- Email: [email protected]
A radioimmunoassay to recognise new brain differentiation antigens
Brain Research, 133 (1977) 139-143 © Elsevier/North-Holland Biomedical Press
139
Short Communications
A radioimmunoassay to recognise new brain differentiation antigens
A. NEIL BARCLAY* MRC Immunochemistry Unit, Department of Biochemistry, University of Oxford, Oxford OX1 3QU (Great Britain)
(Accepted May 25th, 1977)
Many of the specialized functions of the tissues of higher organisms probably involve interactions at the cell surface mediated by tissue-specific cell surface molecules. Very little is known about these interactions, but one possible approach is to use anti° sera to recognise tissue-specific cell surface molecules i.e. differentiation antigens. The antigenicity can then be followed in purification studies leading to the purification and characterization of these molecules. In the long term, the purified antigens and specific high-affinity antisera raised against them could be used to investigate their functions. Several methods are available to study antigens present at the surface of cells, but the variety of cells and their intercellular connections that characterize adult brain also hinder this type of analysis for brain membrane antigens. Thus other forms of brain tissue have been used, e.g. dissociated neonatal cell suspensions 11 or cell lines of neuroectodermal origin 3,10,14, but it would be advantageous to avoid this restriction in target tissue. The aim of this study was to see if homogenized adult brain could be used as target particles in an indirect radioactive binding assay to recognise antigens present on normal brain tissue. The principle of the indirect radioactive binding assay is that target cells bearing the antigen(s) of interest are reacted with antisera which recognise the antigen(s). The cells are washed to remove unbound immunoglobulin and reacted with 125Ilabelled purified anti-(immunoglobulin) antibodies. The cells are washed again, and the amount of labelled antibody bound (which is proportional to the first antibody bound) is quantitated in a g a m m a counter. The amount of antigen in an extract can be quantitated by preincubating the first antibody with the extract and measuring the residual unbound antibody as above. The various applications and advantages of this assay are discussed by Morris and Williams 9 and Williams 16. Unless otherwise stated all procedures were at 0 - 4 °C and a balanced buffered salt solution 1~ was used throughout. * Present address: Institute of Neurobiology, Faculty of Medicine, University of G6teborg, Fack, S-400 33 G6teborg 33, Sweden.
140 The assays were carried out as described in references 6 and 9, except that brain homogenate (which had been fixed with glutaraldehyde) was used as the target particle instead of thymocytes. Brain homogenate was prepared by homogenization of rat brain with 10 vols of buffer in a Potter Elvejham homogenizer and fixed by the addition of an equal volume of 0.25% (w/v) glutaraldehyde (Koch-Light Ltd., Colnbrock, England). After 15 rain at 20 °C, the reaction was stopped by the addition of 1/10 vol. of 10 % (w/v) bovine serum albumin (Sigma Chemical Co., London). The fixed brain homogenate was washed twice by centrifugation and resuspended in 5 % (w/v) bovine serum albumin to the same volume as the original brain homogenate and stored at --70 °C. This brain homogenate was used as target particles in the binding assays in an equivalent manner to thymocytes at l0 s cells/ml (ref. 6). Two antisera used to test this assay system were raised against partially purified rat brain membrane glycoprotein fractions. A crude membrane fraction from rat brain was solubilized with deoxycholate and a glycoprotein fraction separated by affinity chromatography on a lentil