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.     

Estudio comparativo de las reacciones serologicas cuantitativas con un antigeno metabolico de Aspergillus fumigatus

Summary The following quantitative serologic reactions: agar-gel immunodiffusion, complement-fixation, opposite electrophoresis and latex particle agglutination tests have been performed in 38 sera from mycologically proved pulmonary aspergillosis cases. A metabolic antigen from a strain ofAspergillus fumigatus according toAjello et al technic modified by us, has been employed. Sera from 120 subjects suffering from non-mycotic lung conditions, as well as 10 sera from histoplasmosis cases, 10 sera from S. A. blastomycosis and 2 sera from patients with lung aspergillosis produced byA. niger, gave negative results with the above mentioned seroligic reactions.One hundred per cent of positive results were obtained with the complement-fixation test (titre ranging from 1/20 to 1/1280), agar-gel immunodiffusion test (titre up to 1/64) and the opposite immunoelectrophoresis (titre ranging from 1/2 to 1/256). Twenty five per cent negative and 4 non-specific results were registered with the latex particle agglutination test.A correlation of the number of serum precipition bands obtained by the electrophoresis technic with the titre of the quantitative serologic reactions, as well as a correlation of the titre of the circulating antibodies with the severity of the clinical form of aspergillosis seems to be present.Electrophoretic motility of the specific antibody performed in 10 sera showed results like the IgM in 1 instance and an intermediate position between IgA and IgG in 9 samples.     

Comparison of the Reliability and Sensitivity of Three Serological Procedures in Detecting Antibody to Yersinia pestis (Pasteurella pestis)

Three serological procedures, the agar-gel precipitin inhibition, the complement fixation, and the indirect hemagglutination tests, were used to detect and measure antibody to Yersinia pestis in the sera from 383 individuals. Although all three tests were useful in detecting plague antibody, the most reliable and sensitive test procedure was indirect hemagglutination.     

Comparison of Indirect Hemagglutination and Immunodiffusion Tests for Detecting Type II Leukosis (Marek's) Infection in S- and K-Line Chickens

The indirect hemagglutination and immunodiffusion tests were compared for detection of antigen and antibody to JM strain of leukosis virus infection between S- and K-line chickens. The indirect hemagglutination test was more sensitive than the immunodiffusion test for detecting the smallest amount of viral antigen and corresponding antibody in the plasma of infected chickens. The Cornell S-line had higher levels of antigen and antibody as compared with the Cornell K-line during the 20-week experimental period.     

Comparison of Indirect Hemagglutination and Immunodiffusion Tests for Detecting Type II Leukosis (Marek''s) Infection in S- and K-Line Chickens

The indirect hemagglutination and immunodiffusion tests were compared for detection of antigen and antibody to JM strain of leukosis virus infection between S- and K-line chickens. The indirect hemagglutination test was more sensitive than the immunodiffusion test for detecting the smallest amount of viral antigen and corresponding antibody in the plasma of infected chickens. The Cornell S-line had higher levels of antigen and antibody as compared with the Cornell K-line during the 20-week experimental period.     

Rapid, Sensitive Assay for Staphylococcal Enterotoxin and a Comparison of Serological Methods

Reversed passive hemagglutination was used to assay enterotoxin in culture filtrates and in food samples. With cells tanned and then sensitized with antitoxin globulin and preserved with either formaldehyde or pyruvic aldehyde, as little as 0.0007 mug of enterotoxin was detectable. The results of hemagglutination tests compared well with those obtained by quantitative precipitin tests or by immunodiffusion, but hemagglutination was 50 to 100 times more sensitive than the immunodiffusion technique. In addition, results of the hemagglutination test were available within a few hours, and neither elimination of interfering proteins from food extracts nor concentration of the sample, both of which are necessary for immunodiffusion, was required for this procedure.     

Application of agar-gel precipitin test for the detection of Sendai virus antibody in rat sera.

A simplified agar-gel precipitin test was performed for the detection of Sendai virus antibody in rat sera. A close correlation was observed between detection of antibodies by complement-fixation test and agar-gel precipitin test. No correlation was found between results obtained by hemagglutination-inhibition test and agar-gel precipitin test in sera with HI titer of less than 1:8.     

