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Production in ascites fluid of biosynthetically labelled monoclonal antibody to Theileria parva sporozoites
209
Journal of Immunological Methoak, 82 (1985) 209-214
Elsevier JIM 03588
Production in Ascites Fluid of Biosynthetically Labelled Monoclonal Antibody to Theileria parva Sporozoites Dirk A.E. Dobbelaere
1 and
Paul R. Spooner
International Laboratory for Research on Animal Diseases, P.O. Box 30709, Nairobi, Kenya
(Received 30 November 1984, accepted 30 April 1985)
A hybridoma cell line has been previously produced which secretes monoclonal antibodies able to neutralize sporozoites of Theileria paroa, the causative agent of East Coast fever of cattle. Cells from this line were injected intra-peritoneally into pristane-treated BALB/c mice. During the last 4 days of hybridoma cell growth, mice were given 4 daily intraperitoneal injections of a mixture of tritiated amino acids in order to biosynthetic&y radiolabel the monoclonal antibody being produced in ascites fluid. The specific activity of the antibody obtained was 100 mCi/rnmol. The labelled antibody was used to detect, by autoradiography, a surface coat antigen of T. paroa sporozoites in cryostat sections of Theileria-infected tick salivary glands. The method allows the preparation of large quantities of biosynthetically radiolabelled immunological probes for the detection of immunoreactive sites in biological specimens. Key words: ascites tumor - biosynthetic labeling - monoclonal antibody - radio -immunohistochemistry sporozoite
:
- Theileria paroa - tritium
Introduction The use of antibodies as immunological probes of high specificity for the detection and assay of substances in biological specimens was greatly enhanced by the development of hybridoma technology (KBhler and Milstein, 1975). Biosynthetitally labelled radioactive monoclonal antibodies have been used in radio-immunohistochemistry at the light microscopic and electron microscopic level (Cue110 et al., 1982), in studies on secreted immunoglobulins (Levy et al., 1980) and in various tests such as the reciprocal binding inhibition test for monoclonal antibodies (Carrel et al., 1981). Biosynthetically labelled monoclonal antibodies are normally obtained by adding appropriate radioactive amino acids to hybridoma culture fluids during antibody synthesis in vitro, but only pg/ml can be harvested by this method (Cue110 I
’ Present address: Kemforschungszentrum 3640, D-7500 Karlsruhe 1, F.R.G.
Karlsruhe, Institut fur Genetik und Toxikologie, Postfach
0022-1759/85/$03.30 0 1985 Elsevier Science Publishers B.V. (Biomedical Division)
210
and Milstein, 1981). We have investigated the possibility of producing larger amounts of biosynthetically labelled monoclonal antibody in ascites fluid. Recently we described the production of a monoclonal antibody which detected a sporozoite surface antigen of the tick-transmitted protozoan parasite Theileria parva (Dobbelaere et al., 1984). The radio-labelling of this monoclonal antibody in ascites fluid is described in the current paper, and its applicability in light microscopic immuno-autoradiography of T. parva sporozoite antigens is demonstrated. Materials and Methods Mice
BALB/c mice, 10-12 weeks old, reared at ILRAD small animal unit were used. Monoclonal antibody
Monoclonal antibodies against T. parva sporozoites were prepared as described by Dobbelaere et al. (1984), by fusing spleen cells, from mice immunized with crude freeze-thawed sporozoite preparations, with X63-Ag8.653 myeloma cells (Kearney et al., 1979) and cloning antibody-producing cells by limiting dilution (Oi and Herzenberg, 1980). The monoclonal antibody used in this study, MAbD1, is of the IgG 3 isotype and binds to protein A. MAbD1 recognizes an epitope of a sporozoite surface coat antigen and neutralizes T. parva sporozoite infectivity. Production of ascites tumours
BALB/c mice were injected intraperitoneally with 0.5 ml of pristane (2, 6, 10,14-tetramethylpentadecane, Aldrich Chemical Co., Gillingham, Dorset) 10 days before injection of the hybridoma cells (Hoogenraad et al., 1983). MAbDl-producing hybridoma cells were washed twice in Dulbecco's phosphate-buffered saline, pH 7.4 (PBS) and 5 x 1 0 6 cells (in 0.5 ml of PBS) were injected intraperitoneally into each of 25 mice. To produce biosynthetically labelled monoclonal antibody (3H-MAbD1) in ascites fluid, a mixture of 15 tritiated (3H) amino acids (code number TRK.440., The Radiochemical Centre, Amersham) was injected intraperitoneally into 4 of the 25 mice, once daily for 4 days starting on day 7 after inoculation of MAbD1 hybridoma cells. Each inoculum consisted of 0.5 mCi of the 3H-amino acid mixture, made isotonic by addition of 10 x strength Hanks' balanced salt solution (Gibco BioCuh, Paisley, Scotland); the volume of each inoculum was 0.55 ml. Appropriate safety precautions and isolation procedures were observed throughout. Mice were killed 24 h after the last injection of 3H-amino acids and ascites fluid was harvested by aspiration with a Pasteur pipette with the addition of 20 U / m l heparin (Novo, Copenhagen) to the aspirate to prevent clotting. Pooled unlabelled and pooled labelled ascites fluids were clarified by centrifugation at 2500 x g for 10 min. Labelled ascites fluid was dialysed 3 times in 2 litre vol. of 0.1 M phosphate buffer (pH 7.5) containing 0.5 M sodium chloride after which no more tritium could be detected in the dialysis fluid. The dialysate was dispensed into 1 ml and 50 /~1 aliquots and stored at -80°C.
