Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound   Target Concepts: 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)

For double tracing experiments, wheat germ agglutinin (WGA) molecules labeled with two different haptens are desirable. In the present report the suitability of digoxigenylated WGA (DIG-WGA) for retrograde tracing was investigated. For this purpose the new tracer was pressure injected into rat brains and the transported DIG-WGA visualized via its digoxigenyl group with an alkaline phosphatase linked anti DIG antibody in permanently stained sections of high quality. With fixatives containing 2.5% glutaraldehyde only few positive cells were found. However, at milder fixation conditions (4% paraformaldehyde, 0.05% glutaraldehyde 0.2% picric acid, 30 min) retrogradely labeled cells were detected with a sensitivity comparable to tetramethylbenzidine protocols for conventional WGA-HRP (horseradish peroxidase) tracing. Preliminary experiments suggest excellent suitability for double labeling.
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PMID:Digoxigenylated wheat germ agglutinin visualized with alkaline phosphatase-labeled anti-digoxigenin antibodies--a new, sensitive technique with the potential for single and double tracing of neuronal connections. 170 75

A multiple enzyme immunoassay (multi-EIA) was developed to quantify whole-molecule human choriogonadotropin (w-hCG) and free hCG beta-subunits (hCG-beta) simultaneously. A clone of a specific monoclonal antibody was coupled to solid phase; two other clones of different monoclonal antibodies were conjugated to horseradish peroxidase (HRP; EC 1.11.1.7) and alkaline phosphatase (AP; EC 3.1.3.1), respectively. These two enzyme conjugates were mixed together to measure w-hCG or hCG-beta, depending on the selection of the enzyme substrate. To measure w-hCG and hCG-beta simultaneously, both enzyme substrates were used with the blended enzyme conjugates. The assay is simple and reproducible, and can be completed within 2 h with high specificity and sensitivity. We measured w-hCG and hCG-beta in the sera of women with abnormal pregnancies and in patients with tumors of the reproductive system, and observed different hCG-beta/w-hCG ratios in patients with various types of trophoblastic tumors.
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PMID:Simple enzyme immunoassay for the simultaneous measurement of whole choriogonadotropin molecules and free beta-subunits in sera of women with abnormal pregnancies or tumors of the reproductive system. 170 96

The present paper reports immunohistological findings in porcine skin, which were obtained by use of mono- and polyclonal antihuman antibodies and either alkaline phosphatase anti-alkaline phosphatase (APAAP) or peroxidase (POX) technique. Epidermal staining was observed with antibodies to keratins (K 8.12, RSKE 60), filaggrin, and calmodulin (ACAM). Staining of connective tissue and vessels was achieved using antibodies to vimentin (V9(1)), collagen type IV, and fibronectin. In general, these antibodies gave a staining pattern similar to that of normal human skin. The similarities of immunoreactivity to poly- and monoclonal antihuman antibodies in porcine and human skin render porcine skin a reliable model in biomedical research.
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PMID:Immunohistochemistry of porcine skin. 171 Aug 64

Knowledge about B-cell dysfunction and HIV-specific antibody production is necessary for the understanding of both HIV-1-related immunopathology and the (vaccine-induced) humoral immunity involved in protection against AIDS. This paper describes the application of recently developed methods to detect epitope specificity of B cells in lymph-node biopsies with antigen-enzyme conjugates. Cryosections of five lymph-node biopsies from HIV-1-infected individuals and four control tissues were stained with a panel of HIV-1 antigen-enzyme conjugates: recombinant HIV-1 proteins (gp 160, gp 120 and p24), labelled with peroxidase, and synthetic peptides representing neutralizing epitopes from gp120 and gp41, labelled with alkaline phosphatase. Antibody-forming cells (AFCs) were detected in all the HIV-1-infected biopsies with gp160, gp120 and/or p24, in numbers up to 350 per section. AFCs producing specific antibodies against peptide 101 (SP 101), representing the neutralizing epitope 586-608 of gp41, were detected in one patient. These techniques allow correlation of in vivo function of B cells with lymph-node pathology, clinical stage of the disease and serological data. Their potential for the elucidation of HIV-related immunopathogenesis and the development of vaccines is discussed.
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PMID:Immunocytochemical determination of antigen and epitope specificity of HIV-1-specific B cells in lymph-node biopsies from HIV-1-infected individuals. 171 61

