A spectroscopic collagenase assay using peroxidase-labeled collagen

A quantitative collagenase assay detecting soluble collagen fragments is described in this paper. Using the reagent N-succinimidyl 3-(2-pyridyldithio)propionate (SPDP) type I collagen was conjugated with horseradish peroxidase (POD) which was employed as a reporter enzyme. POD was preferentially linked to the TC B fragment in a ratio of 1.4 mol POD/mol collagen. The conjugation product was immobilized on AH-Sepharose via carbodiimide coupling to form the final collagenase substrate used in the assay. POD activity in the supernatants caused by liberated TC B fragments exhibited a linear relationship for collagenase concentrations up to 100 micrograms/ml bacterial collagenase. Over an incubation period of 4 h the lowest detection limits found were 20 ng/100 microliters for bacterial collagenase and 60 ng/100 microliters for human leukocyte collagenase. Incubation of the assay mixture with 5 micrograms trypsin resulted in 3.8% of the activity released by the equivalent amount of leukocyte collagenase. The assay developed here has been shown to be sensitive and specific for collagenase, with the additional advantage that this method is suited for simple and economic handling.     

Radioimmunoassay of soluble and insoluble collagenases in fibrotic liver.

We have postulated that an insufficient active of collagenase relative to increased collagen synthesis may be the cause of the increased collagen accumulation in fibrotic tissues. In the present study, 125I-collagenase and rabbit anti-collagenase immunoglobulin G were used to develop a sensitive radioimmunoassay that detects 0.1 nM (3 ng) of collagenase protein in tissue samples. The assay also can detect collagenase protein that is associated with extracellular-matrix collagen fibrils. Good correlation with an assay of enzyme activity validates the radioimmunoassay for quantification of collagenase. The assay was used to measure amounts of collagenase in relation to fibrotic processes in livers of mice with schistosomiasis. Results indicate that the amounts of collagenase relative to synthesized collagens were significantly lower, and this may contribute to the progressive fibrosis. The occurrence of a maximum amount of collagenase at 7 weeks after infection with Schistosoma mansoni cercariae in concanavalin A-treated animals, as compared with 8 weeks in controls, could account for the large remission of fibrosis in mice so treated. The results emphasize the possible importance of collagenase in controlling or limiting fibrosis.     

Radial diffusion assay of tissue collagenase and its application in evaluation of collagenase inhibitors.

A radial diffusion assay for tissue collagenase (EC 3.4.24.3) has been devised which is simple, sensitive and capable of application to large numbers of samples. The assay employs an agarose matrix containing solubilized lathyritic rat skin collagen as substrate. Fibril formation is induced for 2 h at 37 degrees C subsequent to 41 h digestion at 28 degrees C. The procedure results in sharply defined zones of lysis which may be measured directly or after photography. The characteristics of the procedure are otherwise similar to those reported for other radial diffusion assays. The new method was used to examine the action of 10 compounds which were known or potential inhibitors of tadpole collagenase. The concentration of inhibitor required to produce 50% inhibition is reported for the following compounds: alpha2-macroglobulin, 142 microng/ml; N-acetylcysteine, greater than or equal to 100 mM; cysteine, 8.7 mM; EDTA, 0.46 mM; histidine, greater than or equal to 100 mM; 2,3-dimercaptopropanol, 0.5 mM and mercaptoacetic acid, 70 mM. The procedure also has potential for clinical determinations (e.g. tears, synovial fluid) since assay dishes may be prepared in advance and only 15 micronl of sample is required.     

An evaluation of fluorometric proteinase assays which employ fluorescamine

The sensitivity and utility of proteinase assays employing fluorescamine, a compound which reacts with primary amines to form a fluorescent adduct, was assessed. As little as 1 ng of purified trypsin or clostridiopeptidase A could be detected within 3 h of incubation at 37 degrees C, using casein or gelatin as substrates. Increasing the incubation period to 18 h permitted the detection of 250 pg of each enzyme. When gelled collagen was utilized as substrate, the sensitivity to clostridiopeptidase A was reduced to 2.5 ng at 3 h and 500 pg at 18 h. The techniques could be used to measure the gelatinase, caseinase, and collagenase activities of culture media conditioned by synovial tissue. The main disadvantage of this assay is its susceptibility to interference by compounds which fluoresce or quench. This, in turn, necessitates additional blanks, which may render the assay tedious.     

