A comparison of serological techniques for detecting and identifying honeybee viruses

The sensitivity and specificity of conventional Ouchterlony gel-diffusion, immuno-osmoelectrophoresis (IO), immune serum electron microscopy (ISEM), “decoration,” radioimmunoassay (RIA), and enzyme-linked immunosorbent assay (ELISA) tests for detecting black queen cell virus (BQCV), chronic bee paralysis virus (CBPV), Kashmir bee virus (KBV), and sacbrood virus (SBV) particles in extracts of diseased honeybees were compared. A “slow” ISEM method detected virus particles in extracts of individuals or groups of individuals diluted to 10^?3 and 10^?4, respectively, whereas the IO method and a “fast” ISEM method using protein A were one-tenth as sensitive, and Ouchterlony gel-diffusion tests were only one-thousandth as sensitive. Using the antibody “decoration” technique, mixtures of serologically unrelated virus particles could be resolved. RIA and ELISA were found to be one thousand times more sensitive than ISEM in detecting the particles of BQCV, CBPV, KBV, and SBV; however, nonspecific reactions occurred when using RIA with very dilute particle suspensions, and this made dilution endpoints difficult to assess, but this did not occur when using the ELISA method. There was little difference in the effectiveness of rabbit or hen antisera in the tests, except when protein A was used as it does not combine with hen antibodies.     

The acetylcholinesterase ichthyotoxin is a common component in the extracellular products of Vibrionaceae strains

In previous work, it was reported that a strain of Aeromonas hydrophila (B₃₂) produces the most potent lethal toxin with neurotoxic activity described so far for fish. In the present study, the presence and distribution of this acetylcholinesterase toxin lethal for fish were determined in extracellular products (ECP) of 42 Vibrionaceae strains using both immunological and colorimetric methods. This neurotoxin was shown to be present in the majority of the ECP from the Aeromonas and Vibrio strains tested and is responsible for the specific acetylcholinesterase activity. Also, although the Western blot and Ouchterlony techniques are valid as qualitative methods for the detection of this toxin, the Western blot procedure was 100-fold more sensitive than the Ouchterlony technique.     

The acetylcholinesterase ichthyotoxin is a common component in the extracellular products of Vibrionaceae strains

In previous work, it was reported that a strain of Aeromonas hydrophila (B32) produces the most potent lethal toxin with neurotoxic activity described so far for fish. In the present study, the presence and distribution of this acetylcholinesterase toxin lethal for fish were determined in extracellular products (ECP) of 42 Vibrionaceae strains using both immunological and colorimetric methods. This neurotoxin was shown to be present in the majority of the ECP from the Aeromonas and Vibrio strains tested and is responsible for the specific acetylcholinesterase activity. Also, although the Western blot and Ouchterlony techniques are valid as qualitative methods for the detection of this toxin, the Western blot procedure was 100-fold more sensitive than the Ouchterlony technique.     

Some applications of the surface-spreading technique to virological studies

By application of the surface-spreading technique, virus particles in infected cells and viremia serum, and precipitates in agar plates of the double immunodiffusion technique of Ouchterlony could easily and clearly be visualized without any purification process.     

Enzyme-linked immunosorbent assay (ELISA) as a means of taxonomic analysis of Streptomyces and related organisms

Fourteen Streptomyces strains from various numerical taxonomic classes and representatives of three other genera of actinomycetes were studied using an indirect enzyme-linked immunosorbent assay (IND-ELISA) to determine their serological relationships. The IND-ELISA results agreed with those from previous numerical taxonomic analyses and Ouchterlony double-diffusion studies. The IND-ELISA method is quicker, more quantitative and less subjective than Ouchterlony assays and thus should be useful in Streptomyces taxonomy. The results indicated that Frankia sp. CpI1 was related to Streptomyces.     

