Detection of antibodies to human nerve antigens in sera from leprosy patients by ELISA.

Anti-neural antibodies have been implicated to play a role in the pathogenesis of nerve damage in leprosy patients. To find the relationship between anti-neural antibodies and clinical findings, we attempted to detect antibodies against neurofilament-enriched proteins by ELISA in sera from leprosy patients. Of 289 sera from leprosy patients, 74 (25.6%) had significant anti-neural antibodies; in contrast, 1 (5.0%) of 20 tuberculosis patients and 11 (7.1%) of 154 controls were seroreactive to nerve antigen. When clinical types were considered, a significant level of anti-neural IgG antibodies was detectable in 53 (30.1%) of 176 sera from lepromatous patients compared with 21 (18.6%) of 113 sera from tuberculoid patients, indicating that lepromatous patients were more likely to be seropositive to nerve antigens in ELISA. Some of the ELISA-reactive sera showed antibody reactivity with 38-kD, 40-kD and 43-kD nerve antigens in Western blotting analysis. There was no apparent correlation between seroreactivity to nerve antigens and bacterial load in leprosy patients. Although there was no statistical significance, anti-neural antibodies were detectable more often among the patients on chemotherapy than the untreated and among the patients with erythema nodosum leprosum than without. The results, therefore, suggest that anti-neural antibodies are elicited during the course of leprosy and may be associated with the extensiveness of nerve involvement in the patients.

Anti-neural antibodies in leprosy sera: further characterization of the antigens.

Sera or plasmas from 129 leprosy patients were tested by immunoblotting for antibodies that bound to proteins in a Triton-insoluble fraction enriched in neural intermediate filaments (IF fraction) from human or bovine spinal cord. Sixty samples (47%) showed positive staining of proteins at 35 kDa, 42 kDa or both. The presence of these antibodies appeared to be evenly distributed across the spectrum of disease. The frequency of these antibodies in samples from 12 healthy Ethiopians was similar to that in the leprosy group. Similar antibodies were found in only three of 28 samples from U.S. patients with neurologic diseases and in seven of 35 normal U.S. sera. Sera from U.S. tuberculosis patients stained multiple bands in the 50-30 kDa region of the blots; 11 of 16 stained bands corresponding to the 35 kDa or 42 kDa bands along with a number of other bands in this region. The 35 kDa and 42 kDa antigens do not appear to be breakdown products of neural filaments or glial fibrillary acidic protein, since antibodies to these proteins do not react with the 35 kDa or 42 kDa antigen. Further, the staining pattern with the leprosy sera is unchanged following Ca2+-mediated proteolysis of the IF-enriched fraction. The two antigens differ from one another in isoelectric point: the pI of the 35 kDa antigen is 5.9, and the pI of the 42 kDa antigen is 4.8. Staining of the immunoblots with antibodies against a number of known neural antigens failed to identify the 35 kDa and 42 kDa antigens. The 42 kDa antigen appears to be a component of axolemma, since 42 kDa-positive leprosy sera stained a protein with identical migration in preparations of bovine peripheral nervous system and human central nervous system axolemma. In some sera, antibodies reacting with the 35 kDa antigen were adsorbed by D-O bovine serum albumin, a synthetic analogue of the terminal disaccharide portion of the phenolic glycolipid 1 of Mycobacterium leprae. Antibodies to the 42 kDa antigen were not removed by this treatment.

A patient with lepromatous leprosy and anticytoskeletal antibodies.

Sera from 34 patients with lepromatous leprosy were screened for the presence of autoantibodies by indirect immunofluorescence using two epithelial cell lines, PTK2 and HEp2, as substrates. Indirect immunofluorescence staining of both substrates with the serum of a patient with lepromatous leprosy revealed a cytoplasmic intermediate filament staining pattern. After exposure of PTK2 cells to colchicine, the filaments collapsed into thick perinuclear coils, confirming the presence of intermediate filament reactivity. Immunofluorescence of rat fibroblasts with the same serum also revealed an intermediate filamentous staining pattern. Human keratinocytes exposed to the patient's serum revealed a diffuse cytoplasmic staining pattern. Our study suggests the presence of autoantibodies to cytoskeletal intermediate filaments or to molecules associated with vimentin and possibly keratin subunit proteins in the serum of a patient with lepromatous leprosy.