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. 1982 Nov;79(22):7031–7035. doi: 10.1073/pnas.79.22.7031

Interaction of monoclonal antibodies with mammalian choline acetyltransferase.

G D Crawford, L Correa, P M Salvaterra
PMCID: PMC347269  PMID: 6184721

Abstract

Monoclonal antibodies selective for rat brain choline acetyltransferase (acetyl-CoA: choline O-acetyltransferase, EC 2.3.1.6) were prepared by standard techniques. Five cell lines were isolated from spleen cell-SP/2 hybrids by repetitive cloning with a screening method that used the intrinsic activity of the enzyme. All cell lines secrete immunoglobulin of mouse subclass IgGl, and none inhibit the enzyme activity directly. The size of antibody-enzyme immune complexes formed with different pairs of the monoclonal antibodies was determined by gel filtration with HPLC. By comparing the elution position of choline acetyl-transferase activity after incubation with paired monoclonal antibodies, the spatial relationship of antibody binding domains relative to each other can be defined and classified as independent, mutually exclusive, or overlapping. Immune complexes in excess of Mr 600,000 were formed by some pairs of antibodies with the antigen, indicating independent binding domains on the enzyme. In one case, the paired antibodies formed an immune complex of only Mr 300,000, indicating that they bound in a mutually exclusive fashion. In most cases, pairs of antibodies reacted with the enzyme to give simultaneously both higher and lower Mr immune complexes. We conclude that all five antibodies bind to a relatively localized region of the enzyme surface. Antibodies were screened for usefulness as immunohistochemical markers of choline acetyltransferase-containing neurons by using the indirect immunoperoxidase method. One antibody intensely stains cell bodies of motor neurons and processes in a selective manner in the rat spinal cord and brain stem by using aldehyde-fixed tissue; the remaining antibodies do not react with fixed tissue.

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Selected References

These references are in PubMed. This may not be the complete list of references from this article.