Enzyme-labeled monoclonal antibodies (MAbs) were used in an immunohistochemical, dual-staining study of 10 colon adenocarcinomas. MAbs B72.3 and COL-4, reactive with the high molecular weight tumor-associated glycoprotein-72 (TAG-72) antigen and carcinoembryonic antigen (CEA), respectively, were labeled with horseradish peroxidase or alkaline phosphatase. Dual staining using the two MAbs on a single tissue section (formalin-fixed, paraffin-embedded) showed that greater numbers of carcinoma cells could be detected by using the combination of the two MAbs than could be detected by use of either MAb alone. In many tumors, some carcinoma cells reacted with MAb B72.3, some reacted with MAb COL-4, and some cells reacted with both MAbs. Only 1 of 10 carcinomas showed greater than 75% reactive cells when stained with each MAb individually. In 9 of 10 cases, however, greater than 75% of cells reacted when the combination of MAbs was used. Cell surface and cytoplasmic patterns of reactivity were observed with both MAbs while some pools of extracellular mucin were composed of both TAG-72 and CEA. This study supports the rationale for the use of a combination of anti-TAG-72 and anti-CEA MAbs for in vitro immunologic detection and potential in vivo immunodiagnostic and immunotherapeutic applications for these MAbs in colon adenocarcinoma patients.
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PMID:Complementation of expression of carcinoembryonic antigen and tumor associated glycoprotein-72 (TAG-72) in human colon adenocarcinomas. 171 93

Cryostat sections from rat gracilis muscles were incubated with different biotinylated lectins: Con A (Concanavilin A), WGA (Wheat germ agglutinin), SBA (soybean agglutinin), GS I and GS II (Griffonia simplicifolia agglutinin), LCA (Lens culinaris agglutinin), PNA (peanut agglutinin) and PSA (Pisum sativum agglutinin). The sections were subsequently treated with alkaline phosphatase conjugated avidin. The lectin binding sites were visualized after incubation in substrate media containing: (1) 5-bromo-4-chloro indoxyl phosphate and Nitro Blue tetrazolium or copper sulphate; (2) naphthol AS-MX phosphate or naphthol AS-BI phosphate and various types of diazonium salts; (3) alpha-naphthylphosphate and Fast Blue BB; (4) beta-glycerophosphate according to the method of Gomori. The results obtained with the alkaline phosphatase methods were compared with those seen with a streptavidin-horseradish peroxidase procedure. Several chromogen protocols for visualizing alkaline phosphatase activity showed differences in the ability to detect lectin binding sites. A sarcoplasmic reaction was evident for Con A, GS II, WGA, LCA, and PSA after incubation in the indoxyl phosphate medium. Sarcoplasmic reaction for GS II was also noticed after incubation with naphthol AS-MX Fast Blue BB and beta-glycerophosphate. The latter substrate also gave rise to a sarcoplasmic Con A reaction. With the indoxylphosphate tetrazolium salt method some muscle fibres showed a very strong intracellular reaction after incubation with Con A and GS II while the staining intensity was weak in other fibres. The same muscle fibres were stained with PAS. No sarcoplasmic reactions were observed with either naphthol phosphate media or with the diaminobenzidine peroxidase methods. Further, the staining of the muscle fibre periphery, connective tissue, an capillaries was intensified using the indoxyl method. The indoxylphosphate-tetrazolium salt method seems to be suitable for future investigations of lectin binding sites in muscle sections.
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PMID:Lectin binding in skeletal muscle. Evaluation of alkaline phosphatase conjugated avidin staining procedures. 171 10

