Agar Gel Double Diffusion Tests Research Articles

Purification, composition, and serological characterization of histoplasmin-H and M antigens.

To obtain purified H and M antigens suitable as primary standards in the serological diagnosis of histoplasmosis by agar gel double-diffusion tests, H and M reactive components of histoplasmin were fractionated by column chromatography by using Sephadex G-100, Sephadex G-200, and diethylaminoethyl cellulose. Six fractions from diethylaminoethyl cellulose were reactive in agar gel double-diffusion, complement fixation, and capillary precipitin tests. When examined by electrophoresis on acrylamide gel, one M antigen (fraction 2, molecular weight greater than 200,000) and two H antigens (fractions 5 and 6, molecular weight greater than 200,000) each gave essentially a single protein band. In agar gel double-diffusion and complement fixation tests with sera from patients with proven cases of histoplasmosis, blastomycosis, or coccidioidomycosis, these two fractions of H antigen and the one of M antigen reacted only with sera from proven or suspect cases of histoplasmosis and showed reactivity with those sera known to contain only the anti-H or anti-M antibody, respectively. Fraction 2 (M antigen) and fractions 5 and 6 (H antigens) had carbohydrate-to-protein ratios of 1.60, 0.77 and 0.78, respectively. Both antigens contained galactose, glucose, mannose, and hexosamine, with mannose being the predominant sugar. Fraction 2 was characterized by a high proline and glucose content, whereas fractions 5 and 6 contained higher concentrations of galactose, mannose, glycine, and alanine. Each of these products appeared to separate into two active fractions, one of a molecular weight greater than 200,000 and one of a molecular weight less than 35,000. The M antigen component of fraction 2 was still characterized by a high proline content, whereas the H antigen components of fractions 5 and 6 had a high content of glutamic acid, serine, glycine, and proline.

Untersuchungen zur Therapie virusinfizierten Hopfens (Humulus lupulus L) durch Wärmebehandlung von Fechsern und Triebspitzen

Summary Hot water treatment of dormant hop cuttings at temperatures ranging from 35°C to 65°C in intervals of 5 min. to 4 h. proved to be inadequate for the inactivation of hop viruses. In comparable research works different types of heat chambers have been tested for use in hot air treatments of hop plants. Relatively most satisfying was a stationary chamber in which plants after a period of heat adaptation grew under artificial light conditions in temperatures between 37°C to 40 °C, in relative air humidities between 60 to 80 per cent and in soil temperature of 28°C sufficiently survived a treatment of at least 25 days. From more than 1000 about 2 to 3 cm. long shoot tips of 25 to 118 days heat treated hop plants belonging to 15 varieties, 3 clones and 2 breeding lines, 7401 young hop plants could be developed. RMV (rose mosaic virus), NRSV ( Prunus necrotic ringspot virus), AMV ( arabis mosaic virus), and hop mosaic virus in an extend of 34.0, 27.3, 16.7, and 24.3 per cent have been eliminated. However, elimination of the latent rod shaped hop virus (656 nm) only in 2 varieties extreme rarely succeeded even if the longest heat periods have been used. In order to increase the curing rate the combination of hot air treatment and tip meristem culture techniques is recommended. Healthy or virus-poor hop material has been selected on the basis of 2 to 8 times repeated indexings on test plants, partly repeated agar gel double diffusion tests, using antiseras against RMV, NRSV, and AMV and by the reaction of indicators and electron-microscopical tests in course of several years.

Study of Australia-SH antigen in diluted uman serum by electron microscopy.

The Australia‐SH antigen has been studied by electron microscopy of diluted sera which had not been subject to any purification procedure. Australia‐SH antigen associated particles could be demonstrated in serum dilutions up to 1/10,000, with or without specific antiserum added. Different types of aggregates, depending on the relative amounts of antigen and antibody added to a sample, were observed in checkerboard titration experiments. Australia‐SH antigen was most readily recognized in samples with about equal concentrations of antiserum and antigen‐containing serum, or in samples with moderate antigen excess. For optimal results, serum dilutions of 1/100 and 1/1,000 have been found most suitable. Particles indistinguishable from those of the Australia‐SH antigen were observed in serum of an apparently healthy student selected as a negative control. This serum sample showed negative reaction patterns with antiserum to the Australia‐SH antigen both in agar gel double diffusion tests and in complement fixation tests.

