Monoclonal Antibodies to Substance P: Production, Characterization of Their Fine Specificities, and Use in Immunocytochemistry

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Abstract
Five hybrid clones secreting antibodies to the neuropeptide substance P have been obtained by somatic cell fusion of mouse myeloma cells with splenocytes from immunized mice of the Biozzi strain. To perform rapid and sensitive screening tests as well as to study the fine specificities of each monoclonal antibody, we developed a new enzyme immunoassay of substance P using acetylcholines‐ terase as label. All five monoclonal antibodies were directed to the C‐terminal pentapeptide of substance P, especially to the Phe7 residue. They cross‐reacted with neurokinin A and to some extent with neurokinin B but not with other non‐tachykinin mammalian peptides. One monoclonal antibody (SP 14) was used for immunocytochemical experiments in the rat spinal cord and spinal ganglion, both at the light and electron microscopic levels. A strong specific neurokinin‐like immuno eactivity was observed in cell bodies, nerve fibers, and terminals, with a very low background staining. Finally, the affinities of several analogues of substance P for SP14 manoclonal antibody were shown to be correlated with their biological activities, as measured by their hypotensive effects in vivo. These findings suggested a strong structural resemblance between the combining site of the antibody and that of the physiological substance P receptor.