lectin-Sepharose 4B column, as described in the purification of the Thy-1 antigen from rat brain 1. The glycoprotein fraction was chromatographed on a Sephadex G-100 column (5 cm × 85 cm) in 0.5 % (w/v) Na deoxycholate, 0.01 M Tris.HCl, 0.02 % NAN3, pH 8.0, and two pools made: pool 'A' contained the majority of the glycoproteins (i.e. those that eluted with a Stokes radius of greater than about 4.5 nm (see ref. 1); pool 'B' contained the material which eluted between pool 'A' and those fractions that contained Thy-1. The pools were concentrated by ultrafiltration with an Amicon apparatus using a PM 10 filter and used to raise anti-(A) and anti-(B) sera (see below). Both fractions 'A' and 'B' contained several polypeptide chains when analyzed by polyacrylamide gel electrophoresis in sodium dodecyl sulphate. Protein was determined as described by Hartree 4. Anti-(A) serum was raised in two rabbits by immunizing with fraction 'A' according to the following schedule. Two intramuscular injections of 0.1 mg protein of fraction 'A' in complete Freund's adjuvant and one subcutaneous injection of 1.0 mg in incomplete Freund's adjuvant at weekly intervals, and 4 weeks later an intravenous injection of 0.4 mg 'A'. The bleed analyzed was taken one week later. Anti-(B) serum was raised as for anti-(A) serum except that the immunizations were with 0.l mg, 0.1 mg, 0.4 mg and 0.2 mg of fraction 'B', respectively. 12al-labelled purified horse F(ab')z anti-(rabbit IgG) was prepared as by Williams (ref. 15). Serial dilutions of each antiserum were tested for antibodies binding to brain homogenate by the indirect radioactive binding assay, and both antisera gave good specific binding compared to that obtained with the pre-immune sera. A dilution of 1/750 of each serum was chosen for further analysis, as at this dilution a reasonable amount of specific binding was obtained under conditions where the second (i.e. 125Ilabelled) antibody was not limiting (see ref. 9). The antisera were absorbed with various rat tissues 9 and the effects followed by the indirect radioactive binding assay. Fig. 1 shows that the binding of anti-(A) serum to brain homogenate was completely inhibited by brain homogenate, but no inhibition was obtained with liver, kidney, thymocytes, spleen cells or serum. Thus
141 Inhibiting
104 i
% x 6. d ~ o
cells per a s s a y
10 5
106
i
107
i
e
30
25
~ 2o L
"6
~
5
10-1
I
I
I
10°
101
102
10 3
Inhibitor p r o t e i n per a s s a y ( p g )
Fig. 1. Inhibition of the binding of rabbit anti-(A) serum to brain homogenate. 25-~1 samples of anti-(A) serum diluted 1/750 were absorbed with various rat tissues and the binding of residual antibody to rat brain homogenate was followed with leSI-labelled horse F (ab')2 anti-(rabbit IgG) (100 ~1 at 0.2 ~g/ml and 30 FCi]~g). The inhibitions obtained with homogenates of liver (0), brain (11) and kidney (©) and by rat serum (A), tbymocytes ([]) and spleen cells (A) are shown. One abscissa defines cell numbers and applies to A , [], and the other defines protein and applies to [~, ©, m 0 , A. this antiserum is recognizing antigens present on rat brain h o m o g e n a t e but not on several other rat tissues. Fig. 2 shows that the binding o f anti-(B) serum to brain h o m o genate was completely inhibited by brain h o m o g e n a t e and that the other tissues gave only partial inhibitions even at high concentrations of the inhibiting tissues. Thus the I n h i b i t i n g cells per a s s a y
104
% ~
30
E 6. d
25
105
10 6
107
e
i
i
~ 20 Y
o
m
10-1
I
10°
I
101
i
102
103
Inhibitor p r o t e i n per a s s a y ( ) J g )
Fig. 2. Inhibition of the binding of rabbit anti-(B) serum to brain homogenate. 25-/~] samples of anti-(B) serum diluted 1/750 were assayed as foi anti-(A) serum in Fig. ]. The symbols for the various tissues
are as in Fig. 1.
