Development of quinone linked immunosorbent assay (QuLISA) based on using Folin’s reagent as a non-enzymatic tag: Application to analysis of food allergens

Abstract

Enzyme-labeled immunoassays are widely used for trace analysis of clinically significant compounds owing to their high selectivity. However, utilizing an enzyme for labeling the antibody leads to many problems, including low reproducibility and inhibition of antigen-antibody immuno-reaction due to the enzymes’ instability and bulkiness, respectively. Hence, our research group has been using quinones as stable non-enzymatic tags for antibodies utilizing the redox cycle of quinones. Herein, we aimed to use the broadly available and cost-effective quinone, 1,2-naphthoquinone-4-sulfonate (NQS, Folin’s reagent), for signal tagging of immunoassays. NQS could bond with biotin in a relatively short time and without using a catalyst forming biotin-1,2-naphthoquinone (BT-NQ). The synthesized BT-NQ produces strong chemiluminescence or color upon mixing with reductant and luminol or tetrazolium salts, with sensitivity down to 7.7 nM and 49.0 nM, respectively. Next, BT-NQ was used for developing the first quinone-linked immunosorbent assay (QuLISA) through labeling biotinylated-detection antibodies using avidin and BT-NQ, and it was targeted to detect food allergen, β-casein using sandwich-immunoassay. Our method showed good linearity for determination of β-casein with good sensitivity down to 20.2 ng/mL. The results of our method were compared with ELISA kit results, and QuLISA showed good matching and higher sensitivity. Moreover, we applied Folin’s reagent for direct labeling of avidin, and the resulted compound possessed strong chemiluminescence originating from its quinone content. At last, we can conclude that the use of Folin’s reagent offered a simple, stable, sensitive, and cost-effective approach for labeling immunoassays and direct chemiluminescence labeling of proteins.