The Development of an ELISA Test for the Detection of Mycoplasma bovis Antigen in Milk

Veterinary science, Microbiology

Gunn, John

University:University of Glasgow

关键词: Veterinary science, Microbiology;   

Others  :  http://theses.gla.ac.uk/78353/1/11011420.pdf

来源: University of Glasgow

【 摘 要 】

The objective of the work presented in this thesis was the construction of an ELISA test for the detection of Mycoplasma bovis antigen in milk samples. Consequently, monoclonal antibodies to M. bovis were prepared for use in the ELISA. Whole cell antigen was used to immunise two BALB/c or BALB/c hybrid mice from which serum and spleen cells were harvested. Fusion was carried out with NSO myeloma cell lines and 441 hybridomas were produced. One hundred and fifty-one of these produced antibody to an bovis antigen at levels twice background. The test used for detection and screening of hybridomas was an ELISA constructed with a membrane antigen of approximately 30 kD. Antigen for these bovis screening tests was prepared by the ultrasonic disruption of whole cell suspensions of M. bovis grown in media containing mouse serum. The membrane fractions were harvested by ultracentrifugation at 34 000 g for 30 minutes, resuspended in 10 mM TRIS-HCl buffer, pH 7.8, treated with the chaotropic agent guanidinium thiocyanate (6.0 M) and dialysed against 6.0 M urea. The resulting solubilised antigen was designated antigen "i.p." and used in the screening ELISA . Six antibody-producing hybridomas were cloned further to produce 210 stage-1 clones from which 6 monoclonal antibody lines were produced. Monoclonal antibody (MAb) "5G4" proved to be directed against media constituents but the remaining 5 were directed against M. bovis antigen. MAb "5A10" contained IgG1 with a titre in excess of 1/1000 and was selected for use in the final ELISA. The final ELISA was constructed using MAb "5A10" as capture antibody at a dilution of 1/1000. The same antibody was biotinylated for use at 1/500 as developing antibody. O-phenylenediamine dihydrochloride was used as the substrate and 2.5 M sulphuric acid as the stopping agent. It was evaluated against cultures of 34 isolates from 20 mycoplasma species. Absorption greater than twice background was only noted in the M. bovis and one M. agalactiae isolates. The occurrence of the same antigen in the two species does not prevent the use of this ELISA in the detection of M. bovis in bovine mastitis as M. aqalactiae does not occur in this species. Serial dilutions of broth cultures of M. bovis sc38, the strain against which the monoclonal antibodies were produced, were examined by ELISA and culture. The level of detection was found to be 10 8 cfu/ml. Milk samples from cows which had been infected experimentally with an East German isolate of M. bovis were supplied by Dr H Pfutzner, Jena, in frozen form together with information about the numbers of mycoplasmal cfu present at the time of sampling. 78 milk samples from 6 cows were examined for M. bovis antigen using the ELISA, and for N-acetylglucosaminidase (NAGase) using an ELISA available at Stormont. The ELISA detected bovis in milks which had an original concentration of 10e7 cfu/ml. ELISA results were positive for milks taken between days 3 and 21 (Cow 1); 7 and 38 (Cow 2); 7 and 10 (Cow 3); 7 and 38 (Cow 8) ; and 7 and 10 (Cow 9) . Cow 7 was negative both by ELISA and culture. The NAGase results were positive over a longer period than the M. bovis ELISA results.