Enzyme linked fibrinolytic assay (ELFA). A new method for the measurement of t-PA in plasma using enzyme labelled fibrin.

We present an assay for components of the fibrinolytic system based on hydrolysis of solid-phase associated enzyme-labeled fibrin. This Enzyme-linked Fibrinolytic Assay, (ELFA) permits the measurement of less than 1 IU/ml of t-PA in 50 microliters of plasma diluted to 1:80 within six hours, in a microtiter plate format, with a colorimetric endpoint. High levels (greater than 10 IU/ml) of tissue plasminogen activator can be measured in less than 30 minutes. The assay was approximately 100 times more sensitive than a clot lysis assay performed in microtiter plates and used less reagents. By comparison with the parabolic rate, coupled assay using chromogenic substrates and soluble fibrin, ELFA performed assays with equal sensitivity and in less time. The ELFA assay uses solid phase fibrin as the substrate, activator and indicator for the assay. For this reason, it has the advantage of the clot lysis assays in that it is more analogous to the physiological hydrolysis of fibrin but with the sensitivity and convenience of the parabolic rate, coupled assay.

Myocardial adenylate cyclase activity in ritodrine-treated pregnant rabbits.

To determine whether prolonged exposure to ritodrine results in down-regulation of myocardial adenylate cyclase activity during pregnancy, New Zealand White rabbits with time-dated pregnancies were randomly assigned to a sham-operated control group and to a ritodrine-treated group. Starting at 22 days' gestation (term 31 days), animals received either intravenous ritodrine intermittently or the equivalent volume of vehicle for 3 days. Adenylate cyclase activity was determined in a plasma membrane fraction obtained from ventricular tissue. There was no difference in the basal activity or the basal and guanosine triphosphate activity between the two groups. L-Isoproterenol-dependent stimulation required the presence of guanosine triphosphate. Both maximal and 50% enzymatic stimulation were equivalent in both groups. Furthermore, sodium fluoride-dependent stimulation was similar in both groups. Finally, adenylate cyclase activity in the presence of forskolin and manganese chloride was similar in both groups. From the above, we conclude that intermittent systemic infusions of ritodrine to pregnant rabbits did not result in down-regulation of myocardial adenylate cyclase activity.

Atrial natriuretic peptide and sodium azide dependent guanylate cyclase activities in placentas from normal and severely toxemic patients.

Particulate guanylate cyclase is stimulated by several hormones through receptor-dependent and by nitrosovasodilators through receptor-independent mechanisms. A subtype of atrial natriuretic peptide (ANP) receptors is coupled to guanylate cyclase. It has been shown that there is a down-regulation of the affinity of ANP receptors to alpha-hANP in placental plasma membranes obtained from severely toxemic patients. We have asked the question whether these changes are associated with a down-regulation of ANP-dependent guanylate cyclase activity. Guanylate cyclase was determined by in vitro experiments using a placental plasma membrane fraction obtained from normal and from severely toxemic patients. The presence of ANP-dependent placental guanylate cyclase activity was demonstrated both in normal and toxemic placentas. Although basal guanylate cyclase activity was not influenced by toxemia of pregnancy, there was a significant decrease in the maximum stimulation of this enzyme by alpha-hANP (104.81 +/- 12.02% (n = 4) vs 49.41 +/- 8.73% (n = 7) for normal and toxemics, respectively). Finally, stimulation by a nitrosovasodilator, sodium azide (NaN3), was also lower in toxemic placentas than in normal controls. These observations extend our previously reported results on placental ANP receptor function but also suggest the presence of a possibly receptor-independent decrease in guanylate cyclase activity in toxemic placentas.

Enzyme-linked immunosorbent assay-enzyme-linked coagulation assay for detection of antibodies to Clostridium botulinum neurotoxins A, B, and E and solution-phase complexes.

The solution-phase complex assay for toxins A, B, and E from Clostridium botulinum (Elcatech, Inc., Winston-Salem, N.C.) was modified to measure antibody. The addition of unlabeled polyclonal antibodies to a mixture consisting of toxin with chicken antibody and RVV-XA-labeled horse antibody reduces the sensitivity of detection of neurotoxin. This reduction in sensitivity can be used as a measure of the specific antibody titer.