Evaluation of Agar-Gel Double Diffusion for the Diagnosis of Adenovirus Infection

Agar-gel double diffusion was compared to the Laboratory Branch complement fixation test. The efficacy of the agar-gel double diffusion test for the semiquantitation of group-specific adenovirus antibody and its usefulness as a serodiagnostic aid for the evaluation of adenovirus infection was determined.

Spherules in the serology of Coccidioides immitis. II. Complement fixation tests with human sera.

Soluble and particulate spherule antigens fixed complement in tests with anti—C. immitis sera. However, the soluble antigen proved more active than the particulate one. Cross reactions were noted between the spherule antigens and anti—H. capsulatum and anti—B. dermatitidis sera. Following enzymatic treatment of the particulate preparation a soluble antigen was obtained which formed two bands with anti—C. immitis serum and one band with anti-H. capsulatum serum in an agar gel double diffusion test.

Evaluation of Agar-Gel Double Diffusion for the Diagnosis of Adenovirus Infection

Agar-gel double diffusion was compared to the Laboratory Branch complement fixation test. The efficacy of the agar-gel double diffusion test for the semiquantitation of group-specific adenovirus antibody and its usefulness as a serodiagnostic aid for the evaluation of adenovirus infection was determined.

Rabbit antiserum to rat deciduoma.

Antiserum to rat deciduoma was produced in the rabbit. The antiserum had an inhibitory effect on deciduoma formation when injected into the uterine lumen of pseudopregnant rats immediately after traumatization of the uterus. Normal rabbit serum had no inhibitory effect. The antiserum after absorption with the extract of immature rat uterus indicated the presence of a component specifically reactive to the deciduoma extract by an agar gel double diffusion test. This was shown by a precipitin line between deciduoma extract and the antiserum from which nonspecific antibodies to rat uterine components had been removed. The antiserum after absorption with the extract of immature rat uterus inhibited deciduoma formation when given locally into the uterine lumen. Application of immunological principles

Synovitis induced by killed Erysipelothrix rhusiopathiae: Extended reaction in passively immunized lambs

Pregnant ewes were inoculated twice with a swine erysipelas vaccine. Their lambs, together with an equal number of lambs born to unvaccinated ewes, were inoculated intra-articularly with 0·25 ml. of a heat-killed suspension of E. rhusiopathiae when 24 hours old. Only sera from lambs born to vaccinated ewes showed precipitin lines against the killed suspension of E. rhusiopathiae in agar gel double diffusion tests. The joint reaction in each group of lambs was studied histologically at 1, 2 and 7 days after inoculation. Inoculated joints in non-immunized lambs developed fibrinous synovitis, which was moderate and acute. In contrast, inoculated joints in the passively immunized lambs developed severe fibrinous synovitis, which by 7 days after inoculation had become proliferative and showed similarities to the early stages of E. rhusiopathiae arthritis in pigs, lambs and rats. It is suggested that passively-acquired specific antibody may be a significant factor determining the susceptibility of the newborn animal to infectious arthritis, and that specific antibody may be associated with the pathogenesis of proliferative synovitis in chronic E. rhusiopathiae arthritis.

Role of Biological Mimicry in the Pathogenesis of Rat Arthritis Induced by Mycoplasma arthritidis

