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.     

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.     

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.     

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.     

Comparative Studies of Antigens from Mycoplasma mycoides and Escherichia coli

Extracts of sonically disrupted Mycoplasma mycoides and Escherichia coli were fractionated by sucrose density gradient centrifugation. The presence of antigen in each of the fractions was determined by complement-fixation and agar-gel diffusion precipitin tests, in which cow, pig, and rabbit anti-M. mycoides sera and rabbit anti-E. coli serum were used. Fractions of M. mycoides, with a buoyant density of 1.225 or lower, fixed complement with cow and pig anti-M. mycoides sera. These fractions also formed precipitin lines with pig antiserum. Fractions in the buoyant density range of 1.10 to 1.20 fixed complement with rabbit anti-E. coli serum, but precipitin lines were not formed. All E. coli fractions fixed complement and gave precipitin lines with homologous serum. But fractions in the buoyant density range of 1.10 to 1.20 had minimal complement fixation with heterologous M. mycoides sera. The cross-reacting antigens in M. mycoides and E. coli had a buoyant density of 1.10 to 1.20; the specific antigens were isolated from M. mycoides at a buoyant density of 1.08 to 1.02.     

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.     

Immunological relationships of the South American frog genus Eupsophus (Leptodactylidae)

The agar-gel precipitin technique was used to examine the immunological relationships of the South American frog genus Eupsophus. This taxon shows similarities with the members of the Telmatobiini tribe and presents a characteristic precipitin band, here labelled as A.     

Positive Fluorescent Treponemal Antibody Reactions in Diabetes

In a survey of 129 diabetic patients and 142 normal individuals, a significantly higher percentage of positive reactions in the fluorescent treponemal antibody-200 (FTA-200) test was found among diabetic patients than in the normal population. Absorption of all FTA-200-reactive sera with an extract of Reiter's treponeme eliminated most of the positive reactions in sera from diabetic patients, and three of the five positive reactions detected in sera from apparently normal subjects. On immunoelectrophoresis, precipitin bands developed most frequently between the Reiter sorbent and sera from diabetic patients positive in the FTA-200 test. Serum components responsible for FTA reactivity and precipitin reactions against the sorbent were resistant to treatment with mercaptoethanol, suggesting antibody of the IgG class. Cross-reacting antibodies produced in response to normal treponemal flora, and perhaps acquiring enhanced reactivity by means of nonspecific interacting substances in sera peculiar to the altered physiological state of diabetes, are suggested as possible causes of positive reactions of unabsorbed sera. No correlation could be made between age of the diabetic patient, treatment or duration of the disease, and FTA or precipitin reactivity of the patient's serum.     

Differentiation Between Herpes Simplex Virus Type 1 and Type 2 Strains by Immunoelectroosmophoresis

A method has been elaborated to differentiate between herpes simplex type 1 and type 2 viruses by immunoelectroosmophoresis. With rabbit immune sera cross-absorbed with heterologous virus antigen, a distinct difference was shown between the two virus types. Herpes simplex type 1 virus tested against cross-absorbed type 1 antiserum gave two precipitin lines. Herpes simplex type 2 virus gave one precipitin line when tested against cross-absorbed homologous serum. When the viral antigens were tested against cross-absorbed heterologous immune sera, no or only very weak precipitin reactions were observed. The test is easy and rapid, requires relatively small quantities of antigen and antibody, and is suitable for typing of herpes simplex virus in diagnostic routine work.     

Agar-Gel Precipitin-Inhibition Technique for Histoplasmosis Antibody Determinations

The agar-gel precipitin-inhibition technique has been modified to detect antibodies of Histoplasma capsulatum in sera from human clinical cases and experimental animals infected with this organism. By use of this modified technique, the histoplasmosis can be detected more consistently and reliably than with the direct agar-gel diffusion test, and titers are comparable to those attained by the complement-fixation serological technique.     

