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Cascade immunization: a method of obtaining polyspecific antisera against crude fractions of antigens
Journal of Immunological Methods, 66 (1984) 245-251
245
Elsevier JIM02920
Cascade Immunization: a Method of Obtaining Polyspecific Antisera against Crude Fractions of Antigens Joseph Thalhamer and Johann Freund University of Salzburg, Institute of General Biology, Biochemistry and Biophysics, Department of Biochemistry, Erzabt Klotzstr. 11, 5020 Salzburg, Austria
(Received 11 April 1983, accepted 30 August 1983)
Between 20 and 30 precipitation lines are usually obtained by crossed immune electrophoresis of an Escherichia coli cytoplasmic extract against antisera produced against that extract in individual rabbits.
With a combination of several such antisera, the number of precipitation lines increases to 30-40. Nevertheless, extracts as used in this work contain many antigens in addition to those thus detected. Intermolecular immune competition may be avoided by removing strong immunogens from the extracts. The remaining antigens which give no immune response in the primary immunization are used for further immunization. New antibody production against 8-14 additional antigens occurs after one such 'cascade immunization' step. Separation of strong from weak immunogens is performed by preparative CIE and the use of immuno-affinity columns. The procedure is called cascade immunization because it involves repeated removal of antigens and production of further antisera directed against antigens in the remainder. Key words: cascade immunization - antigenic competition - E. coli antigens
Introduction A n t i g e n i c c o m p e t i t i o n limits the variety a n d yield of a n t i b o d i e s o b t a i n e d following i m m u n i z a t i o n with extracts c o n t a i n i n g m u l t i p l e antigens. A n t i g e n i c c o m p e t i t i o n is the i n h i b i t i o n of the i m m u n e response to one antigen or antigenic d e t e r m i n a n t c a u s e d b y the a d m i n i s t r a t i o n of a n o t h e r antigen or d e t e r m i n a n t . T h e term was i n t r o d u c e d b y L e o n o r Michaelis (1904) in the context of raising a n t i b o d i e s to serum proteins. M i c h a e l i s ' s original o b s e r v a t i o n of c o m p e t i t i o n b e t w e e n globulin a n d a l b u m i n have b e e n r e e x a m i n e d with some interesting results with respect to T cell function, T cell-B cell c o o p e r a t i o n a n d the role of c o m p l e t e F r e u n d ' s a d j u v a n t ( F e l d m a n n a n d Diener, 1970; F e l d m a n n a n d Nossal, 1972; R o w l e y et al., 1973; Taussig, 1973; M o z e s et al., 1974; Dintzis et al., 1976, 1982; A b b a s a n d Klaus, 1978). W e d e s c r i b e here an a t t e m p t to avoid antigenic c o m p e t i t i o n b y r e m o v i n g s t r o n g i m m u n o g e n s f r o m extracts a n d injecting the r e m a i n d e r . This ' c a s c a d e ' 0022-1759/84/$03.00 © 1984 Elsevier Science Publishers B.V.
