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Lipids are co-eluted with immunoglobulins G during purification by recombinant streptococcal protein G affinity chromatography
Journal of Immunological Methods 271 (2002) 107 – 111 www.elsevier.com/locate/jim
Lipids are co-eluted with immunoglobulins G during purification by recombinant streptococcal protein G affinity chromatography Eirini Kitsiouli a,b,1, Marilena E. Lekka b,*, George Nakos c,2, Claude Cassagne a,d,3, Lilly Maneta-Peyret a,4 a
Laboratoire de Biogene`se Membranaire CNRS-UMR 5544, Universite´ Victor Segalen, Bordeaux 2, France b Biochemistry Laboratory, Chemistry Department, University of Ioannina, Ioannina 45110, Greece c Intensive Care Unit, University Hospital of Ioannina, Ioannina 45110, Greece d ESTBB, Universite´ Victor Segalen, Bordeaux 2, France Received 10 April 2002; received in revised form 28 July 2002; accepted 12 August 2002
Abstract The efficiency of recombinant streptococcal protein G (rec-spG) affinity column chromatography in purifying immunoglobulins G (IgG) from lipids has been studied, with particular reference to IgG fractions from bronchoalveolar lavage (BAL) fluid and serum samples from different sources. It was found that the IgG fractions purified by rec-spG affinity column chromatography also contained cholesterol and phospholipids. D 2002 Elsevier Science B.V. All rights reserved. Keywords: Immunoglobulin G; Protein G; Lipids; Cholesterol; BAL
1. Introduction Abbreviations: BAL, bronchoalveolar lavage; BSA, bovine serum albumin; IgG, immunoglobulin G; SDS-PAGE, sodium dodecyl sulfate-polyacrylamide gel electrophoresis; HPTLC, high performance thin layer chromatography; spG, streptococcal protein G; rec-spG, recombinant streptococcal protein G; PC, phosphatidylcholine; PG, phosphatidylglycerol; PS, phosphatidylserine; PI, phosphatidylinositol; PE, phosphatidylethanolamine; Chol, cholesterol; PVDF, polyvinylenedifluoride. * Corresponding author. Tel.: +30-6510-98367; fax: +30-651098774. E-mail addresses: [email protected], [email protected] (E. Kitsiouli), [email protected] (M.E. Lekka), [email protected] (G. Nakos), [email protected] (C. Cassagne), [email protected] (L. Maneta-Peyret). 1 Tel.: +30-6510-22383; fax: +30-6830-51366. 2 Tel./fax: +30-6510-99279. 3 Tel.: +33-557571681; fax: +33-556518361. 4 Tel.: +33-557571681; fax: +33-556518361.
Protein G affinity column chromatography has proven to be a very useful technique for the isolation and purification of IgGs from serum and other biological fluids. Protein G is a multidomain protein expressed on the cell surface of Streptococcus groups C and G. It binds to the Fc region of immunoglobulins from several species by a non-immune mechanism (Guss et al., 1986) exhibiting great affinity for almost all mammalian immunoglobulin G (IgG) classes (Ackerstro¨m and Bjo¨rck, 1986), including all human IgG subclasses (IgG1, IgG2, IgG3 and IgG4). For the isolation of IgGs, streptococcal protein G (spG) was modified by genetic engineering, so that the albumin- and the Fab-binding regions were
0022-1759/02/$ - see front matter D 2002 Elsevier Science B.V. All rights reserved. PII: S 0 0 2 2 - 1 7 5 9 ( 0 2 ) 0 0 3 4 5 - 9
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deleted. There is literature on the degree of separation and purification of IgG from other proteins during protein G affinity chromatography (Guss et al., 1986; Sjo¨bring et al., 1991), but the possible presence of other molecules in the IgG fraction has not been examined. In the present study, we have investigated whether lipids might be recovered together with the IgGs isolated from BAL fluid as well as human and rabbit serum samples by rec-spG affinity column chromatography.
