Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts:   Target Concepts:
Query: EC:1.11.1.7 (peroxidase)
 65,474 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

A sensitive and specific blocking enzyme-linked immunosorbent assay (ELISA) was developed to distinguish infectious bovine rhinotracheitis virus (IBRV)-infected animals from those immunized with a glycoprotein gIII deletion mutant, IBRV(NG)dltkdlgIII. For this ELISA, undiluted test sera are used to block the binding of an anti-IBRV gIII monoclonal antibody (mAbgIII)-horseradish peroxidase (HRPO) conjugate to gIII antigen. TMB substrate is used for color development. Negative S/N values (defined as the absorbance at 650 nm of test sera/absorbance at 650 nm of negative control sera) of > 0.80 were obtained with immune sera from gnotobiotic cattle immunized with several bovine viruses, with bovine antisera to bovine herpesvirus-2, and vesicular stomatitis virus, with porcine antisera to pseudorabies virus and parvovirus, and with normal sera from heterologous species. Negative S/N values were also obtained with sera from rabbits twice vaccinated with IBRV(NG)dltkdlgIII. However, the S/N values became positive (S/N < 0.8) 10 to 17 days after the rabbits were challenge exposed to virulent IBRV(Cooper). Most of 116 sera (84%) from feedlot cattle with virus neutralization (VN) titers of < 1:2 or < 1:4 had negative S/N values > 0.8, but 18 sera with negative VN titers had positive S/N values, consistent with observations indicating that an IBRV outbreak was occurring in one of the feedlot herds. Thirty nine sera (98%) from feedlot cattle with VN titers of 1:2 to 1:128 had positive S/N values (< 0.8). One serum with a VN titer of 1:2 had a borderline (+/-) S/N value of 0.81. After immunization with a commercial gIII-positive IBRV vaccine, 115/116 sera with VN titers of 1:2 to 1:256 had positive S/N values (< 0.8). One serum with a VN titer of 1:2 had a negative S/N value of 0.83. Serum from one vaccinated animal that failed to seroconvert after vaccination (VN < 1:4) showed a strongly positive ELISA S/N of 0.48.
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PMID:Blocking ELISA for distinguishing infectious bovine rhinotracheitis virus (IBRV)-infected animals from those vaccinated with a gene-deleted marker vaccine. 133 Nov 60

Horseradish peroxidase (HRP) was injected, bilaterally, into the rat masseter muscle, subsequent to an intramuscular or intraperitoneal injection of one of five dopamine antagonists (chlorpromazine and haloperidol as the D1 and D2 receptor antagonist, SCH 23390 as the specific D1 receptor antagonist, sulpiride and domperidone as the specific D2 receptor antagonist). Control rats received an injection of a corresponding vehicle solution. After a survival period of 16 h, the brainstem was cut into 60 microns cryosections and processed with the TMB technique. The amount of retrogradely transported HRP was quantitatively measured in terms of the amount of HRP reaction product present in the motoneuron by the method which we have developed using an image processing system combined with a light microscope and a TV camera. Chlorpromazine, haloperidol, SCH 23390 and sulpiride significantly raised the quantity of retrograde transport of HRP. On the contrary, domperidone which can not penetrate the blood-brain barrier showed no significant change in the amount of the retrograde transport. In addition, an intravenous injection of chlorpromazine (8 mg/kg) was found to increase the amplitude of monosynaptic masseteric reflex EMG activity evoked by stimulations of the mesencephalic trigeminal nucleus. These results suggest that a possible regulatory system involving the dopamine receptor in the uptake and retrograde transport of HRP from axon terminals to cell bodies of the masseteric motoneuron exists in higher order neurons which make synaptic contact with the motoneuron.
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PMID:Dopamine receptor antagonists increase markedly the quantity of retrograde transport of HRP in the rat masseteric motoneuron. 135 2

