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1.
A method which can be used to measure the amount of peroxidase antibody which is specifically bound to placental alkaline phosphatase in membrane fragments is described. The technique uses Sepharose 4B chromatography to separate membrane fragments containing bound peroxidase-antibody from unbound peroxidase-antibody. Specificity is demonstrated by nonbinding to rabbit placental membranes, by a strict correlation between membrane-associated phosphatase and bound peroxidase-antibody, and by preventing binding using pure enzyme. The general utility of this method for membrane antigens is discussed.  相似文献   

2.
An enzyme immunoassay was developed for a convenient and sensitive assay of 13,14-dihydro-15-ketoprostaglandin F2 alpha, a metabolite of prostaglandin F2 alpha appearing in human blood. The compound was chemically conjugated to beta-galactosidase from Escherichia coli. The enzyme-labeled antigen was mixed with a sample containing 13,14-dihydro-15-ketoprostaglandin F2 alpha, and the mixture was allowed to react competitively with the antibody immobilized in a polystyrene tube. The activity of beta-galactosidase bound to the antibody was assayed by fluorometry. The enzyme activity was plotted against the amount of authentic 13,14-dihydro-15-ketoprostaglandin F2 alpha to obtain a calibration curve, and the compound was detectable over a range of 10 fmol to 10 pmol. Prostaglandins were extracted from human serum by the use of an octadecylsilyl silica column, and the extract gave an abnormally high level of 13,14-dihydro-15-ketoprostaglandin F2 alpha by enzyme immunoassay due to the presence of unidentified interfering substance(s), which was removed by high-performance liquid chromatography (HPLC). The purified material gave a value in the order of 0.1 pmol per ml of human serum. Validity of the enzyme immunoassay was confirmed by radioimmunoassay and gas chromatography/mass spectrometry (GC-MS) of a methyl ester n-butoximedimethylisopropylsilyl ether derivative.  相似文献   

3.
A highly sensitive and specific solid-phase enzyme immunoassay system for terminal deoxynucleotidyl transferase (TdT, EC 2.7.7.31) has been developed by the use of monospecific antibody against calf thymus TdT and β-d-galactosidase from Escherichia coli as label. The immunoassay system was composed of solid phase (polystyrene beads) with immobilized F(ab′)2 antibody fragments and the antibody Fab′ fragments labeled with β-d-galactosidase. The minimum detectable concentration of calf TdT was 0.1 ng/ml (0.01 ng/assay), making it more sensitive than the radioimmunoassay or enzyme immunoassay methods that use alkaline phosphatase as label, as reported previously. The assay system cross-reacted with human TdT, and TdT in neoplastic cells or sera from leukemic patients was successfully detected by the present immunoassay method.  相似文献   

4.
Phase change enzyme immunoassay   总被引:1,自引:0,他引:1  
A novel enzyme-linked immunoassay employing a partitioning chromophore was developed. The assay system consisted of an aqueous phase and an immiscible organic solvent. Antigen-antibody interaction was indicated by transfer of a chromogenic indicator from the aqueous phase to an organic layer. The indicator employed was a water-soluble phosphate ester of phenylazophenol. Hydrolysis of the ester by acid or alkaline phosphatase produced a water-insoluble phenol that partitioned into toluene. The enzyme employed in this assay format can be covalently linked to antibody or a specific antibody for the phosphatase can be used. Phase change immunoassays were developed for the measurement of alkaline phosphatase, human IgG in whole blood, and the human tumor marker prostatic acid phosphatase. Solid supports of small polystyrene latex particles and Sephadex were employed.  相似文献   

