Thermostability of Antigens Associated with Serotype of Rhizobium japonicum

The antigens associated with serologically distinct strains of Rhizobium japonicum were found to differ in heat sensitivity. Cell preparations from 4 out of 12 strains retained agglutinability, and 1 out of 5 retained antigenicity after they were heated to 120 C. Antigenicity was reduced in most strains after heating to 100 C for 30 min, but agglutinability was not affected by this treatment. This suggests that the antigens are protein-polysaccharide-lipid complexes described for O-type antigens. Cells of strain 46, however, retained both agglutinability and antigenicity after heating to 120 C for 1 hr, and thus a protein in its structure seems improbable. Antigens associated with bacteria from soybean nodules responded to heat treatment in essentially the same manner as those from the same strain grown in nutrient broth. Certain serotypes showed a tendency to agglutinate spontaneously. A heat treatment of 100 C for 30 min, to remove nodule debris and destroy certain blocking antigens, did not interfere with the agglutination reaction. Live cells induced antiserum in rabbit to a higher titer than did heat-treated cells.     

Studies on lectin-induced agglutination of Drosophila embryonic cell lines

The agglutination responses of three Drosophila cell lines to concanavalin A and wheat germ agglutinin have been examined. Although the cell lines were originally derived from late embryonic stages of the Ore-R strain of Drosophila melanogaster, they show quantitative differences in lectin-induced agglutination. Line 1 cells were least agglutinable with both lectins. All three cell lines reached maximum agglutination with concanavalin A concentrations at 25 μg/ml, but the agglutination response to wheat germ agglutinin was biphasic such that an initial rapid increase in agglutination with concentrations up to 25 μg/ml was followed by slower agglutination above this concentration. Cells of lines 1 and 2 from ten-day old cultures exhibited greater lectin-induced agglutination than cells from three-day old cultures. Age-dependent differences were not found for line 3 cells which gave maximum agglutination responses in both young and old cultures. Cell agglutination by concanavalin A was almost completely inhibited by pretreatment of the lectin with methyl-α-d-mannopyranoside, but preincubation of wheat germ agglutinin with N-acetyl-d-glucosamine caused only partial blockage. Lectin-induced agglutination was not reversible by treatment with the monosaccharide inhibitors. These observations have been discussed with reference to the origin of the three cell lines and their cell surface properties.     

Immunochemistry of the surface carbohydrate antigens of Bacteroides fragilis and definition of a common antigen

The components extracted by aqueous phenol from whole cells of Bacteroides fragilis were analysed by SDS-PAGE and immunoblotting and shown to consist of a series of strain-specific, cross-reactive and common antigens. Regularly-spaced ladder patterns on silver-stained gels indicated that in most strains the LPS was present as a predominantly smooth type, but with chain lengths of varying molecular mass, ranging within each particular strain from essentially rough forms to long chain-length smooth forms. The rough form of the LPS at the gel front possessed an antigen common to most of the strains investigated. Another antigen, which migrated behind the rough LPS on SDS gels, was common to all strains of the species. The smooth LPS forms and the other high molecular mass components were strain-specific antigens. Previously published methods are not capable of producing pure LPS or capsular polysaccharide for this organism.     

The agglutinability of cholera O-monoclonal immunoglobulins]

A set of 10 monoclonal antibodies specific for vibrio species and of 5 monoclonal antibodies specific for serovar (Ogawa) was studied. These monoclonal antibodies were directed toward V. cholerae O1 antigen in agglutination reaction and on slide plates. Monoclonal antibodies agglutinating typical strains of cholera vibrios with titration range from 1: 6000 to 1: 25,000 were selected. MA were revealed to interact with cholera vibrio strains with reduced agglutinability. The set of agglutinating O monoclonal immunoglobulins was developed for laboratory identification of cholera O1 vibrios.     

Serological characteristics within the genus Desulfovibrio

Antisera were prepared against one strain each of Desulfovibrio desulfuricans, D. vulgaris and D. salexigens. The antisera were tested for cross reactivity against 36 heterologous Desulfovibrio strains by both agglutination titration and by double immunodiffusion precipitin plates.Generally no cross-reaction was demonstrated by agglutination even between heterologous strains of the same species, suggesting that the surface antigens of Desulfovibrio are highly specific. In immunodiffusion plates a single apparently genus-specific surface antigen could be shown to be present in all but two of the strains tested. Although other common precipitin bands showed the presence of some antigens common between heterologous strains these appeared to be randomly distributed among the strains tested, with the exception of one band shown to be generally specific to strains of D. salexigens. With this exception no other precipitin band could be shown to be consistently specific to any other species, nor consistently common to more than one species.     