Since December 1990, strict limits for pesticide content in drinking water have been in effect in the FRG. These limits have to be enforced and controlled regularly. Because the classical analytical methods, like GC/MS and HPLC are relatively expensive and complicated, concentration and clean up of the samples are necessary. Therefore, the significance of Enzyme Immunosorbent Assays has increased in environmental analysis. The topics of our research are synthesis of suitable Metolachlor conjugates for immunization and as coating antigen, as well as development of an Enzyme Immunosorbent Assay. The chloroacetanilide Metolachlor was coupled with an AMSA-Spacer on Sheep IgG and with AHT-Spacer on BSA. The epitope density was measured photometrically with TNB-derivate. Polyclonal antibodies were produced in white New Zealand rabbits. Metolachlor-AMSA-Sheep IgG conjugate was served as immunogen. The test is indirect and competitive. Microtiter plates are coated with the coating antigen (Metolachlor-AHT-BSA conjugate). In the next step the Metolachlor sample competes with the coating antigen for the antibodies. Detection of bounded antibodies is indirect with a peroxidase-labelled secondary antibody. The chromogenic substrates ABTS, TMB and OPD were tested. TMB showed the best qualities, fast turnover, as well as good and steady colour. Titer of serum and crossreactivity of other chloroacetanilide pesticides and their metabolites were tested. With the exception of Alachlor and Dimethachlor, no pesticide showed a high interaction with serum. Quantitative interpretation was done with the logit/log transformation, which allows comparison of different sera and test series. The antibodies are specific but the affinity is not good enough to detect the limits assigned by the "deutsche Trinkwasserverordnung". Raising the concentration, as is done in classical analytical methods, would solve the problems so that the test can be used as a screening test.
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PMID:[The development of an enzyme immunoassay for the detection of the herbicide metolachlor in water samples]. 145 38

An international collaborative study was conducted at ten sites to examine the performance of enzyme immunoassays (EIAs) for the quantitation of IgG1, IgG2, IgG3, IgG4 and total IgG anti-Haemophilus influenzae type b (Hib) capsular polysaccharide in human serum. All groups used the same reagents: microtiter plates coated with polyribosylribitol phosphate (PRP) conjugated to poly-L-lysine (PLL), reference, control and test human sera, biotin-conjugated International Union of Immunological Societies (IUIS)-documented monoclonal anti-human IgG1-4 and IgG Pan detection antibodies, avidin-peroxidase and TMB substrate. Initial mixing of soluble PRP antigen or an equal volume of buffer with the 20 test sera prior to analysis confirmed PRP antigen specificity in all five EIAs with greater than 80% competitive inhibition at most sites. Positive correlation between the total IgG anti-Hib and sum of IgG1-4 anti-Hib was demonstrated (r2 = 0.99, Y = 1.13X -0.15). Good agreement was shown between the total IgG anti-Hib as measured by EIA and the total Hib-specific antibodies measured by the current radiolabeled antigen binding assay (r2 = 0.97, Y = 4.6X -5.8). Assay parallelism was demonstrated with an average interdilutional %CV of 22% and parallel dose-response curve slopes. The interdilutional %CVs were calculated as an average per sample of the variation of microgram/ml (corrected for dilution) at different dilutions per laboratory for all participating sites. The interlaboratory variation was the only performance parameter studied that exceeded the target level of 35% CV in all IgG1-4 and total IgG anti-Hib assays. IgG subclass distributions in the test sera demonstrated a predominance of IgG1 anti-Hib in the pediatric serum pools and IgG2 anti-Hib in the adult sera, with low but detectable levels of IgG3 and IgG4 anti-Hib in each group.
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PMID:Quantitation of human IgG subclass antibodies to Haemophilus influenzae type b capsular polysaccharide. Results of an international collaborative study using enzyme immunoassay methodology. 156 20

