GATS tag system is compatible with biotin labelling methods for protein analysis.

Polypeptide tags and biotin labelling technologies are widely used for protein analyses in biochemistry and cell biology. However, many peptide tag epitopes contain lysine residues (or amino acids) that are masked after biotinylation. Here, we propose the GATS tag system without a lysine residue and with high sensitivity and low non-specific binding using a rabbit monoclonal antibody against Plasmodium falciparum glycosylphosphatidylinositol (GPI)-anchored micronemal antigen (PfGAMA). From 14 monoclonal clones, an Ra3 clone was selected as it recognized an epitope-TLSVGVQNTF-without a lysine residue; this antibody and epitope tag set was called the GATS tag system. Surface plasmon resonance analysis showed that the tag system had a high affinity of 8.71 × 10-9 M. GATS tag indicated a very low background with remarkably high sensitivity and specificity in immunoblotting using the lysates of mammalian cells. It also showed a high sensitivity for immunoprecipitation and immunostaining of cultured human cells. The tag system was highly sensitive in both biotin labelling methods for proteins using NHS-Sulfo-biotin and BioID (proximity-dependent biotin identification) in the human cells, as opposed to a commercially available tag system having lysine residues, which showed reduced sensitivity. These results showed that the GATS tag system is suitable for methods such as BioID involving labelling lysine residues.

Production of a rabbit monoclonal antibody for highly sensitive detection of citrus mosaic virus and related viruses.

Citrus mosaic virus (CiMV) is one of the causal viruses of citrus mosaic disease in satsuma mandarins (Citrus unshiu). Prompt detection of trees infected with citrus mosaic disease is important for preventing the spread of this disease. Although rabbit monoclonal antibodies (mAbs) exhibit high specificity and affinity, their applicability is limited by technical difficulties associated with the hybridoma-based technology used for raising these mAbs. Here, we demonstrate a feasible CiMV detection system using a specific rabbit mAb against CiMV coat protein. A conserved peptide fragment of the small subunit of CiMV coat protein was designed and used to immunize rabbits. Antigen-specific antibody-producing cells were identified by the immunospot array assay on a chip method. After cloning of variable regions in heavy or light chain by RT-PCR from these cells, a gene set of 33 mAbs was constructed and these mAbs were produced using Expi293F cells. Screening with the AlphaScreen system revealed eight mAbs exhibiting strong interaction with the antigen peptide. From subsequent sequence analysis, they were grouped into three mAbs denoted as No. 4, 9, and 20. Surface plasmon resonance analysis demonstrated that the affinity of these mAbs for the antigen peptide ranged from 8.7 × 10-10 to 5.5 × 10-11 M. In addition to CiMV, mAb No. 9 and 20 could detect CiMV-related viruses in leaf extracts by ELISA. Further, mAb No. 20 showed a high sensitivity to CiMV and CiMV-related viruses, simply by dot blot analysis. The anti-CiMV rabbit mAbs obtained in this study are envisioned to be extremely useful for practical applications of CiMV detection, such as in a virus detection kit.

CF-PA2Vtech: a cell-free human protein array technology for antibody validation against human proteins.

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.