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Hapten-sandwich labeling. IV. Improved procedures and non-cross-reacting hapten reagents for double-labeling cell surface antigens
Journal of ImmunologicaI Methods, 25 (1979) 283--289
283
© Elsevier/North-Holland Biomedical Press
Short communication HAPTEN-SANDWICH LABELING. IV. IMPROVED PROCEDURES AND NON-CROSS-REACTING HAPTEN REAGENTS FOR DOUBLE-LABELING CELL SURFACE ANTIGENS
ELLEN F. WALLACE and LEON WOFSY
Department of Bacteriology and Immunology, University of California, Berkeley, CA 94720, U.S.A. (Received 2 October 1978, accepted 30 October 1978)
New procedures are presented for preparation of hapten-antibody conjugates with the bifunctional amidinating reagent methyl-p-hydroxybenzimidate (HB). Conjugates with improved solubility are effective for hapten-sandwich labeling of cell surface antigens with high sensitivity and specificity. Several non-cross-reacting hapten-antihapten antibody systems are described which are well-suited for simultaneous labeling of different surface antigens.
INTRODUCTION
The use of bifunctional amidinating reagents to couple haptens to antibodies permits hapten-sandwich labeling of cell surface antigens with a degree of amplification equal to or greater than that obtained with standard sandwich procedures (Cammisuli and Wofsy, 1976). The hapten-sandwich technique is suited particularly to the labeling of alloantigens and to doublelabeling where high sensitivity is required, since in these situations the conventional indirect method which uses cross-species anti-Ig may be subject to serious limitations or altogether ruled out. Methods and applications of hapten-sandwich labeling of cell-surface antigens have recently been reviewed (Wofsy et al., 1978). We describe here an improved procedure for use of the amidinating reagent methyl-p-hydroxybenzimidate (HB) to couple azophenyl haptens to antibodies. We report also the selection of additional non-cross-reacting haptens to simplify double-labeling procedures. Our aim has been to overcome more effectively two difficulties encountered in earlier work and previously discussed (Cammisuli and Wofsy, 1976): (1) the original procedure for preparing hapten-amidine antibody conjugates with HB often yielded modified immunoglobulins of poor solubility; and (2) even extremely weak cross-reactions between antibodies to two haptens (e.g., p-azobenzoate and p-azobenzenearsonate) can be revealed in hapten-sandwich labeling of cellsurface antigens; therefore, such antihapten antibodies should not be paired
284 for use in double-labeling experiments. The changes in reagent preparation presented here are small, b u t they result in significantly increased conjugate solubility. The newly employed haptens are p-azobenzoyl glutamate (glut) and p-azobenzoyl glycine (gly), either of which may be coupled to antibodies by reaction with HB. Neither anti-glut nor anti-gly antibodies crossreact with anti-p-azobenzene arsonate (anti-ars), the antihapten antibody used in most of our hapten-sandwich labeling studies to date. MATERIALS AND METHODS
Haptens and reagents p-Aminobenzoyl glutamic acid and p-aminobenzoyl glycine were obtained from ICN Pharmaceuticals, Inc., Plainview, NY, or Sigma, St. Louis, MO. HB hydrochloride was synthesized as before (Cammisuli and Wofsy, 1976) or purchased from Pierce Chemical Co., Rockford, IL. Other haptens and chemical reagents were obtained or prepared as before (Cammisuli and Wofsy, 1976).
Antibodies Rabbit anti-glut and anti-gly antibodies were obtained by immunization with azo-conjugates of keyhole limpet hemocyanin (KLH) and purified by Sepharose affinity chromatography. All procedures were exactly analogous to those used previously to obtain purified rabbit anti-ars antibodies (Wofsy et al., 1974) and resulted in comparable yields of antihapten antibodies. Rabbit anti-mouse brain (anti-MBr) (Golub, 1971) was extensively absorbed with mouse liver, marrow, and red cells and the Thy-1 negative lymphosarcoma Raw 8 (Ralph et al., 1974) until it was specific for thymocytes and T cells. Goat anti-MBr, similarly absorbed, was a gift from Dr. R.I. Mishell (Univ. of Calif., Berkeley). Goat anti-mouse Ig (anti-MIg) was prepared against IgG2a(K) myeloma X5563 (Potter, 1972). Anti-Ia k and Fabanti-Ia k alloantibodies were the same preparations used in another study (Wofsy et al., 1977).
