Journal of Immunological Methods, 134 (1990) 107-112
107
Elsevier JIM 05739
Affinity immunoblotting detection of serum monoclonal immunoglobulins reactive with glycosphingolipids M.O. J a u b e r t e a u , J.M. C o o k , M. D r o u e t a n d J.L. P r e u d ' h o m m e 1 Laboratory of Immunology and CJF I N S E R M 8803, University Hospital, 87042 Limoges Cedex, France, and i Laboratory of Immunology and Immunopathology (CNRS URA 1172), Unioersity Hospital, 86021 Poitiers Cedex, France
(Received20 March 1990, accepted 12 July 1990)
The characterization of serum monoclonal IgM reactive with glycosphingolipids present in the nervous system is of major importance in patients with certain neuropathies. A rapid, sensitive and specific one-step method which permits such a characterization is described. Serum immunoglobulins separated by high resolution agarose electrophoresis are transferred to affinity filters (nitrocellulose sheets coated with glycolipids), and revealed with enzyme-tagged monospecific anti-immunoglobulin antibodies. Key words: Glycosphingolipid;Monoclonalimmunoglobulin; Immunoblot; Affinity filter; Neuropathy
Introduction
Serum monoclonal immunoglobulins (Ig) reactive with glycosphingolipids have been reported in patients with peripheral neuropathies (Ilyas et al., 1984, 1985a,b; Miyatani et al., 1987) and the motor neurone syndrome (Freddo et al., 1986; Ito and Latov, 1988; Nardelli et al., 1988; Jauberteau et al., 1990). Such monoclonal Ig most often belong to the IgM class. In some patients with peripheral neuropathy the monoclonal IgM has been found to react with myelin components. The major antigens recognized by such IgM are the myelin-associated glycoprotein (MAG) (Braun et al., 1982; Ilyas et al., 1984; Brouet et al., 1989),
Correspondence to: M.O. Jauberteau, Laboratory of Immunology, C.H.R.U. Limoges, 2 avenue Alexis Carrel, 87042 Limoges Cedex, France. Abbreoiations: Ig, immunoglobulins; MAG, myelin-associated glycoprotein; SGPG, sulfated glucuronic acid paragloboside; SGLPG, sulfated glucuronic acid lactosaminyl paragloboside; TLC, thin layer chromatography, PBS, phosphate buffered safine.
which is a major glycoprotein of the central nervous system, and two sulfated glycosphingolipids containing glucuronic acid, namely sulfated glucuronic acid paragloboside ( S G P G ) and sulfated glucuronic acid lactosaminyl paragloboside (SGLPG). In adults, these glycolipids are present only in peripheral nerves (Chou et al., 1985, 1986). In certain patients, the monoclonal IgM reacts with an epitope that is carried by the carbohydrate moieties of the glycolipids and which is shared by MAG also. An immunological mechanism has been postulated to explain the peripheral nerve involvement. This hypothesis is supported by the improvement of the neurological symptoms following therapeutic reduction of circulating monoclonal Ig (Lassoued et al., 1985; Haas and Tatum, 1988). It is clearly of interest to search for serum monoclonal IgM with anti-glycolipid antibody activity in patients with a neuropathy of unknown origin. In some patients, the peripheral neuropathy preceded the identification of monoclonal IgM, and 10% of peripheral neuropathies of unknown cause were found to be associated with dysgam-
0022-1759/90/$03.50 © 1990 ElsevierSciencePublishers B.V. (BiomedicalDivision)
108 maglobulinemia (Kelly et al., 1981). In fact, in certain cases, the monoclonal Ig is present at such low levels that it cannot be detected by conventional electrophoresis and immunoelectrophoresis, but requires a more sensitive procedure such as immunoblotting combined with high resolution electrophoresis in order to be identified (Jauberteau et al., 1990). Such an approach involves two independent steps, the characterization of the monoclonal IgM, and the demonstration of its reactivity. It would be very helpful to use a procedure which could provide the same information in a single experiment. Filter affinity transfer is known to be a reliable and specific method for the identification of proteins in gels, provided that the appropriate ligand is bound to cellulose (Erlich et al., 1979). We therefore developed an affinity immunoblotting method based upon the transfer of serum proteins separated by thin-layer agarose electrophoresis to nitrocellulose sheets coated with glycosphingolipids, followed by immunoenzymatic revelation of bound Ig. This procedure has proved to be rapid, sensitive and specific.
