Agar Gel Diffusion Test Research Articles

An epidemic of histoplasmosis on the Isthmus of Panama.

Forty-seven men on the Isthmus of Panama were exposed to histoplasmosis in an old bunker inhabited by bats. The resulting epidemic was studied with serial clinical, serological, and radiological examinations. Thirty-seven (78.7%) of the men showed serological evidence of infection and 26 (70.3%) had symptoms. Incubation periods ranged from 4 to 30 days. A general relationship between severity of illness and degree of exposure was noted. The agar gel diffusion test for precipitin antibodies was more sensitive than the complement-fixation test or slide test in detecting infection with Histoplasma capsulatum. Decontamination procedures and environmental studies are described.

Regulation of the Secretory Process of Granular Components from the Convoluted Tubular Cells of the Mouse Submandibular Gland

Autonomic regulation of the secretion of granule components in the convoluted tubular cells of male mouse submandibular glands was investigated with the use of an agar gel diffusion test using an antisera for male specific components. Whereas the injection of neither a parasympathomimetic agent (pilocarpine) nor a beta-adrenergic agent (isoproterenol) decreased the amount of the components in the glands, the injection of alpha-adrenergic agents (norepinephrine or phenylephrine) significantly decreased the amount of male specific components. Phenoxybenzamine, an alpha-blocker, completely inhibited these actions of norepinephrine and phenylephrine. These facts suggest that the alpha-adrenergic receptor participates in the secretion of male specific components present in the granules in the convoluted tubular cells of mouse submandibular glands.

Precipitating antibodies in cattle infested by Psoroptes ovis (Acarina: Psoroptidae).

Precipitating antibodies, as demonstrated by agar-gel diffusion tests, were found in the sera of 23 of 38 Psoroptes ovis -infested cattle when tested against a P. ovis extract and in the sera of 9 of 17 cattle when tested against a P. cuniculi extract. Sera from 5 of 54 P. ovis -uninfested cattle reacted to the P. ovis extract and 3 of 22 reacted to the P. cuniculi extract. Analysis of the P. ovis and the P. cuniculi extracts indicates that they share common antigens. The reaction between the sera of mite-infested cattle and the above antigens is a specific antigenantibody reaction. Extracts of Hypoderma bovis, Otobius megnini, Haematopinus eurysternus, Demodex bovis , cattle tissue, and cattle blood were made and tested with some of the above sera. Only the H. bovis extract gave a specific reaction with the serum of a grub-infested calf. Reactions with the O. megnini and blood extracts were considered nonspecific.

Purification and properties of M protein extracted from group A streptococci with pepsin: covalent structure of the amino terminal region of type 24 M antigen

M protein was extracted from type 24, group A streptococci with pepsin at pH 5.8 and was further purified by ammonium sulfate precipitation, ribonuclease digestion, ion-exchange chromatography, and isoelectric focusing. The purified pepsin extract of M (pep M) protein was shown to be free of nontype-specific immunoreactivity in (a) complement fixation tests with heterologous M antiserum, (b) skin tests in normal adult guinea pigs, and (c) passive hemagglutination tests for the presence of lipoteichoic acid sensitizing or antigenic activity. The pep M24 was highly immunogenic; two of three rabbits developed opsonic antibody titers of 1:256 and the third a titer of 1:32 6 wk after a single injection of 100-pg doses of pep M24 emulsified in complete Freund's adjuvant. The antisera lacked nontype-specific antibodies and produced single precipitin lines in agar gel diffusion tests against crude HC1 extracts of the homologous M protein. Thus, the type-specific antigenic determinant(s) of type 24 M protein appears to be separable from immunotoxic, cross-reactive antigens without loss of immunogenicity in rabbits. The mobility of pep M24 upon electrophoresis in 10 percent sodium dodecyl sulfate pelyacrylamide gel was consistent with an average mol wt of 33,500 daltons. Amino acid analysis demonstrated a predominance of alanine, followed by glutamic acid, lysine, leucine, and aspartic acid. Pep M24 contained an estimated six to seven methionine residues and approximately ten phenylalanine residues per molecule. No other aromatic amino acids were detected. Automatic Edman degradation of pep M24 yielded the sequence of the first 29 amino acids (the amino terminal amino acid being valine) of the amino terminal region of the molecule. The detection of only one new amino acid at each step of Edman degradation confirmed the homogeneity of the purified pep M24.