Characterization of the Precipitin Bands Detected in the Immunodiffusion Test for Paracoccidioidomycosis

In order to characterize the precipitin bands detected in the immunodiffusion test for paracoccidioidomycosis, a study was undertaken in 54 patients with the disease. On the basis of the pattern of known control sera, the three commonly observed lines of precipitate were designated as 1, 2, and 3 according to their location in the immunodiffusion plate. At time of diagnosis, 28 of the patients exhibited all three bands, 16 gave two bands, and 10 showed only one precipitin line. Over 50 of the sera with three bands had high complement fixation titers (above 1:512), whereas those with one band exhibited lower titers. A similar picture was obtained with the quantitative agar-gel techniques, where titers of 1:64 and above were more commonly observed in sera with three precipitin lines. Follow-up studies carried out in 18 patients revealed that band 3 disappeared first, followed by band 2, and, finally, by band 1. At the end of 2 to 3 years, 85.7% of the patients had lost band 3, 75% band 2, and only 27.7% band 1. Cross-reactions with histoplasmin were found in eight patients who gave the M precipitin line with this antigen. It was found that the latter band and our paracoccidioidin band 3 fused, producing lines of identity. Bands 1 and 2 were specific. The implications of these findings are discussed.     

Experimental paracoccidioidomycosis in the Syrian hamster

Male hamsters (105) received intratesticular injection of suspension of a live yeast phase culture ofParacoccidioides brasiliensis and were sacrificed weekly during 20 weeks. Humoral immunity was studied by the agar-gel immunodiffusion (ID) and indirect immunofluorescence (IF) tests. Cell-mediated immunity was determined by the macrophage migration inhibition test in the presence of phytohemagglutinin (PHA) andParacoccidioides brasiliensis soluble antigen (PbAg). The morphology of the lesions was studied in the inoculation site, lymph nodes, lung, liver, spleen and kidneys.Disseminated paracoccidioidomycosis was observed in 100% of the animals after the first week. The lesions were initially made up of fungi surrounded by polymorphonuclear neutrophils and macrophages. Up to the 10th week the majority of the lesions appeared as compact confluent ephitelioid granulomas containing rare large fungi, some showing signs of degeneration. At this time, the specific antibody titers and the cellular immune response to PHA and PbAg were highest.From the 11th week on the granulomas became less compact, edematous with the epithelioid cells loosely arranged. This change was accompanied by an increase in the number of fungi showing reproductive activity and was associated with renal amyloidosis and progressive decline of cellular immune response both to PHA and PbAg. Contrariwise the titers of circulating antibodies were maintained.In the present model, disseminated paracoccidioidomycosis of the hamster was associated with depression of cellular immunity, change in the pattern of the granuloma, intense fungi proliferation and amyloidosis.     

Serology of paracoccidioidomycosis

The purposes of the present work were: i) to study the positivity indices and compare titers obtained with the indirect immunofluorescence (II), tube precipitation (TP), complement fixation (CF) and double immunodiffusion on agar gel (ID) tests in the sera of 196 patients with paracoccidioidomycosis before treatment, and ii) to compare the initial titers of II with those obtained 1 year or more after treatment. II was the most sensitive serologic reaction (85.2%), and the positivity indices for CF, ID and TP were 67.7%, 66.0% and 50.0%, respectively. The sera tended to show parallel mean titers in II, CF and TP tests. One year after treatment there was a fall in titers of II in 66.2% of patients. The data, taken as a whole, demonstrate the usefulness of the indirect immunofluorescent test and the importance of using 2 or more serologic tests for the diagnosis and monitoring of patients with paracoccidioidomycosis.     

Agar-Gel Precipitin-Inhibition Technique for C-Reactive Protein Determinations: III. Quantitation of C-Reactive Protein in Serum Specimens

A quantitative C-reactive protein serological procedure has been developed. By use of this method, which is performed in agar-gel plates, from 2 to 654 μg of C-reactive protein per ml of titrated human serum can be detected. The method is based on the inhibition of a specific C-reactive protein antigen-antibody precipitate formed in agar-gel by the minimal reactive dilutions of each reagent in 48 hr. It is simple, sensitive, and readily reproducible.     

