Gene/Protein Disease Symptom Drug Enzyme Compound
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Query: EC:3.4.21.69 (APC)
 16,337 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Six monoclonal antibodies for human thrombomodulin (TM) were prepared. All of them recognized an elastase-digested fragment of TM which contains 6 epidermal growth factor (EGF)-like structural domains. We developed a one-step sandwich enzyme immunoassay for soluble TM by using 2 antibodies; one of them, which inhibited thrombin-binding to TM, was fixed to polystyrene balls, and the other, which did not inhibit the thrombin-binding, but inhibited the protein C-activating cofactor activity of TM, was used as peroxidase-labeled conjugate. The sensitivity of this assay was 1 microgram/l for soluble TM. The level of soluble TM was found to be significantly increased in sera of patients with systemic lupus erythematosus in comparison to the level in sera of healthy subjects.
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PMID:One-step sandwich enzyme immunoassay for soluble human thrombomodulin using monoclonal antibodies. 196 42

This study describes a process by which serine proteases that contain an S-1 arginine subsite and active site histidine may be inactivated and subsequently quantitated using a combination of peptidyl chloromethylketone chemistry and immune recognition technology. Active site labeling and inactivation of proteases is attained by modification of the active site histidine with a peptidyl chloromethylketone. In the specific illustrations demonstrated, we used the compound biotinyl-epsilon-aminocaproyl-phenylalanylprolylarginyl chloromethylketone. This reagent reacts quantitatively and specifically with the active site histidine of a wide variety of proteases that are elaborated in the coagulation and fibrinolytic system. The inactivated enzyme(s) may be quantitated by combinations of antiprotein antibodies and avidin binding technology using the biotin moiety on the peptide inhibitor. We have demonstrated the capability of capture of inactivated enzyme products directly on to solid-phase avidin with subsequent quantitation of bound protein using specific antibodies. In the converse system we have captured specific proteases using antiprotein antibodies in the solid phase and have quantitated bound enzyme by using avidin. Subsequent detection and quantitation has been achieved using the enzymatic activity of horseradish peroxidase conjugated either to the antibody or to avidin. Both types of assays are feasible, with avidin capture being the preferred mode when enzyme is evaluated in the presence of excess zymogen, as would be common in the evaluation of most blood-clotting enzymes. Assays are illustrated for tissue plasminogen activator, plasmin, thrombin, factor Xa, and activated protein C, which can measure protease concentrations as low as 50 pmol/L. Specific applications of the assays are provided in studies of the activation of prothrombin by the prothrombinase complex and of factor X with Russell's viper venom factor X activator. These assays measure the mass of active site present in the reaction mixture and are relatively independent of subspecies of enzyme or the environment in which the activity is generated. These assay systems provide powerful tools for elucidating product-precursor relationships in multienzyme feedback reactions involving zymogen activation.
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PMID:Active site-specific immunoassays. 211 28

We have used antibodies to human thrombomodulin isolated from placenta to investigate the distribution of this cofactor for protein C activation in human tissues. Thrombomodulin was found on endothelial cells of arteries, veins, capillaries, and lymphatics by immunocytochemical staining using an avidin-biotin peroxidase method. Thrombomodulin was not detected on sinusoidal lining cells of liver or on postcapillary high-endothelial venules of lymph node, although the latter contained another endothelial antigen, von Willebrand factor. Other cells noted to contain thrombomodulin antigen are those of the syncytiotrophoblast in placenta. The thrombomodulin in syncytiotrophoblast was primarily on the plasma membrane surface that forms the maternal blood sinus. Syncytiotrophoblast also stained with antibodies to von Willebrand factor, which implies that these cells have multiple endothelial functions. Thrombomodulin antigen was found in all organs studied, with the notable exception of brain.
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PMID:Thrombomodulin is found on endothelium of arteries, veins, capillaries, and lymphatics, and on syncytiotrophoblast of human placenta. 299 Dec 98

Thrombomodulin (TM) is the endothelial cofactor of the anticoagulant protein C system. The distribution of TM in the organism was studied in the rabbit using a goat anti TM, affinity-purified antibody and a peroxidase-labelled antigoat immunoglobulin. TM antigen was found on the endothelial surface of all blood vessels: capillaries, arteries and veins. The reaction was specific: connective tissue, smooth and striated muscle bone, cartilage, nerve tissue, secretary epithelia and all parenchyma studied were not strained. Moreover TM antigen was present on the surface of serosa: peritoneum, pericardium and pleura as well as on synovial membranes and on arachnoid, all along the central nervous system. It was absent from pia and dura mater. The antigen was found only after formalin fixation on the vessels and body cavities surface on which it lies. This observation shows that the antigen is easily detached from these surfaces and suggest a possible mobility of this endothelial molecule for which it lies. This observation shows that the antigen is easily detached from these surfaces and suggest a possible mobility of this endothelial molecule for which production and function sites might differ.
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PMID:[Different localization of thrombomodulin]. 303 77