Circulating antigens in infections of mice by tetrathyridia of Mesocestoides corti Hoeppli, 1925

Two circulating antigens were detected in the serum of ICR/Timco female mice infected intraperitonealy with tetrathyridia of the cestode Mesocestoides corti Hoeppli, 1925. One circulating antigen appeared by day 2 postinfection (p.i.) and remained in all mice until at least 90 days p.i. A second antigen appeared in the serum on day 14 p.i. and disappeared from all mice by day 28 p.i. Infected mouse serum also contained antibodies against one secretory/excretory antigen and two antigens in crude homogenate, as judged by double diffusion in two dimensions (Ouchterlony). Immune deposits were observed in the kidney tissue of Rockland mice by transmission electron microscopy, and their identity as products of tetrathyridia was confirmed by immunofluorescence. Further studies showed that the main antibody subclass associated with the mesangial immune deposits was 7S gamma l, and that other subclasses of IgG and IgM were not involved. Antigen was found in the proximal renal tubules of infected mice, as demonstrated by fluorescein-labeled IgG fraction of rabbit antitetrathyridia secretory/excretory antigen antisera. The presence of tetrathyridia antigen in the urine of infected mice was confirmed using the Ouchterlony technique.     

Applications of a Synthetic Neuraminidase Substrate

A rapid and precise assay for neuraminidase using 2-(3'-methoxyphenyl)-N-acetyl-alpha-neuraminic acid (MPN) is described. It is proposed that this substrate be used for the standardization of activity of neuraminidases from viral, bacterial, and mammalian sources. MPN is also used as a chromogenic substrate to localize influenza and parainfluenza virus foci in tissue culture. This technique permits the recovery of infective virus from these stained "plaques." It has also been demonstrated that immunoprecipitin lines containing neuraminidase complexes with antibody in the Ouchterlony test can be observed by a similar staining procedure. No enzyme inhibition occurs in the presence of anti-neuraminidase antibodies or concanavalin A when MPN is used as a substrate in contrast to the results with high-molecular-weight substrates such as fetuin.     

Localization of phosphoprotein C23 to nucleolar structures and to the nucleolus organizer regions

After extraction of Novikoff hepatoma nucleoli with 4 M urea/3 M LiCl, phosphoprotein C23 was isolated by DEAE-cellulose and Bio-Rad AG3-X4A column chromatography. Immunization of rabbits with the highly purified protein C23 resulted in the production of a specific antibody as determined by Ouchterlony diffusion analysis. When the immunoperoxidase method was used to localize protein C23 in cells, it was found in ‘fibrillar centers’ (nucleolonemas) in nucleoli. Protein C23 was also demonstrated to be present on the nucleolus organizer regions (NORs) of metaphase chromosomes.     

Immunohistochemical demonstration of carbonic anhydrase.

Rabbits were immunized using human erythroxyte carbonic anhydrase B (HCA B) purified by the modified methods of Armstrong et al. (1966) and Bernstein and Schraer (1972). The globulin fraction was isolated by ammonium sulphate precipitation. The anti-HCA B globulin was specific, when judged using the double diffusion technique of Ouchterlony and immunoelectrophoresis. No cross reaction with human erythrocyte carbonic anhydrase C was found, but cross reactions with erythrocyte carbonic anhydrase from rat, mouse and guinea pig were observed. Flurorescein isothiocyanate conjugated goat anti-rabbit globulin was used for the localization of HCA B in tissue sections and erythrocytes on slides.     

Immunohistochemical demonstration of carbonic anhydrase

Summary Rabbits were immunized using human erythroxyte carbonic anhydrase B (HCA B) purified. by the modified methods of Armstrong et al. (1966) and Bernstein and Schraer (1972). The globulin fraction was isolated by ammonium sulphate precipitation. The anti-HCA B globulin was specific, when judged using the double diffusion technique of Ouchterlony and immunoelectrophoresis. No cross reaction with human erythrocyte carbonic anhydrase C was found, but cross reactions with erythrocyte carbonic anhydrase from rat, mouse and guinea pig were observed. Fluorescein isothiocyanate conjugated goat anti-rabbit globulin was used for the localization of HCA B in tissue sections and erythrocytes on slides.     

Evolution of alkaline phosphatase in marine species of Vibrio.