142 majority of anti-fbrain) antibodies in the anti-(B) serum were recognizing antigens present on brain homogenate but not expressed on the other tissues. The binding of both anti-(A) and anti-(B) sera to brain homogenate could be almost completely inhibited by both of the glycoprotein fractions (in deoxycholate) used to raise these antisera. 50 ~ inhibition of the binding of anti-(A) serum to brain homogenate was obtained with 0.25 #g fraction ' A ' or 0.54 #g fraction 'B', and 50 ~o inhibition for anti-(B) serum was obtained with 0.60 #g 'A' and 0.36/~g 'B'. Although both anti-(A) and anti-(B) sera must be recognizing antigens present in both fractions ' A ' and 'B', the quantitative differences in the inhibitions indicate that the two antisera differ in their specificities. One major problem in the study of differentiation antigens is in the definition of the specificity of antisera. For instance, one can define an antigen (or antigens) in terms of the nature of the immunogen and the tissue distribution of the antigens recognised 3,8,12, or by the parameters of precipitin lines obtained by crossed immunoelectrophoresis 5. However, apart from alloantisera which can be defined genetically, the only clear method to define and to be able to reproduce an antiserum of a particular specificity is to use the purified antigen as the immunogen. The tissue distribution of the antigens recognized can then be used as confirmation of the specificity. The indirect radioactive binding assay is particularly well suited for following antigenicity during purification studies on membrane antigens 16 and this approach has been successful in the purification of the Thy- 1 antigen from rat thymocytes 7and brain 1,~. The analysis of the two antisera above showed that this assay system could be readily applied to study antigens present on adult homogenized brain, and it should be possible to biochemically characterize and hopefully purify those brain differentiation antigens recognized. ! am grateful to Dr. A. F. Williams and Professor R. R. Porter for advice and encouragement and for support from a Medical Research Council research studentship.
1 Barclay,A. N., Letarte-Muirhead, M. and Williams, A. F., Purification of the Thy-1 moleculefrom rat brain, Bioehem. J., 151 (1975) 699-706. 2 Barclay, A. N., Letarte-Muirhead, M., Williams, A. F. and Faulkes, R. A., Chemical characterisation of the Thy-1 glycoproteinsfrom the membranes of rat tbymocytesand brain, Nature (Lond.), 263 (1976) 563-567. 3 Fields, K. L., Gosling, C., Megson, M. and Stern, P. L., New cell surface antigens in rat defined by tumor s of the nervous system, Proc. nat. A cad. Sci. (Wash.), 72 (1975) 1296-1300. 4 Hartree, E. F., Determination of protein : a modification of the Lowry method that gives a linear photometric response, Analyt. Biochem., 48 (1972) 422-427. 5 Jorgensen, C. S. and Bock, E., Brain specific synaptosomal membrane proteins demonstrated by crossed immunoelectrophoresis, J. Neurochem., 23 (1974) 879-880. 6 Letarte-Muirhead, M., Acton, R. T. and Williams, A. F., Preliminary characterization of Thy-1.1 and Ag-B antigens from rat tissues solubilised in detergents, Biochern. J., 143 (1974) 51-61. 7 Letarte-Muirhead, M., Barclay, A. N. and Williams, A. F., Purification of the Thy-1 molecule, a major cell-surface glycoprotein of rat thymocytes, Biochem. J., 151 (1975) 685-697. 8 Martin, S. E., Mouse brain antigen detected by rat anti-C1300 antiserum, Nature (Lond.), 249 (1974) 71-73. 9 Morris, R. J. and Williams, A. F., Antigens on mouse and rat lymphocytesrecognised by rabbit
143
10 11
12 13
14 15 16
antiserum against rat brain: the quantitative analysis of a xenogeneic antiserum, Europ. J. Immunol., 5 (1975) 274-281. Schachner, M., NS-1 (Nervous System Antigen-l), a glial-cell-specific antigenic component of the surface membrane, Proc. nat. Acad. Sci. (Wash.), 71 (1974) 1795-1799. Schachner, M., Wortham, K. A., Carter, L. D. and Chaffee, J. K., NS-4 (Nervous System Antigen4), a cell surface antigen of developing and adult mouse brain and sperm, Develop. BioL, 44 (1975) 313-325. Schachner, M., Wortham, K. A. and Kincade, P. W., Detection of nervous-system cell surface antigens by heterologous anti-mouse brain antiserum, Cell. ImmunoL, 22 (1976) 369-374. Shortman, K., The separation of different cell classes from lymphoid organs II. The purification and analysis of lymphocyte populations by equilibrium density gradient centrifugation, Aust. J. exp. Biol. med. Sci., 46 (1968) 375-396. Stallcup, W. B. and Cohn, M., Correlation of surface antigens and cell type in cloned cell lines from the rat central nervous system, Exp. Cell Res., 98 (1976) 285-297. Williams, A. F., IgG2 and other immunoglobulin classes on the cell surface of rat lymphoid cells, Europ. J. lmmunol., 5 (1975) 883-885. Williams, A. F., Differentiation antigens of the lymphocyte cell surface. In Contemporary Topics in Molecular Immunology, in press.
