PRODUCTION AND CHARACTERIZATION OF A MONOCLONAL-ANTIBODY AGAINST HUMAN CALCITONIN-GENE-RELATED PEPTIDE (CGRP) AND ITS IMMUNOHISTOCHEMICAL APPLICATION TO SALIVARY-GLANDS

A monoclonal antibody (mAb), 129CD8 was raised against a C-terminal fr agment (aa28-37) of alpha-human calcitonin gene-related peptide (CGRP) coupled to bovine serum albumin. The specificity of the monoclonal an tibody 129CD8 was corroborated by dot immunobinding experiments, enzym e-linked immunoassay and immunostaining of tissue sections. In vitro s tudies showed that the mAb 129CD8 readily recognized the fragment 28-3 7 of alpha-human CGRP and to a slightly lesser degree whole alpha-huma n CGRP and the fragments containing the C-terminal part of the molecul e. The mAb 129CD8 also recognized the beta-human CGRP but not the alph a-rat CGRP. The mAb 129CD8 did not react with substance P, katacalcin, calcitonin, amylin or fragments of alpha-human CGRP lacking the C-ter minal part of the molecule. Immunocytochemical staining was performed on human skin, guinea-pig thyroid and salivary glands and the trigemin al ganglion, and rat thyroid gland. Our findings demonstrate, in keepi ng with previous studies, that in human skin, nerve fibres containing CGRP immunoreactivity are found in both epidermis and dermis. In accor dance with previous investigators, the Merkel cells were immunoreactiv e for CGRP. In the guinea-pig and rat thyroid gland CGRP immunoreactiv ity was localized in the C-cells. The distribution of CGRP immunoreact ivity in the guinea-pig salivary glands is different from that previou sly reported for rat salivary glands. In the guinea-pig trigeminal gan glion, CGRP immunoreactivity was localized mainly in small-sized neuro ns and fibres traversing the ganglion. Double staining with substance P performed on guinea-pig trigeminal ganglion revealed four types of s ensory neurons, those containing both peptides, those containing only substance P or CGRP and those lacking both peptides. Guinea-pig paroti d gland, but not the submandibular or sublingual glands, contained per iacinar fibres exhibiting both immunoreactivities. Substance P-positiv e, CGRP-positive fibres were also seen around parotid and submandibula r, but not around sublingual, gland ducts. All glands received perivas cular innervation showing immunoreactivities for both peptides. The pr esent results support the idea that in the peripheral nervous system o nly a subpopulation of sensory neurons contains both substance P and C GRP. Consequently, colocalization of substance P and CGRP indicates a sensory nerve, while those containing either substance P or CGRP may b e sensory or parasympathetic.