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

Protein A and C, which are major components of the acidic proline-rich proteins in human saliva, were digested, before or after adsorption to hydroxyapatite, with alkaline phosphatase, trypsin, thermolysin and a proteinase preparation from salivary sediment. The results demonstrate that the binding site is located in the proline-poor N-terminal part of the protein, possibly between residues 3 and 25. Phosphoserine is necessary for maximal adsorption of the proteins to hydroxyapatite. When proteins A and C are adsorbed to hydroxyapatite before proteolytic digestion there is a protection of some of the susceptible bonds in the N-terminal part of the proteins and a gradual removal of the proline-rich C-terminal part. Thermolysin can cleave susceptible bonds in the part of the protein that remains bound to hydroxyapatite, but at least some of the resulting peptides are retained on the mineral. Since the ability of the proteins to inhibit hydroxyapatite formation and to bind calcium is located in the N-terminal proline-poor part, it is possible that these activities are retained after proteolytic digestion of the adsorbed proteins.
 ...
PMID:The nature of the hydroxyapatite-binding site in salivary acidic proline-rich proteins. 23 Aug 18

Quantitative interpretation of protein immunoblotting procedures is hampered by a variety of technical liabilities inherent in the use of photographic and densitometric methods. In this paper, we present a novel, simple, and generally applicable alternative procedure to acquire quantitative data from immunoblots. Our strategy employs both the standard alkaline phosphatase color reaction and radiolabelled Protein A. The color reaction is used to localize the polypeptide of interest after transfer to a solid support. The colored bands are then excised and the radioactivity in the colocalized Protein A is quantitated in a gamma counter. In addition to avoiding the problems associated with photographic and densitometric procedures, our assay also overcomes common problems associated with variable gel lane width and individual band distortion. The resulting data is linear over a range of at least 50-fold (10-500 ng of specific protein, for the example used in this study) and is highly reproducible.
 ...
PMID:A general radiochemical-color method for quantitation of immunoblots. 172 52

Protein A of Staphylococcus aureus Cowan I is a powerful immunostimulating agent. Female Swiss Portan rats fed 7,12-dimethylbenz(alpha)anthrancene (DMBA) exhibited increased serum alkaline phosphatase activity, which returned to normal levels following eight weeks of treatment with 12 micrograms protein A subcutaneously. Protein A reduced the potential of tumor induction by DMBA as observed by the noninduction of tumors until three months after discontinuation of protein A administration. The total leukocyte count was not affected. Protein A treatment for six weeks of DMBA-induced mammary adenocarcinoma-bearing rats caused the increased serum alkaline phosphatase activity to decrease but not to normal levels, indicating regression but no disappearance of the tumors. The total leukocyte count of the tumor bearers was stimulated by protein A and increased 24 hours after protein A administration; however, in the fourth week of treatment it returned to normal levels. The leukocytosis suggests that protein A could cause tumor necrosis by an inflammatory reaction, edema, and cell destruction and thus tumor regression.
 ...
PMID:Protective and therapeutic efficacies of protein A on 7,12-dimethylbenz(alpha)anthracene-induced rat mammary adenocarcinoma. 174 Jul 68

Protein A-STb and STb-alkaline phosphatase protein fusions were used as immunogen and antigen, respectively, for the generation and screening of monoclonal antibodies to the Escherichia coli heat-stable enterotoxin STb. Following immunization with immunoglobulin G-Sepharose-purified protein A-STb and hybridoma construction, STb-alkaline phosphatase hybrid protein was used in a labeled antigen capture assay to detect the production of STb-specific monoclonal antibody. STb-specific monoclonal antibodies were characterized by using a combination of immunoblotting and synthetic-peptide-based enzyme immunoassay techniques. Four distinct anti-STb antibodies were identified and characterized.
 ...
PMID:Monoclonal antibodies specific for the Escherichia coli heat-stable enterotoxin STb. 177 22

We present a simple, efficient and rapid method for affinity-purifying antibodies from a relatively crude antiserum in quantities large enough to screen a DNA expression library. The method presents a very convenient way to remove crossreacting or contaminating antibody specificities. The affinity matrix, antigen non-covalently bound to nitrocellulose, is prepared by the electrophoretic separation of antigen by sodium dodecyl sulphate-polyacrylamide gel electrophoresis, followed by the transfer of antigen to nitrocellulose. The matrix can be used repeatedly. A brief wash with 6 M guanidine hydrochloride is included between steps to remove residual antibodies which bind with high affinity to nitrocellulose-bound antigen. Various buffer solutions were assessed as antibody/antigen-dissociating agents. Glycine/HCl buffer, pH 2.5, appeared to be the most efficient in our hands, although a number of other less efficient dissociating reagents, including 4.5 M magnesium chloride, pH 7.5, 6 M urea, pH 7, and 0.05 M diethylamine, pH 11.5, also could be used; these may be the elution conditions of choice for other antibody/antigen combinations. The use of affinity-purified antibody solutions instead of the corresponding antisera gave increased signal-to-noise ratios with the detection systems that are commonly used to identify positive signals in screening expression libraries. Protein A- and goat anti-rabbit-alkaline phosphatase conjugates gave the most sensitive signals.
 ...
PMID:Nitrocellulose-bound antigen repeatedly used for the affinity purification of specific polyclonal antibodies for screening DNA expression libraries. 312 68

