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.     

STUDIES ON MUSCLE DIFFERENTIATION. VI. IMMUNOLOGICAL SPECIFICITY OF TROPOMYOSIN FROM CHICKEN SKELETAL MUSCLES *

The chicken skeletal muscle tropomyosin preparation reacted in agar diffusion test with the anti-chicken skeletal muscle tropomyosin antiserum by forming three precipitin lines which were very close with one another and appeared to be almost a single precipitin line. Three antigens responsible for the formation of these three precipitin lines could not be differentiated in 8 m urea-polyacrylamide gel electrophoresis. These three precipitin lines could be identified to be due to the reaction between authentic tropomyosin molecules and their corresponding antibodies. Further, one of these three antigens was found to be present in the extracts from skeletal and cardiac muscles of various vertebrates so far tested and was identical with the genusand organ-nonspecific antigen as revealed earlier by the immunological study with frog skeletal muscle tropomyosin (Hirabayashi and Hayashi , 1970b). One of the remaining two antigens was clearly found to be present in the skeletal muscle extracts from avian sources. The last antigen was clearly found to be present in the extracts from pectoral and leg muscles, gizzard, anterior stomach, kidney, ovary, oviduct, testis and brain of the chicken. However, the reaction of the antibody against the last antigen with the extract of pectoral muscle of the chicken was very weak.     

Precipitin responses of infected mice to exoantigens and cellular antigens of Trypanosoma musculi.

Plasma were collected from mice which had been immunosuppressed with 650 R from a cobalt-60 gamma radiation source and infected with Trypanosoma musculi. Trypanosomes were also collected from immuno-suppressed mice and from nonirradiated, infected animals. Rabbit antiserum was prepared against trypanosomes fron nonirradiated mice and employed in immunodiffusion analyses to detect trypanosome exoantigens (ExAg) in plasma of irradiated, infected mice and cellular antigens (CAg) in extracts of parasites which had been collected from immunosuppressed and nonirradiated hosts. The rabbit antiserum formed at least 3 precipitin lines with plasma from irradiated, infected mice and 8–9 precipitin lines with extracts of parasites which were obtained from immunosuppressed and untreated mice. Two of the precipitin reactions were against mouse plasma antigens (PAg). Lower levels of PAg appeared to be present in extracts of trypanosomes which were isolated from the irradiated mice than in those from nonirradiated animals.Mice synthesized antibodies against 1 ExAg which was demonstrable in immunodiffusion tests by 14 days after T. musculi infection. A single precipitin reaction was also seen after 21 days. One to 2 precipitin lines were formed with ExAg after 42 days of infection. Two to 3 precipitin lines formed between the ExAg and mouse antisera collected 98, 175 and 341 days after injection of the T. musculi.Similar immunodiffusion reactions were detected with CAg present in both the extracts of T. musculi which had been isolated from irradiated and those from nonirradiated mice and the mouse antisera. One to 2 precipitin lines were found between CAg and antisera from mice which had been infected for 14 days. Two precipitating antigen-antibody systems were seen with antisera collected after 21, 42 and 98 days and 2–3 precipitin reactions were formed between CAg and antisera collected from mice 175 and 341 days after infection.Absorption and immunodiffusion analyses conducted with rabbit and mouse antisera indicated parasite ExAg in plasma of irradiated, T. musculi infected mice were also present in preparations of CAg of the trypanosomes. The persistence of antibody and the increase in the numbers of antigen-antibody systems detected by immunodiffusion during the course of the infection may in part be related to the presence of parasites in capillaries of the kidneys long after they cannot be demonstrated in the peripheral blood of the host.     

Purification of the protein employed in an immunodiffusion test to diagnose infectious bovine rhinotracheitis.

The antigen used in an immunodiffusion test to diagnose infectious bovine rhinotracheitis has been purified by affinity chromatography. The homogeneity of the antigen was indicated by sedimentation rate and sedimentation equilibrium experiments. A So20,w of 0.749 was determined and a molecular weight of 8900 was calculated from sedimentation equilibrium analysis. The purified antigen formed precipitin lines of identity with crude diagnostic antigen. Purified antigen remained serologically active in the immunodiffusion test after lyophilization and subsequent reconstitution.     

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.     

Immunodiffusion test in avian encephalomyelitis. I. Standardization of procedure and detection of antigen in infected chickens and embryos.