The evolution of alkaline phosphatase was studied in marine species of Vibrio. Two antisera prepared against purified alkaline phosphatases from Vibrio splendidus and Vibrio harveyi were used to estimate the amino acid sequence divergence of this enzyme in 51 strains belonging to nine species. The methods used were the quantitative microcomplement fixation technique and the Ouchterlony double-diffusion procedure. There was a high degree of congruence between the measurement of the amino acid sequence divergence of alkaline phosphatase and the percentage of deoxyribonucleic acid homology of the different organisms relative to both reference strains (correlation coefficient of -0.89) as well as between the amino acid sequence divergence of alkaline phosphatase and superoxide dismutase (correlation coefficient of 0.92) relative to V. splendidus. These findings supported the view that the evolution of marine species of Vibrio is primarily vertical and that horizontal evolution (involving genetic exchange between species), if significant, is restricted to a minor fraction of the bacterial genome.     

Antigenic relatedness of immunoglobulins toLegionella pneumophila serogroups 1–6 from humans and the nonhuman primateMacaca fascicularis

The purpose of this study was to determine whether cynomolgus monkey antisera toLegionella pneumophila serogroups 1–6 antigens could be used as positive controls in the indirect immunofluorescence assay (IFA) for legionellosis. Immunoelectrophoretic mobilities and Ouchterlony analyses with heavy chain-specific antisera and IFA titers with immunoglobulin class-specific conjugates were used to show antigenic relatedness of immunized monkey immunoglobulins to those produced as a result of infection in humans. Identical immunoelectrophoretic precipitation patterns were obtained for human and monkey sera with antihuman gamma, mu, and alpha heavy-chain-specific antisera. Ouchterlony analyses showed precipitin bands of partial identity between human and monkey IgG, IgM, and IgA classes. IFA titers in the monkey hyperimmune antisera were >16,000 with antihuman conjugate. These data suggest that hyperimmune cynomolgus monkey antisera are suitable alternatives to human sera for IFA-positive controls.     

Novel immunochemical characteristics of glutathione S-transferase isozymes of rat liver cytosol

Reevaluation of the immunochemical relationships among the individual glutathione S-transferase (GST) isozymes (GSTs 1-1, 1-2, 2-2, 3-3, 3-4, 4-4, and GSTs with isoelectric points of 7.5 and 6.8) of rat liver cytosol was performed utilizing the immunoblot technique. As a result, we found that the respective isozymes of two isozyme classes of rat liver cytosol might possess a common epitope(s) which has been undetected by the Ouchterlony double-diffusion method. The assumption was further supported by the results of the effects of Fab' prepared from some anti-GST antibodies on the enzymatic activity of GSTs.     

Immunochemistry of Biliproteins

Biliproteins were extracted from representatives of the Cyanophyta, Rhodophyta, and Cryptophyta and purified. Both purified and crude biliproteins were used to stimulate rabbit antibody directed specifically against the biliproteins. The antigenic and immunogenic inter-relationships of these proteins were investigated by the Ouchterlony double diffusion technique. C-phycocyanins from all sources were found to be antigenically and immunogenically related and apparently also related to allophycocyanin but not to any of the phycoerythrins. Larger antigenic differences among phycoerythrins from different groups of algae were discovered. The role of aggregation of the individual biliproteins in their immunochemistry was characterized. Attempts were made to determine the phylogenetic significance of these results. The immunochemical aspects of the biliproteins were striking in that protein antigens from vastly different cell types were found to be closely related. This relationship may be interpreted as supporting the suggestion that Rhodophyta evolved from Cyanophyta or from some common ancestral stock.     

Immunochemical characteristics of cholesterol-hydroxylating cytochrome P-450 from adrenal cortex mitochondria

Highly specific antibodies against hemeprotein were obtained by immunizing rabbits with a highly purified cholesterol-hydroxylating cytochrome P-450scc from adrenocortical mitochondria. The antibodies do not specifically interact with other components of the adrenocortical electron transport chain, e. g., adrenodoxin reductase and adrenodoxin. Using double immunodiffusion technique (Ouchterlony method), it was shown that the antibodies did not precipitate the microsomal cytochromes P-450 LM2 and LM4, cytochrome b5 and 11 beta-hydroxylating cytochrome P-450 from adrenocortical mitochondria. Antibodies against cytochrome P-450scc inhibited the cholesterol side chain cleavage activity of cytochrome P-450scc in a reconstituted system. Limited proteolysis with trypsin and immunoelectrophoresis in the presence of specific antibodies revealed that antigenic determinants are present of the heme-containing catalytic domain of cytochrome P-450scc (F1) as well as on the domain responsible for the interaction with the phospholipid membrane (F2).     

