Ouchterlony Tests Research Articles

An assessment of the antigenic homogeneity of Edwardsiella ictaluri using monoclonal antibody

Abstract. Monoclonal antibodies were made against the reference strain of Edwardsiella ictaluri (ATCC 3320). Antibody produced by one of seven anti‐E. ictaluri hybridomas reacted positively by the immunofluorescent antibody technique against 17 other E. ictaluri isolates. All hybridoma antibodies failed to react with six other bacterial species pathogenic to fish including E. tarda. Ouchterlony tests indicated that four anti‐E. ictaluri clones produced only one kind of immunoglobulin. Electrophoresis of 14 different E. ictaluri isolates indicated identical protein bands at 36 and 60 kilodaltons (KD) in all isolates except an isolate from Thailand. Using the immunoblot method, channel catfish anti‐E. ictaluri serum reacted with protein bands at 34 and 60 KD, which indicates that this molecular weight protein in the bacterium may be the dominant immunoprotein.

Proteasomes (multi-protease complexes) as 20 S ring-shaped particles in a variety of eukaryotic cells.

Latent multicatalytic protease complexes, named proteasomes, were purified to apparent homogeneity from various eukaryotic sources, such as human, rat, and chicken liver, Xenopus laevis ovary, and yeast (Saccharomyces cerevisiae), and their functional and structural properties were compared. They showed latency in breakdown of [methyl-3H]casein, but were greatly activated in various ways, such as by addition of polylysine. They all degraded three types of fluorogenic oligopeptides at the carboxyl side of basic, neutral, and acidic amino acids, and the three cleavage reactions showed different spectra for inhibition, suggesting that they had three distinct active sites. The proteasomes all seemed to be seryl endopeptidases with similar pH optima in the weakly alkaline region. Their physiochemical properties, such as their sedimentation coefficients (19 S to 22 S), diffusion coefficients (2.0-2.6 X 10(-7) cm2 s-1), molecular masses (700-900 kDa), and circular dichroic spectra, were similar. Their amino acid compositions were also very similar. Electron microscopy showed that they had similar well-defined symmetrical morphology, appearing to be ring-shaped particles with a small hole in the center. All the proteasomes seemed to be multisubunit complexes consisting of 15-20 polypeptides with molecular masses of 22-33 kDa and isoelectric points of pH 3-10, but they showed species-specific differences in subunit multiplicity. Moreover, they differed immunologically, as shown by Ouchterlony tests and immunoblotting analyses, although cross-immunoreactivities of some subunits or domains were observed. These results indicate that the sizes and shapes of these proteasomes have been highly conserved during evolution, but that they show species-specific differences in immunoreactivities and subunit structures. Thus proteasomes with similar structure and function seem to be ubiquitously distributed in eukaryotic organisms ranging from man to yeast. This distribution implies the general importance of these proteasomes for proteolysis.

Purification and characterization of heat-labile enterotoxin isolated from chicken enterotoxigenicEscherichia coli

The heat-labile enterotoxin (LTc) isolated from chicken enterotoxigenic Escherichia coli was purified to homogeneity and its molecular and antigenic properties were compared with those of purified LTs from porcine and human enterotoxigenic Escherichia coli (LTp, LTh). The A subunit of LTc was identical to that of LTp and the B subunit of LTc was identical to that of LTh but not that of LTp, in mobility on SDS-polyacrylamide gel electrophoresis. Ouchterlony tests demonstrated that LTc is antigenically identical to LTh but not with LTp. The pI point and amino acid composition of LTc were also compared and the results suggest that chicken enterotoxigenic E. coli produced an LT similar to LTh.

Clinical and immunological features of Sjögren's syndrome in patients with primary biliary cirrhosis with emphasis on focal sialadenitis.