A blocking enzyme-linked immunosorbent assay (ELISA) test has been developed to distinguish pseudorabies virus (PRV) (Aujeszky's disease virus) -infected pigs from those immunized with a glycoprotein g92 (gIII) deletion mutant, PRV (dlg92dltk) [OMNIMARK-PRV]. This blocking ELISA test utilizes an anti-PRV gIII monoclonal antibody (mAbgIII)-horseradish peroxidase (HRPO) conjugate, TMB for color development and a cloned PRVg92 (gIII) antigen to coat wells of microtiter test plates. Undiluted sera are used to block the binding of the mAbgIII-HRPO conjugate to the antigen. The gIII blocking ELISA is specific and has a sensitivity comparable to screening ELISA and latex agglutination tests. PRV-negative sera and sera from pigs vaccinated once, twice, or four times with the gIII-negative vaccine all showed negative S/N values of greater than 0.70 (S/N defined as the optical density at 630 nm of test sera/optical density at 630 nm of negative control sera). Sera from PRV-infected herds, sera from pigs experimentally infected with virulent PRV, and sera from pigs vaccinated with modified-live or inactivated gIII+ vaccines were positive for gIII antibodies (S/N less than 0.7). Sera from pigs experimentally infected with 200 PFU virulent PRV seroconverted to gIII+ antibodies 7-10 days postinfection. Sera from pigs vaccinated with gpX- and gI- vaccines seroconverted to gIII+ antibodies 7-8 days after vaccination. The gIII antibodies persisted after gIII+ vaccinated for at least 376 days postvaccination. Sera from pigs protected by vaccination with PRV (dlg92dltk) and then challenge exposed to virulent PRV at 21 days postvaccination showed gIII+ antibodies by 14 days postchallenge. The specificity and sensitivity of the gIII blocking ELISA assay was further demonstrated on the United States Department of Agriculture-National Veterinary Services Laboratory (USDA-NVSL) sera from the 1988 PRV check set and the 1989 gIII PRV check set by comparing the gIII blocking ELISA assay with virus neutralization, screening/verification ELISA and latex agglutination assays.
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PMID:Sensitive glycoprotein gIII blocking ELISA to distinguish between pseudorabies (Aujeszky's disease)-infected and vaccinated pigs. 165 82

GABA-synthesizing neurons were identified in the medulla of the rat by peroxidase-antiperoxidase (PAP) immunohistochemistry for glutamic acid decarboxylase (GAD). Using diaminobenzidine (DAB) either alone or intensified with silver, a relatively large number of GAD-immunoreactive neurons were evident within the reticular formation, raphe nuclei and vestibular nuclei. In all these areas, profuse GAD-immunoreactive varicosities appeared to contact the soma and dendrites of both non-GABA and GABA neurons. These observations suggest that GABA neurons may act as interneurons or local projection neurons within the medulla and accordingly exert a potent inhibitory and/or disinhibitory control on bulbar projection neurons. Within the ventral reticular formation (pars alpha and ventralis of the gigantocellular reticular field) and raphe magnus, large numbers of prominent GAD-immunoreactive neurons resembled in size and morphology and overlapped in distribution the serotonin-immunoreactive neurons of the same regions. However, by sequential double immunostaining utilizing DAB as a chromogen for serotonin (5-HT) and benzidine dihydrochloride (BDHC) for GAD, it was found that GAD-containing neurons were distinct from 5-HT-containing neurons. Following injections of wheat germ agglutinin conjugated to horseradish peroxidase (WGA-HRP) into the upper cervical spinal cord and combined processing for WGA-HRP (using tetramethylbenzidine [TMB] with cobalt) and immunohistochemistry (with DAB), a contingent of spinally projecting neurons were found to contain GAD. The GAD-immunoreactive reticulo- and raphe-spinal neurons were most frequent within the pars alpha and ventralis of the gigantocellular reticular fields and the raphe magnus, where they were approximately equal in number to the coexistent, but distinct 5-HT spinally projecting neurons. GABA neurons of the medulla may thus contribute directly to the bulbar inhibitory influence upon spinal sensory and motor systems.
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PMID:GABA-synthesizing neurons in the medulla: their relationship to serotonin-containing and spinally projecting neurons in the rat. 172 90

We have studied the vestibular efferent fibers pathway, after scratching the peripheral vestibular receptors and horseradish peroxidase injection, and TMB or DAB processing. We have proved the existence of a differentiated bundle in the main vestibular pathway, this bundle crosses the areas where the efferent somas have been situated, some of this fibers have been seen crossing the midline. It has not been possible to connect the efferent somas with the fibers.
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PMID:[A neuroanatomical study of the vestibular efferent system, III: the origin and pathway of the axons]. 190 35

