THE PREPARATION OF TRANSPARENT SOLUTIONS FOR PRECIPITIN TESTS.
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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.
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Recent isolates of Mycoplasma gallisepticum and Mycoplasma synoviae were readily typed by the agar-gel precipitin test with antigens prepared by freezing and thawing, sonic vibration, or sodium dodecyl sulfate. Specific antisera prepared in rabbits or in foot-pad-inoculated chickens were adequate for culture typing. Relatively few sera from chickens and turkeys in naturally infected flocks reacted positively. The precipitin reaction was highly specific, however.
Saline cell free extracts of larvae of the Simulium damnosum complex were used to immunize rabbits for the production of damnosum-antisera. Injections into the lymph nodes produced a more specific antiserum than was obtained by intramuscular injection followed by absorption. This antiserum was used to identify by the precipitin test natural predators of S. damnosum collected from the bandama River in the Ivory Coast. Various trichopteran larvae and odonatan nymphs and adults were found to be the most important predators.
The use of a gel diffusion precipitin (GDP) test for the detection of porcine parvovirus (PPV) infection in pigs is described. The close correlation between gel diffusion precipitin and haemagglutination inhibiting (HI) antibody titres indicates that, with careful standardisation, a high level of sensitivity can be achieved with the GDP test and that it is a simple and relatively inexpensive alternative to the more commonly used HI test. Experimental infection of 2 groups of pigs showed that GDP and HI antibody responses were closely correlated and that GDP antibodies to PPV persisted for at least 41 weeks after infection. In a commercial herd study, serological evidence of declining passive immunity and subsequent acquisition of active immunity was demonstrated by measuring the GDP and HI antibody titres in sequential serum samples of pigs from a known PPV endemic farm. The GDP test described was shown to be less sensitive than haemagglutination (HA) in the detection of viral antigen but was, nevertheless, considered useful as a simple screening test for the amounts of antigen usually present in PPV infected mummified foetuses.
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A capillary procedure for quantitatively determining M protein is described. Capillaries were filled with measured amounts of serum and streptococcal extract. The capillaries were incubated, and then centrifuged to pack the precipitates. The relative sizes of the precipates were compared by a determination of the weights of their paper images (obtained by reflection from a microscope). Meaningful dilution curves were determined by this method. Variations of pH from 6 to 8 had little effect on the M protein precipitin test, and the test was not seriously affected by variations of the NaCl concentration from 0.85 to 4.67%. The addition of divalent ions (Ca(++) and Mg(++)) did not influence the results. This test can be used to make quantitative comparisons of M protein preparations and to titrate type-specific antisera.
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A forty-year-old female from Brazil was admitted to Teikyo Hospital because of easy fatigability, fullness of the abdomen and left hyochondralgia. She was anxious about Schistosoma mansoni infection, because three of her relatives died of the infection. Physical examinations revealed a tenderness at the left hypochondrium. Laboratory data showed no abnormal finding. No egg of S. mansoni was found in the stool. A circumoval precipitin test (COPT) with the serum showed a deposite around the egg. Enzyme-linked immunosorbent assay (ELISA) revealed the presence of antibody against S. mansoni in the serum. A colonoscopy showed no abnormal finding macroscopically. The rectal biopsy showed the existence of mild procitis. The diagnosis was made by finding the characteristic lateral-spined eggs in the biopsy specimens from the rectum. Treatment of 3 g of prazicantel per day for three days was started. She complained of mild nausea at the first dosing. A month later, another three-day-treatment was given. In the case where there are no eggs found in the stool, COPT and ELISA are usefull in detecting the disease, and colonoscopy is recommended in diagnosing the disease.
Anopheles aquasalis has shown local variations in blood-host preference in Brazil: it seems to be anthropophilic in the northeast and zoophilic or opportunistic in the Amazon and other regions. A study was carried out in Guapimirim county, State of Rio de Janeiro, from May to November 1992, for the purpose of identifying the blood meal source of this anopheline by capillary tube precipitin test. A total of 1,366 females were captured at natural resting-places, 725 of which were blood-fed. The gut content of 473 blood fed females reacted to the antisera used (human, cow, horse, pig, dog and chicken). The majority of the females-356 (75.3%)-had blood from only one source. A substantial bovine preference was observed-52.2% had fed on cows, 29.8% on horses, 10.7% on pigs, 4.5% on chickens and 1.7% on dogs, but only few had fed on man (1.1%) and none on rats. On the other hand, 24.7% of the females had fed on more than one host, mainly on cows and horses. It was assumed that An. aquasalis is zoophilic in southeastern Brazil, feeding on a wide variety of animals, the cow being the primary host.
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The recent outbreaks of human fascioliasis in Britain provided the opportunity for employing the double diffusion precipitin test in the diagnosis of this disease. In sensitivity and specificity this test appears to equal the complement-fixation test and has the advantages of simplicity and speed in its performance.
Three hundred fifty human sera were tested by double immunodiffusion, crossed-line electrophoresis, and crossed immuno-affinoelectrophoresis with a concanavalin A intermediate gel for precipitating antibodies to antigens present in cytoplasmic extracts of Candida albicans. Sera from 48 of 287 hospitalized patients at risk of invasive candidiasis contained precipitating antibodies to Candida antigens. Of these 48 sera, 27 had precipitating antibodies only to cell-wall antigens present in the cytoplasmic extract, and 21 sera had precipitating antibodies to both cytoplasmic and cell-wall antigens. The latter sera came from patients who were 2.5 times as likely to have deep-seated candidiasis as those patients with precipitins exclusively to cell-wall antigens. Sera from seven of 22 patients with vaginal candidiasis and 10 of 41 patients with other fungal infections had precipitating antibodies to C. albicans cell-wall antigens; only two of these sera also contained precipitating antibodies to the cytoplasmic antigens. Crossed immunoaffinoelectrophoresis with concanavalin A reduced the number of false-positive results and increased the predictive value positive of the precipitin test for deep-seated candidiasis from 31% to 71%.
The serological response of pigs to Erysipelothrix rhusiopathiae inoculation was monitored by a gel diffusion precipitin test (GDPT) using a crude, serotype-specific, autoclaved antigen and an enzyme-linked immunosorbent assay (ELISA) using a heat-extracted, alcohol precipitated and molecular seived antigen previously shown to react with serum from pigs infected with serotypes 1 or 2. All pigs receiving 3 or 5 weekly intravenous inoculations of either a highly virulent (VRS 229) or a lowly virulent isolate (VRS 252) produced GDPT-reactive antibody within 3 weeks, but only 44% were still reactive at 8 to 9.5 weeks. The ELISA response was significantly higher in pigs inoculated with the highly virulent strain, and was similar in pigs receiving 3 or 5 doses of either strain. In a dose-response trial, after 3 doses of VRS 229, GDPT reactivity occurred earlier and was stronger in pigs given higher doses of E. rhusiopathiae, but the response peaked 3 to 5 weeks after the start of challenge and was short lived. GDPT reactivity correlated with dose, but not with the severity of arthritis. The ELISA demonstrated specific IgG antibody was present by 2 weeks, and persisted to at least 11 weeks. The ELISA reactivity was significantly higher in pigs with arthritis than in pigs that received low doses and were not arthritic. Within groups of pigs with arthritis a significant, dose dependent, linear ELISA response developed but did not correlate with the presence or degree of arthritis at slaughter. Non-arthritic pigs had similar low ELISA responses to uninoculated controls.
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