A collagen film microassay for tissue collagenase.

A quantitative microassay for the detection of bacterial and tissue collagenase is presented. Collagen in acetic acid solution forms a thin, tenacious film upon contact with a dried agar surface. The digestion of the film by collagenase is detected by subsequent staining with Coomassie blue. Undigested film is stained dark blue while areas of collagenase digestion remain clear. The technique can be employed in two ways: with collagen-coated glass coverslips as a rapid screening method or with collagen-lined capillary tubes for precise quantitation. The assay has a sensitivity comparable to those assays using radioactively labeled collagen substrates and requires only 5 to 60 min of incubation.     

Simple cryoprotection and cell dissociation techniques for application of the comet assay to fresh and frozen rat tissues

The single-cell gel electrophoresis (comet) assay has been widely used for genotoxicity studies in cell cultures, but its use in solid tissues is hindered by problems in isolation of cells and in cryopreservation techniques. Here, we used minced liver tissues from rats to compare a homogenization technique for isolation of nuclei with a collagenase digestion method (300 λunits/g liver at 37°C for 20 λmin) for isolation of intact cells for subsequent comet assay. We found that collagenase digestion was preferred to the homogenization technique in fresh tissues, but neither method prevented the extensive DNA damage caused by cryopreservation ( -85°C for 72 λh). To minimize this damage, minced liver (1.0 λg) and kidney (0.5 λg) tissues were added to 20 λml of pre-cooled 10% glycerol or 10% dimethylsulfoxide (DMSO). We showed that cryoprotection with DMSO ( -85°C for 72 λh and 3 weeks), and to a slightly lesser extent with glycerol (72 λh), followed by collagenase digestion led to satisfactory recovery of liver cells with little or no DNA strand breakage. We then used DMSO as a cryoprotective agent to optimize the amount of collagenase and its incubation time in frozen liver and kidney tissues. We showed that the collagenase digestion at 150 λunits/g liver and 300 λunits/g kidney for 10 λmin produced highest cell numbers and minimal DNA strand breaks. We also validated these procedures by injection (i.p.) of rats with a known renal carcinogen, ferric nitrilotriacetate (Fe/NTA). We showed that Fe/NTA strongly induced DNA strand breaks in both rat liver and kidney, while no DNA strand breakage occurred in these tissues from the control rats. In addition, no significant differences in strand breaks were found between fresh tissues and tissues treated with DMSO during freezing at -85°C for 72 λh. Thus, the cryoprotection and the cell dissociation techniques developed here are satisfactory for preparing both fresh and frozen tissues for comet assay. These simple techniques are expected to expand greatly the usefulness and efficacy of the assay.     

Induction of collagenase production in U937 cells by phorbol ester and partial purification of the induced enzyme

The U937 cell line is a monoblast-like cell line that can be induced to differentiate when treated with phorbol ester or a variety of other agents. Collagenase was detected in the media of U937 cell cultures after treatment with phorbol myristate acetate (PMA) at concentrations of 5 ng/mL or greater. In general, no collagenase was detected in the media of untreated cells. The induced collagenase cleaved native type I collagen into the 3/4 and 1/4-length fragments and showed the inhibition by ethylenediaminetetraacetic acid characteristic of the action of mammalian collagenases. Collagenase activity could be detected in the media of treated cells 12-18 h after the addition of PMA. Secretion of collagenase continued for 2-3 days after PMA addition. The production of collagenase by PMA-treated U937 cells was inhibited by actinomycin D and cycloheximide, suggesting that the induction of the enzyme is the result of de novo synthesis. The collagenase secreted by U937 cells induced with PMA has been purified 12-fold by using DEAE-Sephacel followed by wheat germ agglutinin-agarose chromatography. The apparent molecular mass of this U937 collagenase, determined by gel filtration chromatography on the partially purified enzyme, was 29-36 kilodaltons.     

A collagen film for microdetermination of collagenase activity.

