Home

ToxinSensorTM

1. Chromogenic LAL Endotoxin Assay Kit VI Troubleshooting continued Problem Possible Cause Suggestions Endotoxin may be adhering to glass surfaces We recommend dissolving the 6 EU lyophilized Endotoxin standard is not endotoxin standard with 1 ml LAL No linearity mixed well reagent water as stated by the protocol the step 1 in Test Procedure and mixing standard endotoxin dilutions for 15 minutes with a vortex mixer The pH value of samples is not suitable for assay Adjust the pH value of your sample to pH 6 8 as stated by the protocol The negative blank shows a higher OD than that of standards using L00350 The materials e g tips vials etc may be contaminated Proceed to the reagent preparation area in a laminar flow cabinet at room temperature Wear disposable gloves and use endotoxin free materials in order to avoid contamination 13 Protocols continued Semi Quantitative Detection Protocols ToxinSensor Gel Clot Endotoxin Assay Kit Proceed to the Reagent Preparation Area in a laminar flow cabinet at room temperature Wear disposable gloves and use endotoxin free materials in order to avoid contamination I Specimen Preparation pH The pH value of the sample should be at pH 6 8 to ensure good linearity Consequently we recommend adjusting the PH value with HCI or NaOH Dilution Dilution is the most important strategy for dealing with
2. The reconstitution should be vortexed for at least 15 minutes with a vortex mixer Reconstituted E coli endotoxin standard can remain stable for up to 15 days if stored at 20 C III Test procedure Appropriate positive and negative controls are an integral part of each assay LAL Reagent Water can be used as a negative control 1 Carefully dispense 0 1 ml of LAL solution into different Endotoxin free vials Label them negative control positive control and samples 2 Carefully transfer 0 1 ml of positive control negative control and the test samples to the LAL reagent in prepared dispensing vials Cap the vials and mix them thoroughly 15 Protocols continued Semi Quantitative Detection Protocols continued ToxinSensor Gel Clot III Test procedure continued Endotoxin Assay Kit 3 Incubate the incubation rack with all vials in 37 C 1 C continued with non circulating hot water or oven Keep racks standing while incubating 4 Remove the rack after 60 2 minutes of incubation Invert each vial and check whether a gel has formed or not Do not shake vigorously while checking it will break up gel consistency a A positive reaction is characterized by the formation of a firm gel that remains intact when the vial is inverted b A negative reaction is characterized by the absence of a solid clot The lysate may show an increased turbidity or viscosity This is considered a negative result a Calculate o
3. 01EU ml 0 005EU mI Final Endotoxin Concentration III Test Procedure 1 Carefully dispense 100 ul of standards samples and LAL reagent water into different endotoxin free vials and label them as standard 1 2 3 4 5 sample 1 2 etc and blank Sample should be mixed thoroughly for 30 seconds with a vortex mixer too Bubbles must be avoided 2 Add 100 ul of reconstituted LAL to each vial Cap the vials and vortexy for 3 seconds with a vortex mixer 3 Incubate the rack with all vials in a 37 C oven for 45 minutes If the endotoxin concentration is in the range of 0 1 1 EU ml incubate in a 37 C 1 C oven for only 10 minutes iO Endotoxin Assay Kit continued chromogenic substrate solution to each vial Mix gently by swirling Do not shake or invert vortex to avoid foaming then incubate for 6 minutes in a 37 C oven 5 Add 500 ul of reconstituted stop solution color stabilizer 1 to each vial and swirl gently to mix well Do not shake or invert vortex to avoid foaming Then add 500 ul of color stabilizer 2 to each vial and mix well Finally add 500 ul of reconstituted color stabilizer 3 to each vial Gently swirl each vial to mix well for 3 seconds Bubbles must be avoided Read the absorbance of each reaction at 545 nm with distilled water as blank to adjust the photometer to zero absorbance The whole test procedure is also summarized in the following table Stan