5.
Alkaline phosphatase of matrix vesicles isolated from fetal bovine epiphyseal cartilage was purified to apparent homogeneity using monoclonal antibody affinity chromatography. The enzyme from the butanol extract of matrix vesicles bound specifically to the immobilized antibody-Sepharose in the presence of 2% Tween 20 whereas the major portion of nonspecific protein was removed by this single step. Of various agents tested, 0.6 M 2-amino-2-methyl-1-propanol, pH 10.2, was the most effective in eluting 80-100% of the enzyme initially applied. Both Tween 20 and 2-amino-2-methyl-1-propanol associated with the eluted enzyme were effectively removed by the sequential application of DEAE-cellulose and Sepharose CL-6B chromatography. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis of the enzyme preparation treated with sodium dodecyl sulfate and mercaptoethanol showed the presence of a dominant band (using silver staining) corresponding to a molecular weight of 81,000. This molecular weight was nearer reported values for rat liver (Ohkubo, A., Langerman, N., and Kaplan, M. M. (1974) J. Biol Chem. 249, 7174-7180) and porcine kidney (Cathala, G., Brunel, C., Chapplet-Tordo, D., and Lazdunski, M. (1975) J. Biol. Chem. 250, 6040-6045) alkaline phosphatase, than to previously reported values for chicken (Cyboron, G. W., and Wuthier, R. E. (1981) J. Biol. Chem. 256, 7262-7268) and fetal calf (Fortuna, R., Anderson, H. C., Carty, R. P., and Sajdera, S. W. (1980) Calcif. Tissue Int. 30, 217-225) cartilage matrix vesicle alkaline phosphatase. The purified alkaline phosphatase was activated by micromolar Mg2+. The amino acid composition of cartilage alkaline phosphatase was found to be similar to that previously described for porcine kidney (Wachsmuth, E. D., and Hiwada, K. (1974) Biochem. J. 141, 273-282). Double immunoprecipitation data indicated that monoclonal antibody against cartilage alkaline phosphatase cross-reacted with fetal bovine liver or kidney enzyme but failed to react with calf intestinal or rat cartilage enzyme. Thus these observations suggest that alkaline phosphatase of matrix vesicles from calcifying epiphyseal cartilage is a liver-kidney-bone isozyme.  相似文献   

6.
A heterogeneous enzyme immunoassay (EIA), which could be completed within 27 h, was developed for the detection of salmonellae in foods. Samples were subjected to the usual non-selective enrichment for 16–18 at 35°C and to a short (6 h) post-enrichment in a moderately selective broth. The EIA was carried out on polystyrene microtitration plates. A pooled polyvalent Salmonella flagellar antiserum and a protein A-alkaline phosphatase conjugate were used. The sensitivity of the enzyme immunoassay compared favorably with that of the conventional cultural technique for detection of Salmonella in 40 naturally contaminated food and feed samples. No sample was positive only by the cultural technique; samples positive only by the enzyme immunoassay were observed for feeds. Some specimens yielded high background values indicating possible interference from food proteins.  相似文献   

7.
A sensitive and specific enzyme-linked immunoassay for the measurement of ursodeoxycholic acid in human serum was developed. Ursodeoxycholic acid conjugated to alkaline phosphatase (from calf intestine) was used as a tracer. An antiserum to ursodeoxycholic acid serum was raised in rabbits using ursodeoxycholic acid--bovine serum albumin conjugate as an antigen. The binding required 1 hr at 42 degrees C; separation of the bound tracer was achieved by addition of a second antibody, and alkaline phosphatase activity of this bound complex was measured colorimetrically. The ratio of bound to total enzyme activity decreased linearly with a logarithmic increase in ursodeoxycholic acid concentration from 20 to 900 pmol. The specificity and sensitivity of this enzyme-linked immunoassay were similar to those of a radioimmunoassay reported previously. The serum ursodeoxycholic acid levels measured by this method correlated well with those determined by gas--liquid chromatography and radioimmunoassay. Based on these findings, this enzyme-linked immunoassay of bile acid might be useful as a tool for the routine clinical analysis of serum bile acids.  相似文献   

