Gel Double Diffusion Tests Research Articles

Immunohistochemical detection of bovine ruminal carbonic anhydrase isoenzyme.

Rabbits immunized with low-activity ruminal carbonic anhydrase (RCA) isoenzyme, extracted from ruminal epithelial cells isolated by digestion with trypsin, yielded anti-RCA sera which reacted specifically with bovine RCA in double agar gel diffusion and immunoelectrophoretic tests, but failed to cross-react with bovine erythrocyte CA. The localization of RCA was identified in histological sections and isolated ruminal epithelial cell preparations by indirect immunofluorescence and immunoperoxidase tests as the basal, spinosum and granulosum layers of ruminal mucous epithelium.

Stimulation by lincomycin of production of cholera-like enterotoxin inVibrio choleraenon-O1

It has been reported that lincomycin, an inhibitor of protein synthesis, stimulates the production of cholera enterotoxin (CT) and Escherichia coli heat-labile enterotoxin (LT) [1-3]. Lincomycin causes an increase in the rate of synthesis of CT and thus the amounts of both extracellular and intracellular CT were significantly increased [2]. Vibrio cholerae non-O 1 (non-O 1 vibrio or NAG vibrio) produced CT-like enterotoxin. Zinnaka and Carpenter [4] and Ohashi et al. [5] reported that non-O1 vibrios produced enterotoxin which was biologically and immunologically similar to CT. Recently, we reported that V. cholerae non-O1, isolated from the environment, produced CT-like enterotoxin which was biologically similar and immunologically identical to CT by Ouchterlony double gel diffusion test [6]. In this paper we report that the production of CT-like enterotoxin in V. cholerae non-O1 is also stimulated by lincomycin.

Isolation of special antibodies which react only with homologous enterotoxins from Vibrio cholerae and Enterotoxigenic Escherichia coli.

Cholera enterotoxin (CT) and heat-labile enterotoxin (LT) produced by enterotoxigenic Escherichia coli share a common antigenic determinant. In addition, CT and LT each have a unique antigenic determinant. Antisera were prepared by immunoaffinity chromatography against these unique antigenic determinants, that is, antiserum that reacted with CT but not with LT and antiserum that reacted with LT but not with CT. Antiserum against the common antigenic determinant to CT and LT was also prepared by immunoaffinity chromatography. The specificities of these antisera were demonstrated by Ouchterlony double-gel diffusion tests and by neutralization of the activities of CT and LT to cause morphological changes in Chinese hamster ovary cells.

Production of cholera-like enterotoxin by a Vibrio cholerae non-O1 strain isolated from the environment.

Vibrio cholerae non-O1 strain E8498, isolated in 1978 from fresh water in Louisiana, produced a vascular permeability factor when cultured in shallow resting cultures of Casamino Acids-yeast extract-glucose medium for 24 h at 30 degrees C. Undiluted resting culture filtrates contained heat-labile permeability factor activity which was only partially neutralized by cholera antitoxin and GM1 ganglioside. Supernatants concentrated with PM-10 membranes caused hemorrhage and necrosis in rabbits within 1 h after intracutaneous injection, whereas appropriate dilutions of both filtrates and concentrates demonstrated delayed permeability factor activity, without hemorrhage or necrosis, which was indistinguishable in appearance from that caused by purified cholera enterotoxin produced by V. cholerae O1 Inaba strain 569B. Crude E8498 filtrates contained the biological equivalent of about 5 ng/ml of purified enterotoxin. Permeability factor activity in the fraction obtained by 20 to 50% saturation of filtrate concentrate with ammonium sulfate could be completely neutralized by reference standard cholera antitoxin prepared against purified 569 B enterotoxin. Hemorrhagic activity was unaffected by cholera antitoxin. A 5,000-fold concentrate of the culture supernatant yielded a line of identity with purified cholera enterotoxin in an agar gel double-diffusion test against cholera antitoxin purified by affinity column chromatography with BrCN-activated Sepharose 4B-linked purified cholera enterotoxin as the adsorbent. These findings indicate that V. cholerae non-O1 E8498 produces a permeability factor which is immunologically and biologically indistinguishable from that produced by a strain of V. cholerae O1 classical biotype.

Three new serogroups of Neisseria meningitidis

By employing bacterial agglutination absorption, passive hemagglutination, agarose gel double diffusion tests and counterimmunoelectrophoresis technique, the authors have carried out some studies concerning certain meningococcal strains, isolated within recent years in China, which are non-agglutinable with the diagnostic serogrouping seta A, B, C, D, Y(1889), X(1916), 29E(1892), W135(319) and Z of Neisseria meningitidis . As a result of an analysis of serological specificity, it has been tentatively proposed that these strains of N. meningitidis can be divided into three new serogroups. To resolve the problem of nomenclature for these new serogroups, it is suggested that the three new letters and numbers of the representative strains for the respective serological groups i.e. groups H(1890), I(1486) and K(1811) are used.