246 method is frequently used in preparing monoclonal antibodies against unknown mixtures of antigen, which may then be analysed by the monoclonal antibodies they engender. Monoclonal antibodies from the primary immunization step are applied to immunoadsorbent columns, which remove the corresponding antigens. The depleted mixture, enriched in the remaining antigens is injected again (Milstein et al., 1979). In the present work, polyvalent, polyspecific and precipitating antisera were used to prepare immunogens to produce more antibody in cascade fashion. Material and Methods
A n tigens Escherichia coli c600 (Strack and Cox, 1971) was grown in medium containing 1% Bacto Tryptone (Difco) and 0.5% NaC1 adjusted to pH 7. From cultures grown with aeration at 37°C to a density of 108 cells/ml, the organisms were harvested by centrifugation at 6000 rpm (Beckman Rotor SW40), washed twice in tricine buffer IV (Bio-Rad Laboratories) and disrupted in a Braun Melsungen glass bead homogenizer (4000 rpm at 4°C, glass bead diameter 0.12 mm) for 2 min. The extract was cleared by centrifugation at 15,000 rpm for 30 min and stored frozen at - 80°C after addition of 0.015 M sodium azide and 1% aprotinin. The protein concentration was 4 mg/ml. A ntisera Antisera against cell extracts and fractions were produced in rabbits as follows: 2 mg protein was injected subcutaneously at day 0 and again intramuscularly at day 7 and day 21. Following this, 5 mg protein was given subcutaneously at day 22 and intravenously at day 23. Only the first dose was given with complete Freund's adjuvant (CFA), the subsequent injections being given in 0.14 M NaC1. Antisera and antibody fractions were stored frozen at - 8 0 ° C after addition of 0.015 M sodium azide and 1% aprotinin. Separation of immunogenic proteins This was performed by 2 methods. Procedure L Proteins obtained by ammonium sulfate precipitation (Williams and Chase, 1967) were bound to CNBr-activated Sepharose 4B (Gonyea, 1976; Van Eijk and Van Noort, 1976). Some 60 mg IgG were bound to a 10 cm x 1.5 cm column a follows: CNBr-activated Sepharose 4B was washed and re-swollen with 1 mM HC1. The coupling buffer was NaCO 3 (0.1 M, pH 8.3) containing NaC1 (0.5 M). After coupling, remaining active groups were blocked with blocking agent (1 M ethanolamine). Excess adsorbed proteins were washed away with acetate buffer (0.5 M) containing NaC1 (0.5 M). Two milliliters antigen extract were applied and the proteins passing through were collected, concentrated and used for immunizing another rabbit. Procedure 11. Preparative CIE was performed in an agarose gel (100 mm x 200 mm x 5 mm) run at 10°C (tricine buffer IV, pH 8.6, 10 V/cm) for 8 h. The second gel contained 50% antiserum. Proteins reacting with the antiserum were precipitated and immobilized near the cathode. Non-reacting proteins migrated toward the anode
247
~iiii
Fig. 1. CIE analysis of Escherichia coil c600: extract was performed in tricine buffer IV at 5 V / c m for 150 min in the 1st dimension and 1 V / c m for 15 h in the 2nd dimension. Anode to the right for the first dimension and at the top for the 2nd dimension. A (top): precipitation arcs with the extract against an antiserum from one rabbit; B (bottom): against a mixture of antisera from 8 rabbits.
248
and were located in an area separate from that containing precipitation lines at the time the electrophoresis was stopped. The non-reacting proteins were eluted from the gel by homogenizing the corresponding part of the gel and removing the agarose by centrifugation. The supernatant was used for immunizing another rabbit. SDSpolyacrylamide gel electrophoresis of Escherichia coli proteins labeled with 14Clabeled amino acids and autoradiographs were performed as described by O'Farrell (1974). CIE was performed as described by Laurell (1965, 1972). Results
CIE results with an Escherichia coli extract against an antiserum produced by conventional immunization of one rabbit and against pooled antisera from 8 rabbits are shown in Fig. 1.
Fig. 2. Autoradiograph of SDS gel electrophoresis of 14C-labeled Escherichia coil proteins. Lane A shows precipitated proteins; lane B non-precipitated proteins, separated by preparative CIE.
249
Fig. 3. CIE of non-precipitated proteins (nplI) against an antiserum prepared against them (as CII).
Fig. 4. CIE with intermediate gel against E. coli cytoplasmic extract. The first gel contains antibodies obtained by primary immunization (as prim.), the second gel contains antibodies from the first cascade step (as CII).
