Affiliation:
1. Departamento de Inmunología, Facultad de Medicina, Universidad Autónoma de Nuevo León, Monterrey, México. msalinas@ccr.dsi.uanl.mx.
Abstract
We prepared a Nocardia brasiliensis cell extract and purified two immunodominant antigens with molecular weights of 61,000 and 24,000. The isolated proteins were shown to be reasonably pure when analyzed with sodium dodecyl sulfate-polyacrylamide gel electrophoresis (8 to 18% polyacrylamide gradient) and stained with Coomassie blue and silver nitrate. By using an immunoelectrotransfer blot method (Western blotting), we demonstrated that these two purified proteins reacted strongly with serum from N. brasiliensis-infected mycetoma patients. To obtain anti-P61 and anti-P24 monoclonal antibodies (MAbs), we used an N. brasiliensis cell extract as the antigen for the first immunization; 2 weeks later female mice were reimmunized with a semipurified antigen containing the P24 or P61 fraction. A booster injection was given 3 days before the fusion was carried out. Two hybrids that reacted strongly with P24 were cloned by limiting dilution, the generated MAbs were analyzed for isotyping, and their specificity was tested in a Western blot assay with cell extracts from Nocardia asteroides and Mycobacterium tuberculosis cultures. Anti-P24 MAbs were shown to be specific for N. brasiliensis HUJEG-1 and did not cross-react with either the N. asteroides or M. tuberculosis strains used. However, additional studies with several N. asteroides and N. brasiliensis strains are needed to investigate whether there are cross-reactions between strains or species when these MAbs are used. The anti-P61 and anti-24 MAbs were used to locate the antigen in N. brasiliensis cells by immunofluorescence. The lack of reaction with intact cells suggests that the P24 and P61 antigens are not exposed in the complete bacterial cell surface or that the recognized epitopes are different. Only one anti-P61 MAb that reacted specifically with the N. brasiliensis cell extract was obtained.
Publisher
American Society for Microbiology
Subject
Microbiology (medical),Clinical Biochemistry,Immunology,Immunology and Allergy
Reference23 articles.
1. Nocardia species: host-parasite relationships;Beaman B. L.;Clin. Microbiol. Rev.,1994
2. Microimmunodiffusion test for nocardiosis;Blumer S. O.;J. Clin. Microbiol.,1979
3. Monoclonal antibodies against isoelectrically focused Nocardia asteroides proteins characterized by the enzyme-linked immunoelectrotransfer blot method;El-Zaatari F. A.;Diagn. Immunol.,1986
4. Nocardia infections in congenitally athymic (nude) mice and in other inbred mouse strains;Folb P. I.;Infect. Immun.,1977
5. Classification and identification of rhodococci;Goodfellow M.;Zentralbl. Bakteriol.,1990
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