Chemiluminescence enzyme immunoassay enables to measure trace components with high sensitivity and selectivity due to high specificity of antigen-antibody reaction and high sensitivity chemiluminescence test. However, using enzyme-labeled antibodies suffer from problems such as low reproducibility due to the instability of the enzymes and the inhibition of antigen-antibody reaction due to its steric effect. Therefore, here we reported a chemiluminescence immunoassay innovative non-enzymatic labeling reagent through the use of quinone as a tag-signal generator coupled to biotin as a binder, to address the problem of enzymatic labeling.
Biotin-1,4-naphthoquinone (biotin-NQ) are synthesized and characterized and it can produce long lasting chemiluminescence upon mixing with dithiothreitol and luminol popularity quinone redox cycle. Biotin-NQ showed remarkable stability against stress factors of different organizations may face during an immunoassay such as high temperatures, conditions that are very acidic and alkaline, and repeated freeze-thaw cycles.
On the other hand, all of these conditions led to a decrease in the reactivity of the enzyme label because it may denature its protein structure. Finally, the biotin-labeled antibody measurements successfully performed using NQ-biotin and avidin. As a result, the antibody can be detected down to 25.7 nM were 2.5 times sensitive of biotin-HRP-enzyme immunoassay kemiluminesens. In addition, our method is successfully applied to the determination of the immobilized antibody using biotinylated avidin and biotin-NQ, which simulates immunoassay.
Avidin can be detected down to 23.4 nM with excellent linearity (r = 0.996). Accordingly, our advanced reagent, biotin-NQ, can be used as a very stable universal, cost-effective, and steric free non-enzymatic labels for immunoassay.
