Kinetic Chromogenic Bacterial Endotoxin Determination (LAL) – GLP

Protocol # CB129, Turnaround Time: 2-5 Days, Price: $325 per sample (includes testing 6 sample concentrations in duplicates).
The Kinetic Chromogenic Detection Assay for Bacterial Endotoxin utilizes a Limulus Amoebocyte Lysate Chromogenic Substrate. We use reagents provided by Associates of Cape Cod in Massacheusetts. Samples are analyzed undiluted, diluted 1:10, 1:50, 1:100, 1:200, 1:400. Blanks and standards are added to appropriate wells according to a workplan prepared prior to assay initiation. In addition, two weeks of test article spiked with the 0.5 EU/mL standard are analyzed to assess if the test article had any inhibitory effect on the chromogenic substrate. The Chromogenic Substrate is added to all wells. Absorbance of microplate wells is then read at 405 nm at 37 C using the Molecular Devices Thermomax Microplate Reader and Softmax Pro Software. The Kinetic assay typically runs for 60 minutes and collects data from 241 independent reads of the microplate. The standard curve is constructed by regression of the log onset time on the log endotoxin concentration for the standards. The equation for the regression line describes the standard curve. The blank readings were generated using distilled water to adjust the spectrophotometer to zero absorbance and background was subtracted. Standards 5.0, 0.5, 0.05 and 0.005 Endotoxin Units per milliliter (EU/mL) are used to generate the standard curve and calculate the endotoxin concentration in the test article. The linear graph shown below demonstrates a sample of data obtained with an assay that was performed at a linearity range from 0.005 to 5.0 EU/mL. A minimum “R” value of 0.98 is required in order to consider an assay valid.
It has been demostrated that test articles containing carboyhdrates will result in a significant interference with the chromogenic substrate that results in a falsely elevated endotoxin concentration value. The use of B-shield reconstitution buffer reduces the interference but does not elimiate it entirely. We have determined that using dilutions to calculate the final endotoxin value may help in reducing the interference from carbohydrates with the substrate.

It has also been demonstrated that test articles with pH buffer indicators, such as cell culture media, causes an interference with the chromogenic substrate and it is advisable to avoid sending test articles that have a color or that have pH indicators. One way to avoid this issue if one is testing cells in cell culture media is to spin the cells down and resuspend them in sterile Phosphate budffered Saline. Please call Dr. Kilani at the lab. if you need advise or technical assistance with sample submission.

The sponsor will be required to review and approve a protocol prior to study initiation. Please fill out the sample submission form below in either Word or PDF format and send along with the samples.