The tissue and cellular location of a major cross-reactive antigen shared by cotton and soil-borne fungal parasites

A major cross-reactive antigenic substance (CRA), common to cotton ( Gossypium hirsutum ) and certain fungal parasites of cotton roots, was isolated and purified to homogeneity from conidial cultures of Fusarium oxysporum f.sp. vasiufectum (F.o.v,). Agar gel double-diffusion tests indicated the prcscncc of CRA not only in F.o.v. and in cotton roots and seed but also in Thielaviopsis bosicola . Indirect staisung of antibodies using fluorescein isothiocyanate (FITC) indicated that in cross-sections of roots, cut near or just below the root hair zone, the CPA was concentrated mainiy around xylem elements, the endodernais and epidermal cells and was present throughout the cortex tissue. Protoplasts prepared from cross-sections of young cotton roots also contained the CPA which was concentrated in the region of the plasmalemma. Treatment of conidia and mycelia of F.o.v. with antiserum to cotton and using indirect staining with FITC indicated that the CRA was mainly present in hyphal tips and in patch-like aseas on conidia. The possible role of CRA in basic compatibility of host and parasite is discussed.

A newly discovered serotype of Gremmeniella abietina

The Asian, European, and North American serotypes of Gremmeniella abietina were compared with 27 Vermont isolates using gel double diffusion and intragel cross absorption tests. An intermediate serotype, between the North American and European serotypes, was shown by means of cross absorption with antiserum prepared in response to a Vermont isolate. The majority of Vermont isolates resembled the Vermont intermediate in serological reaction, three resembled the European serotype, and two were of unknown serotypes. The serological data support possible hybridization between G. abietina isolates in Vermont as well as the identification of a new serotype in Vermont.

The Trapping of Tymovirus Particles on Electron Microscope Grids by Adsorption and Serological Binding

Summary Many purified tymovirus particles are trapped on buffer-treated grids due to non-specific adsorption. Pre-treatment of grids with serum or addition of crude plant sap to virus preparations greatly inhibited non-specific adsorption. Specific serological binding on grids coated with homologous antiserum produced particle counts comparable to those achieved by non-specific adsorption on buffer-treated grids in the absence of plant sap. Specific serological binding was not influenced by crude sap of Nicotiana clevelandii. For both adsorption and serological binding there was a linear relationship between the log virus concentration and log particle counts at virus concentrations below approx. 10 µg/ml; at higher concentrations the grids were saturated. In the presence of excess virus, antisera at dilutions of less than 10-3 yielded lower particle counts than more dilute antisera. Heterologous reactions were detected only with viruses for which very close serological relationships had been found in the agar gel double-diffusion test. Heterologously bound virus was apparently not replaced by subsequently applied homologous virus. However, adsorbed bottom component virus particles could be replaced by top component particles of the same virus and vice versa.

Serological Studies on the Relationships of Some Isometric Viruses of Gramineae

SUMMARY Particles of 17 isolates of viruses from Gramineae were purified and the virus relationships assessed by different serological techniques. From the reaction end-points of antisera in agar gel double diffusion tests the serological differentiation indices of reciprocal tests were calculated. These indices were used to construct a scheme of serological interrelationship. Spur formation tests were used to distinguish between closely related, non-identical viruses. The isolates were shown to belong to four groups, one of which (the cocksfoot mild mosaicpanicum mosaic virus group) contains most of the isolates and can be subdivided into two main clusters (typified by cocksfoot mild mosaic and panicum mosaic viruses, respectively). Additional information was obtained by immunoelectrophoresis of the viruses with their homologous antisera and by the determination of the mol. wt. of the coat protein subunits using SDS-polyacrylamide gel electrophoresis. The data obtained by both methods agree with the above mentioned results, although some minor differences were found between the immunoelectrophoretic patterns of panicum mosaic virus and its relatives and also the three isolates of Cynosurus mottle virus. Reversed rocket electrophoresis did not provide any information of use in virus grouping. Brome mosaic virus did not react with the antisera to the 17 viruses, but tobacco mosaic virus reacted weakly with all the antisera and also with one of the antisera to brome mosaic virus. These and other recently published results are discussed.

