Abstract
The extracellular organic matrix of developing human enamel is composed of two major classes of proteins, the hydrophobic amelogenins and the acidic enamelins. In order to identify, purify, and characterize the amelogenins from this complex mixture of proteins, and to study their ultrastructural localization and their pathways of synthesis, secretion, and degradation, specific and sensitive probes are needed. In the present paper the production of a monoclonal antibody against human amelogenin employing an intrasplenic primary immunization protocol is described. The monoclonal antibody produced is IgM and recognizes major human amelogenin protein bands in Western immunoblot assays. It also recognizes amelogenin protein bands from other species, specifically bovine and porcine. Indirect immunohistochemical studies showed the monoclonal antibody to react specifically with the extracellular matrix of human developing enamel. It did not react with the underlying dentin layer.
Similar content being viewed by others
References
Termine JD, Belcourt AB, Christner PJ, Conn KM, Nylen M (1980) Proteins of dissociatively extracted fetal tooth matrix proteins. I. Principal molecular species in developing bovine enamel. J Biol Chem 255:9760–9768
Belcourt AB, Fincham AG, Termine JD (1982) Acid-soluble fetal enamelins. J Dent Res 61:1031–1032
Belcourt AB, Fincham AG, Termine JD (1983) Bovine high molecular weight amelogenin proteins. Calcif Tissue Int 35:111–114
Fincham AG (1982) The extracellular matrix of developing dental enamel. In: Anderson W, Sadler W (eds) Perspective in differentiation and hypertrophy. Elsevier Science Publishers, New York, pp 135–156
Lyaruu DM, Belcourt AB, Fincham AG, Termine JD (1982) Neonatal hamster tooth molar development: extraction and characterization of amelogenins, enamelins and soluble dentin proteins. Calcif Tissue Int 34:85–96
Deutsch D, Shapira L, Alayoff A, Leviel D, Yoeli Z, Arad A (1984) Protein and mineral changes during prenatal and postnatal development and mineralization of human deciduous enamel. In: Fearnhead RW, Suga S (eds) Tooth enamel IV. Elsevier Science Publishers, Amsterdam, pp 234–239
Deutsch D, Haisraeli M, Palmon A, Arad A, Alayoff A (1986) Separation, purification and characterization of enamel proteins using HPLC, 2D electrophoresis and specific polyclonal antibodies. In: Sela J, Horowitz S (eds) Current advances in skeletogenesis. Heileger Publishers, Jerusalem, pp 165–178
Slavkin HC, Zeichner-David M, Macdougall M, Bessem C, Bringas P, Honig LS, Lussky J, Vides J (1982) Enamel gene products during murine amelogensis in vivo and in vitro. J Dent Res 61:1467–1478
Robinson C, Kirkham J, Briggs HD, Atkinson PJ (1982) Enamel proteins: from secretion to maturation. J Dent Res 61:1490–1495
Fincham AG, Belcourt AB, Termine JD (1983) The molecular composition of the protein matrix of developing human dental enamel. J Dent Res 62:11–15
Deutsch D, Alayoff A, Ron A, Eilat D (1985) Further characterization of enamel proteins using limited proteolysis and specific polyclonal antibodies. J Dent Res 64:731
Deutsch D, Dafni L, Alayoff A (1987) Preparation of specific polyclonal antibodies against human amelogenin and human enamelin proteins. J Dent Res 66:910
Shimokawa H, Wassmer P, Sobel ME, Termine JD (1984) Characterization of cell-free translation products of mRNA from bovine ameloblasts by monoclonal and polyclonal antibodies. In: Fearnhead RW, Suga S (eds) Tooth enamel IV. Elsevier Science Publishers, Amsterdam, pp 161–166