Materials and methods
Glycosphingolipids The gangliosides GM1 and G D l a and galactocerebrosides purified from bovine brain were obtained from a commercial source (Sigma, St. Louis, MO). Glycosphingolipids were purified from human central (brain white matter) or peripheral (sciatic and crural nerves, cauda equina) nervous tissue obtained by autopsy from patient without neurological disease 6-12 h after death. Briefly, the lipid extract of 1 g of fresh tissue (about 20 ml) dispersed in 20 volumes of chloroform-methanol (2:1; v / v ) according to Folch et al. (1957) was partitioned with 0.2 volume of water. After centrifugation, a volume of watermethanol (1 : 1; v / v ) equal to the removed upper phase was added to the lower phase and separated by centrifugation before adding a similar volume of NaC1 0.15 M-methanol (1 : 1; v / v ) to the lower phase. The gangliosidic fraction was obtained from the pooled upper phases using a reverse-phase C18-silicic column (provided by J. Portoukalian, Lyon, France) as described by Williams and Mc-
Cluer (1980). The neuraminic acid (NeuNAc) content of the purified gangliosidic fraction was determined according to Svennerholm (1957).
Sera Sera were stored frozen at - 8 0 ° C until use. They were obtained from seven patients with demyelinating peripheral neuropathies and monoclonal IgM known to react with S G P G and SGLPG. Another serum having small amounts of a monoclonal IgMX with anti-GM1 and G D l b antibody activity was collected in a patient with motor neurone disease (Jauberteau et al., 1990). The monoclonal IgM present in these sera was characterized by conventional electrophoretic and immunoelectrophoretic analysis of whole sera and IgM separated by gel filtration, and by Western blotting of the serum proteins separated by high resolution agarose electrophoresis (see below). This sensitive procedure permits detection of monoclonal Ig present in small amounts and unrecognized by conventional analysis. These monoclonal IgM had been previously characterized with respect to their reactivity with glycolipids by immunodetection on thin layer chromatography (TLC) plates, following a method based on the procedures of Magnani et al. (1982) and Harpin et al., (1985), as previously described (Jauberteau et al., 1988). Briefly, the various ganglioside preparations were submitted to chromatography on aluminium-backed TLC plates (silica gel 60, Merck, Darmstadt, F.R.G.) in chloroformmethanol-0.25% CaCI2, 50 : 40 : 10 ( v / v / v ) . The plates were saturated with 0.01 M phosphate, 0.15 M NaC1, pH 7.4 phosphate buffered saline (PBS) containing 1% gelatin (Merck) and 10% inactivated horse serum (Gibco, Paisley, Scotland) for 30 min at room temperature. The patients' sera, diluted 1/100 in PBS, were incubated for 2 h at 37°C. After washing, bound Ig was revealed using peroxidase-coupled goat antibodies specific for the various Ig heavy chain classes and light chain types (Cappel, Westchester, PA, U.S.A. and Diagnostics Pasteur, Paris, France). As an example, Fig. 1 shows results obtained with a serum containing a monoclonal IgM reactive with SGPG and SGLPG. Control sera included eight sera from patients with Waldenstr6m's macroglobulinemia without neurological disease, six sera
109
i
SGPG-I~ SGLPG ,,~
Yvelines, France) were immediately applied to the TLC plates, and pressed between two glass plates under a 1 kg weight for 1 h. Nitrocellulose sheets were allowed to air dry until solvent had entirely evaporated. They could be used immediately or kept frozen at - 2 0 ° C prior to saturation. In both instances, free sites were saturated by 5% bovine serum albumin in PBS for 1 h at room temperature with continuous gentle agitation on a rocker platform. Saturation of glycolipid coated nitrocellulose membranes with skimmed milk was not feasible because glycosylate components hid the GM1 epitope hindering affinity blotting of Ig specific for GM1, in contrast to the affinity transfer of Ig specific for SGPG and S G L P G (not shown).
_ _ ~ ...................
Blotting procedure PN
WM
Fig. 1. Reactivity of serum from a patient with demyefinating
neuropathy and a monoclonal IgM with peripheral nerve sulfated glycosphingolipidicantigens on TLC plate. PN, gangliosidic fractions purified from human peripheral nervous tissue. The monoclonal IgM reacts with SGPG and SGLPG (arrows). WM, central white matter gangliosidic fraction, no detectable reactivity.Anti-/.tconjugate.
from patients with monoclonal IgM and peripheral neuropathy but without anti-glycolipid antibodies by immunodetection on TLC plates and two sera from patients with undetermined peripheral neuropathy and no monoclonal IgM.
