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PubMed1981

[Antithrombin III: reference values found with a chromogenic substrate (chromozym TH) (author's transl)].

A new kinetic assay for antithrombin III (heparin-cofactor) in plasma was used to determine reference values. Ranges of 10--15 IU/ml at 25 degrees C and 20--29 IU/ml at 37 degrees C were found for a reference population of 219 men and 204 women 15--93 years of age. There was no evidence that these values vary with age or sex, nor was there any difference between pre- and postprandial antithrombin activities, or between smokers' and non-smokers' values. None of the illnesses present in the reference population nor any related drug therapy affected the antithrombin III level, and there was no statistically significant difference between the values for women taking oral contraceptives and those who did not. However, this latter observation does not preclude the possibility of individual variation.

Adolescent↗
PubMed1988

Use of commercially available monoclonal antibodies for immunoenzyme double staining.

An immunoenzyme double-staining method for the simultaneous detection of two cellular epitopes, using commercially available mouse monoclonal antibodies, is described. The method employs a combination of the suppression of endogenous biotin and two successive indirect techniques with a blocking step in between. The first indirect method involves an unlabelled monoclonal antibody followed by an alkaline phosphatase-conjugated goat anti-mouse immunoglobulin. After a blocking step with normal mouse serum, the second indirect method is applied using a biotinylated monoclonal antibody followed by the visualization of this antibody by avidin-biotinylated peroxidase complex (ABC) or rabbit anti-biotin and peroxidase-conjugated swine anti-rabbit immunoglobulin in successive steps. Using these methods in combination with the introduction of dioctyl sodium sulphosuccinate and tetramethylbenzidine as chromogens for peroxidase activity, two cellular epitopes could be distinguished clearly in tissue sections by the green- and violet-stained peroxidase and alkaline phosphatase activities, respectively. The expression of two epitopes on the same cellular constituent is outlined by the coappearance of both enzyme activities as a bluish-purple colour. This method allows for the simultaneous identification, localization and enumeration of two cellular epitopes. These can serve as parameters for a number of pathological processes.

Animals↗
PubMed1985

Sensitivity of various visualization methods for peroxidase and alkaline phosphatase activity in immunoenzyme histochemistry.

Various chromogen protocols for visualizing peroxidase and alkaline phosphatase activity in immunoenzyme histochemistry were compared with respect to their sensitivity. They were tested on tissue sections of human skeletal muscle and in an antigen spot test using antibodies against slow skeletal muscle myosin. The chromogens included 3-amino-9-ethylcarbazole (AEC), 3, 3'-diaminobenzidine (DAB), p-phenylenediamine-pyrocatechol (PPD-PC) and 4-chloro-1-naphthol (CN) in peroxidase histochemistry, and 5-bromo-4-chloro-3-indolyl phosphate-nitro blue tetrazolium salt (BCIP-NBT), BCIP-tetra nitro blue tetrazolium salt (TNBT) and various combinations of substituted naphthol phosphate-diazonium salt in alkaline phosphatase histochemistry. DAB, CN, and PPD-PC were also employed with imidazole and DAB in addition to Co2+ and Ni2+ ions. The results indicate that DAB-imidazole and DAB-Co2+ and Ni2+ ions are the most sensitive chromogen protocols for visualizing peroxidase activity. Although no large differences were found between the various chromogen protocols for visualizing alkaline phosphatase activity, the protocol BCIP-TNBT is especially recommended. Furthermore, the various chromogen protocols were evaluated as to stability of chromogen solutions and final precipitates, background staining, localization properties, and enhancement of enzyme activity.

Alkaline Phosphatase↗
PubMed1985

Cytochemistry of membrane proteases.

Membrane proteases that are detectable by cytochemical means are the classified exopeptidases, aminopeptidases A and M (or N), gamma-glutamyl transpeptidase (which also acts as transferase), dipeptidyl peptidase IV and the endopeptidase, enteropeptidase (also known as enterokinase). Not yet classified are the possible exopeptidase, tripeptidyl peptidase and endopeptidases I (Ala-endopeptidase) and II (Arg-endopeptidase). All these membrane proteases can be investigated with either chromogenic or fluorogenic procedures using synthetic peptide substrates. The most useful substrates are 4-methoxy-2-naphthylamine amino acids and peptides for cytochemical localizations at the light and electron microscope levels, for cytophotometric quantification and the study of membrane protease isoenzymes after analytical isoelectric focusing. Amino acid or peptide derivatives of naphthylamine AS can be recommended for light microscopical localization and cytofluorometric quantification, and 7-amino-4-methylcoumarin and 7-amino-4-trifluoromethylcoumarin amino acids and peptides for the development of enzyme bands after isoelectric focusing. Cytochemistry reveals the heterogeneity in the distribution and species differences of membrane proteases in adult cells, tissues and organs and during development. It also reveals some common localizations, such as in small intestinal enterocytes and proximal tubule cells. The species and organ differences are substantiated and extended considerably by isoelectric focusing in combination with methods for the cytochemical detection of proteases. In addition, continuous cytophotometry or cytofluorometry (section and cultured cell biochemistry) allows the kinetic characteristics, initial reaction rates and maximum activities of all membrane proteases to be determined. The physiological functions of the endopeptidases and exopeptidases are still a matter of debate. However, from cytochemical inhibition studies with natural peptide substrates, e.g. peptide hormones, there is increasing evidence that the proteases detected with synthetic peptides play a decisive role in many physiological circumstances, e.g. in endocrine regulation mechanisms or the regulation of blood pressure. In this respect, capillary endothelium-linked surface membrane proteases may be especially important.

