Abstract
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-PA2Vtech). 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-PA2Vtech is very useful for validation of antibodies against human protein.
Highlights
Antibodies are widely used for the detection of specific molecules such as peptides, proteins, and chemical compounds
All human recombinant proteins for the array were synthesized as a fusion form of N-terminal FLAG-GST (FG) protein by a wheat cell-free protein production system (Fig. 1A) on 384-well
For construction of a protein array plate, about 14 kinds of FG-proteins were mixed with glutathione-conjugated magnetic beads, and were washed four times with buffer to remove extra proteins from wheat embryo proteins
Summary
Antibodies are widely used for the detection of specific molecules such as peptides, proteins, and chemical compounds. These proteins have a similar epitope, and alanine mutations of these epitope candidates dissolved the reactivity These results indicated that CF-PA2Vtech is very useful for validation of antibodies against human protein. An antibody for detecting a specific virus should be validated by using a wide variety of related viruses to prevent false-positive reactions. The plate was used for validation of two commercially available anti-HA and anti-PD-1 antibodies, and some cross-reactive human proteins with similar epitopes were discovered. These results suggested that our approach using the human protein-array plate for antibody validation, called CF-PA2Vtech, is a useful method for validation of antibodies against human protein
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