A simple, rapid, sensitive, and specific film assay for collagenase activity employing a glass-supported, reconstituted collagen gel is described. Digestion of the collagen film results in sharply defined zones of lysis detectable by staining with Coomassie blue. The assay is semiquantitative and suitable for micro enzyme determination in biological fluids.     

Clofibrate-induced increase in coenzyme A concentration in rat tissues

1. Total, active and latent collagenase activities were determined by direct assay of tissue homogenates. 2. The rate of collagen breakdown during post-partum involution of the rat uterus is correlated with the total activity of collagenase. Both are low at parturition, reach a maximum within 24h and fall slowly to low values of 5 days post partum. This temporal correlation strongly supports the hypothesis that collagenase participates in collagen breakdown in vivo. 3. Further support for this hypothesis is provided by the finding that oestradiol-17 beta (100 micrograms/day, intraperitoneally injected), which inhibits the breakdown of collagen by 36% during the first 4 days of involution, produces a closely corresponding decrease in total collagenase activity. 3. The effect of oestradiol in lowering collagenase activity is not due to alterations in collagen substrate, collagenase kinetic behaviour or latent-to-active enzyme conversion. 4. Of the total assayable collagenase, about 35% is fully active and 65% is in a latent form. 5. About 70% of this latent form can be activated by a serine proteinase found, together with collagenase, in the insoluble fraction of uterine homogenates.     

Amount of Bleeding and Hematoma Size in the Collagenase-Induced Intracerebral Hemorrhage Rat Model

The aggravated risk on intracerebral hemorrhage (ICH) with drugs used for stroke patients should be estimated carefully. We therefore established sensitive quantification methods and provided a rat ICH model for detection of ICH deterioration. In ICH intrastriatally induced by 0.014-unit, 0.070-unit, and 0.350-unit collagenase, the amount of bleeding was measured using a hemoglobin assay developed in the present study and was compared with the morphologically determined hematoma volume. The blood amounts and hematoma volumes were significantly correlated, and the hematoma induced by 0.014-unit collagenase was adequate to detect ICH deterioration. In ICH induction using 0.014-unit collagenase, heparin enhanced the hematoma volume 3.4-fold over that seen in control ICH animals and the bleeding 7.6-fold. Data suggest that this sensitive hemoglobin assay is useful for ICH detection, and that a model with a small ICH induced with a low-dose collagenase should be used for evaluation of drugs that may affect ICH.     

In-gel precipitation of enzymatically released phosphate

The phosphate precipitation reaction using ammonium molybdate and triethylamine under low pH has been applied to gel-based assays for detecting phosphate-releasing enzymes. The sensitivity of the assay is 10 pmol Pi/mm2 of 1.5-mm-thick gel. The assay is applicable to enzymes with a wide range of optimal pH, from acid (pH 4.5) to alkaline phosphatase (pH 9.7), and to enzymes that use acid-labile substrates such as apyrase and glutamine synthetase. Using a negative staining approach, maltose phosphorylase, a phosphate-consuming enzyme, can also be detected. The assay was used to detect glutamine synthetase isoforms, separated by nondenaturing polyacrylamide gel electrophoresis from crude maize extracts. For downstream applications such as staining gels for proteins, the gels with precipitate should be incubated in 10 mM dithiothreitol or beta-mercaptoethanol until the precipitate is dissolved and then thoroughly washed in water. In comparison to calcium phosphate precipitation or the phosphomolybdate-malachite green method, this method is more sensitive. It is a very simple, rapid, versatile, reproducible, and inexpensive method that could be a useful tool in enzymological studies.     

Neutral red uptake assay for the estimation of cell viability/cytotoxicity

The neutral red uptake assay provides a quantitative estimation of the number of viable cells in a culture. It is one of the most used cytotoxicity tests with many biomedical and environmental applications. It is based on the ability of viable cells to incorporate and bind the supravital dye neutral red in the lysosomes. Most primary cells and cell lines from diverse origin may be successfully used. Cells are seeded in 96-well tissue culture plates and are treated for the appropriate period. The plates are then incubated for 2 h with a medium containing neutral red. The cells are subsequently washed, the dye is extracted in each well and the absorbance is read using a spectrophotometer. The procedure is cheaper and more sensitive than other cytotoxicity tests (tetrazolium salts, enzyme leakage or protein content). Once the cells have been treated, the assay can be completed in <3 h.     

