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► We developed ODI chemiluminescent enzyme immunoassays (CLEIAs) using two different enzymes. ► ODI-CLEIAs can rapidly and simultaneously quantify multiple-markers in a single sample. ► ODI CLEIAs will be valuable to a wide variety of research fields such as biochemistry, and pathology.
In order to rapidly and simultaneously quantify and screen trace levels of multiple biomarkers in a single sample, rapid 1,1′-oxalyldiimidazole chemiluminescence (ODI CL) was applied as a biosensor of immunoassays using various enzymes such as alkaline phosphatase (ALP) and horseradish peroxidise (HRP). (1) Fluorescein was formed from the reaction of fluorescein diphosphate (FDP) and immuno-complex conjugated with ALP. (2) Resorufin was formed from the reaction between Amplex Red and H
2O
2 in the presence of immuno-complex conjugated with HRP. When ODI CL reagents (H
2O
2 in isopropyl alcohol, ODI in ethyl acetate) were injected in a test tube or strip-well containing fluorescein and resorufin formed from above two reactions a bright CL emission spectrum having two peaks (518
nm for fluorescein and 602
nm for resorufin) was observed. The two peaks can be independently quantified with an appropriate statistical tool capable of deconvoluting multiple emission peaks. In conclusion, we expect that ODI chemiluminescent enzyme immunoassays (CLEIAs) using a couple of enzymes conjugated with antigen or antibody and substrates can rapidly and simultaneously quantify and screen multiple biomarkers in a single sample.