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The prevalence of serum precipitating antibody to Aspergillus fumigatus in adult cattle in Britain.

The prevalence of precipitating antibody to A. fumigatus in 3509 sera from adult cattle in 44 herds was 25 per cent. Only 19 per cent of sera from grazing or recently housed animals were positive compared with 30 per cent of those taken towards the end of the winter. Mouldy hay was the most likely source of A. fumigatus antigens although a positive correlation between the prevalence of precipitins and either the mouldiness of the hay or the frequency of coughing in the springtime was not established. However, there was a positive correlation between the age of cattle sampled and the prevalence of antibodies. Mycotic abortion had been confirmed in only one of 6 herds in which more than 80 per cent of the cows were seropositive and so it can be inferred that the presence of serum precipitating antibody is of very little value in the diagnosis of mycotic abortion resulting from A. fumigatus infection.

Animal Feed↗

Quantitation of proteins by densitometric scanning of immunoelectrophoretic precipitates.

Densitometric scanning of stained immunoelectrophoretic precipitates of nine human serum proteins showed that the optical density of the precipitates is approximately proportional to the molar concentration of antigen in the sample. Deviations from this correlation were noted for the macroglobulins (mol. wt. greater than 15 x 10(4)). The resolving capacity of the line immunoelectrophoretic procedure permits densitometric valuations of composite patterns.

Blood Proteins↗

New method for production of influenza A virus subtype specific antisera: employment of precipitate obtained by crossed immunoelectrophoretic techniques as antigen.

The crossed immunoelectrophoretic technique was employed for the production of subtype-specific influenza A virus antisera. Glycoprotein-containing precipitates were produced by this technique by using detergent solubilized influenza A virus glycoproteins as antigens in the first dimension gel and rabbit antisera against these antigens in the second dimension gel. By cutting these precipitates out of the gel and using them for the immunization of rabbits, antisera were produced which showed the presence of subtype-specific antibodies in haemagglutination inhibition and immunofluorescent antibody tests but no antibodies in neuraminidase inhibition tests.

Animals↗

The use of polyethylene glycol precipitation to detect low-avidity anti-DNA antibodies in systemic lupus erythematosus.

With a recently introduced method for measurement of low-avidity anti-DNA, the polyethylene glycol (PEG) precipitation assay (Riley et al., 1979), high levels of DNA binding by normal human serum (NHS) were found when circular PM2-DNA was used as antigen. THe nature of this DNA binding was studied. The removal of low-density lipoproteins (LDL) from the serum, e.g., by Aerosil absorption, eliminated DNA binding by NHS. Purified LDL bound DNA to the same extent as NHS. Non-specific binding of NHS or LDL to DNA was prevented by adding dextran sulphate to the incubation mixture. Analysis on sucrose gradients showed that only large DNA-anti-DNA complexes were precipitated by 3.5% PEG. The PEG assay with dextran sulphate is a sensitive assay for low-avidity anti-DNA antibodies. It adds important information to results obtained with the Farr assay, which mainly detects antibodies of high avidity.

Antibodies, Antinuclear↗

Microtiter radioimmunoprecipitation assay of HSV-1 polypeptides with recovery and SDS-PAGE analysis of precipitated proteins: usefulness as screening test for large numbers of specimens including hybridoma supernates.

Immunoprecipitation of radiolabeled polypeptides from complex mixtures of proteins was performed in polystyrene microtiter plates using staphylococcus protein A and various antibody preparations. The method is (1) rapid, (2) uses multichannel micropipettor technology, (3) handles large numbers of specimens easily, (4) requires very small volumes of antigen and antibody (5-50 microliters), (5) provides replicates for statistical analysis and (6) allows recovery of precipitated proteins for direct SDS-PAGE analysis of precipitated proteins. We have shown it is useful as a test to screen large numbers of sera or to characterize monoclonal antibody-containing samples.

Animals↗

A protein A-binding, polyethylene glycol precipitation-based immunoradiometric assay. Application to the detection of immune complexes and C3 in human sera and of private antigens in cross-reacting parasite extracts.