Complement fixation (CF), immunofluorescence, and agar gel double-diffusion tests were used to demonstrate an antigenic relationship between rat tissues and Mycoplasma arthritidis. Rabbit antisera against six strains of M. arthritidis exhibited positive reactions in the CF test with an ethyl alcohol-saline extract of rat muscle, whereas only 6 of 18 antisera against other Mycoplasma species were positive. With the use of gel diffusion techniques, absorption of various M. arthritidis antigens with antiserum against rat muscle removed at least one precipitin band when the absorbed mycoplasma antigens were reacted against homologous antisera. Rabbit antiserum against M. arthritidis was conjugated with fluorescein isothiocyanate and reacted against frozen sections of muscle tissues of various animals. As controls, unlabeled normal rabbit serum and rabbit anti-M. arthritidis serum were included to determine the specificity of the reaction. Rat, hamster, and mouse skeletal muscle exhibited specific fluorescence, whereas chicken, beef, frog, and turtle muscles exhibited no specific fluorescence. Mice injected at birth with rat lymphocytes were found to be more susceptible to subsequent infection by M. arthritidis than were normal mice or mice injected at birth with mouse lymphocytes. These results indicate the occurrence of a heterogenetic antigen(s) common to M. arthritidis and rat tissues. Preliminary evidence suggests that this heterogenetic antigen(s) may enable the mycoplasmas to become established in their host.

Polypeptides of avian RNA tumor viruses: II. Serological characterization

Extraction of AMV with phenol and SDS and electrophoresis of the proteins on acrylamide gels resulted in essentially no loss of gs-CF-antigenicity, and it was possible to test the antigenic properties of the major viral polypeptides. The results indicated that the four major components were detectable in complement fixation with gs-rabbit or gs-hamster sera. The titers of various sera employed against the individual components suggested that these materials were immunologically distinct and the nonidentity of these antigens was further demonstrated by agar gel double diffusion tests. The results of these studies indicated that components 1 and 2 are immunogenically weak, while 3 and 4 are much stronger in this respect. Component 4 was shown to be identical to a material isolated by Tiselius electrophoresis and previously shown to have all the properties of gs antigen ( Bauer and Schäfer, 1965). Whereas the gs-CF-antigenicity was recovered for the most part, a large proportion of the type-specific antigenicity was lost. Nevertheless, enough activity remained to enable testing of various fractions obtained after electrophoresis. The major portion of the Ve antigen activity was found in the region of the main glycoprotein component. However, a direct correlation between this activity and the polysaccharide staining material has thus far not been obtained.

Strain-specific antigen of the avian leukosis sarcoma virus group: I. Isolation and immunological characterization

From each of three strains of avian leukosis sarcoma viruses—avian myeloblastosis virus BAI strain A (AMV), myeloblastosis associated virus strain B (MAV-B), and Schmidt-Ruppin Rous sarcoma virus 1 (SR-RSV-1)—an antigenic component was extracted by treatment with Tween 20 detergent. The isolated antigen is believed to represent strain-specific antigen of the viral envelope (V e antigen) because of various properties: (1) In the agar gel double diffusion test, the antigen reacts with neutralizing chicken antisera to the original virus, but not with normal chicken sera or with chicken antisera to viruses unrelated in their envelope antigenicity to the agent from which the antigen was isolated. (2) The antigen absorbs neutralizing antibody of homologous chicken antiserum, but not of antisera prepared against serologically unrelated virus. (3) A rabbit immune serum against antigen isolated from MAV-B neutralizes MAV-B and other subgroup B viruses, but not subgroup A virus. (4) The antigen obtained from MAV-B induces early interference to subgroup B viruses, but not to subgroup A viruses. The antigenicity of the V e antigen is completely lost after incubation with 0.1 mg of trypsin or 1 mg of α-chymotrypsin per milliliter, and is partially inactivated by treatment with 0.5 mg of papain per milliliter. Some phospholipid- and polysaccharide-digesting enzymes and two other detergents do not cause decrease of the V e antigenicity.

Correlation of Clinical, Parasitological, and Serological Data of Individuals Infected with Entamoeba Histolytica

The Entamoeba histolytica antigens prepared from axenic cultures of the organism gave highly sensitive and good specific serological results when used in the indirect hemagglutination technique on a series of individuals, some of whom had gastrointestinal complaints and some of whom were infected with E. histolytica. The indirect hemagglutination test using these pure E. histolytica antigens was more sensitive and specific than agar gel double diffusion test.