Revised procedure for preparing hapten-HB-antibody conjugates As in our original procedure, diazoniumphenyl hapten is reacted with HB and, after excess diazonium reagent is quenched, the resulting solution is used to amidinate protein. The essential revision in our new protocol is to increase substantially the ratio of diazoniumphenyl hapten to HB, from 1.5 to 5. The object is to eliminate residual free HB (i.e., HB that has n o t been coupled in azo linkage to hapten), since direct amidination with even a few free HB groups renders immunoglobulin insoluble (Cammisuli and Wofsy, 1976). Because of the greater solubility of conjugates prepared by the new procedure, it has been possible to alter for convenience a number of the steps in their purification and subsequent use. (a) Diazonium reagent. To prepare 12 ml of a 0.25 M diazonium solution,
285 dissolve 3 mmole aminophenyl hapten in 9 ml 1 N HC1; at 0°C, with stirring, add 3 mmole sodium nitrite in 3 ml H20. After 30 min, the diazonium reagent may be reacted with HB or aliquoted and stored for several months at --20 ° C. (b) Hapten-azo-HB. To prepare 20 ml of a stock solution of hapten-azoHB, 0.03 M in initial concentration of HB, add 0.6 mmole HB hydrochloride to 3 ml 0.34 M sodium borate buffer adjusted to pH 9.8. (This prevents significant hydrolysis of the HB which would occur with exposure to very acidic conditions.) Add to the mixture, in portions, with stirring at 0°C, 12 ml 0.25 M diazoniumphenyl hapten, maintaining pH at 9.2 + 0.2 with 5 M NaOH. When the addition is complete (about 20 min), allow the reaction to come to room temperature and to continue for a total of 2 h, maintaining pH 9.2 + 0.2 throughout. Add 4 mmole solid imidazole, with stirring, and allow the reagent to stand for 1 h more to quench residual diazonium. Adjust the pH to 8.6 with 2 N HC1 and dilute to a final volume of 20 ml. This reagent may be aliquoted and stored at --20°C for use over a 2 week period, after which unused aliquots should be discarded. (c) Hapten-antibody conjugates. For modification of antibody, 2 vol of the hapten-azo-HB solution (0.03 M in initial HB) are mixed with 1 vol of a 5--20 mg/ml solution of DEAE-purified Ig in 0.34 M borate, pH 8.6, and allowed to react for 15--20 h at room temperature. The modified antibody is concentrated by precipitation at 0°C in 45% saturated a m m o n i u m sulfate (SAS), centrifugation and dissolution in a minim u m volume of deionized water. The reddish-black solution is passed over a Bio-Gel P-100 or P-60 column with 0.017 M sodium borate buffer, pH 8.0, and the purified, orange colored hapten-antibody conjugate is collected at the front. The conjugate solution may be aliquoted, frozen and stored at --20 ° C, or the modified antibody may be precipitated and stored under 45% SAS.
Labeling cell populations Before use, hapten-antibody conjugate solutions in 0.017 M borate, pH 8.0, were adjusted for isotonicity and pH by addition of 0.25 vol of 5 X concentrated phosphate-buffered saline (PBS) so that the final concentration was 0.01 M sodium phosphate, 0.85% NaC1, pH 7.3. Conjugate preparations maintained under 45% SAS were dissolved after centrifugation in distilled water and dialyzed against the 0.017 M borate buffer, pH 8.0, or directly against PBS. Procedures for preparation and fluorescence labeling of mouse spleen cell suspensions were essentially as described previously (Wofsy et al., 1974; Cammisuli and Wofsy, 1976}. Cells were obtained from spleens of unimmunized C3H/diSN, C57BL/6 or BALB/c mice of either sex, age 3--7 months. All antibodies were centrifuged for 10 min at 100,000 X g in a Beckman Airfuge within 2 h of use in labeling procedures. In double-labeling experiments, both first layer reagents were added to cells simultaneously, as
