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Use of a polysulfone membrane support for immunochemical analysid of a glycilipid from Mycobacterium leprae
Journal oflmmunologicalMethods, 79 (1985) 205-211
205
Elsevier JIM03492
Use of a Polysulfone Membrane Support for Immunochemical Analysis of a Glycolipid from
Mycobacterium leprae D o u g l a s B. Y o u n g 1, M e l i n d a J. F o h n 1 a n d T h o m a s M . B u c h a n a n 1,2 1 Immunology Research Laboratory, Pacific Medical Center, University of Washington,, Seattle, WA 98144, and 2 National Hansens Disease Center, Camille, LA 70721, U.S.A.
(Received2 November1984, accepted21 January 1985)
Polysulfonemembraneshave been used as a solid support for chromatographyand immunoblottingof phenolic glycolipid I from Mycobacterium leprae. These membranes have an advantage over other supports such as nitrocellulose and silica gel in that very little non-specificbackground binding of antibodies occurs and assays can readily be carried out with IgM antibodies from human sera. An example of use of the polysulfonechromatographysystem for detection of phenolic glyeolipid I in sera from leprosypatients is described. Key words: polysulfone membrane - glycolipid - Mycobacterium leprae - dot immunoblotting
Introduction Nitrocellulose membranes are frequently employed as a support for protein antigens during immunochemical analysis by techniques such as Western blotting (Towbin et al., 1979) and dot immunoblotting (Hawkes et al., 1982). Several authors have e m p l o y e d an immunoblotting technique for analysis of glycolipid antigens based on a procedure developed by Magnani et al. (1980) involving direct interaction of monoclonal antibodies with lipids separated on silica gel plates following thin layer chromatography (Cheresh et al., 1984; Symington et al., 1984; Young et al., 1984). In comparing antibodies directed to protein and to glycolipid antigens we have frequently found a higher degree of non-specific background binding of the anti-glycolipid antibodies to materials sucla as nitrocellulose. This observation is probably related to t h e ' f a c t that these antibodies frequently belong to the IgM class of immunoglobulins which have been shown to bind non-specifically to a variety of compounds (Wichman and Borg, 1977). While we have been able to show interaction of mouse monoclonal antibodies with phenolic glycolipid I of M y c o b a c t e r i u m leprae on silica gel plates (Young et al., 1984), very high background levels have 0022-1759/85/$03.30 © 1985 ElsevierSciencePublishers B.V. (BiomedicalDivision)
206 prevented a similar demonstration with human sera containing predominantly lgM antibodies to the glycolipid (Young et al., 1984b). In order to simplify the analysis of glycolipid antigens and to develop procedures comparable to those used for protein antigens, we have tested a variety of solid supports with the aim of identifying a material with the ability to bind lipid antigens but having a minimum amount of non-specific binding of IgM antibodies: In this paper we describe the analysis of antibodies to M. leprae phenolic glycolipid I using a polysulfone membrane support. Materials and Methods
Initial screening assay Phenolic glycolipid I purified from infected armadillo tissue (Young and Buchanan, 1983) was dissolved in hexane and spotted onto solid support materials in 2.5 #1 drops containing serial dilutions of lipid. A control spot with hexane only was also applied. After 5 rain, strips were washed for 10 rain each in 2 changes of phosphate-buffered saline pH 7.2 (PBS) and then incubated for 1 h at 37°C with 5% (w/v) bovine serum albumin (BSA) in PBS. Strips were then incubated with human serum samples (diluted 1 : 200) or with monoclonal antibody to phenolic glycolipid (PG2 BSF) (Young et al., 1984) (diluted 1 : 1000) in PBS containing 5% (v/v) fetal calf serum. After 1 h at 37°C, strips were given four 10 min washes in PBS and then incubated for a further 1 h at 37°C with peroxidase-conjugated goat anti-human immunoglobulins (IgA + IgG + IgM) or with peroxidase-conjugated goat anti-mouse IgM (both from Cappel Laboratories, Cochranville, PA) diluted 1 : 1000 in