Two different microtiter plate ELISA tests were devised for the detection of Escherichia coli thermolabile toxin (LTh) either free or extracted from isolated colonies. Both tests used as detection systems purified anti-LTh rabbit immunoglobulins conjugated to biotin, streptavidin peroxidase and TMB. The tests differed by their capture phase which was the GM1-ganglioside for GM1-ELISA and purified anti-LTh rabbit immunoglobulins for sandwich ELISA. The two methods were rapid since they could be performed in less than 2 hours. The detection limits for purified LT were 50 pg/ml and 1.3 ng/ml for sandwich ELISA and GM1-ELISA respectively. For the detection of toxinogenic isolates the extraction buffer containing Triton X-100 was always superior to polymyxin buffer. Using the polymyxin extraction buffer the sandwich ELISA was again more sensitive than the GM1-ELISA since a lower number of isolated colonies could be used for the detection of positive strains. With the Triton X-100 buffer both ELISAs could detect positive strains using a single colony but the sandwich ELISA gave the highest delta OD. We concluded that our sandwich ELISA can rapidly detect either the free Escherichia coli thermolabile toxin or LTh producing strains and could be applied routinely.
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PMID:Comparison of sandwich-ELISA and GM1-ELISA for the detection of Escherichia coli thermolabile enterotoxin. 193 62

This is a quantitative study of the motoneuronal population of the rat's common peroneal nerve following severe crush injury of the sciatic nerve or its component branches. The crush was performed unilaterally under anesthesia for 60 seconds with hemostat jaws covered with tubing to form a smooth, 2 mm long, injured zone. Recovery from injury was allowed for 14 to 188 days. It was measured behaviorally using the sciatic functional index (SFI) and electrophysiologically by comparing the conduction velocity and amplitudes of evoked muscle action potentials prior to injury, and again after injury just before the nerve was labeled with horseradish peroxidase (HRP), and/or its wheat germ agglutinin conjugate (WGA-HRP), 48-72 hours before sacrifice. The motoneurons were retrogradely labeled on both sides so that the uninjured side might serve as a control. On the injured side the nerves were labeled either distal or proximal to the crush site. The tibialis anterior muscles on both sides were removed and weighted. Spinal segments L2 to L6 were cut in serial, frozen cross-sections. HRP reaction products were formed using TMB as the chromogen. The normal peroneal nerve was found to contain 634 +/- 26 motoneurons (22 cases). The number of motoneurons labeled 5-15 mm distal to the injury site (22 cases) was 535 +/- 69 or 84.4% of normal. In 12 cases in which the nerve was labeled 5 mm proximal to the injury normal population numbers (648 +/- 30) were found. These counts demonstrated that the unlabeled 15.6% in the distal labeled cases had not vanished as a result of cell death. Instead, the unlabeled group was composed mainly of small motoneurons whose axons probably had not regenerated distal to the crushed zone. Mean soma size of injured neurons increased to maximum 3-6 weeks after injury and then gradually decreased in size over the following weeks to nearly normal values. This transient increase in size was due to two factors: 1) soma swelling in response to axonal injury, and 2) absence of many small motoneurons, presumably gamma-motoneurons, which were either incapable of, or prevented from, regenerating beyond the injury zone long after larger motoneurons had reinnervated their targets. SFI scores, muscle weights, and amplitude ratios of evoked potentials recovered to control values by 70-80 days post-injury. Conduction velocities remained 20-25% below normal at the end of 80 days.(ABSTRACT TRUNCATED AT 400 WORDS)
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PMID:All peroneal motoneurons of the rat survive crush injury but some fail to reinnervate their original targets. 201 19

The simultaneous presence of homogeneous and heterogeneous reactions at different binding sites of a multiepitope antigen makes the description of the kinetic parameters of the so called "one step" solid phase immunometric assays complex. The authors extended the "one step" approach to the concept of the "soluble sandwich" methodology which differs from the former by the delayed solid phase capture of the biotinylated immunocomplex to a streptavidin coated solid support. Using prolactin monoclonal IEMAs as a model, the equilibria involved in the reactions have been studied on a thermodynamic basis through a description of the kinetics of the interactions between biotinylated Mabs and solid phase streptavidin both in presence and/or in absence of the antigen and HRP-conjugated antibody. A comparative evaluation of models in which the biotinylated antibody was previously insolubilized on the streptavidin solid phase has been performed as well. The experimental work was carried out by using 125I labelled McBiot and Prolactin to trace individual interactions and peroxidase/H2O2/TMB systems to develop the enzymatic analytical signals. A new instrument/data reducing system was also optimized to expand the OD reading range provided by conventional, single wavelength colorimeters. The greater flexibility theoretically expected for the "soluble sandwich" approach and the possibility to extend the analyte working range without detrimental effects on the readability of low doses responses have been experimentally confirmed.
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PMID:The "soluble sandwich" approach for immunoassays: methodological and instrumental implications. 222


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