Synthetic peptide-alkaline phosphatase conjugates can be used to detect the epitope specificity of (i) antibody-forming cells in vivo by immunocytochemistry; (ii) of antibody secreting cells in vitro by spot-ELISA; and (iii) antibodies in solution by capture ELISA. The availability of synthetic peptide-enzyme conjugates using detector enzymes other than alkaline phosphatase would offer several important advantages, for example in double staining approaches. This paper reports the production of synthetic peptide-horseradish peroxidase conjugates and synthetic peptide-beta-galactosidase conjugates. A peptide of 21 amino acids (SP 29) was coupled to peroxidase in seven differing molar ratios of peptide over peroxidase, ranging from 1:3.4 to 1:575, using periodate oxidation of the enzyme. SP 29 was coupled to beta-galactosidase in four molar ratios ranging from 1.25 to 10, using glutaraldehyde pre-activation of the enzyme. The enzyme activity of the different conjugates was determined, the conjugates were tested in direct capture-ELISA with peptide-specific monoclonal antibodies, and the conjugates were tested in immunocytochemistry to detect peptide-specific B cells. The results show that the conjugates perform best if the peptide is coupled to the enzyme at relatively low molar ratios (1-30). The availability of these new peptide-enzyme conjugates broadens the applicability of synthetic peptides for detection purposes in several assay systems.
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PMID:Synthetic peptide conjugates with horseradish peroxidase and beta-galactosidase for use in epitope-specific immunocytochemistry and ELISA. 172 93

Oligonucleotides were end-labelled with digoxigenin (DIG), chemically at the 5'-end or enzymically at the 3'-end. Following specific in situ hybridization of these probes to intracellular rRNA molecules, the hybrids were detected with anti-DIG Fab fragments labelled with fluorescent dyes. The antibody fragments penetrated through the bacterial cell periphery and specifically bound to their antigens. Probe-conferred and non-specific fluorescence per cell were quantified by flow cytometry and compared to values obtained with end-labelled fluorescent probes. The DIG reporter molecules could also be detected in whole fixed cells by antibodies labelled with either alkaline phosphatase or horseradish peroxidase. The penetration of the large antibody-enzyme complexes into the cells required lysozyme/EDTA treatment prior to the hybridization and has so far only been achieved for Gram-negative bacteria. This technique has the potential for significant signal amplification as compared to the fluorescently end-labelled oligonucleotides hitherto used for single cell identification in microbial ecology. Moreover, it can be used instead of fluorescent assays in natural samples showing autofluorescence.
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PMID:Identification of single bacterial cells using digoxigenin-labelled, rRNA-targeted oligonucleotides. 172 65

Enhanced chemiluminescent assays for hydrolase enzymes have been developed using proenhancer and pro-anti-enhancer substrates. Alkaline phosphatase is measured using disodium para-iodophenyl phosphate (proenhancer) which is converted to para-iodophenol and this in turn enhances the light emission from the horseradish peroxidase catalysed chemiluminescent oxidation of luminol by peroxide. An alternative strategy uses para-nitrophenyl phosphate which is converted by alkaline phosphatase to para-nitrophenol which inhibits the enhanced chemiluminescent reaction. The detection limit for the enzyme using the proenhancer and pro-anti-enhancer assays was 100 attomoles and 1 picomole, respectively. The proenhancer strategy was effective in assays for beta-D-galactosidase, beta-D-glucosidase and aryl sulfatase. A limited comparison of the proenhancer and a conventional colorimetric assay for an alkaline phosphatase label in an enzyme immunoassay for alpha-fetoprotein showed good agreement.
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PMID:Chemiluminescent assay of enzymes using proenhancers and pro-anti-enhancers. 172 39

An amplification procedure was developed for the visualization of antigens in human testis using monoclonal antibodies against desmin and vimentin. The technique combines the high sensitive and specific APAAP- and ABAP-methods. Depending on the quality of the antibodies used and the processing of the material prior to the immunocytochemical staining the amplification technique may be applied either as a single APAAP and ABAP- or as a double APAAP and ABAP-combination. Especially after the double amplification reaction a distinct increase of the staining intensity of the vimentin- (in Sertoli cells, myofibroblasts of the lamina propria, and fibroblasts of the interstitium) and desmin- (in myofibroblasts of the lamina propria and smooth muscle cells of the blood vessels) like immunoreactivity was observed. If different diazonium salts were used for the visualization of the alkaline phosphatase activity (e.g. Fast Red TR Salt, Fast Blue BB Salt) desmin- and vimentin-like immunoreactivity can be demonstrated in the same tissue section in a double sequential staining approach. For double staining, the alkaline phosphatase technique may be combined successfully with a technique or a combination that uses peroxidase as a marker.
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PMID:Combination of alkaline phosphatase anti-alkaline phosphatase (APAAP)- and avidin-biotin-alkaline phosphatase complex (ABAP)-techniques for amplification of immunocytochemical staining of human testicular tissue. 172 78


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