2. Materials and methods In this study, the IgG fractions of BAL fluid and serum samples from 10 patients with acute respiratory distress syndrome were isolated by rec-spG affinity chromatography. In addition, sera from two rabbits immunized against phospholipids (Maneta-Peyret et al., 1988) and two non-immunized rabbits were tested. Bronchoalveolar lavage (BAL) fluid was obtained according to Nakos et al. (2001). IgG fractions were isolated from BAL fluid and serum samples using 1 ml protein G (rec-spG) (Sigma, USA), affinity column chromatography, with a binding capacity exceeding 20 mg human IgGs per ml of suspension. BAL fluid (up to 10 mg protein) and 500 Al of each serum sample were diluted to a final volume of 5 ml with 20 mM sodium phosphate buffer, pH 7.2 (equilibration buffer) and loaded on the column. The procedure took place at room temperature. Flow rate was adjusted to 1 ml/min and protein elution was monitored at 280 nm by a UV photometer (ULTROSPEC 1000, Pharmacia Biotech). The IgG fraction was eluted by 0.1 M glycine –HCl buffer, pH 2.7 and it was immediately neutralized with a 1 M Tris-base buffer solution, pH 8.8. Protein analysis was carried out by SDS-PAGE using 12% polyacrylamide gels (Biorad). Proteins were transferred to PVDF membranes (NEN, USA), followed by incubation with anti-human-IgG-peroxidase conjugate (Sigma) diluted 1/10000 with 0.01 M PBS buffer, pH 7.2, containing 0.05% Tween-20. Immunoblot detection was achieved using Chemiluminescent Reagent Rennaissance (NEN). The eluted IgG fraction was subjected to lipid extraction according to Bligh and Dyer (1959) and
analysed by high performance thin layer chromatography (Silica gel 60 F254 plates, 1010 cm, Merck). For the separation of phospholipids and neutral lipids, the following solvent systems were used, respectively: methyl-acetate/n-propanol/chloroform/methanol/potassium chloride 0.25% (12.5:12.5:14:5:3.5, v/v) and hexane/diethyl ether/acetic acid (70:30:2, v/v) (Juguelin et al., 1986). Phosphatidylcholine, phosphatidylglycerol, phosphatidylethanolamine, phosphatidylinositol, phosphatidylserine, sphingomyelin, phosphatidic acid, cholesterol, cholesterol esters as well as mono-, di- and triglycerides (Sigma) were used as reference compounds. The bands were visualized by copper acetate/phosphoric acid charring (Macala et al., 1983) and scanned at 366 nm with a photodensitometer scanner (Camag) operating in reflectance mode and coupled to a computing integrator (Spectra-Physics). Each spot contained 1 –5 Ag of lipid residue.
Fig. 1. Polar and neutral lipid analysis of the IgG fraction: 5 ml of BAL fluid (2 mg protein/ml) was subjected to recombinant streptococcal protein G affinity column chromatography. The total amount of IgG protein isolated was 0.6 mg, as determined by UV absorbance and this isolated IgG fraction was submitted to lipid extraction. The lipid residue was analysed by HPTLC using methylacetate/n-propanol/chloroform/methanol/0.25% potassium chloride (12.5:12.5:14:5:3.5, v/v) as polar solvent system (lane A) and by hexane/diethyl ether/acetic acid (70:30:2, v/v) as neutral solvent system (lane B). PC: phosphatidylcholine, PS: phosphatidylserine, PG: phosphatidylglycerol, PE: phosphatidylethanolamine, Chol: Cholesterol, NL: neutral lipids. SP denotes the starting point, while SF indicates the solvent front.
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Fig. 2. Gas chromatography/mass spectrometry of the neutral lipid migrating to the Rf of cholesterol: (A) human serum sample, (B) rabbit serum sample, (C) standard cholesterol.
