BET METHOD OPTIONS
Methodology & Applications Through free consultation and support we will assist you to determine the method that best suits your needs and sample type. ACC offers all regulatory compliant kinetic and gel-clot BET methods. Introduction Limulus Amebocyte Lysate (LAL) tests detect and quantify bacterial endotoxins derived from the outer cell wall membrane of gram-negative bacteria. The critical component of the LAL reagents used in endo- toxin tests is derived from blood cells (amebocytes) of the horseshoe crab, Limulus polyphemus. Amebocytes contain the proteins of the blood clotting mechanism, which is triggered primarily by endotoxins and also by (1 g 3)- b - D -Glucan. LAL reagents are primarily used to test for endotoxins in injectable pharmaceuticals, bi- ological products, medical devices and renal dialysis centers. Endotoxin tests are described in the Bacterial Endotoxins Test chapter in the United States Pharma- copeia (Chapter <85>) and in the equivalent chapters in the European Pharmacopoeia (Chapter 2.6.14) and the Japanese Pharmacopoeia (General Tests, No. 4.01). Modified LAL reagents can be used for specific detec- tion of (1 g 3)- b - D -Glucans. Selecting a Method Consider the following when deciding which Bacterial Endotoxins Test method to use:
endotoxin concentrations and greater dilution of sam- ples, which is important for overcoming interference. Both kinetic methods utilize software to quantify your test results. The gel-clot method is a simple, positive/ negative, low start-up cost alternative that has been the reference method for years. Kinetic Testing Methods Chromogenic Method The BET reagent is formulated with a synthetic sub- strate which produces a chromophore when cleaved by endotoxin activated enzymes. • Requires either the Pyros Kinetix ® Flex tube reader or an incubating plate reader system such as the BioTek ELX808 IU TM * • Maximum sensitivity to 0.001 EU/mL, highest chro- mogenic sensitivity available in the BET industry when using ACC’s Pyrochrome ® reagent
• Electronically stored data
• Incubation time varies depending on the standard curve range • High sensitivity allows for greater dilution to over- come interference Turbidimetric Method The optical density (turbidity) increase that accompa- nies the clotting reaction is read in our Pyros Kinetix ® Flex tube reader or in an incubating microplate reader. • Requires either the Pyros Kinetix ® Flex tube reader system or an incubating microplate reader such as the BioTek ELX808 IU TM * • Maximum sensitivity to 0.001 EU/mL, highest sensi- tivity available in the BET industry when using ACC’s Pyrotell ® -T reagent • Quantitative test results and electronically stored data • Incubation time varies depending on the standard curve range. Results can be obtained in as little as 15 minutes with ACC reagents
• What are the regulatory requirements, if any?
• What type of product or sample is to be tested?
• What test sensitivity is required? (What is the endotoxin limit specification for the sample?)
• Is quantitative analysis desired?
There are three principal Bacterial Endotoxins Test methods: the chromogenic, turbidimetric and gel-clot methods. The first two may be grouped together as kinetic photometric methods as they require a timed optical reader. Both chromogenic and turbidimetric methods offer the greatest sensitivity, allowing detection of low
*Trademark of BioTek Instruments, Inc.
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