Measurement of Endotoxin
Bacterial endotoxin is probably the most common significant contaminant that might be found in antibody preparations. It is found ubiquitously in normal environments but its pyrogenic effects in vivo can be lethal. It is absolutely vital to control the le
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22 Measurement of Endotoxin Jenny Phillips, Patrick Harrison, and Geoff Hale 1. Introduction Bacterial endotoxin is probably the most common significant contaminant that might be found in antibody preparations. It is found ubiquitously in normal environments but its pyrogenic effects in vivo can be lethal. It is absolutely vital to control the level of endotoxin in therapeutic products, but its significance for experimental work must not be underestimated since it can have numerous confounding effects both in vivo and in vitro. Methods for removing endotoxin from products have been described (1–4), but in our experience, it is much better to avoid it from the beginning by scrupulous control of raw materials and use of good aseptic technique. To grow, bacteria need water; therefore, the most likely sources of endotoxin are water and any process equipment that has been wet. Water must be obtained from a controlled source that is low in endotoxin. If you do not have a suitable dedicated water purification system, then it is best to purchase purified water. Water for irrigation (from a pharmaceutical supplier) is possibly the most economic. Equipment in contact with cells or products should preferably be sterile disposable plastic. Standard tissue culture ware is usually very reliable. Plastic bags for media and process intermediates are now widely available in all sizes (e.g., Stedim, Aubagne, France) and should be used in preference to glass bottles. Silicone tubing (food or medical grade) is suitable for all fluid transfers and should not be reused. If reusable equipment is essential, it can be soaked in 0.5 M NaOH and/or baked in an oven at high temperature (steam sterilization is not sufficient) (1). Whatever precautions are taken to reduce the risk of endotoxin contamination, it is important to carry out routine assays to check that they are effective. For small and inexpensive antibody batches destined for experimental use, it From: Methods in Molecular Medicine, Vol. 40: Diagnostic and Therapeutic Antibodies Edited by: A. J. T. George and C. E. Urch © Humana Press Inc., Totowa, NJ
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may be sufficient only to test the final batch. For larger batches and therapeutic materials, it is much wiser to test all the process intermediates (media, buffers, and so forth) before they are actually used. This will reduce the risk of final failure of a batch. In the past, endotoxins were measured by injecting the test substance into rabbits and measuring the temperature rise. This is costly and an unnecessary use of animals. Now there are commercial test kits based on the clotting reaction induced in horseshoe crabs (animals are still used, but from a lower order!) (5). The test has been refined with an artificial chromogenic substrate, so that the results can be read in a spectrophotometer, which is technically very much more reliable than visual examination of a clot (Bio-Whittaker Inc, Walkersville, MD). At least two versions of the chromogenic test are available, an endpoint met
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