The double immunodiffusion technique was applied to avian encephalomyelitis virus (AEV). Agar gel medium containing such a high concentration of NaCl as 15% was more preferable for highly diluted quantities of reactants than any other NaCl-containing medium. A single precipitin line appeared on the 1st to 7th days of diffusion at room temperature. The specificity of reaction between AEV antigen and homologous immune chicken serum has been demonstrated by no cross reaction between heterologous viruses and specific absorption by homologous virus. The antigen was produced in the brain, viscera, eyeball, whole body and yolk sac of chick embryos inoculated via yolk sac, as well as in the thigh muscles of chicks subcutaneously inoculated at 2 days of age. Antigenicity was detectable in 50% emulsion of these organs with a virus titer more than 10(5.0) per 0.1 g of tissue weight.     

Isolation and characterization of two antigens of Corynebacterium hofmannii

Banach, T. M. (University of Manitoba, Winnipeg, Canada), and R. Z. Hawirko. Isolation and characterization of two antigens of Corynebacterium hofmannii. J. Bacteriol. 92:1304-1310. 1966.-A serologically active substance, extracted from sonically treated cells of Corynebacterium hofmannii with hot HCl, produced two precipitin lines by immunodiffusion tests with a hyperimmune homologous serum. Extracts of other species failed to precipitate with the hofmannii antiserum. The active fraction was eluted from a diethylaminoethyl cellulose column in the third adsorption peak at a linear concentration of 0.5 m KCl, and produced two precipitin lines which corresponded in identity to those formed by the acid extract. Separation of the antigens was achieved by rechromatography on a Sephadex G-200 column; the major antigen was designated A; the minor, B. The homogeneity and purity of each antigen was established by immunoelectrophoresis and, in addition, that of antigen A by disc electrophoresis. Biochemical analyses showed that both antigens were composed of a major protein component with polysaccharide and nucleic acid present in an approximate ratio of 17:3:1, respectively. Glutamic acid, aspartic acid, alanine, glycine, valine, and leucine were the main amino acids present. Antigen A contained 17% less protein and 3.5% less carbohydrate than antigen B. The principal sugars of antigen A were identified as arabinose and glucose. The molecular weight, estimated by gradient centrifugation, was 16,500 for antigen A and 21,000 for antigen B.     

Immunodiffusion studies of the tryptic fragments of bovine nasal-cartilage proteoglycan monomer of high buoyant density

Tryptic fragments of bovine nasal-cartilage proteoglycan, fractionated by dissociative density-gradient ultracentrifugation, were made to react by immunodiffusion against antiserum to a hyaluronidase-digest subfraction of cartilage proteoglycan monomer. This reaction produced two families of partly superimposed precipitin lines. One family was restricted to gradient fractions of medium or low buoyant density and included the immunoprecipitation reaction attributed to the hyaluronic acid-binding region of the cartilage proteoglycan monomer. The second family of precipitin lines was present alone in gradient fractions of high buoyant density. Immunodiffusion studies with antisera to relatively homogeneous keratan sulphate-rich and chondroitin sulphate-bearing fragment subfractions isolated from the gradient fraction of highest density indicated that both subfractions contained the antigenic determinants responsible for the second family of precipitin lines. Additional immunodiffusion studies, with the use of multispecific antisera to chondroitinase ABC digest and hyaluronidase digest of proteoglycan monomer, confirmed that the two subfractions shared antigenic determinants, and, in addition, indicated that these determinants were on one molecular species in the keratan sulphate-rich fragment subfraction and divided among at least three in the chondroitin sulphate-bearing fragment subfraction. Although an unprecedentedly large number of cartilage proteoglycan antigens could be recognized with the antisera employed in this cartilage proteoglycan antigens could be recognized with the antisera employed in this study, it was not possible to identify antigenic determinants unambiguously specific for the three structurally and functionally distinct regions of the cartilage proteoglycan monomer.     

A species-non-specific liver plasma-membrane antigen and its involvement in chronic active hepatitis.

Rabbit liver plasma membranes were isolated and purified by using an aqueous two-phase polymer system. Examination of these preparations with respect to electron-microscopical appearance, distribution of marker enzymes and gross biochemical composition revealed them to be free from contamination by intracellular components. Sera from ten patients with chronic active hepatitis, four with and six without hepatitis B viral markers (HBsAg) in their sera, produced a single precipitin line on immunodiffusion against a detergent extract of the isolated plasma membranes. Sera from HBsAg-positive and HBsAg-negative patients reacted against the same antigen. This antigen was enriched in the plasma membrane preparations compared with whole-liver homogenates and was identical with a species-non-specific antigen in a macromolecular fraction of normal human liver, which has been previously described as liver-specific lipoprotein.     