Laboratory methods of equine pregnancy diagnosis

Rectal examination is a reliable method of diagnosing pregnancy in the mare. Also, test kits are available for the simple quick detection of pregnant mare serum gonadotrophin. Nevertheless there is a considerable demand by practitioners for an independent laboratory service in equine pregnancy diagnosis, particularly during the gestational phase when placental gonadotrophin is concentrated in the blood. An initial attempt to provide a service by means of the agar gel diffusion test was disappointing and alternatives were sought. The primary requirements for an ideal alternative technique were defined as: accuracy, sensitivity, applicability to the optimum request period, elimination of subjective interpretation and a minimal inconclusive rate. Additional considerations included cost, practicality and test duration. The various tests available are summarised and their published accuracies are discussed. It was decided to reverse the current trend from biological to immunological techniques and to modify the test by which Cole and Hart announced the discovery of PMSG in 1930. The utero-ovarian response in the immature female mouse was quantified simply to produce a numerical result. The reason for this is explained, the method is described and its accuracy is evaluated. The primary requirements, defined above, were achieved.

Serologic Tests for Antibody to Sarcocystis in Cattle

Soluble antigen was prepared from Sarcocystis zoites obtained from heart muscle of a bovine inoculated with sporocysts from canine feces and killed 120 days after infection. The antigen was used in an indirect hemagglutination (IHA) test and an agar gel diffusion test to detect antibody to Sarcocystis in experimentally infected cattle. IHA serum titers began to rise 30 to 45 days after infection and reached levels up to 1:39,000 90 days after infection. Sera collected under field conditions from 21 dairy cows had IHA titers ranging from 1:54 to 1:486. Since all cows appeared in good health, titers of 1:486 or less can probably be considered nonsignificant with regard to diagnosis of clinical disease. No positive Sarcocystis IHA titers were obtained with human sera previously found to be IHA positive for toxoplasma, indicating a lack of cross reactivity between antigens. Precipitins in the agar gel diffusion test appeared 30 days postinoculation and became very pronounced at 65 to 90 days.

Evaluation of counter immuno-electrophoresis crossed electro-immunodiffusion and agar gel diffusion for immunodiagnosis of human hydatid disease

Techniques of counter immuno-electrophoresis (CIEP), crossed electro-immunodiffusion (CEID) and agar gel diffusion (AGD) were used for diagnosis of hydatid disease. Sera were obtained from suspected patients before surgery and the antigens used were either human hydatid fluid (HHF) or sheep hydatid fluid (SHF). Out of the 29 suspected cases, 21 were confirmed by recovering the cysts from lung (ten), liver (nine), liver and spleen (two). The experimental results showed that precipitin line(s) with CIEP were obtained with the sera of all 21 confirmed patients using HHF and of 13 using SHF. With CEID, all 19 sera tested demonstrated precipitin patterns with HHF. In the AGD test precipitin lines were obtained in the sera of 15 patients using HHF and of six using SHF. None of the sera from 22 patients suffering from illnesses other than hydatidosis or of the sera from 34 apparently normal individuals gave positive results with CIEP when either HHF or SHF was used as the source of antigen. The CIEP test correlated well with CEID. It is, therefore, suggested that CIEP, which is a rapid and less sophisticated test, can be applied for the diagnosis of hydatid disease and in epidemiological surveys.

Isolation of Infectious Bronchitis Virus from Broiler Chickens in Chile

An infectious-bronchitis-like virus was isolated from 6-week-old broiler chickens unvaccinated with infectious bronchitis virus (IBV). The isolate produced lesions in embryonating chicken eggs (ECE) and clinical signs in birds characteristic of IBV. Serum-neutralization and agar-gel diffusion tests demonstrated the serological relationship of this isolate to IBV.

Some effects of silical treatment on Marek's disease

Treatment of newly hatched chicks with silica by the intraperitoneal route delayed the onset of mortalities due to the JM strain of Marek's disease (MD' virus inoculated at 6 days of age. During the 88-day observation period fewer silica-treated chicks died of MD, but this difference was not usually statistically significant. Silica treatment had no effect on the susceptibility of 4-week-old birds. Silica treatment reduced the antibody response to MD but, in general, not significantly. The antibody response to bovine serum albumin was significantly enhanced if measured by the indirect hemagglutination test but not if measured by the agar gel diffusion test, whereas the response to Brucella abortus was enhanced significantly in N-line (MD-resistant) chicks but not significantly in P-line (MD-susceptible) chicks. Five days after infection, silica-treated chicks had significantly less fluorescing antigen in thymus and bursa than did untreated chicks; no difference was observed in the spleen. After silica treatment the glass-adherent cell population in the buffy coat was increased by up to 10-fold compared with untreated chicks. It is suggested that silica treatment induced macrophage proliferation, with subsequent restriction of MD virus spread, yet allowed an adjuvant-type effect with other antigens.