A highly efficient immunoscreening procedure has been developed to isolate cDNA clones to the calcium-binding protein (CaBP) of the chick embryonic chorioallantoic membrane (CAM). A library of total CAM cDNA was constructed using the expression plasmid vector, pUC 19. Bacterial clones containing plasmids with CaBP cDNA inserts were detected immunohistochemically based on their expression of hybrid CaBP protein sequences. For immunodetection, nitrocellulose bacterial colony replicas were treated with specific antibodies to the CaBP followed by incubation with Staphylococcus aureus Protein A conjugated with alkaline phosphatase (AP) which served as a secondary immunoreagent. Positive clones were then histochemically identified based on AP enzyme activity. The identity of the immunopositive clones was further verified by in vitro translation of mRNA selected by hybridization to the cloned cDNA. The AP-based immunoscreening procedure yields stable reaction products with relatively low background, and should find general application for isolating specific cDNA clones from expression cDNA libraries.
 ...
PMID:Alkaline phosphatase conjugated protein A as a sensitive reagent to immunoscreen an expression cDNA plasmid library: isolation of cDNA to the calcium-binding protein of the chick embryonic chorioallantoic membrane. 382 19

A microplate-adapted enzyme linked immunosorbent assay (ELISA) for detection of C1q-binding immune complexes (IC) and aggregated IgG (delta IgG) is described. Purified human C1q was adsorbed to the wells of flat-bottomed microtiter plates and EDTA-treated serum samples were subsequently introduced. Bound IC was measured by use of alkaline phosphatase-labelled Protein A followed by the substrate para-nitro-phenyl-phosphate. A dose response was found for both delta IgG and BSA anti-BSA complexes, while variations in the concentration of monomer IgG did not affect the optical density. Elevated levels of IC were found in the majority of sera from patients with rheumatoid arthritis and SLE. The described C1q-PA-ELISA is a simple and inexpensive method for detection of C1q-binding immune complexes. The reproducibility is acceptable and the sensitivity is higher than for most IC-methods based on C1q-binding.
 ...
PMID:Estimation of immune complexes by a microplate-adapted C1q-Protein A enzyme-linked-immunosorbent-assay (C1q-PA-ELISA). 633 38

A simple ELISA assay for detecting murine anti-SRBC antibodies of IgG class was developed and the variation of the results according to different experimental conditions was investigated. Erythrocytes were left to settle in flexible plastic microtiter plates, after which they were fixed with glutaraldehyde and the remaining binding sites in the plates saturated with ovalbumin. Serum or monoclonal IgG antibodies were then allowed to react with the erythrocytes. Protein A coupled to alkaline phosphatase caused a color change in the subsequently added enzyme substrate. The results proved to be of good reproducibility, specificity and sensitivity. The assay can be used for measuring IgG concentration, estimating antibody avidity and number of antigenic determinants on the SRBC, as well as screening IgG anti-SRBC hybridomas. The precision of concentration estimates was very good when standard curves were used.
 ...
PMID:An enzyme-linked immunosorbent assay for measuring anti-sheep erythrocyte antibodies. 636 93

Protein A-bearing Staphylococcus aureus was used as a solid-phase matrix in a sandwich-type enzyme immunoassay for urinary albumin. Heat-inactivated, formalin-fixed bacteria were coated with affinity-purified goat anti-human albumin, exposed to solutions containing standard or unknown concentrations of albumin, then challenged with an alkaline phosphatase/anti-human albumin conjugate obtained by periodate oxidation. Alkaline phosphatase activity bound to the bacteria was a function of albumin concentration from 25 to 1000 micrograms/L. This assay was applied to determinations of urinary albumin concentrations between 1.25 and 1000 mg/L. Between-run CV was 2.55 (63.9 mg/L concentration). Within-run CVs for albumin concentrations of 1.9, 38.1, and 638.0 mg/L were 3.7, 3.7, and 2.4%, respectively. Analytical recovery was 95 to 107% across the full working range of the assay. Bence Jones proteins and hemoglobin had no significant effect on the assay. Nonspecific binding of the enzyme-antibody conjugate was 1.3% (SD = 0.7%). Values agreed well with those by radial immunodiffusion.
 ...
PMID:Protein A-bearing Staphylococcus aureus as the solid phase in an enzyme immunoassay and its application to determination of urinary albumin. 681 88

Bovine serum albumin labeled with alkaline phosphatase and antibody have been employed as a model to determine if the use of Protein A bearing Staphylococcus aureus Cowan I strain (SACI) bacteria could be extended to enzyme immunoassay (EIA). SACI do not activate "per se" the enzyme substrate and bind aspectifically minimum amount of enzyme labeled antigen. Experimental conditions are described for the use of SACI both in macro and micro EIA assay which allows the processing of numerous samples with minimum handling. The sensitivity of the EIA is comparable with radioassy (EIA 2ng-RIA 4ng) which uses SACI in place of second antibody. The inhibition test can be performed in 4 hours time. These results suggest that the stability of SACI when combined with that of enzyme labeled antigens can widen the use of EIA, both for investigative and clinical studies.
 ...
PMID:Use of protein A bearing Staphylococcus aureus in enzyme immunoassay (EIA). 699 91


1 2 3 Next >>