Production and characterization of a monoclonal antibody against human calcitonin gene-related peptide (CGRP) and its immunohistochemical application to salivary glands

Summary A monoclonal antibody (mAb), 129CD8 was raised against a C-terminal fragment (aa28–37) of -human calcitonin gene-related peptide (CGRP) coupled to bovine serum albumin. The specificity of the monoclonal antibody 129CD8 was corroborated by dot immunobinding experiments, enzyme-linked immunoassay and immunostaining of tissue sections. In vitro studies showed that the mAb 129CD8 readily recognized the fragment 28–37 of -human CGRP and to a slightly lesser degree whole -human CGRP and the fragments containing the C-terminal part of the molecule. The mAb 129CD8 also recognized the -human CGRP but not the -rat CGRP. The mAb 129CD8 did not react with substance P, katacalcin, calcitonin, amylin or fragments of -human CGRP lacking the C-terminal part of the molecule.Immunocytochemical staining was performed on human skin, guinea-pig thyroid and salivary glands and the trigeminal ganglion, and rat thyroid gland. Our findings demonstrate, in keeping with previous studies, that in human skin, nerve fibres containing CGRP immunoreactivity are found in both epidermis and dermis. In accordance with previous investigators, the Merkel cells were immunoreactive for CGRP. In the guinea-pig and rat thyroid gland CGRP immunoreactivity was localized in the C-cells. The distribution of CGRP immunoreactivity in the guinea-pig salivary glands is different from that previously reported for rat salivary glands. In the guinea-pig trigeminal ganglion, CGRP immunoreactivity was localized mainly in small-sized neurons and fibres traversing the ganglion. Double staining with substance P performed on guinea-pig trigeminal ganglion revealed four types of sensory neurons, those containing both peptides, those containing only substance P or CGRP and those lacking both peptides. Guinea-pig parotid gland, but not the submandibular or sublingual glands, contained periacinar fibres exhibiting both immunoreactivities. Substance P-positive, CGRP-positive fibres were also seen around parotid and submandibular, but not around sublingual, gland ducts. All glands received perivascular innervation showing immunoreactivities for both peptides. The present results support the idea that in the peripheral nervous system only a subpopulation of sensory neurons contains both substance P and CGRP. Consequently, colocalization of substance P and CGRP indicates a sensory nerve, while those containing either substance P or CGRP may be sensory or parasympathetic.