Summary
A highly sensitive and specific monoclonal antibody against the enzyme dopamine β-hydroxylase (DBH) from rat was produced and coded DBH 41. The generated hybridoma secreted immunoglobulins of mouse IgG1 subtype, as determined by radial immunodiffusion. This antibody, characterized by immunoblotting against a crude rat DBH preparation, was found to specifically recognize two bands of molecular weight 70 and 75 kDa corresponding to the soluble and membrane bound forms of the enzyme, respectively. With regard to species specificity, the anti-DBH antibody recognizes only the rat DBH molecule as it exhibits no cross-reactivity with either mouse, human, rabbit, guinea pig, cat or bovine DBH. Comparative immunocytochemical localization of DBH and TOH immunoreactivity was performed in different brain regions and we found that the DBH 41 antibody specifically stained DBH-containing neurons and fibers in the rat central nervous system (CNS). The high sensitivity of the DBH 41 antibody permitted us to detect immunologically the presence of the enzyme even in areas where only scattered DBH-containing fibers were present.
Similar content being viewed by others
References
Blaschko H (1973) Catecholamine synthesis. Br Med Bull 29:105–109
Dahlstrom A, Fuxe K (1964) Evidence for the existence of monoamine-containing neurons in the central nervous system. I. Demonstration of monoamines and cell bodies of brainstem neurons. Acta Physiol Scand 62 [Suppl 232]:1–155
Ebstein RP, Park DH, Freeman LS, Levity SM, Ohuchi T, Goldstein M (1973) A radioimmunoassay of human circulatory dopamine-B-hydroxylase. Life Sci 13:769–774
Eranko O (1952) On the histochemistry of the adrenal medulla of the rat, with special reference to acid phosphatase. Acta Anat (Basel) [Suppl] 17:1–60
Eranko O (1955) Histochemistry of noradrenaline in the adrenal medulla of rats and mice. Endocrinology 57:363–368
Falck B (1962) Observation on the possibilities of the cellular localization of monoamines by a fluorescence method. Acta Physiol Scand 56 [Suppl 197]:1–25
Friedmann S, Kaufman S (1965) 3,4 Dihidroxyphenylethylamine B hydroxilase. J Biochem (Tokio) 240:4763–4773
Furness JB, Costa M, Blessing WW (1977) Simultaneous fixation and production of catecholamine fluorescence in central nervous tissue by perfusion with aldehydes. Histochem J 9:745–750
Furness JB, Health JW, Costa M (1978) Aqueous aldehyde (Faglu) method for fluorescence histochemical localization of catecholamines and for ultrastructural studies of central nervous tissue. Histochemistry 57:285–295
Fuxe K, Goldstein M, Hokfelt T, Joh TH (1970) Immunohistochemical localization of dopamine-β-hydroxylase in the peripheral and central nervous system. Res Commun Chem Pathol Pharmacol 1:627–636
Fuxe K, Goldstein M, Hokfelt T, Joh TH (1971) Cellular localization of dopamine-β-hydroxylase and phenylethanolamine-N-methyl transferase as revealed by immunohistochemistry. In: Eranko O (ed) Progress in brain research, vol 34. Elsevier, Amsterdam, pp 127–138
Geffard M, Buijs RM, Sequela P, Pool CW, Le Moal ML (1984) First demonstration of highly specific and sensitive antibodies against dopamine. Brain Res 294:161–165
Geffen LB, Livett DG, Rush RA (1969) Immunohistochemical localization of protein components of catecholamine storage vesicles. J Physiol (Lond) 204:593–605
Goldstein M (1972) Enzymes involved in the catalysis of catecholamine biosynthesis. In: Ubell R (ed) Methods in neurochemistry. Plenum Press, New York, pp 317–340
Goldstein M, Fuxe K, Hokfelt T, Joh TH (1971) Immunohistochemical studies on phenylethanolamine-N-methyltransferase, dopadecarboxylase and dopamine-β-hydroxylase. Experentia 271:951–952