8.
A procedure for the isolation and separation of three different subfractions of plasma membrane from the cellular slime mould Dictyostelium discoideum is described. The cells were disrupted by freeze-thawing in liquid N(2) and plasma membranes were purified by equilibrium centrifugation in a sucrose gradient. The cell surface was labelled with radioactive iodide by using the lactoperoxidase iodination method. Alkaline phosphatase was identified as a plasma-membrane marker by its co-distribution with [(125)I]iodide. 5'-Nucleotidase, which has been widely described as a plasma-membrane marker enzyme in mammalian tissues, was not localized to any marked extent in D. discoideum plasma membrane. The isolated plasma membranes showed a 24-fold enrichment of alkaline phosphatase specific activity relative to the homogenate and a yield of 50% of the total plasma membranes. Determination of succinate dehydrogenase and NADPH-cytochrome c reductase activities indicated that the preparation contained 2% of the total mitochondria and 3% of the endoplasmic reticulum. When the plasma-membrane preparation was further disrupted in a tight-fitting homogenizer, three plasma-membrane subfractions of different densities were obtained by isopycnic centrifugation. The enrichment of alkaline phosphatase was greatest in the subfraction with the lowest density. This fraction was enriched 36-fold relative to the homogenate and contained 19% of the total alkaline phosphatase activity but only 0.08% of the succinate dehydrogenase activity and 0.34% of the NADPH-cytochrome c reductase activity. Electron microscopy of this fraction showed it to consist of smooth membrane vesicles with no recognizable contaminants.  相似文献   

9.
An enzyme immunoassay (EIA) in which an immunoglobulin A monoclonal antibody from a myeloma (MOPC 467) is used was developed to detect the presence of Salmonella organisms. This myeloma protein binds to a flagellar determinant of the organisms but is not directed toward the H antigens. Of 100 strains tested, 94% were detectable with this antibody. The EIA, used with MOPC 467, is quick, sensitive, and specific, showing virtually no cross-reactivity to other enteric organisms. Initial screening of antibody reactivity was performed by Ouchterlony gel diffusion with the supernatants of heat-treated Salmonella cultures. After this, an EIA was performed on the heat extracts with the myeloma protein, which had been directly coupled to alkaline phosphatase. A positive reaction was indicated by the production of a yellow color after the addition of a substrate (p-nitrophenylphosphate), and this was quantitated by determining the absorbance at 405 nm. The EIA proved to be slightly more sensitive than the Ouchterlony analysis. The sensitivity of the EIA is such that as few as 10(6) Salmonella organisms per ml were detected. This concentration was easily obtained after a 24-h preenrichment incubation of the sample. Mixtures of Salmonella strains with a 10 x concentration of Escherichia coli did not prevent detection of the Salmonella strains. This EIA can be successfully used to detect contamination of foods, as it was used to detect the intentional contamination of infant formula in these studies. Indications are that the EIA is sensitive enough to detect Salmonella strains in M broth subcultures taken directly from a preenrichment culture. Testing of samples could thus be completed 36 h after culture initiation, rather than after 96 h, the time currently needed.  相似文献   

10.
An enzyme immunoassay (EIA) in which an immunoglobulin A monoclonal antibody from a myeloma (MOPC 467) is used was developed to detect the presence of Salmonella organisms. This myeloma protein binds to a flagellar determinant of the organisms but is not directed toward the H antigens. Of 100 strains tested, 94% were detectable with this antibody. The EIA, used with MOPC 467, is quick, sensitive, and specific, showing virtually no cross-reactivity to other enteric organisms. Initial screening of antibody reactivity was performed by Ouchterlony gel diffusion with the supernatants of heat-treated Salmonella cultures. After this, an EIA was performed on the heat extracts with the myeloma protein, which had been directly coupled to alkaline phosphatase. A positive reaction was indicated by the production of a yellow color after the addition of a substrate (p-nitrophenylphosphate), and this was quantitated by determining the absorbance at 405 nm. The EIA proved to be slightly more sensitive than the Ouchterlony analysis. The sensitivity of the EIA is such that as few as 10(6) Salmonella organisms per ml were detected. This concentration was easily obtained after a 24-h preenrichment incubation of the sample. Mixtures of Salmonella strains with a 10 x concentration of Escherichia coli did not prevent detection of the Salmonella strains. This EIA can be successfully used to detect contamination of foods, as it was used to detect the intentional contamination of infant formula in these studies. Indications are that the EIA is sensitive enough to detect Salmonella strains in M broth subcultures taken directly from a preenrichment culture. Testing of samples could thus be completed 36 h after culture initiation, rather than after 96 h, the time currently needed.  相似文献   