Some Properties of Chrysanthemum Mild Mottle Virus, and Comparision of This Virus with Cucumber Mosaic Virus

A virus which is widespread in chrysanthemum in Japan has been identified by Inoue et al. with tomato aspermy virus. In the present study, this virus was found to be serologically unrelated to cucumber mosaic virus (CMV), closely resembling Lawson's chrysanthemum virus. It is considered adequate to separate this virus from tomato aspermy virus of Blencowe type, as suggested by Lawson, and a tentative name chrysanthemum mild mottle virus (CMMV) is proposed.The virus was purified from inoculated leaves of tobacco (Ky-57) by the following procedure: young tobacco plants were inoculated with leaf extract of systemically infected tobacco, ground in 0.1M phosphate buffer (P.B.) containing 0.02M Na2SO3. The inoculated leaves were harvested 3-5 days after inoculation. The leaves were ground in 1 vol. each of 0.15M Na2HPO4 containing 0.01M Na-EDTA and 0.1% thioglycollic acid (pH 7.6), and chloroform-butanol mixture (9:1 vol.), subjected to two cycles of differential centrifugation (9, 000×g15min., and 80, 000×g 150min. or 90, 000×g90min.), and then to density gradient centrifugation in 10-40% sucrose at 24, 000rpm for 180-210min., at 4-6°C. 0.05M P.B. containing 0.005M Na-EDTA was used to resuspend the pellets. The yield of the final preparation was about 30-40mg per 100g of inoculated leaves. For comparision, CMV-Y (yellow strain) was purified by a procedure modified from Scott and Takanami et al. 0.5-1 vol. of 0.005M borate buffer containing 0.005M Na-EDTA pH 8.6-8.9 was used to prepare leaf extract, instead of dialysis (Scott) or treatment with Sephadex (Takanami et al.). Borate buffer containing Na-EDTA was used as the resuspending medium. The yield was about 60mg per 100g of inoculated leaves.The absorbancy of CMMV preparation at 260mμ was 5.6cm2/mg. The UV absorption spectrum between 230 and 300mμ was typical of a nucleoprotein (max. at 260-261, min. at 241-242mμ), and showed 260/280=1.70-1.78 and max./min.=1.46-1.50. CMMV resembles CMV-Y in the UV absorption spectrum, but the ratio of 260/280 showed a slightly larger value in CMMV than in CMV-Y. CMMV preparation yielded about 100 lesions per cowpea leaf at 4.5μg/ml, and about 200 lesions per leaf of Chenopodium amaranticolor at 1.8μg/ml.The purified virus was treated by phenol saturated with 0.1M P.B. containing 0.001M Na-EDTA (pH 7.2) and 5mg/ml bentonite. Sodium-acetate and ethanol were added to the aqueous phase, and the pellet was resuspended in 0.01M P.B. pH 7.2, and ethanol was removed under reduced pressure. Infectivity of the preparation was tested before and after the addition of 0.01 or 0.001μg/ml of RNase-A. The non-RNase preparation yielded more than 250 lesions per cowpea leaf, while the RNase-treated preparation completely lost infectivity.CMMV preparation was examined under electronmicroscope, after being fixed in 2% neutralized-formalin and stained with 1% phosphotungstic acid, or shadowed with Pt-Pd. The virus particles were found to be spherical and about 25-30mμ in diameter (Fig. 2). The sedimentation coefficient of the virus was determined by ultracentrifugation using schlieren optical system, at different virus concentrations (1.2-3.6mg/ml). The value of the sedimentation coefficient of the virus extrapolated to zero concentration was S20, w=97. The mixture of CMMV and CMV-Y showed a single symmetrical schlieren peak (Fig. 3).CMMV preparation (25μg/ml virus dissolved in 0.05M P.B. pH 7.8), completely lost infectivity by 10min. treatment at 55°C, while there was a considerable loss of infectivity by treatment at 50°C (Table 1).Serological comparisons between CMMV and CMV-Y were made in agar gel doublediffusion tests at 4-8°C (1% agar, 0.85% NaCl, 0.0025M Na-EDTA, 0.01% sodium azide, and 0.02M P.B., pH 7.2-7.4). CMMV (0.5-5mg/ml) and CMV-Y

Demonstration of peritrichous flagella of Tyzzer's disease organism.