Preparation of nitrocellulose affinity filters The glycolipids described above were dissolved in methanol and homogeneously dispersed on 10 × 6 cm aluminium-backed TLC plates. Glycolipids dissolved in 2 ml of methanol were gently layered on the plate, the amounts of glycosphingolipids applied being 2.5/~g/cm 2 for the purified GM1, G D l a or galactocerebrosides (150 /~g by plate) and 0.5 #g N e u N A c / c m 2 (30 #g NeuNAc by plate) for the gangliosidic fractions. After air drying, 2 ml of a solution of isopropanol-water (2:1; v / v ) were sprayed on the plates as described by Towbin et al. (1984). Nitrocellulose filters (HAHY 0.45 #m, Millipore, St. Quentin-
Sera were diluted 1 / 5 0 in NaC1 0.15 M and separated by thin layer agarose electrophoresis (Paragon, Beckman, Gagny, France) and first transferred on uncoated nitrocellulose sheets by 1 min pressure blotting as described previously (Aucouturier et al., 1987; Briault et al., 1988) in order to locate monoclonal Ig. A second transfer was immediately carried out on the glycolipidcoated filters by 1 kg pressure for 15 min. After saturation as above, the blots were revealed with alkaline phosphatase-coupled antibodies specific for human -/, a, /z (Biosys, Compirgne, France), K and )~ chains (Sigma) at the appropriate dilutions in PBS containing 2% bovine serum albumin (2 h incubation at room temperature with gentle agitation). The specificity and working dilutions of these various reagents were determined using the same immunoblotting procedure with myeloma sera and urines containing known monoclonal Ig.
Results
Using affinity immunoblotting, specific detection of monoclonal IgM with anti-ganglioside antibody activity was easily obtained. All control sera were repeatedly negative and the experiments performed with affinity filters coated either with gangliosidic fractions or with purified gangliosides yielded the expected results with previously characterized sera from patients with neuropathies.
110
A 4-
B
C
Fig. 2. Affinity transfer of serum Ig from a patient with peripheral neuropathy and a monoclonal IgM with anti-SGPG and anti-SGLPG antibody activity. Immunoblotting of thin layer agarose electrophoresis of serum on normal nitrocellulose (A) (which shows a monoclonal IgM~) and affinity filters coated with gangliosidic fractions from peripheral nerve (B) and brain (C).
A 4B
J
i 1
2
3
4
1
2
I¸ 3
4
Fig. 3. Affinity immunoblotting of sera from patients with monoclonal IgM with antibody activity to SGPG and S G L P G (lanes 1 and 2) or G M I (lanes 3 and 4) transferred to uncoated nitrocellulose (A) or GM1 affinity filter (B). Lanes I and 3 are revealed with anti-/~, lane 2 with anti-K and lane 4 with anti-k conjugates.
111
For instance, a monoclonal IgM~¢ (Fig. 2A) antiSGPG and SGLPG bound affinity filters coated with peripheral nerve gangliosidic fraction (Fig. 2B) but not those coated with gangliosidic fraction purified from central nervous system (Fig. 2C) or with GM1 (Fig. 3B, lanes 1 and 2). In contrast, the monoclonal IgM?~ (Fig. 3A, lanes 3 and 4), known for its anti-GM1 activity, bound GM1 (Fig. 3B, lanes 3 and 4) and central nervous system gangliosidic fractions, but not the ganglioside G D l a and standard galactocerebrosides (not shown). The latter serum contained 2.2 m g / m l IgM by laser nephelometry. Easy detection of its reactivity with GM1 was achieved by affinity immunoblotting up to a serum dilution of 1/800, i.e., with 2.7 n g / m l of IgM of which only a part was monoclonal (Fig. 3A).