211
Purification of MAbD1 3H-MAbD1 used for the determination of specific activity was purified using Protein A-Sepharose CL-4B (Pharmacia Fine Chemicals, Uppsala) following the method described by Sepp~il~i et al. (1981). Immunoglobulin concentrations were calculated from the optical density reading at 280 nm (extinction coefficient at 280 nm for 1 mg/ml = 1.4).
Tritium counts Samples were counted in scintillation fluid (Aquasol-2, New England Nuclear, Boston, MA) using a Packard liquid scintillation spectrometer (model 3330).
Immuno-autoradiography Immuno-autoradiography with 3H-MAbD1 was carried out on cryostat sections of salivary glands of Theileria-infected adult Rhipicephalus appendiculatus ticks by a method similar to that described by Dobbelaere et al. (1984). Five #m cryostat sections of salivary glands were placed on microscope slides and fixed with 0.25% (v/v) glutaraldehyde in distilled water for 5 min at room temperature, followed by a rinse in PBS containing 1% (w/v) bovine serum albumin (PBS/BSA). Slides were stored at - 8 0 ° C until use. To test 3H-MAbD1, sections were thawed at room temperature, delineated with nail varnish and incubated for 45 min with 20 #1 3H-MAbD1 ascites fluid diluted 1/500 (v/v) in PBS and then rinsed 3 times with PBS/BSA. Slides were then stained with Feulgen DNA-stain (Dobbelaere et al., 1984) to reveal the parasite in sections of infected salivary gland acini. Air-dried slides were dipped in Ilford L4 nuclear research emulsion (Ilford, Basildon, Essex), left to dry and exposed for 10-20 days at 4°C and subsequently developed in Kodak D 76 (Eastman Kodak, Rochester, NY). The sections were examined using a light microscope at 500 × magnification, for the presence of silver grains, indicating binding of 3H-MAbD1 over salivary gland acini containing sporozoite antigen.
Results
Ascites tumours and ascites fluid production An average of 3.5 ml of ascites fluid per mouse were collected. There were no significant differences in time for tumour development and quantity of ascites fluid collected, between mice injected with cells and 3H-amino acid mixture, and those injected with cells alone. In both groups, tumours became apparent around day 10; radiolabelled ascites fluid was harvested from 4 mice on day 12 and unlabelled ascites from 21 mice between day 11 and day 14. The amount of protein A-binding immunoglobulin was 4.4 mg/ml in radiolabelled ascites fluid and 5.1 mg/ml in unlabelled ascites fluid.
Specific activity of SH-MAbD1 In the dialysed labelled ascites fluid, over 99% of the 3H counts were precipitable in 20% (w/v) trichloroacetic acid and 23% of the 3H counts measured were in
212
Fig. 1. Autoradiographof a cryostat section(5 ~m) through a Theileriaparva infected tick salivarygland. Strong labelling can be observedover the infected acinus (left) but not over the uninfected acinus (right). (Scale bar = 20 ~m; the autoradiographwas exposed for 20 days). protein A-binding immunoglobulin. The specific activity of protein A-purified 3H-MAbD1 was approximately 100 m C i / m m o l (3.7 G B q / m m o l ) .