Detection and Identification of Diagnostic Histoplasma capsulatum Precipitates by Counterelectrophoresis

Studies were carried out to develop and evaluate a counterelectrophoresis (CEP) technique for the rapid and specific identification of the diagnostically important histoplasmosis H and M precipitin bands. Well-defined and centrally located precipitin bands were produced by using a discontinuous buffer system and a gel matrix composed of agarose and ionagar no. 2. A template was devised which allowed the selective identification of the H and M precipitins. Comparative evaluations were performed with the microimmunodiffusion (ID) and complement fixation tests. In 52 sera from persons with histoplasmosis, either the H or M precipitin, or both, were identified in 42 (81%) of the cases with the CEP technique and in 43 (83%) with the ID test. With sera from 28 persons with heterologous diseases, the CEP technique, like the ID test, failed to react. The specificity of the CEP technique was dependent upon the use of the identity template. The CEP technique is recommended for routine use in laboratories testing moderate numbers of sera. It provides accurate and reproducible results within 90 min, in contrast to the ID test, which requires 18 to 24 h.     

Specificity of the agar gel microimmunodiffusion test in rabies diagnostics.

The agar-gel microimmunodiffusion test (MIDT) with commercially available antirabies sera from different sources was applied to evaluation of rabies infection at about 500 brains from suspected animals. The high nonspecificity of the test and false positive results with nonvirulent materials were stated when compared with the histopathological, biological and FA tests. For evaluation of the nonspecificity and its cause, different antirabies sera and brain antigens from noninfected and rabid animals were used. Absorption of sera with tissue powders or immuno absorbent had a little influence on test specificity. The main causes of nonspecificity was the presence of antibrain antibodies in sera of producers-animals hiperimmunized by brain and spinal cord tissue vaccines. Application of the test to rabies diagnostics without purified control antigens and highly specific sera seems to be unjustified.     

Lectins Specificity to Pathogenesis of Fusarium oxysporum f. sp. vasinfectum

Lectins of cotton were isolated from either resistant or susceptible seed cultivars. In agar-gel double diffusion tests, positive reactions took place between lectins of cotton cultivars and the antisera of their corresponding wilt Fusaria. The number of precipitin bands correlated with the degree of susceptibility of the tested cultivars. On the other hand, no visible reaction was detected when these antisera were subjected to react with either the resistant host or nonhost lectins.     

Use of the Immunodiffusion Test in the Serodiagnosis of Aspergillosis

The diagnostic value of an immunodiffusion (ID) test with standardized precipitinogens derived from five Aspergillus species was determined with sera from 60 proven and 12 suspected cases of aspergillosis. The data demonstrated that the greatest number of aspergillosis cases were detected by the concurrent use of A. fumigatus and A. niger precipitinogens. With these precipitinogens, the ID test permitted the serodiagnosis of aspergillosis in 82% of the 60 proven cases and in 83% of the 12 suspected cases. The presence of one or more precipitins was indicative of aspergilloma, of allergic bronchopulmonary aspergillosis, or of invasive aspergillosis. Precipitins were detected in 93% of the sera from patients with aspergilloma, in 50% of the sera from patients with allergic bronchopulmonary aspergillosis, and in 88% of the sera from patients with invasive aspergillosis. Although the presence of one or two precipitin bands could indicate any form of aspergillosis, the presence of three or four was strong evidence of either aspergilloma or invasive aspergillosis. The ID test was found to be 100% specific in an evaluation of its effectiveness with 65 sera from individuals with other systemic mycotic infections, bacterial or neoplastic diseases, and from apparently normal humans. In diagnosed cases of aspergillosis, the examination of serial serum specimens provided information about the clinical course of the disease. A reduction in the number of precipitin bands and significant titer changes were noted as the patients responded to therapy.     