Discussion Affinity immunoblotting is a sensitive and specific method that readily permits the detection of antibodies, using antigen-coated filters, or of antigens (with antibody-coated filters) in biologic fluids (Erlich et al., 1979). In neurology, it has been used previously for the detection of oligoclonal IgG reactive with viral antigens (D~Srris and Ter Meulen, 1984) or with myelin basic protein (Cruz et al., 1987) in the cerebrospinal fluid. We have developed a procedure based on the same principle, using nitrocellulose sheets coated with glycosphingolipids. Since material separated by the same high resolution electrophoresis is blotted on uncoated and on coated nitrocellulose, a single experiment permits the identification of monoclonal Ig and the demonstration of antibody activity. The method proved to be both specific and sensitive. It can be used initially with filters coated with gangliosidic fractions in order to screen sera from patients with neuropathy of undetermined origin, the precise antibody activity then being further characterized using nitrocellulose filters coated with purified gangliosides. Preliminary experiments with sera from patients with various neurological diseases suggest that the method may also be used for the detection of polyclonal antibodies.
Acknowledgements We thank Dr. P. Aucouturier for his participation in the study and Dr. J. Portoukalian for the gift of the silicic column. This work was supported by INSERM and 'Fondation pour la Recherche Mrdicale'.
References Aucouturier, P., Capella, M., Briault, S., Danon, F., Intrator, L. and Preud'homme, J.L. (1987) Caract~risation des immunoglobulines monoclonales dans les liquides biologiques par immunoempreinte sur nitrocellulose. Rev. Inst. Pasteur (Lyon) 20, 147. Braun, P.E., Frail, D.E. and Latov, N. (1982) Myelin associated-glycoprotein is the antigen for a monoclonal IgM in polyneuropathy. J. Neurochem. 39, 1261. Briault, S., Courtois-Capella, M., Duarte, F., Aucouturier, P. and Preud'homme, J.L. (1988) Isotypy of serum monoclonal immunoglobulins in human immunodeficiency virus-infected adults. Clin. Exp. Immunol. 74, 182. Brouet, J.C., Dellagi, K., Gendron, M.C., Chevalier, A., Schmitt, C. and Mihaesco, E. (1989) Expression of a public idiotype by human monoclonal IgM directed to myelin-associated glycoprotein and characterization of the variability subgroup of their heavy and light chains. J. Exp. Med. 170, 1551. Chou, K.H., llyas, A.A., Evans, J.E., Quarles, R.H. and Jungalwala, F.B. (1985) Structure of a glycolipid reacting with monoclonal IgM in neuropathy and with HNK1. Biochem. Biophys. Res. Commun. 128, 383. Chou, K.H,, Ilyas, A.A., Evans, J.E., Costello, E., Quarles, R.H. and Jungalwala, F.B. (1986) Structure of sulfated glucuronyl glycolipids in the nervous system reacting with HNK1 antibody and some IgM paraproteins in neuropathy. J. Biol. Chem. 261, 11717. Cruz, M., Olsson, T., Ernerudh, J., H~Sjeberg, B. and Link, H. (1987) Immunoblot detection of oligoclonal anti-myelin basic protein IgG antibodies in cerebrospinal fluid in multiple sclerosis. Neurology 37, 1515. DiSrris, R. and Ter Meulen, V. (1984) Detection and identification of virus-specific oligoclonal IgG in unconcentrated cerebrospinal fluid by immunoblot technique. J. Neuroimmunol. 7, 77. Erlich, H.A., Levinson, J.R., Cohen, S.N. and McDevitt, H.O. (1979) Filter affinity transfer. A new technique for the in situ identification of proteins in gels. J. Biol. Chem. 254, 12240. Freddo, L., Yu, R.K., Latov, N., Donofrio, P.D., Hays, A.P., Greenberg, H.S., Albers, J.W., Allessi, A.D. and Keren, D. (1986) Gangliosides GM1 and G D l b are antigens for IgM M-protein in a patient with motor neuron disease. Neurology 36, 454.
