Gel Double Diffusion Tests Research Articles

沈降抗体検出用アスペルギルス粗抗原に関する若干の検討 補遺

We reported earlier on culture filtrate antigens from A. fumigatus that had been incubated at 25°C for 1-10 weeks (Jpn J Med Mycol 34: 439-444, 1993). Additionally, we prepared five culture filtrate antigens from A. fumigatus ATCC26430 grown by shaking at 25°C for 11-15 weeks in Sabouraud liquid medium. We also prepared a polysaccharide antigen from the same strain by extracting mycelia and conidia. The growth of the fungi became weaker in cultures after 12 weeks, and by stopped at 14 weeks. Sixteen antigens were then employed in agar gel double diffusion (DD) tests against the sera of 18 aspergilloma patients. The results of DD showed three types (A, B, C) of precipitin pattern, with 9 patients belonging to type A, 6 to type B and 3 to type C. In all cases, precipitin lines were formed with the antigens obtained after 6-15 weeks of culture. To avoid contamination, inferior cultures and accumulation of waste, the incubation time should be as short as possible. Thus, as noted in our previous letter, we found that the antigen obtained from 6-week culture filtrates at 25°C is the most useful for demonstrating precipitin reactions against the sera of aspergilloma patients.

Use of the double immuno gel diffusion test and the enzyme-linked immunosorbent assay to distinguish false from true reactors in the complement fixation test forBrucella ovis

A gel diffusion test with sonicated Brucella ovis antigen and an enzyme-linked immunosorbent assay based on heat-extracted antigen were used to distinguish false from true reactions in a complement fixation test based on heat-extracted antigen. Of 142 complement fixing reactors (occurring in supposedly Brucella ovis-free, accredited flocks), the gel diffusion test correctly identified the status of 139 animals as compared to 128 with the enzyme-linked immunosorbent assay. A combination of the two methods resulted in a correct identification of 141 animals. The procedures provide an easy, cheap and quick way to determine the true status of reactors that show up during routine use of the complement fixation test in Brucella ovis re-accreditation procedures.

Heterogeneity among heat-labile enterotoxins produced by porcine enterotoxigenic Escherichia coli.

The heat-labile enterotoxins (LT) produced by various porcine strains (LTp) of enterotoxigenic Escherichia coli were purified to homogeneity and their molecular properties were compared with each other. By double gel diffusion and rabbit skin permeability test, LTps from WT-1 (LTp-WT-1) and BO-149 (LTp-BO-149) strains were antigenically and biologically identical. By polyacrylamide gel electrophoresis with sodium dodecyl sulfate (SDS), the mobilities of A and B subunits of LTp (BO-149) were identical to those of LTp (WT-1). However, on polyacrylamide gel electrophoresis without SDS, LTp (BO-149) and LTp (WT-1) differed in mobility, suggesting their molecular differences. Though the pI value of the B subunit of LTp (BO-149) was identical to that of LTp (WT-1), the pI value of the A subunit of LTp (BO-149) was higher than that of LTp (WT-1). These data suggest that there is molecular heterogeneity among LTps produced by the two porcine enterotoxigenic Escherichia coli strains.

A simple purification method of Vibrio cholerae non-O1 hemagglutinin/protease by immunoaffinity column chromatography using a monoclonal antibody.

A new simple purification method (I) for Vibrio cholerae non-O1 hemagglutinin/protease (NAG-HA/P) was developed. The method (I) requires only an immunoaffinity column chromatography using a monoclonal antibody against NAG-HA/P. The method (I) is much simpler than previously reported purification method (II) (Honda, T. et al, Infection and Immunity 57: 2799-2803, 1989) which required four or more complicated chromatographic procedures. Method (I) also gave an improved recovery rate (about 27%) compared with (II). The molecular weight of NAG-HA/P purified by method (I) was mainly 34 kilodaltons (kDa) with a little of 32 kDa, whereas that of NAG-HA/P purified by (II) was usually 32 kDa. Immunological analysis by the Ouchterlony double gel diffusion test and Western blotting test using polyclonal antibody against 32 kDa protein revealed that the 34 and 32 kDa proteins are immunologically indistinguishable and thus it is supposed that 34 K protein is an isoform or a preform of the 32 K protein.