Serological purification of polysaccharide antigens from Streptococcus mutans serotypes a and d: characterization of multiple antigenic determinants.

The polysaccharide antigen preparations from serotype a and serotype d strains of Streptococcus mutans contained both a serotype-specific antigenic determinant and a common a-d antigenic determinant, as demonstrated by agar gel diffusion studies and a quantitative cross-precipitin assay. The chromatographically purified antigens were isolated by a method which depended on their serological specificity to determine if these two antigenic determinants were located on the same molecule. The a and d polysaccharides were recovered from specific antigen-antibody complexes and characterized with respect to their immunological specificity and chemical composition. Agar gel diffusion tests demonstrated that, in both the a and d preparations, the serotype-specific antigenic determinant and the common a-d antigenic determinant were present in one molecule.

Studies on viral arthritis (teno‐synovitis) in Italy

Three of several virus isolates made from different groups of chickens affected with arthritis were serially passaged in chicken embryos, and then cultured on chicken kidney cells with the production of cytopathic effects and development of syncytia. Resistance to inactivating agents and electron microscopic features showed their relationship to reoviruses. By the agar gel diffusion test the isolates were shown to be serologically related to each other and to 2 American strains. A tissue suspension from infected embryos when inoculated into the footpad of one to 15 day-old chicks reproduced arthritis.

Purification and serology of bean yellow mosaic virus.

Isolate No.30 of bean yellow mosaic virus (BYMV) was purified from broad bean leaves by extraction with 0.1M Tris-HCl buffer, pH7.0, containing 0.05M EDTA and 1% 2-mercaptoethanol; repeated clarification with carbon tetrachloride and precipitation with 4% polyethylene glycol (#6, 000) followed by differential centrifugation and sucrose density-gradient centrifugation. ISCO fractionator scanning patterns indicated a high degree of purity but serological results revealed the presence of some host contaminates. The yield of virus was about 2mg/100g of broad bean leaves.The titer of the antiserum against isolate No.30 was 1/2048 by ring precipitin tests. Although the intact virus particles could not diffuse in agar gel plates, a clear precipitin line was observed when 0.5% SDS (or LIS) was added to the agar or virus suspension. Isolate No.30 was compared serologically with a necrotic strain and a chlorotic spot strain of BYMV in agar gel diffusion tests. Spur reactions were observed between isolate No.30 and these strains.

Experimentelle xenogene Immunpankreatitis Immunhistologische, enzymhistochemische und ultrastrukturelle Untersuchungen

Repeated intraperitoneal injections of anti-mouse pancreas rabbit serum or of anti-mouse pancreas guinea pig serum produce a chronical sclerotizing pancreatitis. This study has the aim to contribute to the further elucidation of the changes which occur in the acinar cells, as well as to the etiology and pathogenesis of immune pancreatitis, by means of immunohistological, enzyme histochemical and electron microscopic studies. Anti-mouse pancreas rabbit serum was obtained by sensitization of rabbits with an admixture of AB-mouse pancreas extract (100,000 g - supernatant) and complete Freund's adjuvant [details see NIZZE, Exp. Path. (1975a)]. The presence of precipitating mouse pancreas antibodies in the rabbit serum was ascertained by the agargel diffusion test according to Duchterlony (1958). The experiments were performed with 54 adult male white mice (AB colony strain) of 22 to 30 g.b.s. (averagely 26 g). The animals were divided into 4 groups which were treated as follows: 1. 24 mice with anti-mouse pancreas rabbit serum, 2. 12 mice with rabbit normal serum, 3. 12 mice with physiological saline, 4. 6 mice remained untreated (controls) Always 4 animals of the group 1 as well as each 2 of the groups 2 and 3 were administered in total 1, 3, 5, 9, 17 or 33 intraperitoneal injections of 0.3 ml of the correspondent serum or with physiological saline within 3 hours, 1, 2, 4, 8 or 16 days. The last injection was regularly applied 3 hours before sacrification by decapitation. The time of sacrification was always at 11.00 o'clock a.m. For immunohistological and enzyme histochemical investigations 10 mum thick cryostat sections were prepared consisting of pancreatic specimens piled up to a bloc. In each case the tissue samples were taken from the experimental animals and from one control animal sacrificed at the same day. The sections were incubated in FITC-labelled anti-rabbit globulin goat serum at room temperature for 30 min in a moist chamber. For control of specificity were employed: a) initial incubation of equal sections with unlabelled anti-rabbit globulin goat serum for 30 min (""blocking test''), b) pancreatic tissue specimens of each one untreated control animal present in the cryostat sections and thus incubated like the pancreatic tissue of the experimental animals, c) native nonincubated cryostat sections from the same bloc to exclude nonspecific autofluorescence. Evaluation of the sections was done in a Zeiss-Lg-microscope with HBO-50 high pressure mercury lamp. Exciter filters were UG 1/3.5 and 1/1.5, the eyepiece was screened with a GG 9/1 filter photographs were taken on ORWO X-ray film RS 2 (VEB Filmfabrik Wolfen). The enzyme histochemical studies were performed on cryostat sections of the same tissue bloc using the following methods: lead nitrate- or calcium-Co-method after GOMORI (1952) for demonstration of acid and alkaline phosphatase, naphthylacetate method (NACHLAS and SELIGMAN 1945) for nonspecific esterase, MTT-co-method (PEARSE et al...