286 were b o t h second layer a n t i h a p t e n reagents a f t e r the intervening washes. F l u o r e s c e n c e was observed in a Zeiss m i c r o s c o p e with Ploem illumination and a p p r o p r i a t e filters. RESULTS
Conjugate properties Using our revised p r o c e d u r e for h a p t e n c o n j u g a t i o n to i m m u n o g l o b u l i n , the following conjugates were p r e p a r e d : goat anti-MIg m o d i f i e d with glut-, gly-, ars-, b e n z o a t e - ( b e n z - ) , or p h e n y l @ l a c t o s i d e - ( l a c - ) HB; ars-HB and lacHB rabbit anti-MBr; glut-HB goat anti-MBr; ars-HB m o u s e anti-Ia k, ars-HB and lac-HB m o u s e Fab anti-Ia k. In a d d i t i o n m o u s e anti-5 chain antibodies derived f r o m the h y b r i d o m a s 11-6.3 (anti-Ig5 b a l l o t y p e ) and 10-4.22 (antiIg5 a a l l o t y p e ) (Oi et al., 1978) were m o d i f i e d with ars-HB in o u r l a b o r a t o r y b y Dr. William C. Raschke (Salk Institute, La Jolla, CA) and Dr. J o h n R. N o r t h (University of Bristol Medical School, Bristol, U.K.). With the exception of the tac-HB conjugates with whole i m m u n o g l o b u l i n , all p r e p a r a t i o n s were soluble in PBS at or above c o n c e n t r a t i o n s used for labeling cells. Most samples, at c o n c e n t r a t i o n s o f a b o u t 2 mg/ml, could be dialyzed against PBS at 4°C for h o u r s or, in some cases, days w i t h o u t substantial precipitation. While c o n j u g a t e solubility varied s o m e w h a t in d i f f e r e n t preparations, ars-HB and glut-HB conjugates were m o r e soluble than gly-HB and benz-HB conjugates, while lac-HB conjugates were satisfactory o n l y with Fab. We did n o t a t t e m p t q u a n t i t a t i v e c o m p a r i s o n s o f the e f f i c i e n c y as labeling reagents o f conjugates p r e p a r e d b y the old and new procedures. H o w e v e r , in no case did the sensitivity and specificity o f labeling achieved with the new
|
Fig. 1. Double-labeling of mouse spleen cells to distinguish B and T lymphoeytes. Cells were treated with glut-HB anti-MIg and ars-HB anti-MBr, washed, and then labeled with Fl-anti-glut and Rh-anti-ars. The same field was photographed in phase (left) and, with appropriate filters, for fluoreseein (center) and rhodamine (right) fluorescence.
287 : ~1%
~ , ~i~, ~ , ~ ,,~ ~
~" • i
Fig. 2. Double-labeling to detect expression of Ia k antigens on C3H/diSN mouse T cell blasts. Spleen cells were depleted of B lymphocytes and cultured for 2 days with concanavalin A. Harvested cells were treated with ars-HB-Fab anti-Ia k and glut-HB-anti-MBr, washed, and then labeled with Fl-anti-ars and Rh-anti-glut. The photograph shows a field of cells in phase, left; fluorescein, center; rhodamine, right. Some cells with T cell label, also label for Ia k.
conjugates a p p e a r l o w e r t h a n with t h e p r e p a r a t i o n s used in the earlier studies. Bright f l u o r e s c e n c e labeling was achieved w i t h the m o d i f i e d m o u s e alloantibodies. Ars-HB anti-5-Ig5 a labeled 4 5 - - 5 0 % of B A L B / c (Ig5 a) spleen l y m p h o c y t e s while failing to label C 5 7 B L / 6 (Ig5 b) l y m p h o c y t e s ; the reverse result was o b t a i n e d with ars-HB anti-6-Ig5 b.
