How sensitive is the HRP chemiluminescent substrate?

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Chemiluminescence immunoassay, represented by luminescent reagents such as luminol and acridinium esters, is a very popular biochemical detection and analysis method in recent years. This method is known for its high degree of automation and ultra-high sensitivity. The sensitivity of chemiluminescence analysis is much higher than that of immunoturbidimetric and enzyme-linked immunoassay.


Compared with radioimmun在·oassay, immunoturbidimetry, colloidal gold, and enzyme-linked immunoassay are more advanced, and the sensitivity is already high. The detection range is milligrams to micrograms, and some detection limits or quantification limits can reach 0.1μg/L , That is, tens to one hundred nanograms; and the sensitivity of chemiluminescence is higher, which can reach the level of picograms, femtograms or even hundreds of gram. An egg is 50 grams, 1 gram is 1,000 milligrams, and 1 milligram is 1,000 nanograms. Then, by analogy, Pike, Feike, Ake and so on are all 1,000 times. This can see the gap between the sensitivity.


HRP chemiluminescence substrate sensitivity

Commonly used chemiluminescent substrates include horseradish peroxidase HRP substrate and alkaline phosphatase AP substrate.

1. Chemiluminescence substrate of HRP

HRP has a molecular weight of about 40kDa and has become a conventional enzyme for chemiluminescence western blot detection. Its chemiluminescence substrates include luminol and isoluminol, also called luminol. The luminescence performance of isoluminol can be significantly improved after modification. The improvement and development of HRP chemiluminescent substrates make it more sensitive to Western blotting than AP substrates. Its stability and smaller molecular weight make its application gradually popular. By labeling more molecules per IgG, higher detection sensitivity can be achieved.

The chemiluminescent substrate used for horseradish peroxidase (HRP) is mostly a two-component system, consisting of a stable peroxide solution and an enhanced luminol solution.


2. AP chemiluminescent substrate

Alkaline phosphatase (AP, 86kDa) is often used as a Western blot detection. The substrate is dephosphorylated by AP to produce a metastable dioxetane phenate anion intermediate, which is decomposed and synthesized at a wavelength of 475nm. Emit the light with maximum intensity, the emitted light can remain stable within 24-96 hours, allowing multiple exposures.


In addition to HRP substrates luminol and isoluminol, Desheng also has a direct chemiluminescence reagent acridinium ester. Compared with HRP substrates and AP substrates, it can directly emit light without enzymatic catalysis. It has more advantages in quantitative detection.