title: Qualitative and quantitative studies on human B7.1-Fc fusion protein and the application in pharmacokinetic study in rhesus monkeys.
abstract: 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).
authors.affiliation: State Key Laboratory of Pathogen and Biosecurity, Beijing 100071, PR China.
authors.forename: Zhihang
authors.initials: Z
authors.lastname: Chen
authors.forename: Ruorui
authors.initials: R
authors.lastname: Liu
authors.forename: Jinjing
authors.initials: J
authors.lastname: Che
authors.forename: Hongjiao
authors.initials: H
authors.lastname: Shi
authors.forename: Yunan
authors.initials: Y
authors.lastname: Hou
authors.forename: Yunlong
authors.initials: Y
authors.lastname: Liu
authors.forename: Lili
authors.initials: L
authors.lastname: Sun
authors.forename: Li
authors.initials: L
authors.lastname: Tan
authors.forename: Chengqi
authors.initials: C
authors.lastname: Shan
authors.forename: Peisheng
authors.initials: P
authors.lastname: Hu
authors.forename: Yuanguo
authors.initials: Y
authors.lastname: Cheng
mesh_terms: 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
chemical_list: 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