The colorimetric method is a quantitative technique for detecting endotoxins, relying on the relationship between the intensity of a color reaction and the endotoxin concentration. This method utilizes the reaction between bacterial endotoxin (lipopolysaccharide, LPS) and a specialized horseshoe crab reagent.
When endotoxin reacts with the horseshoe crab reagent, the following cascade occurs:
Factor C activation: The endotoxin activates factor C in the reagent.
Sequential activation: Activated factor C triggers factor B, which subsequently activates procoagulant proteins.
Coagulase generation: The activated procoagulant proteins are converted into biologically active coagulase.
The coagulase cleaves synthetic chromogenic substrates, producing a measurable color change. The rate of color change and the color intensity are directly proportional to the endotoxin concentration in the sample solution.
To quantify endotoxins:
The change in absorbance at a specific wavelength is monitored during the reaction.
A standard curve, established from solutions with known endotoxin concentrations and their corresponding absorbance changes, is used to calculate the endotoxin content in the sample.