139
Short Communications
A radioimmunoassay to recognise new brain differentiation antigens
A. NEIL BARCLAY* MRC Immunochemistry Unit, Department of Biochemistry, University of Oxford, Oxford OX1 3QU (Great Britain)
(Accepted May 25th, 1977)
Many of the specialized functions of the tissues of higher organisms probably involve interactions at the cell surface mediated by tissue-specific cell surface molecules. Very little is known about these interactions, but one possible approach is to use anti° sera to recognise tissue-specific cell surface molecules i.e. differentiation antigens. The antigenicity can then be followed in purification studies leading to the purification and characterization of these molecules. In the long term, the purified antigens and specific high-affinity antisera raised against them could be used to investigate their functions. Several methods are available to study antigens present at the surface of cells, but the variety of cells and their intercellular connections that characterize adult brain also hinder this type of analysis for brain membrane antigens. Thus other forms of brain tissue have been used, e.g. dissociated neonatal cell suspensions 11 or cell lines of neuroectodermal origin 3,10,14, but it would be advantageous to avoid this restriction in target tissue. The aim of this study was to see if homogenized adult brain could be used as target particles in an indirect radioactive binding assay to recognise antigens present on normal brain tissue. The principle of the indirect radioactive binding assay is that target cells bearing the antigen(s) of interest are reacted with antisera which recognise the antigen(s). The cells are washed to remove unbound immunoglobulin and reacted with 125Ilabelled purified anti-(immunoglobulin) antibodies. The cells are washed again, and the amount of labelled antibody bound (which is proportional to the first antibody bound) is quantitated in a g a m m a counter. The amount of antigen in an extract can be quantitated by preincubating the first antibody with the extract and measuring the residual unbound antibody as above. The various applications and advantages of this assay are discussed by Morris and Williams 9 and Williams 16. Unless otherwise stated all procedures were at 0 - 4 °C and a balanced buffered salt solution 1~ was used throughout. * Present address: Institute of Neurobiology, Faculty of Medicine, University of G6teborg, Fack, S-400 33 G6teborg 33, Sweden.
140 The assays were carried out as described in references 6 and 9, except that brain homogenate (which had been fixed with glutaraldehyde) was used as the target particle instead of thymocytes. Brain homogenate was prepared by homogenization of rat brain with 10 vols of buffer in a Potter Elvejham homogenizer and fixed by the addition of an equal volume of 0.25% (w/v) glutaraldehyde (Koch-Light Ltd., Colnbrock, England). After 15 rain at 20 °C, the reaction was stopped by the addition of 1/10 vol. of 10 % (w/v) bovine serum albumin (Sigma Chemical Co., London). The fixed brain homogenate was washed twice by centrifugation and resuspended in 5 % (w/v) bovine serum albumin to the same volume as the original brain homogenate and stored at --70 °C. This brain homogenate was used as target particles in the binding assays in an equivalent manner to thymocytes at l0 s cells/ml (ref. 6). Two antisera used to test this assay system were raised against partially purified rat brain membrane glycoprotein fractions. A crude membrane fraction from rat brain was solubilized with deoxycholate and a glycoprotein fraction separated by affinity chromatography on a lentil lectin-Sepharose 4B column, as described in the purification of the Thy-1 antigen from rat brain 1. The glycoprotein fraction was chromatographed on a Sephadex G-100 column (5 cm × 85 cm) in 0.5 % (w/v) Na deoxycholate, 0.01 M Tris.HCl, 0.02 % NAN3, pH 8.0, and two pools made: pool 'A' contained the majority of the glycoproteins (i.e. those that eluted with a Stokes radius of greater than about 4.5 nm (see ref. 1); pool 'B' contained the material which eluted between pool 'A' and those fractions that contained Thy-1. The pools were concentrated by ultrafiltration with an Amicon apparatus using a PM 10 filter and used to raise anti-(A) and anti-(B) sera (see below). Both fractions 'A' and 'B' contained several polypeptide chains when analyzed by polyacrylamide gel electrophoresis in sodium dodecyl sulphate. Protein was determined as described by Hartree 4. Anti-(A) serum was raised in two rabbits by immunizing with fraction 'A' according to the following schedule. Two intramuscular injections of 0.1 mg protein of fraction 'A' in complete Freund's adjuvant and one subcutaneous injection of 1.0 mg in incomplete Freund's adjuvant at weekly intervals, and 4 weeks later an intravenous injection of 0.4 mg 'A'. The bleed analyzed was taken one week later. Anti-(B) serum was raised as for anti-(A) serum except that the immunizations were with 0.l mg, 0.1 mg, 0.4 mg and 0.2 mg of fraction 'B', respectively. 