Bacterial surface antigen-specific monoclonal antibodies used to detect beer spoilage pediococci.

Fourteen monoclonal antibodies (Mabs) were isolated that react with surface antigens of Pediococcus beer spoilage organisms, including P. damnosus, P. pentosaceous, P. acidilactici, and unspeciated isolates. Immunoblotting, enzyme immunoassays (EIAs) of protease- and neuraminidase-treated surface antigen extracts, carbohydrate competition EIAs, and cardiolipin EIAs were used to characterize the bacterial antigens involved in Mab binding. Antigen stability in situ was tested by protease treatment or surface antigen extraction of washed bacteria. In most cases, the Mabs bind to Pediococcus surface antigens that appear to be covalently bound cell wall polymers resistant to alteration or removal from the bacterial surface. These bacterial surface antigen reactive Mabs show good potential for rapid, sensitive, and specific immunoassay detection of Pediococcus beer spoilage organisms.     

Serogrouping of oral Streptococcus intermedius

Employing twenty fresh oral isolates of Streptococcus intermedius, studies were carried out to characterize serological relations among the isolates and also between the isolates and the strains of bacterial species closely related to S. intermedius. The Rantz-Randall extracts from the cells were used as antigens. The anti-rabbit serum raised against S. intermedius ATCC 27335T reacted with the cell extracts from only three strains of the isolates, which were designated serogroup I strains. The other isolates were classified into four serogroups, I, III, IV, and V, which specifically reacted with the cell extracts from the homologous serogroup strains. However, the serogroup II antiserum formed in immunodiffusion a common precipitin line between the extracts from the cells of serogroups II and I. The serogroups I, III, IV, and V antisera reacted with none of the extracts from the bacterial cells closely related to S. intermedius, which included Streptococcus anginosus ATCC 33397T, Streptococcus constellatus ATCC 27823T, three NCTC strains of "Streptococcus milleri," and three ATCC strains of Streptococcus MG. The precipitin line formed by the homologous reaction of the serogroup II antiserum was found to be a reaction of identity with that formed by the extract from "S. milleri" NCTC 10708. Conversely, the antiserum against NCTC 10708 strain did not react with the cell extracts of serogroup II.     

Specificity of an antiserum against Erwinia carotovora subsp. atroseptica in indirect ELISA

Attempts to differentiate Erwinia carotovora subsp. atroseptica (Eca) from Erwinia carotovora subsp. carotovora (Ecc) by indirect ELISA using polyclonal antisera against the former bacterium were unsuccessful. However, when bacterial cells were preincubated with an antiserum against Eca serogroup I and excess serum washed away prior to coating on micro-ELISA plates, specificity was improved. This modified indirect ELISA was able to separate Eca serogroups I, XVIII and XXII from all the Ecc serogroups tested. Cross adsorption of the antiserum with Ecc serogroup XXIX resulted in greatly reduced absorbance values for all strains/serogroups except Eca serogroups I and XXII. Cross adsorption with the homologous Eca strain reduced absorbance values for all strains/serogroups. It is suggested that the differentiation of Eca serogroups I and XXII obtained with the modified indirect ELISA could be attributed to the removal of antibodies cross reacting to soluble antigens and the retention of antibodies to specific cell surface antigens.     

Epitope mapping of monoclonal antibodies specific for the directly cross-linked mesodiaminopimelic acid peptidoglycan found in the anaerobic beer spoilage bacterium Pectinatus cerevisiiphilus.