We describe the simple and rapid enzyme immunoassay of protein C in human plasma with use of a Cobas Fara centrifugal analyzer. The antibody, labeled with horseradish peroxidase, is reacted with antigen (protein C) for 15 min. The peroxidase activity of the resulting antigen-antibody conjugate is measured at 500 nm for 5 min in the presence of excess H2O2, phenol, and 4-aminoantipyrine, as compared with that of free conjugates. Results are calculated from a stored standard curve and expressed as a percentage of the value determined for a pooled specimen of normal adult plasma. The standard curve is linear from 0% to 200%. The CV is generally less than 4% for different concentrations of protein C. In liver cirrhosis, hepatocellular carcinoma, therapy with warfarin, thrombosis, and disseminated intravascular coagulation, protein C concentrations are about 40-70% of normal. Results obtained with the present homogeneous enzyme immunoassay correlated well with those by enzyme-labeled immunosorbent assay (r = 0.97).
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PMID:Protein C in human plasma determined by homogeneous enzyme immunoassay with use of a centrifugal analyzer. 304 78

We have developed a variation of the solid-phase enzyme-linked immunosorbent assay (ELISA) to enable measurement of the activity and antigen levels of protein C (PC) in human plasma. With this assay it is possible to do both tests with the same sample and same microtiter plate coated with anti-PC monoclonal antibody (MCA)JTC-4, which inhibited neither activation of PC nor activity of activated PC (APC). Even in patients undergoing heparin treatment for severe disseminated intravascular coagulation, there were no detectable differences between amidolytic activity and antigen levels of PC in patients' plasma. In addition, there was a strong correlation between the immunologic levels of PC in patients' plasma determined both by polyclonal ELISA using peroxidase-labeled immunopurified antiprotein C-IgG and those found with MCA ELISA using peroxidase-labeled MCAJTC-5, which does not bind to APC. In contrast, when oral anticoagulation therapy was started, immunologic levels of plasma PC estimated by peroxidase-labeled MCAJTC-1, a MCA that recognizes a gamma-carboxyglutamic acid domain-related conformational change of PC induced by metal ions, decreased more rapidly than did either the PC level determined by polyclonal ELISA or the percent prothrombin time. This suggested that comparison of MCAJTC-1-recognized PC levels and prothrombin time may be necessary at the beginning of oral anticoagulation therapy to treat patients safely.
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PMID:Level of protein C determined by combined assays during disseminated intravascular coagulation and oral anticoagulation. 358 May 75

We isolated protein C from a barium citrate-adsorbed fresh plasma and human factor IX concentrate by immunoaffinity chromatography on a column of Sepharose coupled with monoclonal antibodies to protein C. The antibodies used were conformation-specific monoclonal antibodies to the calcium-induced structure of protein C. Protein C was bound to antibodies coupled with Sepharose in the presence of calcium ions and was eluted with EDTA. This immunopurification resulted in a 13,000-fold purification of the fully functional zymogen from plasma. The immunoaffinity-isolated protein C was found to have higher amounts of single-chain protein C than conventionally isolated protein C when analyzed by sodium dodecyl sulfate-polyacrylamide gels under reduced conditions. The factor IX concentrate was applied to this Ca2+-dependent antibody JTC-3-immobilized Sepharose in the presence of 5 mM CaCl2, and protein C with its gamma-carboxyglutamic acid (Gla) domain intact was firstly bound to this column and then eluted by metal chelation with EDTA. When flow-through fractions were applied again in the presence of Ca2+ to this column, modified protein C which had lost its N-terminal 42-residue peptide was weakly bound to this column. It was eluted in the absence of Ca2+. However, only a low percentage of modified protein C was detectable by an enzyme-linked immunosorbent assay using Ca2+-dependent monoclonal antibody JTC-3 and peroxidase-labeled immunopurified polyclonal antibody. These results indicate that factor IX concentrate has both Gla-domain-intact and Gla-domainless protein C. Moreover, it suggests that Ca2+-dependent monoclonal antibody JTC-3 may recognize the coupled conformational change of protein C induced by the combined effect of Ca2+ binding to the Gla domain and to other parts of protein C.
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PMID:Immunoaffinity purification of protein C by using conformation-specific monoclonal antibodies to protein C-calcium ion complex. 362 Apr 98

