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Agar-gel precipitin-inhibition test for coccidioidomycosis. I. Preliminary evaluation of the complement-fixation and agar-gel precipitin tests in the serodiagnosis of human coccidioidomycosis.

The agar-gel precipitin-inhibition serological test for coccidioidomycosis was a more sensitive indicator of Coccidioides immitis antibodies than the tube precipitin, the agar-gel immunodiffusion, in the complement-fixation tests in assaying monkey sera, whether these sera were from prechallenge-vaccinated or postchallenged animals. When applying this technique to the assay of human sera, an analogous finding generally persisted. However, some human sera were positive by the complement-fixation test and negative by the agar-gel precipitin-inhibition test. These sera were diffused in agar-gel against various coccidioidin complement-fixation, tube precipitin, and agar-gel precipitin-inhibition test antigens with essentially negative results.

Animals

Immunoaffinity isolation and partial characterization of the Coccidioides immitis antigen detected by the tube precipitin and immunodiffusion-tube precipitin tests.

The antigen participating in the tube precipitin (TP) serologic test for coccidioidomycosis was isolated from mycelial-phase antigen (coccidioidin) by immunoaffinity and characterized by various analytical procedures. This was accomplished by first preparing the antigen-antibody precipitate by using antigen and human serum positive for TP (immunoglobulin M) antibody and then liberating the antigen by digestion with pronase. This protease destroyed the antibody and left the antigen intact as indicated by immunodiffusion-TP. The coccidioidal antigen was isolated from the proteolytic digest by using size exclusion chromatography. DEAE chromatography of this antigen yielded two fractions with immunodiffusion-TP reactivity which had average molecular sizes of 225 and 140 kilodaltons, respectively. The presence of carbohydrate and amino acids indicated that the antigen(s) is a glycopeptide. Compositional analysis showed that one fraction contained 3-O-methylmannose, mannose, and glucose in a ratio of 8:1.2:1, whereas the second fraction contained 3-O-methylmannose, mannose, glucose, and galactose in a ratio of 1:1:1:1. The amino acids glycine, alanine, serine, threonine, aspartic acid plus asparagine, and glutamic acid plus glutamine constituted 60 to 70% of the amino acids in both glycopeptides. Neither antigen could be detected entering the gel in sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Lectin affinity provided evidence of a high-mannose asparagine-linked glycopeptide in the first peak and an asparagine-linked glycopeptide with a biantennary complex-type structure in the second peak.

Amino Acids

[Comparison of immunoenzymatic staining technique (IEST), double gluing strip circumoval precipitin test (DGS-COPT) and conventional circumoval precipitin test (CV-COPT) for diagnosis of schistosomiasis japonica].

IEST, DGS-COPT and CV-COPT using lyophilized ova of schistosoma japonicum were performed on sera from 120 cases of schistosomiasis japonica, 120 cases of schistosomiasis japonica 3-8 years after being cured with praziquantel and 120 healthy individuals by single-blind method. The sensitivity and specificity of IEST was 91.7% and 95.8% respectively which were significantly higher than that of both DGS-COPT and CV-COPT. The negative conversion rate of cured patients was 70.8% with IEST, 80.8% with DGS-COPT and 81.7% with CV-COPT. The results showed that IEST has higher diagnostic value for schistosomiasis than both COPT. DGS-COPT has the same diagnostic value as CV-COPT, however, it was easy to perform and time-saving, thus it might be applied in the fields for practical purposes.

Animals

Precipitins and specific IgG antibody to Aspergillus fumigatus in a chest unit population.