11.
A microtechnique has been developed for the measurement of alkaline phosphatase in minute amounts of renal tissue. This microtechnique utilizes the known fluorescent property of 4-methylumbelliferyl phosphate following enzymatic hydrolysis. The reaction is sensitive and reproducible and is inhibited by l-bromotetramisole, a specific alkaline phosphatase inhibitor. The microdetermination of alkaline phosphatase activity in the various segments of the mouse nephron allowed the localization of the enzyme in the glomeruli, and in the proximal convoluted tubule where the activity progressively decreases from the capsule of Bowman to the more distal segments. The enzyme was absent from the pars recta or S3 and from the rest of the nephron. This technique is applicable to very small amounts (0.1 μg of protein) of any tissue containing alkaline phosphatase.  相似文献   

12.
Interleukin-6 (IL-6), an inflammatory cytokine, is one of the most important mediators of fever, the acute phase response, and inflammatory conditions. Described here is an integrated microfluidic immunosensor capable of detecting the concentration of IL-6 in human serum samples by use of an electrochemical method in a microfluidic biochip format. The detection of IL-6 was carried out using a sandwich immunoassay method based on the use of anti-IL-6 monoclonal antibodies, immobilized on a 3-aminopropyl-modified controlled-pore glass (APCPG) packet in a central channel (CC) of the microfluidic system. The IL-6 in the serum sample is allowed to react immunologically with the immobilized anti-IL-6 and biotin-labeled second antibodies specific to IL-6. After washing, the streptavidin–alkaline phosphatase conjugate is added. p-Aminophenyl phosphate is converted to p-aminophenol by alkaline phosphatase, and the electroactive product is quantified on a gold electrode at 0.10 V. For electrochemical detection and enzyme immunoassay, the LOD was 0.41 and 1.56 pg mL−1, respectively. Reproducibility assays employed repetitive standards of IL-6, and the intra- and inter-assay coefficients of variation were below 6.5%. Compared with the traditional IL-6 sensing method, the integrated microfluidic immunosensor required smaller amounts of sample to perform faster detection.  相似文献   

13.
A sensitive heterologous enzyme immunoassay for prostaglandin E2 was developed using 9-deoxy-9-methylene-prostaglandin F2 alpha as a stable prostaglandin E2 mimic. beta-Galactosidase was conjugated to the hapten mimic. Anti-prostaglandin E2 IgG was bound to a polystyrene tube. The enzyme-labeled hapten mimic mixed with unlabeled prostaglandin E2 was allowed to react in a competitive manner with the immobilized antibody. Then, the beta-galactosidase specifically bound to the antibody was assayed fluorometrically, and the enzyme activity was correlated with the amount of unlabeled prostaglandin E2. According to the calibration curve thus obtained, prostaglandin E2 could be determined in a range of 1.2-430 fmol. Prostaglandin E2 was extracted from human urine by the use of an octadecylsilyl silica column. The crude extract contained a substance(s) which disturbed the enzyme immunoassay and gave an apparently high content of prostaglandin E2. The interfering substance was separated from prostaglandin E2 by reverse-phase high-performance liquid chromatography. The purified urinary extract was examined by the enzyme immunoassay for prostaglandin E2, and the validity of the results was confirmed by gas chromatography-selected ion monitoring.  相似文献   