Nineteen monoclonal antibodies (Mabs) were isolated based on reactivity with disrupted Pectinatus cerevisiiphilus cells. All of the Mabs reacted with cells from which the outer membrane had been stripped by incubation with sodium dodecyl sulphate, suggesting the peptidoglycan (PG) layer was involved in binding. Mab reactivity with purified PG confirmed this. Epitope mapping revealed the Mabs in total recognize four binding sites on the PG. Mabs specific for each of the four sites also bound strongly to disrupted Pectinatus frisingensis, Selenomonas lacticifix, Zymophilus paucivorans, and Zymophilus raffinosivorans cells, but weakly to disrupted Megasphaera cerevisiae cells. No antibody reactivity was seen with disrupted cells of 11 other species of Gram-negative bacteria. These results confirm that a common PG structure is used by several species of anaerobic Gram-negative beer spoilage bacteria. These results also indicate that PG-specific Mabs can be used to rapidly detect a range of anaerobic Gram-negative beer spoilage bacteria, provided the bacterial outer membrane is first removed to allow antibody binding.     

Brewing spoilage Lactobacilli detected using monoclonal antibodies to bacterial surface antigens.

A panel of thirteen monoclonal antibodies (Mabs) was assembled that reacts with surface antigens on eight of eleven Lactobacillus brewing spoilage organisms, including one or more of L. brevis, L. buchneri, L. casei-alactosus, L. plantarum, or unspeciated isolate(s). Immunoblotting was done to identify the antigens involved in Mab binding. Antigen stability in situ was tested by protease treatment and by surface antigen extraction of washed bacteria. Protease susceptibility of extracted surface antigens was also examined. In most cases, Lactobacillus surface antigens detected by the Mabs appear to be noncovalently bound proteins readily altered or removed from the bacterium by various environmental conditions. This research identifies brewing conditions that need to be tested to ascertain whether bacterial surface antigen-reactive Mabs can be used for the rapid, sensitive, and specific detection of Lactobacillus brewing spoilage organisms.     

Antigenic variability of the outer membrane antigens of Legionella pneumophila serogroups 1 to 8

The outer membrane proteins of Legionella pneumophila serogroups 1 to 8 were prepared from broken cells by selective solubilization using sodium lauryl sarcosinate. The isolated proteins were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and transferred to nitrocellulose sheets. Rabbit antisera against each of the eight serogroups of L. pneumophila were obtained by immunizing each animal with live bacteria. The transferred proteins were revealed using these antisera and peroxidase-labeled swine anti-rabbit immunoglobulins. Antigenic determinants common to all eight serogroups were found in at least three outer membrane antigens (19, 29, and 45 kilodaltons (kDa)). However, cross-absorption experiments revealed that these three antigens were immunologically related, but not identical among serogroups. The antigenic relationships observed with two of these three antigens correlated well with cross-reactions observed in immunofluorescence. When a monoclonal antibody directed against L. pneumophila serogroup 1 lipopolysaccharide was used to reveal a blot of serogroup 1 outer membrane antigens, the 29- and 45-kDa bands appeared. This demonstrates a strong association between lipopolysaccharide and outer membrane proteins.     

Screening and characterization of monoclonal antibodies to the surface antigens of Listeria monocytogenes serotype 4b

Aims:  This study aims to develop and characterize monoclonal antibodies (Mabs) with high specificity and affinity for surface antigens of an epidemiologically important serotype 4b of Listeria monocytogenes .
Methods and Results:  Hybridoma clones were derived from B lymphocytes of mice immunized with L. monocytogenes serotype 4b and screened against this strain by an enzyme-linked immunosorbent assay. Twenty-nine clones secreting Mabs reactive with formalin-killed bacteria were obtained; 15, 8, 5 and 1 Mabs were immunoglobulin subclasses IgG2a, IgG2b, IgM and IgG1, respectively. Immuofluoresence or immunogold labelling demonstrated all except five IgM and one IgG2a Mabs bound to the surface of a live L. monocytogenes serotype 4b. The majority of the 23 surface-binding Mabs recognized linear epitopes on a 77-kDa protein. These surface-binding Mabs exhibited little or no cross-reactivity with non-4b serotypes (1/2a, 1/2b, 3a, etc.) of L. monocytogenes , five other Listeria species, Escherichia coli O157:H7 and Salmonella enterica serovar Typhimurium.
Conclusions:  The Mabs recognizing a 77-kDa surface protein are novel antibodies with specificity and affinity for L. monocytogenes serotype 4b.
Significance and Impact of the Study:  These anti-77 kDa surface protein Mabs may be explored as reagents for the development of Mabs-based diagnostic immunoassays for L. monocytogenes serotype 4b strains.     