Preparatory Immunodiffusion for Production of Specific Anti-Adult Southern Pine Beetle Serum

Preparatory immunodiffusion was used to produce an antiserum specific for the southern pine beetle, Dendroctonus frontalis (Zimm.), and the black turpentine beetle, D. terebrans (Olivier). Precipitin bands were excised from two-dimensional electroimmunodiffusion agar gel plates of crude antigen and antiserum, extracted in saline, emulsified with Freund's incomplete adjuvant, and injected subcutaneously near the inguinal lymph nodes of rabbits. The specific antiserum did not cross react with Ips avulsus Eichhoff, I. calligraphus Germar, or I. grandicollis (Eichhoff) when tested at high antigen concentrations by agar gel double diffusion, Crowle's template micro-double diffusion, two-dimensional electroimmunodiffusion, counter immunoelectrophoresis, or cellulose acetate membrane electrophoresis with immunofixation. The antiserum is genus specific in all tests, and species specific when run in the agar gel double diffusion test against a standard Dendroctonus antigen (7.0 mg protein/ ml).

Purification and characterization of potato leafroll virus

Potato leafroll virus (PLRV) was purified from infected potato ( Solanum tuberosum L.) with yields of 0.4–0.6 mg/kg of foliage. The virus sedimented as a single component of 127 S. An antiserum prepared against purified virus had a maximum titer of 1:1024 in agar gel double diffusion tests. PLRV had a buoyant density of 1.39 g/ml and an estimated nucleic acid content of 28%. The nucleic acid had a molecular weight of 2.0 × 10 6 and was degraded by RNase but not by DNase, indicating that the PLRV nucleic acid is RNA. The sedimentation coefficient of the RNA molecule was 34.5 S and after treatment with formaldehyde, 20.7 S. Dissociated coat protein migrated as a single band in polyacrylamide gel electrophoresis and the average subunit molecular weight was 26,300. PLRV should be considered a member of the luteovirus group.

Purification and some physicochemical properties of staphylococcal enterotoxin D.

A method was developed for the isolation of staphylococcal enterotoxin D in highly purified form from cultures of Staphylococcus aureus strain 1151m. The method involves removal of the toxin from the culture supernatant fluid with the ion-exchange resin CG-50 followed by chromatography on carboxymethylcellulose (twice) and by gel filtration on Sephadex G-75 (twice). The purified toxin is homogeneous by polyacrylamide gel and sodium dodecyl sulfate-polyacrylamide gel electrophoresis and double gel diffusion tests. It is a simple, colorless, antigenic protein with an isoelectric point of 7.4 as determined by isoelectric focusing. Its molecular weight was determined to be 27 300 +/- 700 by molecular sieve chromatography on Sephadex G-100 and sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Its serological activity is stable over a wide range of pH values (1.2--10.7). The enterotoxin consists of 236 amino acid residues and contains no free sulfhydryl groups. End-group analysis showed serine to be the NH2-terminal amino acid and lysine to be the COOH-terminal amino acid.

Attempt to detect bovine antibody against Fusobacterium necrophorum by the agar gel double diffusion test.

In order to detect bovine serum antibody against Fusobacterium necrophorum, serological investigation was made on bovine isolates of this species by the agar gel double diffusion test. Precipitin lines were observed between most of the HC1-heat extracts prepared from these strains and four anti-F. necrophorum sera. Precipitin was developed in rabbits inoculated experimentally with the organism, in the late stage of infection. Precipitin lines appeared between concentrated antigen prepared from the VPI 2891 strain and 16 of 23 sera from cattle affected with hepatic abscess, and between this antigen and 4 of 88 sera from healthy cattle.

Developmentally regulated lectins from chick muscle, brain, and liver have similar chemical and immunological properties

Developmentally regulated lectins in extracts from brain, liver and muscle of 16-day-old chick embryos and liver of 7-day-old chicks have been purified by affinity chromatography. The purified preparations from the different tissues were indistinguishable in molecular weight and isoelectric point. The lectins could also not be distinguished when tested as antigens with antiserum raised against highly purified muscle lectin. This apparent identity was indicated both in double gel diffusion tests and by determination of the antibody-mediated inhibition of hemagglutination activity of the various lectins. Thiodigalactoside and lactose were potent inhibitors of the lectins from all sources. Galactose was a less potent inhibitor, especially with preparations from embryonic liver. After isoelectric focusing of these purified preparations, they all showed reduced and equivalent galactose sensitivity. Since the lectins from the different tissues appear identical, there is presently no basis to infer that they impart qualitative uniqueness to these tissues during differentiation.

Saponin Adjuvants. V. Precipitation Of Serum Components by Non-Purified Saponin Adjuvants in Agar Gel Diffusion.

