Monoclonal antibodies to P24 and P61 immunodominant antigens from Nocardia brasiliensis.

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.

Investigation of the temporal humoral immune response in a murine model of actinomycetoma.

The murine model of actinomycetoma offers the potential of studying many unknown aspects of this infection. In this work, the model was used to investigate the temporal humoral immune response to actinomycetoma agents. Groups of 7- to 9-week-old female BALB/c mice were inoculated in one of the hind footpads with one of four different Nocardia strains. To mimic the constant exposure of infected humans to the virulent soil inhabiting agents, a second injection consisting of live nocardiae in incomplete Freund's adjuvant was administered five months after the first one. Murine serum samples were collected throughout the study and their IgM and IgM titers were determined by ELISA and the Western blot assay. The results obtained indicate that the ELISA titers increased as the infection progressed and this correlated with a greater number of antigen bands being recognized in the blots. Overall, however, the ELISA titers were lower for the N. brasiliensis infected mice than those of the N. asteroides ones. This observation may be indicative of an immunosuppressive state and is worthy of further investigation.

Investigation of the temporal humoral immune response in a murine model of actinomycetoma

The murine model of actinomycetoma offers the potential of studying many unknown aspects of this infection. In this work, the model was used to investigate the temporal humoral immune response to actinomycetoma agents. Groups of 7- to 9-week-old female BALB/c mice were inoculated in one of the hind footpads with one of four different Nocardia strains. To mimic the constant exposure of infected humans to the virulent soil inhabiting agents, a second injection consisting of live nocardiae in incomplete Freund's adjuvant was administered five months after the first one. Murine serum samples were collected throughout the study and their IgM and IgM titers were determined by ELISA and the Western blot assay. The results obtained indicate that the ELISA titers increased as the infection progressed and this correlated with a greater number of antigen bands being recognized in the blots. Overall, however, the ELISA titers were lower for the N. brasiliensis infected mice than those of the N. asteroides ones. This observation may be indicative of an immunosuppressive state and is worthy of further investigation

Monoclonal antibodies to Nocardia asteroides and Nocardia brasiliensis antigens.

Nocardia asteroides and Nocardia brasiliensis whole-cell extracts were used as antigens to generate monoclonal antibodies (MAbs). Six stable hybrid cell lines secreting anti-Nocardia spp. MAbs were obtained. These were characterized by enzyme-linked immunosorbent assay, Western blot (immunoblot), and immunofluorescence assay. Although all the MAbs exhibited different degrees of cross-reactivity with N. asteroides and N. brasiliensis antigens as well as with culture-filtrate antigens from Mycobacteria spp., they have the potential for use as reagents in the purification of Nocardia antigens.