Qualitative and quantitative studies on human B7.1-Fc fusion protein and the application in pharmacokinetic study in rhesus monkeys.
A sensitive, accurate, and precise enzyme immunoassay (EIA) for the quantification of intact human B7.1-Fc in rhesus monkey serum was validated, and the characteristics of B7.1 and Fc moiety of fusion protein were identified by surface plasmon resonance (SPR) and flow-cytometric method, respectively. B7.1-Fc bound to CD28 and CTLA-4 with K(d) values of 45.1 and 9.58 nM, respectively, which were very closed to the previous reports and the function of Fc moiety of fusion protein was also confirmed by Fc receptor binding assay and IL-8 releasing assay. To monitor the intact protein, the EIA method employed a sandwich scheme in which a multiclonal anti-human IgG (Fc specific) antibody and a monoclonal anti-human B7.1 antibody were served as capture and detection antibody, respectively. This EIA has a range of reliable response of 0.5-32 ng/ml. The LLOQ was established at 0.5 ng/ml. The intra-assay precision and accuracy were 6.1-8.8% and (3.0-9.0)%, respectively with the inter-assay precision and accuracy were 5.7-11.5% and (10.7-9.1)%, respectively. Stability was established under certain conditions and no significant differences were found. This validated EIA assay was then successfully employed in the assessment of pharmacokinetic behavior of B7.1-Fc in rhesus monkeys after intravenous infusion, and a non-linear characteristics was established across the investigated dosage range (32-320 μg/kg).
Chen
Zhihang
Z
State Key Laboratory of Pathogen and Biosecurity, Beijing 100071, PR China.
Liu
Ruorui
R
Che
Jinjing
J
Shi
Hongjiao
H
Hou
Yunan
Y
Liu
Yunlong
Y
Sun
Lili
L
Tan
Li
L
Shan
Chengqi
C
Hu
Peisheng
P
Cheng
Yuanguo
Y
D000818:Animals; D000911:Antibodies, Monoclonal / Q000737:chemistry; D000915:Antibody Affinity; D015703:Antigens, CD / Q000737:chemistry; D018122:B7-1 Antigen / Q000737:chemistry*; D018106:CD28 Antigens / Q000737:chemistry; D060908:CTLA-4 Antigen; D002626:Chemistry, Pharmaceutical / Q000379:methods*; D006801:Humans; D007124:Immunoenzyme Techniques / Q000379:methods; D007141:Immunoglobulin Fc Fragments / Q000737:chemistry*; D016209:Interleukin-8 / Q000378:metabolism; D007700:Kinetics; D008253:Macaca mulatta; D051379:Mice; D015203:Reproducibility of Results
D000911:Antibodies, Monoclonal; D015703:Antigens, CD; D018122:B7-1 Antigen; D018106:CD28 Antigens; D060908:CTLA-4 Antigen; C556706:CTLA4 protein, human; C556707:Ctla4 protein, mouse; D007141:Immunoglobulin Fc Fragments; D016209:Interleukin-8