Antibody detection and cross-reactivity among species and serogroups of Legionella by indirect immunofluorescence test

The purpose of the present study was to determine possible cross-reactivity between different serogroups or between different species by means of bacterial smears of individual L. pneumophila serogroups and several non-pneumophila Legionella spp. Using BIOCHIP slide we found a considerable frequency of cross-reactions with L. pneumophila serogroup 12 (88.4%) therefore BIOCHIP slide proved as useful method for identification of cross-reactivity between members of the Legionnellaceae.     

Production and characterization of monoclonal antibodies to Aeromanas sobria surface antigens

Abstract A panel of eight murine monoclonal antibodies (Mabs) was raised against the surface antigens of Aeromonas sobria isolated from a patient with diarrhoea. Antibodies to the LPS core and O-antigen and to protein antigens were generated. Three Mabs of the IgM isotype against either protein or LPS agglutinated 34/75 Aeromonas isolates from clinical and environmental sources. Similar numbers of A. hydrophila, A. sobria and A. caviae isolates were agglutinated. The Mabs were screened for their ability to inhibit A. sobria adhesion to HEp-2 cells, and haemagglutination (HA). Two Mabs directed against conformationally dependent epitopes on a 43-kDa protein blocked both functions. Of the anti-LPS Mabs one blocked adhesion only, and another blocked HA but not adhesion. Immunoprecipitation studies suggested that LPS-protein complexes may be involved in these potential virulence functions of A. sobria .     

Production and characterization of monoclonal antibodies to Aeromonas sobria surface antigens

A panel of eight murine monoclonal antibodies (Mabs) was raised against the surface antigens of Aeromonas sobria isolated from a patient with diarrhoea. Antibodies to the LPS core and O-antigen and to protein antigens were generated. Three Mabs of the IgM isotype against either protein or LPS agglutinated 34/75 Aeromonas isolates from clinical and environmental sources. Similar numbers of A. hydrophila, A. sobria and A. caviae isolates were agglutinated. The Mabs were screened for their ability to inhibit A. sobria adhesion to HEp-2 cells, and haemagglutination (HA). Two Mabs directed against conformationally dependent epitopes on a 43-kDa protein blocked both functions. Of the anti-LPS Mabs one blocked adhesion only, and another blocked HA but not adhesion. Immunoprecipitation studies suggested that LPS-protein complexes may be involved in these potential virulence functions of A. sobria.     

Characterization of rhizobia nodulating Lotus subbiflorus from Uruguayan soils

Generation times, acid production, carbon utilization, immunological properties, plasmid content, protein profile and symbiotic properties of 15 isolates of rhizobia nodulating Lotus subbiflorus were studied. Based on specific growth rates, carbon source utilization and acid production, 13 out of the 15 isolates could be assigned to the slow-growing group of rhizobia (bradyrhizobia). Using antisera against whole cells of three isolates, we separated the 15 isolates into three serogroups. Only the slow-growing isolate Ls4 and the fast-growers Ls5 and Ls552 lacked cross-reactivity with any of the sera tested. Electrophoretic mobilities of whole cell protein from seven out of the eight isolates included in the serogroup represented by strain Ls31 were identical. Similarly, isolates Ls1B3 and Ls1B4, both in serogroup Ls1B3, had the same pattern of cell proteins. In contrast, isolates Ls3 and Ls7, belonging to serogroup Ls7, differed in protein profile. Plant growth experiments carried out under bacteriologically controlled conditions revealed that all of the isolates effectively nodulated L. subbiflorus and L. pedunculatus, but were unable to form effective nodules on L. tenuis and L. corniculatus. All isolates showed similar effectiveness in symbiosis with L. subbiflorus, except isolate Ls7, which gave significantly higher plant dry weight.Abbreviations ELISA enzyme linked immuno-sorbent assay - kDa kiloDalton - MM mineral medium - PBS phosphate-buffered saline - RE relative efficiency - SDS-PAGE sodium dodecyl sulfate-polyacrylamide gel electrophoresis - YEM deyeast extract mannitol     

Acetohydroxy acid synthase I, a required enzyme for isoleucine and valine biosynthesis in Escherichia coli K-12 during growth on acetate as the sole carbon source.