Purification and immunoreactivity of monoclonal antibodies against myeloma-λ chain

In this study, two murine IgM-monoclonal antibodies (IgM-McAbs) against lambda chain of myeloma protein from hybridoma ascites were purified by Sephadex G200 chromatography. The eluate of the first peak on absorption at 280 nm formed one precipitation band with rabbit anti-mouse IgM or anti-mouse immunoglobulin in agar gel double diffusion test; it also formed a detectable precipitation line in the IgM reaction area in immunoelectrophoresis. These findings showed that the eluate of the first peak on absorption at 280 nm contained purified IgM-McAbs. It was found in indirect ELISA that the immunoreactivity of the eluate of the first peak was four to five fold higher than that of the original ascites with equimolar protein concentration. Double antibody sandwich ELISA analyses of the immunoreactivity of HRP-conjugated IgM-monoclonal antibodies with lambda chain denoted that the purified IgM-McAb-HRP-conjugate might be of practical value in quantitative as well as qualitative assay of lambda chains.

Characterization of a New Fimbrial Antigen Present in Escherichia coli strains Isolated from Calves*

Thirteen Escherichia coli strains isolated from calves with diarrhoea, supposed to carry a common antigen were examined for their hemagglutinating activity and compared by bacterial agglutination, double diffusion in two dimensions and by crossed immunoelectrophoresis (CIE). Two of the strains were examined also in the electron microscope. Most of the strains agglutinated red blood cells of horse, ox, guinea pig and chicken, of which the agglutination of ox erythrocytes was mannose-resistant (MRHA). None of the strains agglutinated human erythrocytes. All strains with MRHA of ox red blood cells, regardless to their O:K:H antigens could be agglutinated in unabsorbed or absorbed antisera produced against cultures C1209 (020:K-:H9) and C1213 (09:K36:H-) when live cells as antigens were used. None of these sera agglutinated reference strains carrying K88, K99, 987P, F41 or FY (Att25) antigen respectively. By the double gel diffusion test and by CIE in extracts (60 degrees C) of the strains a common heat labile antigen, responsible for the MRHA of ox red blood cells was identified. Electron microscopy revealed that this common antigen was represented by thin, long, hair-like fimbriae on cells of E. coli C1213, and that specific homologous antibodies attached to these fimbriae.

The use of DNA probes for confirming enterotoxin production by Staphylococcus aureus and micrococci

DNA-DNA colony hybridization was employed to evaluate the results obtained by different immunological methods for detection of staphylococcal enterotoxin. Staphylococcus aureus strains tested for staphylococcal enterotoxin production by immuno-assays and micrococci not previously tested for staphylococcal enterotoxin production were examined for presence of the genes encoding for staphylococcal enterotoxin A, B, C and E by using three corresponding DNA probes. The staphylococcal enterotoxin A probe also detected staphylococcal enterotoxin E gene because of 100% homology. The optimal sensitivity plate method showed the best accordance between the immuno-assay and the hybridization reactions. The enzyme-linked immunosorbent assay detected 12.5 to 17% staphylococcal enterotoxin producers without hybridization reactions. The microslide gel double diffusion test and the reversed passive latex agglutination test showed rather poor accordance with the hybridization reactions. All 17 strains of different micrococci investigated were negative in hybridization with all three DNA probes.