An immunoradiometric assay, based on the precipitation of antigen-antibody complexes by polyethylene glycol (PEG) and on the subsequent binding of PEG-soluble radiolabelled staphylococcal protein A to the PEG-insoluble complexes, is described. The assay can be applied to the detection of naturally occurring, circulating immune complexes, and of complexes artificially created by mixing antigen and antibody solutions, which makes it of potential use for the detection of either antigen or antibody in several situations. Pre-treatment of the antibody-containing sera with 3% PEG greatly reduced the background values and increased the sensitivity of the assay. The assay was also applied to the detection and isolation of Leishmania donovani antigens that did not cross-react with antigens of the related parasite Trypanosoma cruzi (private antigens) and private antigens of insect-derived metacyclic trypomastigotes of T. cruzi in relation to culture-derived metacyclic trypomastigotes of T. cruzi. A simple and extremely effective procedure for washing precipitates with just one centrifugation is also described.

Animals↗

A sensitive double-diffusion microassay suitable for the detection of idiotype-antiidiotype precipitates.

Using agarose gel coated on GelBond film sheets and using Coomassie blue stain followed by silver stain, a sensitive double-diffusion microassay has been developed for detecting small amounts of precipitate forming during idiotype-antiidiotype reactions. The sensitivity of the method is 10-100 times greater than classical immunodiffusion tests. Other advantages include the need for minimal amounts of unconcentrated sample, the absence of radioactive or toxic substrates, no interference due to a second or third antibody coat such as are used in immunoenzymatic techniques, and the possibility of a direct evaluation of qualitative data such as identity, cross-reactivity or non-identity. As little as 40 ng antibody could be detected, corresponding to an antibody concentration of 8 micrograms/ml, making the microassay useful for rapid screening of idiotype-antiidiotype precipitates during routine analysis of hybridoma supernatants.

ABO Blood-Group System↗

Dose-dependent and stereoselective antagonism by diltiazem of naloxone-precipitated morphine abstinence after acute morphine-dependence in vivo and in vitro.

The effects of two enantiomers of diltiazem (l-cis and d-cis) on naloxone-precipitated morphine abstinence after acute morphine dependence were evaluated in vivo in mice and in vitro in isolated pieces of rat terminal ileum. d-cis-diltiazem (10-40 mg/kg) produced a dose-dependent inhibition of the jumping and body weight loss induced by naloxone in acutely morphine dependent mice. By contrast, l-cis-diltiazem 40 mg/kg did not significantly inhibit jumping and produced a much lower inhibition of body weight loss than the same dose of d-cis-diltiazem. In addition, d-cis-diltiazem (0.01-1 microM) produced a concentration-dependent inhibition of naloxone-induced contracture in morphine dependent ilea, whereas l-cis-diltiazem, even at 1 microM, did not inhibit this contracture. These results suggest that calcium channels may play a similar role in naloxone-precipitated morphine abstinence in vivo and in vitro.

Animals↗

Effects of beta-carboline-ethyl ester on plasma corticosterone--a parallel with antagonist-precipitated diazepam withdrawal.

Diazepam has been shown to produce physical dependence based on observations of behavioral stimulation or, in our laboratory, by increases in plasma corticosterone (CS) during antagonist-precipitated withdrawal. The behavioral excitation appears similar to that observed following the administration of beta-carboline esters--agents reported to interact with benzodiazepine receptors and termed "inverse agonists." The focus of the present study is to correlate the occurrence of changes in CS with behavioral excitation previously observed by others. Further, these studies are designed to show a parallel between the manifestations of benzodiazepine withdrawal and the pharmacologic effects of beta-carboline ethyl ester. Experiments were done in conscious unrestrained male Sprague-Dawley rats, with chronic i.v. catheters, using sound-attenuated one-way vision boxes. These studies compared the hormonal and behavioral changes induced by beta-carboline ethyl ester (beta CCE) with CGS-8216-precipitated withdrawal in rats treated with diazepam for 8 days. Rats treated chronically with diazepam (5 mg/kg/day), showed a significant increase in plasma (CS) following CGS-8216. Behavioral abstinence scores were also significantly elevated. beta CCE (0.5-5.0 mg/kg) showed a significant dose-related increase in plasma CS. Behavioral scores were also increased at doses of 0.5 and 2.0 mg/kg. beta CCE-induced plasma CS increases were antagonized by CGS-8216 at doses of 1.0 and 2.0 mg/kg but not by 0.5 mg/kg. In animals chronically treated with diazepam, beta CCE evoked a more prolonged plasma CS elevation than in vehicle-treated animals suggesting a dual agonist/antagonist effect. These data suggest a parallel between CS elevations and behavioral effects during withdrawal as well as similarities between the action of beta CCE and the manifestations of this withdrawal.