Serological and pathological findings in 21 patients with primary Sjögren's syndrome (primary SS) were compared with those in 32 patients with primary biliary cirrhosis (PBC). In ELISA, anti-SS-B/La antibodies were detected in sera from 14 (67%) of the patients with primary SS, but only from 12 (38%) of those with PBC. With the Ouchterlony test, anti-SS-A/Ro antibodies were found in sera from 15 (71%) of the primary SS patients, but in no PBC patient. Of those PBC patients investigated prospectively with objective tests, four of 11 (36%) had keratoconjunctivitis sicca, and five of 15 (33%) had pathological sialometry results. In contrast, all PBC patients but one (i.e., 14 of 15 or 93%) showed evidence of focal sialadenitis. In immunochemical study of PBC patients, IgM immunoreactivity was found in the stroma, particularly adjacent to excretory ducts and acini in salivary glands (5 of 5), whereas no such IgM deposits were observed in patients with primary SS (3 of 3), nor in healthy controls (n = 20). We conclude that the frequency of anti-SS-A/Ro and anti-SS-B/La antibodies in serum is lower in PBC patients than in patients with primary SS. The incidence of focal sialadenitis is high in PBC, though only one third of the PBC patients studied here showed clinical evidence of glandular dysfunction. With immunochemical techniques, sialadenitis associated with PBC is distinguishable by its significant IgM reaction from sialadenitis in primary SS.

Lens neutral endopeptidase occurs in other bovine and human tissues.

Lens neutral endopeptidase (EC 3.4.24.5) was previously thought to be unique to the eye lens. We report here the finding of a neutral endopeptidase, in a variety of bovine and human tissues, which is very similar both biochemically and immunologically to the lens endopeptidase. SDS/polyacrylamide-gel electrophoresis of partially purified enzyme fractions from various bovine tissues shows the characteristic pattern of at least eight bands with Mr values ranging from 24,000 to 32,000 which was described for the bovine-lens neutral endopeptidase. The relative activity of the enzyme varies from tissue to tissue with lung having the highest activity. Partially purified enzyme fractions from these tissues cross-react with antiserum raised in rabbit against bovine lens endopeptidase showing apparent identity when examined side by side in Ouchterlony double-diffusion tests. The human enzyme also cross-reacts with the antiserum but when tested by double-diffusion against the bovine enzyme the precipitin lines show spurring at the joining edges indicating a structural difference between the human and the bovine enzymes. It was also found by Western blot experiments, after denaturing polyacrylamide-gel electrophoresis of the enzyme, that the polypeptide components of the human and bovine enzymes show somewhat different banding patterns.

Host immunoglobulin G titre and antibody activity in haemolymph of the tick, Ornithodoros moubata.

Immunoglobulin G (IgG) in tick haemolymph was analysed immunochemically and biochemically for its antigenicity, antibody activity and relative concentration in a soft tick, Ornithodoros moubata (Murray) sensu Walton 1962 (Acari: Argasidae). Ouchterlony immunodiffusion tests showed that haemolymph from a tick engorged on rabbit IgG (or human IgG) through an artificial membrane, reacted with anti-rabbit IgG (anti-human IgG) but not with anti-human IgG (anti-rabbit IgG). This indicates that haemolymph of the fed tick contains IgG with a similar antigen specificity to host blood IgG. IgG from tick haemolymph was demonstrated by enzyme immunoassay to have the same antibody activity as ingested IgG. The IgG concentration in tick haemolymph was measured by a quantitative single immunodiffusion test. Changes of IgG titre after a bloodmeal were correlated with IgG activity, which was low for 5 days after a bloodmeal and then suddenly increased. The IgG titre reached a maximum 7 days post-engorgement, and remained high for over 4 months during and after oviposition. 125I-labelled IgG was injected into the tick haemocoel to determine the persistence of IgG in the haemolymph. Recovery of labelled IgG was low at 1 and 3 days, and high at 5, 8 and 16 days after engorgement. The data suggest that IgG in haemolymph disappears quickly soon after engorgement possibly by degradation and/or absorption (adhesion to tissues).

β Trace-protein as marker for cerebrospinal fluid fistula

The detection of beta trace-protein with the help of an easy-to-perform Ouchterlony test is strongly indicative of liquorrhea. The protein has been purified from pooled cerebrospinal fluid and an antiserum has been produced in rabbits. An open CSF fistula was diagnosed in 24 cases and there was no false positive result.

