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Flag antibody cross reactivity11/4/2023 For example, an antibody for detecting a specific virus should be validated by using a wide variety of related viruses to prevent false-positive reactions. Since the antibody is widely used for multiple applications as described above, the validation methods are based on these applications. However, it is not easy to validate antibody specificity 9. In these utilizations of the antibody, especially for antibody drugs, the specificity of the antibody is the most important feature because the antibody is expected to recognize a single specific target molecule only. In addition, the monoclonal antibody (mAb) has also been used in many medical therapies such as cancer immunotherapies 4, 5 and infectious disease treatments 6, 7, 8. Utilizing these features, the antibody is widely used to detect specific molecules such as proteins, peptides, nucleotides, or small chemical compounds in a broad range of biotechnological applications such as ELISA (enzyme-linked immunosorbent assay), immunoblotting, immunohistochemistry, and immunoprecipitation. These results indicated that CF-PA 2Vtech is very useful for validation of antibodies against human protein.Īn antibody molecule has three major features: 1) high specificity, 2) high affinity, and 3) a high variety of recognition molecules 1, 2, 3. These proteins have a similar epitope, and alanine mutations of these epitope candidates dissolved the reactivity. The cross-reactivity of these proteins was also confirmed by immunoblotting. Using this protein array plate, commercially available anti-HA or anti-PD-1 antibody reacted to 13 or three human proteins, respectively. 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. Full-length human cDNAs were fused to N-terminal FLAG-GST and then synthesized by the wheat cell-free system. Here, we demonstrate a novel protein array technology for antibody validation (CF-PA 2Vtech). 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. However, it is very difficult to confirm antibody specificity. The specificity of an antibody is therefore its most important feature. Antibodies are widely used for the detection of specific molecules such as peptides, proteins, and chemical compounds.
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