112 Folch, J., Lees, M. and Sloane-Stanley, G.H. (1957) A simple method for the isolation and purification of total lipids from animals tissues. J. Biol. Chem. 226, 497. Haas, D.C. and Tatum, A.H. (1988) Plasmapheresis alleviates neuropathy accompanying IgM anti-myelin-associated glycoprotein paraproteinemia. Ann. Neurol. 23, 394. Harpin, M.L., Coulon-Morelec, M.J., Yeni, P., Danon, F. and Baumann, N. (1985) Direct sensitive immunocharacterization of gangliosides on plastic thin-layer plates using peroxidase staining. J. Immunol. Methods 78, 135. Ilyas, A.A., Quarles, R.H., Mclntosh, T.D., Dobersen, M.J., Trapp, B.D., Dalakas, M.C. and Brady, R.O. (1984) IgM in a human neuropathy related to paraproteinemia binds to a carbohydrate determinant in the myelin-associated glycoprotein and to a ganglioside. Proc. Natl. Acad. Sci. U.S.A.. 81, 1225. Ilyas, A.A., Quarles, R.H., Dalakas, M.C. and Brady, R.O. (1985a) Polyneuropathy with monoclonal gammopathy: glycolipids are frequently antigens for IgM paraproteins. Proc. Natl. Acad. Sci. U.S.A. 82, 6697. Ilyas, A.A., Quarles, R.H., Dalakas, M.C., Fishman, P.H. and Brady, R.O. (1985b) Monoclonal IgM in a patient with paraproteinemic polyneuropathy binds to gangliosides containing disialosyl groups. Ann. Neurol. 18, 655. Ito, M. and Latov, N. (1988) Monoclonal IgM in two patients with motor neuron disease bind to the carbohydrate antigens Gal (ill-3) GalNAc and Gal (ill-3) GIcNAc. J. Neuroimmunol. 19, 245. Jauberteau, M.O., Henin, D., Bouche, P., Vallat, J.M., Dumas, M., Dellagi, S., Leger, J.M., Harpin, M.L., Ratihanirana, H., Chaunu, M.P., Quarles, R.H. and Baumann, N. (1988) Etude des anticorps antiglycolipides au cours des dysglobulin6mies monoclonales ~ IgM associ4es ~ une neuropathie p6riph6rique. Rev. Neurol. (Paris) 144, 474. Jauberteau, M.O., Gualde, N., Preud'homme, J.L., Rigaud, M.,
Gil, R., Vallat, J.M. a n d Baumann, N. (1990) Human monoclonal IgM with autoantibody activity against two gangliosides (GM1 and G D l b ) in a patient with motor neuron syndrome. Clin. Exp. Immunol., in press. Kelly, J.J., Kyle, R.A., O'Brien, P.C. and Dyck, P.J. (1981) Prevalence of monoclonal protein in peripheral neuropathy. Neurology 31, 1480. Lassoued, K., Dellagi, K., Brouet, J.C., Clauvel, J.P., Bussel, A. and Seligmann, M. (1985) Effect of plasma exchange in nine patients with peripheral neuropathy and monoclonal IgM directed to myelin-associated glycoprotein. Plasma Ther. Transfus. Technol. 6, 449. Magnani, J.L., Nilsson, B., Brockhaus, M., Zopf, D., Steplewski, Z., Koprowski, H. and Ginsburg, V. (1982) A monoclonal antibody-defined antigen associated with gastrointestinal cancer in a ganglioside containing sialylated lactoN-fucopentaose II. J. Biol. Chem. 257, 14365. Miyatani, N., Baba, H., Sato, S., Nakamura, K., Yuasa, T., and Miyatake, T. (1987) Antibody to sialosyllactosaminylparagloboside in a patient with IgM paraproteinemia and polyradiculoneuropathy. J. Neuroimmunol. 14, 189. Nardelli, E., Steck, A.J., Barkas, T., Schuep, M. and Jerusalem, F. (1988) Motor neuron syndrome and monoclonal IgM with antibody activity against gangliosides GM1 and GDlb. Ann. Neurol. 23, 524. Svennerholm, L. (1957) Quantitative estimation of sialic acids. Biochim. Biophys. Acta, 24, 604. Towbin, H., Schoenenberger, C., Ball, R., Braun, D.G. and Rosenfelder, G. (1984) Glycosphingolipid-blotting: an immunological detection procedure after separation by thinlayer chromatography. J. Immunol. Methods 72, 471. Williams, M.A. and McCluer, R.H. (1980) The use of Sep-Pak C18 cartridges during the isolation of gangliosides. J. Neurochem. 35, 266.
