Characterisation of epitopes of type 1 fimbriae of Salmonella using monoclonal antibodies specific for SEF21 fimbriae of Salmonella enteritidis.

Monoclonal antibodies (mAbs) were used to identify and characterise epitopes of type 1 (SEF21) fimbriae of Salmonella enteritidis. The distribution of the epitopes among salmonellas and other enterobacteria was investigated, as well as the influence of growth media and temperatures on their expression. At least four different epitope clusters were identified on SEF21 fimbriae of S. enteritidis. Two of these clusters were associated with fimbrial haemagglutinins that were either common to all salmonellae tested, or restricted only to S. enteritidis and S. dublin. The four epitope clusters were identified on type 1 fimbriae of most Salmonella serotypes, as well as non-haemagglutinating type 2 fimbriae of S. pullorum and S. gallinarum, and on many other enterobacterial species. The expression of the epitopes was affected by growth conditions.

Development and application of enzyme-linked immunosorbent assay for specific detection of Salmonella enteritidis infections in chickens based on antibodies to SEF14 fimbrial antigen.

A double-antibody sandwich enzyme-linked immunosorbent assay (ELISA) was developed to detect antibodies to the SEF14 fimbrial antigen (SEF14-DAS ELISA) and was evaluated for its use in the specific detection of chicken flocks infected with Salmonella enteritidis. The SEF14-DAS ELISA successfully discriminated between chickens experimentally infected with S. enteritidis and those infected with S. panama or S. typhimurium, although the SEF14 responses in adult birds infected with S. enteritidis were detectable but low. In contrast, ELISAs used to detect antibodies to lipopolysaccharide (LPS) and flagella were unable to discriminate between the infected groups of chicks and adult birds infected with different Salmonella serotypes. LPS and flagellar responses were low and variable in chicks, whereas in adult hens they were found to be consistently strong. When flocks naturally infected with S. enteritidis were tested by the SEF14-DAS ELISA and ELISAs to detect LPS and flagellar antibodies, it was found that they could all identify the infected flocks, although there was little correlation between individual serum samples. The study shows that the SEF14-DAS ELISA may offer advantages over existing assays with comparable sensitivities coupled with higher specificities for the serological detection of S. enteritidis-infected chicken flocks.

Characterisation of monoclonal antibodies specific to SEF 21 fimbriae of Salmonella enteritidis and their reactivity with other Salmonellae and Enterobacteria.

A panel of monoclonal antibodies (mAbs) specific to type 1 (SEF 2) fimbriae of S. enteritidis was produced using crude and HPLC purified preparations of SEF 21 fimbriae. Sixteen mAbs were selected by indirect ELISA using both purified SEF 21 antigen and whole cells of S. enteritidis. Eight mAbs were confirmed by immunoprecipitation assay to react specifically with SEF 21 fimbriae. These mAbs were further characterised for their reactivity patterns by the "whole cell" ELISA and latex agglutination test with a number of strains of Salmonella and other enterobacteria. Not all SEF 21 mAbs reacted in both ELISA and latex agglutination tests with whole bacterial cells. mAb 611 was the only one suitable for use in both tests. Unexpectedly these mAbs reacted with the type 1 fimbriae of many of the tested strains of enterobacteria. mAb 721 reacted with most strains of Salmonella (89.1%) and enterobacteria (71.4%) tested. mAb 611 reacted with 61%-75% of strains of Salmonella and with 6.9%-17.6% of enterobacteria in ELISA and latex tests respectively. These mAbs will be useful reagents for further characterisation of type 1 fimbriae expressed by members of the family Enterobacteriaceae.

The use of latex particle agglutination to specifically detect Salmonella enteritidis.

This paper reviews the development and evaluation of a latex particle agglutination test to specifically identify cultured Salmonella enteritidis organisms. The test is based on the use of two monoclonal antibody-coated latex reagents, one of which detects the recently discovered SEF14 fimbriae expressed predominantly by S. enteritidis and S. dublin organisms, while the second reagent detects the H'p' antigen of S. dublin flagella. In a series of field trials 141 out of 142 strains of S. enteritidis from eighteen phage types were correctly identified by the latex test. A further 175 salmonella isolates representing 35 serotypes were tested and only two false-positives (S. dublin) in the latex test were recorded. This is the first rapid serotype specific test for S. enteritidis to be developed, and highlights the potential advantage of fimbrial antigens as novel diagnostic antigens of the future.

