title: Determination of the affinity constant of streptavidin-coupled magnetic particles and a biotinylated antibody for high performance of magnetic solid carrier in immunoassays.
abstract: Streptavidin-coupled magnetic particles (SAMPs) usually serve as a separation tool in immunoassays to specifically recognize and capture biotinylated protein. Determining the affinity constant (KA) of SAMPs and biotinylated proteins and further evaluating the performance of SAMPs as a solid-phase carrier are of great importance. Towards this goal, we present a simple method to detect the KA of SAMPs and a biotinylated antibody (Ab). A combined equilibrium equation derived from fluorescence labeling experiments was employed to evaluate the binding capacity between SAMPs and a biotinylated antibody. The main experimental conditions were optimized, and the KA of SAMPs and a biotinylated antibody was determined to be 3.64±0.31×10(7)M(-1) with a coefficient of variation (CV) of 8.38%. The initial effective biotinylated antibody binding sites of as-synthesized SAMPs were found to be 132pmol/mg SAMPs. In addition, the KA of Ab towards SAMPs with four different streptavidin coating densities (16-83μg/mg MPs) was studied, and the results showed no significant difference for the KA among these four SAMPs. This suggests that the binding activity of SAMPs with Ab would not be affected by the streptavidin coupling density at least below 83μg/mg MPs. However, when the four SAMPs were used as the capture reagent in sandwich immunoassays, the SAMPs with streptavidin coating density of 83μg/mg MPs had a lower affinity than the other three SAMPs with less streptavidin coating density.
authors.affiliation: Division of Cardiology, Shanghai Sixth Hospital, Shanghai Jiao Tong University School of Medicine, State Key Discipline Division, Shanghai 200233, PR China; Nano Biomedical Research Center, School of Biomedical Engineering, Med-X Research Institute, Shanghai Jiao Tong University, Shanghai 200030, PR China.
authors.forename: Xiaoyan
authors.initials: X
authors.lastname: Dai
authors.forename: Hong
authors.initials: H
authors.lastname: Xu
authors.forename: Xin
authors.initials: X
authors.lastname: Zhang
authors.forename: Wei
authors.initials: W
authors.lastname: Zhu
authors.forename: Hongchen
authors.initials: H
authors.lastname: Gu
authors.forename: Meng
authors.initials: M
authors.lastname: Wei
mesh_terms: D000906:Antibodies / Q000378:metabolism*; D001710:Biotin / Q000378:metabolism; D019921:Biotinylation; D002264:Carboxylic Acids / Q000737:chemistry; D007118:Immunoassay / Q000379:methods*; D007700:Kinetics; D008163:Luminescent Measurements; D060328:Magnetic Phenomena; D019809:Streptavidin / Q000378:metabolism*
chemical_list: D000906:Antibodies; D002264:Carboxylic Acids; D001710:Biotin; D019809:Streptavidin
keywords: Affinity constant; Biotinylated antibody; Effective binding sites; Streptavidin-coupled magnetic particles