Monoclonal antibodies to P24 and P61 immunodominant antigens from Nocardia brasiliensis

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-P24 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.

Immune Response toNocardia brasiliensisAntigens in an Experimental Model of Actinomycetoma in BALB/c Mice

“…SDS-PAGE analysis of N. brasiliensis antigens. The CCE obtained as described above and resolved by SDS-PAGE on an 8 to 18% gradient polyacrylamide gel shows a complex mixture of bands with molecular mobility from 10 to 66 kDa as previously published (16); CCE and purified fraction were used for Western blot identification of immunodominant antigens. The purified fraction was used to stimulate in vitro lymphocyte proliferation.…”

“…After being reconstituting with 1 ml of phosphatebuffered saline (pH 7.2), it was incubated for 2 h at 37°C with 150 g of DNase I (Sigma Chemical Co., St. Louis, Mo.) and then applied to a Sephadex G-100 column as previously described (16,18). Fractions containing the immunodominant antigen P24 were collected and used as purified antigen for ELISA and for lymphocyte stimulation in culture.…”

“…Even though many persons are accidentally inoculated, few develop the actinomycetoma lesion; host mechanisms that control and heal the lesion are unknown. Anti-N. brasiliensis antibodies have been demonstrated both in human patients and in experimental animals (15,16). The role of these antibodies in host protection is not clear (2,17); in humans, the presence of anti-N. brasiliensis antibodies has been helpful in serodiagnosis and has recently been introduced for use in routine clinical laboratories (18).…”