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E. Kitsiouli et al. / Journal of Immunological Methods 271 (2002) 107–111
GC-MS was performed on a Varian 3400 gas chromatograph coupled with a Finnigan Mat TSQ700 mass spectrometer controlled by a Dec Station 5000/ 33. Samples were analysed on a fused silica 25-m OV1 capillary column (30 m, i.d. 0.25 mm) with helium as carrier gas. The column temperature was programmed from 170 to 300 jC (10 jC/min) with the injector temperature fixed at 220 jC. The mass spectrometer operated in the electron impact mode with an electron energy of 70 eV. BAL fluid was tested for the presence of anticholesterol IgG antibodies using ELISA according to Maneta-Peyret et al. (1988). Various amounts, ranging from 5 to 200 Ag of cholesterol/100 Al hexane, were deposited on each well of a microtiter plate (maxisorp, Nunc) and absorbance was measured at 490 nm in a MR 5000 Dynatech spectrophotometer.
3. Results Analysis of the samples by rec-spG affinity column chromatography revealed two peaks; the first representing the nonretained protein, while the second represented the IgG fraction retained by the column. The total protein content of IgGs isolated from 5 ml BAL, 2 mg prot/ml ranged from 0.3 to 1.6 mg, as determined by UV absorbance at 280 nm. SDS-PAGE of the reduced IgG fraction revealed two bands at 50 and 25 kDa, corresponding to the heavy and light chains of IgG, respectively. After immunoblotting, these bands were labeled with anti-IgG antibodies. The eluted material representing the IgG fraction from BAL or serum samples was submitted to lipid extraction. The HPTLC of neutral lipids revealed that all the samples contained a lipid migrating at the Rf of cholesterol. Small amounts of PC, PS, PG and PE were also detected (Fig. 1). The cholesterol/IgG ratio was found to be approximately 1/50 to 1/150 mol/ mol. The area of the HPTLC plate corresponding to the Rf of cholesterol was extracted and subjected to GCMS analysis (Fig. 2). The mass spectra, interpreted according to Hamming and Foster (1972), showed that the lipid derived from the isolated IgG fractions was identical to that of standard cholesterol. It is noteworthy, however, that the isolated antiphospholipid antibodies of the IgG class from BAL and various
sera did not show any reaction with cholesterol as target antigen when tested by ELISA.
4. Discussion In previous studies we have detected and isolated IgGs from bronchoalveolar lavage fluids and serum samples of patients with acute respiratory distress syndrome (ARDS), by rec-spG affinity column chromatography. These purified IgG fractions contained auto-antibodies against lipids, as evidenced from their ability to bind to lipid target antigens, by ELISA (Maneta-Peyret et al., 2001; Nakos et al., 2001). In the present work, we investigated whether lipids were co-eluted in the fraction of the isolated IgGs during the affinity chromatographic procedure, since they could already have been associated with the IgG molecules and in that case, might not be detected by ELISA. The presence of cholesterol in the IgG fractions from sera of non-immunized and phospholipidimmunized rabbits suggests that it was a general phenomenon characterizing IgGs from different sources and not only the anti-phospholipid antibodies of the IgG class. To the best of our knowledge, the presence of lipids in the IgG fractions of BAL and serum samples purified by rec-spG affinity column chromatography has not been reported previously. The nature and significance of these IgG –lipid interactions are still unclear. However, this association was consistently observed in all the analysed samples. Whether this represents a structural ligand recognised by immunoglobulins or is merely an additional mechanism of cholesterol transport in the aqueous environment of blood or alveolar fluid needs further investigation. In either case, the presence of lipids in the IgG fraction could complicate the study of antiphospholipid antibodies, especially as far as it concerns their binding constants with lipid-antigens.
Acknowledgements This work was supported by: the CNRS-University Victor Segalen Bordeaux-2 and ‘‘Ministe`re des Affaires Etrange`res-Programmes PLATON’’. It was also supported by the ‘‘French Embassy’’ in Athenes, the University of Ioannina and by ‘‘Air Liquid Hellas’’.
