Gene/Protein Disease Symptom Drug Enzyme Compound
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Query: EC:2.7.7.49 (reverse transcriptase)
 31,746 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Antibody immunoglobulin G (IgG) to human immunodeficiency virus type 1 (HIV-1) in serum was detected by ultrasensitive enzyme immunoassays (immune complex transfer enzyme immunoassays) with recombinant reverse transcriptase (rRT), p17 (rp17) and p24 (rp24) of HIV-1 as antigens and beta-D-galactosidase from Escherichia coli as the label. The immune complex, comprising 2,4-dinitrophenyl-bovine serum albumin-recombinant protein conjugate, antibody IgG to HIV-1, and recombinant protein-beta-D-galactosidase conjugate, was trapped on polystyrene beads coated with affinity-purified (anti-2,4-dinitrophenyl group) IgG, eluted with epsilon N-2,4-dinitrophenyl-L-lysine, and transferred to polystyrene beads coated with affinity-purified (anti-human IgG gamma-chain) IgG. Bound beta-D-galactosidase activity was assayed by fluorometry. The assays were highly reproducible with no serious serum interference, and they were much more sensitive than Western immunoblotting for the corresponding antigens. Signals with rRT, rp17, and rp24 for asymptomatic carriers were at least 56,000-, 680-, and 22-fold higher, respectively, than those for seronegative individuals, and neither indeterminate nor false-positive results were observed, whereas some serum samples were false negative or false positive by Western blotting for p17 and/or p24 antigen. In some cases, seroconversion was detected earlier than by conventional methods. Therefore, these assays are suggested to be more useful than conventional methods not only for the confirmation of antibody IgGs to RT, p17, and p24 of HIV-1 in serum but also for the detection of seroconversion.
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PMID:Immune complex transfer enzyme immunoassay that is more sensitive and specific than western blotting for detection of antibody immunoglobulin G to human immunodeficiency virus type 1 in serum with recombinant pol and gag proteins as antigens. 854 31

Serum samples of four HIV-1 seroconversion serum panels were subjected in a single assay tube simultaneously to ultrasensitive enzyme immunoassays (immune complex transfer enzyme immunoassays) for p24 antigen of HIV-1 and for antibody IgGs to p17 and reverse transcriptase (RT) of HIV-1. Signals became positive 7-15 days earlier than the detection of antibodies to HIV-1 by conventional methods and remained strongly positive even after levels of p24 antigen declined. Thus the simultaneous detection of p24 antigen and antibody IgGs to p17 and RT made possible both as early a diagnosis of HIV-1 infection as the appearance of p24 antigen in the circulation, shortening "the window period," and as reliable a diagnosis of the infection as that by the detection of antibodies to HIV-1 from the time of seroconversion until late stages of the infection, since the serum level of antibody IgG to RT was high not only in asymptomatic carriers but also in patients with AIDS-related complex and AIDS.
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PMID:Earlier diagnosis of HIV-1 infection by simultaneous detection of p24 antigen and antibody IgGs to p17 and reverse transcriptase in serum with enzyme immunoassay. 881 65

Following HIV infection, there is a window period of 6-8 weeks, during which HIV antibodies are not detectable and the infection cannot be diagnosed by methods for detecting HIV antibodies. However, HIV antigens are detectable in the latter part of the window period, although the level of HIV antigens declines as the level of HIV antibodies increases. We developed an ultrasensitive enzyme immunoassay (immune complex transfer enzyme immunoassay) for the simultaneous detection of both p24 antigen of HIV-1 and antibody IgGs to p17 and reverse transcriptase of HIV-1 in a single assay tube and tested 11 HIV-1 seroconversion serum panels and serum samples randomly collected from 79 HIV-1 seropositive subjects and 100 HIV-1 seronegative subjects. The simultaneous detection was shown not only to shorten the window period significantly as compared with conventional methods for HIV-1 antibody detection but also to make possible a reliable diagnosis of HIV-1 infection from the time of seroconversion until late stages of the infection.
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PMID:Shortening of the window period in diagnosis of HIV-1 infection by simultaneous detection of p24 antigen and antibody IgG to p17 and reverse transcriptase in serum with ultrasensitive enzyme immunoassay. 891 Jun 47