THE INFLUENCE OF THE MOLECULAR WEIGHT ANTIGEN ON THE PROPORTION OF ANTIBODY TO ANTIGEN IN PRECIPITATES

The assumption that, at the equivalence point in specific precipitin reactions, the antigen molecule is completely covered with a single layer of antibody-globulin molecules has been shown to account fairly well for the antibody-antigen ratios of some representative native single proteins, and the pneumococcus S III hapten.     

Human granulocyte-specific nuclear antigen(s). II. Detection of antigens in human proliferative syndromes

Antisera raised to dehistonized chromatin from isolated normal human granulocytes revealed the presence of chromatin-associated antigens specific for the human neutrophils that appear during late stages of myeloid cellular differentiation. Immunological specificity was demonstrated by C fixation, immunodiffusion, and immunocytochemical reactions. Chromatin prepared from both normal granulocytes and specimens of myeloid leukemia showed immunologic reactivity. Although the normal antigens were detected in a specimen of CML, the position of immunodiffusion precipitin lines was different from that obtained with normal granulocyte chromatin. In addition, chromatin prepared from the myeloid leukemic cell line HL-60 expressed only one of the three precipitin bands normally found in immunodiffusion. The immunocytochemical staining reaction was confined to the nucleus of mature neutrophils in normal peripheral blood smears. Greater than 90% of cells in peripheral blood specimens of CML showed positive immunocytochemical nuclear staining. In other types of leukaemia, the normal mature granulocyte reacted with antiserum, but the nonmyeloid leukemic cells in these specimens did not. The specificity of immunologic reactions described here suggests the usefulness of nuclear antigens as cell markers.     

Nonspecific Precipitation of Serum Proteins by Sodium Lauryl Sulfate in Agar Diffusion and Immunoelectrophoresis

The anionic detergent sodium lauryl sulfate (SLS), in a final concentration of 0.1% and greater, reacted with whole serum in agar diffusion and immunoelectrophoresis to form artifactual precipitin lines. These lines occurred when either Ionagar or agarose was used as the supporting gel and were not affected by the presence of urea and 2-mercaptoethanol. Analytic chemical tests confirmed that the precipitating agent is SLS, and staining techniques showed that the detergent precipitates both protein and lipoprotein components of whole serum. Multiple artifactual precipitin lines occurred with a wide variety of animal sera, and a single line formed with human 7S immunoglobulin. Hence, in agar diffusion studies in which SLS is present in the test system, these artifactual lines may be easily misinterpreted as true antigen-antibody precipitin reactions.     

Myosin chains of myocardial tissue. I. Purification and immunological properties of myosin heavy chains

An antiserum specific to dog myocardial myosin has been developed against highly purified myosin heavy chains. The antiserum is specific for the heavy chains of myosin, giving a single precipitin line in an immunodiffusion assay for either the heavy chains of myosin or native myosin, and does not react with any other myocardial proteins. In such assays myosin acts as a single, uniform antigen. Using this antiserum, a radioimmunoassay has been developed to quantitate myosin in a homogenate of myocardial tissue containing free myosin dissociated from other cellular components.     

Further observations on the specificity of antigen 5 of Echinococcus granulosus.

The presence of IgE antibodies to antigen 5 of Echinococcus granulosus was detected by means of radioimmunoelectrophoresis in the sera of two of six patients infected with E. multilocularis. Sera from three of these patients gave a precipitin band in gel diffusion tests identical to that produced by a monospecific rabbit anti-E. granulosus antigen 5 serum, when tested against whole hydatid fluid. Sera from 19 individuals infected with Fasciola hepatica, 20 with Schistosoma mansoni, and 5 with with Taenia saginata showed no detectable antibodies against antigen 5 of E. granulosus, The monospecific rabbit anti-E. granulosus antigen 5 serum did not react in immunodiffusion with homologous antigen when absorbed with either 4 mg/ml of whole hydatid fluid or with 200 mg/ml of a soluble E. multilocularis extract. Absorption of the monospecific antiserum with crude antigens of either F. hepatica, Onchocerca volvulus, S. mansoni, or T. saginata did not abolish the reaction with antigen 5. It appears, therefore, that antigen 5 can no longer be considered specific for E. granulosus, but is also present in E. multilocularis. In the light of this observation, some reevaluation of immunodiagnostic tests in hydatid disease will be necessary.     