E. Kitsiouli et al. / Journal of Immunological Methods 271 (2002) 107–111
References Ackerstro¨m, B., Bjo¨rck, L., 1986. A physicochemical study of protein G, a molecule with unique immuloglobulin G-binding properties. J. Biol. Chem. 261, 10240. Bligh, E.G., Dyer, W.J., 1959. A rapid method of total lipid extraction and purification. Can. J. Biochem. Physiol. 37, 911. Guss, B., Eliasson, M., Olsson, A., Uhlen, M., Frej, A.K., Jornvall, H., Flock, J.I., Lindberg, M., 1986. Structure of the IgG-binding regions of streptococcal protein G. EMBO J. 5, 1567. Hamming, M.C., Foster, N.G., 1972. Interpretation of mass spectra compounds. Academic Press, New York. Juguelin, H., Heape, A., Boiron, F., Cassagne, C., 1986. A quantitative developmental study of neutral lipids during myelinogenesis in the peripheral nervous system of normal and trembler mice. Dev. Brain Res. 25, 249.
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Macala, L.J., Yu, R.K., Ando, S., 1983. Analysis of brain lipids by high performance thin-layer chromatography and densitometry. J. Lipid Res. 24, 1243. Maneta-Peyret, L., Bessoule, J.J., Geffard, M., Cassagne, C., 1988. Demonstration of high specificity antibodies against phosphatidylserine. J. Immunol. Methods 108, 123. Maneta-Peyret, L., Kitsiouli, E., Lekka, M.E., Nakos, G., Cassagne, C., 2001. Autoantibodies to lipids of patients with acute respiratory distress syndrome. Crit. Care Med. 29, 1950. Nakos, G., Kitsiouli, E., Maneta-Peyret, L., Cassagne, C., Tsianos, E., Lekka, M.E., 2001. The characteristics of bronchoalveolar lavage from patient with antiphospholipid syndrome who developed ARDS. Clin. Rheumatol. 20, 91. Sjo¨bring, U., Bjo¨rck, L., Kastern, W., 1991. Streptococcal protein G. Gene structure and protein binding properties. J. Biol. Chem. 266, 399.
Lipids are co-eluted with immunoglobulins G during purification by recombinant streptococcal protein G affinity chromatography Eirini Kitsiouli a,b,1, Marilena E. Lekka b,*, George Nakos c,2, Claude Cassagne a,d,3, Lilly Maneta-Peyret a,4 a
Laboratoire de Biogene`se Membranaire CNRS-UMR 5544, Universite´ Victor Segalen, Bordeaux 2, France b Biochemistry Laboratory, Chemistry Department, University of Ioannina, Ioannina 45110, Greece c Intensive Care Unit, University Hospital of Ioannina, Ioannina 45110, Greece d ESTBB, Universite´ Victor Segalen, Bordeaux 2, France Received 10 April 2002; received in revised form 28 July 2002; accepted 12 August 2002
Abstract The efficiency of recombinant streptococcal protein G (rec-spG) affinity column chromatography in purifying immunoglobulins G (IgG) from lipids has been studied, with particular reference to IgG fractions from bronchoalveolar lavage (BAL) fluid and serum samples from different sources. It was found that the IgG fractions purified by rec-spG affinity column chromatography also contained cholesterol and phospholipids. D 2002 Elsevier Science B.V. All rights reserved. Keywords: Immunoglobulin G; Protein G; Lipids; Cholesterol; BAL
1. Introduction Abbreviations: BAL, bronchoalveolar lavage; BSA, bovine serum albumin; IgG, immunoglobulin G; SDS-PAGE, sodium dodecyl sulfate-polyacrylamide gel electrophoresis; HPTLC, high performance thin layer chromatography; spG, streptococcal protein G; rec-spG, recombinant streptococcal protein G; PC, phosphatidylcholine; PG, phosphatidylglycerol; PS, phosphatidylserine; PI, phosphatidylinositol; PE, phosphatidylethanolamine; Chol, cholesterol; PVDF, polyvinylenedifluoride. * Corresponding author. Tel.: +30-6510-98367; fax: +30-651098774. E-mail addresses: [email protected], [email protected] (E. Kitsiouli), [email protected] (M.E. Lekka), [email protected] (G. Nakos), [email protected] (C. Cassagne), [email protected] (L. Maneta-Peyret). 1 Tel.: +30-6510-22383; fax: +30-6830-51366. 2 Tel./fax: +30-6510-99279. 3 Tel.: +33-557571681; fax: +33-556518361. 4 Tel.: +33-557571681; fax: +33-556518361.