Ultrasensitive enzyme immunoassays (immune complex transfer enzyme immunoassays) were developed for antibody IgGs to HIV-1 using recombinant reverse transcriptase (rRT), p17 (rp17), and p24 (rp24) as antigens. Antibody IgGs were reacted with 2,4-dinitrophenyl-recombinant antigens and recombinant antigen-beta-D-galactosidase conjugates, and the immune complexes formed, comprising the three components, were trapped onto polystyrene beads coated with (anti-2,4-dinitrophenyl group) IgG. After washing, the immune complexes were eluted from the polystyrene beads with excess of epsilon N-2,4-dinitrophenyl-L-lysine and were transferred to clean polystyrene beads coated with (antihuman IgG gamma-chain) IgG. beta-D-Galactosidase activity bound to the last polystyrene beads was assayed by fluorometry. By transfer of the immune complexes from one solid phase to another, the nonspecific binding of the beta-D-galactosidase conjugates was minimized and the sensitivity was markedly improved. The immune complex transfer enzyme immunoassays using rRT, rp17, and rp24 as antigens were 300-1,000-fold, 1,000-3,000-fold, and 30-100-fold, respectively, more sensitive than Western blotting for the corresponding antigens and 10-300-fold more sensitive than a conventional ELISA and a gelatin particle agglutination test. For urine (100 microliters), whole saliva (1 microliter), and serum (1 microliter) samples, the sensitivity and specificity of the immune complex transfer enzyme immunoassay using rRT as antigen were both 100%. However, for urine samples in which the specific activities of antibody IgG to RT, p17, and p24 were much lower than those in serum samples probably due to degradation by the kidney, a longer assay of bound beta-D-galactosidase activity or/and a concentration process for urine was required. The use of more than 1 microliter of whole saliva was recommended for reliable diagnosis of the infections, whereas 1 microliter of serum was sufficient for the purpose. The positivity with rRT as antigen could be confirmed by demonstration of antibody IgGs to p17 and p24 in most of the urine, whole saliva, and serum samples. In HIV-1 seroconversion serum panels, antibody IgG to p17 was detected as early as or even earlier than antibodies to HIV-1 by a conventional ELISA or/and a gelation particle agglutination test, whereas antibody IgGs to RT and p24 were detected as early as or later than antibody IgG to p17. Thus the uses of rRT and rp17 as antigens were advantageous over that of the other antigens for randomly collected serum samples probably long after the infection and serum samples at early stages of the infection, respectively. On the basis of these results and other reports, the immune complex transfer enzyme immunoassay was developed for simultaneous detection of p24 antigen and antibody IgGs to RT and p17 in a single assay tube, and the window period (8 weeks, although widely variable), during which diagnosis of HIV-1 infection is not possible due to the absence of detectable antibodies to HIV-1, was shortened by 2 weeks. As a result, the simultaneous detection made possible not only as early diagnosis as that by detection of p24 antigen, but also as reliable diagnosis as that by detection of antibodies to HIV-1. Finally, the immune complex transfer enzyme immunoassay has been recently improved so as to be performed within shorter periods of time (2-3 hr) with higher sensitivity, and testing many samples has become easy.
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PMID:More reliable diagnosis of infection with human immunodeficiency virus type 1 (HIV-1) by detection of antibody IgGs to pol and gag proteins of HIV-1 and p24 antigen of HIV-1 in urine, saliva, and/or serum with highly sensitive and specific enzyme immunoassay (immune complex transfer enzyme immunoassay): a review. 929 94