BACKGROUND: The enzyme linked immunosorbent assay (ELISA) for detecting IgG antibodies to Aspergillus fumigatus is more sensitive than the measurement of Aspergillus precipitins. The relation of the results from both techniques to the clinical pattern of disease in a large unselected group of patients from a large referral centre is unknown. METHODS: The clinical relation of precipitins to Aspergillus fumigatus to clinical disease was determined retrospectively in 98 patients attending a primary referral centre. Precipitin results were compared with the specific IgG antibody to A fumigatus in 88 of the sera. Precipitins were determined by the agar gel double diffusion test and specific IgG antibody to A fumigatus by a quantitative ELISA. RESULTS: Precipitins were detected in the unconcentrated serum of 51 patients. Thirty nine of these had a mycetoma or allergic bronchopulmonary aspergillosis, 34 having specific IgG antibody to A fumigatus more than the control range. Forty seven patients had precipitins only after threefold concentration of serum or to only one of the four A fumigatus antigen extracts. Most of these had specific IgG in or near the control range. Thirty of these had A fumigatus skin test negative asthma or bronchiectasis, in which aspergillus was probably not pathogenic. There was a close relation between the level of antibody detected by the ELISA and the number of precipitin lines. CONCLUSIONS: This study reaffirmed the supportive role of aspergillus precipitins in the diagnosis of pulmonary aspergillosis. No additional benefit in the routine use of the ELISA was seen. It also showed that care should be taken in interpreting positive precipitin results from concentrated serum and that using several rather than one A fumigatus antigen extract is helpful for identifying allergic aspergillosis.

Antibodies, Fungal

Incidence of serum anti-DNA precipitins in patients with systemic lupus erythematosus by counterimmunoelectrophoresis.

The technique of counterimmunoelectrophoresis (CIE) has been adapted for detection of serum precipitins to calf thymus (CT) DNA in patients with SLE, discoid LE, miscellaneous connective tissue and infectious diseases, and control populations. Of seventy-eight LE patients, 58% demonstrated anti-ss DNA precipitins, and 20% exhibited anti-ds DNA precipitins. Good correlation was noted between the presence of ss DNA precipitins and ss DNA binding values determined by the more sensitive ammonium sulphate precipitation assay. Depressed total serum haemolytic complement activity in CH50 mu/ml was noted in 64% of sera exhibiting ss DNA precipitins and 38% of those with negative ss DNA precipitins. There was a strong association, however, between ds DNA precipitins and depressed serum complement levels. Although less sensitive than primary binding assays, CIE can be used as a rapid and simple screening test for detection of circulating anti-native and denatured CT DNA precipitins. CT DNA serum precipitins are present in a significantly higher percentage of SLE patients when compared with other disease states and normal control populations.

Ammonium Sulfate

Candida precipitins in pregnant women: validity of the test systems used.

Sera from 200 pregnant women, with symptoms suggestive of vaginitis and harbouring yeast in the vagina, were examined for precipitating antibodies to three antigens of C. albicans, using a gel double diffusion test. A high overall incidence of precipitin-positive sera (47.5%) was found compared with an incidence of 18% in the unselected pregnant population previously studied (Stanley, Hurley, and Carroll 1972). Using the clinicopathological criteria of Carroll, Hurley, and Stanley (1973), a final aetiological diagnosis of C. albicans mycosis was reached in 75 cases and precipitins were demonstrated in 64%. Forty-eight women harbouring C. albicans responded favourably to a single course of antifungal treatment, and probably had mycotic vaginitis. The incidence of precipitins in this group was 42%. C. albicans was isolated from a further 55 of 62 patients, in whom the incidence of precipitins was 32%.;Booking' sera were investigated from 50 of the 200 women studied. Sixty-four per cent of women had symptoms of vaginitis at booking and 32% were precipitin positive. Twenty-eight per cent had precipitins on both occasions, and a further 24% acquired candida precipitins during pregnancy. None of the seven newborn with oral or skin thrush had precipitins to C. albicans.The results indicate that the detection of precipitating antibodies to C. albicans, particularly to all three of the antigens described in this paper, would be a useful additional criterion in the diagnosis of candida vaginitis, particularly if the vaginitis were persistent, recurrent, or unresponsive to therapy. The sensitivity of the test system used was 64%, and its specificity 87%; as such, the test is valid and may be reasonably useful as a screening procedure.