Immuno-autoradiography Pooled 3H-MAbD1 ascites fluid showed the same specificity for T. parva sporozoite a n t i g e n as unlabelled MAbD1 ascites fluid. Autoradiographs of Theileria-infected salivary gland sections incubated with 3H-MAbD1 revealed clearly defined selective binding of the biosynthetically labelled 3H-MAbD1 to the infected acinar cells (Fig. 1). Background labelling was lower than on autoradiographs with unlabelled MAbD1 and 125I-labelled anti-mouse immunoglobulin as second antibody (not shown). Exposure for 10 days was sufficient for the detection of 3HMAbD1 binding to parasite antigen, but labelling was more pronounced with no significant increase in background after 20 days exposure.
Discussion These results demonstrate that it was possible to produce biosynthetically radiolabelled monoclonal antibody in ascites fluid, which could be used for the detection of immunoreactive sites. Biosynthetic labelling with a mixture of 3H-amino
213
acids did not noticeably affect binding or specificity of MAbD1 to Theileria sporozoites as demonstrated by immuno-autoradiography. Using tritiated MAbD1, background labelling was considerably lower than that observed by Dobbelaere et al. (1984) in autoradiographs with unlabelled MAbD1 and 125I-labelled anti-mouse immunoglobulin as second antibody. The advantage of biosynthetic labelling over chemical/enzymatic labelling have been discussed (Cuello and Milstein, 1981). One of the most important dangers of chemical/enzymatic labelling of antibodies is the possibility of causing configurational changes in the antigen-antibody combining site that can result in reduced or abolished affinity for the epitope. This is avoided by biosynthetic labelling. The usual methods by which hybridoma-secreted antibodies are biosynthetically labelled in vitro, depend on incorporation of radiolabelled amino acids (e.g., [14C]or [3H]-lysine) added to a hybridoma culture medium which is free of the cold amino acid (e.g., lysine-free medium). By this method only /~g amounts/ml of labelled immunoglobulin can be harvested from the culture supernatant. The ascites tumours described in this work on the other hand secreted mg amounts/ml of labelled MAbD1. There is an obvious advantage in being able to rely on a uniform source of radiolabelled reagent with the same specific activity throughout a series of related experiments. Because of the long half-lives of 3H or 14C, prolonged storage of antibody labelled with these isotopes is possible without serious loss in radioactivity. Although the specific activity of the labelled antibodies obtained by biosynthetic labelling in ascites fluid was lower than can be obtained in vitro, the activity was sufficient for autoradiography and, in our experiments, exposure times longer than routinely necessary for autoradiography using tritium were not required.. The technique described here is easy to perform and, considering the:yield of labelled immunoglobulin the costs of producing quantities of labelled antibody from ascites are much lower than the costs of producing equivalent amounts of biosynthetically labelled antibody by in vitro methods. Tritiated monoclonal antibodies have become a useful tool in immunocytochemistry. Cuello et al. (1982) reported the successful application of biosynthetically tritiated monoclonal antibodies in immunocytochemical studies at both light and electron microscopic levels, for direct localization of immunoreactive sites on neuroactive substances in the central nervous system of the rat. They, and others (Van Rooijen, 1981), have also described the combined use of radio-immunohistochemistry with immunoenzymatic methods or immunofluorescence for simultaneous detection of 2 antigenic sites. We have been able to follow by autoradiography the synthesis of a Theileria sporozoite surface coat antigen, during development of the parasite in the feeding tick, using 3H-MAbD1 and cryostat sections of Theileria-infected tick salivary glands (Dobbelaere et al., 1985). The production of biosynthetically radiolabelled monoclonal antibodies may also find application in in vivo experiments (e.g., passive transfer experiments) where the need might arise to demonstrate the presence of antibody in different tissues after dilution in body fluids. Furthermore, biosynthetically labelled monoclonal antibody could also be used in radioimmunoassay systems for detection or quantitation of antigens carrying the epitope recognized by the labelled antibody.
