Anti-peptide antibodies against the human blood platelet thromboxane A2/prostaglandin H2 receptor. Production, purification and characterization.
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.
Borg
C
C
Department of Pharmacology, University of Illinois, Chicago 60607.
Lam
S C
SC
Dieter
J P
JP
Lim
C T
CT
Komiotis
D
D
Venton
D L
DL
Le Breton
G C
GC
D000595:Amino Acid Sequence; D000818:Animals; D000906:Antibodies / Q000302:isolation & purification* / Q000494:pharmacology*; D000917:Antibody Formation / Q000187:drug effects; D000918:Antibody Specificity; D001665:Binding Sites; D001792:Blood Platelets / Q000276:immunology / Q000502:physiology / Q000648:ultrastructure*; D002846:Chromatography, Affinity; D004797:Enzyme-Linked Immunosorbent Assay; D000939:Epitopes / Q000032:analysis / Q000276:immunology; D006801:Humans; D015151:Immunoblotting; D007074:Immunoglobulin G / Q000276:immunology / Q000302:isolation & purification; D008244:Lysophosphatidylcholines / Q000494:pharmacology; D008969:Molecular Sequence Data; D010455:Peptides / Q000276:immunology* / Q000302:isolation & purification / Q000494:pharmacology*; D010974:Platelet Aggregation / Q000187:drug effects; D011463:Prostaglandins H / Q000378:metabolism; D011817:Rabbits; D011982:Receptors, Prostaglandin / Q000276:immunology* / Q000378:metabolism / Q000502:physiology; D017482:Receptors, Thromboxane / Q000276:immunology* / Q000378:metabolism / Q000502:physiology; D044244:Receptors, Thromboxane A2, Prostaglandin H2
D000906:Antibodies; D000939:Epitopes; D007074:Immunoglobulin G; D008244:Lysophosphatidylcholines; D010455:Peptides; D011463:Prostaglandins H; D011982:Receptors, Prostaglandin; D017482:Receptors, Thromboxane; D044244:Receptors, Thromboxane A2, Prostaglandin H2