14.
DNA-dependent RNA polymerase B has been extensively purified from the larval fat body of the tobacco hornworm (Manduca sexta) by employing chromatography on ion-exchange columns of DEAE-Sephadex, DEAE-cellulose and phosphocellulose and centrifugation on glycerol gradients. The isolated enzyme after electrophoresis on acrylamide gels shows one main band and one minor band, both having enzyme activity sensitive to alpha-amanitin. The catalytic and physicochemical properties of the enzyme are similar to those of other eucaryotic B-type RNA polymerases. The enzyme has an apparent molecular weight of 530000, is inhibited 50% by alpha-amanitin at 0.04 microgram/ml and shows maximum activity on denatured DNA at 5 mM Mn2+ and 100 mM ammonium sulfate. An antibody was obtained that cross-reacts with the pure enzyme and forms a precipitin line. This antibody does not cross react with either Escherichia coli RNA polymerase or with wheat germ RNA polymerase but does react with one of the B polymerases isolated from wing tissue of the silkmoth, Antheraea pernyi.  相似文献   

15.
A simple, sensitive, and inexpensive method is presented for measuring the binding of actinomycin D (AMD) to either chromatin or DNA. The procedure gives results which are comparable with those obtained by the optical titration procedure, but is superior to that method in that it does not require continuous attention, can be used with turbid chromatin preparations, and allows simultaneous processing of several samples. Chromatin or DNA that has been allowed to react with AMD is subjected to sucrose density gradient centrifugation to remove unbound antibiotic. The AMD-DNA or AMD-chromatin complex is then dissociated by incubation with 5m urea, and the released AMD is extracted with ether and estimated spectrophotometrically.  相似文献   

16.
The cholesterol sulphate sulphohydrolase (CHS-ase) exhibiting molecular weight of 30 kDa was purified from human placenta microsomes. The microsomal proteins were extracted with 0.5% Triton X-100. The DEAE-cellulose chromatography of the solubilized microsomal proteins, performed at pH 7.6 allowed to separate two enzymatically active fractions. One of them was associated with the protein fraction unbound by DEAE-cellulose, the other was tightly bound by ion exchanger. The 30 kDa cholesterol sulphate sulphohydrolase was purified to homogenity from the protein fraction tightly bound by DEAE-cellulose. The highly purified enzyme preparation (specific activity 385 nmol min(-1)mg(-1) of protein) exhibited optimal activity at pH 6.4, the K(m) was established to be 6.7 x 10(-6)M, the pI value was 7.4. The 30 kDa cholesterol sulphate sulphohydrolase, in contrast to the CHS-ase form originated from the protein fraction unbound by DEAE-cellulose, was not sensitive to alkaline phosphatase treatment and phosphohydrolase inhibitors. The effects of steroids, -SH reacting agents and sulphohydrolase inhibitors on the enzyme activity were tested.  相似文献   

17.
Canalicular plasma membranes were isolated from rat liver homogenates using nitrogen cavitation and calcium precipitation methods. Compared with homogenates, the membranes were enriched 55- to 56-fold in gamma-glutamyltransferase, aminopeptidase M, and alkaline phosphatase activities and showed very low enrichment in markers of other membranes. By electron microscopy, the membrane preparation contained neither junctional complexes nor contaminating organelles and consisted exclusively of vesicles. The presence of vesicles was also evident from the osmotic sensitivity of D-[6-3H]glucose uptake into the membrane preparation. Antisera obtained from rabbits immunized with highly purified rat kidney gamma-glutamyltransferase inhibited the transferase activity of intact or Triton X-100-solubilized membranes by 45-55%. Treatment of vesicles with anti-gamma-glutamyltransferase antisera and anti-rabbit IgG antisera increased the apparent density of the membranes during sucrose density gradient centrifugation. gamma-Glutamyltransferase and aminopeptidase M activities were selectively removed from the vesicles by limited proteolysis with papain without changing the intravesicular space or alkaline phosphatase activity of the membranes. Specific binding of anti-gamma-glutamyltransferase antibody to the outer surface of isolated hepatocytes was observed as measured by the antisera and 125I-labeled protein A; binding followed saturation kinetics with respect to antibody concentration. These data indicate that the isolated canalicular membrane vesicles are exclusively oriented right-side-out and that gamma-glutamyltransferase and aminopeptidase M are located on the luminal side of rat liver canalicular plasma membranes.  相似文献   