Protein G affinity column chromatography has proven to be a very useful technique for the isolation and purification of IgGs from serum and other biological fluids. Protein G is a multidomain protein expressed on the cell surface of Streptococcus groups C and G. It binds to the Fc region of immunoglobulins from several species by a non-immune mechanism (Guss et al., 1986) exhibiting great affinity for almost all mammalian immunoglobulin G (IgG) classes (Ackerstro¨m and Bjo¨rck, 1986), including all human IgG subclasses (IgG1, IgG2, IgG3 and IgG4). For the isolation of IgGs, streptococcal protein G (spG) was modified by genetic engineering, so that the albumin- and the Fab-binding regions were
0022-1759/02/$ - see front matter D 2002 Elsevier Science B.V. All rights reserved. PII: S 0 0 2 2 - 1 7 5 9 ( 0 2 ) 0 0 3 4 5 - 9
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deleted. There is literature on the degree of separation and purification of IgG from other proteins during protein G affinity chromatography (Guss et al., 1986; Sjo¨bring et al., 1991), but the possible presence of other molecules in the IgG fraction has not been examined. In the present study, we have investigated whether lipids might be recovered together with the IgGs isolated from BAL fluid as well as human and rabbit serum samples by rec-spG affinity column chromatography.
2. Materials and methods In this study, the IgG fractions of BAL fluid and serum samples from 10 patients with acute respiratory distress syndrome were isolated by rec-spG affinity chromatography. In addition, sera from two rabbits immunized against phospholipids (Maneta-Peyret et al., 1988) and two non-immunized rabbits were tested. Bronchoalveolar lavage (BAL) fluid was obtained according to Nakos et al. (2001). IgG fractions were isolated from BAL fluid and serum samples using 1 ml protein G (rec-spG) (Sigma, USA), affinity column chromatography, with a binding capacity exceeding 20 mg human IgGs per ml of suspension. BAL fluid (up to 10 mg protein) and 500 Al of each serum sample were diluted to a final volume of 5 ml with 20 mM sodium phosphate buffer, pH 7.2 (equilibration buffer) and loaded on the column. The procedure took place at room temperature. Flow rate was adjusted to 1 ml/min and protein elution was monitored at 280 nm by a UV photometer (ULTROSPEC 1000, Pharmacia Biotech). The IgG fraction was eluted by 0.1 M glycine –HCl buffer, pH 2.7 and it was immediately neutralized with a 1 M Tris-base buffer solution, pH 8.8. Protein analysis was carried out by SDS-PAGE using 12% polyacrylamide gels (Biorad). Proteins were transferred to PVDF membranes (NEN, USA), followed by incubation with anti-human-IgG-peroxidase conjugate (Sigma) diluted 1/10000 with 0.01 M PBS buffer, pH 7.2, containing 0.05% Tween-20. Immunoblot detection was achieved using Chemiluminescent Reagent Rennaissance (NEN). The eluted IgG fraction was subjected to lipid extraction according to Bligh and Dyer (1959) and
analysed by high performance thin layer chromatography (Silica gel 60 F254 plates, 1010 cm, Merck). For the separation of phospholipids and neutral lipids, the following solvent systems were used, respectively: methyl-acetate/n-propanol/chloroform/methanol/potassium chloride 0.25% (12.5:12.5:14:5:3.5, v/v) and hexane/diethyl ether/acetic acid (70:30:2, v/v) (Juguelin et al., 1986). Phosphatidylcholine, phosphatidylglycerol, phosphatidylethanolamine, phosphatidylinositol, phosphatidylserine, sphingomyelin, phosphatidic acid, cholesterol, cholesterol esters as well as mono-, di- and triglycerides (Sigma) were used as reference compounds. The bands were visualized by copper acetate/phosphoric acid charring (Macala et al., 1983) and scanned at 366 nm with a photodensitometer scanner (Camag) operating in reflectance mode and coupled to a computing integrator (Spectra-Physics). Each spot contained 1 –5 Ag of lipid residue.
