Summary
The serotonin antigen (5-HT-BSA formaldehyde conjugate) used for obtaining anti-5-HT antibodies was studied to obtain additional data concerning the nature of its immunogen. Dialysis against 0.1 M acetic acid and then against distilled water proved to be the best way of removing 5-HT condensation products not bound to BSA. The hapten has the configuration of a tetrahydro-β-carboline (THBC) ring structure that is coupled to protein most probably via the carbon(s) ortho to the phenolic hydroxyl group and the indole nitrogen. The cyclic secondary amine of the THBC remained unsubstituted and was not involved in the bridging to BSA. This functional group was effectively blocked by acetylation and was unreactive to glutaraldehyde. On the other hand, in 5-HT conjugates synthesized using glutaraldehyde as the coupling agent, no cyclization to THBC occurred, and the amino groups were blocked. The chemical reactivity of the secondary amino group of the hapten in the synthesized conjugates was compared to the immunoreactivity of 5-HT conjugates formed in tissues. Immunostaining of formaldehyde-fixed serotoninergic neurons of the raphe of rats was suppressed by acetylation and the use of glutaraldehyde as the primary fixative, but the staining was unaffected when glutaraldehyde was reacted with formaldehyde-fixed 5-HT neurons. It is concluded that the cyclic secondary amine of the THBC structure is not conjugated to protein and forms part of the 5-HT-antibody-binding site in immunogens formed in vitro and in tissues.
This is a preview of subscription content, log in via an institution to check access.
Similar content being viewed by others
References
Abramovitch, RA, Spencer ID (1964) The carbolines. In: Advances in heterocyclic chemistry. Academic Press, New York London, pp 79–207
Bader H, Oroshinik W (1957) Synthetic oxytocics. Synthesis and reactions of 3-Indolyl-2′-pyridylcarbinols and of 2,3-(2′,3′-Indolo)-hexahydroquinolizines. J Am Chem Soc 79:5686–5689
Brusco A, Peressini S, Oliva D, Pecci Saavedra J (1982) Serotonin antigenicity in nerve cells: specificity of antisera and preservation with aldehyde fixatives. Proc. III Congreso Braileiro de Biología Celular, 36 B, July 19–25, Sao Paulo, Brasil
Brusco A, Peressini S, Pecci Saavedra J (1983) Serotonin-like immunoreactivity and anti-5-hydroxytryptamine (5-HT) antibodies: Ultrastructural application in the central nervous system. J Histochem Cytochem 31:524–530
Fraenkel-Conrat N, Brandon BA, Olcott HS (1947) The reaction of formaldehyde with proteins. IV Participation of indole groups. Gramicidin. J Biol Chem 168:99–118
Fraenkel-Conrat H, Olcott HS (1948) Reaction of formaldehyde with proteins. VI. Cross-linking of amino groups with phenol, imidazole or indole groups. J Biol Chem 174:827–843
Grota LJ, Brown GM (1974) Antibodies to indolealkylamines: serotonin and melatonin. Can J Biochem 52:196–202
Grota LJ, Snieckus V, DeSilva SO, Brown GM (1983) Antibodies to indolealkylamines II: site of conjugation of melatonin to protein using formaldehyde. Can J Biochem Cell Biol 61:1096–1101
Harvey DG, Miller EJ, Robson W (1941) The Adamkiewics, Hopkins and Cole, and Rosenheim tests for tryptophan. An investigation of the configuration of the organic molecule responsible for the colour formation and its bearing on the constitution of Yohimbine, with a note on the action of formaldehyde on tryptophan. J Chem Soc (April) 1941, pp 153–159
Holcengerg J, Benditt EP (1959) A new histochemical technique for demonstration of enterochromaffin cells: a reaction for indole-ethylamines. J Histochem Cytochem 7:303–304
Jepson JB, Stevens BJ (1953) A fluorescence test for serotonin and other tryptamines. Nature 172:772
Joule JA, Smith GF (1978) Heterocyclic Chemistry, 2nd edn. Van Nostrand Reinhold, London, pp 257–287