The fimbriae of Bacteroides nodosus play a major role in protective immunity against ovine footrot and are an important determinant in the serological classification system that divides field isolates into at least eight serogroups and 16 serotypes. Purified fimbriae contain two polypeptide antigens, the structural subunit of the fimbrial strand (molecular weight about 17,000) and a basal protein (molecular weight about 80,000), both of which exhibit structural variation. Fimbriae were prepared from all prototype strains, as well as from a number of other isolates representative of each of the B. nodosus serotypes, and analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Substantial variation was observed in the electrophoretic mobility of the fimbrial subunits from the prototypes of each of the eight serogroups. With the exception of serogroup H, which is an unusual case, the apparent molecular weights of the fimbrial subunits ranged from about 16,500 in serogroup D to 19,000 in serogroup F (serotype 1); in serogroup A, B, C and E, the apparent molecular weights were clustered in the range of 17,000 to 17,500, whereas serogroup G was about 18,500. Serogroup H fimbriae appeared to consist of two smaller polypeptides, which in the prototype (H1) had apparent molecular weights of about 6,000 and 10,000 and which seem to have arisen as a consequence of an internal proteolytic nick in the original subunit. Electrophoretic variation in the fimbrial subunit was also observed between different serotypes, although with the exceptions of serogroups F and H, this was not as pronounced as between the serogroups. Examination of a number of isolates classified within the same serotypes showed that some variation, although minor, also occurred at this level. The basal antigen exhibited significant variation at all levels of the serotypic hierarchy in a manner apparently unrelated to the classification system. Among the range of isolates examined, the apparent molecular weight of this antigen varied from about 77,000 to 88,000.     

Western blot (immunoblot) analysis of the fimbrial antigens of Bacteroides nodosus

The roles of the fimbrial subunit and the putative basal protein antigens in the serological classification of Bacteroides nodosus have been examined by Western blot (immunoblot)-antibody binding studies of fimbriae isolated from a wide range of strains representative of different serogroups and serotypes. Fimbrial subunits were recognized by antiserum against the homologous serogroup but not generally by heterologous antisera, whereas recognition of the basal antigen was independent of serological classification. Secondary cross-reaction patterns among fimbrial subunits indicated that some serogroups may be more closely related than others. Examples include serogroups C and G and serogroups D and H. Similar analyses of isolates classified within serotypes A1 and A2, with serotype-specific antisera, showed that this subdivision is also determined by the fimbrial subunit and that significant variation does occur even at this level. These studies suggest that the various serogroups and serotypes of B. nodosus comprise a series of overlapping sets of antigenically related strains.     

Level of IgG antibodies to bacteria of 12 species in the blood sera of rabbits, immunized with preparations obtained from Neisseria meningitidis, grown under the stress conditions

The blood sera of rabbits, immunized with preparations obtained from N. meningitidis of serogroups A, B or C, cultivated under the stress conditions, were studied. These sera were found to contain IgG antibodies not only to N. meningitidis antigens, but also to the bacterial antigens of 12 species. The sera of rabbits, immunized with meningococcal preparation of serogroup A, were found to have the elevated levels of IgG antibodies, in comparison with the control, to the antigens of 3 other bacterial species; the blood sera of rabbits, immunized with meningococcal preparation of serogroup B, were found the elevated levels of IgG antibodies to the antigens of 11 other bacterial species; and the blood sera of rabbits, immunized with meningococcal preparation of serogroup C, to the antigens of 9 other bacterial species. The study of serogroup B meningococci, used as an example, revealed the influence of the growth phase of the culture on the content of cross-reacting antigens. Their greatest amount was determined at the stationary phase when the stressor effect on the culture reached its maximum and their least amount, at the exponential phase when the stressor effect on the culture was minimal. It was, therefore, found to be expedient to obtain immunodiagnostic and test systems from N. meningitidis cultures, grown to middle of the exponential phase of growth.     

