Figure 6
Autoantibody detection by the second-designed protein chip, to which, 1 hr-incubation was applied for the cross-linking of antigen proteins. (A-L) Full color fluorescence images of the chips by GenePi×4000B. Detected channel for Alexa555 or Alexa633 are indicated in left bottom of each figure. The abbreviations are same as for the first protein chip in Figure 2. Additionally, in the top row, two 64 fmol-spots of mouse IgG (m, detected in A, C and G), rabbit IgG (r, in B and D), and human IgG (h, in E-F, H-L) were immobilized for the secondary antibody detection control. (A-D) The protein spots certification. After denaturation step, the spots were detected by anti-GFP antibody and following anti-mouse IgG-Alexa633 secondary antibodies (A), anti-HSP70 and anti-rabbit IgG-Alexa555 (B), anti-SOD2 and anti-mouse IgG-Alexa633 (C), anti-PRDX6 and anti-rabbit IgG-Alexa555 (D). (E-L) Autoantibody detection on the chips. Autoandibody mode by ×5 diluted sera and anti-human IgG-Alexa633 (E-F, H-L), or total protein mode by anti-GFP antibody and anti-mouse-Alexa555 (G only) on the same chip of F. Chip images of the anti-human IgG antibody only (E), or six serum samples tested in Figure 5, C7 (F and G), No.150 (H), No. 193 (I), No. 222c (J), No. 419 (K), and No. 259 (L) are shown. (M) Quantitative results of the chips. Autoantibodies/ spot (%) in the y-axis is showing percentage of proteins on each spot bound to autoantibody for sera, calculated after normalization of each signal mode by human IgG and mouse IgG control spots. Statistical significance of the detection from each GFP-fused recombinant antigen protein was compared with that from GFP on the same chip by the same sera, shown as *** < 0.0001, 0.0001 < ** < 0.001, 0.001 < * <0.01 by Student-t test. (h), (+/+), (+/−) indicates serum samples from healthy individuals, HCV+/HCC + patients, HCV+/HCC- individuals, respectively.