Clin. exp. Immunol. (1992) 87, 368-372

Detection of antibodies to human nerve antigens in sera from leprosy patients by ELISA J. Y. PARK, S. N. CHO, J. K. YOUN, D. I. KIM*, R. V. CELLONAt, T. T. FAJARDO Jrt, G. P. WALSHt & J. D. KIM Department of Microbiology, Yonsei University College of Medicine, Seoul, *Korean Institute for Leprosy Research, Anyang, Korea, and tLeonard Wood Memorial Centre for Leprosy Research, Cebu, Philippines

(Acceptedfor publication 2 October 1991)

SUMMARY to play a role in the pathogenesis of nerve damage in implicated been have Anti-neural antibodies 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 1 13 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.



antigens antibodies leprosy autoimmunity

On the other hand, Mshana et al. [4] could not demonstrate any significant association between seroreactivity to bovine myelin proteins and leprosy. Recently, further conflicting results have appeared in the literature. Itty et al. [5] reported that all leprosy patients examined had significant antibodies to human peripheral nerve antigens with a specificity of almost 100%. However, Ghaswala et al. [6] could not find any significant difference in seroreactivity to human peripheral nerve antigens between controls and leprosy patients. It may thus be important to determine the association of anti-neural antibodies with the extent and degree of nerve damage in leprosy patients and to identify the antigenic molecules associated with nerve damage before looking into the role of anti-neural antibodies in the pathogenesis of nerve damage in leprosy. In this study, therefore, we attempted to prepare neurofilament-enriched nerve antigens from human peripheral nerves and spinal cords and to use the nerve antigens in immunoblotting to examine the antigenic molecules and in ELISA for detecting antibodies in sera from leprosy patients and controls. In addition, the prevalence of anti-neural antibodies was analysed based on clinical types, bacterial indices,

INTRODUCTION Nerve damage is a major manifestation of leprosy. Although the pathogenic mechanisms of nerve damage remain to be determined, anti-neural antibodies have been explored as a possible cause of nerve destruction. To determine the association between anti-neural antibodies and nerve damage in leprosy patients, there have been many attempts to detect antibodies to nerve antigens. The results from previous studies were, however, mixed. Wright et al. [1] reported anti-axonal antibodies in 39% of leprosy patients by the indirect immunofluorescent antibody (IFA) test. This was comparable to the report that about 35% of leprosy patients had significant antibodies to neurofilament proteins originating from axons [2]. Subsequently, Benjamins et al. [3] reported that the major antigenic components of the neurofilament-enriched fractions were the 35-kD and 42-kD proteins and that about 47% of leprosy patients had antibodies bound to 35 kD, 42 kD, or to both antigens. Correspondence: Dr Sang-Nae Cho, Department of Microbiology, Yonsei University College of Medicine, C.P.O. Box 8044, Seoul 100680, Republic of Korea.



Anti-neural antibodies in leprosy 1*0

chemotherapy, and the history of erythema nodosum leprosum (ENL) reactions in leprosy patients. PATIENTS AND METHODS Serum specimens Serum specimens were obtained from 289 leprosy patients from the Catholic Skin Disease Clinic, Taegu, Korea and from the Skin Clinic at the Leonard Wood Memorial Centre, Cebu, Philippines. Patients were classified according to the criteria established by Ridley & Jopling [7]. For controls, serum samples were obtained from 154 staff members at a teaching hospital with no apparent nerve damage. Serum specimens from 20 patients with tuberculosis meningitis were also examined for the presence of anti-neural antibodies. Nerve antigens Nerve antigens were prepared from human cervical spinal cords (from CI and C2 segments) of persons who died by accident or trauma and using the protocols described by Schlaepfer et al. [8] and Eustis-Turf et al. [2] with minor modifications, which yielded mostly intermediate filament-enriched fractions. Briefly, the spinal cords were stripped of the outer connective tissues, minced, and macerated by soaking in 2-5 mm EGTA and 1 0 mM sodium phosphate buffer solution, pH 7-0, for I h at room temperature. An equal volume of0-25 M NaCi was then added to the mixture, and homogenized using a Ten Broek Tissue Grinder (Corning, Corning, NY). After centrifugation at 12 000 g for 20 min, the supernatant was saved. The pellet was then rehomogenized in a double volume of 0-1 M NaCl and centrifuged.