PBS with 1% BSA. Strips were washed a further 4 times and bound antibody was detected by incubation for 10 rain at room temperature in 0.1 M citrate buffer pH 5 containing 0.5 mg/ml 3,3'-diaminobenzidine (Sigma Chemical Co., St. Louis, MO) and 0.03% H 202. The reaction was stopped by washing in distilled water. Assay on polysulfone membrane The low degree of non-specific binding to the polysulfone membrane allowed the above assay to be simplified as follows. Antigen was applied in hexane to strips of polysulfone supplied as Tuffryn® membrane filters HT-200 (0.2 /~m pore size) by Gelman Sciences (Ann Arbor, MI). Strips were rinsed briefly in PBS then incubated for 15 min with 5% BSA in PBS on a rotary shaker. Antibody was then added directly to the BSA solution and incubation continued for a further 1 h at room temperature. Strips were washed as above and secondary antibody was then added in PBS with 1% BSA. After 1 h at room temperature, strips were washed and color development was carried out as above. Alternatively, peroxidase-conjugated secondary antibody was substituted by radiolabeled secondary antibody and washed strips were exposed to X-ray film overnight to form autoradiographs. Antibodies were labeled with 125I using the chloramine-T method described by Greenwood et al. (1963). Chromatography on polysulfone membrane Phenolic glycolipid in hexane was spotted onto the bottom of polysulfone strips
207 and allowed to dry. Separating solvent was added to a chromatography tank and, after a few hours to allow saturation of the tank, polysulfone strips were placed inside with their ends dipping in the solvent. Solvent was allowed to run up the strips for 30 min in a manner identical to that used for conventional thin-layer chromatography on silica gel plates. The strips were removed from the tank, allowed to dry, and screened with antibodies as described above.
Analysis of lipids from human sera Lipids were extracted from serum samples by Bligh-Dyer monophasic extraction (Bligh and Dyer, 1959). Serum sample (0.5 ml) was mixed with distilled water (0.5 ml), methanol (2.5 ml) and chloroform (1.25 ml) and incubated for 2 h at room temperature with occasional mixing. Chloroform (1.25 ml) and water (1.25 ml) were then added with further mixing and 2 phases were separated by centrifugation at 5000 × g for 15 min. The lower (chloroform) layer was removed and the upper layer re-extracted with a further 2.5 ml of chloroform. The 2 chloroform extracts were combined and evaporated to dryness under a stream of nitrogen. Phenolic glycolipid was partially purified from the chloroform extracts by a method based on that of Hunter and Brennan (1981). Silicic acid and celite were mixed in a 2 : 1 ratio and 100 mg of the dry powder was placed in a pasteur pipette plugged with glass wool. This was washed with 5 ml of chloroform and lipid extracts from human sera were then applied in 2 portions of 0.5 ml of chloroform. This was followed by a wash with 2 ml of chloroform and then a final elution with 2 ml of 2.5% (v/v) methanol in chloroform. By applying gentle pressure with a pipette bulb to the top of the pasteur pipette, the partial purification procedure could be accomplished in 5-10 min. The 2.5% methanol eluates were evaporated under nitrogen and then analyzed by chromatography on polysulfone membranes. Results
Screening of solid supports A variety of potential support media were screened for their ability to bind the glycolipid antigen and their resistance to non-specific background binding of IgM antibodies. These included nitrocellulose, cellulose acetate, regenerated cellulose and polysulfone membranes, paper and glass fiber filters, glass slides and silica gel thin-layer chromatography plates. The polysulfone membrane was found to bind the glycolipid antigen with very lit.tie background binding of the antibody. Fig. 1 compares the binding of human sera to phenolic glycolipid spotted onto nitrocellulose and polysulfone. In both cases binding of antibodies to the glycolipid could be shown using sera from leprosy patients while sera from uninfected individuals were negative. With the nitrocellulose membrane, however, a considerable amount of background staining of the strip was observed after incubation with human sera (strips l(b) and 2(b) in Fig. 1) while the polysulfone membrane remained white. Chromatography on polysulfone membrane Phenolic glycolipid was found to migrate on polysulfone strips in a manner