Antigenic characteristics of various strains of Campylobacter jejuni. Preliminary note: technic of extraction of soluble thermostable antigens

Immune serum from rabbit has been obtained against Campylobacter jejuni ATCC no 29428 from the same strain an antigen of the outer Envelope was prepared by EDTA extraction. The specificity of antibodies against the outer antigen has been observed by direct agglutination, C.F., passive haemoagglutination and houcherlony test in agar. In eight other strains of Campylobacter isolated from patients, cross reactions with the ATCC no 29428 strain have been stored, by direct agglutination: only one strain (H) cross-reacted with the immune serum in use.     

Mating reaction inSaccharomyces cerevisiae

The effect of proteolytic enzymes on sexual agglutinability of haploid cells of the yeastSaccharomyces cerevisiae was examined. Sexual agglutinability of cells of botha and α types was lost on treatment with alkaline protease and two kinds of neutral proteases ofBacillus subtilis, pronase and α-chymotrypsin. Agglutinability of α type cells was lost after treatment with acid protease ofRhizopus chinensis and trypsin, but that ofa type cells was not. These results indicate that the sex-specific substance responsible for the sexual agglutination (agglutination factor) ina type cells differs from that in α type cells. Agglutination factors were solubilized from cell-wall fractions of both mating types by Glusulase treatment. These crude factors specifically inhibited the agglutinability of cells of the opposite mating type with little effect on the agglutinability of cells of the same mating type.     

Concanavalin A-mediated agglutination of 3T3 cells after exposure to UV-irradiated herpes simplex virus.

Studies were made to determine the effect of UV-irradiation of herpes simplex virus (HSV) on Concanavalin A (Con-A)-mediated agglutination of 3T3 cells. There were three different phases of agglutination by Con-A of cells infected with HSV. The agglutinability began to increase from 3 or 4 hr, or 72 hr after exposure of cells to HSV. The early-appearing agglutinability was further divided into two phases, based on its sensitivity to metabolic inhibitors. These were tentatively called "Early 1 or inhibitor sensitive", "Early 2 or inhibitor insensitive" and "Late" agglutinability. "Early 1" agglutination, detected from 3 hr post infection (pi), was induced by treating cells with HSV, either active or UV-irradiated for less than 5 min and was inhibited when actinomycin D (1 microgram/ml) or cycloheximide (50 microgram/ml) was added to the cultures. "Early 2" agglutination began to increase from 4 hr pi when cells were inoculated with HSV irradiated for 7 to 20 min and was not affected by either inhibitor. HSV irradiated for 6 min failed to induce either agglutinability. "Late" agglutination, observed 72 hr pi, was detected in cultures which had been treated with HSV irradiated for 4 to 15 min. Among those, virus irradiated for 6 to 8 min was most efficient. HSV-transformed cells were also agglutinated without exception by low concentrations of Con-A.     

Regulation of concanavalin A agglutination by the extracellular matrix

The agglutinability of rat C₆ glioma cells by concanavalin A (Con A) depends upon cell density. From sparse density to near confluency agglutinability increases as cell density rises. Both the half-maximal concentration and the maximum amplitude of agglutination by Con A are functions of cell density, but are separate cell parameters differing in the extent to which they are affected by density and the point at which they become insensitive to further density increases. Both trypsin and EDTA reduce cell agglutinability. The similarity in recovery kinetics between low density cells and cells dissociated with EDTA or trypsin suggests that low density cells may lose the same surface agglutination component(s) removed by trypsin and EDTA. Density-dependent regulation of Con A agglutinability is anchorage dependent; cells grown in suspension display no such phenomenon. The cooperative cell regulation of agglutinability is mediated by the extracellular matrix, or micro-exudate. The matrix contains two activities: low density cultures produce a matrix inhibitor of Con A agglutinability, while high density cultures produce a matrix promotor.     

