Development of a Spacer-optimized Quenchbody against Tumor Necrosis Factor Alpha

Abstract

Tumor necrosis factor alpha (TNFα) is a proinflammatory cytokine, and its overproduction causes infectious diseases and autoimmune disorders. Therefore, rapid and accurate detection of TNFα is important, and the need for a convenient detection system has increased. In this study, single-chain variable fragment (scFv)-type Quenchbody (Q-body) was developed that could detect TNFα by mixing the Q-body with TNFα, followed by measuring its fluorescence intensity. Recombinant scFvs were constructed with several different lengths of flexible GGGS repeat spacers between dye and scFv. The fluorescent responses of Q-bodies increased in an antigen-dose-dependent manner. The fluorescence intensity in the presence of a maximum concentration of antigen was compared to that of the denatured Q-body, resulting in the Q-body with the (GGGS)5 spacer showing a fully de-quenched signal, whereas other Q-bodies with shorter spacer lengths showed insufficient de-quenching efficiency. These results indicated that adjusting the spacer length is important for improving the response of the Q-body. The Q-body with the optimized spacer length showed a maximum 4.5-fold increase in fluorescence intensity with a broad antigen concentration range, and nanomolar order of detection limit, indicating the usefulness of this Q-body for one-step detection of TNFα.