Gene/Protein Disease Symptom Drug Enzyme Compound
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Query: EC:3.1.3.1 (alkaline phosphatase)
 47,916 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Human parvovirus B19 is a cause of aplastic crises in patients with haemolytic anaemias, prolonged bone marrow failure in the immunosuppressed, and fetal death secondary to non-immune hydrops. The immunohistological detection of parvovirus B19 in formalin-fixed, paraffin-embedded tissues has not previously been reported, and definitive diagnosis of infection in such specimens has relied on the use of specialized DNA hybridization and amplification techniques. A new monoclonal antibody to B19 capsid proteins, R92F6, was found to be capable of labelling infected cells in paraffin-embedded tissues from all 19 cases of parvovirus-related fetal hydrops tested, and in bone marrow from a child with congenital immunodeficiency and chronic parvovirus infection. Viral antigen was detected both in cytoplasmic and in nuclear distributions using the alkaline phosphatase anti-alkaline phosphatase (APAAP) technique without preceding proteolytic digestion. The viral epitope recognized appears to be highly conserved, as specimens were obtained over a 13-year period from widely spaced locations in the U.K. Antibody R92F6 should facilitate rapid diagnosis of parvovirus B19 infection in routinely processed and archival specimens.
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PMID:Immunohistological detection of human parvovirus B19 in formalin-fixed, paraffin-embedded tissues. 131 62

A dot-blot hybridization immunoenzymatic assay with a chemiluminescent endpoint was developed for the rapid and sensitive detection of viral and plasmid DNAs. Digoxigenin-labeled probes were used to detect cytomegalovirus, parvovirus B19, and plasmid pBR328 DNAs. Hybridized probes were immunoenzymatically visualized by anti-digoxigenin Fab fragments labeled with alkaline phosphatase, and adamantyl 1,2-dioxetane phenyl phosphate was used as chemiluminescent substrate. Results were recorded by instant photographic films. The chemiluminescent hybridization assay was performed in about 8 hr and was able to detect as little as 50-10 fg of homologous target DNA.
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PMID:Chemiluminescent assay for the detection of viral and plasmid DNA using digoxigenin-labeled probes. 165 May 41

A chemiluminescence dot blot hybridization assay was used for the detection of B19 parvovirus DNA in human sera by using digoxigenin-labeled probes. The probes were revealed immunoenzymatically by use of anti-digoxigenin Fab fragments conjugated with alkaline phosphatase. The chemiluminescence signal was obtained by reacting the labeled probe-target complex with an enzyme-triggerable dioxetane substrate. The emitted photons were detected with instant photographic films. In the search for B19 parvovirus DNA, 2,808 serum samples were analyzed.
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PMID:Chemiluminescence dot blot hybridization assay for detection of B19 parvovirus DNA in human sera. 177 33

A non-radioactive dot-blot hybridization assay for the detection of B19 parvovirus infections was developed using a digoxigenin-labelled probe both on nylon and nitrocellulose filters. A 700 bp BamHI HindIII fragment of B19 DNA was used to construct the probe. Probe labelling was carried out by incorporating deoxyuridine triphosphate labelled with digoxigenin. The dot-blot hybridization assay was visualized by an immunoenzymatic reaction using antidigoxigenin Fab fragments labelled with alkaline phosphatase. The specificity and sensitivity of digoxigenin-labelled B19 DNA probe was compared with the results obtained with 32P-labelled B19 DNA probe. Out of the 504 serum samples tested, 3 samples were positive in all the hybridization assays performed and 494 were negative, 7 serum samples gave a weak positive reaction when Dig-B19 probe was used on nitrocellulose filters. The 77 pharyngeal swabs tested were negative in all the hybridization assays performed. Our hybridization assay showed a high sensitivity and reproducibility and it appears to be a rapid, practical and reliable test for routine screening of B19 parvovirus DNA in large numbers of clinical specimens.
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PMID:Detection of B19 parvovirus infections by a dot-blot hybridization assay using a digoxigenin-labelled probe. 215 76

A nonradioactive dot blot hybridization assay for human parvovirus B19 DNA was set up by using a biotin-labeled DNA probe and streptavidin-alkaline phosphatase conjugate. The assay was used to examine 4,895 specimens referred for B19 virus diagnosis during 1987. Of 48 specimens that gave positive reactions for B19 DNA, 41 were confirmed virus positive by electron microscopy (n = 36), radioimmunoassay (n = 26), or counterimmunoelectrophoresis (n = 20). In 7 samples which were not confirmed and in 11 samples giving weak reactions for B19 DNA, there was serological or epidemiological evidence of recent B19 infection. A further 70 specimens gave weak, apparently false-positive reactions. By electron microscopy, 13 of 16 were contaminated by bacteria, and plasmid DNA was demonstrated in one specimen. Of 55 specimens tested, 52 reacted with streptavidin-alkaline phosphatase conjugate alone. These were probable sources of nonspecificity in an otherwise practical and economic screening method for B19 virus.
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PMID:Dot blot hybridization assay of B19 virus DNA in clinical specimens. 254 Nov 66