Double labeling In tests f o r a n t i h a p t e n a n t i b o d y cross-reactivity, spleen cells were labeled with e i t h e r glut-HB-, gly-HB-, or ars-HB-anti-MIg, w a s h e d twice and t h e n t r e a t e d with m a t c h i n g or n o n - m a t c h i n g F l - a n t i h a p t e n a n t i b o d i e s . In each case w h e n the c o r r e s p o n d i n g s e c o n d - l a y e r a n t i h a p t e n a n t i b o d y was used, 4 5 - - 5 0 % o f the l y m p h o c y t e s were stained. L a b e l i n g did n o t o c c u r witb unmatched antihapten antibodies. D o u b l e - l a b e l i n g e x p e r i m e n t s were t h e n p e r f o r m e d with glut-HB r a b b i t anti-MIg and ars-HB r a b b i t anti-MBr, w i t h F l - a n t i - g l u t and r h o d a m i n e (Rh)anti-ars as s e c o n d l a y e r reagents. With m o u s e spleen l y m p h o c y t e s , 44% disp l a y e d the F I - and 34% the Rh-label; less t h a n 2% of t h e cells s h o w e d b o t h labels. Fig. 1 shows cells stained b y this p r o c e d u r e , clearly distinguishing T and B cells. Similar results were o b t a i n e d with gly-HB-anti-MIg and ars-HBanti-MBr: 50% o f the cells stained as B cells, 30% at T cells; less t h a n 3% s h o w e d b o t h labels. In c o n n e c t i o n with a s e p a r a t e s t u d y o f Ia e x p r e s s i o n on c o n c a n a v a l i n - A s t i m u l a t e d T cell blasts, a r s - H B - F a b anti-Ia k and glut-HB-anti-MBr were paired. Fig. 2 s h o w s labeled T cells, s o m e o f w h i c h are also labeled f o r Ia k. This s t u d y will be d e s c r i b e d in full s e p a r a t e l y (C. H e n r y , B. M a y h e w Doe, J. K i m u r a , J.R. N o r t h and L. Wofsy, m a n u s c r i p t in p r e p a r a t i o n ) . DISCUSSION H a p t e n - s a n d w i c h m e t h o d s , especially f o r d o u b l e - l a b e l i n g cell surface antigens, are m a d e m o r e c o n v e n i e n t and versatile as a result of: (1) i m p r o v e d p r o c e d u r e s t h a t yield m o r e soluble h a p t e n - H B - a n t i b o d y c o n j u g a t e s , and (2)
288
the availability of several anti-hapten antibodies that do n o t cross-react in double-labeling experiments. The problem of preparing soluble, non-cross-reacting conjl,gates for hapten-sandwich double-labeling was previously accomplished in a more elaborate way. Resort was made to another bifunctional amidinating reagent, methyl-3,5-dihydroxybenzimidate (DHB), to prepare conjugates that were more soluble, b u t otherwise less satisfactory than those coupled with HB (Cammisuli and Wofsy, 1976). Also, hapten-HB Fab conjugates were successfully used since these were more soluble than conjugates made with whole antibody (Cammisuli and Wofsy, 1976; Wofsy et al., 1977). Double-labeling was performed with ars-HB- and lac-DHB conjugates, b u t efforts to pair ars hapten-sandwich reagents with benzoate- or benzenesulfonate conjugates revealed an unacceptable level of cross-reaction. Non-specific cross-labeling was observed when oppositely charged ars- and p-trimethylammonium aniline conjugates were paired for double-labeling (Wofsy et al., 1978). The study reported here shows that ars-HB, glut-HB, and gly-HB conjugates can be used in any combination with complete discrimination for simultaneous labeling of multiple cell surface antigens. A single bifunctional amidinating reagent, HB, and uniform preparative procedures are used for each of the conjugates. The cost and availability of the glut- and glyreagents, as well as the effectiveness of the immunization and antibody purification procedures, make these hapten systems as practical as the well established anti-ars system. The accessibility of at least 3 non-cross-reacting hapten combinations is of value, since hapten-sandwich procedures are not limited to fluorescence labeling of antigens, but can also be used to attach electron microscopic or radioactive markers as well as for receptor-specific fractionation of cells (Wofsy et al., 1978). In this report, we have cited additional examples of the use of the haptensandwich m e t h o d to obtain highly amplified, specific labeling of ailoantigens. This would appear to be of particular value in connection with the use of hybridoma-derived monoclonal alloantibodies, which may be of low affinity and are restricted to the recognition of a single antigenic determinant. ACKNOWLEDGEMENTS
This work was supported by National Institute of Health Grants CA24436 and CA-9179 and by The Miller Institute for Basic Research in Science, Berkeley, CA. REFERENCES Cammisuli, S. and L. Wofsy, 1976, J. Immunol. 117, 1695. Golub, E.S., 1971, Cell. Immunol, 2, 353. Oi, V.T., P.P. Jones, J.W. Goding, L.A. Herzenberg and L.A. Herzenberg, 1978, in: Current Topics in Microbiology and Immunology, Vol. 81, eds. F. Melchers, M. Potter and
289 N. Warner (Springer, New York) pp. 115--129. Potter, M., 1972, Physiol. Rev. 52, 631. Ralph, P., J. Nakoinz and W.C. Raschke, 1974, Biochem. Biophys. Res. Commun. 61, 1268. Wofsy, L., P.C. Baker, K. Thompson, J. Goodman, J. Kimura and C. Henry, 1974, J. Exp. Med. 140, 523. Wofsy, L., H.O. McDevitt and C. Henry, 1977, J. Immunol. 119, 61. Wofsy, L., C. Henry and S. Cammisuli, 1978, in: Contemporary Topics in Molecular Immunology, Vol. 7, eds. F.P. Inman and R.A. Reisfeld (Plenum Press, New York) pp. 215--237.