Influence of adjuvants on immunity in rabbits vaccinated with infective larval somatic proteins of Trichostrongylus colubriformis

Adult New Zealand rabbits were vaccinated at 2-week intervals with three doses of 100 μg of infective larval somatic proteins (L 3SP) administered subcutaneously with Freund's complete adjuvant (FCA) or beryllium hydroxide and then challenged orally with 10 000 L 3. Groups of rabbits immunized orally with three doses of 5000 or 2000 L 3 served as vaccination controls. Intestinal worm burdens on Day 21 after challenge revealed that beryllium hydroxide effectively potentiates the protective immunogenicity of L 3SP. The level of protection obtained using the beryllium adjuvant (94.8%) was nearly as high as that in rabbits immunized with three doses of 5000 L 3 (99.8%). Rabbits vaccinated using FCA showed very poor immunity (29.5%). Local and systemic antibody levels detected by radioimmunoprecipitation tests using 125I-L 3-SP showed very little correlation with the degree of protection. The beryllium hydroxide-treated group demonstrated significantly higher bile IgA antibody levels than other experimental rabbits. FCA-treated rabbits developed a much higher serum precipitating antibody response, detectable using gel double diffusion tests, than the beryllium group. Also, mucosal IgA antibody levels detected on Day 21 after challenge were significantly higher in the FCA group than in other groups.

Serological Comparison of Isolates of Cherry Leaf Roll Virus from Diseased Beech and Birch Trees in a Forest Decline Area in Germany with Other Isolates of the Virus

Abstract Two isolates of cherry leaf roll virus (CLRV), one from diseased beech and one from diseased birch trees in an area with forest decline near Bonn in West Germany, were found to be serologically closely related, but not indentical as assessed by spurformation of precipitin lines in agarose gel double diffusion tests. Such tests also distinguished these German CLRV isolates from ten other distinct CLRV isolates obtained from different natural hosts and from various countries. The German beech isolate was most similar serologically to isolates from walnut and from birch in England and the German birch isolate to an English cherry isolate and an isolate from Sambucus racemosa in Finland. These results provide further evidence of the antigenicdiversity of CLRV.

A comparison of two enzyme linked immunosorbent assays for enzootic bovine leucosis serology

Abstract Extract The double gel diffusion test (GDT) is the internationally accepted standard method for detection of antibodies against bovine leukemia virus (BLV)(1). The method has been standardised (within certain limits) and a reference serum has been introduced by the European Commission to control the level of sensitivity of the test.

Simplified procedure for purification ofBordetella bronchisepticadermonecrotic toxin

Bordetella bronchiseptica dermonecrotic toxin was purified by a simplified method. The method consisted of SP Toyopearl 650M chromatography and high performance liquid chromatography on a TSK gel G3000SW column. 47.5% of the activity of the crude cell extract was recovered. The purified toxin behaved as a homogeneous protein in sodium dodecyl sulfate polyacrylamide gel electrophoresis, high performance liquid chromatography, and agar gel double diffusion tests.

Simplified procedure for purification of Bordetella bronchiseptica dermonecrotic toxin

Bordetella bronchiseptica dermonecrotic toxin was purified by a simplified method. The method consisted of SP Toyopearl 650M chromatography and high performance liquid chromatography on a TSK gel G3000SW column. 47.5% of the activity of the crude cell extract was recovered. The purified toxin behaved as a homogeneous protein in sodium dodecyl sulfate polyacrylamide gel electrophoresis, high performance liquid chromatography, and agar gel double diffusion tests.

Detection of Petunia Asteroid Mosaic, Carnation Ringspot and Tobacco Necrosis Viruses in Ditches and Drainage Canals in a Grapevine‐Growing Area in West Germany

AbstractA number of virus isolates were obtained from ditches and drainage canals in the Palatinate grapevine‐growing area (‘Rheinpfalz’) in West Germany. By means of the immunoelectron microscopical decoration and the agar gel double diffusion tests these isolates were identified as petunia asteroid mosaic virus, carnation ringspot virus and tobacco necrosis virus strain D, respectively.