Genetic relatedness of Legionella longbeachae isolates from human and environmental sources in Australia.

The genetic relatedness of Legionella longbeachae isolated in Australia since 1987 was investigated by restriction fragment length polymorphism (RFLP) analysis and allozyme electrophoresis. Three radiolabeled probes were used in Southern hybridizations for the RFLP studies. They were Escherichia coli 16S and 23S rRNA and cloned fragments of L. longbeachae selected empirically from genomal banks in lambda and a cosmid. The legionellae included in the study comprised 11 Legionella longbeachae serogroup 1 organisms isolated from humans, 28 L. longbeachae serogroup 1 isolates from environmental sources, and 3 L. longbeachae serogroup 2 environmental isolates. These were compared with the American Type Culture Collection reference strains of both serogroups and some other related Legionella species. Results of allozyme and RFLP analysis showed that all the isolates from humans and all but three of the environmental L. longbeachae serogroup 1 isolates were closely related. They were also closely related to L. longbeachae serogroup 1 ATCC 33462. There was wider variation among the three L. longbeachae serogroup 2 environmental isolates. One of these was closely related to L. longbeachae serogroup 2 ATCC 33484. RFLP studies with the rRNA probe provided the most discrimination among isolates but did not distinguish between the two serogroups.     

Enzyme-linked immunosorbent assays for the detection of Neisseria gonorrhoeae specific antibodies.

An indirect enzyme-linked immunosorbent assay (ELISA) using rigid polystyrene microtiter plates was adapted to detect specific gonococcal antibodies against outer membrane-complex antigens extracted from Neisseria gonorrhoeae. The concentration of antigen to obtain maximum coating of the well was 10 micrograms protein per millilitre. The optimal binding of the primary antibody and enzyme-conjugated antimmunoglobulin was achieved after 1 h at 37 degrees C. Under these conditions using gonococcal antisera, no cross-reactivity was observed with outer membrane antigens extracted from Neisseria meningitidis serogroups B, C, X, Y, and W135. Neisseria meningitidis serogroup A demonstrated low levels of cross-reactivity. All the non-pathogenic Neisseria spp. tested were negative (absorbance value at 400 nm/30 min less than 0.15). The reaction of immune serum against outer membrane complex absorbed to the microwells was completely inhibited with soluble-specific antigen but not with purified N. gonorrhoeae lipopolysaccharide. Quantitative inhibition permitted the measurement of low levels of antigen (0.5 microgram/ml). The detection of N. gonorrhoeae antibody with ELISA is specific and highly sensitive.     

Antigenic cross-reactivity of major outer membrane proteins in enterobacteriaceae species.

The protein constituents in the outer membrane (OM) of several serotypes of Escherichia coli and some other Enterobacteriaceae cross-reacted antigenically. Solubilized OM preparations of these bacteria were applied in interfacial precipitin tests to antisera elicited in rabbits against whole bacterial cells, absorbed with their appropriate lipopolysaccharide before testing. The resulting immunecomplexes were analysed on polyacrylamide gels. Protein profiles of the immunoprecipitates showed a considerable antigenic cross-reactivity of outer membrane proteins between most E. coli serotypes. Cross-reactivity, though substantially lower, was also found with OM from three other Enterobacteriaceae species, but was not detectable with Pseudomonas aeruginosa OM. When OM preparations were solubilized at room temperature, the peptidoglycan-bound proteins in the molecular weight range 37,000 to 41,000 predominated in the protein profiles of the immunecomplexes. In profiles of immunecomplexes obtained with boiled OM preparations, a heat-modifiable protein (mol. wt 33,000) predominated. The major OM proteins of the Gram-negative bacterium may therefore play a role as common surface antigens of the family of Enterobacteriaceae.     