Detecting the Enterotoxigenicity of Staphylococcus aureus Strains

An optimal sensitivity plate method for examining large number of staphylococcal strains for production of the known enterotoxins (A-E) is presented. Small volumes of relatively concentrated enterotoxin are produced by the semi-solid agar, cellophane-over-agar, or sac culture techniques. Detection of the enterotoxin in the supernatant fluid is accomplished with the optimal sensitivity plate method. In this method small plastic petri dishes (50 mm) were used for a modified Ouchterlony of high sensitivity.     

Subunit structure and immunological properties of wheat carboxypeptidase

Wheat carboxypeptidases I, II, III and IV from wheat seeds with isoelectric points of 4.8, 5.6, 6.0 and 6.5, respectively, were found to be homogeneous by the Ouchterlony double immunodiffusion technique using an antiserum of the enzyme III. In a previous paper [1], the native enzyme III (MW = 118 k) was separated into two 58 k subunits (MW = 58 k) and further divided into the 35 k and 25 k fragments (MW = 35 k and 25 k, respectively). The native enzyme III and the 58 k subunit produced a single precipitin line against the antiserum. The 35 k and 25 k fragments did not cross-react with the antiserum. The amino acid compositions of the 35 k and 25 k fragments were similar to each other. Amino-terminal amino acids of the 35 k and 25 k fragments were both glutamic acid. Carboxy-terminal groups of the 35k and 25k fragments were determined to be -(Gly, Ser)-Glu-OH and -Thr-Pro-Glu-OH, respectively. The Ouchterlony double immunodiffusion technique revealed the presence of a common antigen in a carboxypeptidase from wheat seeds and one from germinated wheat. Comparison of both enzymes is discussed.     

Serological Studies on Actinobacillus actinomycetem-comitans

One hundred strains of Actinobacillus actinomycetem-comitans were examined serologically by determining their agglutination reactions with six absorbed antisera. Twenty-four reaction patterns were found, and the results were reproducible. The agglutinating antigens were heat-stable. Precipitation reactions were also studied by means of the Ouchterlony agar-gel diffusion technique with Fuller and acetone extracts. The agglutination test was considered preferable for epidemiological and ecological investigations on A. actinomycetem-comitans.     

Purification and Characterization of a Protease Produced by Streptococcus Faecalis Var. Liquefaciens

Abstract

An extracellular protease produced by Streptococcus faecalis var. liquefaciens has been purified 535 fold by standard purification procedures. The homogeneity of the enzyme preparation was demonstrated by disc electrophoresis, isoelectrofocusing, sedimentation equilibrium and the Ouchterlony technique. The enzyme has a molecular weight of approximately 30,000 with an isoelectric point of 4.7. Kinetic values with casein as a substrate, show it to have a K of 9.1 × 10 ^?2mmoles with a V_max of 0.454 ΔO.D. max     

Serological studies of Bratislava mosaic virus of grape vine

Bratislava mosaic virus was transmitted mechanically toChenopodium quinoa. The virus was isolated by means of gradient ultracentrifugation and the, proteins were distributed on an automatic recording photometer. Single fractions were taken with a fraction collector and used as antigen. At the same time, antigen from leaves of the variety Sylván zelený infected with the Bratislava mosaic virus was prepared by means of the gradient ultracentrifugation. The antisera were obtained from rabbits immunized with individual antigens. The antisera were tested with the saps ofC. quinoa infected with the Bratislava mosaic virus, by the method of double diffusion into agar according to Ouchterlony, and with adjusted saps from suckers infected with the Bratislava mosaic virus, by the method of double diffusion into agar according to Oudin. A specific reaction can be obtained in both cases only under the assumption that all conditions mentioned in the study are strictly kept.