Equine retained placenta: technique for and tolerance to umbilical artery injections of collagenase

Under laboratory conditions and in clinical experiments, bacterial collagenase has proven to be effective in hydrolyzing placenta and detaching cotyledon from caruncle in the bovine species. Laboratory studies in which placental samples were incubated with collagenase have also demonstrated that collagenase is 3.7 times more effective in hydrolyzing equine placenta than bovine placenta. This led to the hypothesis that collagenase may be a potential treatment for mares with retained placenta. However, that collagenase may hydrolyze the uterine wall and perforate the uterus was a concern. It was the purpose of this study thus to determine any adverse effects of collagenase on the equine uterus and to develop a method for intraplacental injection of collagenase. Three normally expelled intact placentas from Arabian mares, 10 cyclic mixed-breed mares, and 4 mares of various breeds with retained placenta were used. Fluoroscein dye and latex were used to study the placental vasculature and to determine a suitable dose of collagenase; placentas were hydrolyzed by collagenase solution in vitro. Bacterial collagenase solution (40,000 units, 200 ml) was infused into the uterine lumen of each cyclic mare. Uterine biopsies were obtained from the mares before collagenase infusion and again at 16 h and 26 d after infusion. In the mares with retained placenta, each placenta was infused via its umbilical cord vessels with 200,000 units of bacterial collagenase in 1 L of saline. Results showed that none of the uteri from cyclic mares were damaged by collagenase treatment. During a 4-wk period of monitoring (including endoscopy) mares with retained placenta did not show any abnormalities. Retained placentas were expelled in less than 6 h after collagenase treatment. It was concluded that intraplacental injections of collagenase are a safe and potentially effective treatment for retained placenta in mares.     

Validation of a reverse transcription nested polymerase chain reaction (RT-nPCR) to detect bovine viral diarrhea virus (BVDV) associated with in vitro-derived bovine embryos and co-cultured cells

Sensitive RT-nPCR assays can be used for the rapid detection of viruses. The objective of this research was to validate an RT-nPCR assay for detection of BVDV associated with various samples collected from an IVF system. In 12 research replicates, we maintained matured COCs as negative controls or exposed them to 1 of 4 noncytopathic strains (SD-1, NY-1, CD-87, or PA-131) of BVDV for 1 h immediately before IVF. After 4 d of IVC, we harvested groups of 5 nonfertile ova or degenerated embryos (NFD) and some associated cumulus cells and transferred developing embryos and the remaining cumulus cells into secondary IVC drops. On the seventh d of IVC, cumulus cells, groups of 5 washed NFD and groups of 5 developed, washed embryos were harvested. We also collected single developed embryos after washing, washing with trypsin, washing and cryopreservation in ethylene glycol, or washing with trypsin and cryopreservation in ethylene glycol. All washes were performed according to International Embryo Transfer Society standards. Developed embryos and NFD were sonicated prior to assay. All samples were assayed for BVDV using virus isolation and RT-nPCR. The virus isolation and RT-nPCR assays determined that all negative control samples were BVDV-free. Virus was detected in association with all exposed cumulus cells and groups of developed embryos using both virus isolation and RT-nPCR. Results from viral assays of other exposed samples indicate enhanced sensitivity of the RT-nPCR assay. The RT-nPCR assay used in this research exhibited acceptable sensitivity, specificity, predictive value and repeatability for rapid detection of BVDV associated with the various samples obtained from an IVF system.     

A luminescence-based test for determining ornithine decarboxylase activity

A sensitive chemiluminescence-based method for the assay of ornithine decarboxylase (ODC) has been developed. This method, which permits the detection of putrescine (the product of ODC) at a picomolar range, can be used to determine ODC activity in cellular extracts. Extracts are incubated with ornithine and spotted onto p81 phosphocellulose paper strips. After drying, the papers are washed with ammonium hydroxide to remove contaminants, which may interfere with the assay. Putrescine is next eluted from the paper by shaking in an elution buffer containing magnesium sulfate. Partially purified hog kidney diamine oxidase is then used to oxidize putrescine in the eluate. The hydrogen peroxide formed during the oxidation is determined by chemiluminescence using luminol and peroxidase. This simple analytical method has the sensitivity of conventional assays based on the use of radioactive ornithine.     