Biochemical and serological properties of Actinobacillus pleuropneumoniae biotype 2 strains isolated from swine

The biochemical and serological properties of 21 strains of Actinobacillus pleuropneumoniae biotype 2 isolated from haemorrhagic necrotic pleuropneumonia of swine were examined. For serologic typing, the indirect haemagglutination (IHA) and the double gel-diffusion tests were used. On the basis of their soluble surface antigens, our A. pleuropneumoniae biotype 2 isolates could be assigned to two proposed serotypes. Serotype 1 comprised 11 strains and serotype 2 comprised 10 strains. All strains contained two surface antigen components. In the strains belonging to serotype 1, one of the antigens was identical with the serotype-specific antigen of Pasteurella haemolytica T4. Both antigens of serotype 2 strains proved to be type-specific. Four strains received from Switzerland, including the holotype strain of A. pleuropneumoniae biotype 2, and three strains isolated from swine in the G.D.R. belonged to serotype 2. Both the double gel diffusion and the IHA tests detected a 2-way cross-reaction between biotype 1, serotype 2 and biotype 2, serotype 2 strains of A. pleuropneumoniae, which could be eliminated using cross-absorbed sera.

Purification and some properties of a Vero toxin from a human strain of Escherichia coli that is immunologically related to Shiga-like toxin II (VT2)

A cytotoxin to Vero cells (Vero toxin), which was immunologically related to Shiga-like toxin II (SLT-II) (or VT2), was purified from a strain of Escherichia coli isolated from a patient with hemolytic uremic syndrome. The toxin was active on Vero cells but much less active on HeLa cells, a property similar to that of the recently identified SLT-II variant from E. coli strains that caused edema disease of swine. Thus the toxin purified in this report was tentatively named Shiga-like toxin II variant (Vero toxin 2 variant). The purification procedures consisted of ammonium sulfate fractionation, DEAE-Sepharose CL-6B column chromatography, chromatofocusing column chromatography, and repeated high performance liquid chromatography (HPLC) on TSK-gel G-2000SW column and on TSK-gel DEAE-5PW columns. About 90 μg of purified toxin was obtained from 45 I of the culture supernatant with a yield of about 16%. The purified toxin consisted of A and B subunits of molecular sizes similar to those of SLT-II (VT2). The isoelectric point of the purified toxin was 6.1, which was different from that of SLT-II (VT2) (pl = 4.1). In an Ouchterlony double gel diffusion test, purified toxin and SLT-II (VT2) formed precipitin lines with spur formation against anti-purified toxin and anti-SLT-II (anti-VT2), respectively. The purified toxin was cytotoxic to Vero cells, about 6 pg of the toxin killing 50% of the Vero cells, and showed lethal toxicity to mice when injected intraperitoneally, the LD 50 being about 2.7 ng per mouse.

Characterization of Dogwood Mosaic Nepovirus fromCornus florida

A virus serologically related to Arabis mosaic virus was isolated from dogwood (Cornus florida) growing wild in South Carolina in two areas about 0.4 km apart. The virus reacted with antisera to four Arabis mosaic virus strains in gel double-diffusion tests and formed spurs with three strains of Arabis mosaic virus and with grape fanleaf virus. Based on epitope similarity indices, this virus was different from Arabis mosaic virus; therefore it was designated dogwood mosaic virus, a new member of the Arabis mosaic virus subgroup of the nepoviruses

A Strain of Eggplant Mosaic Virus Causing a Severe Disease of Tomato in Argentina

AbstractA virus causing a severe disease of tomatoes in Argentina was identified as a strain of eggplant mosaic virus (EMV). It resembles the type, Abelia latent and various Andean potato latent strains of EMV in its host range and its transmissibility at allow rate by Epitrix sp. It differs from these strains, however, serologically and in some of its cytopathic effects. In serological agar gel double diffusion tests it proved to be closely related to the tomato white necrosis isolate of EMV studied by Barradas (1983) in Brazil.

Antigenic characteristics of larval Paragonimus westermani.