214
Acknowledgements S p e c i a l t h a n k s are e x p r e s s e d to J a m e s N . K i a r i e for skillful t e c h n i c a l a s s i s t a n c e a n d D r . W o l f V o i g t for the s u p p l y of tick m a t e r i a l . D . A . E . D o b b e l a e r e is s p o n s o r e d b y the D e p a r t m e n t of D e v e l o p m e n t C o - o p e r a t i o n of the B e l g i a n G o v e r n m e n t .
References Carrel, S., R.S. Accolla, N. Gross and J.-P. Mach, 1981, in: Monoclonal Antibodies and T-Cell Hybridomas, eds. G.J. H~immerling, U. H~rmaerling and J.F. Kearney (Elsevier, Amsterdam) p. 174. Cuello, A.C. and C. Milstein, 1981, in: Physiological Peptides and New Trends in Radioimmunology, ed. Ch. A. Bizollon (Elsevier, Amsterdam) p. 293. Cuello, A.C., J.V. Priestley and C. Milstein, 1982, Proc. Natl. Acad. Sci. U.S.A. 79, 665. Dobbelaere, D.A.E., P.R. Spooner, W.C. Barry and A.D. Irvin, 1984, Parasite Immunol. 6, 361. Dobbelaere, D.A.E., P. Webster, B.L. Leitch, W.P. Voigt and A.D. Irvin, 1985, Exp. Parasititol. August issue. Hoogenraad, N., T. Helman and J. Hoogenraad, 1983, J. Immunol. Methods 61, 317. Kearney, J.F., A. Radbruch, B. Liesegang and K. Rajewski, 1979, J. Immunol. 123, 1548. K6hler, G. and C. Milstein, 1975, Nature (London) 256, 495. Levy, R., K. Dilley, S. Brown and Y. Bergrnan, 1980, in: Monoclonal Antibodies, Hybridomas: A New Dimension in Biological Analysis, eds. R.H. Kennet, T.J. McKearn and K.B. Bechtol (Plenum Press, New York) p. 137. Oi, V.T. and L.A. Herzenberg, 1980, in: Selected Methods in Cellular Immunology, eds. B.B. Mischell and S.M. Shiigi (Freeman, San Francisco, CA) p. 351. Sepp~il~i, I., H. Sarvas, F. P6terfy and O. M~kel~, 1981, Scand. J. Immunol. 14, 335. Van Rooijen, N., 1981, J. Immunol. Methods 40, 247.
Journal of Immunological Methoak, 82 (1985) 209-214
Elsevier JIM 03588
Production in Ascites Fluid of Biosynthetically Labelled Monoclonal Antibody to Theileria parva Sporozoites Dirk A.E. Dobbelaere
1 and
Paul R. Spooner
International Laboratory for Research on Animal Diseases, P.O. Box 30709, Nairobi, Kenya
(Received 30 November 1984, accepted 30 April 1985)
A hybridoma cell line has been previously produced which secretes monoclonal antibodies able to neutralize sporozoites of Theileria paroa, the causative agent of East Coast fever of cattle. Cells from this line were injected intra-peritoneally into pristane-treated BALB/c mice. During the last 4 days of hybridoma cell growth, mice were given 4 daily intraperitoneal injections of a mixture of tritiated amino acids in order to biosynthetic&y radiolabel the monoclonal antibody being produced in ascites fluid. The specific activity of the antibody obtained was 100 mCi/rnmol. The labelled antibody was used to detect, by autoradiography, a surface coat antigen of T. paroa sporozoites in cryostat sections of Theileria-infected tick salivary glands. The method allows the preparation of large quantities of biosynthetically radiolabelled immunological probes for the detection of immunoreactive sites in biological specimens. Key words: ascites tumor - biosynthetic labeling - monoclonal antibody - radio -immunohistochemistry sporozoite
:
- Theileria paroa - tritium
Introduction The use of antibodies as immunological probes of high specificity for the detection and assay of substances in biological specimens was greatly enhanced by the development of hybridoma technology (KBhler and Milstein, 1975). Biosynthetitally labelled radioactive monoclonal antibodies have been used in radio-immunohistochemistry at the light microscopic and electron microscopic level (Cue110 et al., 1982), in studies on secreted immunoglobulins (Levy et al., 1980) and in various tests such as the reciprocal binding inhibition test for monoclonal antibodies (Carrel et al., 1981). Biosynthetically labelled monoclonal antibodies are normally obtained by adding appropriate radioactive amino acids to hybridoma culture fluids during antibody synthesis in vitro, but only pg/ml can be harvested by this method (Cue110 I
’ Present address: Kemforschungszentrum 3640, D-7500 Karlsruhe 1, F.R.G.
