Design, synthesis and studies of triphosphate analogues for the production of anti AZT-TP antibodies.

Triphosphates anabolites are the active chemical species of nucleosidic reverse transcriptase inhibitors in HIV-therapy. Herein, we describe (i) the design of stable triphosphate analogues of AZT using molecular modelling, (ii) their synthesis and (iii) their use for producing anti AZT-TP antibodies in the aim of developing an immunoassay for therapeutic drug monitoring.

The production and evaluation of antibodies for enzyme immunoassay of AZTTP.

We describe the development of the first enzyme immunoassay for quantifying AZTTP that does not use of radioactive labeling. Anti-AZTTP antibodies were raised in rabbits by immunizing with an AZTTP-kelhoyle limpet hemocyanin (KLH) conjugate. Competitive immunoassays indicated a nanomolar sensitivity to AZTTP. One of the antisera produced was specific for AZTTP.

Monitoring of intracellular levels of 5'-monophosphate-AZT using an enzyme immunoassay.

We have developed a competitive enzyme immunoassay suitable for routine monitoring of intracellular levels of 5'-monophosphate-AZT (AZT-MP). This assay is performed in 96-well microtiter plates coated with anti-rabbit immunoglobulin antibodies and is based on the use of rabbit polyclonal antibodies raised against an AZT-MP analog and of an AZT-MP/acetylcholinesterase conjugate as tracer. It is very sensitive, with a detection limit close to 0.1 ng/ml (0.2 pmol/ml), and precise (CV < 20% from 20 to 0.3 ng/ml). Very low cross-reactivities were observed with AZT and the corresponding di- and triphosphate derivatives as well as with other related nucleotides and nucleosides. The validity of the assay was demonstrated by measuring intracellular concentrations of AZT-MP in peripheral blood mononuclear cells (PBMCs) and in monocyte-derived macrophages (MDMs) cultured in the presence of various concentrations of AZT (from 0.01 microM to 10 microM). We observed very high levels of AZT-MP in stimulated (PHA + IL2) PBMCs (> 100 pmol/10(6) cells) while, as expected, much lower concentrations were measured in resting PBMCs or MDMs (0.1 to 2 pmol/10(6) cells). The assay constitutes a very convenient tool permitting easy, precise studies of the first step of the intracellular metabolism of AZT leading to the formation of AZT-TP in cultured cells.

Stimulation of human gamma delta T cells by nonpeptidic mycobacterial ligands.

Most human peripheral blood gamma delta T lymphocytes respond to hitherto unidentified mycobacterial antigens. Four ligands from Mycobacterium tuberculosis strain H37Rv that stimulated proliferation of a major human gamma delta T cell subset were isolated and partially characterized. One of these ligands, TUBag4, is a 5' triphosphorylated thymidine-containing compound, to which the three other stimulatory molecules are structurally related. These findings support the hypothesis that some gamma delta T cells recognize nonpeptidic ligands.

Characterization of a monoclonal antibody to thymidine glycol monophosphate.

A monoclonal antibody specific for thymine glycol (TG) in irradiated or OsO4-treated DNA was obtained by immunizing with thymidine glycol monophosphate (TMP-glycol) conjugated to bovine serum albumin by a carbodiimide procedure. Screening by dot-immunobinding and enzyme-linked immunosorbant assay (ELISA) procedures gave eight clones that bound OsO4- treated DNA. One of them, 2.6F.6B.6C, an IgG2a kappa, was characterized further. Hapten inhibition studies with OsO4-treated DNA showed that the antibody was specific for TMP-glycol. Among the various inhibitors tested, inhibition was in the order TMP-glycol greater than 5,6-dihydrothymidine phosphate greater than TMP greater than thymidine glycol greater than TG. Inhibition by 5,6-dihydrothymidine, thymidine, thymine, AMP, and CMP was negligible. In OsO4-treated DNA, as few as 0.5 TG per 10,000 bp were detectable by direct ELISA. Inhibition assays could detect as few as 1.5 TG per 10,000 bp. The antibody was equally reactive with native or denatured DNA containing TG. Among the X-irradiated homopolymers dC, dA, dG, and dT, only dT reacted with the antibody. Using an ELISA, the antibody could detect damage in irradiated DNA at the level of 20 Gy. Thus the antibody is of potential use in assays for DNA damage caused by X rays or other agents that damage DNA by free radical interactions.

TMP-reactive autoantibodies in human SLE sera demonstrate thymine-dependent oligonucleotide specificity.

Autoantibodies present in the sera of lupus patients and specific for single-stranded (ss) DNA were fractionated into subsets based upon their reactivity towards 5' nucleotide haptens. As evaluated by ELISA testing, antibodies retained by TMP-agarose bound to TMP-BSA and ssDNA but not to other nucleotide-BSA conjugates or to double-stranded (ds) DNA. Competition-inhibition studies further revealed that TMP-enriched oligo- and polynucleotides were the preferred antigens for these affinity purified antibodies. Similar assays with sequence- or size- defined oligonucleotides further implied that those oligonucleotides comprised entirely of TMP residues were most antigenic and that antigenicity increased with size (length). These results document the existence of a TMP-dependent oligonucleotide specificity among a diverse population of autoanti-ssDNA antibodies.

[Antibodies against the photoproduct of thymidylyl (3'-5')-ribothymidine dinucleoside monophosphate]. / Anticorps anti le photoproduit du dinucléoside monophosphate thymidylyl (3'-5')-ribothymidine.

The synthesis of thymidylyl (3'-5')-ribothymidine (Tp5meU) is described. After ultraviolet irradiation., the dinucleoside was linked to serum albumin and then injected into rabbits. Antibodies against irradiated Tp5meU were purified by affinity chromatography. The specificity of the antibodies has been studied by radioimmunoassay.

Uracil-specific anti-R.N.A. antibodies in scleroderma.

Antibodies to single-stranded R.N.A. were found by counter immunoelectrophoresis in all of 40 sera from patients with scleroderma. These antibodies were specific to the uracil bases of R.N.A. Antibodies to R.N.A. were also found in 20 of 40 sera from patients with systemic lupus erythematosus (S.L.E.), but in none of forty controls. Antibodies to R.N.A. found in S.L.E. sera could be differentiated immunochemically from those found in scleroderma in that they were more heterogeneous and could react selectively with either uridine or uridine monophosphate. Antibodies ot D.N.A. were more frequent in S.L.E. than in scleroderma. That antibodies to D.N.A. are actually present in scleroderma and precipitin lines are not the result of cross reactivity with anti-R.N.A. antibodies is indicated by the finding that 10 of the 18 scleroderma sera which reacted with D.N.A. also reacted with thymidine, a base present in D.N.A. but not in R.N.A.