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© Elsevier/North-Holland Biomedical Press
Short communication HAPTEN-SANDWICH LABELING. IV. IMPROVED PROCEDURES AND NON-CROSS-REACTING HAPTEN REAGENTS FOR DOUBLE-LABELING CELL SURFACE ANTIGENS
ELLEN F. WALLACE and LEON WOFSY
Department of Bacteriology and Immunology, University of California, Berkeley, CA 94720, U.S.A. (Received 2 October 1978, accepted 30 October 1978)
New procedures are presented for preparation of hapten-antibody conjugates with the bifunctional amidinating reagent methyl-p-hydroxybenzimidate (HB). Conjugates with improved solubility are effective for hapten-sandwich labeling of cell surface antigens with high sensitivity and specificity. Several non-cross-reacting hapten-antihapten antibody systems are described which are well-suited for simultaneous labeling of different surface antigens.
INTRODUCTION
The use of bifunctional amidinating reagents to couple haptens to antibodies permits hapten-sandwich labeling of cell surface antigens with a degree of amplification equal to or greater than that obtained with standard sandwich procedures (Cammisuli and Wofsy, 1976). The hapten-sandwich technique is suited particularly to the labeling of alloantigens and to doublelabeling where high sensitivity is required, since in these situations the conventional indirect method which uses cross-species anti-Ig may be subject to serious limitations or altogether ruled out. Methods and applications of hapten-sandwich labeling of cell-surface antigens have recently been reviewed (Wofsy et al., 1978). We describe here an improved procedure for use of the amidinating reagent methyl-p-hydroxybenzimidate (HB) to couple azophenyl haptens to antibodies. We report also the selection of additional non-cross-reacting haptens to simplify double-labeling procedures. Our aim has been to overcome more effectively two difficulties encountered in earlier work and previously discussed (Cammisuli and Wofsy, 1976): (1) the original procedure for preparing hapten-amidine antibody conjugates with HB often yielded modified immunoglobulins of poor solubility; and (2) even extremely weak cross-reactions between antibodies to two haptens (e.g., p-azobenzoate and p-azobenzenearsonate) can be revealed in hapten-sandwich labeling of cellsurface antigens; therefore, such antihapten antibodies should not be paired
284 for use in double-labeling experiments. The changes in reagent preparation presented here are small, b u t they result in significantly increased conjugate solubility. The newly employed haptens are p-azobenzoyl glutamate (glut) and p-azobenzoyl glycine (gly), either of which may be coupled to antibodies by reaction with HB. Neither anti-glut nor anti-gly antibodies crossreact with anti-p-azobenzene arsonate (anti-ars), the antihapten antibody used in most of our hapten-sandwich labeling studies to date. MATERIALS AND METHODS
Haptens and reagents p-Aminobenzoyl glutamic acid and p-aminobenzoyl glycine were obtained from ICN Pharmaceuticals, Inc., Plainview, NY, or Sigma, St. Louis, MO. HB hydrochloride was synthesized as before (Cammisuli and Wofsy, 1976) or purchased from Pierce Chemical Co., Rockford, IL. Other haptens and chemical reagents were obtained or prepared as before (Cammisuli and Wofsy, 1976).
Antibodies Rabbit anti-glut and anti-gly antibodies were obtained by immunization with azo-conjugates of keyhole limpet hemocyanin (KLH) and purified by Sepharose affinity chromatography. All procedures were exactly analogous to those used previously to obtain purified rabbit anti-ars antibodies (Wofsy et al., 1974) and resulted in comparable yields of antihapten antibodies. Rabbit anti-mouse brain (anti-MBr) (Golub, 1971) was extensively absorbed with mouse liver, marrow, and red cells and the Thy-1 negative lymphosarcoma Raw 8 (Ralph et al., 1974) until it was specific for thymocytes and T cells. Goat anti-MBr, similarly absorbed, was a gift from Dr. R.I. Mishell (Univ. of Calif., Berkeley). Goat anti-mouse Ig (anti-MIg) was prepared against IgG2a(K) myeloma X5563 (Potter, 1972). Anti-Ia k and Fabanti-Ia k alloantibodies were the same preparations used in another study (Wofsy et al., 1977).
