Evaluation of microarray surfaces and arraying parameters for autoantibody profiling

Abstract

Autoantigen microarrays are being used increasingly to study autoimmunity. Significant variation has been observed when comparing microarray surfaces, printing methods, and probing conditions. In the present study, 24 surfaces and several arraying parameters were analyzed using >500 feature autoantigen microarrays printed with quill pins. A small subset of slides, including FAST, PATH, and SuperEpoxy2, performed well while maintaining the sensitivity and specificity of autoantigen microarrays previously demonstrated by our laboratory. By optimizing the major variables in our autoantigen microarray platform, subtle differences in serum samples can be identified that will shed light on disease pathogenesis.

Figure 1

Autoantigen microarray analysis using a histidine-specific mAb. (A) Intraslide CVs and (B) interslide CVs were compared for a panel of recombinant human his-tagged antigens printed on FAST, PATH, and SuperEpoxy2 slides at 200 µg/mL using a robotic microarrayer. (C) MFI-Bs were compared for U1-A on FAST, PATH, SuperEpoxy2, SuperEpoxy, and poly-l-lysine slides printed at 400, 200, 100, and 50 µg/mL. (D) MFI-Bs were compared for a panel of recombinant human his-tagged antigens printed at 200 µg/mL on FAST, PATH, and SuperEpoxy2 slides. Six microarrays of each slide surface were probed with a histidine-specific His-Tag mAb (Novagen^®) and Cy3-conjugated GAM IgG and IgM secondary antibody (Jackson ImmunoResearch Laboratories). For (A), the CV for each antigen was calculated from eight replicate features on each slide, and the median intraslide CV for six slides was calculated for each antigen. For (B), the median MFI-B for each antigen was calculated from eight replicate features on each slide, and the interslide CV among six slides was calculated. The “median” data points represent the median of CVs for all 18 his-tagged antigens for each surface. For (C) and (D), each data point represents the median MFI-B of 48 features calculated from eight replicate features on six slides of each surface. CV = (SD/mean)100 of the MFI-B β2GP1, β2 glycoprotein 1; CENP-B, centromere protein B; Jo-1, histidyl-tRNA synthetase; Ku, Ku (p70/p80); La, La/SS-B; LC1, liver cytosol type I-antigen; PCNA, proliferating cell nuclear antigen; PL-12, alanyl-tRNA synthetase; PL-7, threonyl-tRNA synthetase; Ribo P, ribosomal phosphoprotein P0; Ro52, Ro/SS-A 52 kDa; Scl-70, DNA topoisomerase I; TPO, thyroid peroxidase; U1–68, U1-snRNP-68 protein; U1-A, U1-snRNP-A protein; U1-C, U1-snRNP-C protein.

Figure 2

Autoantigen microarray analysis using human positive control sera. Autoantigens were printed on FAST, PATH, and Super- Epoxy2 slides using a robotic microarrayer, probed with human positive control sera (ImmunoVision) and Cy3-conjugated GAH IgG and IgM secondary antibody (Jackson ImmunoResearch Laboratories). (A) Intraslide CVs for each serum sample were calculated for relevant antigens on the microarray. For Jo-1, TPO, ssDNA, Ribo P, RNP, Smith, and La the relevant antigens are the same as the sera named. Relevant antigens for centromere include CENP-A and CENP-B; for mitochondrial include α-ketoglutarate dehydrogenase and pyruvate dehydrogenase; and for histone include histones H1, H2a and H4, H2b, and H3. (B) Titration of the ribo P-specific human positive control serum on FAST slides revealed decreasing fluorescence of ribo P and other positive antigens with higher dilutions of the sample. All microarrays were probed with Cy3-conjugated GAH IgG and IgM secondary antibody (Jackson ImmunoResearch Laboratories) at 1:2000 except for a control microarray with no secondary added. Antigens were printed in replicates of eight. For (A), each human positive control sera data point represents the median CV for eight replicates of all representative antigens on one microarray and the median data point represents the median of CVs for the 11 different sera. For Fig. 2B, each data point represents the median of eight replicate features on one microarray. RNP, ribonuclear protein.