A Simple Method for Assessment of Human Anti-Neu5Gc Antibodies Applied to Kawasaki Disease
Figure 3
Specific detection of anti-Neu5Gc IgG in normal human serum.
A. Plates were coated with WT or Cmah-KO mouse sera and Anti-Neu5Gc IgG of human serum S34 [16] tested by ELISA. B. Plates were coated with WT or Cmah-KO mouse sera, then human serum S34 IgG binding tested by ELISA (0) or by EIA after pre-incubation of S34 with Cmah-KO mouse sera (as depicted in Fig. 1B). EIA specifically inhibited anti-Cmah-KO (anti-mouse) reactivity, resulting in Neu5Gc-specific reactivity on WT coated plates. For S34, the optimal Cmah-KO inhibitor is at 1∶4000 dilution. C. Sialic acid specific binding demonstrated by mock versus mild periodate pre-treatment of coated wells. Human serum S34 (1∶100) tested over WT mouse serum without inhibitor (0) or by EIA method with diluted Cmah-KO mouse sera. In the presence of Cmah-KO inhibitor, S34 binding to WT was abolished compared to mock treated wells hence specificity is towards sialylated-(Neu5Gc)-glycans. Numbers 1–4 refer to the specific steps predicted by the model, as described in Fig. 1D. Each figure is representative of at least two independent experiments. Bars are mean ± s.d. of triplicates (A–C).