250
Fig. 5. CIE of non-precipitatedproteins (np I) obtained in procedure I against antiserum from the first cascade step (as CI). Proteins 1-4 were not removed completelyby the separation procedure I, and are the same as peaks 1-4 in Fig. 1A. Precipitated proteins (labeled with t4C-labeled amino acids) were recovered from the gel by cutting out the precipitation area. The agarose gel containing the precipitated proteins was mixed with an equal volume of O'Farrell sample buffer, boiled for 5 min and applied to the SDS gel. Fig. 2 compares the autoradiographs of precipitated and non-precipitated 14Clabeled Escherichia coli cytoplasmic proteins separated by preparative CIE against a primary antiserum. Immunization with the proteins in lane B (Fig. 2) elicited antibody production against them (Fig. 3). CIE with an intermediate gel was performed to demonstrate the difference between a primary antiserum and an antiserum of the first cascade step (Fig. 4). Separation of non-precipitated from precipitated proteins was according to procedure II. This was preferred to procedure I since the latter (Fig. 5) did not completely remove the strong immunogens.
Discussion
The method described here of improving limited immune responses against extracts containing multiple antigens should be of general use (e.g. for plant extracts and pollen extracts). Cascade immunization elicits antibodies not produced by conventional immunization procedures. The number of antigens in the E. coli extract
251 revealed by C I E was increased from the 2 0 - 3 0 obtained on primary immunization to 6 0 - 1 0 0 by cascade immunization. A further advantage of this technique is that antisera produced at different stages of the cascade do not share antibodies of the same specificity. Antibodies against high concentration proteins in the extract are present only in the antisera of the first and possibly second cascade steps, whereas antisera from subsequent cascade steps contain antibodies against weak immunogens or immunogens present in low concentration. The use of cascade antisera in parallel avoids the difficulty of heavy precipitation lines obscuring weak ones. The relative inefficiency of the i m m u n o a d s o r b e n t technique c o m p a r e d with preparative C I E was p r o b a b l y due to overloading the column with antigens present at high concentrations. Some antibodies obtained at the first immunization step seem to have lower affinities and successful removal of the corresponding antigens m a y require repeated i m m u n o a d s o r p t i o n resulting in very dilute antigen solutions. Removal of antigens by preparative C I E is a better method.
Acknowledgements We thank Prof. Dr. Strack for helpful discussions.
References Abbas, A.K. and G.G. Klaus, 1978, Eur. J. Immunol. 8, 217. Dintzis, H.M., R.Z. Dintzis and B. Vogelstein, 1976, Proc. Natl. Acad. Sci. U.S.A. 73, 3671. Dintzis, R.Z., B. Vogelstein and H.M. Dintzis, 1982, Proc. Natl. Acad. Sci. U.S.A. 79, 884. Feldmann, M. and E. Diener, 1970, J. Exp. Med. 131, 247. Feldmann, M. and G.J. Nossal, 1972, Transplant. Rev. 13, 3. Gonyea, L.M., 1976, Clin. Chem. 23, 234. Laurell, C.B., 1965, Anal. Biochem. 10, 358. Laurell, C.B., 1972, Scand. J. Clin. Lab. Invest. 29, 124. Michaelis, L., 1904, Dtsch. Med. Wochenschr. 30, 1240. Milstein, C., G. Galfre and D.S. Secher, 1979, Ciba Found. Symp. no. 66, (Elsevier, Amsterdam) p. 251. Mozes, E., M. Sela and M.J. Taussig, 1974, Immunology 27, 641. O'Farrell, P.H., 1974, J. Biol. Chem. 250, 4007. Rowley, D.A., F.W. Fitch, F.P. Stuart, H. K6hler and H. Consenza, 1973, Science 181, 1133. Strack, H.B. and J.H. Cox, 1971, Virology 43, 719. Taussig, M.J., 1973, Curt. Top. Microbiol. Immunol. 60, 125. Van Eijk, H.G. and W.L. Van Noort, 1976, Eur. J. Bioehem. 54, 411. Williams, C. and M. Chase (eds.), 1967, Methods in Immunology and Immunochemistry, No. I (Academic Press, New York) pp. 315-321.