Fig. 1. Polar and neutral lipid analysis of the IgG fraction: 5 ml of BAL fluid (2 mg protein/ml) was subjected to recombinant streptococcal protein G affinity column chromatography. The total amount of IgG protein isolated was 0.6 mg, as determined by UV absorbance and this isolated IgG fraction was submitted to lipid extraction. The lipid residue was analysed by HPTLC using methylacetate/n-propanol/chloroform/methanol/0.25% potassium chloride (12.5:12.5:14:5:3.5, v/v) as polar solvent system (lane A) and by hexane/diethyl ether/acetic acid (70:30:2, v/v) as neutral solvent system (lane B). PC: phosphatidylcholine, PS: phosphatidylserine, PG: phosphatidylglycerol, PE: phosphatidylethanolamine, Chol: Cholesterol, NL: neutral lipids. SP denotes the starting point, while SF indicates the solvent front.
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Fig. 2. Gas chromatography/mass spectrometry of the neutral lipid migrating to the Rf of cholesterol: (A) human serum sample, (B) rabbit serum sample, (C) standard cholesterol.
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E. Kitsiouli et al. / Journal of Immunological Methods 271 (2002) 107–111
GC-MS was performed on a Varian 3400 gas chromatograph coupled with a Finnigan Mat TSQ700 mass spectrometer controlled by a Dec Station 5000/ 33. Samples were analysed on a fused silica 25-m OV1 capillary column (30 m, i.d. 0.25 mm) with helium as carrier gas. The column temperature was programmed from 170 to 300 jC (10 jC/min) with the injector temperature fixed at 220 jC. The mass spectrometer operated in the electron impact mode with an electron energy of 70 eV. BAL fluid was tested for the presence of anticholesterol IgG antibodies using ELISA according to Maneta-Peyret et al. (1988). Various amounts, ranging from 5 to 200 Ag of cholesterol/100 Al hexane, were deposited on each well of a microtiter plate (maxisorp, Nunc) and absorbance was measured at 490 nm in a MR 5000 Dynatech spectrophotometer.
3. Results Analysis of the samples by rec-spG affinity column chromatography revealed two peaks; the first representing the nonretained protein, while the second represented the IgG fraction retained by the column. The total protein content of IgGs isolated from 5 ml BAL, 2 mg prot/ml ranged from 0.3 to 1.6 mg, as determined by UV absorbance at 280 nm. SDS-PAGE of the reduced IgG fraction revealed two bands at 50 and 25 kDa, corresponding to the heavy and light chains of IgG, respectively. After immunoblotting, these bands were labeled with anti-IgG antibodies. The eluted material representing the IgG fraction from BAL or serum samples was submitted to lipid extraction. The HPTLC of neutral lipids revealed that all the samples contained a lipid migrating at the Rf of cholesterol. Small amounts of PC, PS, PG and PE were also detected (Fig. 1). The cholesterol/IgG ratio was found to be approximately 1/50 to 1/150 mol/ mol. The area of the HPTLC plate corresponding to the Rf of cholesterol was extracted and subjected to GCMS analysis (Fig. 2). The mass spectra, interpreted according to Hamming and Foster (1972), showed that the lipid derived from the isolated IgG fractions was identical to that of standard cholesterol. It is noteworthy, however, that the isolated antiphospholipid antibodies of the IgG class from BAL and various
sera did not show any reaction with cholesterol as target antigen when tested by ELISA.