Oligosaccharide sequence of human breast cancer cell heparan sulfate with high affinity for laminin.

Laminin-1 is a basement membrane glycoprotein implicated in tumor-host adhesion, which involves the cell-binding domain(s) of laminin-1 and tumor cell surface heparan sulfate (HS). The specific tumor cell surface HS oligosaccharide sequences that are necessary for binding to laminin-1 have not been characterized. To identify this laminin-binding oligosaccharide sequence, GlcNSO4-rich oligosaccharides terminating with [3H]2,5-anhydromannitol (AManR) residues were isolated from human breast cancer cell (MCF-7)-derived HS through hydrazinolysis/high pH (4.0) nitrous acid treatment/[3H]NaBH4 reduction. These oligosaccharides were chromatographed on a laminin-1 affinity column. A high affinity dodecasaccharide was isolated and characterized. Disaccharide analysis yielded IdoA(2-SO4) --> AManR(6-SO4) as the only disaccharide upon treatment of this dodecasaccharide with nitrous acid at low pH (1.5). The sequence of laminin-binding high affinity oligosaccharide is therefore [IdoA(2-SO4) --> GlcNSO4(6-SO4)]5[IdoA(2-SO4) --> AManR(6-SO4)]. Low affinity dodecasaccharides composed of [IdoA(2-SO4) --> GlcNSO4(6-SO4)]5, [IdoA(2-SO4) --> GlcNSO4] were also isolated by laminin-1 affinity chromatography. Molecular modeling studies indicate that a heparin-binding peptide sequence corresponding to amino acid residues 3010-3031 (KQNCLSSRASFRGCVRNLRLSR) in the G domain of laminin-1, modeled as a right-handed alpha-helix, carries an array of basic residues well placed to bind to clusters of sulfate groups on the high affinity dodecasaccharide.

Influence of glucose on production and N-sulfation of heparan sulfate in cultured adipocyte cells.

Altered lipoprotein lipase regulation associated with diabetes leading to the development of hypertriglyceridemia might be attributed to possible changes in content and the fine structure of heparan sulfate and its associated lipoprotein lipase. Adipocyte cell surface is the primary site of synthesis of lipoprotein lipase and the enzyme is bound to cell surface heparan sulfate proteoglycans via heparan sulfate side chains. In this study, the effect of diabetes on the production of adipocyte heparan sulfate and its sulfation (especially N-sulfation) were examined. Mouse 3T3-L1 adipocytes were exposed to high glucose (25 mM) and low glucose (5.55 mM) in the medium and cell-associated heparan sulfate was isolated and characterized. A significant decrease in total content of heparan sulfate was observed in adipocytes cultured under high glucose as compared to low glucose conditions. The degree of N-sulfation was-assessed through oligosaccharide mapping of heparan sulfate after chemical cleavages involving low pH (1.5) nitrous acid and hydrazinolysis/high pH (4.0) nitrous acid treatments; N-sulfation was found to be comparable between the adipocyte heparan sulfates produced under these glucose conditions. The activity and message levels for N-deacetylase/N-sulfotransferase, the enzyme responsible for N-sulfation in the biosynthesis of heparan sulfate, did not vary in adipocytes whether they were exposed to low or high glucose. While most cells or tissues in diabetic situations produce heparan sulfate with low-charge density concomitant with a decrease in N-sulfation, adipocyte cell system is an exception in this regard. Heparan sulfate from adipocytes cultured in low glucose conditions binds to lipoprotein lipase by the same order of magnitude as that derived from high glucose conditions. It is apparent that adipocytes cultured under high glucose conditions produce diminished levels of heparan sulfate (without significant changes in N-sulfation). In conclusion, it is possible that the reduction in heparan sulfate in diabetes could contribute to the decreased levels of heparan sulfate associated lipoprotein lipase, leading to diabetic hypertriglyceridemia.

Enzyme-linked immunosorbent assay and enzyme-linked coagulation assay for detection of Clostridium botulinum neurotoxins A, B, and E and solution-phase complexes with dual-label antibodies.