The characteristics of the antigens in larval flukes of Paragonimus westermani were examined. The agar gel double-diffusion test and competitive enzyme-linked immunosorbent assay, using the antiserum against the antigen extracted from larval flukes, showed cross-reactivity between the larval and adult antigens. In immunoblotting analysis, there was a decrease in reactivity of the antiserum absorbed with the adult fluke antigen against the band with a molecular weight of 26, 000. However, the bands corresponding to 34, 000 or higher molecular weights were still detected. The immunoperoxidase-staining technique revealed that the antigens reacting to the unabsorbed and absorbed antisera were both located on the surface of the gut epithelium and in the luminal contents of larval flukes. These results suggest that the extract of larval P. westermani possesses at least two antigens, originating probably from the gut, one being common to the adult and the other characteristic of the larva.

Properties of a new strain of tobacco streak virus from Clematis vitalba (Ranunculaceae)

SUMMARYA distinct strain of tobacco streak virus (TS V/Cle), isolated in Yugoslavia from wild Clematis vitalba showing chlorotic spots or yellow netting of the leaves and from many symptomless shrubs, is described.TSV/Cle was seed transmitted in C. vitalba (70%), and in the experimental hosts Chenopodium quinoa (80%), Nicotiana benthamiana and N. megalosiphon. It was also detected in the pollen of infected C. quinoa. Purified virus preparations mostly contained quasi‐spherical particles measuring 24–26 × 28, 26–28 × 28–30 and 28–31 × 32–36 mn, and sedimented in sucrose density gradient and analytical centrifugation as three components with sedimentation coefficients of 76S, 87S and 98S. The virus contained a single polypeptide species of mol. wt of c. 25 000. Unfractionated TSV/Cle preparations contained four RNA species with mol. wts, estimated by gel electrophoresis in agarose, of 1.1 × 106, 0.9 × 106, 0.7 × 106 and 0.3 × 106. In comparative experiments, TSV/Cle differed from four reference strains of TSV (TSV/B, TSV/HF, TSV/RN, and TSV/Ro) in host range and in symptoms induced in some common hosts. In agar gel double diffusion tests it was more closely related to TSV/B and TSV/M (SDI = 5) than to TSV/HF (SDI = 7), TSV/RN (SDI = 7) or TSV/Ro (SDI = 5–8). Immunoelectrophoresis experiments clearly distinguished TSV/Cle from the reference strains. TSV/Cle strain was detected in C. vitalba plants from distant and climatically different regions in Yugoslavia.

Purification and some properties of a Vero toxin from Escherichia coli O157:H7 that is immunologically unrelated to Shiga toxin

A cytotoxin to Vero cells (Vero toxin) was purified from Escherichia coli O157:H7 isolated from a patient with hemorrhagic colitis by ammonium sulfate fractionation, DEAE-cellulose column chromatography, repeated chromatofocusing column chromatography and repeated high performance liquid chromatography. About 440 μg of purified Vero toxin was obtained from 12 liters of culture with a yield of about 22%. The purified Vero toxin showed similar cytotoxic activity to that of Shiga toxin to Vero cells and killed about 50% of the Vero cells at 1 pg. The activity was lost on heating the toxin at 80 °C for 10 minutes, but not at 60 °C for 10 minutes. The toxin also showed lethal toxicity to mice when injected intraperitoneally, the LD 50 being 1 ng per mouse. The purified Vero toxin consisted of A and B subunits with molecular weights of about 35000 and 10700, respectively, which were slightly larger than those of Shiga toxin. On polyacrylamide gel disc electrophoresis, the mobility of the purified Vero toxin differed from that of Shiga toxin. The isoelectric point of the toxin was 4.1, which was also different from that of Shiga toxin (pl = 7.0). Furthermore, Vero toxin and Shiga toxin were found to be immunologically unrelated; anti-Vero toxin did not react with Shiga toxin, and similarly anti-Shiga toxin did not react with the Vero toxin in either the Ouchterlony double gel diffusion test or enzyme-linked immunosorbent assay. The Vero toxin purified in this work was found to be immunologically identical to VT2 and Shiga-like toxin II reported previously.