A unique amino acid sequence of the B subunit of a heat-labile enterotoxin isolated from a human enterotoxigenic Escherichia coli

The purified B subunit of heat-labile enterotoxin produced from a human strain, 240-3, of enterotoxigenic Escherichia coli (LTh(240-3)) was carboxymethylated, succinylated, digested with chymotrypsin and subjected to high performance liquid chromatography (HPLC), and the amino acid compositions of the peptide peaks from the column were analyzed and compared with the data reported by Yamamoto and Yokota ( J. Bacteriol. 155, 728.1983), who deduced the amino acid sequence of LTh(H10407) from the DNA sequence of a human strain H10407. Only one fraction differed in amino acid composition from that reported by them. This fraction was found to consist of peptides with the sequences Arg-Asn-Thr-Gln-Ile-Tyr and Arg-Ile-Ala-Tyr. Yamamoto and Yokota reported the sequence of the latter peptide as Arg-Ile-Thr ∗-Tyr, which corresponds to the peptide from 73rd to 76th from amino (N-) terminus. Thus amino acid residue 75 from the N-terminus of LTh-B(240-3) is alanine, not threonine. The B subunit of cholera toxin also has alanine at position 75. LTh(240-3) appeared similar to LTh(H10407) in an Ouchterlony test, vascular permeability test and GMI ganglioside ELISA. These data show that substitution of threonine for alanine at position 75 from the N-terminus does not affect the immunological and biological characteristics of LTh.

Purification and Characterization of Two Endoxylanases from Clostridium acetobutylicum ATCC 824

Two endoxylanases produced by C. acetobutylicum ATCC 824 were purified to homogeneity by column chromatography. Xylanase A, which has a molecular weight of 65,000, hydrolyzed larchwood xylan randomly, yielding xylohexaose, xylopentaose, xylotetraose, xylotriose, and xylobiose as end products. Xylanase B, which has a molecular weight of 29,000, also hydrolyzed xylan randomly, giving xylotriose and xylobiose as end products. Xylanase A hydrolyzed carboxymethyl cellulose with a higher specific activity than xylan. It also exhibited high activity on acid-swollen cellulose. Xylanase B showed practically no activity against either cellulose or carboxymethyl cellulose but was able to hydrolyze lichenan with a specific activity similar to that for xylan. Both xylanases had no aryl-beta-xylosidase activity. The smallest oligosaccharides degraded by xylanases A and B were xylohexaose and xylotetraose, respectively. The two xylanases demonstrated similar K(m) and V(max) values but had different pH optima and isoelectric points. Ouchterlony immunodiffusion tests showed that xylanases A and B lacked antigenic similarity.

The coming of the clone

Six antigenic components of murine leukemia viruses (MuLV) were identified by Ouchterlony tests after degradation of the viruses with Tween 80 and ether. A type-specific antigen (IIv), three gs-species (I, IIgs, and IV), and one gs-interspecies (V) antigen were recognized. The range of viral specificity of a further minor antigen (III) is not yet known. For five MuLV antisera, the reactivity spectra were evaluated by complement fixation, Ouchterlony, and neutralization tests. By Ouchterlony and, preferably, by complement fixation tests, it could be shown that the L cell-virus does not belong to either the FMR or the G type; it apparently is still another serotype of MuLV.

Purification and partial characterization of a non-O1 Vibrio cholerae hemolysin that cross-reacts with thermostable direct hemolysin of Vibrio parahaemolyticus.

