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Recombinant Factor C Assay Infographic
- 1. An Animal Free Alternative to Endotoxin Testing Recombinant Factor C Assay It can cause inflammation, fever, septic shock, organ failure or even death. They can easily contaminate samples and products. Endotoxins can survive sterilisation process. Endotoxins testing is part of the regulatory requirement for validation. What is Recombinant Factor C? Recombinant Factor C (rFC) is a recombinantly manufactured protein used for the detection of bacterial endotoxins It strongly binds to the LPS and detect their presenceWhat is endotoxin? Type of pyrogen Present in cell wall of gram negative bacteria Lipid A is responsible for the endotoxin activity Why test for endotoxins? Endotoxins Limits 0.2 EU/kg ( Intrathecal ) 5.0 EU/kg ( Intravenous & intramuscular ) ICH Q4B annex 14 (Bacterial Endotoxins Test GeneralChapter) Pharmacopoei a (Ph. Eur.), (JP) & (USP) Parenteral pharmaceutical products Medical devices Water for injection What is it tested on? Control bioburden in the food industry Raw materials Procedure Binding Step 1 Step 2 Wash Detection Step 3 90 minutes 37 degrees Celsius Shaking Repeat three times 90 minutes 37 degrees Celsius Fluorescence EndoLISA is new method for demonstration of endotoxin (LPS) levels where high affinity binding of endotoxin by LPS- specific phage protein is followed by endpoint fluorescence measurement This assay enables the user to precisely determine the endotoxin level 192 tests, all reagents needed for running the assay included in the kit EndoLISA plates, pre coated microtiter strips Endotoxin-Free Water Binding Buffer Endotoxin Standard Wash Buffer Enzyme Substrate Assay Buffer Cover Foils Product Format without matrix effects without the need for dilution with a very broad measurement range Water Used in rFC Assays Ultrapure Water Sterile Water For Injection Water For Injection Reproducibility of the assay Greater control which results in a product with a greater lot- to-lot consistency Displays consistency and more reproducible results Less variable recovery values for positive product control EndoLISA Sensitivity Is the rFC Assay Better than the LAL-Based Assays? Simple to use Rapid Various compendial test methods available Requires a small amount of test sample Overall inexpensive method Highly standardized LAL test doesn't discriminate between living and dead bacteria High initial expenses False positives possible Maximum valid dilution differs between test methods Nanoparticles may interfere with one or more formats of the standard limulus amoebocyte lysate Lot-to-lot variability Advantages Limitations LAL-Based Assay Endotoxin specific Sustainable resource Sensitivity range from 0.001 to 500 EU/ml Lot-to-lot reproducibility Approved by FDA Comparable to other quantitative LAL methods Higher tolerance for chaotropic agents, organic solvents and detergents Not a compendial test Expensive Only one test method available rFC-Based Assay EU Directive 2010/63 demands The 3R - Replacement, Refinement and Reduction of animals used for scientific purposes Do you know? Advantages Limitations