Evaluation of purified H and M antigens of histoplasmin as reagents in the complement fixation test.

Complement-fixation (CF) tests were performed with purified H and M antigens, histoplasmin, and Histoplasma capsulatum whole cell yeast phase antigen using sera of 126 patients with proven or suspected histoplasmosis. Specific titers for either H or for M antibody were obtained with the individual purified antigens; the highest titers were comparable to those obtained with histoplasmin. However, in sera containing only anti-M antibody, the titers obtained with the purified M antigen were 2 to 16 times those obtained with the histoplasmin or yeast phase antigens. The CF test for either H or M antibody was 4 to 32 times as reactive as the agar-gel microimmunodiffusion test; in general precipitin lines were obtained with either H or M antigens from sera with CF titers greater than or equal to 8. With sera containing H antibody, there was an excellent correlation between the CF titers obtained with purified M antigen and histoplasmin. The correlations of CF titers with H antigen and either histoplasmin or yeast phase antigen were very low.     

Echinococcus granulosus: comparison between antigens in scolices and hydatid fluid.

The sheep hydatid fluid and scolex antigens of Echinococcus granulosus were precipitated by increasing ammonium sulphate concentrations. The antigenic profiles, obtained by complement fixation and indirect hemagglutination inhibition tests on the ammonium sulphate precipitates after linear sucrose gradient ultracentrifugation, were different comparing the hydatid fluid and the scolex extracts. Antigenic non-identity was found between sheep hydatid fluid and scolex extracts by immunodiffusion and indirect hemagglutination inhibition tests. The ammonium sulphate precipitates of hydatid fluid and scolex extracts revealed several different bands by slab-gel examination.     

Comparative evaluation of various immunodiagnostic tests for the diagnosis of Taenia solium cysticercosis in pigs, using fractionated antigens

The sensitivity and specificity of double immunodiffusion (DID), indirect haemagglutination test (IHA), immunoelectrophoresis (IEP), counterimmunoelectrophoresis (CIEP) and enzyme-linked immunosorbent assay (ELISA) were evaluated and compared using saline extracted of Taenia solium larval scolex and its Sephadex G-200 fractionated 1st and 2nd peak as antigens. Various immunodiagnostic tests gave different results with different antigens. Highest sensitivity (92.5%) was obtained with 84.6% sensitivity was obtained with IHA and CIEP respectively using scolex antigen. CIEP gave better results as compared to IEP. Crude antigen gave high sensitivity but less specificity. It was concluded that CIEP can be used as a field test for the anti-mortem diagnosis and ELISA can be employed for laboratory confirmation of T. solium cysticercosis in pigs using fractionated 1st peak antigen.     

Enzyme-linked immunosorbent assay for efficient detection of antibody to bluetongue virus in pronghorn (Antilocapra americana)

An indirect enzyme-linked immunosorbent assay (ELISA), using cell-associated viral antigen, was developed for detection of antibody to bluetongue virus (BTV) in field-collected pronghorn (Antilocapra americana) sera. To test the applicability of the ELISA to seroepizootiologic studies, pronghorn serum samples from three Wyoming counties (USA) were tested. Bluetongue virus ELISA results were compared to those of the bluetongue immunodiffusion assay. Discrepant serum samples were retested for reaction to either BTV or epizootic hemorrhagic disease virus. The pronghorn BTV ELISA gave rapid, quantitative, objective results and should facilitate testing large numbers of sera for BT diagnostic and seroepizootiologic studies.     

A simple procedure for isolation and purification of A-type staphylococcus enterotoxin

A new procedure is presented for the isolation and purification of A-type staphylococcus enterotoxin. Homogeneous enterotoxin preparation was obtained by purification in 2 phases. In radial double agar-gel immunodiffusion the smallest precipitating dose of the isolated and purified enterotoxin was found to be 1.4-0.7 micrograms protein and 0.4-0.1 micrograms nitrogen. In cat experiments the dose giving a positive reaction was 2 micrograms protein or 0.5 micrograms nitrogen calculated for kg body weight.     