208 analogous to that observed during thin-layer chromatography on silica gel plates. Fig. 2 shows an autoradiograph prepared from 6 polysulfone strips which were spotted with glycolipid (100 ng) and subjected to chromatography using increasingly polar solvents with diethyl ether added to hexane at 0, 5, 10, 20, 30 and 40% (v/v). The resulting position of the antigen was then located using monoclonal antibody. The glycolipid migrated approximately half way up the strip in the presence of 10% ether in hexane. Phenolic glycolipid in human sera The chromatography procedure on polysulfone was used to analyze lipid extracts from serum samples obtained from leprosy patients and from uninfected individuals. Lipid samples equivalent to an original serum volume of 0.25 ml were spotted onto polysulfone in 20/~1 of hexane and chromatographed using 10% ether in hexane. Fig. 3 shows an autoradiograph of such a chromatogram after development with monoclonal antibody and 12SI-labeled secondary antibody. Lanes 1 and 10 contained standard samples of purified phenolic glycolipid I (100 ng each) and lane 2 contained an extract from a pooled sample of normal human sera. A lipid with a
2 (~)
3 (a)
]] 2. (b)
3 (b)
Fig. 1. Comparison of polysulfone and nitrocellulose membranes. Strips of polysulfone (l(a), 2(a)) and nitrocellulose (l(b), 2(b)) were spotted with 2.5 ~l drops of hexane containing (left to right) 200, 100, 50, 25, 12, 6 and 0 ng of phenolic glycolipid. Strips labeled '1' were then reacted with pooled sera from lepromatous leprosy patients, while strips labeled '2' were reacted with a pool of normal human sera. Bound antibody was detected using peroxidase-conjugatedsecondary antibody as described in the text. Strips 3(a) and 3(b) show control portions of polysulfone and nitrocellulose respectively incubated without primary antibody.
209
0
5
1 0 .....
30
40
Fig. 2. Chromatography of phenolic glycolipid on polysulfone. Phenolic glycolipid was spotted onto polysulfone strips and subjected to chromatography as described in the text. The figure shows an autoradiograph of the membrane after reaction with monoclonal antibody and radiolabded secondary antibody. 'O' marks the point of application of the samples, 'S' marks the solvent front. The numbers below each strip indicate the percentage volume of ether present in the hexane developing solvent.
0".,
" 1
2
3
~e e 4
5
6
7
8
9
10
Fig. 3. Detection of phenolic glycolipid in sera by polysulfone chromatography. Lipid extracts from human sera were analyzed by polysulfone chromatography as described in the text. 'O' marks the point of application of the samples, 'S' marks the solvent front. Lanes 1 and 10 contained standard samples of phenolic glycolipid. Lane 2 contained a sample from a pool of normal human sera. The remaining lanes contained samples from leprosy patients classified according to the Ridley-Jopling scale as 'BT' (lanes 3 and 4), 'BB' (5 and 6) and 'BL' (7, 8 and 9). The serum sample analyzed in lane 9 was from the same patient as that in lane 8, but was taken after 3 years of chemotherapy.
210 migration pattern identical to phenolic glycolipid I was recognized by the monoclonal antibody in extracts from sera of 2 patients with borderline lepromatous (BL) leprosy (lanes 7 and 8) as defined by the Ridley-Jopling scale (Ridley and Jopling, 1966). Samples from borderline (BB) and borderline tuberculoid (BT) patients were negative as was a second sample from the same BL patient shown in lane 8 but taken after 3 years of treatment (lane 9). Some radioactivity was also associated with material remaining at the origin (particularly in lanes 6, 7 and 8) and with material migrating at the solvent front. This may result from non-specific interaction of the monoclonal antibody with other serum lipids or, in lanes 7 and 8, from incomplete separation of the phenolic glycolipid from other lipids during chromatography.