Identification of Isolates of Clostridium perfringens Types C and D by Agglutination and Fluorescent-Antibody Methods

Agglutination and fluorescent-antibody methods were employed for screening Clostridium perfringens types C and D from 393 isolates of this organism. All of 50 strains which were isolated in Japan and were agglutinable with an antiserum prepared against a stock strain of type C no. 3182 toxigenically belonged to type C, but the antiserum showed no cross-agglutination with any of type C strains isolated in Denmark. All of the latter strains, however, were agglutinated by an antiserum prepared against a Danish strain, CWC11. Of 64 strains, showing heat-labile agglutinability by type D antiserum L9, 22 strains were toxigenically identified as type D strains which can be divided into three groups by the heat-stable antigens; no strains which were L-agglutination-positive but O-agglutination-negative were epislon-toxigenic. All of 13 strains, the heat-stable antigen of which was agglutinable by a type D antiserum VX81, were toxigenically type D strains. The results of fluorescent-antibody tests were almost in agreement with those of agglutination test with type C strains and completely with those of the O-agglutination test with type D strains. No beta-, epsilon- or delta-toxigenicity could be demonstrated in strains which were not agglutinated by our test sera for types C and D strains. Further examination of cultural properties of Japanese and Danish type C strains revealed that the two groups were considerably different in urease production, capsule formation, and delta- and alpha-toxigenicities.     

Lipopolysaccharide heterogeneity in strains ofSerratia marcescens agglutinated by O14 antiserum

Lipopolysaccharides (LPS) from 71 strains ofSerratia marcescens that were agglutinated by O14 antiserum were examined by SDS-PAGE. Four major profiles were found, designated LPS1 to LPS4. These groups accounted for 51, 7, 5, and 3 strains respectively. Five strains were unclassified. Immunoblotting showed that O14 antibodies bound only to LPS1 and not to LPS2, 3, or 4. LPS1 also bound antibodies in O1, O4, O12, and O23 antisera. LPS2 reacted specifically with O8 antiserum, LPS3 with O6, and LPS4 with O2, O3, O6, O12, and O21 antisera. These reactions were not found in agglutination tests with boiled, whole-cell antigens. However, tests with autoclaved antigens (45 min at 121°C) corroborated the immunoblotting classifications; LPS1 strains belonged to serotype O14, LPS2 to serotype O8, LPS3 to serotype O6, and LPS4 to serotype O21. We conclude that there is a heat-stable antigen on many clinical strains ofS. marcescens that masks the expression of O-specific LPS antigens and which binds with nonspecific antibody in serum O14. We propose that O-antigens should be prepared from autoclaved cultures and that the H-reference strain O14H9 CDC 1783-57 (LPS2) should be reclassified as serotype O8.     

Temperature-sensitive loss of sexual agglutinability in Saccharomyces cerevisiae

Temperature dependency of sexual agglutination in Saccharomyces cerevisiae was found. Of 31 strains tested, which showed normal agglutination when cultured at 25°C, 29 strains lost their sexual agglutinability when they were grown at 37°C.     

Surface antigens of Proteus mirabilis revealed by electroblotting from sodium dodecyl sulphate-polyacrylamide gels

Western Blotting of whole cell preparations of three strains of Proteus mirabilis after separation by electrophoresis on SDS-polyacrylamide gels revealed a complex pattern of antigens. Similar antigen profiles were obtained with isolated outer membranes indicating that the majority of cell surface antigens are located in the outer membrane. Major outer membrane proteins were strongly antigenic and cross-reactive. The highly immunogenic flagella were detected in whole cell preparations and visible in isolated outer membranes. Whereas the protein and flagellar antigens were cross-reactive, lipopolysaccharide (LPS) could only be detected as immunoreactive material using homologous antisera for each strain. The LPS appeared as two broad bands (high and low Mr, respectively) in immunoblots of whole cells, isolated outer membranes and purified LPS. However, isolated LPS could be resolved into multiple sharp bands when 4 M urea was included in the gel system. These discrete bands are assumed to represent differing O antigen chain lengths of the LPS as reported for other Gram-negative organisms.     

