Preferential detection of pro-carboxypeptidase R by enzyme-linked immunosorbent assay.
We generated two monoclonal antibodies (mAbs), 2A16 and 10G1, against pro-carboxypeptidase R (proCPR), also known as thrombin activatable fibrinolysis inhibitor (TAFI). By use of these mAbs, we developed a sandwich enzyme-linked immunosorbent assay (ELISA) system to detect proCPR. Since the amount of the antigen detectable by the ELISA was essentially the same in fresh plasma and serum incubated at 37 C for 1 hr, we concluded that the ELISA system detected not only proCPR, but also inactivated CPR generated from proCPR. However, an appreciable amount of proCPR remained unactivated in serum. For extensive activation of proCPR in plasma, thrombin and thrombomodulin complexes (TTM) can be used together with CaCl2. Following extensive conversion of proCPR to CPR by T-TM and CaCl2, converting plasma to serum (T-TM serum), antigenicity became undetectable by ELISA. Further analysis revealed that 2A16 reacts only with proCPR although 10G1 reacts with proCPR, active CPR and inactivated CPR. Therefore, we concluded that the ELISA system preferentially detects proCPR and not CPR. Our sandwich ELISA system utilizing 2A16 and 10G1 provides a suitable method for detecting proCPR and can be used to determine levels of proCPR in plasma samples from patients.
Tani
Sayuri
S
Department of Molecular Biology, Nagoya City University Graduate School of Medical Sciences, Nagoya, Aichi 467-8601, Japan.
Akatsu
Hiroyasu
H
Ishikawa
Yasushige
Y
Okada
Noriko
N
Okada
Hidechika
H
D000911:Antibodies, Monoclonal; D025901:Carboxypeptidase B2 / Q000097:blood*; D004305:Dose-Response Relationship, Drug; D004792:Enzyme Precursors / Q000097:blood*; D004797:Enzyme-Linked Immunosorbent Assay; D006801:Humans; D011485:Protein Binding; D013917:Thrombin / Q000378:metabolism; D018180:Thrombomodulin / Q000378:metabolism
D000911:Antibodies, Monoclonal; D004792:Enzyme Precursors; D018180:Thrombomodulin; D025901:Carboxypeptidase B2; D013917:Thrombin