Christner PJ, Lally ET, Ads AH, Herold RC (1983) Production of a monoclonal antibody to bovine enamel proteins. Arch Oral Biol 18:773–779
Christner PJ, Lally ET, Miller RD, Leontzwich P, Rosenbloom J, Herold RC (1985) Monoclonal antibodies to different epitopes in amelogenin from fetal bovine teeth recognize higher molecular weight components. Arch Oral Biol 30:849–854
Deutsch D, Palmon A, Catalano-Sherman J, Laskov R (1987) Production of monoclonal antibodies against enamelin and against amelogenin proteins of developing bovine enamel matrix. Adv Dent Res 1:282–288
Rosenbloom GJ, Lally E, Dixon M, Spencer A, Herold R (1986) Production of a monoclonal antibody to enamelins which does not cross-react with amelogenins. Calcif Tissue Int 39:412–415
Spitz M, Spitz L, Thorpe R, Eugui E (1984) Intrasplenic primary immunization for the production of monoclonal antibodies. J Immunol Methods 70:39–43
Galfre G, Milstein G (1981) Preparation of monoclonal antibodies: strategies and procedures. Methods Enzymol 73:3–46
Kearney JE, Radbruch A, Liesengang B, Rajewsky K (1979) A new mouse myeloma line which has lost immunoglobulin expression but permits the construction of antibody secreting hybrid cell lines. J Immunol 123:1548–1550
Eilat D, Hochberg M, Fishel R, Laskov R (1982) Antibodies to RNA from autoimmune NZB/NZW mice recognize a similar antigenic determinant and show a large idiotypic diversity. Proc Natl Acad Sci USA 79:3818–3822
Fisher LW, Termine JD (1985) Purification of the noncollagenous proteins from bone: technical pitfalls and how to avoid them. In: Ornoy A, Harell A, Sela J (eds) Current advances in skeletogenesis. Elsevier Science Publishers B.V., Amsterdam, pp 467–472
Dan-Goor M, Silberstein L, Kessel M, Muhlard A (1990) Localization of epitopes and functional effects of two novel monoclonal antibodies against skeletal muscle. J Muscle Res Cell Motil 11:216–226
Fazekas De St Groth S, Scheidegger D (1980) Production of monoclonal antibodies: strategy and tactics. J Immunol Methods 35:1–21
Ouchterlony Q (1949) Antigen-antibody reactions in gels II. Factors determining the site of precipitate. Arkiv Kemi 1:43–48
Towbin H, Staechelin T, Gordon J (1979) Electrophoretic transfer of proteins from polyacrylamide gels to nitrocellulose sheets: procedure and applications. Proc Natl Acad Sci 76: 4350–4354
Matsurdaira P (1987) Sequence from picomole quantities of proteins electroblotted onto polyvinylidene difluoride membranes. J Biol Chem 262:10035–10038
Catalano-Sherman J, Palmor, A, Burstein Y, Deutsch D (in press) Amino acid sequence of a major human amelogenin protein employing Edman degradation and cDNA sequencing. J Dent Res
Fincham AG, Belcourt AB, Termine JD, Butler WT, Cothran C (1983) Amelogenins: sequence homologies in enamel matrix protein from three mammalian species. J Biochem 211:149–154
Fincham AG, Ha Y, Pavlova Z, Slavkin HC, Snead ML (1989) Human amelogenins: sequences of “TRAP” molecules. Calcif Tissue Int 45:243–250
Slavkin HC, Zeichner-David M, Snead ML, Samuel N, Bessem C, Bringas P (1984) Genetic and phylogenetic aspects of enamel. In: Belcourt AB, Ruch JV (eds) Workshop on tooth morphogenesis and differentiation. INSERM Publications, Strasbourg
Author information
Authors and Affiliations
Rights and permissions
About this article
Cite this article
Catalano-Sherman, J., Laskov, R., Palmon, A. et al. Production of a monoclonal antibody against human amelogenin. Calcif Tissue Int 54, 76–80 (1994). https://doi.org/10.1007/BF00316294
Received:
Accepted:
Issue Date:
DOI: https://doi.org/10.1007/BF00316294