Karlsruhe, Institut fur Genetik und Toxikologie, Postfach
0022-1759/85/$03.30 0 1985 Elsevier Science Publishers B.V. (Biomedical Division)
210
and Milstein, 1981). We have investigated the possibility of producing larger amounts of biosynthetically labelled monoclonal antibody in ascites fluid. Recently we described the production of a monoclonal antibody which detected a sporozoite surface antigen of the tick-transmitted protozoan parasite Theileria parva (Dobbelaere et al., 1984). The radio-labelling of this monoclonal antibody in ascites fluid is described in the current paper, and its applicability in light microscopic immuno-autoradiography of T. parva sporozoite antigens is demonstrated. Materials and Methods Mice
BALB/c mice, 10-12 weeks old, reared at ILRAD small animal unit were used. Monoclonal antibody
Monoclonal antibodies against T. parva sporozoites were prepared as described by Dobbelaere et al. (1984), by fusing spleen cells, from mice immunized with crude freeze-thawed sporozoite preparations, with X63-Ag8.653 myeloma cells (Kearney et al., 1979) and cloning antibody-producing cells by limiting dilution (Oi and Herzenberg, 1980). The monoclonal antibody used in this study, MAbD1, is of the IgG 3 isotype and binds to protein A. MAbD1 recognizes an epitope of a sporozoite surface coat antigen and neutralizes T. parva sporozoite infectivity. Production of ascites tumours
BALB/c mice were injected intraperitoneally with 0.5 ml of pristane (2, 6, 10,14-tetramethylpentadecane, Aldrich Chemical Co., Gillingham, Dorset) 10 days before injection of the hybridoma cells (Hoogenraad et al., 1983). MAbDl-producing hybridoma cells were washed twice in Dulbecco's phosphate-buffered saline, pH 7.4 (PBS) and 5 x 1 0 6 cells (in 0.5 ml of PBS) were injected intraperitoneally into each of 25 mice. To produce biosynthetically labelled monoclonal antibody (3H-MAbD1) in ascites fluid, a mixture of 15 tritiated (3H) amino acids (code number TRK.440., The Radiochemical Centre, Amersham) was injected intraperitoneally into 4 of the 25 mice, once daily for 4 days starting on day 7 after inoculation of MAbD1 hybridoma cells. Each inoculum consisted of 0.5 mCi of the 3H-amino acid mixture, made isotonic by addition of 10 x strength Hanks' balanced salt solution (Gibco BioCuh, Paisley, Scotland); the volume of each inoculum was 0.55 ml. Appropriate safety precautions and isolation procedures were observed throughout. Mice were killed 24 h after the last injection of 3H-amino acids and ascites fluid was harvested by aspiration with a Pasteur pipette with the addition of 20 U / m l heparin (Novo, Copenhagen) to the aspirate to prevent clotting. Pooled unlabelled and pooled labelled ascites fluids were clarified by centrifugation at 2500 x g for 10 min. Labelled ascites fluid was dialysed 3 times in 2 litre vol. of 0.1 M phosphate buffer (pH 7.5) containing 0.5 M sodium chloride after which no more tritium could be detected in the dialysis fluid. The dialysate was dispensed into 1 ml and 50 /~1 aliquots and stored at -80°C.
211
Purification of MAbD1 3H-MAbD1 used for the determination of specific activity was purified using Protein A-Sepharose CL-4B (Pharmacia Fine Chemicals, Uppsala) following the method described by Sepp~il~i et al. (1981). Immunoglobulin concentrations were calculated from the optical density reading at 280 nm (extinction coefficient at 280 nm for 1 mg/ml = 1.4).
Tritium counts Samples were counted in scintillation fluid (Aquasol-2, New England Nuclear, Boston, MA) using a Packard liquid scintillation spectrometer (model 3330).
