In this study, with the aim of adapting an enzyme-linked immunosorbent assay (ELISA) system for point-of-care testing (POCT), we propose an image analysis method for ELISAs using a centrifugal microfluidic device that automatically executes the assay. The developed image analysis method can be used to quantify the color development reaction on a TMB (3,3',5,5'-tetramethylbenzidine) substrate. In a conventional ELISA, reaction stopping reagents are required at the end of the TMB reaction. In contrast, the developed image analysis method can analyze color in the color-developing reaction without a reaction stopping reagent. This contributes to a reduction in total assay time. The microfluidic devices used in this study could execute reagent control for ELISAs by steady rotation. In the demonstration of the assay and image analysis, a calibration curve for mouse IgG detection was successfully prepared, and it was confirmed that the image analysis method had the same performance as the conventional analysis method. Moreover, the changes in the amount of color over time confirmed that a calibration curve equal to the endpoint analysis was obtained within 2 min from the start of the TMB reaction. As the assay time before the TMB reaction was approximately 7.5 min, the developed ELISA system could detect TMB in just 10 min. In conventional methods using a plate reader, the assay required a time of 90 min for manual handling using microwell plates, and in the case of using automatic microfluidic devices, 30 min were required. The time of 10 min realized by this proposed method is equal to the time required for detection in an immunochromatographic assay with a lateral flow assay; therefore, it is expected that ELISAs can be performed sufficiently to adapt to POCT.
Keywords: ELISA; POCT; centrifugal microfluidics.