A NEW RAPID TECHNIQUE FOR SALMONELLA ANTIGEN DETECTION USING A PARTICLE CONCENTRATION FLUORESCENCE IMMUNOASSAY (PCFIA)

A fluorescent assay employing polystyrene beads coated with antibody to a common structural antigen (CSA) of salmonellae has been developed to detect the presence of Salmonella. This sensitive technique is very fast, relatively simple and with further testing may be suitable for screening food products as well as clinical diagnostics. Comparison was made with a commercially available Salmonella antigen capture enzyme immunoassay (EIA). As little as one single colony-forming unit (CFU) could be detected by fluorescence after only 16 h of enrichment. the procedure requires only 1 h to run and uses proportionately smaller volumes of sample and reagents than EIA.     

The extraction of a tissue collagenase associated with ovulation in the rat

A method has been developed to assay collagenase in ovarian extracts in the presence of tissue inhibitors. Rat ovarian tissue is first extracted with Triton X-100 and then heated to 60 degrees C in 50 mM Tris buffer containing 100 mM CaCl2. This extract contains collagenase activity and putative inhibitor(s). The inhibitory activity is removed by reduction with dithiothreitol and alkylation with iodoacetamide. Collagenase is then activated with aminophenylmercuric acetate and assayed using 3H-acetylated collagen from which the telopeptides have been removed. Identification of this activity as collagenase was performed by using the metalloprotease inhibitors EDTA and o-phenanthroline and by demonstration of the typical collagen cleavage fragments on sodium dodecyl sulfate-gel electrophoresis. To investigate the changes in collagenase activity associated with ovulation, immature rats received 20 IU of pregnant mare's serum gonadotropin and 52 h later 10 IU of human chorionic gonadotropin (hCG). After hCG administration, ovaries were removed at intervals from 0 to 20 h. Collagenase activity rose from 4.9 +/- 1.4% digestion of the 3H-collagen at 0 time to a maximum of 24.7 +/- 1.5% digestion at 8 h after hCG and remained high at 12 h (time of ovulation) and up to 20 h (18.7 +/- 1.9% and 16.1 +/- 1.6% digestion, respectively). These findings support a role of collagenase in the rupture of the follicle and they suggest a further role for this enzyme in the events following ovulation.     

Simultaneous analyses of clostridium collagenase and neutral proteinase activities with a single synthetic substrate

Two simple methods are presented for simultaneously detecting the activities of both Clostridium histolyticum collagenase and a neutral proteinase that contaminates commercial preparations of collagenase, using a single common substrate carbobenzoxy-Gly-Pro-Leu-Gly-Pro. This pentapeptide is cleaved only once either at the Leu-Gly bond by collagenase or at the Pro-Leu bond by the neutral proteinase. Both products, Gly-Pro and Leu-Gly-Pro, can be analyzed not only semiquantitatively by thin-layer chromatography on cellulose sheets, but also quantitatively by direct application of the digest to an automatic amino aicd analyzer for the calculation of initial velocities. These methods have an advantage over the conventional caseinolysis assay in the time required for incubation. A tetrapeptide, carbobenzoxy-Gly-Pro-Leu-Gly, is also cleaved by the neutral proteinase, but less effectively than the pentapeptide. It is proposed that this proteinase has thermolysin-like specificity.     

A rapid microassay for detecting hepatitis B surface antigen by dot enzyme immunoassay

This study describes a dot enzyme immunoassay (Dot-EIA) for detecting hepatitis B surface antigen (HBsAg). The results demonstrated that the detection level of this assay for HBsAg was 1.5 ng/ml; no false-positive or -negative readings were observed. Also, this Dot-EIA had some advantages over standard EIA: (1) antiserum could be directly and immediately bound on nitrocellulose paper set into microfiltration apparatus, (2) the paper could be easily washed under reduced pressure using a water aspirator, (3) all assay steps could be performed at room temperature within 2 h, (4) the well-defined brown spots could be evaluated by both visual observation and densitometric reading. The Dot-EIA reported here may be useful for rapid diagnosis and screening of HBsAg in serum.