Anti-peptide antibodies against the human blood platelet thromboxane A 2/prostaglandin H 2 receptor : Production, purification and characterization

Abstract

Two anti-peptide antibodies have been raised against the human blood platelet thromboxane A 2/prostaglandin H 2 (TXA 2/PGH 2) receptor. Based on the published sequence of the placental TXA 2/ PGH 2 receptor, two decapeptide segments were selected as potential antigens: one in the first extra- cellular 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 immuno- reactivity toward their synthetic antigens, solubilized platelet membranes and affinity-purified TXA 2/ PGH 2 receptor protein. Furthermore, Western blot analysis revealed that: (1) each antibody reacted with the purified platelet TXA 2/PGH 2 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 TXA 2/PGH 2 receptor protein. Functional analysis demonstrated that neither antibody interfered with ADP- or U46619-induced platelet aggregation or [ 3H]SQ29,548 binding to the solubilized receptor. These results suggest that the antibody epitopes are separate from the TXA 2/PGH 2 binding domain. In summary, two specific anti-peptide antibodies have been raised against the human platelet TXA 2/PGH 2 receptor. These antibodies should prove to be of value in the further investigation of the platelet TXA 2/PGH 2 receptor.