A newly identified hemolysin (NAG-rTDH), which is related to the thermostable direct hemolysin (Vp-TDH) of Vibrio parahaemolyticus produced by non-O1 Vibrio cholerae, was studied. NAG-rTDH was purified by successive column chromatographies on DEAE-cellulose and an immunoaffinity column coupled with anti-Vp-TDH immunoglobulin. The molecular weight of NAG-rTDH was estimated as 18,500, similar to that of Vp-TDH, as judged by sodium dodecyl sulfate slab gel electrophoresis, but its charge or molecular shape was different, judging from its electrophoretic mobility. The lytic activities of NAG-rTDH on erythrocytes of most animals were essentially similar to those of Vp-TDH, but that on sheep erythrocytes was different. The hemolytic activity of NAG-rTDH was stable on heating at 100 degrees C for 10 min, as was that of Vp-TDH. Immunological cross-reactivity between NAG-rTDH and Vp-TDH was demonstrated by both the Ouchterlony test and the neutralization test. Thus, we conclude that non-O1 V. cholerae produce a new type of hemolysin that is similar but not identical to the thermostable direct hemolysin of V. parahaemolyticus.

Fractionation of antigen for ELISA of bovine fascioliasis

In order to obtain the most specific and sensitive antigen from crude antigens of Fasciola hepatica for the immunodiagnosis of bovine fascioliasis by the enzyme linked immunosorbent assay(ELISA), phosphate buffered saline extract of F. hepatica was prepared. The crude extract was fractionated into 7 antigens using Sephadex G-100 column chromatography. Seven fractionated antigens were applied to ELISA, precipitation test and intradermal test, respectively. Results obtained are as follows: The specificity (95 per cent confidence interval in negative sera of bovine fascioliasis; Mean+2 x SD of absorbance ) of the first (MW>150,000) and the second antigens (MW 120,000) were 93.7 per cent, but those of others including crude antigen showed 100 per cen.t. The sensitivity (positive sera of bovine fascioliasis having higher values with compared to the criterion) of the first, the sixth (MW 16,000) and the seventh antigen (MW<5,000) were 91.6 per cent, 87.5 per cent and 0 per cent, respectively, but those of others showed all 100 per cent. The absorbance by ELISA using the fifth antigen (MW 26,000) was 8.43-folds higher in the positive sera than that in the negative sera. This could be used as one of the most specific antigens for the immunodiagnosis of bovine fascioliasis. In Ouchterlony test, precipitin lines were not found in the sera naturally infected with F. hepatica, but some were found in the sera of rabbits immunized with the crude antigens. The numbers of precipitin lines in the sera of rabbits were different in the different fractionated antigens. They were 6 in the crude, 2 in the second and the third antigens, 1 in the forth, the fifth and the sixth antigens and absent in the seventh antigen, respectively. The wheal size for the bovine infected with F. hepatica was 2.46+/-0.15 cm in the intradermal test antigen (saline extract of F. hepatica) supplied by the Veterinary Research Institute, Rural Development Administration, Korea. The wheal size of the first, the second and the third antigens were larger than that of intradermal test antigen, whereas those of the forth, the fifth, the sixth and the seventh antigens showed smaller than that of the intradermal test antigen. The results suggest that the fifth antigen may be specific antigen for the immunodiagnosis of bovine fascioliasis.

Assay of possible formation of antigenic components in heat-treated plasma protein preparations.

To eliminate the risk of hepatitis infections, human plasma protein preparations can be heated in solution to 60 degrees C for 10 h thus inactivating several viruses. Preclinical safety experiments were performed in order to exclude the possibility of the formation of antigenic components, not present in normal human plasma, through this pasteurization step. Rabbits were immunized with either the heated or the unheated product. Sera were investigated in the Ouchterlony test against the homologous antigen to demonstrate precipitating antibodies. Antisera of rabbits immunized with the heated product were adsorbed with the preparation without the heating step. After centrifugation, the incubates were retested in the Ouchterlony assay. In passive cutaneous anaphylaxis assays guinea-pigs received the adsorbed antisera i.v., and both preparations were injected intracutaneously. The diameters of skin reactions were measured and did not show differences between those treated with the heated and non-heated product. No neoantigens could be detected with the following heated plasma protein fractions: prothrombin, coagulation factors VIII, IX, X, XIII, antithrombin III, fibrinogen, and Cl-inactivator. These results did not apply to heat-denaturated rabbit serum in a validation experiment.