245
Elsevier JIM02920
Cascade Immunization: a Method of Obtaining Polyspecific Antisera against Crude Fractions of Antigens Joseph Thalhamer and Johann Freund University of Salzburg, Institute of General Biology, Biochemistry and Biophysics, Department of Biochemistry, Erzabt Klotzstr. 11, 5020 Salzburg, Austria
(Received 11 April 1983, accepted 30 August 1983)
Between 20 and 30 precipitation lines are usually obtained by crossed immune electrophoresis of an Escherichia coli cytoplasmic extract against antisera produced against that extract in individual rabbits.
With a combination of several such antisera, the number of precipitation lines increases to 30-40. Nevertheless, extracts as used in this work contain many antigens in addition to those thus detected. Intermolecular immune competition may be avoided by removing strong immunogens from the extracts. The remaining antigens which give no immune response in the primary immunization are used for further immunization. New antibody production against 8-14 additional antigens occurs after one such 'cascade immunization' step. Separation of strong from weak immunogens is performed by preparative CIE and the use of immuno-affinity columns. The procedure is called cascade immunization because it involves repeated removal of antigens and production of further antisera directed against antigens in the remainder. Key words: cascade immunization - antigenic competition - E. coli antigens
Introduction A n t i g e n i c c o m p e t i t i o n limits the variety a n d yield of a n t i b o d i e s o b t a i n e d following i m m u n i z a t i o n with extracts c o n t a i n i n g m u l t i p l e antigens. A n t i g e n i c c o m p e t i t i o n is the i n h i b i t i o n of the i m m u n e response to one antigen or antigenic d e t e r m i n a n t c a u s e d b y the a d m i n i s t r a t i o n of a n o t h e r antigen or d e t e r m i n a n t . T h e term was i n t r o d u c e d b y L e o n o r Michaelis (1904) in the context of raising a n t i b o d i e s to serum proteins. M i c h a e l i s ' s original o b s e r v a t i o n of c o m p e t i t i o n b e t w e e n globulin a n d a l b u m i n have b e e n r e e x a m i n e d with some interesting results with respect to T cell function, T cell-B cell c o o p e r a t i o n a n d the role of c o m p l e t e F r e u n d ' s a d j u v a n t ( F e l d m a n n a n d Diener, 1970; F e l d m a n n a n d Nossal, 1972; R o w l e y et al., 1973; Taussig, 1973; M o z e s et al., 1974; Dintzis et al., 1976, 1982; A b b a s a n d Klaus, 1978). W e d e s c r i b e here an a t t e m p t to avoid antigenic c o m p e t i t i o n b y r e m o v i n g s t r o n g i m m u n o g e n s f r o m extracts a n d injecting the r e m a i n d e r . This ' c a s c a d e ' 0022-1759/84/$03.00 © 1984 Elsevier Science Publishers B.V.