The immune complex transfer enzyme immunoassays for antibody IgGs to p17, p24, and reverse transcriptase (RT) of HIV-1 were tested under various conditions. Antibody IgGs to HIV-1 were reacted for up to 20 hr with 2,4-dinitrophenyl-bovine serum albumin-recombinant HIV-1 protein conjugates and recombinant HIV-1 protein-beta-D-galactosidase conjugates, and the immune complexes formed, comprising the three components, were trapped onto polystyrene beads coated with (anti-2,4-dinitrophenyl group) IgG by incubation at 4-30 degrees C for up to 2 hr with shaking and were transferred onto polystyrene beads coated with (antihuman IgG gamma-chain) IgG in the presence of excess of epsilon N-2,4-dinitrophenyl-L-lysine by incubation at 4-30 degrees C for up to 2 hr with shaking. When serum randomly collected from an HIV-1 seropositive subject and serum included in an Western blot kit were tested, the formation of the immune complex was almost completed within 1 hr for antibody IgG to p17, within 1-2 hr for antibody IgG to p24 and within 4 hr for antibody IgG to RT. Even for antibody IgG to p17, however, the immune complex continued to be formed for at least 2 hr, when serum samples at early stages of HIV-1 infection were tested. Trapping and transferring of the immune complexes were faster at higher temperatures and were almost completed within 0.5-1.5 hr, although the amount of the immune complexes trapped and transferred at 25 and/or 30 degrees C increased for 0.5-1 hr, but subsequently tended to decline. When the formation, trapping, and transferring of the immune complexes were performed for 0.5, 1, and 1 hr, respectively, with shaking followed by 1 hr assay of bound beta-D-galactosidase activity, the sensitivities for antibody IgGs to p17, p24, and RT using 10 microliters of serum samples were similar to or significantly higher than those of the corresponding previous immune complex transfer enzyme immunoassays using 10 microliters of serum samples, in which the formation, trapping, and transferring of the immune complexes were performed for 3, 16, and 3 hr, respectively, without shaking, followed by 2.5 hr assay of bound beta-D-galactosidase activity, and the sensitivities for antibody IgGs to p17, p24, and RT using 100 microliters of serum samples were 21-22-fold, 5.5-6.3-fold, and 5.3-6.0-fold, respectively, higher. When each period of time for the formation, trapping, and transferring of the immune complexes was prolonged to up to 4 hr, the sensitivities for antibody IgGs to p17, p24, and RT using 100 microliters of serum samples were improved 88-93-fold, 15-17 fold and 20-24-fold, respectively, as compared with those of the previous ones.
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PMID:Optimal conditions of immune complex transfer enzyme immunoassays for antibody IgGs to HIV-1 using recombinant p17, p24, and reverse transcriptase as antigens. 952 94