107
Elsevier JIM 05739
Affinity immunoblotting detection of serum monoclonal immunoglobulins reactive with glycosphingolipids M.O. J a u b e r t e a u , J.M. C o o k , M. D r o u e t a n d J.L. P r e u d ' h o m m e 1 Laboratory of Immunology and CJF I N S E R M 8803, University Hospital, 87042 Limoges Cedex, France, and i Laboratory of Immunology and Immunopathology (CNRS URA 1172), Unioersity Hospital, 86021 Poitiers Cedex, France
(Received20 March 1990, accepted 12 July 1990)
The characterization of serum monoclonal IgM reactive with glycosphingolipids present in the nervous system is of major importance in patients with certain neuropathies. A rapid, sensitive and specific one-step method which permits such a characterization is described. Serum immunoglobulins separated by high resolution agarose electrophoresis are transferred to affinity filters (nitrocellulose sheets coated with glycolipids), and revealed with enzyme-tagged monospecific anti-immunoglobulin antibodies. Key words: Glycosphingolipid;Monoclonalimmunoglobulin; Immunoblot; Affinity filter; Neuropathy
Introduction
Serum monoclonal immunoglobulins (Ig) reactive with glycosphingolipids have been reported in patients with peripheral neuropathies (Ilyas et al., 1984, 1985a,b; Miyatani et al., 1987) and the motor neurone syndrome (Freddo et al., 1986; Ito and Latov, 1988; Nardelli et al., 1988; Jauberteau et al., 1990). Such monoclonal Ig most often belong to the IgM class. In some patients with peripheral neuropathy the monoclonal IgM has been found to react with myelin components. The major antigens recognized by such IgM are the myelin-associated glycoprotein (MAG) (Braun et al., 1982; Ilyas et al., 1984; Brouet et al., 1989),
Correspondence to: M.O. Jauberteau, Laboratory of Immunology, C.H.R.U. Limoges, 2 avenue Alexis Carrel, 87042 Limoges Cedex, France. Abbreoiations: Ig, immunoglobulins; MAG, myelin-associated glycoprotein; SGPG, sulfated glucuronic acid paragloboside; SGLPG, sulfated glucuronic acid lactosaminyl paragloboside; TLC, thin layer chromatography, PBS, phosphate buffered safine.
which is a major glycoprotein of the central nervous system, and two sulfated glycosphingolipids containing glucuronic acid, namely sulfated glucuronic acid paragloboside ( S G P G ) and sulfated glucuronic acid lactosaminyl paragloboside (SGLPG). In adults, these glycolipids are present only in peripheral nerves (Chou et al., 1985, 1986). In certain patients, the monoclonal IgM reacts with an epitope that is carried by the carbohydrate moieties of the glycolipids and which is shared by MAG also. An immunological mechanism has been postulated to explain the peripheral nerve involvement. This hypothesis is supported by the improvement of the neurological symptoms following therapeutic reduction of circulating monoclonal Ig (Lassoued et al., 1985; Haas and Tatum, 1988). It is clearly of interest to search for serum monoclonal IgM with anti-glycolipid antibody activity in patients with a neuropathy of unknown origin. In some patients, the peripheral neuropathy preceded the identification of monoclonal IgM, and 10% of peripheral neuropathies of unknown cause were found to be associated with dysgam-
0022-1759/90/$03.50 © 1990 ElsevierSciencePublishers B.V. (BiomedicalDivision)
108 maglobulinemia (Kelly et al., 1981). In fact, in certain cases, the monoclonal Ig is present at such low levels that it cannot be detected by conventional electrophoresis and immunoelectrophoresis, but requires a more sensitive procedure such as immunoblotting combined with high resolution electrophoresis in order to be identified (Jauberteau et al., 1990). Such an approach involves two independent steps, the characterization of the monoclonal IgM, and the demonstration of its reactivity. It would be very helpful to use a procedure which could provide the same information in a single experiment. Filter affinity transfer is known to be a reliable and specific method for the identification of proteins in gels, provided that the appropriate ligand is bound to cellulose (Erlich et al., 1979). We therefore developed an affinity immunoblotting method based upon the transfer of serum proteins separated by thin-layer agarose electrophoresis to nitrocellulose sheets coated with glycosphingolipids, followed by immunoenzymatic revelation of bound Ig. This procedure has proved to be rapid, sensitive and specific.
Materials and methods
Glycosphingolipids The gangliosides GM1 and G D l a and galactocerebrosides purified from bovine brain were obtained from a commercial source (Sigma, St. Louis, MO). Glycosphingolipids were purified from human central (brain white matter) or peripheral (sciatic and crural nerves, cauda equina) nervous tissue obtained by autopsy from patient without neurological disease 6-12 h after death. Briefly, the lipid extract of 1 g of fresh tissue (about 20 ml) dispersed in 20 volumes of chloroform-methanol (2:1; v / v ) according to Folch et al. (1957) was partitioned with 0.2 volume of water. After centrifugation, a volume of watermethanol (1 : 1; v / v ) equal to the removed upper phase was added to the lower phase and separated by centrifugation before adding a similar volume of NaC1 0.15 M-methanol (1 : 1; v / v ) to the lower phase. The gangliosidic fraction was obtained from the pooled upper phases using a reverse-phase C18-silicic column (provided by J. Portoukalian, Lyon, France) as described by Williams and Mc-
Cluer (1980). The neuraminic acid (NeuNAc) content of the purified gangliosidic fraction was determined according to Svennerholm (1957).