Discussion
The applicability of nitrocellulose membranes for immunochemical analysis is due in part to their ability to form a strong bond with protein antigens. This property is not required for glycolipid antigens and it is advantageous therefore to select a support medium which shows a minimum amount of interaction with proteins in order to reduce background binding of antibodies. Polysulfone was found to be a suitable support for analysis of the phenolic glycolipid from Mycobacterium leprae. The technique involving spotting the glycolipid onto the polysulfone membrane for antibody interaction is analogous to the dot-immunoblotting technique described by Hawkes et al. (1982) for spotting protein antigens onto nitrocellulose. The advantage of the polysulfone support is that it can conveniently be used to monitor binding of IgM antibodies from human sera. An additional property of the polysulfone system is that lipids can be separated by chromatography and then detected by direct interaction with antibody. Again the low background binding of the polysulfone is an advantage as compared to previous assays using silica gel plates (Cheresh et al., 1984; Symington et al., 1984; Young et al., 1984). The utility of the polysulfone chromatography assay has been shown in a preliminary analysis of lipid extracts from human sera. A lipid was identified in 2 serum samples from lepromatous leprosy patients which is identical to M. leprae phenolic glycolipid I both in its chemical properties - - as seen by its migration during polysulfone chromatography - - and in its antigenic properties - - as defined by its interaction with a monoclonal antibody to the glycolipid. The absence of this lipid from normal sera along with its specific chemical and immunological properties strongly suggest that it is in fact phenolic glycolipid I. The structure of phenolic glycolipid I is based on study of the lipid isolated from infected armadillo tissues (Hunter and Brennan, 1981; Hunter et al., 1982) but a similar glycolipid has previously been found in human tissues during analysis of skin biopsies from leprosy patients (Young, 1981). The detection of the glycolipid in sera from lepromatous leprosy patients but not from tuberculoid leprosy patients is consistent with the fact that the former group is characterized by a high antigen load as seen by staining for acid-fast bacteria (Ridley and Jopling, 1966).
211
Acknowledgements This research was supported in part by the IMMLEP and THELEP components of the UNDP/World Bank/WHO Special Program for Research and Training in Tropical Diseases, and by the Rockefeller Foundation Program for Research on Great Neglected Diseases.
References Bligh, E.G. and W.J. Dyer, 1959, Can. J. Biochem. Physiol. 37, 911. Cheresh, D.A., A.P. Varki, N.M. Varki, W.B. Stallcup, J. Levine and R.A. Reisfeld, 1984, J. Biol. Chem. 259, 7453. Greenwood, F.C., W.M. Hunter and J.S. Glover, 1963, Biochem. J. 89, 114. Hawkes, R., E. Niday and J. Gordon, 1982, Anal. Biochem. 119, 142. Hunter, S.W. and P.J. Brennan, 1981, J. Bacteriol. 147, 728. Hunter, S.W., T. Fujiwara and P.J. Brennan, 1982, J. Biol. Chem. 257, 15072. Magnani, J.L., S. Smith and V. Ginsberg, 1980, Anal. Biochem. 109, 399. Ridley, D.S. and W.H. Jopling, 1966, Int. J. Lepr. 34, 255. Symington, F.W., I.D. Bernstein and S.I. Hakomori, 1984, J. Biol. Chem. 259, 6008. Towbin, H., T. Staehelin and J. Gordon, 1979, Proc. Natl. Acad. Sci. U.S.A. 76, 4350. Wichman, A. and H. Borg, 1977, Biochim. Biophys. Acta 490, 363. Young, D.B., 1981, Int. J. Lepr. 49, 198. Young, D.B. and T.M. Buchanan, 1983, Science 221, 1057. Young, D.B., S.R. Khanolkar, L.L. Barg and T.M. Buchanan, 1984a, Infect. Immun. 43, 183. Young, D.B., S. Dissanayake, R.A. Miller, S.R. Khanolkar and T.M. Buchanan, 1984b, J. Infect. Dis. 149, 870.