Bradyrhizobium japonicum Serocluster 123 and Diversity among Member Isolates

Diversity was examined within a group of 79 isolates of Bradyrhizobium japonicum reactive to fluorescent antibodies (FAs) prepared against B. japonicum USDA 123. Analyses were by means of cross-adsorbed FAs, bacteriophage typing, and endonuclease restriction digest patterns. Serogroups 127 and 129 shared antigenic determinants with serogroup 123 but not with each other. Bacteriophage and DNA digest patterns reflected more common features between serogroups 123 and 127 than between 123 and 129. Serogroups 129 and 122 showed FA cross-reactivity. The term serocluster was proposed to reflect interrelationships observed among the serogroups.     

Towards the Immunoproteome of Neisseria meningitidis

Despite the introduction of conjugated polysaccharide vaccines for many of the Neisseria meningitidis serogroups, neisserial infections continue to cause septicaemia and meningitis across the world. This is in part due to the difficulties in developing a, cross-protective vaccine that is effective against all serogroups, including serogroup B meningococci. Although convalescent N. meningitidis patients develop a natural long-lasting cross-protective immunity, the antigens that mediate this response remain unknown. To help define the target of this protective immunity we identified the proteins recognized by IgG in sera from meningococcal patients by a combination of 2D protein gels, western blots and mass spectrometry. Although a number of outer membrane antigens were identified the majority of the antigens were cytoplasmic, with roles in cellular processes and metabolism. When recombinant proteins were expressed and used to raise sera in mice, none of the antigens elicited a positive SBA result, however flow cytometry did demonstrate that some, including the ribosomal protein, RplY were localised to the neisserial cell surface.     

Diversity among Field Populations of Bradyrhizobium japonicum in Poland

Genetic structure in field populations of Bradyrhizobium japonicum isolated in Poland was determined by using several complementary techniques. Of the 10 field sites examined, only 4 contained populations of indigenous B. japonicum strains. The Polish bradyrhizobia were divided into at least two major groups on the basis of protein profiles on polyacrylamide gels, serological reaction with polyclonal antisera, repetitive extragenic palindromic PCR fingerprints of genomic DNA, and Southern hybridization analyses with nif and nod gene probes. Serological analyses indicated that 87.5% of the Polish B. japonicum isolates tested were in serogroups 123 and 129, while seven (12.5%) of the isolates tested belonged to their own unique serogroup. These seven strains also could be grouped together on the basis of repetitive extragenic palindromic PCR fingerprints, protein profiles, and Southern hybridization analyses. Cluster analyses indicated that the seven serologically undefined isolates were genetically dissimilar from the majority of the Polish B. japonicum strains. Moreover, immuno-cross-adsorption studies indicated that although the Polish B. japonicum strains reacted with polyclonal antisera prepared against strain USDA123, the majority failed to react with serogroup 123- and 129-specific antisera, suggesting that Polish bradyrhizobia comprise a unique group of root nodule bacteria which have only a few antigens in common with strains USDA123 and USDA129. Nodulation studies indicated that members of the serologically distinct group were very competitive for nodulation of Glycine max cv. Nawiko. None of the Polish serogroup 123 or 129 isolates were restricted for nodulation by USDA123- and USDA129-restricting soybean plant introduction genotypes. Taken together, our results indicate that while genetically diverse B. japonicum strains were isolated from some Polish soils, the majority of field sites contained no soybean-nodulating bacteria. In addition, despite the lack of long-term soybean production in Poland, field populations of unique B. japonicum strains are present in some Polish soils and these strains are very competitive for nodulation of currently used Polish soybean varieties.