Sera Sera were stored frozen at - 8 0 ° C until use. They were obtained from seven patients with demyelinating peripheral neuropathies and monoclonal IgM known to react with S G P G and SGLPG. Another serum having small amounts of a monoclonal IgMX with anti-GM1 and G D l b antibody activity was collected in a patient with motor neurone disease (Jauberteau et al., 1990). The monoclonal IgM present in these sera was characterized by conventional electrophoretic and immunoelectrophoretic analysis of whole sera and IgM separated by gel filtration, and by Western blotting of the serum proteins separated by high resolution agarose electrophoresis (see below). This sensitive procedure permits detection of monoclonal Ig present in small amounts and unrecognized by conventional analysis. These monoclonal IgM had been previously characterized with respect to their reactivity with glycolipids by immunodetection on thin layer chromatography (TLC) plates, following a method based on the procedures of Magnani et al. (1982) and Harpin et al., (1985), as previously described (Jauberteau et al., 1988). Briefly, the various ganglioside preparations were submitted to chromatography on aluminium-backed TLC plates (silica gel 60, Merck, Darmstadt, F.R.G.) in chloroformmethanol-0.25% CaCI2, 50 : 40 : 10 ( v / v / v ) . The plates were saturated with 0.01 M phosphate, 0.15 M NaC1, pH 7.4 phosphate buffered saline (PBS) containing 1% gelatin (Merck) and 10% inactivated horse serum (Gibco, Paisley, Scotland) for 30 min at room temperature. The patients' sera, diluted 1/100 in PBS, were incubated for 2 h at 37°C. After washing, bound Ig was revealed using peroxidase-coupled goat antibodies specific for the various Ig heavy chain classes and light chain types (Cappel, Westchester, PA, U.S.A. and Diagnostics Pasteur, Paris, France). As an example, Fig. 1 shows results obtained with a serum containing a monoclonal IgM reactive with SGPG and SGLPG. Control sera included eight sera from patients with Waldenstr6m's macroglobulinemia without neurological disease, six sera
109
i
SGPG-I~ SGLPG ,,~
Yvelines, France) were immediately applied to the TLC plates, and pressed between two glass plates under a 1 kg weight for 1 h. Nitrocellulose sheets were allowed to air dry until solvent had entirely evaporated. They could be used immediately or kept frozen at - 2 0 ° C prior to saturation. In both instances, free sites were saturated by 5% bovine serum albumin in PBS for 1 h at room temperature with continuous gentle agitation on a rocker platform. Saturation of glycolipid coated nitrocellulose membranes with skimmed milk was not feasible because glycosylate components hid the GM1 epitope hindering affinity blotting of Ig specific for GM1, in contrast to the affinity transfer of Ig specific for SGPG and S G L P G (not shown).
_ _ ~ ...................
Blotting procedure PN
WM
Fig. 1. Reactivity of serum from a patient with demyefinating
neuropathy and a monoclonal IgM with peripheral nerve sulfated glycosphingolipidicantigens on TLC plate. PN, gangliosidic fractions purified from human peripheral nervous tissue. The monoclonal IgM reacts with SGPG and SGLPG (arrows). WM, central white matter gangliosidic fraction, no detectable reactivity.Anti-/.tconjugate.
from patients with monoclonal IgM and peripheral neuropathy but without anti-glycolipid antibodies by immunodetection on TLC plates and two sera from patients with undetermined peripheral neuropathy and no monoclonal IgM.