205
Elsevier JIM03492
Use of a Polysulfone Membrane Support for Immunochemical Analysis of a Glycolipid from
Mycobacterium leprae D o u g l a s B. Y o u n g 1, M e l i n d a J. F o h n 1 a n d T h o m a s M . B u c h a n a n 1,2 1 Immunology Research Laboratory, Pacific Medical Center, University of Washington,, Seattle, WA 98144, and 2 National Hansens Disease Center, Camille, LA 70721, U.S.A.
(Received2 November1984, accepted21 January 1985)
Polysulfonemembraneshave been used as a solid support for chromatographyand immunoblottingof phenolic glycolipid I from Mycobacterium leprae. These membranes have an advantage over other supports such as nitrocellulose and silica gel in that very little non-specificbackground binding of antibodies occurs and assays can readily be carried out with IgM antibodies from human sera. An example of use of the polysulfonechromatographysystem for detection of phenolic glyeolipid I in sera from leprosypatients is described. Key words: polysulfone membrane - glycolipid - Mycobacterium leprae - dot immunoblotting
Introduction Nitrocellulose membranes are frequently employed as a support for protein antigens during immunochemical analysis by techniques such as Western blotting (Towbin et al., 1979) and dot immunoblotting (Hawkes et al., 1982). Several authors have e m p l o y e d an immunoblotting technique for analysis of glycolipid antigens based on a procedure developed by Magnani et al. (1980) involving direct interaction of monoclonal antibodies with lipids separated on silica gel plates following thin layer chromatography (Cheresh et al., 1984; Symington et al., 1984; Young et al., 1984). In comparing antibodies directed to protein and to glycolipid antigens we have frequently found a higher degree of non-specific background binding of the anti-glycolipid antibodies to materials sucla as nitrocellulose. This observation is probably related to t h e ' f a c t that these antibodies frequently belong to the IgM class of immunoglobulins which have been shown to bind non-specifically to a variety of compounds (Wichman and Borg, 1977). While we have been able to show interaction of mouse monoclonal antibodies with phenolic glycolipid I of M y c o b a c t e r i u m leprae on silica gel plates (Young et al., 1984), very high background levels have 0022-1759/85/$03.30 © 1985 ElsevierSciencePublishers B.V. (BiomedicalDivision)
206 prevented a similar demonstration with human sera containing predominantly lgM antibodies to the glycolipid (Young et al., 1984b). In order to simplify the analysis of glycolipid antigens and to develop procedures comparable to those used for protein antigens, we have tested a variety of solid supports with the aim of identifying a material with the ability to bind lipid antigens but having a minimum amount of non-specific binding of IgM antibodies: In this paper we describe the analysis of antibodies to M. leprae phenolic glycolipid I using a polysulfone membrane support. Materials and Methods
Initial screening assay Phenolic glycolipid I purified from infected armadillo tissue (Young and Buchanan, 1983) was dissolved in hexane and spotted onto solid support materials in 2.5 #1 drops containing serial dilutions of lipid. A control spot with hexane only was also applied. After 5 rain, strips were washed for 10 rain each in 2 changes of phosphate-buffered saline pH 7.2 (PBS) and then incubated for 1 h at 37°C with 5% (w/v) bovine serum albumin (BSA) in PBS. Strips were then incubated with human serum samples (diluted 1 : 200) or with monoclonal antibody to phenolic glycolipid (PG2 BSF) (Young et al., 1984) (diluted 1 : 1000) in PBS containing 5% (v/v) fetal calf serum. After 1 h at 37°C, strips were given four 10 min washes in PBS and then incubated for a further 1 h at 37°C with peroxidase-conjugated goat anti-human immunoglobulins (IgA + IgG + IgM) or with peroxidase-conjugated goat anti-mouse IgM (both from Cappel Laboratories, Cochranville, PA) diluted 1 : 1000 in PBS with 1% BSA. Strips were washed a further 4 times and bound antibody was detected by incubation for 10 rain at room temperature in 0.1 M