Effects of ionic and nonionic detergents on antigen-antibody reactions.

Using highly sensitive and quantitative radioimmunoassay procedures we have measured the effects of different concentrations of three commonly used detergents, SDS, DOC, and Triton X-100, on antibody-antigen reactions. Triton X-100, had a relatively mild effect on primary antigen-antibody bindings, the precipitin reaction, and a double antibody RIA as evidenced by only an 8 to 10% inhibition of binding or precipitation. These results were not detergent concentration dependent, as Triton concentrations ranging from 5 to 0.1% had virtually no differential effects. Sodium deoxycholate (DOC) had a more profound effect on both primary antigen-antibody binding and the precipitin reaction than did Triton X-100, and its effects, unlike those of Triton X-100, were concentration dependent. There was a direct relationship between concentration of DOC and degree of inhibition of both primary binding and immune precepitation especially in antigen excess. Sodium dodecylsulfate (SDS), at concentrations 10- to 100-fold less than either Triton X-100 or DOC, had profound inhibitory effects on primary antigen-antibody binding, the precipitin reaction, and a double antibody radioimmunoassay. Generally, at concentrations greater that 0.01% SDS, almost all immunochemical reactivity is destroyed.     

The application of immunodiffusion test to the estimation of hydrolysis of mycobacteria proteins by proteolytic enzymes of mice peritoneal cells

The possibility of applying the immunodiffusion (ID) test in agarose gel to detect the degradation of the somatic proteins of mycobacteria BCG-Poland by proteolytic enzymes of non activated and activated mice peritoneal cells was investigated. Degradation of those proteins was determined by disappearance of some precipitin lines resulting from the hydrolysis. The immunodiffusion test was found to be useful to show the degradation of protein of mycobacteria by cathepsins of mice peritoneal cells. Disappearance of 4 precipitin lines in ID test occurred after 3 h incubation of mycobacteria proteins with extracts of non activated mice peritoneal cells at pH 3, which gives evidence of degradation of some mycobacteria antigens. The extention of the time of incubation up to 24 h gave the same results. At the incubation at pH 4.5 no disappearance of precipitin lines was observed. It was found that cathepsins no specifically activated mice peritoneal cells degradate mycobacteria proteins at pH 3 and pH 4.5. Degradation occurred more rapidly at pH 3 achieving maximal effect after 6 h. The degree of degradation at pH 4.5 increased during the whole period of examination, i.e. up to 24 h of incubation time after which a disappearance of 5 precipitin lines was found. No degradation was found after incubation at pH 6 and pH 8.     

Immunochemical analysis of a Smith-like antigen isolated from two human strains of Staphylococcus aureus.

A surface antigen consisting of aminoglucuronic acid and N-acetyl-L-alanine was isolated from the culture filtrates of two human strains of Staphylococcus aureus. Double diffusion analysis in agar suggested that the antigen is immunologically similar to the alanyl-aminoglucuronic acid capsule of the Smith strain of S. aureus. Quantitative precipitin inhibition studies indicated that N-acetyl-L-alanine is the immunodominant determinant of the acidic antigen. In addition, conjugates consisting of N-acetyl-L-alanine coupled to bovine serum albumin gave a significant precipitin reaction with anti-staphylococcal serum which is rich in alanyl-aminoglucuronic acid polymer antibodies. Antibodies with N-acetyl-L-alanine specificity were isolated from N-acetyl-L-alanine-Sepharose immunoabsorbent columns. Double diffusion analysis in agar indicated that the eluted antibodies were serologically reactive and belonged to the IgG class of immunoglobulins.     

