The key methodological principle of chromogenic assays is to reveal the presence of the analyte in a test sample via chemically-induced visible color changes. The resulting color is then measured using spectrophotometric methods to reveal the concentration of the analyte in the sample.
This method forms the basis of the chromogenic LAL assay. The LAL reagent is mixed with a chromogenic reagent (a peptide connected to p-nitroaniline, a yellow colorant) to produce a synthetic chromogenic substrate. This is added to the test sample to create the test solution, which is then incubated.
If endotoxins are present in the sample, the subsequent enzymatic reactions of the LAL reagent break the peptide bonds connected to the p-nitroaniline molecules, releasing the yellow color into the solution. The more endotoxin present, the more yellow the solution will become. This can be quantitated using a spectrophotometer or absorbance plate reader to reveal the specific endotoxin concentration. The chromogenic nature of the assay makes it the most appropriate choice for testing small volume parenterals, vaccines, antibiotics and biologicals. It is also ideally suited to testing inhibitory products, which may interfere with the clotting mechanism in turbidimetric and gel clot LAL assays.