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

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-reactivity 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.

Intracellular concentration of protease inhibitors in HIV-1-infected patients: correlation with MDR-1 gene expression and low dose of ritonavir.

BACKGROUND: Protease inhibitors (PIs) are substrates for the P-glycoprotein (P-gp/170) encoded by the multi-drug resistance gene (MDR-1). HIV infection is associated with increased expression of P-gp. The role of MDR gene overexpression in clinical pharmacokinetics is not known. METHOD: We determined by HPLC, at trough and peak levels, the current PI concentrations in plasma (P) and in peripheral blood mononuclear cells (PBMCs) (intracellular concentration [IC]) from 49 HIV-infected patients receiving different treatment combinations: nelfinavir ([NFV] n = 12); indinavir ([IDV] n = 10); amprenavir ([APV] n = 5); ritonavir (RTV) 100 bid/IDV 800 mg bid (n = 6); RTV 400 bid/IDV 400 mg bid (n = 3); RTV 100 bid/saquinavir (SQV) 600 mg tid (n = 9); APV 600 bid/RTV 100 mg bid (n = 4). We determined the mean ratio of intracellular/plasma PI concentration for each treatment group. The MDR-1 gene expression was determined by a semi-quantitative reverse transcriptase polymerase chain reaction (RT-PCR). HIV viral load was simultaneously measured. RESULTS: 49 patients (mean age 41 +/- 8.7 years; mean CD4 cell count 418 [57-972]; mean HIV RNA 2.1 +/- 0.8 log(10)) were included in the study. Patients who overexpressed the MDR-1 gene had significantly lower trough intracellular PI levels (p =.02) or lower intracellular accumulation of PI (p =.042). Patients treated with low-dose RTV in combined regimens with detectable RTV intracellular concentration showed lack of MDR-1 gene expression (p =.01). Patients with HIV RNA < 40 copies/mL had significantly higher RTV intracellular accumulation (p =.029). CONCLUSION: In HIV-infected patients, IC of PI is inversely correlated with MDR-1 gene overexpression. Undetectable viral load was associated with the use of low-dose RTV, probably linked to better intracellular accumulation of the drug. Nevertheless, further investigation is needed to confirm these results.

Quantitation of Leishmania infantum in tissues of infected BALB/c mouse by sandwich ELISA.

In this report, a sandwhich ELISA was developed to quantify spleen and liver burdens from L. infantum-infected BALB/c mice. Amastigote antigens obtained following Nonidet P40 extraction of parasite-harbouring tissues were captured by anti-L. infantum human IgG insolubilized onto microtiter plate and subsequently revealed with anti-L. infantum F(ab)' fragments labelled with peroxidase. The method was easy to perform, precise and capable to specifically and accurately detect 5 x 10(4) amastigotes/100 mg tissue. Parasite burdens from infected BALB/c mice, in various conditions, were measured by ELISA and Giemsa-stained touch imprint reference methods, and compared. Both techniques agreed well with close values for liver burdens, but the spleen loads measured by the ELISA were, on average, 10.7 times higher than those calculated from imprints. This difference was attributed partly to the underestimation brought by Stauber's formula. However, it did not preclude the usefulness of this newly developed test, since results obtained in kinetics studies and evaluation of the efficiency of leishmanicidal drugs allowed us to draw identical conclusions.

Quantitation of 3'-amino-3'-deoxythymidine (AMT), a toxic catabolite of 3'-azido-3'-deoxythymidine (AZT), by competitive ELISA.

In the present study, a competitive ELISA technique was developed to specifically quantitate 3'-amino-3'-deoxythymidine (AMT), a toxic catabolite of 3'-azido-3'-deoxythymidine (AZT) detected in serum from AZT-treated patients. In order to eliminate cross-reacting AZT, serum sample was extracted with ethylacetate and then AMT was acetylated (Ac-AMT). A 5'-hemisuccinate-AMT-horseradish peroxidase conjugate was used as a tracer in the presence of anti-AMT rabbit antibodies which were raised against a 5' hemisuccinate-AMT-bovine serum albumin immunogen. Bound/free separation was achieved with an anti-rabbit IgG mouse monoclonal antibody insolubilized onto a microtiter plate. The limit of quantification of Ac-AMT was as low as 0.4 ng/ml in serum samples. This ELISA technique was applied for monitoring AMT plasma levels in patients receiving AZT therapy. The intra and inter-individual variations of the AZT/AMT plasma concentration ratios underlined the need for such a specific test in studying the formation of this toxic catabolite.