Antipeptide antibodies that recognize a lymphocyte substance P receptor.
In an effort to investigate the presence of substance P (SP) receptors on lymphocytes, polyclonal antibodies against SP receptors were developed. The immunogen used to generate these antibodies was a peptide encoded by an RNA complementary to the mRNA for SP. The rationale for using this SP complementary peptide (termed SP CP) as an immunogen resulted from the observation that 3H-SP bound to microtiter wells coated with SP CP in a dose dependent and saturable fashion. Furthermore, binding was blocked with excess unlabeled SP or SP antagonist, D-Pro2-D-Phe7-D-Trp9-SP. Inasmuch as the peptide, SP CP, specifically bound 3H-SP, we hypothesized that antibodies against this peptide might recognize a SP receptor binding site. Using the SP receptor positive lymphoblast cell line, IM-9, affinity-purified antibodies against SP CP but not antibodies against keyhole limpet hemocyanin recognized a molecule on the surface of IM-9 cells. Anti-SP CP binding to IM-9 cells was blocked with excess SP antagonist, suggesting that the antibody and the SP antagonist were competing for the same binding site. In support of this possibility, anti-SP CP antibodies blocked 3H-SP binding to IM-9 cells. An immunoaffinity column coupled with antibodies against SP CP bound protein from solubilized IM-9 cells. This isolated protein bound 125I-Tyr8-SP and binding was specifically blocked with SP as well as by SP antagonist, neurokinin A, and eledoisin. Passthrough material did not bind SP suggesting that a SP receptor had been purified. Western blot analysis of solubilized IM-9 cell proteins using anti-SP CP antibodies but not preimmune IgG recognized a single protein of 58,000 D. Taken together, these results demonstrate that antibodies against SP CP recognize a SP receptor present on the lymphocyte cell line, IM-9.
Pascual
D W
DW
Department of Physiology and Biophysics, University of Alabama, Birmingham 35294.
Blalock
J E
JE
Bost
K L
KL
D000818:Animals; D000906:Antibodies / Q000378:metabolism*; D000918:Antibody Specificity; D001666:Binding Sites, Antibody; D002846:Chromatography, Affinity; D003432:Cross-Linking Reagents; D005260:Female; D008214:Lymphocytes / Q000276:immunology / Q000378:metabolism*; D009842:Oligopeptides / Q000276:immunology* / Q000378:metabolism; D011817:Rabbits; D011869:Radioligand Assay; D018040:Receptors, Neurokinin-1; D017981:Receptors, Neurotransmitter / Q000276:immunology*; D013373:Substance P / Q000378:metabolism*
D000906:Antibodies; D003432:Cross-Linking Reagents; D009842:Oligopeptides; D018040:Receptors, Neurokinin-1; D017981:Receptors, Neurotransmitter; D013373:Substance P