Photometric Quantitative Endotoxin Testing

CA Analytical provides three distinct endotoxin testing methods, offering flexibility to select the most appropriate technique based on the specific properties and requirements of the material being tested.

• Kinetic Turbidimetric Technique – This assay is based on the activation of an enzymatic cascade in the LAL reagent by endotoxins, leading to the formation of a gel-like clot. In this method, the increase in turbidity (cloudiness) due to clot formation is measured over time using a spectrophotometer.
  • Kinetic: Measures changes over time (rather than a single endpoint).
  • Turbidimetric: Based on light scattering caused by turbidity (formation of a clot).
  • As the reaction progresses, the solution becomes increasingly turbid. The time required to reach a certain level of turbidity is inversely proportional to the endotoxin concentration: the more endotoxin present, the faster the turbidity increases.
• Kinetic Chromogenic Technique – This assay combines the kinetic (time-based) measurement of endotoxin activity with a color-producing (chromogenic) reaction.
  • The presence of endotoxins activates a cascade of enzymes in the LAL reagent.
  • One of the enzymes, coagulase, cleaves a synthetic chromogenic substrate.
  • This releases p-nitroaniline (pNA), a yellow-colored compound.
  • The rate of color formation is directly proportional to the endotoxin concentration in the sample.
  • Absorbance is measured spectrophotometrically over time.
  • Faster color development indicates a higher concentration of endotoxin.
• Recombinant Cascade Reagent (rCR) – rCR stands for recombinant Cascade Reagent. It is a synthetic, recombinant version of the LAL enzyme cascade, produced using genetically engineered cells (e.g., insect or bacterial cells). This recombinant system mimics the natural LAL enzymatic reaction but without using any animal products. The assay works by mimicking the native LAL cascade using recombinant enzymes:
  • Endotoxin binds to and activates recombinant Factor C (rFC).
  •  Activated rFC triggers the activation of recombinant Factor B.
  • This leads to the activation of recombinant proclotting enzyme, which cleaves a chromogenic substrate.
  • The cleavage releases p-nitroaniline (pNA), which turns yellow.

The rate of color development is proportional to endotoxin concentration.