Japanese Journal of MicrobiologyVolume 12, Issue 3 p. 361-363 NoteFree Access Demonstration of Peritrichous Flagella of Tyzzer's Disease Organism Kôsaku Fujiwara, Kôsaku Fujiwara Department of Animal Pathology, Institute of Medical Science, University of Tokyo, Minato-ku, Tokyo Department of Pathology, National Institute of Health, Shinagawa-ku, TokyoSearch for more papers by this authorHiroaki Kurashina, Hiroaki Kurashina Department of Animal Pathology, Institute of Medical Science, University of Tokyo, Minato-ku, Tokyo Department of Pathology, National Institute of Health, Shinagawa-ku, TokyoSearch for more papers by this authorNaochika Matsunuma, Naochika Matsunuma Department of Animal Pathology, Institute of Medical Science, University of Tokyo, Minato-ku, Tokyo Department of Pathology, National Institute of Health, Shinagawa-ku, TokyoSearch for more papers by this authorReiji Takahashi, Reiji Takahashi Department of Animal Pathology, Institute of Medical Science, University of Tokyo, Minato-ku, Tokyo Department of Pathology, National Institute of Health, Shinagawa-ku, TokyoSearch for more papers by this author Kôsaku Fujiwara, Kôsaku Fujiwara Department of Animal Pathology, Institute of Medical Science, University of Tokyo, Minato-ku, Tokyo Department of Pathology, National Institute of Health, Shinagawa-ku, TokyoSearch for more papers by this authorHiroaki Kurashina, Hiroaki Kurashina Department of Animal Pathology, Institute of Medical Science, University of Tokyo, Minato-ku, Tokyo Department of Pathology, National Institute of Health, Shinagawa-ku, TokyoSearch for more papers by this authorNaochika Matsunuma, Naochika Matsunuma Department of Animal Pathology, Institute of Medical Science, University of Tokyo, Minato-ku, Tokyo Department of Pathology, National Institute of Health, Shinagawa-ku, TokyoSearch for more papers by this authorReiji Takahashi, Reiji Takahashi Department of Animal Pathology, Institute of Medical Science, University of Tokyo, Minato-ku, Tokyo Department of Pathology, National Institute of Health, Shinagawa-ku, TokyoSearch for more papers by this author First published: March 1968 https://doi.org/10.1111/j.1348-0421.1968.tb00407.xCitations: 10AboutPDF ToolsRequest permissionExport citationAdd to favoritesTrack citation ShareShare Give accessShare full text accessShare full-text accessPlease review our Terms and Conditions of Use and check box below to share full-text version of article.I have read and accept the Wiley Online Library Terms and Conditions of UseShareable LinkUse the link below to share a full-text version of this article with your friends and colleagues. Learn more.Copy URL No abstract is available for this article. REFERENCES 1Craigie, J. 1966. ‘Bacillus piliformis’ (Tyzzer) and Tyzzer's disease of the laboratory mouse. I. Propagation of the organism in embryonated eggs. Proc. Roy. Soc. B165: 35– 60. 2Fujiwara, K. 1967. Complement fixation reaction and agar gel double diffusion test in Tyzzer's disease of mice. Japan. J. Microbiol. 11: 103– 117. 3Fujiwara, K., Fukuda, S., Takagaki, Y., and Tajima, Y. 1963. Tyzzer's disease of mice. Electron microscopy of the liver lesions. Japan. J. Exp. Med. 33: 203– 212. 4Fujiwara, K., Kurashina, H., Maejima, K., Tajima, Y., Takagaki, Y., and Naiki, M. 1965. Actively induced immune resistance to the experimental Tyzzer's disease of mice. Japan. J. Exp. Med. 35: 259– 275. 5Fujiwara, K., Takagaki, Y., Maejima, K., Kato, K., Naiki, M., and Tajima, Y. 1963. Tyzzer's disease of mice. Pathologic studies on experimentally infected animals. Japna. J. Exp. Med. 33: 183– 202. 6Fujiwara, K., Takagaki, Y., Naiki, M., Maejima, K., and Tajima, Y. 1964. Tyzzer's disease in mice. Effects of corticosteroids on the formation of liver lesions and the level of blood transaminases in experimentally infected animals. Japan. J. Exp. Med. 34: 59– 75. 7Tyzzer, E. E. 1917. A fatal disease of the Japanese waltzing mouse caused by a spore-bearing bacillus (Bacillus piliformis, N. sp.). J. Med. Res. 38: 307– 338. Citing Literature Volume12, Issue3March 1968Pages 361-363 ReferencesRelatedInformation

