Anti-peptide antibodies against the human blood platelet thromboxane A2/prostaglandin H2 receptor. Production, purification and characterization.

C Borg, S C Lam, J P Dieter, C T Lim, D Komiotis, D L Venton, G C Le Breton

文献索引：Biochem. Pharmacol. 45(10) , 2071-8, (1993)

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摘要

Two anti-peptide antibodies have been raised against the human blood platelet thromboxane A2/prostaglandin H2 (TXA2/PGH2) receptor. Based on the published sequence of the placental TXA2/PGH2 receptor, two decapeptide segments were selected as potential antigens: one in the first extracellular loop corresponding to residue 89 through 98, and the other in the C-terminal region of the intracellular domain corresponding to residue 314 through 323. Rabbits were immunized with each peptide, and the antisera were subjected to a two-step purification procedure. The IgG fraction was purified using a DEAE Affi-Gel Blue column, and the peptide-specific IgG was further purified by affinity chromatography employing each peptide as the immobilized ligand. The combined purification factor for both procedures was approximately 60-fold. By ELISA, both antibodies displayed immunoreactivity toward their synthetic antigens, solubilized platelet membranes and affinity-purified TXA2/PGH2 receptor protein. Furthermore, Western blot analysis revealed that: (1) each antibody reacted with the purified platelet TXA2/PGH2 receptor protein (55 kDa); and (2) each antibody recognized a single band (55 kDa) in solubilized platelet membranes. These findings establish antibody specificity for the human platelet TXA2/PGH2 receptor protein. Functional analysis demonstrated that neither antibody interfered with ADP- or U46619-induced platelet aggregation of [3H]SQ29,548 binding to the solubilized receptor. These results suggest that the antibody epitopes are separate from the TXA2/PGH2 binding domain. In summary, two specific anti-peptide antibodies have been raised against the human platelet TXA2/PGH2 receptor. These antibodies should prove to be of value in the further investigation of the platelet TXA2/PGH2 receptor.