Animals↗
PubMed1985

Fluorescence methods for localizing proteinases and proteinase inhibitors in skeletal muscle.

Proteinases and proteinase inhibitors have become suspect in a wide variety of muscle wasting conditions that might be treatable if knowledge of the cellular locale and function of these molecules were known. Fluorescent probes have been useful in the localization of proteinases in muscle samples from human and animal specimens. These include the histochemical localization of proteinases based on the specific fluorescence of hydrolysis product derivatives, but this approach has been limited to the lysosomal proteinases because of the acidic requirements of the trapping reaction of the primary reaction product. Immunohistochemical techniques do not have the same restrictions and a number of lysosomal and nonlysosomal proteinases have been identified in muscle by this means. Unfortunately, they do not yield any information as to the activity of the enzymes. This is an important consideration since the extracellular environment contains a number of proteinase inhibitors, some of which may be internalized by the cell.

Animals↗
PubMedDate not supplied

Quantitation of endotoxin in blood from patients with meningococcal disease using a limulus lysate test in combination with chromogenic substrate.

The levels of endotoxin in blood were determined using the Limulus lysate test in combination with a chromogenic substrate. Plasma was analyzed from four patients with fatal meningococcal septicaemia and from one patient who survived meningococcal meningitis. All septicaemia patients showed high levels of endotoxin in their blood during the early stage of their disease. In two of these patients, blood samples collected at intervals of two days revealed a gradual disappearance of measurable endotoxin from the circulation. The patient with meningitis had no clinical signs of circulatory deficiency or coagulopathy and was consistently negative for endotoxin using this test procedure. Pretreating the plasma with heat and alkali and combining the Limulus lysate test with a chromogenic substrate seem to provide a sensible method for the detection and quantitation of endotoxin in blood.

Adolescent↗
PubMedDate not supplied

Reaction rate measurements of proteases and glycosidases with chromogenic methods.

Simultaneous azo-coupling and indigogenic methods were evaluated for the quantitative histochemical assay of the plasma membrane proteases gamma-glutamyl transpeptidase (EC 2.3.2.2) and dipeptidyl peptidase IV (EC 3.4.14.5) and the glycosidases maltase-glucoamylase and glucoamylase (EC 3.2.1.20) in decidual cells, jejunal enterocytes and renal proximal tubulocytes. Using kinetic (continuous) microdensitometry, a linear increase in the final reaction product was found from 3 up to 10 min, depending on the substrate concentration and the plasma membrane glycosidase or protease under investigation. Combined continuous and end point (static) microdensitometry revealed a linear relationship between the section thickness (enzyme concentration) and final reaction product up to 12 microns for the proteases and up to 16 microns for the glycosidases. Apparent Km and Vmax values were calculated with a computerized version of the direct linear plot and compared with the results obtained with the linear transformations according to Lineweaver-Burk, Eadie-Hofstee and Hanes. Apparent Km and Vmax values for the proteases were calculated separately for each animal and were 1.82 mM and 1.02 mM and 2.43 arbitrary units (a.u.) and 1.67 a.u. (gamma-glutamyl transpeptidase, decidua) and 0.42 mM and 0.38 mM and 0.29 and 0.26 a.u. (dipeptidyl peptidase IV, decidua). For the alpha-D-glucosidases, the corresponding values were 0.23 mM and 0.15 a.u. (kidney) and 0.55 mM and 0.20 a.u. (jejunum). The results show the suitability of the indigogenic methods for quantitative histochemical measurements of plasma membrane alpha-D-glucosidases, whereas the simultaneous azo-coupling procedures seemed to be less suitable for the quantification of surface membrane proteases, due to, for example, interactions of diazonium salts with amino acid or peptide substrates, reaction products and peptide activators.

Animals↗
PubMed1994

A simple activity assay for thrombin and hirudin.

Cloning of the thrombin cDNA has made it possible to study thrombin function by site-directed mutagenesis. Quantitative results from studies of thrombin mutants are often hindered by difficulties in assaying the enzyme activity. The high enzyme concentrations required for activity determination by standard methods limit their usefulness to thrombin mutants that cannot be readily produced in large quantities. We have developed a novel method using the synthetic substrate S-2238 and hirudin, a tight-binding inhibitor of thrombin, that allows for the active-site titration of thrombin at concentrations as low as 20 pM, with an error of < or = 5%. In addition, hirudin activity can be determined by this method to concentrations as low as 40 pM, with an error of < or = 5%.