Antigens

Characterization of the glycoprotein antigens which mediate the Schistosoma japonicum circumoval precipitin reaction.

The circumoval precipitin test is a serological test used for diagnosis of schistosomiasis japonica. Soluble egg antigens of Schistosoma japonicum block the formation of the circumoval precipitin by serum from infected humans. Consequently, circumoval precipitin inhibition was used to monitor purification of the soluble egg antigens of S. japonicum. Crude egg antigens were separated into protein and glycoprotein fractions by lectin chromatography on concanavalin A Sepharose. The glycoprotein fraction produced two intense precipitin lines upon immunodiffusion analysis with human chronic infection sera. The protein fraction produced two faint precipitin lines which did not cross-react with those of the glycoprotein fraction. The glycoprotein fraction contained 90% of the circumoval precipitin inhibitory activity. Isoelectric focusing of (125)I-labeled concanavalin A Sepharose fractions revealed at least four groups of potential S. japonicum antigens, termed JAG I, II, and III, and a JAG IV complex. These had isoelectric points ranging from 3.2 to 6.7. In these respects, the S. japonicum egg antigen glycoproteins are similar to those of Schistosoma mansoni. The glycoproteins were further separated by diethylaminoethyl ion-exchange chromatography. On immunodiffusion analysis it was found that one of the strong Ouchterlony precipitin lines was associated with glycoproteins that did not adsorb to diethyl-aminoethyl columns, whereas the second Ouchterlony precipitin was heterodisperse, being present in the first, second, and third of the four peaks eluted from the diethylaminoethyl column. Immunoelectrophoresis of the diethylaminoethyl fractions demonstrated that the antigen present in highest concentration in soluble egg antigen glycoproteins, JAG II, was extremely heterodisperse in its behavior on diethylaminoethyl columns. This is unlike the S. mansoni antigens which can be easily separated by diethylaminoethyl ion-exchange chromatography.

Animals

Micro diffusion precipitin tests for enteroviruses and influenza B virus.

Middleton, G. K., Jr. (Bowman Gray School of Medicine, Winston-Salem, N.C.), H. G. Cramblett, H. L. Moffet, J. P. Black, and H. Shulenberger. Micro diffusion precipitin tests for enteroviruses and influenza B virus. J. Bacteriol. 87:1171-1176. 1964.-A simple micro precipitin gel diffusion test has been adapted to the study of viral antigens. As far as is known from a review of recent literature, this is the first use of the ECHO viruses in precipitin tests and the first attempt to demonstrate by the gel diffusion technique precipitins in a patient's serum after natural virus infection rather than artificial immunization. The principal value of this technique in virology is the rapid identification or qualitative analysis of viral antigen preparations by use of pooled or specific hyperimmune sera. Virus concentrations of 10(7)tcid(50) per 0.1 ml are required for reliable results, but only 0.015 ml of serum is necessary for each test. Virus-serum precipitin reactions were type-specific except for reciprocal precipitation of ECHO 1 and ECHO 8 by their hyperimmune sera. No viral antigens have been found common to two or more virus types among those tested. Precipitins for viral antigens occur frequently in serum of patients after a viral infection and are readily detected by micro precipitin gel diffusion tests. However, this precipitin test remains at present a tool for virus and antigen identification and offers an approach for research appraisal of host response to infections.

Animals

Steep gradients of inert substances as supports for precipitin reactions.