Immunodiffusion test for diagnosing basidiobolomycosis

An immunodiffusion test was developed for the diagnosis of basidiobolomycosis. When culture filtrate antigen (CFA) from Basidiobolus ranarum was reacted against two human patient and two rabbit antisera, 2 precipitin bands, inner (N) and outer (Y), were revealed for both patient and rabbit antisera. A line of identity was also observed between precipitin bands obtained with patient and rabbit sera. When CFA from B. ranarum (B CFA) was reacted against rabbit sera which contained antibody to Conidiobolus coronatus and Pythium insidiosum, 1 precipitin band corresponding to inner band (N) was observed. This finding showed that B. ranarum, C. coronatus and P. insidiosum shared at least one common antigen. After B CFA was absorbed with Pythium rabbit antiserum, the inner precipitin line that occurred between B CFA and rabbit antisera of Pythium and Conidiobolus disappeared. However, with Basidiobolus rabbit antiserum, the result did not change. The antigens which could be demonstrated by inner (N) and outer (Y) precipitin bands were heat stable at 56 ° C for 30 min. The titer of the antibodies specific to these antigens decreased as the lesions subsided. When B. ranarum CFA was reacted against sera from 20 apparently normal persons, 20 diabetes mellitus patients, 5 aspergillosis patients, 2 candidosis patients and 3 pythiosis patients, no precipitin band was found. B. ranarum CFA was also treated with each rabbit antiserum specific to Candida albicans, Malassezia furfur and Aspergillus fumigatus. No precipitin bands occurred with any of these antisera. Thus, this test was found to be practical, sensitive and specific, and can be used to monitor patients infected with Basidiobolus ranarum.     

Agar-Gel Precipitin-Inhibition Technique for C-Reactive Protein Determinations: II. Degree of Sensitivity and Reproducibility

The agar-gel precipitin-inhibition test (AGPI) for C-reactive protein determinations proved more sensitive than the capillary tube-precipitin procedure. Furthermore, titers of positive CRP sera were obtained by this AGPI technique. This technique was compared with other indices of infectivity, namely, the white blood cell count and the erythrocyte sedimentation rate. The ease and reproducibility of this recommended test procedure were demonstrated by technicians unfamiliar with this procedure.     

Agar-Gel Precipitin-Inhibition Test for Coccidioidomycosis: II. Serological Test Antigen Studies in Agar-Gel

In this study of saprophytic and parasitic growth-phase extracts of Coccidioides immitis, an antigen from the spherule culture supernatant fluid detected a specific antibody in heretofore serologically negative suspect coccidioidomycosis human sera when diffused in agar-gel. This antigen-antibody reaction occurred also in some of the serologically positive human coccidioidomycosis sera. This study indicates that this antigen-antibody reaction should be utilized as a possible routine serological test for complete serodiagnosis.     

Quantitative Measurement of Precipitating Antibodies in Streptococcal Grouping Antisera by the Single Radial Immunodiffusion Technique

A comparative study was made of the single radial immunodiffusion test and the classical quantitative precipitin test for determining the amount of precipitable antibodies present in streptococcal groups A and C antisera. The potency of 21 group A and 54 group C antisera was determined by both methods; purified group-specific carbohydrates were used as antigens. The coefficient of correlation between the results from the two methods was 0.976 for group A antisera and 0.946 for group C antisera. When the concentration of antigen, the volume of antiserum used, and the depth of the antigen-agar mixture are kept constant, the diameter of the precipitin disc is directly related to the concentration of precipitable antibodies present in the antiserum. The use of the radial immunodiffusion test for evaluating and standardizing streptococcal grouping antisera is discussed as well as the advantages and disadvantages of using a concentrated vaccine for producing these antisera.     

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.     

Group-specific and type-specific gel diffusion precipitin tests for bluetongue virus serotype 20 and related viruses

Using antigens prepared from cell cultures infected by bluetongue (BLU) virus type 20 (BLU-20), and sera from cattle which had recovered from experimental infection by that virus, two distinct precipitin reactions were demonstrated by immunodiffusion. Two distinct gel diffusion precipitin tests were developed based on these reactions. The antigen of one was common to BLU-20 and two other Australian BLU isolates, CSIRO 154 (BLU-21) and CSIRO 156 (BLU-1). It was therefore concluded to be a group-specific test. The antigen of the second appeared to be unique to BLU-20. The test based on this antigen correlated well with the virus neutralization test for BLU-20 and it was therefore concluded to be type-specific. Similar methods applied to a virus of the Palyam (PAL) group demonstrated two precipitin reactions of similar broad (group) and narrow (type) specificity.