Immunogenicity of the delta-haemolysin of Staphylococcus aureus.

SUMMARY Antibody to δ-lysin of Staphylococcus aureus was produced in rabbits given injections of purified antigen. Removal of normal serum inhibitors was effected by ammonium-sulphate fractionation and ion-exchange chromato-graphy. The purified anti-δ-lysin showed a single line of precipitation with purified antigen in Ouchterlony agar gel-diffusion tests and when subjected to immunoelectrophoresis. The line of precipitation also coincided with the zone of haemolysis caused by δ-lysin when the agar gel-diffusion test was performed in blood-agarose plates. A plot of the data obtained in a quantitative precipitin test performed with purified antibody and δ-lysin showed a characteristic curve confirming their interaction; in control tests with non-immune sera in place of δ-antibody, no precipitation was evident.

Characterization and evaluation of a soluble antigen complex prepared from the yeast phase of Histoplasma capsulatum.

Soluble yeast-phase (YPS) antigen preparations (Reeves et al., 1972) were obtained from three strains of Histoplasma capsulatum. These were analyzed by agar-gel diffusion and complement fixation tests with human sera from known cases of histoplasmosis. Two of the preparations contained the H and M antigens normally found in histoplasmin, whereas the third preparation contained the H but not the M antigen. In addition, a group of unknown antigens, designated as the Y (yeast) antigens, were demonstrated in all three YPS preparations. On the basis of Sephadex gel filtration data, the molecular weight of the YPS M antigen was estimated to range from 117,000 to greater than 200,000; that of the YPS H antigen was estimated to range from about 60,000 to greater than 200,000; and that of the Y antigens was estimated to range from less than 10,000 to 100,000. Complement fixation tests with human homologous and heterologous sera showed that the Y antigens were specific for H. capsulatum; Y antigens were not detected in the single lot of histoplasmin used in this study. When 60 human histoplasmosis sera and serum samples from nine animals experimentally infected with H. capsulatum were analyzed, it was found that antibodies to the Y antigens occurred with about the same frequency as antibodies to the H antigen but with less frequency than that of antibodies to the M antigen. When used in rabbits as immunogens for the preparation of specific antisera to H. capsulatum, the components of the YPS preparations caused the formation of numerous cross-reacting antibodies. The data from this study show that the value of the YPS preparations for the serological diagnosis of histoplasmosis rests on the specificity of the H, M, and Y antigens and on the fact that the primary production of antibodies is restricted to these antigens in the course of natural infections. The YPS preparations were found to be stable for a period of at least 11 months under a variety of storage conditions and temperatures. Data obtained with various killing agents and metabolic inhibitors suggest an improved method for preparing the YPS antigens by using a suitable strain and killing the cells with iodoacetate.

Serological Studies on Francisella tularensis, Francisella novicida, Yersinia philomiragia, and Brucella abortus

A study was made to reveal serological relationships among Brucella abortus, Francisella tularensis, Francisella novicida, and Yersinia philomiragia in bacterial agglutination and double agar-gel diffusion tests. All antisera used for the experiments were prepared in rabbits. In agglutination tests, cross-reaction occurred only between F. tularensis and B. abortus. Acetone-precipitated material showed common antigens for three speciesF. tularensis, F. novicida, and Y. philomiragia in agar-gel diffusion tests whereas polysaccharide preparations were shown to be species-specific for each of the four organisms. B. abortus acetone material reacted only with F. tularensis antiserum in agar-gel diffusion. F. tularensis antiserum, after absorption by intact B. abortus organisms, failed to react with B. abortus both in agar-gel diffusion and agglutination tests. The results show that B. abortus has surface antigens in common only with F. tularensis and that F. tularensis, F. novicida, and Y. philomiragia are antigenically related.