Revised procedure for preparing hapten-HB-antibody conjugates As in our original procedure, diazoniumphenyl hapten is reacted with HB and, after excess diazonium reagent is quenched, the resulting solution is used to amidinate protein. The essential revision in our new protocol is to increase substantially the ratio of diazoniumphenyl hapten to HB, from 1.5 to 5. The object is to eliminate residual free HB (i.e., HB that has n o t been coupled in azo linkage to hapten), since direct amidination with even a few free HB groups renders immunoglobulin insoluble (Cammisuli and Wofsy, 1976). Because of the greater solubility of conjugates prepared by the new procedure, it has been possible to alter for convenience a number of the steps in their purification and subsequent use. (a) Diazonium reagent. To prepare 12 ml of a 0.25 M diazonium solution,
285 dissolve 3 mmole aminophenyl hapten in 9 ml 1 N HC1; at 0°C, with stirring, add 3 mmole sodium nitrite in 3 ml H20. After 30 min, the diazonium reagent may be reacted with HB or aliquoted and stored for several months at --20 ° C. (b) Hapten-azo-HB. To prepare 20 ml of a stock solution of hapten-azoHB, 0.03 M in initial concentration of HB, add 0.6 mmole HB hydrochloride to 3 ml 0.34 M sodium borate buffer adjusted to pH 9.8. (This prevents significant hydrolysis of the HB which would occur with exposure to very acidic conditions.) Add to the mixture, in portions, with stirring at 0°C, 12 ml 0.25 M diazoniumphenyl hapten, maintaining pH at 9.2 + 0.2 with 5 M NaOH. When the addition is complete (about 20 min), allow the reaction to come to room temperature and to continue for a total of 2 h, maintaining pH 9.2 + 0.2 throughout. Add 4 mmole solid imidazole, with stirring, and allow the reagent to stand for 1 h more to quench residual diazonium. Adjust the pH to 8.6 with 2 N HC1 and dilute to a final volume of 20 ml. This reagent may be aliquoted and stored at --20°C for use over a 2 week period, after which unused aliquots should be discarded. (c) Hapten-antibody conjugates. For modification of antibody, 2 vol of the hapten-azo-HB solution (0.03 M in initial HB) are mixed with 1 vol of a 5--20 mg/ml solution of DEAE-purified Ig in 0.34 M borate, pH 8.6, and allowed to react for 15--20 h at room temperature. The modified antibody is concentrated by precipitation at 0°C in 45% saturated a m m o n i u m sulfate (SAS), centrifugation and dissolution in a minim u m volume of deionized water. The reddish-black solution is passed over a Bio-Gel P-100 or P-60 column with 0.017 M sodium borate buffer, pH 8.0, and the purified, orange colored hapten-antibody conjugate is collected at the front. The conjugate solution may be aliquoted, frozen and stored at --20 ° C, or the modified antibody may be precipitated and stored under 45% SAS.
