Abstract
At least nine monoclonal antibodies against phytochrome from Pisum sativum L. and 20 against phytochrome from Avena sativa L. have been obtained from mouse hybridomas that were produced by fusion of spleen cells with SP 2/O-Ag14 myeloma cells. Hybridomas were selected and cloned in a single step by plating on a semisolid methylcellulose medium. Eight antibodies to Pisum and one to Avena phytochrome were immunopurified from hybridoma medium or ascitic fluid. When necessary, secreted antibodies were verified to be against phytochrome by demonstrating to be against phytochrome by demonstrating immunoadsorption of phytochrome, detected as loss of photoactivity and-or by appearance of the approx. 120,000-dalton phytochrome band upon sodium dodecyl sulfate polyacrylamide gel electrophoresis.
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Abbreviations
- ELISA:
-
enzyme-linked immunosorbent assay
- IgG:
-
immunoglobulin G
- PBS:
-
10 mM sodium phosphate, 140 mM NaCl, pH 7.4
- RIA:
-
radioimmunoassay
- SAR:
-
specific absorbance ratio, A 667/A280 with phytochrome in its redabsorbing form
References
Briggs, W.R., Rice, H.V. (1972) Phytochrome: chemical and physical properties and mechanism of action. Annu. Rev. Plant Physiol. 23, 293–334
Cordonnier, M.-M., Pratt, L.H. (1982) Immunopurification and initial characterization of dicotyledonous phytochrome. Plant Physiol. 69, 360–365
Cundiff, S.C., Pratt, L.H. (1975) Immunological and physical characterization of the products of phytochrome proteolysis. Plant Physiol. 55, 212–217
Davis, J.M., Pennington, J.E., Kubler, A.-M., Conscience, J.-F. (1982) A simple, single-step technique for selecting and cloning hybridomas for the production of monoclonal antibodies. J. Immunol. Methods 50, 161–171
Fazekas de St. Groth, S., Scheidegger, D. (1980) Production of monoclonal antibodies: strategy and tactics. J. Immunol. Methods 35, 1–21
Gardner, G., Pike, C.S., Rice, H.V., Briggs, W.R. (1971) “Disaggregation” of phytochrome in vitro — a consequence of proteolysis. Plant Physiol. 48, 686–693
Goding, J.W. (1980) Antibody production by hybridomas. J. Immunol. Methods 39, 285–308
Hopkins, D.W., Butler, W.L. (1970) Immunochemical and spectroscopic evidence for protein conformational changes in phytochrome transformations. Plant Physiol. 45, 567–570
Hunt, R.E., Pratt, L.H. (1979a) Phytochrome immunoaffinity purification. Plant Physiol. 64, 332–336
Hunt, R.E., Pratt, L.H. (1979b) Phytochrome radioimmunoassay. Plant Physiol. 64, 327–331
Kennett, R.H. (1980) Freezing of hybridoma cells. In: Monoclonal antibodies, p. 375, Kennett, R.H., McKearn, T.J., Bechtol, R.H., eds. Plenum Press, New York
Kennett, R.H., McKearn, T.J., Bechtol, K.B., eds. (1980) Monoclonal antibodies. Plenum Press, New York
Köhler, G., Milstein, C. (1975) Continuous cultures of fused cells secreting antibody of predefined specificity. Nature (London) 256, 495–497
Littlefield, J.W. (1964) Selection of hybrids from matings of fibroblasts in vitro and their presumed recombinants. Science 145, 709–710
Pratt, L.H. (1973) Comparative immunochemistry of phytochrome. Plant Physiol. 51, 203–209
Pratt, L.H. (1982) Phytochrome: the protein moiety. Annu. Rev. Plant Physiol. 33, 557–582
Pratt, L.H. (in press) Assay of photomorphogenic photoreceptors. In: Encyclopedia of plant physiology, N.S., Mohr, H., Shropshire, W., Jr., eds. Springer, Berlin Heidelberg New York
Pratt, L.H., Coleman, R.A. (1971) Immunocytochemical localization of phytochrome. Proc. Natl. Acad. Sci. USA 68, 2431–2435
Rice, H.V., Briggs, W.R. (1973a) Immunochemistry of phytochrome. Plant Physiol. 51, 939–945
Rice, H.V., Briggs, W.R. (1973b) Partial characterization of oat and rye phytochrome. Plant Physiol. 51, 927–938
Rice, R.H., Means, G.E. (1971) Radioactive labelling of proteins in vitro. J. Biol. Chem. 246, 831–832
Roux, S.J., McEntire, K., Brown, W.E. (1982) Determination of extinction coefficients of oat phytochrome by quantitative amino acid analysis. Photochem. Photobiol. 35, 537–543
Shulman, M., Wilde, C.D., Köhler, G. (1978) A better cell line for making hybridomas secreting specific antibodies. Nature (London) 276, 269–270
Sober, H.A., ed. (1970) CRC handbook of biochemistry: selected data for molecular biology, 2nd edn., p. C-80. CRC Press, Cleveland, O.
Staehelin, T., Durrer, B., Schmidt, J., Takacs, B., Stocker, J., Miggiano, V., Stähli, C., Rubinstein, M., Levy, W.P., Hershberg, R., Pestka, S. (1981a) Production of hybridomas secreting monoclonal antibodies to the human leukocyte interferons. Proc. Natl. Acad. Sci. USA 78, 1848–1852
Staehelin, T., Hobbs, D.S., Kung, H., Lai, C.-Y., Pestka, S. (1981b) Purification and characterization of recombinant human leukocyte interferon (IFLrA) with monoclonal antibodies. J. Biol. Chem. 256, 9750–9754
Voller, A., Bidwell, D., Bartlett, A. (1980) Enzyme-linked immunosorbent assay. In: Manual of clinical immunology, 2nd edn., pp. 359–371, Rose, N.R., Friedman, H., eds. American Society of Microbiology, Washington, D.C.
Weber, K., Osborn, M. (1969) The reliability of molecular weight determination by dodecyl sulfate-polyacrylamide gel electrophoresis. J. Biol. Chem. 244, 4406–4412
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Cordonnier, MM., Smith, C., Greppin, H. et al. Production and purification of monoclonal antibodies to Pisum and Avena phytochrome. Planta 158, 369–376 (1983). https://doi.org/10.1007/BF00397340
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DOI: https://doi.org/10.1007/BF00397340