citrate buffer pH 5 containing 0.5 mg/ml 3,3'-diaminobenzidine (Sigma Chemical Co., St. Louis, MO) and 0.03% H 202. The reaction was stopped by washing in distilled water. Assay on polysulfone membrane The low degree of non-specific binding to the polysulfone membrane allowed the above assay to be simplified as follows. Antigen was applied in hexane to strips of polysulfone supplied as Tuffryn® membrane filters HT-200 (0.2 /~m pore size) by Gelman Sciences (Ann Arbor, MI). Strips were rinsed briefly in PBS then incubated for 15 min with 5% BSA in PBS on a rotary shaker. Antibody was then added directly to the BSA solution and incubation continued for a further 1 h at room temperature. Strips were washed as above and secondary antibody was then added in PBS with 1% BSA. After 1 h at room temperature, strips were washed and color development was carried out as above. Alternatively, peroxidase-conjugated secondary antibody was substituted by radiolabeled secondary antibody and washed strips were exposed to X-ray film overnight to form autoradiographs. Antibodies were labeled with 125I using the chloramine-T method described by Greenwood et al. (1963). Chromatography on polysulfone membrane Phenolic glycolipid in hexane was spotted onto the bottom of polysulfone strips
207 and allowed to dry. Separating solvent was added to a chromatography tank and, after a few hours to allow saturation of the tank, polysulfone strips were placed inside with their ends dipping in the solvent. Solvent was allowed to run up the strips for 30 min in a manner identical to that used for conventional thin-layer chromatography on silica gel plates. The strips were removed from the tank, allowed to dry, and screened with antibodies as described above.
Analysis of lipids from human sera Lipids were extracted from serum samples by Bligh-Dyer monophasic extraction (Bligh and Dyer, 1959). Serum sample (0.5 ml) was mixed with distilled water (0.5 ml), methanol (2.5 ml) and chloroform (1.25 ml) and incubated for 2 h at room temperature with occasional mixing. Chloroform (1.25 ml) and water (1.25 ml) were then added with further mixing and 2 phases were separated by centrifugation at 5000 × g for 15 min. The lower (chloroform) layer was removed and the upper layer re-extracted with a further 2.5 ml of chloroform. The 2 chloroform extracts were combined and evaporated to dryness under a stream of nitrogen. Phenolic glycolipid was partially purified from the chloroform extracts by a method based on that of Hunter and Brennan (1981). Silicic acid and celite were mixed in a 2 : 1 ratio and 100 mg of the dry powder was placed in a pasteur pipette plugged with glass wool. This was washed with 5 ml of chloroform and lipid extracts from human sera were then applied in 2 portions of 0.5 ml of chloroform. This was followed by a wash with 2 ml of chloroform and then a final elution with 2 ml of 2.5% (v/v) methanol in chloroform. By applying gentle pressure with a pipette bulb to the top of the pasteur pipette, the partial purification procedure could be accomplished in 5-10 min. The 2.5% methanol eluates were evaporated under nitrogen and then analyzed by chromatography on polysulfone membranes. Results
Screening of solid supports A variety of potential support media were screened for their ability to bind the glycolipid antigen and their resistance to non-specific background binding of IgM antibodies. These included nitrocellulose, cellulose acetate, regenerated cellulose and polysulfone membranes, paper and glass fiber filters, glass slides and silica gel thin-layer chromatography plates. The polysulfone membrane was found to bind the glycolipid antigen with very lit.tie background binding of the antibody. Fig. 1 compares the binding of human sera to phenolic glycolipid spotted onto nitrocellulose and polysulfone. In both cases binding of antibodies to the glycolipid could be shown using sera from leprosy patients while sera from uninfected individuals were negative. With the nitrocellulose membrane, however, a considerable amount of background staining of the strip was observed after incubation with human sera (strips l(b) and 2(b) in Fig. 1) while the polysulfone membrane remained white. Chromatography on polysulfone membrane Phenolic glycolipid was found to migrate on polysulfone strips in a manner