  1. Berzofsky J. A., Hicks G., Fedorko J., Minna J. Properties of monoclonal antibodies specific for determinants of a protein antigen, myoglobin. J Biol Chem. 1980 Dec 10;255(23):11188–11191. [PubMed] [Google Scholar]
  2. Chao L. P. Choline acetyltransferase: purification and characterization. J Neurosci Res. 1980;5(2):85–115. doi: 10.1002/jnr.490050202. [DOI] [PubMed] [Google Scholar]
  3. Chao L. P., Kan K. S., Hung F. M. Immunohistochemical localization of choline acetyltransferase in rabbit forebrain. Brain Res. 1982 Mar 4;235(1):65–82. doi: 10.1016/0006-8993(82)90196-2. [DOI] [PubMed] [Google Scholar]
  4. Cozzari C., Hartman B. K. Preparation of antibodies specific to choline acetyltransferase from bovine caudate nucleus and immunohistochemical localization of the enzyme. Proc Natl Acad Sci U S A. 1980 Dec;77(12):7453–7457. doi: 10.1073/pnas.77.12.7453. [DOI] [PMC free article] [PubMed] [Google Scholar]
  5. Crawford G., Slemmon J. R., Salvaterra P. M. Monoclonal antibodies selective for Drosophila melanogaster choline acetyltransferase. J Biol Chem. 1982 Apr 10;257(7):3853–3856. [PubMed] [Google Scholar]
  6. Darnall D. W., Klotz I. M. Subunit constitution of proteins: a table. Arch Biochem Biophys. 1975 Feb;166(2):651–682. doi: 10.1016/0003-9861(75)90432-4. [DOI] [PubMed] [Google Scholar]
  7. Dietz G. W., Jr, Salvaterra P. M. Purification and peptide mapping of rat brain choline acetyltransferase. J Biol Chem. 1980 Nov 25;255(22):10612–10617. [PubMed] [Google Scholar]
  8. Eng L. F., Uyeda C. T., Chao L. P., Wolfgram F. Antibody to bovine choline acetyltransferase and immunofluorescent localisation of the enzyme in neurones. Nature. 1974 Jul 19;250(463):243–245. doi: 10.1038/250243a0. [DOI] [PubMed] [Google Scholar]
  9. Ey P. L., Prowse S. J., Jenkin C. R. Isolation of pure IgG1, IgG2a and IgG2b immunoglobulins from mouse serum using protein A-sepharose. Immunochemistry. 1978 Jul;15(7):429–436. doi: 10.1016/0161-5890(78)90070-6. [DOI] [PubMed] [Google Scholar]
  10. Fonnum F. A rapid radiochemical method for the determination of choline acetyltransferase. J Neurochem. 1975 Feb;24(2):407–409. doi: 10.1111/j.1471-4159.1975.tb11895.x. [DOI] [PubMed] [Google Scholar]
  11. Kimura H., McGeer P. L., Peng F., McGeer E. G. Choline acetyltransferase-containing neurons in rodent brain demonstrated by immunohistochemistry. Science. 1980 May 30;208(4447):1057–1059. doi: 10.1126/science.6990490. [DOI] [PubMed] [Google Scholar]
  12. LOWRY O. H., ROSEBROUGH N. J., FARR A. L., RANDALL R. J. Protein measurement with the Folin phenol reagent. J Biol Chem. 1951 Nov;193(1):265–275. [PubMed] [Google Scholar]
  13. Laemmli U. K. Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature. 1970 Aug 15;227(5259):680–685. doi: 10.1038/227680a0. [DOI] [PubMed] [Google Scholar]
  14. Levey A. I., Aoki M., Fitch F. W., Wainer B. H. The production of monoclonal antibodies reactive with bovine choline acetyltransferase. Brain Res. 1981 Aug 10;218(1-2):383–387. doi: 10.1016/0006-8993(81)91316-0. [DOI] [PubMed] [Google Scholar]
  15. Malte-Sørenssen D. Recent progress in the biochemistry of choline acetyltransferase. Prog Brain Res. 1979;49:45–58. doi: 10.1016/S0079-6123(08)64620-8. [DOI] [PubMed] [Google Scholar]
  16. McGeer P. L., McGeer E. G., Singh V. K., Chase W. H. Choline acetyltransferase localization in the central nervous system by immunohistochemistry. Brain Res. 1974 Dec 6;81(2):373–379. doi: 10.1016/0006-8993(74)90955-x. [DOI] [PubMed] [Google Scholar]
  17. Medgyesi G. A., Füst G., Gergely J., Bazin H. Classes and subclasses of rat immunoglobulins: interaction with the complement system and with staphylococcal protein A. Immunochemistry. 1978 Feb;15(2):125–129. doi: 10.1016/0161-5890(78)90052-4. [DOI] [PubMed] [Google Scholar]
  18. Rossier J. Immunohistochemical localization of choline acetyltransferase: real or artefact? Brain Res. 1975 Nov 21;98(3):619–622. doi: 10.1016/0006-8993(75)90381-9. [DOI] [PubMed] [Google Scholar]
  19. Rossier J. Purification of rat brain choline acetyltransferase. J Neurochem. 1976 Mar;26(3):543–548. doi: 10.1111/j.1471-4159.1976.tb01509.x. [DOI] [PubMed] [Google Scholar]
  20. Ryan R. L., McClure W. O. Purification of choline acetyltransferase from rat and cow brain. Biochemistry. 1979 Nov 27;18(24):5357–5365. doi: 10.1021/bi00591a016. [DOI] [PubMed] [Google Scholar]
  21. Shulman M., Wilde C. D., Köhler G. A better cell line for making hybridomas secreting specific antibodies. Nature. 1978 Nov 16;276(5685):269–270. doi: 10.1038/276269a0. [DOI] [PubMed] [Google Scholar]
  22. Slemmon J. R., Salvaterra P. M., Crawford G. D., Roberts E. Purification of choline acetyltransferase from Drosophila melanogaster. J Biol Chem. 1982 Apr 10;257(7):3847–3852. [PubMed] [Google Scholar]
  23. Smith C. P., Carroll P. T. A comparison of solubilized and membrane bound forms of choline-O-acetyltransferase (EC 2.3.1.6) in mouse brain nerve endings. Brain Res. 1980 Mar 10;185(2):363–371. doi: 10.1016/0006-8993(80)91074-4. [DOI] [PubMed] [Google Scholar]
  24. Tzartos S. J., Rand D. E., Einarson B. L., Lindstrom J. M. Mapping of surface structures of electrophorus acetylcholine receptor using monoclonal antibodies. J Biol Chem. 1981 Aug 25;256(16):8635–8645. [PubMed] [Google Scholar]

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