Goldstein M, Fuxe K, Hokfelt T (1972) Characterization and tissue localization of catecholamine synthesizing enzymes. Pharmacol Rev 24:293–309
Goldstein M, Anagnoste B, Freedman LS, Roffman M, Ebstein RP, Park DH, Fuxe K, Hokfelt T (1973) Characterization, localization and regulation of catecholamine synthesis enzymes. In: Usdin E, Snyder S (eds) Frontiers in catecholamine research. Pergamon Press, New York, pp 69–78
Grzanna R, Coyle JT (1976) Rat adrenal dopamine-β-hydroxylase: purification and immunological characteristics. J Neurochem 27:1091–1096
Grzanna R, Morrison JH, Coyle JT, Molliver ME (1977) The immunohistochemical demonstration of noradrenergic neurons in the rat brain. The use of homologous antiserum to dopamine-β-hydroxylase. Neurosci Lett 4:127–134
Grzanna R, Molliver ME, Coyle JT (1978) Visualization of central noradrenergic neurons in thick sections by the unlabelled antibody method. A transmitter-specific Golgi image. Proc Natl Acad Sci USA 75:2502–2506
Hartman BK (1973) Immunofluorescence of dopamine-β-hydroxylase. Application of improved methodology to the localization of the peripheral and central noradrenergic nervous system. J Histochem Cytochem 4:312–332
Hartman BK, Udenfriend S (1970) Immunofluorescent localization of dopamine-β-hidroxilase in tissues. Mol Pharmacol 6:85–94
Hartman BK, Zide D, Udenfriend S (1972) The use of dopamine-β-hydroxylase as a marker for the central noradrenergic nervous system in the rat brain. Proc Natl Acad Sci USA 69:2722–2726
Helle B, Fillenz M, Stanford C, Pihl KB, Srebro B (1979) A simplified method for raising antibodies to rat dopamine-β-hydroxylase. J Neurochem 32:1351–1355
Helle KB, Serck-Hanssen G (1981) Different forms of dopamine-β-hydroxylase in vesicles? In: Sjarne I, Hedquist P, Langercrantz H, Wennmalm A (eds) Chemical neurotransmission 75 years. Academic Press. London, pp 85–90
Hokfelt T, Fuxe K, Goldstein M, Joh TH (1973) Immunohistochemical studies of three catecholamine synthesis enzymes: aspects on methodology. Histochemie 33:231–254
Kenigsberg RL, Cuello AC (1990) Production of a bi-specific monoclonal antibody recognizing mouse kappa light chains and horseradish peroxidase. Applications in immunoassays. Histochemistry 95:155–163
Kohler G, Milstein C (1975) Continuous cultures of fused cells secreting antibodies of pre-defined specificity. Nature 256:495–497
Kohler G, Milstein C (1976) Derivation of specific antibody-producing tissue culture and tumour lines by cell fusion. Eur J Immunol 6:511–519
Labatut R, Buda M, Berod A (1988) Long term changes in rat brain tyrosine hydroxylase following reserpine treatment: a qualitative immunochemical analysis. J Neurochem 50:1375–1380
Lamourox A, Vigny A, Biquet NF, Darmon MC, Franck R, Heny JP, Mallet J (1987) The primary structure of human dopamine-beta-hydroxylase: insights into the relationship between the soluble and the membrane bound forms of the enzyme. EMBO J 6:7931–7937
Lindvall O, Björklund A (1974) The glyoxilic acid fluorescence histochemical method: a detailed account of the methodology for the visualization of central catecholamine neurons. Histochemistry 39:97–127
Margolis RK, Finne J, Krusius T, Margolis RV (1984) Structural studies on glycoprotein oligosaccharides of chromaffin granule membranes and dopamine-beta-hydroxylase. Arch Biochem Biophys 228:443–449
Markey KA, Kondo S, Shenkman L, Goldstein M (1980) Purification and characterization of tyrosine hydroxylase from a clonal pheochromocytoma cell line. Mol Pharmacol 17:79–85