Labeling cell populations Before use, hapten-antibody conjugate solutions in 0.017 M borate, pH 8.0, were adjusted for isotonicity and pH by addition of 0.25 vol of 5 X concentrated phosphate-buffered saline (PBS) so that the final concentration was 0.01 M sodium phosphate, 0.85% NaC1, pH 7.3. Conjugate preparations maintained under 45% SAS were dissolved after centrifugation in distilled water and dialyzed against the 0.017 M borate buffer, pH 8.0, or directly against PBS. Procedures for preparation and fluorescence labeling of mouse spleen cell suspensions were essentially as described previously (Wofsy et al., 1974; Cammisuli and Wofsy, 1976}. Cells were obtained from spleens of unimmunized C3H/diSN, C57BL/6 or BALB/c mice of either sex, age 3--7 months. All antibodies were centrifuged for 10 min at 100,000 X g in a Beckman Airfuge within 2 h of use in labeling procedures. In double-labeling experiments, both first layer reagents were added to cells simultaneously, as
286 were b o t h second layer a n t i h a p t e n reagents a f t e r the intervening washes. F l u o r e s c e n c e was observed in a Zeiss m i c r o s c o p e with Ploem illumination and a p p r o p r i a t e filters. RESULTS
Conjugate properties Using our revised p r o c e d u r e for h a p t e n c o n j u g a t i o n to i m m u n o g l o b u l i n , the following conjugates were p r e p a r e d : goat anti-MIg m o d i f i e d with glut-, gly-, ars-, b e n z o a t e - ( b e n z - ) , or p h e n y l @ l a c t o s i d e - ( l a c - ) HB; ars-HB and lacHB rabbit anti-MBr; glut-HB goat anti-MBr; ars-HB m o u s e anti-Ia k, ars-HB and lac-HB m o u s e Fab anti-Ia k. In a d d i t i o n m o u s e anti-5 chain antibodies derived f r o m the h y b r i d o m a s 11-6.3 (anti-Ig5 b a l l o t y p e ) and 10-4.22 (antiIg5 a a l l o t y p e ) (Oi et al., 1978) were m o d i f i e d with ars-HB in o u r l a b o r a t o r y b y Dr. William C. Raschke (Salk Institute, La Jolla, CA) and Dr. J o h n R. N o r t h (University of Bristol Medical School, Bristol, U.K.). With the exception of the tac-HB conjugates with whole i m m u n o g l o b u l i n , all p r e p a r a t i o n s were soluble in PBS at or above c o n c e n t r a t i o n s used for labeling cells. Most samples, at c o n c e n t r a t i o n s o f a b o u t 2 mg/ml, could be dialyzed against PBS at 4°C for h o u r s or, in some cases, days w i t h o u t substantial precipitation. While c o n j u g a t e solubility varied s o m e w h a t in d i f f e r e n t preparations, ars-HB and glut-HB conjugates were m o r e soluble than gly-HB and benz-HB conjugates, while lac-HB conjugates were satisfactory o n l y with Fab. We did n o t a t t e m p t q u a n t i t a t i v e c o m p a r i s o n s o f the e f f i c i e n c y as labeling reagents o f conjugates p r e p a r e d b y the old and new procedures. H o w e v e r , in no case did the sensitivity and specificity o f labeling achieved with the new
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Fig. 1. Double-labeling of mouse spleen cells to distinguish B and T lymphoeytes. Cells were treated with glut-HB anti-MIg and ars-HB anti-MBr, washed, and then labeled with Fl-anti-glut and Rh-anti-ars. The same field was photographed in phase (left) and, with appropriate filters, for fluoreseein (center) and rhodamine (right) fluorescence.
287 : ~1%
~ , ~i~, ~ , ~ ,,~ ~
~" • i
Fig. 2. Double-labeling to detect expression of Ia k antigens on C3H/diSN mouse T cell blasts. Spleen cells were depleted of B lymphocytes and cultured for 2 days with concanavalin A. Harvested cells were treated with ars-HB-Fab anti-Ia k and glut-HB-anti-MBr, washed, and then labeled with Fl-anti-ars and Rh-anti-glut. The photograph shows a field of cells in phase, left; fluorescein, center; rhodamine, right. Some cells with T cell label, also label for Ia k.
conjugates a p p e a r l o w e r t h a n with t h e p r e p a r a t i o n s used in the earlier studies. Bright f l u o r e s c e n c e labeling was achieved w i t h the m o d i f i e d m o u s e alloantibodies. Ars-HB anti-5-Ig5 a labeled 4 5 - - 5 0 % of B A L B / c (Ig5 a) spleen l y m p h o c y t e s while failing to label C 5 7 B L / 6 (Ig5 b) l y m p h o c y t e s ; the reverse result was o b t a i n e d with ars-HB anti-6-Ig5 b.
