Chemiluminescent ELISAs

Luminescent immunoassays combine great sensitivity with broad dynamic range. One of the more effective luminescent indicators is Luminol, which, with proper enhancers, gives a bright and stable light emission.

Figure 1. Enhanced Luminol Reaction

Luminol (3-aminophthal hydrazide) was first synthesized as a chemiluminescent substrate in 1928. Unfortunately, the reaction required a harsh reaction conditions, and the light emission was dim and of short duration. The addition of enhancers, however, led to an intense long lasting light output, leading to the wide use of enhanced luminol in immunoassays. The development of 96-well-plate luminometers led the way for manual chemiluminescent immunoassays.

The luminol reaction involves oxidation of HRPO (FeIII) to compound I (FeIV free radical) by hydrogen peroxide. Compound I is then reduced by luminol in a two step process to yield first Compound II (FeIV), then native HRPO and two luminol radicals. The luminol radicals undergo further reactions to yield an electronically excited 3-aminophthalate dianion, which emits light as it returns to the ground state. Many substituted phenols, 6-hydroxybenzothiazoles, napthols and aromatic amines react with compound II with a much higher rate constant, thereby enhancing the chemiluminescent reaction.