Preparation of nitrocellulose affinity filters The glycolipids described above were dissolved in methanol and homogeneously dispersed on 10 × 6 cm aluminium-backed TLC plates. Glycolipids dissolved in 2 ml of methanol were gently layered on the plate, the amounts of glycosphingolipids applied being 2.5/~g/cm 2 for the purified GM1, G D l a or galactocerebrosides (150 /~g by plate) and 0.5 #g N e u N A c / c m 2 (30 #g NeuNAc by plate) for the gangliosidic fractions. After air drying, 2 ml of a solution of isopropanol-water (2:1; v / v ) were sprayed on the plates as described by Towbin et al. (1984). Nitrocellulose filters (HAHY 0.45 #m, Millipore, St. Quentin-
Sera were diluted 1 / 5 0 in NaC1 0.15 M and separated by thin layer agarose electrophoresis (Paragon, Beckman, Gagny, France) and first transferred on uncoated nitrocellulose sheets by 1 min pressure blotting as described previously (Aucouturier et al., 1987; Briault et al., 1988) in order to locate monoclonal Ig. A second transfer was immediately carried out on the glycolipidcoated filters by 1 kg pressure for 15 min. After saturation as above, the blots were revealed with alkaline phosphatase-coupled antibodies specific for human -/, a, /z (Biosys, Compirgne, France), K and )~ chains (Sigma) at the appropriate dilutions in PBS containing 2% bovine serum albumin (2 h incubation at room temperature with gentle agitation). The specificity and working dilutions of these various reagents were determined using the same immunoblotting procedure with myeloma sera and urines containing known monoclonal Ig.
Results
Using affinity immunoblotting, specific detection of monoclonal IgM with anti-ganglioside antibody activity was easily obtained. All control sera were repeatedly negative and the experiments performed with affinity filters coated either with gangliosidic fractions or with purified gangliosides yielded the expected results with previously characterized sera from patients with neuropathies.
110
A 4-
B
C
Fig. 2. Affinity transfer of serum Ig from a patient with peripheral neuropathy and a monoclonal IgM with anti-SGPG and anti-SGLPG antibody activity. Immunoblotting of thin layer agarose electrophoresis of serum on normal nitrocellulose (A) (which shows a monoclonal IgM~) and affinity filters coated with gangliosidic fractions from peripheral nerve (B) and brain (C).
A 4B
J
i 1
2
3
4
1
2
I¸ 3
4
Fig. 3. Affinity immunoblotting of sera from patients with monoclonal IgM with antibody activity to SGPG and S G L P G (lanes 1 and 2) or G M I (lanes 3 and 4) transferred to uncoated nitrocellulose (A) or GM1 affinity filter (B). Lanes I and 3 are revealed with anti-/~, lane 2 with anti-K and lane 4 with anti-k conjugates.
111
For instance, a monoclonal IgM~¢ (Fig. 2A) antiSGPG and SGLPG bound affinity filters coated with peripheral nerve gangliosidic fraction (Fig. 2B) but not those coated with gangliosidic fraction purified from central nervous system (Fig. 2C) or with GM1 (Fig. 3B, lanes 1 and 2). In contrast, the monoclonal IgM?~ (Fig. 3A, lanes 3 and 4), known for its anti-GM1 activity, bound GM1 (Fig. 3B, lanes 3 and 4) and central nervous system gangliosidic fractions, but not the ganglioside G D l a and standard galactocerebrosides (not shown). The latter serum contained 2.2 m g / m l IgM by laser nephelometry. Easy detection of its reactivity with GM1 was achieved by affinity immunoblotting up to a serum dilution of 1/800, i.e., with 2.7 n g / m l of IgM of which only a part was monoclonal (Fig. 3A).
Discussion Affinity immunoblotting is a sensitive and specific method that readily permits the detection of antibodies, using antigen-coated filters, or of antigens (with antibody-coated filters) in biologic fluids (Erlich et al., 1979). In neurology, it has been used previously for the detection of oligoclonal IgG reactive with viral antigens (D~Srris and Ter Meulen, 1984) or with myelin basic protein (Cruz et al., 1987) in the cerebrospinal fluid. We have developed a procedure based on the same principle, using nitrocellulose sheets coated with glycosphingolipids. Since material separated by the same high resolution electrophoresis is blotted on uncoated and on coated nitrocellulose, a single experiment permits the identification of monoclonal Ig and the demonstration of antibody activity. The method proved to be both specific and sensitive. It can be used initially with filters coated with gangliosidic fractions in order to screen sera from patients with neuropathy of undetermined origin, the precise antibody activity then being further characterized using nitrocellulose filters coated with purified gangliosides. Preliminary experiments with sera from patients with various neurological diseases suggest that the method may also be used for the detection of polyclonal antibodies.
Acknowledgements We thank Dr. P. Aucouturier for his participation in the study and Dr. J. Portoukalian for the gift of the silicic column. This work was supported by INSERM and 'Fondation pour la Recherche Mrdicale'.
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