208 analogous to that observed during thin-layer chromatography on silica gel plates. Fig. 2 shows an autoradiograph prepared from 6 polysulfone strips which were spotted with glycolipid (100 ng) and subjected to chromatography using increasingly polar solvents with diethyl ether added to hexane at 0, 5, 10, 20, 30 and 40% (v/v). The resulting position of the antigen was then located using monoclonal antibody. The glycolipid migrated approximately half way up the strip in the presence of 10% ether in hexane. Phenolic glycolipid in human sera The chromatography procedure on polysulfone was used to analyze lipid extracts from serum samples obtained from leprosy patients and from uninfected individuals. Lipid samples equivalent to an original serum volume of 0.25 ml were spotted onto polysulfone in 20/~1 of hexane and chromatographed using 10% ether in hexane. Fig. 3 shows an autoradiograph of such a chromatogram after development with monoclonal antibody and 12SI-labeled secondary antibody. Lanes 1 and 10 contained standard samples of purified phenolic glycolipid I (100 ng each) and lane 2 contained an extract from a pooled sample of normal human sera. A lipid with a
2 (~)
3 (a)
]] 2. (b)
3 (b)
Fig. 1. Comparison of polysulfone and nitrocellulose membranes. Strips of polysulfone (l(a), 2(a)) and nitrocellulose (l(b), 2(b)) were spotted with 2.5 ~l drops of hexane containing (left to right) 200, 100, 50, 25, 12, 6 and 0 ng of phenolic glycolipid. Strips labeled '1' were then reacted with pooled sera from lepromatous leprosy patients, while strips labeled '2' were reacted with a pool of normal human sera. Bound antibody was detected using peroxidase-conjugatedsecondary antibody as described in the text. Strips 3(a) and 3(b) show control portions of polysulfone and nitrocellulose respectively incubated without primary antibody.
209
0
5
1 0 .....
30
40
Fig. 2. Chromatography of phenolic glycolipid on polysulfone. Phenolic glycolipid was spotted onto polysulfone strips and subjected to chromatography as described in the text. The figure shows an autoradiograph of the membrane after reaction with monoclonal antibody and radiolabded secondary antibody. 'O' marks the point of application of the samples, 'S' marks the solvent front. The numbers below each strip indicate the percentage volume of ether present in the hexane developing solvent.
0".,
" 1
2
3
~e e 4
5
6
7
8
9
10
Fig. 3. Detection of phenolic glycolipid in sera by polysulfone chromatography. Lipid extracts from human sera were analyzed by polysulfone chromatography as described in the text. 'O' marks the point of application of the samples, 'S' marks the solvent front. Lanes 1 and 10 contained standard samples of phenolic glycolipid. Lane 2 contained a sample from a pool of normal human sera. The remaining lanes contained samples from leprosy patients classified according to the Ridley-Jopling scale as 'BT' (lanes 3 and 4), 'BB' (5 and 6) and 'BL' (7, 8 and 9). The serum sample analyzed in lane 9 was from the same patient as that in lane 8, but was taken after 3 years of chemotherapy.
210 migration pattern identical to phenolic glycolipid I was recognized by the monoclonal antibody in extracts from sera of 2 patients with borderline lepromatous (BL) leprosy (lanes 7 and 8) as defined by the Ridley-Jopling scale (Ridley and Jopling, 1966). Samples from borderline (BB) and borderline tuberculoid (BT) patients were negative as was a second sample from the same BL patient shown in lane 8 but taken after 3 years of treatment (lane 9). Some radioactivity was also associated with material remaining at the origin (particularly in lanes 6, 7 and 8) and with material migrating at the solvent front. This may result from non-specific interaction of the monoclonal antibody with other serum lipids or, in lanes 7 and 8, from incomplete separation of the phenolic glycolipid from other lipids during chromatography.