鸭细小病毒04Nb株的分离鉴定与rep基因测序与分析

采集浙江宁波地区以腹泻、呼吸困难为主要症状的病鸭肝组织,接种正常鸭胚尿囊腔增殖病毒。雏鸭感染试验显示发病症状及病理变化明显,死亡率为75%。电镜下可见纯化病毒直径约20nm左右的球形病毒粒子。免疫琼脂扩散实验结果显示与鸭细小病毒(duckparvovirusDPV)标准株阳性血清有明显沉淀线。经SDS-PAGE呈现3条结构蛋白带,与DPV标准株一致;参照GenBankDPV非结构蛋白基因序列设计引物,PCR扩增反应获得目的条带,克隆测序后,与DPV代表株序列同源性达98%。根据上述实验结果,确定引起本次鸭场疫病的病原为DPV。为进一步研究该分离株rep基因的序列特征,对其rep基因克隆测序,与GenBank中两株DPV、两株鹅细小病毒(GPV)进行序列比对,结果显示rep基因核苷酸序列与DPV参考毒株同源性为98%以上,与GPV同源性为80%左右。     

Comparative Evaluation of Five Serological Methods for the Diagnosis of Sporotrichosis

The diagnosis of sporotrichosis can be time consuming. Serological procedures could facilitate the rapid and accurate diagnosis of this disease. A slide latex agglutination (SLA) test for sporotrichosis was developed and compared with the tube agglutination (TA), complement fixation (CF), and immunodiffusion (ID) tests in the serological study of 80 proven human cases of sporotrichosis representing the cutaneous, subcutaneous, and extracutaneous forms of the disease. In addition, the indirect fluorescent antibody (IFA) technique was applied to 61 case sera. In the SLA test, latex particles sensitized with culture filtrate antigens from the yeast form of Sporothrix schenckii (B 959) detected 94% of the cases, as compared to 96% of the cases detected by the TA test, 68% by the CF test, and 56% by the ID test. The IFA test detected 90% of the 61 cases. The SLA and ID tests were specific, showing no reactions with sera from 86 persons with no disease or with diseases other than sporotrichosis. Because of its sensitivity, specificity, ease of performance, and ability to provide results in 5 min, the SLA test is highly recommended for routine use in the clinical laboratory.     

Identification of Actinomyces israelii and Actinomyces naeslundii by Fluorescent-Antibody and Agar-Gel Diffusion Techniques

This study was an attempt to develop a fluorescent-antibody (FA) test to differentiate Actinomyces israelii and A. naeslundii as an aid in their laboratory identification. Two strains of A. israelii (X522 and A601) and two strains of A. naeslundii (X454 and X600), which had received intensive study by several investigators, were used for the immunization of rabbits. Working titers, based on tests with antigens prepared from the homologous strains and from well-established heterologous strains, were determined for each labeled antibody preparation. These conjugates and their normal serum control conjugates were used separately to stain 85 cultures of Actinomyes species and 23 strains of other species that might be confused with them. Acetone-precipitated soluble antigens from these same strains were tested with different antisera in the agar-gel diffusion test. Results showed that A. israelii (X522 and A601) and A. naeslundii (X454 and X600) labeled antiglobulins, when used at their working titers, stained most strains of their homologous species. Agar-gel diffusion results showed general agreement with those of the FA tests. The two tests appear to be equal in sensitivity, but the FA test is more specific, since several cross-reactions were noted with the agar-gel diffusion test whereas no cross-reactions were obtained with the FA reagents. Agar-gel and FA studies suggest that at least two serotypes of A. israelii may be associated with human disease. Although the majority of strains tested in this study appear to belong to a common serotype, "serotype 1," two strains of an apparent second serotype, "serotype 2," were encountered. FA staining of tissue impression smears from experimentally infected mice was successful when a counterstain, Evans Blue dye, was used.     

Specific Immunodiffusion Test for Blastomycosis

A specific immunodiffusion test for blastomycosis has been developed. The test permitted the detection of approximately 80% of 113 proven cases of blastomycosis. Two diagnostically important precipitins designated A and B were frequently recognized in patients with blastomycosis. Routine use of reference sera containing the A and B precipitins in immunodiffusion tests would permit the specific diagnosis of blastomycosis without the need for parallel tests with coccidioidin and histoplasmin.