246 method is frequently used in preparing monoclonal antibodies against unknown mixtures of antigen, which may then be analysed by the monoclonal antibodies they engender. Monoclonal antibodies from the primary immunization step are applied to immunoadsorbent columns, which remove the corresponding antigens. The depleted mixture, enriched in the remaining antigens is injected again (Milstein et al., 1979). In the present work, polyvalent, polyspecific and precipitating antisera were used to prepare immunogens to produce more antibody in cascade fashion. Material and Methods
A n tigens Escherichia coli c600 (Strack and Cox, 1971) was grown in medium containing 1% Bacto Tryptone (Difco) and 0.5% NaC1 adjusted to pH 7. From cultures grown with aeration at 37°C to a density of 108 cells/ml, the organisms were harvested by centrifugation at 6000 rpm (Beckman Rotor SW40), washed twice in tricine buffer IV (Bio-Rad Laboratories) and disrupted in a Braun Melsungen glass bead homogenizer (4000 rpm at 4°C, glass bead diameter 0.12 mm) for 2 min. The extract was cleared by centrifugation at 15,000 rpm for 30 min and stored frozen at - 80°C after addition of 0.015 M sodium azide and 1% aprotinin. The protein concentration was 4 mg/ml. A ntisera Antisera against cell extracts and fractions were produced in rabbits as follows: 2 mg protein was injected subcutaneously at day 0 and again intramuscularly at day 7 and day 21. Following this, 5 mg protein was given subcutaneously at day 22 and intravenously at day 23. Only the first dose was given with complete Freund's adjuvant (CFA), the subsequent injections being given in 0.14 M NaC1. Antisera and antibody fractions were stored frozen at - 8 0 ° C after addition of 0.015 M sodium azide and 1% aprotinin. Separation of immunogenic proteins This was performed by 2 methods. Procedure L Proteins obtained by ammonium sulfate precipitation (Williams and Chase, 1967) were bound to CNBr-activated Sepharose 4B (Gonyea, 1976; Van Eijk and Van Noort, 1976). Some 60 mg IgG were bound to a 10 cm x 1.5 cm column a follows: CNBr-activated Sepharose 4B was washed and re-swollen with 1 mM HC1. The coupling buffer was NaCO 3 (0.1 M, pH 8.3) containing NaC1 (0.5 M). After coupling, remaining active groups were blocked with blocking agent (1 M ethanolamine). Excess adsorbed proteins were washed away with acetate buffer (0.5 M) containing NaC1 (0.5 M). Two milliliters antigen extract were applied and the proteins passing through were collected, concentrated and used for immunizing another rabbit. Procedure 11. Preparative CIE was performed in an agarose gel (100 mm x 200 mm x 5 mm) run at 10°C (tricine buffer IV, pH 8.6, 10 V/cm) for 8 h. The second gel contained 50% antiserum. Proteins reacting with the antiserum were precipitated and immobilized near the cathode. Non-reacting proteins migrated toward the anode
247
~iiii
Fig. 1. CIE analysis of Escherichia coil c600: extract was performed in tricine buffer IV at 5 V / c m for 150 min in the 1st dimension and 1 V / c m for 15 h in the 2nd dimension. Anode to the right for the first dimension and at the top for the 2nd dimension. A (top): precipitation arcs with the extract against an antiserum from one rabbit; B (bottom): against a mixture of antisera from 8 rabbits.
248
and were located in an area separate from that containing precipitation lines at the time the electrophoresis was stopped. The non-reacting proteins were eluted from the gel by homogenizing the corresponding part of the gel and removing the agarose by centrifugation. The supernatant was used for immunizing another rabbit. SDSpolyacrylamide gel electrophoresis of Escherichia coli proteins labeled with 14Clabeled amino acids and autoradiographs were performed as described by O'Farrell (1974). CIE was performed as described by Laurell (1965, 1972). Results
CIE results with an Escherichia coli extract against an antiserum produced by conventional immunization of one rabbit and against pooled antisera from 8 rabbits are shown in Fig. 1.
Fig. 2. Autoradiograph of SDS gel electrophoresis of 14C-labeled Escherichia coil proteins. Lane A shows precipitated proteins; lane B non-precipitated proteins, separated by preparative CIE.
249
Fig. 3. CIE of non-precipitated proteins (nplI) against an antiserum prepared against them (as CII).
Fig. 4. CIE with intermediate gel against E. coli cytoplasmic extract. The first gel contains antibodies obtained by primary immunization (as prim.), the second gel contains antibodies from the first cascade step (as CII).
