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  • Volume 69 , Number 1
  • Page: 46–8
CORRESPONDENCE

A study on the reproducibility of two serological assays for detection of Mycobacterium leprae infection

Om Parkash






To the Editor:

The detection of Mycobacterium lepraeinfection is one of the important tools for effective control of leprosy. It is known that phenolic glycolipid-I (PGL-I) and 35-kDa protein are the two components of M. leprae which are major targets for antibody response in leprosy patients. Employing PGL-I and a monoclonal antibody against 35-kDa antigen, two M. leprae -specific serological assays-the PGL-I-based indirect enzyme linked immunosorbent assay (PGL-ELISA) and the monoclonal antibody inhibition test (MAIT)-have been developed (4,8,10,12). These assays are highly specific and sensitive for detection of M. leprae infection, mainly in lepromatous patients. Apart from the diagnosis of M. leprae infection, PGL- ELISA might also be useful for application in monitoring the response of the patients to chemotherapy (2,9), to measure the magnitude of M. leprae infection (17), to identify the emergence of relapse (6) and lepra reactions (1). On the other hand, the MAIT has been studied mainly for diagnostic applications (13) and for monitoring the efficacy of chemotherapy (5,11). It is well understood that accuracy and reliability of any test depend upon its reproducibility. Therefore, reproducibility is considered as an essential characteristic of any ideal diagnostic assay. To our knowledge, regarding PGL-ELISA and MAIT there is no documented information about their reproducibilities. Therefore, this study was carried out to investigate in tra-assay and inter-assay variability for these two assays.

Measurement of anti-PGL-I antibodies by ELISA. IgM anti PGL-I antibodies were measured by indirect ELISA following the major steps described by Sinha, et al. (11) with slight modifications where required. In brief, the wells of a round-bottom microtiter plate (Immunoplate; Nunc, Raskilde, Denmark) were coated (by incubating the wells overnight at 4°C) with 50 µl/well (containing required concentration of antigen, i.e., 2 ng/50 µl) of trisaccharide conjugated to bovine serum albumin (NT-P-BSA). Uncoated antigen was removed by dumping, followed by washing (three times) with phosphate buffered saline (PBS) containing 0.1% Tween-20 (PBST) and patting of the plates on tissue paper to make them free of washing buffer. Wells of the plate were then blocked with 2.0% bovine serum albumin (150 µl/ml) in phosphate buffered saline (BSA-PBS) for 1.5 hr. After washing the wells, respective patients' sera, diluted 1:300 with BSA-PBS, were put into wells (50 µl/well). The wells were then incubated for 2 hr at 37°C and washed. This was followed by the addition of 1:1000 diluted proxidase labelled antihuman IgM. The wells were incubated at 37°C for 1 hr. The washing step was repeated and then the wells were incubated (at room temperature for 20 min) with ortho-phenylene di­amine dihydrochloride (Sigma Chemical Co., St. Louis, Missouri, U.S.A.) supplemented with hydrogen peroxide. Finally, the reaction was stopped by adding 7.0% H,S04 (50 µl/well) and the optical density (OD) values were recorded at 492 nm in a microplate ELISA reader. Serum samples with an ab- sorbance of greater than or equal to 0.2 were considered positive.

Measurement ofanti-M. leprae antibod­ies by monoclonal antibody based assay. The anti-M leprae antibodies were measured by a competitive inhibition type of ELISA. The main guidelines for performing this test were obtained from the previously described method (11) with slight modifications. Briefly, the round-bottom wells of microtiter plates (Nunc) were coated with 50 pl/well of M. leprae antigen (containing 5 pg/50 pi of anti­gen) by overnight incubation at 4°C. After this, unbound antigen was removed by washing with PBST. The wells were then blocked with 2.0% BSA-PBS for 1.5 hr at 37°C. The wells were then emptied and washed. Next, serum samples (diluted 1:10 in BSA-PBS) were added (25 µl/well) to the wells and incubated at 37°C for 1 hr. This was then followed by the addition of 25 µl/well of diluted (1:1000) peroxidase labelled ML04. Incubation was further carried out for 2 hr at 37°C. All the wells were then washed with PBST and blotted dry before the addition of substrate (orthophenylene diamine dihydrochloride) for 20 min. Finally, the enzymatic reaction was stopped with H,S04 and readings were recorded by an ELISA reader. The results were expressed as percent inhibition, by anti-M. leprae antibodies in patients' sera, of binding of labelled monoclonal antibody ML04 to M. leprae antigen coated on the wells. The serum causing ≥50% inhibition was taken as positive.

The reproducibilities of the assays were determined by testing three serum specimens for each assay, having varying quantities of anti-M. leprae antibodies. Serum samples with high levels of antibody were strongly positive. Samples with medium levels of antibodies were close to the cut-off point. Low antibody level sera had antibodies below the cut-off value. As described above, the PGL- ELISA readings were recorded as OD values; whereas for MAIT the results were expressed as percent inhibition of binding of peroxidase labelled anti-M. leprae monoclonal antibody (ML04) to coated M. leprae antigen. The variability in test readings was expressed as a percent coefficient of variation (%CV). Data obtained by PGL-ELISA are shown in Table 1 and MAIT data are given in Table 2. The results indicate that intra-assay variations of both the assays were smaller than 12% for sera having high and medium levels of antibodies and >28% for sera containing low levels of antibodies. The overall intra-assay variation (12.9%) for PGL-ELISA was slightly lower than that for MAIT (19.6%). However, inter-assay variations with all the sera were moderately high (ranging from 21.3% to 31.9%) for both the assays. Another interesting observation was that with sera having high levels of antibodies variation was less. Contrary to this, low antibody containing serum showed the highest variation. Thus, a kind of gradation, depending upon the antibody levels, in variation was seen.

 

 

 

Having observed these variations in readings of the assays, it became tempting to investigate the reproducibilities (intra-assay as well as inter-assay) of the approach to score the test results as positive or negative. According to our findings, the reproducibility rates were better with the PGL-ELISA (Table 3) than with the MAIT (Table 4). All sera having various levels of anti-M. leprae antibody showed promising reproducibility (90%-100%) with the PGL-ELISA. On the other hand, with the MAIT, sera having high and low levels of anti-M. leprae antibodies showed 100% reproducibility; whereas sera having medium levels of antibody showed comparatively lower reproducibility (i.e., 79% for intra-assay and 78% for inter-assay). Nevertheless, the overall reproducibilities of both the assays were remarkably high (almost above 92%).

 

 

 

In light of the above findings, I wish to suggest that sera showing values near the cut-off point must be repeated at least three times, particularly when using the MAIT in order to get accurate results. Contrarily, testing for samples having high and low levels of antibodies need not be repeated.

 

- Om Parkash, M.Sc.; Ph.D.

Senior Research Officer (Immunology)
Central JALMA Institute for Leprosy
Taj Ganj
Agra 282 001, India

Acknowledgment. I thank LEPRA. United Kingdom, for its support in acquiring some of the reagents/chemicals used for the study. Thanks to Dr. M. J. Colston, National Institute for Medical Research, London, U.K.; Dr. S. Izumi, Tokyo. Japan and Dr. J. Ivanyi, U.K., for providing M. leprae sonicated antigen. natural trisaccharide conjugated to bovine serum albumin (NT-P-BSA) and monoclonal antibody against M. leprae , respectively. Thanks to Dr. Mohan Natrajan. Assistant Director, Central JALMA Institute for Leprosy, for his suggestions.

 

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