Kinetic Endotoxin Chromogenic
November 20, 2012Download
Related Products: Gen5 for Detection
Gen5 version required: 1.0 or higher
Basis for the Assay:
Endotoxins are a class of compounds found in the outer layer of some gram-negative bacteria and are prevalent in the environment. Chromogenic endotoxin assays typically use a modified Limulus Amoebocyte Lysate (LAL) and a colored substrate to detect endotoxins. There are endpoint and kinetic methods for the assay. This sample describes a typical quantitative kinetic protocol wherein the intensity of color produced during the reaction is proportional to the amount of endotoxin in the wells. This protocol is set up to: a) find the concentration of endotoxin in samples by comparing the sample’s Onset Time to the Onset Time of the standards and b) to calculate the amount of endotoxin recovered from sample controls (also known as “spikes” or “positive product controls”) which have had a known concentration of endotoxin added to them initially.
The assay is run at 370 C, with a shake prior to the kinetic measurement. Kinetic measurements are taken in intervals of about 19 seconds for just over one hour at 405 nm. Data Reduction steps include a Well Analysis to find the Onset OD (and Onset Time), Curve Analysis of (Onset Time v Concentration) and a Transformation formula to find the percent recovery of endotoxin in sample wells.
The experiment file contains the absorbance measurements used in the data reduction results to determine the Onset OD, the standard curve, and percent recovery of endotoxin.
This sample layout includes 5 standards (50 to 0.005 eu/ml), each in triplicate. Samples 1 through 7 are in duplicate, as are the sample control wells. Samples 1-4 and sample controls 1-4 are diluted x 40.
This report includes a matrix containing the Well IDs, Onset Time and Percent Recovery. A second matrix includes the kinetic curves of al wells. The standard curve is presented as well as statistics tables for the standards, samples and sample control concentration results.