Discussion
The applicability of nitrocellulose membranes for immunochemical analysis is due in part to their ability to form a strong bond with protein antigens. This property is not required for glycolipid antigens and it is advantageous therefore to select a support medium which shows a minimum amount of interaction with proteins in order to reduce background binding of antibodies. Polysulfone was found to be a suitable support for analysis of the phenolic glycolipid from Mycobacterium leprae. The technique involving spotting the glycolipid onto the polysulfone membrane for antibody interaction is analogous to the dot-immunoblotting technique described by Hawkes et al. (1982) for spotting protein antigens onto nitrocellulose. The advantage of the polysulfone support is that it can conveniently be used to monitor binding of IgM antibodies from human sera. An additional property of the polysulfone system is that lipids can be separated by chromatography and then detected by direct interaction with antibody. Again the low background binding of the polysulfone is an advantage as compared to previous assays using silica gel plates (Cheresh et al., 1984; Symington et al., 1984; Young et al., 1984). The utility of the polysulfone chromatography assay has been shown in a preliminary analysis of lipid extracts from human sera. A lipid was identified in 2 serum samples from lepromatous leprosy patients which is identical to M. leprae phenolic glycolipid I both in its chemical properties - - as seen by its migration during polysulfone chromatography - - and in its antigenic properties - - as defined by its interaction with a monoclonal antibody to the glycolipid. The absence of this lipid from normal sera along with its specific chemical and immunological properties strongly suggest that it is in fact phenolic glycolipid I. The structure of phenolic glycolipid I is based on study of the lipid isolated from infected armadillo tissues (Hunter and Brennan, 1981; Hunter et al., 1982) but a similar glycolipid has previously been found in human tissues during analysis of skin biopsies from leprosy patients (Young, 1981). The detection of the glycolipid in sera from lepromatous leprosy patients but not from tuberculoid leprosy patients is consistent with the fact that the former group is characterized by a high antigen load as seen by staining for acid-fast bacteria (Ridley and Jopling, 1966).
211
Acknowledgements This research was supported in part by the IMMLEP and THELEP components of the UNDP/World Bank/WHO Special Program for Research and Training in Tropical Diseases, and by the Rockefeller Foundation Program for Research on Great Neglected Diseases.
References Bligh, E.G. and W.J. Dyer, 1959, Can. J. Biochem. Physiol. 37, 911. Cheresh, D.A., A.P. Varki, N.M. Varki, W.B. Stallcup, J. Levine and R.A. Reisfeld, 1984, J. Biol. Chem. 259, 7453. Greenwood, F.C., W.M. Hunter and J.S. Glover, 1963, Biochem. J. 89, 114. Hawkes, R., E. Niday and J. Gordon, 1982, Anal. Biochem. 119, 142. Hunter, S.W. and P.J. Brennan, 1981, J. Bacteriol. 147, 728. Hunter, S.W., T. Fujiwara and P.J. Brennan, 1982, J. Biol. Chem. 257, 15072. Magnani, J.L., S. Smith and V. Ginsberg, 1980, Anal. Biochem. 109, 399. Ridley, D.S. and W.H. Jopling, 1966, Int. J. Lepr. 34, 255. Symington, F.W., I.D. Bernstein and S.I. Hakomori, 1984, J. Biol. Chem. 259, 6008. Towbin, H., T. Staehelin and J. Gordon, 1979, Proc. Natl. Acad. Sci. U.S.A. 76, 4350. Wichman, A. and H. Borg, 1977, Biochim. Biophys. Acta 490, 363. Young, D.B., 1981, Int. J. Lepr. 49, 198. Young, D.B. and T.M. Buchanan, 1983, Science 221, 1057. Young, D.B., S.R. Khanolkar, L.L. Barg and T.M. Buchanan, 1984a, Infect. Immun. 43, 183. Young, D.B., S. Dissanayake, R.A. Miller, S.R. Khanolkar and T.M. Buchanan, 1984b, J. Infect. Dis. 149, 870.