Precipitin reactions were conducted in the wells of micro titer plates and the light "absorbance" at 405nm plotted as a function of the neg log2 dilutions of the antigens using the Titertek Multiscan apparatus. The procedure followed was to incorporate antibody in 20% sucrose and to diffuse the mixture into serial two-fold dilutions of antigen overlaid on the sugar-antibody columns in the wells. Where the antigen met its antibody in equivalence precipitin bands formed. The bands of precipitates remained suspended. The precipitates formed by mixing the antibody sugar mixture with the serial two-fold dilutions of the antigen were also recorded in the Titertek Multiscan apparatus. Tobacco mosaic virus and its IgY type antibody produced simoidal precipitin curves with the layering as well as the mixing techniques. The haemocyanin of the whelk and its IgY antibody yielded a precipitin diagram with the layering technique which gave evidence to three antigens. The three antigens in the haemocyanin was confirmed by two dimentional Laurell-electrophoresis. Semi-purified human colostral dimeric IgA and its IgY antibody produced a precipitin diagram which showed evidence of four of five antigens. With the mixing technique a single precipitin maximum was obtained preceded by a prozone. Rabbit Ig and its IgY antibody gave a precipitin curve with two maxima. The precipitin curve obtained when the reagents were mixed had one peak preceded by a prozone.

Animals

Serum precipitins against microbes in mouldy hay with respect to the geographical location of the farm and to the work of farmers.

This study was based on a sample of 3,065 farmers from a larger survey population of 12,056 Finnish farmers. Data were gathered in a postal survey conducted by the Social Insurance Institution of Finland. Serum precipitins were determined by a microplate method of immune diffusion. The antigen panel consisted of mycelial antigens of Micropolyspora faeni, Thermoactinomyces vulgaris, Aspergillus fumigatus, and Aspergillus umbrosus. Geographical variation in the prevalence of precipitins was statistically significant, evidently due to climatic differences. The more intensive the cattle raising, the more commonly were precipitins found in the sera of farmers. For participation in animal tending (cattle, pigs, or poultry) or in plant cultivation work only, the prevalence of precipitins was largest among farmers who tended cattle or swine. According to earlier studies, serum precipitins to Thermoactinomyces vulgaris are associated with farmer's lung. Precipitins to this microbe were most commonly found in farmers who tended pigs and were also very common in farmers who worked only in plant cultivation. These findings imply that farmer's lung may also develop among pig farmers and grain producers. Precipitins to Thermoactinomyces vulgaris were clearly related to the type of grain drier used on the farm. The study failed to identify detailed tasks in farming associated with large prevalence of precipitins, probably owing to considerable overlap in the exposure encountered in different tasks.

Agriculture

Reactivity of alkali-soluble, water-soluble cell wall antigen of Coccidioides immitis with anti-Coccidioides immunoglobulin M precipitin antibody.

The alkali-soluble, water-soluble cell wall antigen of Coccidioides immitis (C-ASWS) mycelia and spherules was shown to react with anti-Coccidioides immunoglobulin M (IgM) precipitin antibody, both in the classical tube precipitin test and in the immunodiffusion assay for tube precipitin antibody (IDTP). The reactions obtained between C-ASWS and reference IgM precipitin antibody were identical to the reaction obtained when reference coccidioidin (CDN) was used. Definitive proof that C-ASWS extracts contain antigenic determinants that are reactive with IgM tube precipitin antibody was obtained by solid-phase immunoadsorption. Elution of reference IDTP antiserum over a column containing mycelium C-ASWS coupled to Sepharose 4B completely adsorbed precipitin antibody; i.e., reactivity in the IDTP was demonstrable in the column eluate but not in the column effluent fraction. The antigenic composition of C-ASWS extracts was evaluated and compared with that of CDN by two-dimensional immunoelectrophoresis against burro anti-CDN. The results established that both mycelium and spherule C-ASWS contain antigenic determinants in common with only one antigen present in CDN. The latter, designated antigen 2, is a large polymer which is predominant among the antigenic components in CDN. On a dry weight comparison, antigen 2 determinants were most concentrated in spherule C-ASWS, followed by mycelium C-ASWS and reference IDTP antigen. The finding that C-ASWS extracts are reactive with IgM tube precipitin antibody and are antigenically identical to antigen 2 in CDN suggests that antigen 2 is the biologically active component of CDN in tube precipitin assays.