A note on the production and use of antisera for the detection of part‐digested sticklebacks in predator stomach‐content samples

Preliminary experiments indicate that serological techniques provide a useful method of identifying specific prey antigens from the stomach contents of fish predators. Problems with cross‐reacting antigens from different prey species can be reduced by absorbing anti‐sera with tissue extracts of the cross‐reacting species. Very small volumes of part‐digested prey tissue are sufficient for identification of the prey species when using agarose diffusion plates (Ouchterlony tests).

Fungal fimbriae. IV. Composition and properties of fimbriae from Ustilago violacea

The fimbriae of Ustilago violacea consist of long protein fibrils of 7-nm diameter. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis of the fimbriae of this species and of several other species of Ustilago and Rhodotorula demonstrated that they are composed of a protein of 74, 000 Da which can spontaneously assemble into 7-nm fibrils. No carbohydrate moiety was detected. Fimbrial protein retained both its fibrillar structure and antigenicity when exposed to a variety of chemical treatments, and even when autoclaved. Concentrations of cations greater than 10 −1 M (monovalent cations) or 5 × 10 −2 M (divalent cations) resulted in a loss of fimbriae, while the effect of chelators suggested that calcium is important to the structural integrity of fimbriae. Ouchterlony tests using antisera prepared against the fimbriae of U. violacea and R. rubra indicated that while there are differences in the antigenic sites, the fimbrial protein of different basidiomycete species is highly conserved. Fimbrial protein did not react with several mammalian antisera directed against cytoskeletal proteins.

Purification of neutral lens endopeptidase: close similarity to a neutral proteinase in pituitary.

A neutral endopeptidase (EC 3.4.24.5) that degrades alpha- and beta-crystallins occurs in mammalian lens. A procedure for purification of this enzyme from bovine lens is described. The enzyme appears to have a high molecular weight (Mr approximately equal to 700,000) and under denaturing conditions dissociates into at least eight polypeptide subunits with Mrs ranging from 24,000 to 32,000. A neutral proteinase in bovine pituitary has been reported previously to have similar structural characteristics. We have found that this enzyme purified from bovine pituitary is indistinguishable in molecular weight and in subunit composition from bovine lens endopeptidase. In addition, antiserum raised in rabbit against the purified lens enzyme crossreacts with bovine pituitary enzyme. When examined side by side in Ouchterlony double-diffusion tests, the two enzymes give a continuous precipitin line with no spurring. It is concluded that lens neutral endopeptidase and pituitary neutral proteinase are structurally closely similar, if not identical. This is a surprising result because it had been thought previously that the lens endopeptidase was unique to lens, where its crystallin substrates comprise a large proportion of the total tissue protein. In other tissues, crystallin is either absent or occurs, at most, in trace amounts.

Exposure to microorganisms, febrile and airway-obstructive symptoms, immune status and lung function of Swedish farmers.

A questionnaire was sent to 512 farmers, members of a local farmer's health association. Eighty were interviewed, and their serum precipitins and total serum immunoglobulin E (IgE) values were determined. Forty-five underwent extensive pulmonary function tests. On the basis of the clinical evaluation it was estimated that 19% of the farmers had experienced febrile reactions (fever and/or shivering) following exposure to organic dust, 50% of the 19% having been exposed within the last 2.5 years. Common causes were moldy grain, hay, and woodchips. The pulmonary function, gas exchange, and chest radiographs of those who had previously had febrile reactions were normal. Only 13% showed positive precipitin reactions in Ouchterlony double-diffusion tests. With more sensitive tests, positive precipitins were found in 59%, but they were negatively correlated with febrile episodes. Air samples collected during work with hay and grain on 21 farms contained between 10(7) to 2 X 10(9) microorganisms/m3. The highest values were associated with symptoms of alveolitis. Eleven percent of the farmers reported obstructive chest symptoms following exposure to organic dust. This group showed decreased pulmonary function and elevated serum IgE levels and included a high proportion of smokers and exsmokers. No correlation was found between febrile and obstructive reactions.