Viruses isolated from Narcissus (Narcissus spp.) in Japan V

A virus was isolated from a large cupped narcissus plant which showed yellow stripe symptoms collected in Niigata Prefecture in 1971. The virus was readily transmitted by juice inoculation. Among the test plants of 46 species in 14 families, 37 species in 13 families, namely, Chenopodiaceae, Amaranthaceae, Aizoaceae, Caryophylaceae, Papaveraceae, Cruciferae, Leguminosae, Violaceae, Solanaceae, Pedaliaceae, Cucurbitaceae, Compositae, Amaryllidaceae, were found susceptible to the virus.The virus was not transmitted by Myzus persicae, but was transmitted by nematode (Xiphinema bakeri). An evidence of soil transmission was provided by greenhouse tests using soil which was infested with nematodes including Xiphinema bakeri. Lots of 25 X. bakeri sieved from soil in which infected petunia had been grown for 2 to 3 weeks were placed near the roots of potted healthy petunia. After 6 weeks the virus was detected by juice inoculation and by serological test in all inoculated petunia plants. No infection occurred in control petunia plants grown in soil free from X. bakeri. The virus was proved to be transmitted also through seeds of soybean.The virus in vitro withstood heating at 60C for 10 minutes, but not 65C, and 21 days of storage at 22C, but not 28 days. The virus particles were found to be spherical about 25nm in diameter.Antiserum prepared injecting rabbit with a semi-purified virus preparation showed homologous precipitin tube titre of 1/1024. By agar gel diffusion tests, the virus showed negative reaction to antisera against tomato black ring virus (kindly sent from Dr. B.D. Harrison) and against tobacco ringspot virus and tomato ringspot virus (kindly sent from Dr. R. Stace-Smith), but showed positive reaction to the antiserum against arabis mosaic virus (kindly sent from Dr. B.D. Harrison).From the above results, the virus was identified as arabis mosaic virus, which has hitherto not been reported in Japan.Virus-free narcissus seedling juice-inoculated with the virus remained apparently healthy for 15 months, although the virus was recovered from these plants.

Studies on Reactive Conditions for the Discrimination of Animal Species of Meat by Agar Gel Diffusion Test

Studies on Reactive Conditions for the Discrimination of Animal Species of Meat by Agar Gel Diffusion Test

YURDUMUZDA İZOLE EDİLEN M. capri SUŞLARININ ÜREME- İNHİBİSYON VE AGAR-JEL DİFFUSYON TEKNİKLERİ İLE ANTİJENİK ANALİZLERİ ÜZERİNDE ARAŞTIRMALAR

Suınınary: In this study, 9 M. capri strains isolated fi'om several parl~ of Turkey were used. Accoding to the results of the experiment, it was uııderstood that there were no antigenie differences between isolates. All hyperimmun sera were reacted ",ith homolog and heterolog antigens both in the growth-inhibition and agar-gel diffusion tests. Özet: Bu çalışmada, yurdumıızun çeşitli yöreleriilden izole edilmiş 9 M. caprİ suşu kullanıldı. Araştırmanın sonuçlarına göre, suşlar arasında antijenik bir ayrılık saptanamadı. Bütün hiperİmmun serumlar, homolog ve hcteroıog antijenler ilc, hem üreme-İnhibisyon ve hem de agar-jel diffusyon testlerİnde reaksiyon verdiler.

Type-A Influenza Viruses Isolated from Wild Free-Flying Ducks in California

From 6 October 1972 to 3 December 1972, 41 type-A influenza virus isolants were recovered from free-flying wild ducks, and 7 isolants from domestic ducks in southern California. The typespecific antigen (ribonucleoprotein) was identified by the agar-geldiffusion test, and tentative identification of one strain-specific antigen (hemagglutinin) was attempted by the hemagglutinationinhibition test. These isolants support the theories that wild birds play an important role in the dissemination of type-A influenza viruses and may provide optimum conditions for genetic interaction of type-A influenza viruses, resulting in new hybrid strains.