In order to reduce the nonspecific signal of noncompetitive solid phase immunoassays and to improve their sensitivities, the immune complex transfer enzyme immunoassay has been developed. Antigens to be measured were reacted with 2,4-dinitrophenyl-biotinyl-antibody Fab' and antibody Fab'-beta-D-galactosidase conjugate, and antibody IgGs to be measured were reacted with 2,4-dinitrophenyl-antigen and antigen-beta-D-galactosidase conjugate. The immune complexes formed comprising the three components were trapped onto colored polystyrene beads coated with affinity-purified (anti-2,4-dinitrophenyl group) IgG. After washing, the immune complexes were eluted from the colored polystyrene beads with epsilonN-2,4-dinitrophenyl-L-lysine, and the eluates were incubated with white polystyrene beads coated with streptavidin for antigens and coated with affinity-purified (anti-human IgG gamma-chain) IgG for antibody IgGs to transfer the immune complexes. By this method, ultrasensitive enzyme immunoassays have been developed for HIV-1 p24 antigen and antibody IgGs to HIV-1 p17 and reverse transcriptase (RT). The nonspecific signals in the absence of the antigen and the antibody IgGs were reduced 300 to 15,000-fold by the immune complex transfer process, but the amounts of the immune complexes decreased only 1.8 to 3.1-fold by the immune complex transfer. As a result, the sensitivities for HIV-1 p24 antigen and antibody IgGs to HIV-1 p17 and RT were improved 100 to 5,600-fold by the immune complex transfer. The detection limit of HIV-1 p24 antigen by 20 hr assay of beta-D-galactosidase activity (10 zmol) was 4,000 to 17,000-fold lower than those obtained with currently available commercial kits. The improved sensitivities for antibody IgGs to p17 and RT by 20 hr assay of beta-D-galactosidase activity were 1 x 10(5) to 3 x 10(5)-fold higher than those of Western blotting for p17 and p66 bands. However, the nonspecific signals in the absence of antigens and antibody IgGs were enhanced to various degrees by two factors. In order to transfer the immune complexes more efficiently within shorter periods of time, the colored polystyrene beads were incubated with the white polystyrene beads in the presence of epsilonN-2,4-dinitrophenyl-L-lysine. Such direct contact between solid phases for trapping and transferring of the immune complexes significantly enhanced the nonspecific signals. In addition, the presence of human serum samples containing neither antigens to be measured nor antibody IgGs to be measured also enhanced the nonspecific signals to various extents. Namely, these two factors limited the effect of the immune complex transfer to improve the sensitivity by 20 hr assay of beta-D-galactosidase activity. By 1 hr assay of beta-D-galactosidase activity, the detection limit of HIV-1 p24 antigen using 10 microl of serum samples (0.24 pg/ml) was 40 to 80-fold lower than those obtained with currently available commercial kits using 100 to 200 microl of serum samples (10 to 20 pg/ml) and the detection limits of antibody IgGs to HIV-1 pl7 and RTwere 1 x 10(4) to 3 x 10(4)-fold lower than those by Western blotting for p17 and p66 bands. Finally, the immunoreactions involved in the immune complex transfer enzyme immunoassays--the formation, trapping, and transferring of the immune complexes--will be performed within 15 to 30 min.
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PMID:Potential of the immune complex transfer enzyme immunoassay for antigens and antibodies to improve the sensitivity and its limitations. 959 2

In order to perform the immune complex transfer enzyme immunoassays for HIV-1 p24 antigen and antibody IgGs to HIV-1 p17, reverse transcriptase and gp41 antigens as rapidly as possible, methods for rapid formation of the immune complexes on solid phase are described. HIV-1 p24 antigen was reacted with monoclonal anti-p24 Fab'-beta-D-galactosidase conjugate at a high concentration and subsequently with polystyrene beads coated successively with affinity-purified (anti-2,4-dinitrophenyl group) IgG and 2,4-dinitrophenyl-biotinyl bovine serum albumin-affinity-purified anti-p24 Fab' conjugate. Antibody IgGs to HIV-1 were reacted with polystyrene beads coated successively with affinity-purified (anti-2,4-dinitrophenyl group) IgG and 2,4-dinitrophenyl-HIV-1 antigen conjugates and subsequently with HIV-1 antigen-beta-D-galactosidase conjugates. The periods of time used for the formation of the immune complexes comprising the three components on the polystyrene beads (15-30 min) were much shorter than those used in the previous immune complex transfer enzyme immunoassays (90-300 min), and the sensitivities of the present and previous immune complex transfer enzyme immunoassays were similar. The detection limit of the HIV-1 p24 antigen by the present and previous methods were similar (3 to 10 zmol/assay).
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PMID:Rapid formation of the immune complexes on solid phase in the immune complex transfer enzyme immunoassays for HIV-1 p24 antigen and antibody IgGs to HIV-1. 967 Nov 75

Human immunodeficiency virus type 1 (HIV-1) is considered to infect nondividing cells because nuclear localization signals (NLS) in matrix (MA, p17(Gag)) and Vpr allow active nuclear transport of the preintegration complex. Previous studies demonstrated that HIV-1 reverse transcription is successful only in cells with proliferative potential, thus restricting HIV-1 replication to cycling cells. To sort out this apparent discrepancy we compared the phenotype of a chimeric HIV-1 variant lacking a functional Vpr and MA-NLS (R7. deltaVpr.deltaNLS), and previously described to lack replicative capacity in macrophages and growth-arrested cells, with a chimera lacking a functional Vpr (R7.deltaVpr). Both variants replicated efficiently in primary macrophages, with only minimal differences in the kinetics of reverse transcription, integration, or p24 production. In agreement with our previous observation, elongation of reverse transcription was restricted to the proliferating subpopulation of macrophages. Replication of R7.deltaVpr and R7.deltaVpr.deltaNLS could also be demonstrated in aphidicolin-treated macrophages, indicating efficient nuclear transport in G1/S phase-arrested cells. In conclusion, our results confirm the dependency of the process of HIV-1 reverse transcriptase on cell proliferation in primary macrophages and exclude an important role of MA-NLS and Vpr in macrophage infection.
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PMID:Phenotype of HIV-1 lacking a functional nuclear localization signal in matrix protein of gag and Vpr is comparable to wild-type HIV-1 in primary macrophages. 991 76

