C. Borg et al., ANTIPEPTIDE ANTIBODIES AGAINST THE HUMAN BLOOD-PLATELET THROMBOXANE-A2 PROSTAGLANDIN-H2 RECEPTOR - PRODUCTION, PURIFICATION AND CHARACTERIZATION/, Biochemical pharmacology, 45(10), 1993, pp. 2071-2078
Two anti-peptide antibodies have been raised against the human blood p
latelet thromboxane A2/prostaglandin H-2 (TXA2/PGH2) receptor. Based o
n the published sequence of the placental TXA2/PGH2 receptor, two deca
peptide segments were Selected as potential antigens: one in the first
extracellular loop corresponding to residue 89 through 98, and the ot
her 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 chromatograp
hy employing each peptide as the immobilized ligand. The combined puri
fication factor for both procedures was approximately 60-fold. By ELIS
A, both antibodies displayed immunoreactivity toward their synthetic a
ntigens, solubilized platelet membranes and affinity-purified TXA2/PGH
2 receptor protein. Furthermore, Western blot analysis revealed that:
(1) each antibody reacted with the purified platelet TXA2/PGH2 recepto
r protein (55 kDa); and (2) each antibody recognized a single band (55
kDa) in solubilized platelet membranes. These findings establish anti
body specificity for the human platelet TXA2/PGH2 receptor protein. Fu
nctional analysis demonstrated that neither antibody interfered with A
DP- or U46619-induced platelet aggregation or [H-3]SQ29,548 binding to
the solubilized receptor. These results suggest that the antibody epi
topes are separate from the TXA2/PGH2 binding domain. In summary, two
specific anti-peptide antibodies have been raised against the human pl
atelet TXA2/PGH2 receptor. These antibodies should prove to be of valu
e in the further investigation of the platelet TXA2/PGH2 receptor.