Moore RY (1981) Fluorescence histochemical methods: neurotransmitter histochemistry. In: Heimer, Robards M (eds) Neuroanatomical tract-tracing methods. Plenum Press, New York, pp 441–482
Moore RY, Card JP (1984) Noradrenaline-containing neurons systems. In: Björklund A, Hokfelt T (eds) Handbook of chemical neuroanatomy, vol. 2, part I, Elsevier, Amsterdam New York Oxford, pp 123–156
Morrison JH, Molliver ME, Grzanna R, Coyle JT (1981) The intracortical trajectory of the coeruleus-cortical projection in the rat: a tangentially organized cortical afferent. Neuroscience 6:139–158
Nolan J, Fonseca R, Hogue Angeletti R (1985) A monoclonal antibody to dopamine β-monooxygenase: detection of biosynthetic intermediates. Arch Biochem Biophys 240:257–264
Park DH, Hashimoto T, Ebstein RP, Goldstein M (1976) Purification and immunochemical characterization of dopamine-β-hydroxylase from human pheochromocytoma. Mol Pharmacol 12:73–81
Ribeiro-da-Silva A, Kenigsberg RL, Mazzoni IE, Cuello AC (1989) NGF receptor immunoreactivity in the skin and its relationship to sensory and sympathetic fibers (abstract). Soc Neurosci 15:346.5
Saxena A, Fleming PJ (1983) Isolation and reconstitution of the membrane bound form of dopamine-beta-hydroxylase. J Biol Chem 258:4147–4152
Semenenko FM, Bramwell S, Sidebottom E, Cuello AC (1985) Development of a mouse antiperoxidase secreting hybridoma for use in the production of a mouse PAP complex for immunocytochemistry and as a parent line in the development of hybrid-hybridoma. Histochemistry 83:405–408
Semenenko FM, Cuello AC, Goldstein M, Lee KY, Sidebottom E (1986) A monoclonal antibody against tyrosine hydroxylase: application in light and electron microscopy. J Histochem Cytochem 34:817–821
Slater EP, Zaremba S, Hogue-Angeletti RA (1981) Purification of membrane bound dopamine-beta-monooxygenase from chromaffin granule: relation to soluble dopamine-beta-monooxygenase. Arch Biochem Biophys 211:288–296
Sperk G, Galhaup I, Schlogle E, Hortnagl H, Horykiewicz O (1980) A sensitive and reliable assay of dopamine-β-hydroxylase in tissue. J Neurochem 35:972–976
Spitz S, Spitz L, Thorpe L, Eugui E (1984) Intraspleen primary immunization for the production of monoclonal antibodies. J Immunol Methods 70:39–43
Steinbush HWM, Verhofstad AAY, Penile B, Varga Y, Yoosten HWJ (1981) Immunohistochemical characterization of monoamine containing neurons in the central nervous system by antibodies to serotonin and noradrenaline. A study in the rat and the lamprey. Acta Histochem [Suppl] 24:107–122
Sternberger LA, Hardy PH, Cuculis JJ, Meyer HG (1970) The unlabelled antibody enzyme method of immunocytochemistry: preparation and properties of soluble antigen-antibody complex (horseradish peroxidase) and its use in identification of spirochetes. J Histochem Cytochem 18:315–333
Vaitukaitis J, Robbins JB, Nieschlog E, Ross GT (1971) A method for producing antisera with small doses of immunogen. J Clin Endocrinol Metab 33:988–991
Wu H-J, Parmer RJ, Koop AH, Rozarsly DJ, O'Connor DT (1990) Molecular cloning, structure and expression of dopamine-B-hydroxylase from bovine adrenal medula. J Neurochem 55:97–105
Zaremba S, Hogue-Angeletti R (1981) Transmembrane nature of chromaffin granule dopamine-β-monooxygenase. J Biol Chem 256:12310–12315
Author information
Authors and Affiliations
Rights and permissions
About this article
Cite this article
Mazzoni, I.E., Jaffe, E. & Cuello, A.C. Production and immunocytochemical application of a highly sensitive and specific monoclonal antibody against rat dopamine-β-hydroxylase. Histochemistry 96, 45–50 (1991). https://doi.org/10.1007/BF00266760
Accepted:
Issue Date:
DOI: https://doi.org/10.1007/BF00266760