LAL – Bacteria Endotoxin Testing

Background

The discovery of the horseshoe crab’s most significant biological role in recent medicine was made by Frederick Bang in the early 1950′s. Bang discovered that the horseshoe crab’s blood cells, called amoebocytes, contain a clotting agent that attaches to dangerous endotoxins produced by gram negative bacteria. The test was accepted by the United States Food and Drug Administration (FDA) in 1983 as a standard test for endotoxins. In 1987, the FDA established guidelines for LAL testing of pharmaceuticals and medical devices.

Principle of the Test

LAL Chromogenic endotoxin assay utilizes a modified Limulus Amoebocyte Lysate and a synthetic color-producing substrate to detect endotoxin presence. This assay is quantitative and the color intensity developed upon addition of the sample to the LAL supplied with the kit is proportional to the amount of endotoxin present in the sample and can be calculated from a standard curve. The kinetic chromogenic quantitative assay for bacterial endotoxin can be performed at the low assay range (0-5 EU/ml) or the higher range (5-50 EU/ml) depending on sponsor’s request. The assay is performed using a Molecular Devices Microplate reader controlled by Softmax Pro software. During the 60 minute incubation, 241 readings are collected from all wells. The standard curve is deduced from the 241 readings and sample concentrations are calculated automatically based on the standard curve. The assay is highly sensitive.

Sample Requirements

USP requires 3% of the production lot with a minimum of 3 and maximum of 10 devices to be pooled and tested. For liquid samples, a minimum of 0.6 ml is required and powder sample require enough material to reconstitute into a minimum of 1 ml pyrogen-free water. Initial validation on 3 lots must be performed in order to validate the method of testing for a given product.

Samples containing B-Glucans (carbohydrates, mold, starch, plant material, food supplements) interfere with the pyrochrome and produce falsely elevated endotoxin levels. Please inform the laboratory if your sample contains carbohydrates or B-glucans. A special buffer is used to resuspend the pyrochrome in order to overcome the nonspecific interference. There is an additional $30 per sample for samples requiring the special re suspension buffer.

 

The Charles River Endosafe PTS System:

The Endosafe®-PTS™ was used to determine the level of endotoxin in the test article.   The PTS™ utilizes LAL reagents in an FDA-licensed disposable test cartridge with a handheld reader for a completely contained, real-time endotoxin testing system.

Under GLP Conditions Price: $250 – Protocol # CB129A
Under Non-GLP Conditions Price: $195 – Protocol # CB130A

Turnaround time

24-72 hours for LAL performed under cGMP conditions and 48-96 hours for LAL tests performed under GLP conditions.

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