A new solid phase enzyme-linked immunosorbent assay (ELISA) was developed for the quantitation of human protein C antigen. Anti-protein C F(ab')2 fragments were adsorbed to polystyrene plates. The binding of serial dilutions of control or test plasma, containing protein C, was detected by incubation with peroxidase-labeled anti-protein. C-IgG followed by the addition of hydrogen peroxyde and 0-phenylenediamine. This ELISA is specific, sensitive (detection limit: 0.02%) and accurate (variation coefficient: 3 to 10%). When results are compared to those obtained by the Laurell technique (electroimmunodiffusion, EID), the correlation coefficient is 0.95 in all tested plasmas. Protein C antigen was measured by ELISA and EID in plasma from 40 controls, 14 patients with congenital protein C deficiency, 15 patients with liver cirrhosis and 40 dicoumarol-treated cases. In normal plasma, protein C ranged from 70 to 126%. In congenital deficiency, protein C was between 35 and 58% in 13 cases and 9% in one of them. In patients with liver cirrhosis and dicoumarol-treated cases, levels of protein C antigen were compared to those of other vitamin K dependent factors, i.e. Factors II and IX measured by EID and Factor X assayed by EID and ELISA. In liver cirrhosis, the amount of protein C was significantly lower than that of Factors II, IX and X. In short-term and long-term dicoumarol-treated patients, the highest correlation (r = 0.72) was observed between protein C and Factor X levels. In the plasma of patients undergoing oral anticoagulant therapy, protein C decreased more rapidly than Factors X or II and migrated in presence of calcium as a double peak, one with a normal mobility and one more anodal corresponding to the non carboxylated form of protein C.
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PMID:A new method for the estimation of protein C by ELISA. 608 75

We investigated possible explanations for the common occurrence of perivenular lesions in liver allografts of patients on FK506 within a few weeks to several months after OLT. Hematoxylin and eosin-stained sections of pre- and postperfusion biopsy specimens and day 7 post-transplant protocol biopsy specimens from 31 patients, randomly assigned to either FK506 or CsA as primary immunosuppressive agent, were reviewed, and immunohistochemical stains for HLA-DR antigen and S-100 protein were performed by the avidin-biotin peroxidase complex method. The histologic features of cellular rejection in the portal tracts of day 7 posttransplant allograft biopsy specimens from patients on FK506 were milder than those from patients on CsA. Immunohistochemical stains for HLA-DR showed intense positivity in a variety of cell types in day 7 posttransplant specimens from both groups, including sinusoidal-lining cells, bile duct epithelial cells, vascular endothelial cells, inflammatory cells, and occasional injured hepatocytes. Although diffuse lobular staining was seen in the majority of cases in both groups, either with or without rejection, liver biopsy specimens from patients on FK506 showed concentration of positively stained cells in perivenular regions more often, and at a lower overall histologic grade of rejection, than specimens from patients on CsA. There were no differences in the number and distribution of S-100 protein-positive dendritic APC between biopsy specimens from FK506 versus CsA-treated patients, or between specimens with and without cellular rejection in either group. It is suggested that the development of perivenular injury, which is seen frequently in allograft biopsy specimens from patients on FK506 obtained at various intervals after transplantation, may be related to drug toxicity rather than to the process of allograft rejection.
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PMID:FK506 versus cyclosporine as primary immunosuppressive agent for orthotopic liver allograft recipients. Histologic and immunopathologic observations. 750 52

C4b-binding protein (C4BP), a regulatory component in the complement system, binds to an anticoagulant vitamin K-dependent plasma protein S (PS) which acts as a cofactor of activated protein C. We raised monoclonal antibodies against C4BP and PS, and developed two different one-step sandwich enzyme immunoassay (EIA) systems for human total C4BP (assay A) and PS-C4BP complex (assay B) by using a solid phase monoclonal antibody and a horseradish peroxidase-labeled monoclonal antibody (Fab'). The reaction time of the assay was 45 min in both EIA systems: 30 min for the immunoreaction and 15 min for the color reaction. The sensitivities were 12 and 20 mg/l in assays A and B, respectively. Linearity was obtained between 31 and 500 mg/l in both EIA systems. Assay A could detect both uncomplexed C4BP and PS-C4BP complex with equal efficiency so that total C4BP level was not affected by PS. The levels of total C4BP and PS-C4BP complex were found to significantly increase in sera from patients with membranous nephropathy and decrease with liver cirrhosis in comparison with the levels in normal subjects. On the other hand, a difference in the total C4BP and PS-C4BP complex levels was not shown between IgA nephropathy and normal subjects. Affinity column analysis and difference of total C4BP and PS-C4BP complex levels showed that most of C4BP in sera exists as PS-C4BP complex.
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PMID:One-step sandwich enzyme immunoassays for human C4b-binding protein (C4BP) and protein S-C4BP complex using monoclonal antibodies. 775 11


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