Gene/Protein Disease Symptom Drug Enzyme Compound
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Query: UMLS:C0021051 (immunodeficiency)
 71,517 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

We developed a non-radioisotopic (non-RI) reverse transcriptase assay (RTA). The reverse transcriptase (RT) incorporates biotin-11-deoxyuridine-triphosphate (bio-dUTP) using a poly(rA) template hybridized with oligo(dT) primer that is immobilized on the surface of a 96-well microtiter plate. This assay is thus semi-automated by adapting it to an ELISA testing format. The incorporation of bio-dUTP was enhanced by adding cold dTTP to the reaction mixture, optimally in a molar ratio 4:1 (dTTP:bio-dUTP). This non-RI RTA is more sensitive than the conventional RI assay for the detection of purified Rous-associated virus 2 (RAV-2) and of human immunodeficiency virus type 1 (HIV-1) lysate. Because of its simple procedure, higher sensitivity and non-use of RI materials, the assay can be utilized not only for virological studies but also for routine safety screening of biological products for retroviral contamination.
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PMID:A non-radioisotopic reverse transcriptase assay using biotin-11-deoxyuridinetriphosphate on primer-immobilized microtiter plates. 128 Jun 40

A one-step procedure which uses enzymes in a crude extract of herpes simplex virus (HSV) type 1-infected cells to synthesize 5-[125I]iodo-2'-deoxyuridine triphosphate [( 125I]dUTP) from [125I]dU is described. The design of a one-step procedure for the purification of the product is also presented. The recovery of [125I]dUTP from [125I]dU varied between 50 and 75%, the radiochemical purity of the product was greater than 90%, and both synthesis and purification were completed within 8 h. The sensitivity and specificity of [125I]dUTP as a substrate for both DNA-dependent DNA polymerase (DNAp) and RNA-dependent DNA polymerase (reverse transcriptase, RT) were evaluated and compared to those of [3H]dTTP for the following specimens: purified cloned Klenow fragment, crude extracts of HeLa-, BHK-, and HSV-2-infected BHK cells, purified avian myeloblastosis virus RT, and purified cloned human immunodeficiency virus (HIV) RT. The [125I]dUTP was accepted as a substrate equally as well [3H]dTTP by all of the specimens at all of the concentrations tested. When the same amount of radiolabel was used, [125I]dUTP gave a sensitivity 10- to 25-fold higher than that of [3H]dTTP. The gain in sensitivity was due to the higher specific activity and a higher counting efficiency of the 125I-label compound. The use of [125I]dUTP also offered technical advantages over alternative substrates available, such as product separation without acid precipitation and exclusion of the need for scintillation cocktails. The half-life of the nucleic also gives a reasonable shelf-life for use in routine assays. Activity of less than 0.3 pg of HIV RT could be detected when the new substrate was used, and this made it possible to quantitate HIV RT antibodies (abs) in diluted serum samples without purifying the immunoglobulin. Analysis of 31 HIV-infected individuals showed that all of them had anti-HIV RT ab and that the amount of serum needed for 50% inhibition of the HIV RT activity corresponded to an amount of immunoglobulin 100-fold smaller (i.e., 0.02-31.4 micrograms) than has been previously reported. With the substrate it was also possible to detect DNAp activity in serum from healthy individuals, although a long-duration assay was required. In a long-duration assay the DNAp activity found in sera from healthy individuals was linear with respect to time, whereas the DNAp activity found in many sera from tumor patients was not. [125I]dUTP is judged to be an excellent substrate for detecting and quantifying the activity of various DNA-synthesizing enzymes and their blocking abs.
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PMID:Improved assays for DNA-polymerizing enzymes by the use of enzymatically synthesized 5-[125I]iodo-2'-deoxyuridine triphosphate, illustrated by direct quantitation of anti-HIV reverse transcriptase antibody and by serum DNA polymerase analyses. 169 11