The measurement of toxins A, B, and E from Clostridium botulinum was accomplished by use of a modified sandwich enzyme-linked immunosorbent assay (ELISA) employing labeled horse antibody and either chicken antibody or biotinylated horse antibody. The complexes formed in solution phase were captured onto solid phases coated with rabbit anti-chicken immunoglobulin G (chicken antibody) or avidin (biotinylated antibody). The assay was brought to the sensitivity of the mouse bioassay (5 to 10 pg/ml, or 0.03 to 0.07 pM) by employing as labeling enzyme the factor X activator of Russell's viper venom (RVV-XA) and a sensitive coagulation-based assay amplification system known as enzyme-linked coagulation assay. Complex formation was found to be a slower reaction than binding to the capture plate, and so the assay used a preincubation step to produce the solution-phase complexes before they were bound to the solid phase. Keeping the concentrations of Russell's viper venom factor X activator antibody and capture antibody constant for diluted samples and diluting complexes into buffer without keeping labeled antibody concentrations constant were equivalent in allowing the detection of low neurotoxin concentrations. This ELISA-enzyme-linked coagulation assay procedure is a convenient alternative to the mouse bioassay, which shows complete resolution of the neurotoxins in addition to the requisite sensitivity.

Sensitive enzyme-linked immunosorbent assay for detection of Clostridium botulinum neurotoxins A, B, and E using signal amplification via enzyme-linked coagulation assay.

A new immunoassay amplification method has been applied to the measurement of toxins A, B, and E from Clostridium botulinum. The technique is a modified enzyme-linked immunosorbent assay (ELISA) which relies on the detection of sandwich complexes on microtiter plates by a solid-phase coagulation assay known as ELCA, or enzyme-linked coagulation assay. In the method, a coagulation activating enzyme (RVV-XA) isolated from the venom of Russell's viper is conjugated to affinity-purified horse antibodies specific for toxin type A, B, or E. Plates are coated with affinity-purified antibodies, and standard captag (capture-tag) protocols using labeled antibody are employed to bind the toxin from solution. Complexes are detected by adding a modified plasma substrate which contains all the coagulation factors mixed with alkaline phosphatase-labeled fibrinogen and solid-phase fibrinogen; deposition of solid-phase, enzyme-labeled fibrin on the solid phase is then a reflection of formation of toxin-RVV-XA-antibody complexes on the solid phase. Because of the ability to detect RVV-XA by this coagulation assay at concentrations < 0.1 pg/ml, it was possible to measure C. botulinum toxins A, B, and E at mouse bioassay levels (< 10 pg/ml, or < 0.07 pM) for both purified neurotoxin and crude culture filtrates obtained from strains known to produce appropriate single toxins. ELISA-ELCA should be applicable to measurement of toxins in most of the materials (contaminated food, blood, and excreta) for which the comparably sensitive mouse bioassay is currently employed. This method has the potential of broad application to the measurement of low concentrations of any antigen for which appropriate immunochemical reagents are available, in a color test format.

Amplified Immunoassay ELISA-ELCA for Measuring Clostridium botulinum Type E Neurotoxin in Fish Fillets.

The measurement of Clostridium botulinum type E toxin in fish was accomplished using an amplified immunoassay (enzyme-linked immunosorbent assay-enzyme-linked coagulation assay [ELISA-ELCA]) based on the coagulation cascade. Fresh catfish fillets inoculated with a mixture of spores from five strains of C. botulinum type E were packaged in high barrier film with air, vacuum and modified atmosphere and stored at 4, 8 or 16°C for up to 75 days. Toxin production was monitored during storage by both mouse bioassay (trypsin and non-trypsin treated) and ELISA-ELCA on the non-trypsinized samples. All 26 inoculated products that were positive by the mouse bioassay were also positive by ELISA-ELCA. Of 35 uninoculated samples which were not toxic in mouse bioassay, none were positive by ELISA-ELCA; of 73 inoculated samples which were not toxic by mouse bioassay, 14 had toxin measurable by the ELISA-ELCA. The position of these immunoassay-positives in the sampling sequence indicated that the toxin was identified by the immunoassay before it was found in the mouse bioassay. These results suggest that the ELISA-ELCA technique is a usable alternative to the mouse bioassay for monitoring C. botulinum type E toxin production in fish challenge studies.