Ascites of 62,208 PETAL mAbs were prepared in 162 384-well plates and printed onto nitrocellulose-coated slides (FAST Maine Manufacturing, no. 10486111) in a high-density microarray format (named as PETAL array) using the Marathon System (Arrayjet Ltd., UK). Approximately 100 pl of ascites was printed for each antibody per spot. The array and block layout are shown in Fig. 1C. A total of 110 blocks were aligned into 10 rows and 11 columns. Each block contains a subarray of 48 × 12 = 576 individual antibody spots, except the subarrays in the last row were printed with 40 × 12 = 480 (three blocks) or 39 × 12 = 368 (eight blocks) spots. Additional control rows, including a positioning fluorescent spot (Cy3) and a biotin–bovine serum albumin (BSA) gradient (0.4 to 50 pg) of eight spots, were also printed for each block similar to previous antibody arrays (48, 49). Biotin-BSA was prepared by saturated labeling of BSA with Thermo Fisher Sulfo-NHS-LC-Biotin (no. 21336) labeling reagent. PETAL arrays were stored at −80°C.

To evaluate PETAL array quality, the slides were blotted directly with a mixture of a Streptavidin-Cy3 (Sigma, no. S6402) and a Cy5-labeled goat anti-mouse IgG (Jackson ImmunoResearch, no. 115-175-146), both at a dilution of 1:3000 in 1× PBS. Fluorescence of Cy3 and Cy5 was recorded using 532- and 635-nm channels by the GenePix 4200A Microarray Scanner (Molecular Devices LLC). Images were analyzed using GenePix Pro 6.0 software to give fluorescent intensities of each spot and its corresponding background. Missing or distorted spots, typically controlled under 5% of the total spots, were automatically marked by the software.

Reproducibility of array experiments was evaluated by incubating a triplicate of the same sample with three PETAL arrays. The fluorescent intensity of each spot was normalized using biotin-labeled BSA signal in each block and array. The normalized fluorescent intensities were plotted between every experimental pair. Pearson product-moment correlation coefficient value (R value) was calculated for each data pair in which the r value of +1 means total positive correlation and 0 is no correlation (50).

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