Animals↗

The NMDA antagonist dizocilpine (MK801) attenuates motivational as well as somatic aspects of naloxone precipitated opioid withdrawal.

The non-competitive N-methyl-D-aspartate (NMDA) receptor antagonist dizocilpine has recently been reported to antagonize certain overt withdrawal signs in morphine dependent rats. The purpose of the present study was to reassess this response and examine the effect of this drug in a model presumably reflective of the motivational impact of withdrawal using the place conditioning technique. Rats were made opiate dependent by the subcutaneous implantation of a 75 mg morphine pellet. Three-4 days later withdrawal was precipitated by naloxone 0.5 mg/kg. Dizocilpine (0.1-0.5 mg/kg) attenuated many of the subsequent behaviours elicited by naloxone, notably diarrhoea, mouth movements, paw shakes and ptosis. In a separate group of morphine dependent rats, naloxone (0.05 mg/kg) precipitated withdrawal produced a clear place aversion. This place aversion was blocked by dizocilpine (0.02-0.1 mg/kg) pre-treatment prior to conditioning. Therefore dizocilpine may modify both motivational and somatic aspects of opioid withdrawal.

Animals↗

Subcutaneous injection of an analog of neuropeptide FF precipitates morphine abstinence syndrome.

Neuropeptide FF (NPFF) has been shown to exert various antiopiate actions, including precipitation of opiate abstinence syndrome by third ventricle injection in morphine dependent rats. In the present study, dansyl-Pro-Gln-Arg-Phe-amide, a lipophilic analog of NPFF, was injected into morphine dependent rats and appropriate sham controls at a dose of 9 mg/kg s.c. Comparison groups were injected with ethanol/water vehicle alone. The NPFF analog precipitated a vigorous opiate abstinence syndrome in morphine dependent rats, but not in sham controls.

Amino Acid Sequence↗

Precipitation of morphine withdrawal syndrome in rats by administration of mu-, delta- and kappa-selective opioid antagonists.

The acute effects of opioid drugs are generally hypothesized to be mediated by multiple receptors, for which three types of binding sites have been established. In order to evaluate the selective participation of each type of opioid receptor in opiate withdrawal, the opiate withdrawal syndrome, precipitated by the intraventricular acute administration of mu-, delta- and kappa-selective opioid antagonists was investigated. After implantation of the cannula into the lateral ventricle, rats were made physically dependent by subcutaneous insertion of two 75-mg pellets of morphine (base). D-Phe-Cys-Tyr-D-Trp-Arg-Thr-Pen-Thr-NH2 (CTAP) (5-5000 ng), a mu-selective opioid antagonist, naltrindole (62-2000 ng), a delta-selective antagonist or nor-binaltorphimine (nor-BNI) (600-20,000 ng), a kappa-selective antagonist, were administered 72 hr after implantation of the pellets. All three drugs elicited some signs of morphine withdrawal but they differed in both their potency and their efficacy. The most efficacious and the most potent was CTAP, eliciting 8 of the 14 withdrawal signs at doses of 5-5000 ng. Nor-BNI was less efficacious and less potent, eliciting a significant increase in 5 of the 14 withdrawal signs in a dose range of 600-20,000 ng. Naltrindole was the least potent and least efficacious of the three drugs, eliciting a significant increase of only 2 withdrawal signs after intraventricular administration of 2000 ng. In a second experiment, the withdrawal syndrome was precipitated by the combined administration of CTAP+naltrindole or CTAP+nor-BNI. The severity of withdrawal, obtained with these two combinations, was similar to that observed with CTAP alone. These results support the importance of the mu receptor in the expression of central opiate dependence and suggest a minor role for delta and kappa receptors.