Immuno-autoradiography Immuno-autoradiography with 3H-MAbD1 was carried out on cryostat sections of salivary glands of Theileria-infected adult Rhipicephalus appendiculatus ticks by a method similar to that described by Dobbelaere et al. (1984). Five #m cryostat sections of salivary glands were placed on microscope slides and fixed with 0.25% (v/v) glutaraldehyde in distilled water for 5 min at room temperature, followed by a rinse in PBS containing 1% (w/v) bovine serum albumin (PBS/BSA). Slides were stored at - 8 0 ° C until use. To test 3H-MAbD1, sections were thawed at room temperature, delineated with nail varnish and incubated for 45 min with 20 #1 3H-MAbD1 ascites fluid diluted 1/500 (v/v) in PBS and then rinsed 3 times with PBS/BSA. Slides were then stained with Feulgen DNA-stain (Dobbelaere et al., 1984) to reveal the parasite in sections of infected salivary gland acini. Air-dried slides were dipped in Ilford L4 nuclear research emulsion (Ilford, Basildon, Essex), left to dry and exposed for 10-20 days at 4°C and subsequently developed in Kodak D 76 (Eastman Kodak, Rochester, NY). The sections were examined using a light microscope at 500 × magnification, for the presence of silver grains, indicating binding of 3H-MAbD1 over salivary gland acini containing sporozoite antigen.
Results
Ascites tumours and ascites fluid production An average of 3.5 ml of ascites fluid per mouse were collected. There were no significant differences in time for tumour development and quantity of ascites fluid collected, between mice injected with cells and 3H-amino acid mixture, and those injected with cells alone. In both groups, tumours became apparent around day 10; radiolabelled ascites fluid was harvested from 4 mice on day 12 and unlabelled ascites from 21 mice between day 11 and day 14. The amount of protein A-binding immunoglobulin was 4.4 mg/ml in radiolabelled ascites fluid and 5.1 mg/ml in unlabelled ascites fluid.
Specific activity of SH-MAbD1 In the dialysed labelled ascites fluid, over 99% of the 3H counts were precipitable in 20% (w/v) trichloroacetic acid and 23% of the 3H counts measured were in
212
Fig. 1. Autoradiographof a cryostat section(5 ~m) through a Theileriaparva infected tick salivarygland. Strong labelling can be observedover the infected acinus (left) but not over the uninfected acinus (right). (Scale bar = 20 ~m; the autoradiographwas exposed for 20 days). protein A-binding immunoglobulin. The specific activity of protein A-purified 3H-MAbD1 was approximately 100 m C i / m m o l (3.7 G B q / m m o l ) .
Immuno-autoradiography Pooled 3H-MAbD1 ascites fluid showed the same specificity for T. parva sporozoite a n t i g e n as unlabelled MAbD1 ascites fluid. Autoradiographs of Theileria-infected salivary gland sections incubated with 3H-MAbD1 revealed clearly defined selective binding of the biosynthetically labelled 3H-MAbD1 to the infected acinar cells (Fig. 1). Background labelling was lower than on autoradiographs with unlabelled MAbD1 and 125I-labelled anti-mouse immunoglobulin as second antibody (not shown). Exposure for 10 days was sufficient for the detection of 3HMAbD1 binding to parasite antigen, but labelling was more pronounced with no significant increase in background after 20 days exposure.
Discussion These results demonstrate that it was possible to produce biosynthetically radiolabelled monoclonal antibody in ascites fluid, which could be used for the detection of immunoreactive sites. Biosynthetic labelling with a mixture of 3H-amino
213
acids did not noticeably affect binding or specificity of MAbD1 to Theileria sporozoites as demonstrated by immuno-autoradiography. Using tritiated MAbD1, background labelling was considerably lower than that observed by Dobbelaere et al. (1984) in autoradiographs with unlabelled MAbD1 and 125I-labelled anti-mouse immunoglobulin as second antibody. The advantage of biosynthetic labelling over chemical/enzymatic labelling have been discussed (Cuello and Milstein, 1981). One of the most important dangers of chemical/enzymatic labelling of antibodies is the possibility of causing configurational changes in the antigen-antibody combining site that can result in reduced or abolished affinity for the epitope. This is avoided by biosynthetic labelling. The usual methods by which hybridoma-secreted antibodies are biosynthetically labelled in vitro, depend on incorporation of