Characterisation of monoclonal antibodies against a fimbrial structure of Salmonella enteritidis and certain other serogroup D salmonellae and their application as serotyping reagents.

A panel of 13 monoclonal antibodies from different hybridomas was produced against a novel salmonella fimbrial antigen expressed predominantly by Salmonella enteritidis strains. The specificity of the monoclonal antibodies to this antigen (SEF14) was confirmed by enzyme-linked immunosorbent assay (ELISA) using purified SEF14, immune electron microscopy and, with 11 monoclonal antibodies, the identification of a repeating protein subunit (14,300kDa) on the antigen. Blocking-ELISA with the monoclonal antibodies identified epitopes in at least three, non-overlapping clusters which appeared evenly distributed on SEF14 in immune electron microscopy. The use of the monoclonal antibodies in direct-binding ELISA on a range of salmonella serotypes suggested that the epitopes on SEF14 are highly conserved and were expressed by all the S enteritidis strains examined; some strains of S dublin and the only strain of S moscow available were the only other serotypes that expressed SEF14. A latex agglutination reagent based on a monoclonal antibody was developed and used to test for SEF14 on 280 strains (representing 120 serotypes in 24 serogroups of salmonellae) that had been grown on Sensitest agar for 18 hours at 37 degrees C. All S enteritidis strains (64) and most S dublin strains (28 of 33) produced SEF14 as did the two strains representing S blegdam and S moscow. SEF14 was not detected in any other strains of serotypes from serogroup D or from any other serogroup examined.

An interlaboratory trial of a latex agglutination kit for rapid identification of Salmonella enteritidis.

An interlaboratory trial was conducted of a latex agglutination kit for the rapid identification of Salmonella enteritidis, to assess the stability of the components and its performance with respect to a panel of three coded salmonellas and 243 field isolates. Two of the components of the kit deteriorated with time. All 62 isolates of S enteritidis were correctly identified by the kit; only two false positives were recorded and no false negatives.

Detection of a novel fimbrial structure on the surface of Salmonella enteritidis by using a monoclonal antibody.

A fimbrialike structure expressed on the surface of Salmonella enteritidis was identified by using a monoclonal antibody (69/25) produced against intact S. enteritidis cells. Fimbriae were less than 5 nm in diameter and carried a protein consisting of subunits with a molecular weight of 14,300. No hemagglutinating activity associated with the fimbriae was detected. An epitope on the fimbrial antigen identified by monoclonal antibody 69/25 was expressed by all 58 S. enteritidis strains, 12 of 36 S. dublin strains, and a single strain of S. moscow examined. None of 169 other isolates tested from 17 salmonella serogroups expressed this epitope.

The detection of Clostridium perfringens epsilon antitoxin in rabbit serum by monoclonal antibody based competition ELISA.

A competitive enzyme-linked immunosorbent assay (CELISA) has been developed, standardized and compared with the toxin neutralization (TN) test performed in mice for the measurement of antibody responses in rabbits vaccinated with clostridial vaccines. In CELISA, sera were tested at a single dilution for their ability to compete with the reaction between a monoclonal antibody, which neutralizes epsilon toxin, and epsilon toxoid coated on to a solid phase. The results of the two tests correlated well. CELISA was specific, rapid, reproducible and simple to perform and offered an alternative to the TN test that reduced the requirement for experimental animals in the potency testing of clostridial vaccines.

The production and evaluation of monoclonal antibodies to Clostridium perfringens type D epsilon toxin.

The production of five monoclonal antibodies to the epsilon prototoxin of Clostridium perfringens is described. All five monoclonal antibodies located three proteins in a trypsinized preparation of epsilon prototoxin. These proteins were located at 37.6 kDal, 35.6 kDal and 33.7 kDal by Western blots. Two of the monoclonal antibodies, M26/2 and M27/12, neutralized epsilon toxin in the mouse lethality assay. Four of the five monoclonal antibodies are considered suitable as reagents to detect epsilon toxin in assay procedures.

Immunoelectrophoresis in quality control of veterinary clostridial products.

Each year the pharmaceutical industry produces 150-250 batches of anti-Clostridial vaccines and sera for veterinary use in the U.K. Under Medicines Act requirements these products must be tested for potency before release to the public, such tests being performed in animals. The Ministry of Agriculture, as licensing authority, also carries out potency test on representative samples of these products. To avoid unnecessary use of animals we employ immunoelectrophoresis (IEP) as a screening test. Results obtained so far show a close correlation between values obtained by IEP and the statutory tests and offer a potential alternative to the use of animals in potency tests.