左きょう部肺吸虫症の１例

A 39-year old man had had painful awelling of the left cheek for 20 days, but no pulmonary symptoms or signs.His cheek was not tender, but there was a 10mm induration in the center of the diffuse swelling. Laboratory examinations revealed no inflammation but indicated an allergic reaction: 10% eosinophils and 8.290 units/ml of IgE.This induration was excised through the left buccal mucosa for diagnosis. One worm 5mm×7mm, was found in the muscle plate and indentified pathalogically as Paragonimus westermani (P. w.) in the masseter muscle. A skin test with veronal buffer saline Paragonimus antigen gave a positive reaction 29mm×30mm. The specific antibody test was more use than the Ouchterlony test or immunoelectrophoresis in the differential diagnosis of this species of fluke.Precipitin bands and a specific band for P.w. were confirmed by these methods.Bithionol therapy 30mg-50mg/kg by mouth every other day for ten doses, as reported in 1961 by Yokogawa M. et al., is effective in almost all cases of P. w.

Chemical and immunological characterization of lipopolysaccharides from phase I and phase II Coxiella burnetii.

Lipopolysaccharides (LPSs) isolated from phase I and phase II Coxiella burnetii (LPS I and LPS II, respectively) were analyzed for chemical compositions, molecular heterogeneity by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and immunological properties. The yields of crude phenol-water extracts from phase I cells were roughly three to six times higher than those from phase II cells. Purification of LPSs by ultracentrifugation gave similar yields for both LPS I and LPS II. Purified LPS I and LPS II contained roughly 0.8 and 0.6% protein, respectively. The fatty acid constituents of the LPSs were different in composition and content, with branched-chain fatty acids representing about 15% of the total. beta-Hydroxymyristic acid was not detected in either LPS I or LPS II. A thiobarbituric acid-periodate-positive compound was evident in the LPSs; however, this component was not identified as 3-deoxy-D-mannooctulosonic acid by gas and paper chromatographies. LPS II contained D-mannose, D-glucose, D-glyceromannoheptose, glucosamine, ethanolamine, 3-deoxy-D-mannooctulosonic acid-like material, phosphate, and fatty acids. LPS I contained the unique disaccharide galactosaminuronyl glucosamine and nine unidentified components in addition to the components of LPS II. The hydrophobic, putative lipid A fraction of LPS I and LPS II contained the above constituents, but the hydrophilic fraction was devoid of ethanolamine. The LPS I disaccharide galactosaminuronyl glucosamine was found in both fractions of the acetic acid hydrolysates. Analysis of LPSs by sodium dodecyl sulfate-polyacrylamide gel electrophoresis followed by silver staining indicated that LPS II was composed of only one band, whereas LPS I consisted of six or more bands with irregular spacing. Ouchterlony immunodiffusion tests demonstrated that LPS I reacted with phase I but not with phase II whole-cell hyperimmune antibody, and LPS II reacted neither with phase I nor phase II hyperimmune antibody. From these results, it was concluded that the chemical structures of LPSs from C. burnetii were different from those of the LPSs of gram-negative bacteria; however, the LPS structural variation in C. burnetii may be similar to the smooth-to-rough mutational variation of saccharide chain length in gram-negative bacteria.

Non-O1 Vibrio cholerae hemolysin: purification, partial characterization, and immunological relatedness to El Tor hemolysin.

Hemolysin of a non-O1 Vibrio cholerae strain was purified and characterized. The purified hemolysin gave a single protein band on conventional and sodium dodecyl sulfate-gel electrophoresis. Its molecular weight was estimated as 60,000 by sodium dodecyl sulfate-polyacrylamide slab gel electrophoresis. It had a pI of 5.7. The purified hemolysin caused increased vascular permeability of rabbit skin and rapid death of mice on intravenous injection and also lysed erythrocytes of various animal species. An Ouchterlony double gel diffusion test using antiserum against the purified hemolysin indicated that the hemolysin from non-O1 V. cholerae was immunologically related, but not identical, to the hemolysin from El Tor V. cholerae. Antiserum against the purified hemolysin neutralized the hemolytic activity of the hemolysins from not only non-O1 but also El Tor V. cholerae.