18.
Two enzyme-linked amperometric immunosensors specific for salmonellas were developed as rapid methods for quantifying and detecting these organisms in pure cultures and foods. Both used alkaline phosphatase as the enzyme reporter molecule but one system used phenyl phosphate as the substrate followed by the electrochemical detection of phenol at a polarized platinum electrode. The other system incorporated an enzyme amplification step and relied on the electrochemical detection of a reduced mediator, ferrocyanide. Both assays were rapid (4 h) and specific and generated salmonella-dependent signals above 10(4) cfu/ml (phenyl phosphate system) or 10(5) cfu/ml (enzyme amplified system) in pure cultures and samples of several foods, although the results with beef samples showed considerable variation. Both systems were able to detect low (1-5 cfu/g or /ml) numbers of salmonellas in foods after non-selective (18 h) and selective (22 h) enrichment steps but four samples, out of 147, gave false positive results. False positive results were eliminated by reducing the enrichment steps to 6 h and 18 h respectively (90 samples).  相似文献   

19.
In the present study we developed an immunoenzymatic double staining technique allowing the simultaneous detection of two neuroactive substances with primary antibodies of the same species and their simultaneous visualization in semithin sections of epoxy-embedded material. For this purpose, primary antibodies against glutamate, GABA, and serotonin were either biotinylated or labeled with the trinitrophenyl (TNP) group. The latter was visualized by a detection system here referred to as the hapten-anti-hapten bridge (HAB) technique. The HAB technique consists of anti-TNP antibodies, serving as bridges between the TNP-ylated primary antibody, and a TNP-ylated marker enzyme, such as alkaline phosphatase. The single components of the HAB technique were optimized by use of a dot-blot assay and an "artificial tissue" system. The optimal staining sequence consisted of TNP-ylated primary antibody with a molar TNP:antibody ratio of 12:1, followed by anti-TNP antibody and TNP-ylated alkaline phosphatase (molar TNP:enzyme ratio of 20:1). No further improvement of detection sensitivity could be obtained when soluble immunocomplexes between anti-TNP antibody and TNP-ylated alkaline phosphatase on the side of phosphatase excess were prepared and used instead of simple TNP-ylated alkaline phosphatase. When compared with other established procedures, such as avidin-conjugated alkaline phosphatase or the ABC method, the HAB technique revealed a similar detection sensitivity. The TNP-ylated primary antibody, however, had to be used at higher concentration than the corresponding unlabeled primary antibody. The suitability of the HAB technique in combination with a modified three-step ABC technique for the simultaneous demonstration of glutamate-like and GABA-like immunoreactivity in the rat brain was demonstrated. The advantages of the new technique in comparison with existing double staining methods are discussed.  相似文献   

20.
The development of rapid methods for detection of viable Listeria monocytogenes is crucial to prevent listeriosis and product recalls. While immunomagnetic separation has been used for isolating Listeria spp., lack of specificity and pathogenicity determination render this method unsatisfactory. A two-step method using Protein A agarose beads (Immunobeads) coated with a more specific antibody, monoclonal antibody (MAb)-C11E9 for L. monocytogenes was developed. Immunobeads were allowed to capture Listeria cells from a variety of samples and tested for cytopathogenic action on a murine hybridoma B-lymphocyte, Ped-2E9 cell line by Trypan blue staining, and by an alkaline phosphatase (AP)-based cytotoxicity assay. The two-step method was used to test uninoculated hotdogs, bologna, and raw beef, chicken, and pork samples, following selective enrichment in half-Fraser broth. Pure culture studies proved the assay to be specific for L. monocytogenes, while a similar assay with Dynal Anti-Listeria immunomagnetic beads was positive for L. monocytogenes, L. ivanovii, and L. seeligeri. Detection and confirmation of cytopathogenicity of Listeria cells from food samples after 24-h selective enrichment were completed in 2-4 h. Isolates were further analyzed by the CAMP test for hemolytic activity and RiboPrinter for genomic patterns. Using immunoseparation and cytotoxicity as a two-step rapid method, viable L. monocytogenes could be isolated, detected, and confirmed as cytopathogenic in 28 h or less.  相似文献   

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