In this quantitative dot-blot hybridization assay for detecting B19 parvovirus DNA, we used three different chemiluminescent substrates [adamantyl-1,2-dioxetane phenyl phosphates (PPD and the new PPD-Plus) and the chloro-5-substituted adamantyl-1,2-dioxetane phosphate (CSPD) plus Emerald enhancer] and a high-performance, low-intensity-light imaging luminograph apparatus. The hybridization test uses digoxigenin-labeled DNA probes, which are immunoenzymatically revealed by anti-digoxigenin Fab fragments conjugated with alkaline phosphatase. All the detection systems with the various chemiluminescent substrates gave sensitive and reproducible results for calibrators and positive or negative reference clinical samples, with high reproducibility (CV 4-17%). The signal was measured after 45 min of incubation. The luminograph apparatus could detect 10 fg of homologous DNA with the PPD-Plus substrate, whereas the detection limit with the CSPD and PPD substrates was 20 fg and 20-50 fg, respectively. Analysis of 26 samples with the three substrates showed good sensitivity and specificity for viral detection.
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PMID:Application of a low-light imaging device and chemiluminescent substrates for quantitative detection of viral DNA in hybridization reactions. 749 7

A cocktail of 10 oligonucleotides selected at intervals along the length of the genome of human parvovirus B19 was labelled enzymically with digoxigenin and chemically with either digoxigenin (DIG) or dinitrophenyl (DNP). Chemical labelling was easier and more practical for the production of large quantities of probe. Pools labelled with either digoxigenin or DNP could detect 10 fg of B19 DNA in a dot blot reaction using an alkaline phosphatase antibody conjugate and colorimetric detection. Formalin fixed tissue from 11 consecutive cases of fetal hydrops were examined by in situ hybridisation (ISH). Both probe cocktails detected human parvovirus B19 DNA in 3 cases, with positive cells in all tissues examined and with equal sensitivity. The DNP pool is significantly cheaper and simpler to produce and could provide an inexpensive reagent suitable for diagnostic detection of viral nucleic acid in histopathological material.
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PMID:Synthetic oligonucleotide cocktails as probes for detection of human parvovirus B19. 763 29

A method for the direct detection of human parvovirus DNA in serum samples that uses a digoxigenin-labeled RNA probe to hybridize with target B19 DNA, followed by capture of the hybrid onto a microtiter plate wells previously coated with a second oligonucleotide probe was developed. The captured hybrid is then detected with anti-digoxigenin-alkaline phosphatase conjugate and chemiluminescent substrate and the reaction read on a scintillation counter. The relative sensitivities of the microwell and standard dot blot hybridization assays were compared. The chemiluminescent microwell hybridization assay was more sensitive than dot-blot hybridization and could be performed in a few hours. This format, therefore, permits rapid and sensitive detection of parvovirus DNA suitable for the clinical setting.
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PMID:Chemiluminescent microwell hybridization assay for direct detection of human parvovirus B19 DNA. 771 57

A dot-blot hybridization assay was developed to detect B19 DNA using strand-specific RNA probes labelled with digoxigenin. The sensitivity of the assays was evaluated either using 'plus' and 'minus' sense RNA probes in two different hybridization assays, or in two successive reactions of the same assay. The hybridized probes were revealed immunoenzymatically using anti-digoxigenin Fab fragments conjugated with alkaline phosphatase. The enzyme was visualized by colorimetric reaction. Since 'minus' sense RNA probe gave the best results in the dot-blot procedures, we increased the sensitivity of the hybridization assay visualizing the 'minus' sense digoxigenin-labelled RNA probe by chemiluminescent reaction. In these experimental conditions up to 20 fg of target B19 DNA could be visualized. In the search for B19 DNA, 4656 serum samples were analyzed by chemiluminescent reaction of 'minus' sense digoxigenin-labelled RNA probe and for comparison with the digoxigenin-labelled DNA probe. Positive results were confirmed by Southern blotting. Out of 4656 serum samples analyzed, 4648 gave negative results, 1 resulted positive to all the hybridization assays, 6 only using RNA probe and 1 only by DNA probe.
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PMID:Evaluation of strand-specific RNA probes visualized by colorimetric and chemiluminescent reactions for the detection of B19 parvovirus DNA. 811 43

An in situ hybridization assay using a digoxigenin-labelled probe was developed to detect B19 DNA in bone marrow erythroid elements of immunodeficient patients with hypoplastic anaemia. A 700 bp Bam HI-Hin dIII fragment of B19 DNA was used to construct the probe by incorporating deoxyuridine triphosphate labelled with digoxigenin. The in situ hybridized B19 DNA probe was visualized by an immunoenzymatic reaction using antidigoxigenin Fab fragments labelled with alkaline phosphatase. Dark blue coloured inclusions at the enzyme site were detected in the nuclei of B19 infected erythroid cells at different stages of cell differentiation. Six out of the nine patients studied showed a positive reaction by in situ hybridization assay. The assay we developed proved highly specific and sensitive and it appears to be a suitable diagnostic test for investigating the possible role of B19 infection as a cause of haematopoietic disorders in immunocompromised hosts.
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PMID:In situ detection of B19 DNA in bone marrow of immunodeficient patients using a digoxigenin-labelled probe. 838 13


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