Lillie RD (1964) Studies on histochemical acylation procedures. I Phenols. J Histochem Cytochem 12:522–529
McCaldin DJ (1960) The chemistry of ninhydrin. Chem Rev 60:39–51
Milstein C, Wright B, Cuello C (1983) The discrepancy between the crossreactivity of a monoclonal antibody to serotonin and its immunohistochemical specificity. J Mol Immunol 20:113–123
Pearse AGE (1972) Histochemistry. Theoretical and applied, vol 2, 3rd edn. Little Brown, Boston, pp 1291–1292
Pecci Saavedra J, Brusco A, Peressini S, Oliva ED (1982) Serotonin antigenicity in the central nervous system. J Histochem Cytochem 30:543
Pecci Saavedra J, Brusco A, Tandler CJ (1983) Tetrahydro-β-carboline as the true immunogen in the production of anti-5-HT like antibodies. Comun Biol 2:49–59
Pecci Saavedra J, Pasik T, Pasik P (1983) Immunocytochemistry of serotoninergic neurons in the central nervous system of monkeys. In: Caputto R, Marsan CA (eds) Proc Symp on Molecular Aspects of Nervous Stimulation, Transmission and Learning and Memory. Raven Press, New York, pp 81–96
Peressini S, Brusco A, Pecci Saavedra J (1984) Basis for the specificity of anti-5-HT-like antisera in immunocytochemistry applied to the central nervous system. Histochemistry 80:597–601
Ranadive NS, Sehon AN (1967) Antibodies to serotonin. Can J Biochem 45:1701–1710
Schipper J, Tilders FJH (1983) A new technique for studying specificity of immunocytochemical procedures: Specificity of serotonin immunostaining. J Histochem Cytochem 31:12–18
Smith I (1969) Chromatographic and electrophoretic techniques. 3rd edn. Pitman Press, Bath, Great Britain
Smuckler EA, Erksen N, Benditt EP (1960) The chemical nature of the chromation reaction in the enterochromaffin cells. J Histochem Cytochem 8:314–315
Solcia E, Sampietro R (1967) Indole nature of enterochromaffin substance. Nature 214:196–197
Steinbusch HWN, Verhofstad AAJ, Joosten HWJ (1978) Localization of serotonin in the central nervous system by immunohistochemistry: description of a specific and sensitive technique and some applications. Neuroscience 3:811–819
Sternberger LA (1979) Immunocytochemistry, 2nd edn. John Wiley, New York
Tandler CJ, Solari AJ (1982) Methanol-acetic anhydride: an efficient blocking agent for electron microscope cytochemistry. Its application to mouse testis and other tissues. Histochemistry 76:351–361
Tandler CJ, Pecci Saavedra J (1984) Bound tetrahydro-β-carboline as the true antigen in the production of anti-5-HT-like antibodies. Sites of conjugation of serotonin to protein using formaldehyde. Comun Biol 3:55–69
Thompson BB (1968) The Mannich reaction. J Pharm Sci 57:715–733
Udenfriend S, Weissbach H (1963) 5-hydroxytryptophan and derivatives. In: Colowick SP, Kaplan NO (eds) Methods in enzymology, vol VI. Academic Press, New York London, pp 598–605
Vanable JW (1963) A ninhydrin reaction giving a sensitive quantitative fluorescence assay for 5-hydroxytryptamine. Anal Biochem 6:393–403
Verhofstad AAJ, Steinbusch HWM, Joosten HWJ, Penke B, Varga J, Goldstein M (1983) Immunocytochemical localization of noradrenaline, adrenaline and serotonin. In: Polak JM, Van Noorden S (eds). Immunocytochemistry. Practical applications in pathology and biology. Wright, Bristol, pp 143–168
Wilcox PE (1967) Determination of amide residues by chemical methods. In: Colowick SP, Kaplan NO (eds) Methods in enzymologie, vol XI. Academic Press, New York London, pp 63–76
Author information
Authors and Affiliations
Rights and permissions
About this article
Cite this article
Tandler, C.J., Brusco, A., Peressini, S. et al. Further evidence for the specificity of anti-5-HT (serotonin)-like antisera in immunocytochemistry. Histochemistry 85, 67–72 (1986). https://doi.org/10.1007/BF00508655
Received:
Accepted:
Issue Date:
DOI: https://doi.org/10.1007/BF00508655