Scheme 1. Luminol chemiluminescence of the antigen-binding antibody conjugated with HRP in All-in-One Immunoassays.
Scheme 2. Luminol chemiluminescence of the antigen-binding antibody conjugated with ABEI in All-in-One immunoassays.
Luminol chemiluminescence can be applied to as a detection method of the all-in-one immunoassay. As shown in Scheme 1, the antigen-binding antibody conjugated with horseradish peroxidase (HRP) formed in the All-in-One immunoassay emits blue chemiluminescence with the addition of luminol (or isoluminol) and hydrogen peroxide in Tris-HCl (pH 8 – 9) containing an enhancer, such as 4-iodophenol, 4-( midazole-1-yl)phenol, 4-bromophenol, 4-phenylphenol, 3-(10’-phenothiazinyl) propane-1-sulfonate, and 4-morpholinopyridine. Also, the all-in-one immunoassay can be operated with the antigen-binding antibody conjugated with N-(4-aminobutyl)-N-ethylisoluminol (ABEI) to emit bright luminol chemiluminescence with the addition of HRP, hydrogen peroxide in Tris-HCl (pH 8 – 9) containing an enhancer as shown in Scheme 2. As examples, All-in-One Immunoassay using 2 different luminol chemiluminescence detections quantified trace levels of D-dimer (< 0.5 ng/ml) in human serum based on 2 reaction mechanisms in Schemes 1 and 2.