Endotoxin Testing by Limulus Amoebocyte Lysate (LAL) Chromogenic Assay

Principle of Chromogenic test

The chromogenic method was first developed in 1977 by investigators from Japanese, who determined that endotoxin-activated LAL would cleave amino acid cleavage sites containing chromophore pNA (p-nitroanilide)[1]. This reaction causes the release of pNA, which gives its distinctive yellow color to the assay. The chromogenic assay measures the absorbance of light at 405 nm, where the pNA molecules are absorbed. The amount of endotoxin within the sample is directly proportional to the degree and rate of light is absorbed. [2,3]

The basic principle flow is as follows:

Endotoxin testing By Limulus Amoebocyte Lysate (LAL) Chromogenic Assay

Method of Chromogenic test

Both the kinetic chromogenic method and the endpoint chromogenic method require a standard curve to quantify the endotoxin concentration presents in a sample. For the kinetic chromogenic method calculates the concentration of endotoxin by comparing sample's reaction time with the reaction time of the endotoxin standard curve. The endpoint chromogenic method measures the pNA after a set incubation period to determine the endotoxin concentration. [3][4]

Advantages of Chromogenic test:

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  1. T. Morita et al., Jpn J Med Sci Biol. PMID,1978, 31(2):178-81.
  2. Wenqiong Song, Xianting Ding. Methods of Endotoxin Detection, Journal of Laboratary Automation, 2015, Vol.20(4) 354-364.
  3. Alex Wheeler. Comparing Endotoxin Detection Methods, Pharmaceutical Technology, 2017 Vol.41(11), 58-62
  4. Timothy J. Joiner. Comparison of Endotoxin Testing Methods for Pharmaceutical Products, International Journal of Pharmaceutical Compounding, 2002, Vol. 6 No. 6 November/December.

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