Application of Chromogenic Technique to Bacterial Endotoxins Test

Chromogenic technique is among the three techniques which also contain gel-clot technique and turbidimetric technique to detect or quantify endotoxins from Gram-negative bacteria by using amoebocyte lysate extracted from the blue blood of horseshoe crab (Limulus polyphemus or Tachypleus tridentatus). It could be classified as an endpoint-chromogenic assay or a kinetic-chromogenic assay based on the particular assay principle employed.

The reaction principle is that: the amebocyte lysate contains a cascade of serine protease enzymes (proenzymes) which can be activated by bacterial endotoxins. Endotoxins activate the proenzymes to produce activated enzymes (termed coagulase), the latter catalyzes the split of the colorless substrate, releasing a yellow-colored product pNA. The released pNA can be measured photometrically at 405nm. And the absorbance is positively correlated with the endotoxin concentration, then the endotoxin concentration could be quantified accordingly.