PCR is the most sensitive and direct method for detecting blood-borne viruses, as well as an efficient means for producing vector-free probes. However, the application of PCR, especially in the laboratory diagnosis of human immunodeficiency virus (HIV) infection, is impeded by the current use of radiolabeled oligonucleotide probes. Therefore, we have developed a nonisotopic PCR immunoreactive bead (PCR-IRB) assay to detect HIV type 1 proviral DNA from peripheral blood mononuclear cells (PBMC). We used a biotinylated primer in a set of three oligonucleotides selected from the HIV long terminal repeat region for heminested PCR amplification. An internal probe was synthesized by PCR with incorporation of digoxigenin-labeled dUTP. After solution hybridization of the probe with PCR-amplified products (amplicons), the hybridized DNA was captured with streptavidin-coated magnetic beads. For the detection of hybrids, flow cytometric analyses were carried out by two procedures: (i) direct detection with fluorescein isothiocyanate (FITC)-labeled antidigoxigenin immunoglobulin G (IgG) antibody and (ii) indirect detection with antidigoxigenin sheep IgG antibody followed by FITC-labeled anti-sheep IgG antibody. Both procedures in the PCR-IRB assay detected two to three copies of HIV proviral DNA sequences, a sensitivity that is comparable with that of the conventional radioactive detection of amplicons following probe hybridization and electrophoresis. To compare the PCR-IRB assay with the conventional method, we tested 53 pedigreed PBMC specimens from blood donors and newborns; the results obtained were identical. This nonisotopic PCR-IRB assay can also be automated for potential application in laboratory diagnosis of HIV infection, blood bank screening, and therapeutic monitoring of viremia and perinatal transmission.
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PMID:Flow cytometric immunodetection of human immunodeficiency virus type 1 proviral DNA by heminested PCR and digoxigenin-labeled probes. 749 17

An assay for detection of the reverse transcriptase (RT) of the human immunodeficiency virus (HIV) was developed using poly A linked to microtiter plate with colorimetric detection of incorporated biotin deoxyuridine triphosphate (biotin-dUTP). During the RT reaction, biotin-dUTP was incorporated into oligodeoxythymidylic acid (oligo-dT) which had been hybridized with poly A. At the detection step, horseradish peroxidase conjugated streptavidin was added, followed by the reaction of a colorimetric substrate for this enzyme. This method was contrasted with the two standard isotopic RT assays. There was excellent correlation between the colorimetric RT assay and each of two isotopic RT assays for both detection and quantification of avian myoblastosis virus reverse transcriptase (AMV-RT) and of HIV RT in human lymphocytes infected in vitro with HIV-1. The total assay required for performing the colorimetric assay, including the RT reaction, was 40 min.
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PMID:Poly A-linked colorimetric microtiter plate assay for HIV reverse transcriptase. 750 83

A new method is described which is suitable for reliably analysing apoptotic fragmentation in small amounts of DNA. After isolation, DNA was labelled with biotin-4-dUTP using Klenow polymerase. Then DNA was size-separated by agarose gel electrophoresis, blot transferred and subsequently visualized by the streptavidin alkaline phosphatase-BCIP/NBT procedure. This non-radioactive method was used to detect apoptotic DNA in rat pheochromocytoma PC12 cells, treated with tributyltin (1 nM). While only 30 ng of DNA is required for analysis of apoptotic DNA using the new blot technique, 100-fold more material is needed to identify the fragmentation of DNA after separation by agarose gel electrophoresis and direct staining with ethidium bromide. In a further set of experiments, rat cortical cells were incubated with human immunodeficiency virus type 1 viral glycoprotein of M(r) of 120 kDa (gp120) to induce apoptosis. More than 0.3 ng of gp 120/ml are required to detect apoptotic DNA by the direct procedure; only 0.1 ng gp120/ml or less were sufficient to document clear DNA fragmentation using the non-radioactive blotting technique described here. These results demonstrate that the new procedure can be used to analyse very small amounts of apoptotic DNA and shows that gp120-induced apoptosis can be measured at low concentrations of the viral protein.
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PMID:A non-radioactive, sensitive method for the detection of DNA fragmentation in apoptotic cells (rat pheochromocytoma PC12 and rat cortical cells). 752 53

An enzyme-linked immunosorbent assay (ELISA) using biotin-labelled oligo-dT primer and digoxigenin (Dig)-dUTP was designed to measure the reverse transcriptase (RT) activity of human immunodeficiency virus type 1 (HIV-1). The ELISA system involves the selective detection step of a newly synthesized cDNA by two specific bindings, biotin-streptavidin binding and alkaline phosphatase (AP)-conjugated anti-Dig-Dig binding, and the enzymatic amplification step to increase coloring generated by AP. This method was used to measure the activity of RT in the culture supernatants of peripheral leukocytes obtained from four anti-HIV-1-positive persons cocultivated with those from four anti-HIV-1-negative persons. RT activity was detected in all of four anti-HIV-1-positive culture supernatants but not in those cultivated with anti-HIV-1-negative supernatants alone. Thus, our improved ELISA for detection of HIV-1 appears to be sensitive enough and useful for routine laboratory work. This non-radioactive method will also be useful for detecting other retroviruses and for screening of RT inhibitors.
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PMID:Detection of reverse transcriptase activity by enzyme-linked immunosorbent assay in human immunodeficiency virus type 1. 754 28