Animals↗

REM sleep deprivation (REMSD) antagonizes naloxone-precipitated withdrawal in acute morphine-dependent rats.

In rats allowed undisturbed sleep (control and stress) the administration of naloxone (10 mg/kg, s.c.) to morphine (7.5 mg/kg, s.c. 90 min prior) pretreated animals precipitated a jumping behaviour. REM sleep deprivation (REMSD 96 h, prior) significantly decreased the frequency of the naloxone-precipitated jumping behaviour compared with control and stressed animals. In second animal model for morphine withdrawal, naloxone (10 mg/kg, s.c.) provoked myoclonic twitch activity (MTA) in rats previously exposed to morphine (7.5 mg/kg, s.c., 90 min prior). The intensity of naloxone-induced MTA in REM sleep deprived rats was significantly lower compared to stressed animals, but it is not different from the control group. It is suggested that REMSD interferes with a neural mechanism involved in the development of acute dependence. Results are discussed in light of a possible functional insufficiency of a mu-opioid system during REMSD.

Animals↗

Antibody to host factor precipitates poliovirus RNA polymerase from poliovirus-infected HeLa cells.

An antibody to a host cell protein (host factor) which inhibited the host factor-dependent transcription of poliovirion RNA was used to characterize the immunoprecipitated proteins from both uninfected and poliovirus-infected HeLa cells. The antibody specifically precipitated a major protein of approximately 67,000 Da and a 40,000-Da minor protein from uninfected HeLa cells. The immune serum also specifically precipitated poliovirus RNA polymerase (P63, NCVP4) from poliovirus-infected HeLa cells indicating an association of the viral RNA polymerase with the host factor in vivo. A physical interaction between purified host factor and P63 could also be demonstrated in vitro. The antibody was further used to determine the distribution of host factor among different subcellular fractions of HeLa cells. Most of the cellular host factor was present in the cytoplasmic fraction (70%). Approximately 30% of the total host factor was found to be associated with the ribosomes.

Antibodies↗

The beta-carboline derivatives ZK 93426 and FG 7142 fail to precipitate abstinence signs in diazepam-dependent cats.

The aim of the present study was to investigate the ability of different benzodiazepine recognition site antagonists (Ro 15-1788 and ZK 93426) and inverse agonists (Ro 15-4513, FG 7142 and CGS 8216) to induce abstinence signs in diazepam-dependent cats. Different groups of cats were challenged with each of the benzodiazepine recognition site ligands under investigation 24 hours after the last dose of chronic treatment with diazepam (7 mg/kg, IP at 8.00 a.m. and 8.00 p.m. for 21 consecutive days). The benzodiazepine derivatives Ro 15-4513 and Ro 15-1788 precipitated an abstinence syndrome within minutes after IP administration. The pyrazoloquinoline derivative CGS 8216 also induced withdrawal signs that were less severe and had a longer latency than those elicited by Ro 15-4513 and Ro 15-1788. Abstinence signs included tremors, increased muscle tone, irritability, fear, arched-back posture, pupillary dilation and vocalizations. On the other hand, the beta-carboline derivatives ZK 93426 and FG 7142 failed to precipitate abstinence signs in diazepam-dependent cats when given at doses that prevented the acute effects of diazepam. Our results demonstrate that the ability to induce withdrawal signs in diazepam-dependent cats depends on the chemical structure of the challenge drug (i.e., benzodiazepine or pyrazoloquinoline), since beta-carboline antagonists like ZK 93426 and partial inverse agonists like FG 7142 lack this property.

Animals↗

Naloxone-precipitated changes in biogenic amines and their metabolites in various brain regions of butorphanol-dependent rats.

