A comparative evaluation of serological assays for lepromatous leprosy
A comparative antibody analysis of sera from 26 patients with lepromatous leprosy showed consistently high titres to the phenolic glycolipid I disaccharide and to the ML04 epitope of the 35 kD protein antigen of My cobacterium leprae. Antibody titres of these two specificities were positively correlated (p < 0·0 I) and both declined after chemotherapy, although this trend was apparent earlier after the onset of therapy for the anti-35kD antibody response. Two healthy subjects (out of 18 tested)
... (out of 18 tested) from the leprosy endemic area had pronounced anti-PGL-I but no demonstrable anti-35kD antigen activities. In contrast with the above results, antibody levels to lipoarabinomannan were much lower and with great individual variation between the LL patients. Finally, antibody levels to the M. leprae-specific IIIE9 epitope (peptide 422-436) of the 65kD protein antigen were not demonstrable in the majority of LL patients. Serological studies in leprosy have been pursued for the past 80 years! with interest in diagnosis, classification within the spectrum of disease, monitoring of chemotherapy, prognosis of subclinical infection2 and response to vaccination.3 Recent progress in the molecular definition of My cobacter ium leprae-specific, antigenic determinants provided a new impetus to these studies. The phenolic glycolipid I (PGL-I) and its terminal disaccharide have been used in a solid-phase binding enzyme linked assay,4-6 whereas a competition test has been applied using monoclonal antibodies (MAB) to the 35 kD antigen and other protein or polysaccharide antigens,1-!2 These assays confirmed results from previous studies which showed that serum antibody levels are profoundly increased in multi bacillary (lepromatous) but not in paucibacillary (tuberculoid) leprosy. The relative immunodominance of the respective antigens has not as yet been fully evaluated. The purpose of this paper has been to compare antibody levels to distinct epitopes in individual patients with lepromatous leprosy. 0305-75 1 8/88/059 195 +05 $01.00 © British Leprosy Relief Association 195 196 J Mwatha et al. Materials and Methods SERA Thirty patients, attending the clinic of the JALMA Institute for Leprosy (Agra, India) were classified as lepromatous leprosy (LL) according to the Ridley-Jopling scale. The 18 control sera were from healthy subjects living in the Agra region. Sera were collected from patients before and at various times after chemotherapy which was given in accordance with WHO recommendations. 13 ANTI 35 KD PROTEIN, ML04-COMPETITION ASSAY Microtitre plates (Immulon, M 129B Dynatech) were coated with 50 Ilg/ml (25 Ill/well) M. leprae soluble extract (MLSE) for 20 hr at 4°C. After washing 3 times with phosphate buffered saline (PBS) the plates were blocked with 3% bovine serum albumin (BSA) in PBS for I hr at 20°C and serum samples, serially diluted (100-5 1 200) in 3% BSA-0·05% Tween -20 were added to wells. After 30 min incubation at 37°C in a humidified chamber, peroxide-labelled ML04 MAB (25 Ill/well) in BSA-Tween was added and incubated for I hr at 37°C. Plates were washed and incubated with 3,3',5'5' tetramethyl benzidine (TMB)-H 2 0 2 substrate (100 Ill/well, ofa fresh solution containing 50 Ilg/ml TMB and 0·006% H 2 0 2 in 0·08 M Na Citrate pH 5·0) for 30 min at 20°e. The reaction was stopped with 50 Ill/well of 0·5 M H 2 S0 4 and the optical density read at 450 nm in a titertek multiscan reader. Antibody titres are expressed as reciprocal serum dilutions giving 50% inhibition of ML04 binding to MLSE coated wells (ID s o ). ANTI-LIPOARABINOMANNAN (LAM) ML34-COMPETITION ASSAY This test was performed with MAB ML34 which binds to an epitope expressed on (LAM). IS.16 The competition assay was done essentially as described in the previous paragraph, but using 1 2s l_ labelled ML34 as in the original technique.17 PHENOLIC GL YCOLIPID BINDING AS SA Y Microtitre plates were coated with 5 Ilg/ml phenolic glycolipid I disaccharide (PGDS) (3,6-Dimethyl-fJ-D-Glucosyl (1-4) 2,3-Dimethyl-IX-L-Rhamnosyl) conjugated to BSA (from R Gigg) in PBS (50 Ilg/well) for 20 hr at 4°C, washed with PBS and blocked with 3% BSA for I hr at 37°C in a humid chamber. Serial doubling dilutions (100-5 1 200) of test sera in 1% BSA Tween (50 J.tl/well) were added to PGDS-coated or uncoated plates and incubated for 2 hr at 37°C. Plates were washed with TweenjPBS, then incubated with 50 J.tl/well of I: 1000 diluted peroxidase coupled goat anti human IgM (Sigma Chern. Co. UK) for I hr at 37°e. After washes with Tween/PBS, substrate was added and the reaction read as described above. Relative binding values were calculated over the range of serum dilutions with OD values corrected for binding to the uncoated plate, using a positive reference serum as 100%. Antibody titres were expressed as reciprocal serum dilutions giving 15% binding (ABT I s ). ANTI-65kD ANTIGEN TESTS Microtitre plates were coated overnight at 4°C with 50 Ill/well of the M. leprae-specific IIIE9 peptidel8 dissolved in 0· 1 M Na bicarbonate at 0· 1 Ilg/ml concentration. Plates were washed with PBS and blocked for I hr at room temperature with BSA/Tween. After incubation with sera for 2 hr and washing with PBS, plates were incubated for I hr with 1/1000 dilution of peroxidase conjugated rabbit anti-human IgG (Sigma Chemical Co) in BSA/Tween, washed, and developed as described above. The IIIE9 MAB was used as the positive control. Comparative evaluation of serological assays fo r LL 197 Additional tests were performed with radio labelled IVD8 antibodyI 2 using the competition assay as described before.'7 STATISTICAL ANALYSIS Correlations betweeen antibody titres of the various specificities and for antibody titres related to years of chemotherapy were analysed using correlation coefficients.