12al-labelled purified horse F(ab')z anti-(rabbit IgG) was prepared as by Williams (ref. 15). Serial dilutions of each antiserum were tested for antibodies binding to brain homogenate by the indirect radioactive binding assay, and both antisera gave good specific binding compared to that obtained with the pre-immune sera. A dilution of 1/750 of each serum was chosen for further analysis, as at this dilution a reasonable amount of specific binding was obtained under conditions where the second (i.e. 125Ilabelled) antibody was not limiting (see ref. 9). The antisera were absorbed with various rat tissues 9 and the effects followed by the indirect radioactive binding assay. Fig. 1 shows that the binding of anti-(A) serum to brain homogenate was completely inhibited by brain homogenate, but no inhibition was obtained with liver, kidney, thymocytes, spleen cells or serum. Thus
141 Inhibiting
104 i
% x 6. d ~ o
cells per a s s a y
10 5
106
i
107
i
e
30
25
~ 2o L
"6
~
5
10-1
I
I
I
10°
101
102
10 3
Inhibitor p r o t e i n per a s s a y ( p g )
Fig. 1. Inhibition of the binding of rabbit anti-(A) serum to brain homogenate. 25-~1 samples of anti-(A) serum diluted 1/750 were absorbed with various rat tissues and the binding of residual antibody to rat brain homogenate was followed with leSI-labelled horse F (ab')2 anti-(rabbit IgG) (100 ~1 at 0.2 ~g/ml and 30 FCi]~g). The inhibitions obtained with homogenates of liver (0), brain (11) and kidney (©) and by rat serum (A), tbymocytes ([]) and spleen cells (A) are shown. One abscissa defines cell numbers and applies to A , [], and the other defines protein and applies to [~, ©, m 0 , A. this antiserum is recognizing antigens present on rat brain h o m o g e n a t e but not on several other rat tissues. Fig. 2 shows that the binding o f anti-(B) serum to brain h o m o genate was completely inhibited by brain h o m o g e n a t e and that the other tissues gave only partial inhibitions even at high concentrations of the inhibiting tissues. Thus the I n h i b i t i n g cells per a s s a y
104
% ~
30
E 6. d
25
105
10 6
107
e
i
i
~ 20 Y
o
m
10-1
I
10°
I
101
i
102
103
Inhibitor p r o t e i n per a s s a y ( ) J g )
Fig. 2. Inhibition of the binding of rabbit anti-(B) serum to brain homogenate. 25-/~] samples of anti-(B) serum diluted 1/750 were assayed as foi anti-(A) serum in Fig. ]. The symbols for the various tissues
are as in Fig. 1.
142 majority of anti-fbrain) antibodies in the anti-(B) serum were recognizing antigens present on brain homogenate but not expressed on the other tissues. The binding of both anti-(A) and anti-(B) sera to brain homogenate could be almost completely inhibited by both of the glycoprotein fractions (in deoxycholate) used to raise these antisera. 50 ~ inhibition of the binding of anti-(A) serum to brain homogenate was obtained with 0.25 #g fraction ' A ' or 0.54 #g fraction 'B', and 50 ~o inhibition for anti-(B) serum was obtained with 0.60 #g 'A' and 0.36/~g 'B'. Although both anti-(A) and anti-(B) sera must be recognizing antigens present in both fractions ' A ' and 'B', the quantitative differences in the inhibitions indicate that the two antisera differ in their specificities. One major problem in the study of differentiation antigens is in the definition of the specificity of antisera. For instance, one can define an antigen (or antigens) in terms of the nature of the immunogen and the tissue distribution of the antigens recognised 3,8,12, or by the parameters of precipitin lines obtained by crossed immunoelectrophoresis 5. However, apart from alloantisera which can be defined genetically, the only clear method to define and to be able to reproduce an antiserum of a particular specificity is to use the purified antigen as the immunogen. The tissue distribution of the antigens recognized can then be used as confirmation of the specificity. The indirect radioactive binding assay is particularly well suited for following antigenicity during purification studies on membrane antigens 16 and this approach has been successful in the purification of the Thy- 1 antigen from rat thymocytes 7and brain 1,~. The analysis of the two antisera above showed that this assay system could be readily applied to study antigens present on adult homogenized brain, and it should be possible to biochemically characterize and hopefully purify those brain differentiation antigens recognized. ! am grateful to Dr. A. F. Williams and Professor R. R. Porter for advice and encouragement and for support from a Medical Research Council research studentship.