Cell Wall

California arbovirus (La Crosse) infections. II. Precipitin antibody tests for the diagnosis of California encephalitis.

Precipitin antibodies to California group arboviruses were studied by double agar gel immunodiffusion (ID) and counterimmunoelectrophoresis (CEP). Forty children with California encephalitis (CE), 12 patients with other forms of meningoencephalitis, and 120 residents of endemic CE areas were tested. Precipitin antibodies were detected only in the 40 patients with CE, whereas hemagglutination-inhibiting, neutralizing, and complement-fixing antibodies were also found in persons without a history of CE. Precipitin antibodies did not persist for more than a year, in contrast to the other three antibodies which may be detected for 2 or more years. Of the children with CE, 36% had precipitin antibodies in their acute sera by CEP and 44% by ID. Precipitin responses were subtype-specific, and the patients reacted most strongly with La Crosse virus. The results indicated that precipitin techniques would be useful for the early detection of CE in some patients and that the presence of precipitin antibodies in a patient with acute central nervous system infection is strongly suggestive of CE. In addition, precipitin tests can be used to distinguish the California subtype responsible for CE in a given area.

Antibodies, Viral

Precipitins against microbes in mouldy hay in the sera of farmers with farmer's lung or chronic bronchitis and of healthy farmers.

The aim of this study was to determine which precipitins against four antigens in 2,440 farmers are associated with the occurrence of farmer's lung (FL). The antigen panel consisted of mycelial antigens of Micropolyspora faeni, Thermoactinomyces vulgaris, Aspergillus fumigatus and Aspergillus umbrosus. As reference groups we used healthy farmers and those with chronic bronchitis. For the occurrence of precipitins against the four antigens there was a statistically significant difference between farmers with FL and healthy farmers but not between farmers with chronic bronchitis and healthy farmers. In a stepwise logistic linear regression analysis, farmers with FL and chronic bronchitis were compared to healthy farmers with respect to precipitins to the four antigens. Precipitins against Thermoactinomyces vulgaris differentiated farmers with FL (p less than 0.05) but not farmers with chronic bronchitis from healthy farmers. In Finland the occurrence of FL seems to be associated mainly with precipitins against Thermoactinomyces vulgaris, not with precipitins against Micropolyspora faeni as in Great Britain, and not with precipitins of Aspergillus umbrosus, which occurred most frequently in the sera of Finnish farmers. This association is in accordance with the exposure to spores of airborne moulds in farmers' work environment, where spore concentrations of Thermoactinomyces vulgaris have been measured to be about six times higher than those of Micropolyspora faeni.

Antibodies, Bacterial

Precipitins to an aflatoxin-producing strain of Aspergillus flavus in patients with malignancy.

Serum samples from 121 patients in whom malignant disease had been diagnosed, were assayed for precipitins to fungal isolates from leukemia-associated environments. Control sera were from age-, sex-, and race-matched patients with no history of malignant disease. Sera from 36 (30%) malignancy patients and seven (6%) controls yielded a precipitin band to an aflatoxin-producing Aspergillus flavus isolate from a leukemia-associated house (x2 = 222, p less than 0.05%). No significant numbers of precipitins were obtained to either of the other fungal isolates from that and another such house. Although A. fumigatus has frequently been incriminated as a source of infection in patients with malignancy, only 9% of malignancy patients had a precipitin response to it, as did 1.6% of controls. Also, the presence of the precipitins to A. flavus was not connected with past radiation or immunosuppressive therapy. However, among patients with precipitins to A. fumigatus there was a higher death rate in the year following the study. Precipitins to A. flavus may be related to heavy environmental exposure possibly leading to aflatoxin exposure which may contribute to development of malignancy though immunosuppressive effects.

Adolescent