Peptide/MHC tetrameric complexes were used to enumerate the frequency of HLA class I-restricted epitope-specific CD8+ T cells in 18 HLA-A*0201 HIV type 1-infected asymptomatic patients. HLA-A*0201 molecules were complexed to HIV Gag p17 (amino acids 77-85) and reverse transcriptase (amino acids 464-472) peptides, biotinylated, and bound to streptavidin-phycoerythrin to form tetramers. We show in this study that 17 of 18 HIV-1-infected asymptomatic patients have circulating frequencies of 1/50-1/1000 CD8+ T cells that recognize both Gag and Pol CTL epitopes or either epitope alone. The functional nature of these cells is open to interpretation, as we show that despite relatively high frequencies of fresh epitope-specific CD8+ T cells, variant epitope sequences in viral plasma progeny were rare. In addition, the majority of tetramer-positive cells did not display discernible fresh CTL activity; only after restimulation with specific peptide in culture was there an expansion of epitope-specific CD8+ cells, correlating with high CTL activity. These data suggest that fresh tetramer-stained cells probably represent memory precursors; we demonstrate, with the application of highly active antiretroviral therapy, that the interruption of chronic antigenic stimulation causes significant reductions in the frequency of these cells in five of six patients. In conclusion, this study provides evidence that persistently replicating viral populations are probably required to maintain high frequencies of HIV-1 epitope-specific CD8+ T cells in asymptomatic chronically infected individuals
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PMID:Frequency of class I HLA-restricted anti-HIV CD8+ T cells in individuals receiving highly active antiretroviral therapy (HAART). 997 42

For earlier diagnosis of human immunodeficiency virus type 1 (HIV-1) infection, the sensitivities of immune complex transfer enzyme immunoassays for HIV-1 p24 antigen and antibody immunoglobulin G (IgG) to HIV-1 p17 antigen were improved approximately 25- and 90-fold, respectively, over those of the previous immunoassays by performing solid-phase immunoreactions with shaking and increasing the serum sample volumes, and immune complex transfer enzyme immunoassay of antibody IgM to p17 antigen was also performed in the same way as the improved immunoassay of antibody IgG to p17 antigen. By the improved immunoassays, p24 antigen and antibody IgG to p17 antigen were detected earlier in 32 and 53%, respectively, of the HIV-1 seroconversion serum panels tested than before the improvements, and p24 antigen was detected as early as or earlier than HIV-1 RNA by reverse transcriptase-PCR (RT-PCR) in all of the panels tested. In 4 panels out of 19 tested, antibody IgG to p17 antigen or both antibodies IgG and IgM to p17 antigen were detected earlier than p24 antigen and RNA, although the antibody levels declined slightly before their steep increases usually observed after p24 antigen and RNA. Thus, the window period in diagnosis of HIV-1 infection can be shortened by detection of p24 antigen with the improved immunoassay as much as by detection of RNA with RT-PCR and, in some cases, more by detection of antibodies IgG and IgM to p17 antigen with the improved immunoassays than by detections of p24 antigen with the improved immunoassay and RNA with RT-PCR.
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PMID:Earlier detection of human immunodeficiency virus type 1 p24 antigen and immunoglobulin G and M antibodies to p17 antigen in seroconversion serum panels by immune complex transfer enzyme immunoassays. 1106 90


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