1 Barclay,A. N., Letarte-Muirhead, M. and Williams, A. F., Purification of the Thy-1 moleculefrom rat brain, Bioehem. J., 151 (1975) 699-706. 2 Barclay, A. N., Letarte-Muirhead, M., Williams, A. F. and Faulkes, R. A., Chemical characterisation of the Thy-1 glycoproteinsfrom the membranes of rat tbymocytesand brain, Nature (Lond.), 263 (1976) 563-567. 3 Fields, K. L., Gosling, C., Megson, M. and Stern, P. L., New cell surface antigens in rat defined by tumor s of the nervous system, Proc. nat. A cad. Sci. (Wash.), 72 (1975) 1296-1300. 4 Hartree, E. F., Determination of protein : a modification of the Lowry method that gives a linear photometric response, Analyt. Biochem., 48 (1972) 422-427. 5 Jorgensen, C. S. and Bock, E., Brain specific synaptosomal membrane proteins demonstrated by crossed immunoelectrophoresis, J. Neurochem., 23 (1974) 879-880. 6 Letarte-Muirhead, M., Acton, R. T. and Williams, A. F., Preliminary characterization of Thy-1.1 and Ag-B antigens from rat tissues solubilised in detergents, Biochern. J., 143 (1974) 51-61. 7 Letarte-Muirhead, M., Barclay, A. N. and Williams, A. F., Purification of the Thy-1 molecule, a major cell-surface glycoprotein of rat thymocytes, Biochem. J., 151 (1975) 685-697. 8 Martin, S. E., Mouse brain antigen detected by rat anti-C1300 antiserum, Nature (Lond.), 249 (1974) 71-73. 9 Morris, R. J. and Williams, A. F., Antigens on mouse and rat lymphocytesrecognised by rabbit
143
10 11
12 13
14 15 16
antiserum against rat brain: the quantitative analysis of a xenogeneic antiserum, Europ. J. Immunol., 5 (1975) 274-281. Schachner, M., NS-1 (Nervous System Antigen-l), a glial-cell-specific antigenic component of the surface membrane, Proc. nat. Acad. Sci. (Wash.), 71 (1974) 1795-1799. Schachner, M., Wortham, K. A., Carter, L. D. and Chaffee, J. K., NS-4 (Nervous System Antigen4), a cell surface antigen of developing and adult mouse brain and sperm, Develop. BioL, 44 (1975) 313-325. Schachner, M., Wortham, K. A. and Kincade, P. W., Detection of nervous-system cell surface antigens by heterologous anti-mouse brain antiserum, Cell. ImmunoL, 22 (1976) 369-374. Shortman, K., The separation of different cell classes from lymphoid organs II. The purification and analysis of lymphocyte populations by equilibrium density gradient centrifugation, Aust. J. exp. Biol. med. Sci., 46 (1968) 375-396. Stallcup, W. B. and Cohn, M., Correlation of surface antigens and cell type in cloned cell lines from the rat central nervous system, Exp. Cell Res., 98 (1976) 285-297. Williams, A. F., IgG2 and other immunoglobulin classes on the cell surface of rat lymphoid cells, Europ. J. lmmunol., 5 (1975) 883-885. Williams, A. F., Differentiation antigens of the lymphocyte cell surface. In Contemporary Topics in Molecular Immunology, in press.