Immunological cross-reactivity between chicken slow skeletal and ventricular muscle myosin

Antibodies elicited in rabbits against chicken slow skeletal anterior latissimus dorsi and ventricular myosin were analyzed by double immunodiffusion for their ability to react with homologous and heterologous antigen at different stages of immunization (1--12 months). Each anti myosin antiserum formed a single, strong precipitin line with its immunogen after short time of immunization. This reaction was specific for myosin heavy chains as determined by GEDELISA (gel electrophoresis derived enzyme lined immunosorbent assay) test. In rabbits injected with ventricular myosin after long time of immunization a second, fainter precipitin line has generally been observed. The antigenic determinants responsible for this precipitin line have been localized on the light myosin subunits. By comparing the two types of anti myosin antisera with heterologous antigen we have obtained evidence for partial immunological cross-reactivity between slow skeletal and ventricular muscle myosins. In particular, all anti ventricular myosin antisera displayed a marked immunological reactivity with anterior latissimus dorsi myosin whereas most of anti anterior latissimus dorsi myosin antisera showed absence of reciprocity. By means of immunofluorescence and immunoabsorption techniques both common and unique slow skeletal and ventricular antigenic determinants have been demonstrated.     

Immunodiffusion Analysis of Yaba Poxvirus Structural and Associated Antigens

Yaba poxvirus virions were extracted and purified from Rhesus monkey tumors. A saline-soluble virion fraction (Y-xp), obtained by mechanical fractionation of purified virions with an X-press, contained seven components in acrylamide gel electrophoresis; five of these components were reactive in immunodiffusion with whole virion and Y-xp antisera produced in rabbits and monkeys. The saline-insoluble residue remaining after X-press treatment was hydrolyzed with sodium dodecyl sulfate, urea, and 2-mercaptoethanol (SUM). This fraction, Y-sum, contained five components, four of which were demonstrable by immunodiffusion. There was no evidence of antigenic relationships between Y-xp and Y-sum antigens in immunodiffusion. In acrylamide gel electrophoresis, one Y-xp and one Y-sum component had similar mobilities. Y-xp but not Y-sum antisera contained viral-neutralizing antibodies. Virus-free saline extracts of Yaba tumor prepared with Genetron (YS) were essentially devoid of virion structural antigens. They failed to induce precipitating antibodies for virion antigens, were nonreactive in immunodiffusion with virion antisera, and gave low complement-fixation titers with virion antisera. Yaba virion antigens were recovered from the Genetron tumor sediment by SUM and alkaline hydrolysis. Antisera prepared to YS extracts gave a maximum of 17 precipitin lines in immunodiffusion with YS extracts; none was identified as a virion structural antigen. Saline extracts of tumor prepared without Genetron contained immunogenic amounts of 5 virion antigens and 12 to 14 associated antigens. Animals immunized with infected cell culture extracts (virus-free) formed antibodies to six to seven virion antigens. The implications of using extracts of Yaba poxvirus-infected tissues in complement-fixation tests to measure virion antibodies were discussed.     

Effects of polyethylene glycol and dextrans on immunoprecipitations: a two-cross immunodiffusion study

Precipitating titers and immunochemical titers obtained in a wide range of antigen-to-antibody concentration ratios by the two-cross immunodiffusion technique are compared with the corresponding laser light scatter precipitin curves. The two-cross immunodiffusion technique has also been applied to investigate whether polyethylene glycol of molecular mass 6000 and dextrans of molecular masses from 10,000 to 2,000,000 enhance the immunoprecipitation processes of the system human serum IgG-rabbit immune serum at pH 5.5 and 8.1 at 20 degrees C. It was found that the significant increase of precipitating titers of both precipitating components in the presence of polyethylene glycol is a consequence of a strong decrease of solubility of the primary antigen-antibody complex. The decrease of solubility does not affect the immunochemical titer of the immune serum, indicating stoichiometrical invariance of the precipitate at the equivalence. The apparent strong decrease of diffusion coefficients of both antigen and antibody in 20- and 40-g/liter polyethylene glycol solution is attributed to increase of viscosity of the solutions and to a partial self-association of protein molecules due to steric exclusion. In 40-g/liter polyethylene glycol solutions at pH 5.5 every fourth molecular entity of antigen and every third molecular entity of antibody are present in the form of a two-molecular self-associate, whereas in 20-g/liter polyethylene glycol solutions only 1% of antigen molecules and 8% of antibody molecules are associated. With the increase of pH to 8.1 the self-association of protein molecules is strongly further enhanced. Dextrans in 20-g/liter solutions, without regard to their relative molecular masses, do not influence precipitating titers and solubility of the antigen-antibody system at equivalence and do not enhance self-association of protein molecules. The strong decrease of diffusion coefficients of immunoglobulin G antigen and antibodies in dextran solutions is solely attributed to the increase of viscosity of the dextran solutions; hence there was no evidence of interaction of dextrans with serum IgG proteins.