Selected article for: "assay format and avidity effect"

Author: Chu, Ruiyin; Reczek, David; Brondyk, William
Title: Capture-stabilize approach for membrane protein SPR assays
  • Document date: 2014_12_8
  • ID: ueofl0wn_2
    Snippet: However, since antibodies are dimeric molecules, it is possible that one antibody molecule could bind to two peptides on the chip surface thereby resulting in artificially high affinity through the avidity effect. Affinity (KD) data generated by the reverse format indeed were higher than the data generated by the conventional assay format. Therefore, the higher affinity data for the same antibody, at least in part, is attributed to the avidity ef.....
    Document: However, since antibodies are dimeric molecules, it is possible that one antibody molecule could bind to two peptides on the chip surface thereby resulting in artificially high affinity through the avidity effect. Affinity (KD) data generated by the reverse format indeed were higher than the data generated by the conventional assay format. Therefore, the higher affinity data for the same antibody, at least in part, is attributed to the avidity effect. In order to evaluate the avidity effect in more detail, different levels (5, 20, and 100 RU, equivalent to 0.78, 3.1 and 15.6 fM/mm 2 , respectively) of biotinylated peptides were captured on a SA chip surface and kinetics assays were performed. As shown in Figure 2a , although the overall antibody binding RUs correlated to the peptide level captured, the shape of the sensorgrams for each antibody at different peptide densities were similar. As shown in Figure 2b , at three different peptide immobilization levels, MAB190 showed similar affinity results whereas clone 16D7 was dramatically different. The highest level of peptide (100 RU) did indeed alter the overall affinity (KD), mainly due to artificial Koff (9.85 3 10 28 , Table 2 ), which was outside of the Biacore accuracy range (Koff $ 10 25 S 21 ).

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