Influence of a naloxone (an opioid receptor antagonist) challenge (5 mg/kg, IP) on levels of biogenic amines and their metabolites in various brain regions of rats infused continuously with butorphanol (a mu/delta/kappa mixed opioid receptor agonist; 26 nmol/microliter/h) or morphine (a mu-opioid receptor agonist; 26 nmol/microliter/h) was investigated using high-performance liquid chromatography with electrochemical detection (HPLC-ED). Naloxone precipitated a withdrawal syndrome and decreased the levels of: dopamine (DA) in the cortex and striatum, 3,4-dihydroxyphenylacetic acid (DOPAC) in the striatum, homovanilic acid (HVA) in the striatum, limbic, midbrain, and pons/medulla regions in butorphanol-dependent rats. However, the levels of norepinephrine (NE), serotonin (5-hydroxytryptamine; 5-HT), and 5-hydroxyindoleacetic acid (5-HIAA) in the regions studied were not affected by naloxone-precipitated withdrawal. In addition, naloxone increased the HVA/DA ratio in the cortex, while this ratio was reduced in the limbic, midbrain, and pons/medulla. The reduction of 5-HIAA/5-HT ratio was also detected in the limbic area. In the animals rendered dependent on morphine, the results obtained were similar to those of butorphanol-dependent rats except for changes of 5-HIAA levels in some brain regions. These results suggest that an alteration of dopaminergic neuron activity following a reduction of DA and its metabolites in specific brain regions (e.g., striatum, limbic, midbrain, and pons/medulla) play an important role in the expression of the opioid withdrawal syndrome.

Animals↗

Immunoglobulin E-mediated asthma and hypersensitivity pneumonitis with precipitating anti-hapten antibodies due to diphenylmethane diisocyanate (MDI) exposure.

Two workers are presented who were exposed to diphenylmethane diisocyanate (MDI) while coating pipes with a polyurethane foam. After a latent period of exposure, worker A developed immediate-onset asthma and worker B developed a clinical picture of hypersensitivity pneumonitis for which he was hospitalized. The antibody response of these workers to a conjugate of MDI with human serum albumin (MDI-HSA) was measured by gel precipitation, total antibody binding of 125I MDI-HSA, and specific IgG and IgE antibody by polystyrene-tube radioimmunoassay (PTRAI). Worker B had precipitating antibody to MDI-HSA by double immunodiffusion in gel. Both workers had high levels of IgG antibody specific for MDI-HSA which had some cross-reactivity with a conjugate or toluene diisocyanate and HSA. Total serum antibody binding of 125I MDI-HSA was 15 microgram/ml in worker A and 900 microgram/ml in worker B. Both workers had serum IgE antibody specific for MDI-HSA as measured by two PTRIA techniques. These results indicate that a marked immunologic response to MDI is possible in exposed workers and that hypersensitivity pneumonitis can occur subsequent to the inhalation of a low-molecular-weight chemical in the industrail setting.

Adult↗

N-glycanase treatment of F(ab')2 derived from asymmetric murine IgG3 mAb determines the acquisition of precipitating activity.

The aim of this study was to analyse four anti-DNP asymmetrically glycosylated monoclonal IgG3 antibodies (194/2, 194/5, 194/6 and 194/12) before and after carbohydrate manipulation. Microheterogeneity in the composition of the carbohydrate moiety involved in Fab' glycosylation was detected using lectins. Additional O-glycosidic carbohydrate chains were detected within the Fc region of two monoclonal antibodies. Fab' glycosylation produced a difference in the binding constants (Ka) in each paratope of two orders of magnitude, as determined by means of primary ligand-antibody interaction. The difference in binding affinity and the importance of Fc-Fc interaction was evidenced by a lack of BSA-DNP precipitation by the F(ab')2 fragments. The oxidation of the antibodies with sodium periodate caused the disappearance of the low affinity binding site as determined by fluorescence quenching. Furthermore, the enzymatic removal of the carbohydrate with N-glycanase determined the acquisition of precipitating activity by the F(ab')2 fragments.

Animals↗