Binding Sites↗
PubMed1988

Evaluation of a ten-minute chromogenic substrate test for identification of pathogenic Neisseria species and Branhamella catarrhalis.

A ten-minute chromogenic substrate test was evaluated for its ability to rapidly identify pathogenic Neisseria spp. and Branhamella catarrhalis. Identifications obtained with this system were compared to those obtained using conventional procedures. The test correctly identified 98.9% of 90 Neisseria gonorrhoeae, 98.3% of 60 Neisseria meningitidis, 96.2% of 26 Neisseria lactamica, and 100% of 36 Branhamella catarrhalis strains. Eight Neisseria subflava strains that grew on modified Thayer-Martin agar were prolyl aminopeptidase positive and were misidentified as Neisseria gonorrhoeae. Other strains of saprophytic Neisseria spp. also reacted with the chromogenic substrates. The system was accurate and reliable for identifying the commonly encountered pathogenic species. In light of recent reports describing new species and atypical Neisseria strains, however, careful attention to the salient features of both common and atypical organisms is necessary for proper use of rapid enzymatic identification tests.

Chromogenic Compounds↗
PubMed1983

Cleavage of p-nitroanilides of N-acylated tri- and tetrapeptides by alanine endopeptidase from the brush border membranes of rat enterocytes.

The activity of the alanine endopeptidase from the intestinal brush border was studied using chromogenic substrates of the general formula Sc-Ala2-X-pNA. Sc-Y-Z-Ala-pNA and W-Ala3-pNA respectively. Substrates with C-terminal Leu or Nle are hydrolyzed more readily than Ala-analogues. At least one Ala-residue in one of the positions adjacent to the C-terminus is necessary for the enzyme activity. An Na-substituent has no effect on the activity.

Animals↗
PubMed1985

An improved spectrophotometric assay for histamine and diamine oxidase (DAO) activity.

Diamine oxidase or its substrates can be measured using a coupled reaction with peroxidase and the chromogen 3-methyl-2-benzothiazolone hydrazone (MBTH) with an appropriate acceptor such as 3-(dimethylamino)benzoic acid (DMAB). This method provides a more rapid and sensitive method of measuring histamine than current colorimetric assays; using this system, histamine was quantitated at concentrations of 10 to 400 mumol/l. The Km of DAO was determined to be 2.9 X 10(-5) M, a lower value than previously reported.

Amine Oxidase (Copper-Containing)↗
PubMed1994

Evaluation of two new chromogenic media for detection of Salmonella in stools.

A clinical collaborative study was conducted to compare two new chromogenic agar media, Rambach agar and the Salmonella Detection and Identification Medium (SMID) (bioMérieux, France), with two conventional media, Salmonella-Shigella agar and Hektoen agar. Thirty-nine Salmonella strains involving 14 serotypes were isolated from 1,454 stool specimens. After enrichment in a selective broth, 100% sensitivity was obtained with each medium. The SMID and Rambach agars are considerably more specific than the conventional media. Although SMID agar detects all Salmonella serotypes, it is not as specific as Rambach agar, which requires a complementary test (C8 esterase test) to detect all serotypes.

Agar↗
PubMed1984

Rapid enzymatic characterization of clinically encountered anaerobic bacteria with the API ZYM system.

The purpose of this study was to determine if enzyme profiles of anaerobic bacteria, obtained with the API ZYM system, are sufficiently distinctive and reproducible to merit future development of the system and an expanded data base for the four major groups of clinically encountered anaerobes. Of the total 155 clinical isolates and reference strains that were tested, 88% had distinctive patterns. It was possible to differentiate between 89% of the anaerobic gram-negative bacilli, 64% of anaerobic cocci, 100% of anaerobic gram-positive non-sporeforming bacilli and 100% of the Clostridium spp. using only the API ZYM plus Gram reaction, morphology and relation to oxygen. Overall reproducibility for 1,330 enzyme-substrate reactions was 97%. It was concluded that the API ZYM could be a practical system for rapid identification of clinical anaerobe isolates provided that an expanded data base is developed and that certain additional recommended tests are used.

Bacteria↗
PubMed1984

Pyridinium 2-azo-p-dimethylaniline chromophore, a chromogenic reagent for beta-lactamase testing compared to nitrocefin.

Pyridinium-2-azo-p-dimethylaniline chromophore was evaluated as a test tube, filter paper and spectrophotometric assay for detection of beta-lactamases from gram-positive and gram-negative organisms. Although useful for detection of TEM beta-lactamases in Haemophilus influenzae and Neisseria gonorrhoeae, it was a poor agent for detecting TEM, OXA and PSE enzymes in Enterobacteriaceae. It also proved poor for detecting cephalosporinases in Pseudomonas aeruginosa and Enterobacteriaceae, and penicillinases in Staphylococcus aureus when compared to nitrocefin. As a spectrophotometric substrate it was equivalent to nitrocefin and cephaloridine with various beta-lactamases.

Anti-Bacterial Agents↗