radiolabelled amino acids (e.g., [14C]or [3H]-lysine) added to a hybridoma culture medium which is free of the cold amino acid (e.g., lysine-free medium). By this method only /~g amounts/ml of labelled immunoglobulin can be harvested from the culture supernatant. The ascites tumours described in this work on the other hand secreted mg amounts/ml of labelled MAbD1. There is an obvious advantage in being able to rely on a uniform source of radiolabelled reagent with the same specific activity throughout a series of related experiments. Because of the long half-lives of 3H or 14C, prolonged storage of antibody labelled with these isotopes is possible without serious loss in radioactivity. Although the specific activity of the labelled antibodies obtained by biosynthetic labelling in ascites fluid was lower than can be obtained in vitro, the activity was sufficient for autoradiography and, in our experiments, exposure times longer than routinely necessary for autoradiography using tritium were not required.. The technique described here is easy to perform and, considering the:yield of labelled immunoglobulin the costs of producing quantities of labelled antibody from ascites are much lower than the costs of producing equivalent amounts of biosynthetically labelled antibody by in vitro methods. Tritiated monoclonal antibodies have become a useful tool in immunocytochemistry. Cuello et al. (1982) reported the successful application of biosynthetically tritiated monoclonal antibodies in immunocytochemical studies at both light and electron microscopic levels, for direct localization of immunoreactive sites on neuroactive substances in the central nervous system of the rat. They, and others (Van Rooijen, 1981), have also described the combined use of radio-immunohistochemistry with immunoenzymatic methods or immunofluorescence for simultaneous detection of 2 antigenic sites. We have been able to follow by autoradiography the synthesis of a Theileria sporozoite surface coat antigen, during development of the parasite in the feeding tick, using 3H-MAbD1 and cryostat sections of Theileria-infected tick salivary glands (Dobbelaere et al., 1985). The production of biosynthetically radiolabelled monoclonal antibodies may also find application in in vivo experiments (e.g., passive transfer experiments) where the need might arise to demonstrate the presence of antibody in different tissues after dilution in body fluids. Furthermore, biosynthetically labelled monoclonal antibody could also be used in radioimmunoassay systems for detection or quantitation of antigens carrying the epitope recognized by the labelled antibody.
214
Acknowledgements S p e c i a l t h a n k s are e x p r e s s e d to J a m e s N . K i a r i e for skillful t e c h n i c a l a s s i s t a n c e a n d D r . W o l f V o i g t for the s u p p l y of tick m a t e r i a l . D . A . E . D o b b e l a e r e is s p o n s o r e d b y the D e p a r t m e n t of D e v e l o p m e n t C o - o p e r a t i o n of the B e l g i a n G o v e r n m e n t .
References Carrel, S., R.S. Accolla, N. Gross and J.-P. Mach, 1981, in: Monoclonal Antibodies and T-Cell Hybridomas, eds. G.J. H~immerling, U. H~rmaerling and J.F. Kearney (Elsevier, Amsterdam) p. 174. Cuello, A.C. and C. Milstein, 1981, in: Physiological Peptides and New Trends in Radioimmunology, ed. Ch. A. Bizollon (Elsevier, Amsterdam) p. 293. Cuello, A.C., J.V. Priestley and C. Milstein, 1982, Proc. Natl. Acad. Sci. U.S.A. 79, 665. Dobbelaere, D.A.E., P.R. Spooner, W.C. Barry and A.D. Irvin, 1984, Parasite Immunol. 6, 361. Dobbelaere, D.A.E., P. Webster, B.L. Leitch, W.P. Voigt and A.D. Irvin, 1985, Exp. Parasititol. August issue. Hoogenraad, N., T. Helman and J. Hoogenraad, 1983, J. Immunol. Methods 61, 317. Kearney, J.F., A. Radbruch, B. Liesegang and K. Rajewski, 1979, J. Immunol. 123, 1548. K6hler, G. and C. Milstein, 1975, Nature (London) 256, 495. Levy, R., K. Dilley, S. Brown and Y. Bergrnan, 1980, in: Monoclonal Antibodies, Hybridomas: A New Dimension in Biological Analysis, eds. R.H. Kennet, T.J. McKearn and K.B. Bechtol (Plenum Press, New York) p. 137. Oi, V.T. and L.A. Herzenberg, 1980, in: Selected Methods in Cellular Immunology, eds. B.B. Mischell and S.M. Shiigi (Freeman, San Francisco, CA) p. 351. Sepp~il~i, I., H. Sarvas, F. P6terfy and O. M~kel~, 1981, Scand. J. Immunol. 14, 335. Van Rooijen, N., 1981, J. Immunol. Methods 40, 247.