G-->A hypermutation is a remarkable phenomenon resulting from retroviral reverse transcription in the presence of highly biased dNTP concentrations. Of the three reverse transcriptases (RTases) available, those of human immunodeficiency virus type 1 (HIV-1), avian myeloblastosis virus (AMV) and Moloney murine leukemia virus (MoMLV), the HIV-1 enzyme showed the greatest sensitivity to biased [dCTP]/[dTTP] ratios. The HIV-1 RTase was able to discriminate between dUTP, dITP and the four DNA precursors and was insensitive to pH. There was little preference for nucleotide contexts. A few exceptionally modified sequences were found presumably resulting from G-->A hypermutation and multiple strand transfer. This particular predilection of the HIV-1 and, by extrapolation, the lentiviral RTases towards G-->A hypermutation suggests that the phenomenon may have contributed to the remarkably elevated A content of these retroviral genomes.
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PMID:Reverse transcriptase and substrate dependence of the RNA hypermutagenesis reaction. 754 58

The human immunodeficiency virus-1 reverse transcriptase (HIV-1 RT) heterodimer (M(r) = 66,000 and M(r) = 51,000) has been photoaffinity labeled using 4-thiodeoxyuridine triphosphate (S4-dUTP) as a probe. A nascent polymerization complex was assembled from a single-stranded DNA template, a 12-mer DNA primer, and the necessary dNTPs (one of which was alpha-32P-labeled) to extend the primer to produce the n-1 product. The photoaffinity probe was then uniquely added at the 3'-terminal position of the extended primer bound at the catalytic site and photolyzed. The larger subunit (p66) was exclusively derivatized. The unique radioactive peptide resulting from proteolysis was isolated and identified by amino acid sequencing.
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PMID:Active site labeling of HIV-1 reverse transcriptase. 768 13

A colorimetric assay for detection of reverse transcriptase (RT) of the human immunodeficiency virus (HIV) was developed using oligodeoxythymidylic acid (oligo-dT)-linked magnetic beads and digoxigenin-deoxyuridine triphosphate (dig-dUTP). During the RT reaction, dig-dUTP was incorporated into oligo-dT which had been hybridized to polyadenylic acid [poly (A)]. At the detection step, an alkaline phosphatase-conjugated antibody to digoxigenin was added, followed by the addition of a colorimetric substrate for this enzyme. This method showed excellent correlation with the isotopic RT assay, which used tritiated thymidine triphosphate ([3H]dTTP), for detection of purified avian-myeloblastosis-virus RT (AMV-RT). This assay also demonstrated close correlation with the isotopic RT assay using human peripheral-blood lymphocytes infected in vitro with HIV. This colorimetric RT assay offers important advantages over the conventional radioactive RT assays with respect to its simplicity, safety and cost. The total assay time, including the RT reaction step, was less than 1 h, and therefore provides a reliable rapid assay for detection and quantification of HIV.
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PMID:Detection of human immunodeficiency virus (HIV) by colorimetric assay for reverse transcriptase activity on magnetic beads. 769 Oct 79

We have developed a new non-isotopic polymerase chain reaction (PCR) based method for the detection of the human immunodeficiency virus type 1 (HIV-1) DNA in clinical samples. In this method a two step PCR is used to amplify and label HIV-1 DNA segments by incorporation of digoxigenin-11-dUTP (dig-dUTP). Digoxigenin labelled amplified products are hybridized to membrane immobilized complementary DNA probes. Hybridization is detected non-radioactively by incubating the filters with antidigoxigenin antibody conjugated with alkaline phosphatase followed by a standard phosphatase assay. With this method the detection limit was between 1 and 10 HIV-1 DNA copies in a background of 1 microgram of human genomic DNA. Furthermore, we were able to detect HIV-1 DNA in 41 out of 41 HIV-1 antibody positive individuals while 10 out of 10 HIV-1 seronegative individuals gave consistently negative results. Our data indicate that this simple non-isotopic technique is sensitive and specific for the detection of HIV-1 DNA in clinical samples and can constitute a good alternative to other non-isotopic methods described to date.
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PMID:Detection of HIV-1 DNA by polymerase chain reaction incorporation of digoxigenin-11-dUTP and hybridization to immobilized probes. 796 98


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