Generation of Q-bead against bone Gla protein with simplified preparation steps.

Quenchbody (Q-body)-based immunoassays enable the detection of antigen within a few minutes with high sensitivity and specificity, thereby revealing their applicability as biosensors for quantifying several biomolecules of interest; however, while producing a Q-body, it is necessary to eliminate the unconjugated dye after labeling to separate the Q-body from the capturing bead and to change the buffer using ultrafiltration, which is time-consuming and leads to yield reduction. In this study, we generated a recombinant single chain variable fragment against bone Gla protein as a model antibody. We labeled the antibody with a dye to generate a Q-body and subsequently added affinity beads to the Q-body mixture. After washing, we directly added antigen without extracting the Q-body from the bead and then measured the fluorescence intensity. As a result, the antigen-dependent fluorescence response was obtained from "Q-bead", which was almost the same as that of the Q-body generated according to the conventional method. The Q-bead was generated within only 2.5 h, thus requiring an hour and two steps less than those required for the generation of the traditional Q-body. No expensive Flag peptide was required to recover the total antibody from beads. Moreover, the ultra-filtration step was eliminated in this bead-based method, leading to improved convenience and cost- and time-saving attributes. The Q-bead-based assay can be used as a standard protocol for simple and rapid analysis of antibody-based molecular detection.

Generation of a novel antibody against BxPrx, a diagnostic marker of pine wilt disease.

BACKGROUND: Bursaphelenchus xylophilus is a pathogenic nematode that causes pine wilt disease (PWD). To prevent the rapid spread of this pathogen, developing a method for rapid and accurate detection of B. xylophilus is required. METHODS AND RESULTS: In this study, we produced a B. xylophilus peroxiredoxin (BxPrx), which is a protein that is overexpressed in B. xylophilus. Using recombinant BxPrx as an antigen, we generated and selected a novel antibody that binds to BxPrx via phage display and biopanning. We subcloned the anti-BxPrx single-chain variable fragment-encoding phagemid DNA to mammalian expression vector. We transfected the plasmid into mammalian cells and produced a highly sensitive recombinant antibody that enabled nanogram order detection of BxPrx. CONCLUSION: The sequence of anti-BxPrx antibody as well as the rapid immunoassay system described here can be applied for rapid and accurate diagnosis of PWD.