4. Discussion In previous studies we have detected and isolated IgGs from bronchoalveolar lavage fluids and serum samples of patients with acute respiratory distress syndrome (ARDS), by rec-spG affinity column chromatography. These purified IgG fractions contained auto-antibodies against lipids, as evidenced from their ability to bind to lipid target antigens, by ELISA (Maneta-Peyret et al., 2001; Nakos et al., 2001). In the present work, we investigated whether lipids were co-eluted in the fraction of the isolated IgGs during the affinity chromatographic procedure, since they could already have been associated with the IgG molecules and in that case, might not be detected by ELISA. The presence of cholesterol in the IgG fractions from sera of non-immunized and phospholipidimmunized rabbits suggests that it was a general phenomenon characterizing IgGs from different sources and not only the anti-phospholipid antibodies of the IgG class. To the best of our knowledge, the presence of lipids in the IgG fractions of BAL and serum samples purified by rec-spG affinity column chromatography has not been reported previously. The nature and significance of these IgG –lipid interactions are still unclear. However, this association was consistently observed in all the analysed samples. Whether this represents a structural ligand recognised by immunoglobulins or is merely an additional mechanism of cholesterol transport in the aqueous environment of blood or alveolar fluid needs further investigation. In either case, the presence of lipids in the IgG fraction could complicate the study of antiphospholipid antibodies, especially as far as it concerns their binding constants with lipid-antigens.
Acknowledgements This work was supported by: the CNRS-University Victor Segalen Bordeaux-2 and ‘‘Ministe`re des Affaires Etrange`res-Programmes PLATON’’. It was also supported by the ‘‘French Embassy’’ in Athenes, the University of Ioannina and by ‘‘Air Liquid Hellas’’.
E. Kitsiouli et al. / Journal of Immunological Methods 271 (2002) 107–111
References Ackerstro¨m, B., Bjo¨rck, L., 1986. A physicochemical study of protein G, a molecule with unique immuloglobulin G-binding properties. J. Biol. Chem. 261, 10240. Bligh, E.G., Dyer, W.J., 1959. A rapid method of total lipid extraction and purification. Can. J. Biochem. Physiol. 37, 911. Guss, B., Eliasson, M., Olsson, A., Uhlen, M., Frej, A.K., Jornvall, H., Flock, J.I., Lindberg, M., 1986. Structure of the IgG-binding regions of streptococcal protein G. EMBO J. 5, 1567. Hamming, M.C., Foster, N.G., 1972. Interpretation of mass spectra compounds. Academic Press, New York. Juguelin, H., Heape, A., Boiron, F., Cassagne, C., 1986. A quantitative developmental study of neutral lipids during myelinogenesis in the peripheral nervous system of normal and trembler mice. Dev. Brain Res. 25, 249.
111
Macala, L.J., Yu, R.K., Ando, S., 1983. Analysis of brain lipids by high performance thin-layer chromatography and densitometry. J. Lipid Res. 24, 1243. Maneta-Peyret, L., Bessoule, J.J., Geffard, M., Cassagne, C., 1988. Demonstration of high specificity antibodies against phosphatidylserine. J. Immunol. Methods 108, 123. Maneta-Peyret, L., Kitsiouli, E., Lekka, M.E., Nakos, G., Cassagne, C., 2001. Autoantibodies to lipids of patients with acute respiratory distress syndrome. Crit. Care Med. 29, 1950. Nakos, G., Kitsiouli, E., Maneta-Peyret, L., Cassagne, C., Tsianos, E., Lekka, M.E., 2001. The characteristics of bronchoalveolar lavage from patient with antiphospholipid syndrome who developed ARDS. Clin. Rheumatol. 20, 91. Sjo¨bring, U., Bjo¨rck, L., Kastern, W., 1991. Streptococcal protein G. Gene structure and protein binding properties. J. Biol. Chem. 266, 399.