Rabbit anti-neural antiserum Two New Zealand White rabbits were immunized intramuscularly with 1 mg of the nerve antigens mixed with Freund's incomplete adjuvant (Sigma Chemical Co, St Louis, MO). After an additional two injections with a 4-week interval, blood samples were obtained from the ear vein. Serum was separated and stored at -20 C until used. ELISA ELISA described by Voller et al. [9] was employed with minor modifications. Briefly, 50 ,ul of diluted nerve antigen (2 5 gg/ml) in carbonate buffer, pH 9-6, were added to the wells of flatbottomed ELISA plates (Costar, Cambridge, MA), and incubated overnight at 37C in a moist chamber. The wells were then washed with phosphate-buffered saline, pH 7 4, containing 0-05% Tween 20 (PBST), and blocked with PBST containing 0-5% bovine serum albumin (BSA) for I h. After emptying the wells, 50 pl of serum diluted 1/300 in PBST containing 5% normal goat serum (NGS) (GIBCO, Grand Island, NY) were added to the wells and incubated for 90 min at 370C. After washing the wells, 50 p1 of affinity-purified peroxidase-conjugated goat anti-human IgG (Behring Diagnostics, La Jolla, CA) diluted 1/5000 in PBST-5% NGS were added and incubated for 90 min at 370C. Finally, after another washing, 50 pl of the mixture of o-phenylenediamine and H202 in citrate buffer, pH 5-0, were added and incubated at room temperature for about 10 min. The reaction was then stopped with 50 p1 of 2-5 N H2SO4 and the absorbance was read at 490 nm. Each serum specimen was tested in duplicate and the mean absorbance in wells

0 *8

0.6 _



41 CD '-

0.4 2*


Leprosy patients

Healthy controls

Fig. 1. Scatter diagram of seroreactivity to nerve antigens in sera from leprosy patients and healthy controls. Each point represents one person. The arrows indicate the mean absorbance for each group, and all points above the dashed line (the absorbance: 0 205) are considered seropositive.

without antigens was subtracted from the mean absorbance in wells with nerve antigens before analysis. Western blotting Nerve antigens were separated on 10% gel by a discontinuous SDS-PAGE as reported by Laemmli [10]. The nerve antigen molecules separated on gel were then transferred to nitrocellulose membrane in a electrophoretic transfer apparatus by the method described by Towbin et al. [11]. After blocking the membrane with PBST containing 3% BSA, the nerve antigen molecules were reacted with serum diluted 1/100, followed by the rest of the ELISA steps described above. Finally, antigen bands reacting with anti-neural antibodies were visualized with 4-chloro-l-naphthol (Bio-Rad, Richmond, CA) solution. The molecular weight of the nerve antigens was determined by comparing with the pre-stained molecular markers (Bio-Rad).

Statistical analysis The X2-test was employed to examine the significance in difference in the prevalence of anti-neural antibodies between leprosy patients and controls. RESULTS

Seroreactivity of each individual in the patient and control groups to neurofilament antigens in ELISA are shown in Fig. 1. The mean absorbance (± s.d.) was 0-187 + 0-1 10 for 289 leprosy

J. Y. Park et al.


Table 1. Prevalence of anti-neural antibodies among leprosy patients and controls by ELISA Seropositive*

Subjects Leprosy patients Lepromatous Tuberculoid Untreated Treated TB meningitis Healthy subjects *

Number assayed



289 176 113 86 203 20 154

74 53 21 16 58 1 11

25 6 30 1 18 6 18 6 28 6 50 7-1

Criteria for seropositivity: A490

>0 205.

patients and 0 140 + 0 032 for 1 54 healthy controls, respectively. With the ELISA conditions used in this study, the cut-off value for the seroreactivity was determined as 0-205 by adding 2 x s.d. to the mean absorbance of the healthy controls who had no apparent neurological damage. Of 289 sera from leprosy patients, 74 (25 6',o) had significant anti-neural antibodies; in contrast, one (5 0%)) of 20 tuberculosis patients and 11 (7-1"/,) of 154 controls had significant to antibodiesnervantntigen (Table 1). Thus, leprosy patients had significantly greater chance of exposure to nerve antigens (P

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 a...
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