The immunologically active adjuvant Quil A does not induce precipitating antibodies in rabbits. This was tested by immunodiffusion of serum samples taken after repeated injections of Quil A. Quil A does not react non-specifically with any of 6 different animal sera tested (rabbit, guinea pig, horse, sheep, cattle, and pig). Two commercially available saponins with known adjuvant activity (Saponin MT, E. Merck, and Saponin P 3, Food Industries) produced non-specific precipitation in double gel diffusion tests with all the sera, as did crude extracts of the South-American tree Quillaja saponaria Molina.} This phenomenon in relation to the local tissue damage caused by non-purified saponin preparations is discussed.

IMMUNOCHEMICAL INVESTIGATIONS OF HUMAN PITUITARY β‐LIPOTROPIN

Antiserum to human beta-lipotropin has been raised in rabbits and characterized by gel double diffusion, quantitative precipitin, microcomplement fixation and biological neutralization tests. A sensitive radioimmunoassay for human beta-lipotropin has been developed. It was demonstrated that the antiserum shows specificity for this hormone. Human beta-melanotropin and human beta-endorphin showed only a minimal degree of cross-reaction, while ovine beta-lipotropin showed a partial but significant degree of cross-reactivity.

The diagnosis of Aspergillus fumigatus infection of the nasal chambers of the dog with particular reference to the value of the double diffusion test

ABSTRACTTen case examples are used to show that the agar gel double diffusion test is a reliable technique in the diagnosis of nasal A. fumigatus infection in the dog. Radiography is also helpful but culture and direct smear testing are unreliable.

小児喘息患児のダニに対する免疫反応とその屋内塵中のダニ相について

Immunological reactions were performed on asthmatic children living in and around Tokyo with the extract of house dust mite, Dermatophagoides pteronyssinus, as antigen. Mite fauna in house dusts of the patients was examined and relation between the mite fauna and immunological findings was studied. Higher than 70% or more positive rates were obtained in skin test and radioallergosorbent test (RAST) either to house dust or the mite antigens. On the other hand, passive hemagglutination test and double gel diffusion test against the mite extract were all negative. House dust was collected from the residences of 17 skin test and RAST positive patients and 10 negative asthmatic children. Pyroglyphid mites constituted 70-90% of the mite fauna and constitutional pattern of other mites did not differ in both groups. However, number of mites found in dusts per square meter for a week differed significantly (p<0.05). This study comfirms the importance of the house dust mite in children's asthma and shows that absolute number of mites in the dust plays an important role in the sensitization of children with substantial figure.

Antigenicity of bovine milk xanthine oxidase in guinea pigs.

Six female and four male albino guinea pigs were immunized with active purified xanthine oxidase of bovine milk mixed with equal volume of Freund's complete adjuvant. One male and one female were immunized with heat-inactivated xanthine oxidase mixed with equal volume of adjuvant. Two males and four females were controls and received a phosphate buffer mixed with equal volume of adjuvant. The mixtures were administered intradermally and subcutaneously at weekly intervals for 6 consecutive wk and blood samples collected weekly. The enzyme was antigenic by the coated tanned red blood cell method. After the third weekly immunization, precipitating antibodies were in the sera of animals that received the active enzyme. Hemagglutination titers increased during subsequent weeks and reached a maximum after the sixth weekly immunization. Antisera from animals immunized with heat-inactivated xanthine oxidase gave a positive response similar to that with animals immunized with the active enzyme. However, when the same antisera were tested with sheep red blood cells coated with heat-inactivated enzyme, no hemagglutination was observed. Ouchterlony double gel-diffusion tests showed that it may be possible to differentiate between antibodies elicited to active and heat-inactivated xanthine oxidase.

Collaborative Study of the Serological Identification of Staphylococcal Enterotoxins by the Microslide Gel Double Diffusion Test

A collaborative study was conducted, using the microslide gel double diffusion test for enterotoxin detection by determining the staphylococcal enterotoxigenicity of 7 strains of Staphylococcus aureus. Two strains produced staphylococcal enterotoxin A (SEA), 2 strains produced staphylococcal enterotoxin B (SEB), one strain produced both SEA and SEB, and 2 strains produced neither SEA nor SEB. To ascertain the effectiveness of this method for the detection of enterotoxins, strains of enterotoxigenic staphylococci which produced small or relatively large amounts of toxin were used in the study. The cultures were grown on semi-solid brain-heart infusion agar and the culture fluids were assayed serologically for SEA and SEB. The qualitative results of this study by 16 collaborators indicated that this technique, in general, demonstrated a high degree of specificity as well as simplicity and reproducibility in the identification of the enterotoxins. Of the 16 participating laboratories, 14 reported the correct diagnoses for the 7 staphylococcal strains studied and 2 laboratories determined 6 of the 7 strains correctly. Based on these results, this method has been adopted as official first action.