressure liquid; PBMC; BSA; HIV; D4T-HS; AZT; 2′,3′-Didehydro-2′,3′-dideoxythymidine; HPLC; ELISA; Human; Cell culture; Plasma; Retroviridae; HPLC chromatography; Thymidine; Monoclonal antibody; Infection; Virus; Viral disease; Antiviral; Lentivirinae; Human immunodeficiency virus; ELISA assay
• ISSN: 0022-1759; 1872-7905
• DOI: 10.1016/0022-1759(94)90354-9

2′,3′-didehydro-2′,3′-dideoxythymidine (D4T) is a thymidine analogue with potent anti-HIV activity in vitro and is currently being investigated therapeutically in patients with advanced HIV infection. We describe a first one-step competitive ELISA method developed for D4T measurement. Anti-D4T rabbit antibodies were raised against a D4T hemisuccinate-bovine serum albumin immunogen. A D4T-hemisuccinate-horseradish peroxidase conjugate and a monoclonal anti-rabbit IgG antibody insolubilized onto a microtiter plate were used as a tracer and capture system, respectively. The method was capable of detecting 2 ng/ml of D4T in cell cultures and 20 ng/ml of D4T in plasma samples previously separated in microconcentrator devices. Cross-reactivit y analysis showed that thymidine, D4T monophosphate, or azidothymidine, were weakly recognized by the ELISA and that thymine or other nucleosides were unreactive. The test was successfully used for the quantification of D4T in cell extracts from CEM or Molt 4 cell lines cultured with D4T and in the plasma of patients with advanced HIV infection, receiving D4T therapy. Moreover this ELISA could be used for the indirect quantification of D4T phosphorylated intracellular metabolites previously separated by reverse phase HPLC and hydrolyzed with alkaline phosphatase.