CellFree Sciences. Co. Ltd., 3 Bunkyo-cho, Matsuyama, Ehime, 790-8577, Japan.
Proteo-Science Center, 3 Bunkyo-cho, Matsuyama, Ehime, 790-8577, Japan.
Sci Rep. 2019 Dec 18;9(1):19349. doi: 10.1038/s41598-019-55785-5.
Antibodies are widely used for the detection of specific molecules such as peptides, proteins, and chemical compounds. The specificity of an antibody is therefore its most important feature. However, it is very difficult to confirm antibody specificity. Recently, we made a human protein array consisting of 19,712 kinds of recombinant human proteins produced by a wheat cell-free protein production system. Here, we demonstrate a novel protein array technology for antibody validation (CF-PAVtech). Full-length human cDNAs were fused to N-terminal FLAG-GST and then synthesized by the wheat cell-free system. To construct a 20 K human protein array, about 10 to 14 kinds of human proteins were mixed and captured in each well by glutathione-conjugated magnetic beads in 12 plates or one plate with 384- or 1536-well format, respectively, using a strong magnetic device. Using this protein array plate, commercially available anti-HA or anti-PD-1 antibody reacted to 13 or three human proteins, respectively. The cross-reactivity of these proteins was also confirmed by immunoblotting. These proteins have a similar epitope, and alanine mutations of these epitope candidates dissolved the reactivity. These results indicated that CF-PAVtech is very useful for validation of antibodies against human protein.
抗体被广泛用于检测特定的分子，如肽、蛋白质和化合物。因此，抗体的特异性是其最重要的特征。然而，抗体特异性的确认非常困难。最近，我们使用小麦无细胞蛋白生产系统生产的 19712 种重组人蛋白制作了一个人类蛋白阵列。在这里，我们展示了一种新的抗体验证的蛋白质阵列技术（CF-PAVtech）。全长人 cDNA 与 N 端 FLAG-GST 融合，然后通过小麦无细胞系统合成。为了构建 20K 人类蛋白质阵列，大约 10 到 14 种人类蛋白质在每个孔中混合，并用谷胱甘肽偶联的磁珠捕获，在 12 个板或一个 384 孔或 1536 孔的板中，分别使用强磁场装置。使用这个蛋白质阵列板，市售的抗 HA 或抗 PD-1 抗体分别与 13 或 3 个人类蛋白质反应。这些蛋白质的交叉反应性也通过免疫印迹得到了证实。这些蛋白质具有相似的表位，这些表位候选物的丙氨酸突变溶解了反应性。这些结果表明，CF-PAVtech 对于验证针对人类蛋白质的抗体非常有用。