Serological Characterization of Actinobacillus pleuropneumoniae Biotype 1 Strains Antigenically Related to both Serotypes 2 and 7

Nine Danish Actinobacillus pleuropneumoniae biotype 1 isolates were shown by latex agglutination and indirect haemagglutination to possess capsular polysaccharide epitopes identical to those of serotype 2 strain 1536 (reference strain of serotype 2) and strain 4226 (Danish serotype 2 strain). Immunodiffusion confirmed the antigenic relationship with serotype 2 and further demonstrated an antigenic relationship with strain WF83 (reference strain of serotype 7). SDS-PAGE with LPS from strains 1536, 4226, WF83 and strain 7317 (representative of the 9 isolates examined) showed that strains WF83 and 7317 had an identical smooth ladder pattern whereas LPS from strains 1536 and 4226 showed a distinctly different pattern. The antigenic similarities of the LPS of strains WF83 and 7317 were confirmed by immunoblots using rabbit or pig antisera prepared against the 3 strains. No antigenic similarities in the LPS of strains 1536 and 7317 were revealed. Since an antigenic determinant specific for the 9 isolates could not be demonstrated with the methods used, the strains are proposed to be designated K2:O7.     

Chemical Characterization of the Lipopolysaccharide of Pseudomonas solanacearum

The carbohydrates present in lipopolysaccharide (LPS) from Pseudomonas solanacearum are rhamnose, xylose, 2-amino-2-deoxyglucose, glucose, heptose, and 2-keto-3-deoxyoctonate. LPS extracted from cultures grown on either glycerol or glucose (as the major source of carbon) and extracted after various incubation periods had similar compositions. The LPS from several strains of the bacterium contained the same component sugars, but the amounts of each sugar varied considerably. It was observed, however, that xylose and 2-amino-2-deoxyglucose increased proportionately with rhamnose, the major component. Phenol-water-extracted LPS contained measurable amounts of nucleic acid, protein, and arabinan, but none of these polymers were detected in LPS extracted with phenol-chloroform-petroleum ether. Polysaccharides liberated from LPS by mild acid hydrolysis were purified by gel filtration. Carbohydrate analysis of the LPS from a virulent, fluidal strain (K60) showed that the O-specific antigen consisted of rhamnose, xylose, and 2-amino-2-deoxyglucose in the proportions 4:1:1. The LPS of an avirulent, afluidal strain (B1) lacked the O-specific antigen; the R-core region consisted of rhamnose, glucose, heptose, and 2-keto-3-deoxyoctonate. Methylation analysis indicated that the K60 O-specific antigen was composed of a hexasaccharide repeating unit containing 3-, 2-, and 3,4-substituted rhamnopyranosyl residues, 3-substituted 2-amino-2-deoxyglucose, and terminal xylopyranose in the molar ratios 2:1:1:1:1.     

Trypanosoma brucei: loss of variable antigens during transformation from bloodstream to procyclic forms in vitro.

The loss of variable antigen from Trypansoma brucei during transformation from the bloodstream to the procyclic form in vitro has been monitored by agglutination and immunofluorescence reactions using antisera against both forms. Maximum agglutination of transforming trypanosomes with anti-culture form sera was obtained in 36–48 hr coinciding with loss of the surface coat as seen by electron microscopy. Agglutination with antisera against homologous bloodstream forms, however, reached a constant minimum but still positive level after 7–9 days: absorption of such antisera with culture or heterologous bloodstream forms reduced this period of persistent agglutinability to 72–84 hr, suggesting that the sera contained antibodies to “common” (surface membrane) antigens which became exposed when the surface coat was lost during transformation. The indirect immunofluorescence reaction provided a direct correlation of loss of antigen with loss of coat. The majority of trypanosomes lost detectable variable antigen by 36–48 hr, but a few flagellates, morphologically resembling bloodstream forms, retained the coat and capacity for labeling up to 84 hr; the numbers of such persistent bloodstream forms were shown to be sufficient to give a positive agglutination reaction for the population as a whole up to this time. Variable antigen appeared to be lost by dilution over the entire trypanosome surface rather than in patches or caps and the relevance of this observation to the process of antigenic variation is discussed.     

