- Volume 66 , Number 2
- Page: 235–8
Serologic assays using the 18-kDa antigen of Mycobacterium leprae expressed in the yeast Saccharomyces cerevisiae
To the Editor:
Leprosy diagnosis is mainly based on clinical data and is confirmed by the detection of Mycobacterium leprae in infected tissues by direct bacilloscopy. In tuberculoid (TT) cases, the bacilli are seldom found and the diagnosis can only be confirmed by histopathologic examination. The development of specific serological assays for easier etiological diagnosis would allow the realization of seroepidemiological assays and would improve the follow up of patients, which possibly would imply in the installation of earlier and more effective control measures (8).
Among different M. leprae antigens, special attention was given to the 18-kDa antigen (p18), since it was involved in the immune response in mice and in humans (4, 7) and was initially considered restricted to two mycobacteria species, because cross-reaction using a specific monoclonal antibody was only found with M. simiae serovar 1 (12). Our group have developed a system for the expression of p18 in Saccharomyces cerevisiae utilizing an inducible promoter and a secretion cassette that produces more than 100 ing/L of pi 8 (18). This antigen was purified and used for delayed-type hypersensitivity assays in mice (19). In the present report, the use of this purified recombinant antigen in enzyme immunoassays (ELISA) was verified for the detection of M. leprae infections.
We studied seven leprosy outpatients followed up at the Department of Dermatology from São Paulo State Health Secretary at São Paulo, Brazil. The bacterial index (BI) (1-1-5+) was determined by analysis of the earlobe exudate and the clinical form was classified according to the criteria proposed by Ridley and Jopling (20). For our control group, we used eight confined individuals from a psychiatric hospital at Juqueri (about 30 km from São Paulo) for whom no previous contact with leprosy patients had been reported. The Brazilian Ministry of Health ethics guidelines were followed.
RESULTS AND DISCUSSION
Converse to previous serological assays, in this study we analyzed the use of an antigen corresponding to the authentic p18 expressed in S. cerevisiae for the detection of specific antibodies. This study is also the first to analyze the detection of anti-p18 antibodies among Brazilian patients.
The results of the ELISAs for the detection of anti-p18 antibodies (total and classes IgG, IgM and IgA) in the leprosy patients and the control group are shown in The Table. For total antibodies, IgA and IgM classes, patient sera were diluted 1/10.
For the IgG class, serum dilution ranged from 1/100 to 1/1000. The means of the OD492 values obtained from leprosy patients were always higher than those obtained from the control group, independent of the antibody class analyzed, but no statistically significant differences were found (Student's t test). The largest difference between the mean value in patients and in the control group was obtained when the patient sera was diluted 1/1000. Some individuals in the control group also showed higher OD492 values.
Other studies have shown that p18 would not be suitable for a routine serological assay for the diagnosis or follow up of leprosy patients since many patients (especially at the tuberculoid pole) did not show anti-p18 antibodies but some control patients also showed these antibodies (9, 11, 21, 22).
These previous results led us to first check only patients closer to the lepromatous pole of the disease. We observed that the average levels of antibodies against p18 in these patients were generally higher than those of the control group. However, some leprosy patients did not show anti-p18 anti-bodies and some individuals in the control group did have high antibodies levels against p18.
Initial reports describe this antigen restricted to a few mycobacterial species, such as M. siniiae serovar 1 (12) and M. smegmatis (15). Later, it was found that crossreaction could also be found with M. tuberculosis and M. bovis BCG (5, 6, 21, 22). These data were not confirmed by other authors, especially at the T-cell level (16, 17, 18), and no homologous gene was found in M. tuberculosis even when using polymerase chain reaction (PCR) with degenerated primers (3). Interestingly, the presence of T-cell clones recognizing specifically p18 from M. leprae (but not from other mycobacteria species) was recently reported from one subject who was not exposed to leprosy (1). The nature of the antigen stimulating this apparent specific response could not be determined.
On the other hand, the absence of detectable anti-p18 antibodies in some leprosy patients can be explained by the fact that the response against this protein in the natural infection seems to be more important at the cellular level (17) and is not an early event (13).
It is noteworthy that the patient who showed the highest antibody levels for all of the immunoglobulins tested (except IgM, for which he showed the second highest antibody level tested) was also the only one analyzed who showed a negative result on the bacilloscopy assay, although he was classified as LL. This result agrees with some data previously published suggesting that p18 would elicit a protective response against M. leprae infection (6). It was recently shown that immunization with recombinant BCG expressing p18 was capable of inducing antibodies, a lymphocyte proliferative response, and some protection against infection (2).
In conclusion, the 18-kDa recombinant antigen of M. leprae obtained in S. cerevisiae does not seem useful for routine serological assays for the diagnosis or follow up of leprosy patients.
- João R. R. Pinho, M.D., Ph.D.
Vanda A. U. F. de Souza, Ph.D.
Heitor F. Andrade, Jr., M.D., Ph.D.
Ana Clara G. Schenberg, Ph.D.
Laboratório de Biologia Molecular Instituto Adolfo Lutz
Laboratórios de Virologia e de Protozoologia, Instituto de Medicina Tropical de Sao Paulo, FMUSP and Centro de Pesquisas em Biotecnologia-ICBUSP
Sao Paulo, SP, Brasil
Acknowledgment. This work was supported by the World Health Organization/Tropical Diseases Research Program/World Hank, the Centro Brasileiro Argentino de Biotecnologia, LIMHCFMUSP and CAPES.
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Reprint requests to Dr. J. R. R. Pinho, Laboratório de Biologia Molecular, Instituto Aldolfo Lutz, Avenida Dr. Arnaldo 355, 012246-902 Sao Paulo, SP, Brasil, email: jrrpinho@usp.br; fax: 55-11-853-3505.