250
Fig. 5. CIE of non-precipitatedproteins (np I) obtained in procedure I against antiserum from the first cascade step (as CI). Proteins 1-4 were not removed completelyby the separation procedure I, and are the same as peaks 1-4 in Fig. 1A. Precipitated proteins (labeled with t4C-labeled amino acids) were recovered from the gel by cutting out the precipitation area. The agarose gel containing the precipitated proteins was mixed with an equal volume of O'Farrell sample buffer, boiled for 5 min and applied to the SDS gel. Fig. 2 compares the autoradiographs of precipitated and non-precipitated 14Clabeled Escherichia coli cytoplasmic proteins separated by preparative CIE against a primary antiserum. Immunization with the proteins in lane B (Fig. 2) elicited antibody production against them (Fig. 3). CIE with an intermediate gel was performed to demonstrate the difference between a primary antiserum and an antiserum of the first cascade step (Fig. 4). Separation of non-precipitated from precipitated proteins was according to procedure II. This was preferred to procedure I since the latter (Fig. 5) did not completely remove the strong immunogens.
Discussion
The method described here of improving limited immune responses against extracts containing multiple antigens should be of general use (e.g. for plant extracts and pollen extracts). Cascade immunization elicits antibodies not produced by conventional immunization procedures. The number of antigens in the E. coli extract
251 revealed by C I E was increased from the 2 0 - 3 0 obtained on primary immunization to 6 0 - 1 0 0 by cascade immunization. A further advantage of this technique is that antisera produced at different stages of the cascade do not share antibodies of the same specificity. Antibodies against high concentration proteins in the extract are present only in the antisera of the first and possibly second cascade steps, whereas antisera from subsequent cascade steps contain antibodies against weak immunogens or immunogens present in low concentration. The use of cascade antisera in parallel avoids the difficulty of heavy precipitation lines obscuring weak ones. The relative inefficiency of the i m m u n o a d s o r b e n t technique c o m p a r e d with preparative C I E was p r o b a b l y due to overloading the column with antigens present at high concentrations. Some antibodies obtained at the first immunization step seem to have lower affinities and successful removal of the corresponding antigens m a y require repeated i m m u n o a d s o r p t i o n resulting in very dilute antigen solutions. Removal of antigens by preparative C I E is a better method.
Acknowledgements We thank Prof. Dr. Strack for helpful discussions.
References Abbas, A.K. and G.G. Klaus, 1978, Eur. J. Immunol. 8, 217. Dintzis, H.M., R.Z. Dintzis and B. Vogelstein, 1976, Proc. Natl. Acad. Sci. U.S.A. 73, 3671. Dintzis, R.Z., B. Vogelstein and H.M. Dintzis, 1982, Proc. Natl. Acad. Sci. U.S.A. 79, 884. Feldmann, M. and E. Diener, 1970, J. Exp. Med. 131, 247. Feldmann, M. and G.J. Nossal, 1972, Transplant. Rev. 13, 3. Gonyea, L.M., 1976, Clin. Chem. 23, 234. Laurell, C.B., 1965, Anal. Biochem. 10, 358. Laurell, C.B., 1972, Scand. J. Clin. Lab. Invest. 29, 124. Michaelis, L., 1904, Dtsch. Med. Wochenschr. 30, 1240. Milstein, C., G. Galfre and D.S. Secher, 1979, Ciba Found. Symp. no. 66, (Elsevier, Amsterdam) p. 251. Mozes, E., M. Sela and M.J. Taussig, 1974, Immunology 27, 641. O'Farrell, P.H., 1974, J. Biol. Chem. 250, 4007. Rowley, D.A., F.W. Fitch, F.P. Stuart, H. K6hler and H. Consenza, 1973, Science 181, 1133. Strack, H.B. and J.H. Cox, 1971, Virology 43, 719. Taussig, M.J., 1973, Curt. Top. Microbiol. Immunol. 60, 125. Van Eijk, H.G. and W.L. Van Noort, 1976, Eur. J. Bioehem. 54, 411. Williams, C. and M. Chase (eds.), 1967, Methods in Immunology and Immunochemistry, No. I (Academic Press, New York) pp. 315-321.