Effects of growth phase and boiling of enteropathogenic Escherichia coli strains on their interaction with Pseudomonas aeruginosa lectins

Escherichia coli strains from' serotypes O86, 0128 and O111 varied in their reactivity with Pseudomonas aeruginose lectins (PA-I with D-galactose specificity and PA-II which binds L-fucose, D-mannose, L-galactose and D-fructose). Generally, cells of O86 strains were agglutinated by PA-I, but not by PA-II, and those of O128 serotype were agglutinated by PA-II, and not by PA-I. Adsorption tests showed that cells of E. coli O86 strains adsorb PA-I to a greater extent than PA-II, while most E. coli O128 strains adsorbed higher amounts of PA-II. Cells of E. coli O111B4 which were not agglutinated by either Pseudomonas lectin could still adsorb both. Boiling of O86 and O128 cells frequently enhanced their agglutinability as well as their lectin adsorption capacity. The agglutinability enhancement was somewhat more prominent in boiled stationary phase cells than in log phase cells probably due to late synthesis of the O antigen components concomitantly with the heat-sensitive components (K antigens) which masked them. PA-I agglutinating activity was inhibited by the lipopolysaccharide (LPS) extracted from E. coli O86 cells, while PA-II was inhibited by the LPS extracted from E. coli O128 cells. These findings indicate that the receptors to the Pseudomonas lectins probably reside in the terminal part of the O-specific-polysaccharide of the LPSs of these bacteria.     

α-mating-type-specific suppression and a-mating-type-specific enhancement by tunicamycin of sexual agglutinability in the yeast Saccharomyces cervisiae

Effects of tunicamycin (TM) on the sexual agglutinability and zygote formation of Saccharomyces cerevisiae were studied using the two kinds of haploid strains, inducible and constitutive for sexual agglutinability. Induction of sexual agglutinability by opposite mating type sex pheromone of inducible strains was inhibited by TM in mating type but not in a mating type. The recovery by temperature-shift-down from the temperature-suppressed sexual agglutinability of constitutive strains was enhanced by TM in a mating type but rather inhibited in mating type. Pretreatment with TM of constitutive strains enhanced sexual agglutinability in a mating type but not in mating type. The above-mentioned a-mating-type-specific agglutinability-enhancing actions of TM were discussed in relation to the action mechanism of pheromone which induces or enhances the sexual agglutinability of a cells.Zygote formation was inhibited by TM in both constitutive and inducible strains at concentrations which showed only partially inhibitory effect on sexual agglutinability.Abbreviations AI agglutination index - TM tunicamycin     

Specific activity of colored variants of N. meningitidis colonies

The character of fluorescence of the colonies formed by serogroup A meningococci (229 strains) in oblique light and their activity in the agglutination test with group specific S and RD antisera to the meningococcal dissociant of the same serogroup were studied. Orange and orange-green colonies were found to have pronounced group-specific activity, and faint gray colonies changed the character of their agglutination with the group-specific antiserum up to the loss of agglutinability; and simultaneously their capacity for agglutination with the antiserum to the dissociant was revealed. The study of the character of fluorescence and the group-specific activity of meningococcal colonies belonging to other serogroups provided similar results.     

Mutants inducible for sexual agglutinability in Saccharomyces cerevisiae

Summary We have isolated the mutants, T55s-41(a) and T562s-161 () which have no sexual agglutinability when cultured at 28°C, but become sexually agglutinable by the action of the sex pheromone produced by respective opposite mating type. The sex-specific glycoproteins responsible for sexual agglutination were detected in the mutants treated with the opposite mating type pheromone, but not in those treated with the same mating type pheromone.The induction of sexual agglutinability by the pheromone required both nitrogen and carbon sources and was inhibited by cycloheximide. The induction by the pheromone of sexual agglutinability was much more sensitive to osmotic shock and Triton X-100 in T55s-41 than in H1-0, an inducible a strain found in our stock cultures. When cultured at 22°C both T55s-41 and T562s-161 produced respective agglutination substances without the sex pheromones.H1-0 carried more than one genes responsible for the inducibility (inducible genes). The inducible genes carried by T55s-41 and T562s-161 were recessive, possibly linked to none of the mating type locus, thr4 and his 4, and shown to be identical. The inducible gene in H22, an inducible a strain found in our stock cultures and at least one of the inducible genes in H1-0 were linked to the mating type locus. All the inducible genes observed so far were not specific to the mating type in their action.