Characterization of Mycoplasma pulmonis variants isolated from rabbits. I. Identification and properties of isolates.

Mycoplasma showing at least two colony types were isolated from the nares and oropharynx of New Zealand white rabbits. Two strains were purified by single-colony passages and characterized. Morphology by phase-contrast and electron microscopy was typical of Mycoplasmataceae. Both grew anaerobically as well as aerobically, caused hemolysis of guinea pig, sheep, and horse red blood cells, and fermented glucose. These characteristics are shared by members of the species M. pulmonis, commonly isolated from the respiratory tracts of laboratory rats and mice. By use of the growth-inhibition test and agar-gel double-diffusion tests, the two strains were found to be serologically related to each other and to M. pulmonis ATCC 14267 but not to other representative Mycoplasma species from man and animals.

Complement fixation reaction and agar gel double diffusion test in Tyzzer's disease of mice.

Complement fixation reaction and agar gel double diffusion test were made using as antigens, water or phosphate buffer saline extracts of liver from mice severely infected with Tyzzer's disease. The supernatant of the extract heated at 56 C for 30 minutes was highly reactive with immune mouse serum in the complement fixation test, while an intense precipitate band was observed only with unhealed extracts in agar gel double diffusion tests. Cross reactions were observed in both tests between liver extracts from mice infected with different strains of organisms originating from different sources and showing varied degrees of virulence. Such reactions were not shown between Tyzzerimmune serum and liver extracts from uninfected or murine hepatitis-infected mice, nor were they shown between the Tyzzer-antigen and normal or anti-murine hepatitis virus serum. The complement fixation test was used to test retired breeder mouse sera for disease contamination in mouse colonies. Positive reactions were observed in test sera collected from some breeding colonies that subsequently proved to be contaminated by the cortisone provocation test in young animals.

Antigenic similarity between isolates of Trametes versicolor (linnaeus ex fries) lloyd and other related species

An antiserum against Trametes versicolor isolate D4 has been developed. The use of agar-gel double-diffusion tests has revealed at least eight antigenic components in the fungus. Some antigens identical to those of D4 have been detected in other related fungi using both unabsorbed and absorbed antiserum. The overall serological data seem to agree with the conventional classification of these fungi and it is suggested that serological methods may be useful in identifying various Trametes species.

RELATIONSHIP OF THE ADENOVIRUS ERYTHROCYTE-RECEPTOR-MODIFYING FACTOR TO THE TYPE-SPECIFIC COMPLEMENT-FIXING ANTIGEN.

SummaryThe similarity in elution characteristics of the ERM factor and type-specific CF antigen following fractionation of adeno-virus type 1 and type 2 suspensions by chromatography with DEAE-cellulose; the formation of only a single precipitation line in agar-gel double diffusion tests with purified material containing both activities; and the type-specific serologic reaction of both antigens indicated that the type-specific CF antigen was the carrier of the ERM factor.

EVIDENCE THAT TOMATO ASPERMY AND CUCUMBER MOSAIC VIRUSES ARE SEROLOGICALLY UNRELATED.

Abstract A procedure for the partial purification of tomato aspermy virus was developed and antisera were produced. Antisera specific for tomato aspermy and cucumber mosaic viruses were reacted separately and in combination with the respective homologous and heterologous antigens in agar gel double-diffusion tests. Results from these tests indicate that the viruses are serologically unrelated.