Double labeling In tests f o r a n t i h a p t e n a n t i b o d y cross-reactivity, spleen cells were labeled with e i t h e r glut-HB-, gly-HB-, or ars-HB-anti-MIg, w a s h e d twice and t h e n t r e a t e d with m a t c h i n g or n o n - m a t c h i n g F l - a n t i h a p t e n a n t i b o d i e s . In each case w h e n the c o r r e s p o n d i n g s e c o n d - l a y e r a n t i h a p t e n a n t i b o d y was used, 4 5 - - 5 0 % o f the l y m p h o c y t e s were stained. L a b e l i n g did n o t o c c u r witb unmatched antihapten antibodies. D o u b l e - l a b e l i n g e x p e r i m e n t s were t h e n p e r f o r m e d with glut-HB r a b b i t anti-MIg and ars-HB r a b b i t anti-MBr, w i t h F l - a n t i - g l u t and r h o d a m i n e (Rh)anti-ars as s e c o n d l a y e r reagents. With m o u s e spleen l y m p h o c y t e s , 44% disp l a y e d the F I - and 34% the Rh-label; less t h a n 2% of t h e cells s h o w e d b o t h labels. Fig. 1 shows cells stained b y this p r o c e d u r e , clearly distinguishing T and B cells. Similar results were o b t a i n e d with gly-HB-anti-MIg and ars-HBanti-MBr: 50% o f the cells stained as B cells, 30% at T cells; less t h a n 3% s h o w e d b o t h labels. In c o n n e c t i o n with a s e p a r a t e s t u d y o f Ia e x p r e s s i o n on c o n c a n a v a l i n - A s t i m u l a t e d T cell blasts, a r s - H B - F a b anti-Ia k and glut-HB-anti-MBr were paired. Fig. 2 s h o w s labeled T cells, s o m e o f w h i c h are also labeled f o r Ia k. This s t u d y will be d e s c r i b e d in full s e p a r a t e l y (C. H e n r y , B. M a y h e w Doe, J. K i m u r a , J.R. N o r t h and L. Wofsy, m a n u s c r i p t in p r e p a r a t i o n ) . DISCUSSION H a p t e n - s a n d w i c h m e t h o d s , especially f o r d o u b l e - l a b e l i n g cell surface antigens, are m a d e m o r e c o n v e n i e n t and versatile as a result of: (1) i m p r o v e d p r o c e d u r e s t h a t yield m o r e soluble h a p t e n - H B - a n t i b o d y c o n j u g a t e s , and (2)
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the availability of several anti-hapten antibodies that do n o t cross-react in double-labeling experiments. The problem of preparing soluble, non-cross-reacting conjl,gates for hapten-sandwich double-labeling was previously accomplished in a more elaborate way. Resort was made to another bifunctional amidinating reagent, methyl-3,5-dihydroxybenzimidate (DHB), to prepare conjugates that were more soluble, b u t otherwise less satisfactory than those coupled with HB (Cammisuli and Wofsy, 1976). Also, hapten-HB Fab conjugates were successfully used since these were more soluble than conjugates made with whole antibody (Cammisuli and Wofsy, 1976; Wofsy et al., 1977). Double-labeling was performed with ars-HB- and lac-DHB conjugates, b u t efforts to pair ars hapten-sandwich reagents with benzoate- or benzenesulfonate conjugates revealed an unacceptable level of cross-reaction. Non-specific cross-labeling was observed when oppositely charged ars- and p-trimethylammonium aniline conjugates were paired for double-labeling (Wofsy et al., 1978). The study reported here shows that ars-HB, glut-HB, and gly-HB conjugates can be used in any combination with complete discrimination for simultaneous labeling of multiple cell surface antigens. A single bifunctional amidinating reagent, HB, and uniform preparative procedures are used for each of the conjugates. The cost and availability of the glut- and glyreagents, as well as the effectiveness of the immunization and antibody purification procedures, make these hapten systems as practical as the well established anti-ars system. The accessibility of at least 3 non-cross-reacting hapten combinations is of value, since hapten-sandwich procedures are not limited to fluorescence labeling of antigens, but can also be used to attach electron microscopic or radioactive markers as well as for receptor-specific fractionation of cells (Wofsy et al., 1978). In this report, we have cited additional examples of the use of the haptensandwich m e t h o d to obtain highly amplified, specific labeling of ailoantigens. This would appear to be of particular value in connection with the use of hybridoma-derived monoclonal alloantibodies, which may be of low affinity and are restricted to the recognition of a single antigenic determinant. ACKNOWLEDGEMENTS
This work was supported by National Institute of Health Grants CA24436 and CA-9179 and by The Miller Institute for Basic Research in Science, Berkeley, CA. REFERENCES Cammisuli, S. and L. Wofsy, 1976, J. Immunol. 117, 1695. Golub, E.S., 1971, Cell. Immunol, 2, 353. Oi, V.T., P.P. Jones, J.W. Goding, L.A. Herzenberg and L.A. Herzenberg, 1978, in: Current Topics in Microbiology and Immunology, Vol. 81, eds. F. Melchers, M. Potter and
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