4. an example The dilution of endotoxin standards and the incubation temperature are major factors that influence the OD value so the endotoxin standards should be dissolved well in LAL water and the incubation temperature should be 37 1 C 11 ToxinSensor Endotoxin Detection System www genscript com Protocols continued Quantitative Detection Protocols continued ToxinSensor Chromogenic LAL Endotoxin Assay Kit continued V Performance Characteristics Linearity The linearity of the standard curve within the concentration range used to measure endotoxin values must be verified At least 4 endotoxin standards spanning the expected concentration range should be assayed along with a blank in duplicate The absolute value of the coefficient of correlation r for the individual mean absorbance of the standards vs their corresponding endotoxin concentration should be 20 980 Reproducibility Replicate samples should be run in order to establish good technique and low coefficient of variation The coefficient of variation C V equals 100 times the standard deviation of a group of values divided by the mean and is expressed as a percent The C V absorbance should be less than 10 2 5 ToxinSensor Endotoxin Detection System www genscript com ToxinSensor Endotoxin Detection System www genscript com Protocols continued Quantitative Detection Protocols continued ToxinSensor
5. GenScript The Biology CRO GenScript USA Inc 860 Centennial Ave Piscataway NJ 08854 Tel 732 885 9188 732 885 9688 Fax 732 210 0262 732 885 5878 Email product genscript com ToxinSensor Endotoxin Detection System Version 12172010 User Manual GenScript The Biology CRO ToxinSensor Endotoxin Detection System www genscript com Table of Contents Intended Use 1 Warning 20 2200 e cece cece reece 1 Background 1 Product Overview 3 Protocols 7 Customer References 18 Related Products 19 ToxinSensor Endotoxin Detection System www genscript com ToxinSensor Endotoxin Detection System www genscript com a i Intended Use GenScript ToxinSensor Endotoxin Detection System is After checking factors such as budget amounts of protein intended for use as an in vitro end product endotoxin test sample test frequency and assay sensitivity you can for human and animal parenteral drugs biological products select one endotoxin detection kit from GenScript and medical devices The system is not intended for use in the detection of endotox
6. ays 110 00 L00408 ToxinEraser Endotoxin Removal Advanced Kit 3 5 Assays 220 00 L00402 ToxinEraser Endotoxin Removal Resin 1ml 60 00 M01053 ToxinEraser Regeneration Buffer 125 ml 25 00 M01054 ToxinEraser Equilibration Buffer 125 ml 25 00 19 Technical Support Visit the GenScript Web site at www genscript com for 1 Technical resoures including manuals MSDS FAQ etc 2 Online 2010 2011 Product Catalog 3 Additional promotions and special offers Any question about Products please email us at product genscript com 20
7. dards Samples Blank Standards ml 0 1 Samples ml 0 1 LAL Reagents Water ml 0 1 LAL ml 0 1 0 1 0 1 Mix well and incubate at 37 C 1 0 C min 45 45 45 Substrate solution ml 0 1 0 1 0 1 Mix well and incubate at 37 C 1 0 C min 6 6 6 Stop Solution ml 0 5 0 5 0 5 Color stabilizer 2 ml 0 5 0 5 0 5 Color stabilizer 3 ml 0 5 0 5 0 5 Mix well and read the absorbance at 545 nm gt 10 ToxinSensor Endotoxin Detection System www genscript com Protocols continued Quantitative Detection Protocols continued ToxinSensor Chromogenic LAL Endotoxin Assay Kit continued IV Calculation Concentration Under the standard conditions the absorbance at 545 nm is linear in the concentration range of 0 005 to 0 1 or 0 1 to 1 EU ml endotoxin Plot the mean absorbance for the four standards on the x axis the corresponding endotoxin concentration in EU ml on the y axis Draw a best fit line among these points and determine endotoxin concentrations of samples graphically y 0 2778x 0 0032 0 8 R 0 9964 0 6 0 4 0 2 Endotoxin Concentration EU ml 1 1 0 2 0 4 0 6 ga L OD 545 nm If the mean absorbance value of a sample is x the endotoxin concentration of the sample will be 0 2778x 0 0032 EU ml All incubations were performed for 45 min Note the OD values of standards may be differ in different endotoxin assays the curve as above is only
8. f endotoxin level In this test the endotoxin level in the positive sample is equal or higher than 0 25 EU ml while in the negative sample is lower than 0 25 IV Example 1 Sample 1 mg ml Protein A provided in PBS pH 7 4 The Protein A is purified from E coli sonicate by Ni NTA Resin 2 Prepare dilution in LAL reagent water according to following dilution times 1 200 000 1 400 000 1 800 000 16 ToxinSensor Endotoxin Detection System www genscript com ToxinSensor Endotoxin Detection System www genscript com Protocols continued Semi Quantitative Detection Protocols continued ToxinSensor Gel Clot IV Example continued Endotoxin Assay Kit 3 The test is performed as above protocol states and the continued assay result is Positive control Negative conirol 1 200 000 1 400 000 1 800 000 4 Endotoxin concentration value of this sample is in the range of 50 000 100 000 EU ml Endotoxin Concentration Value Dilution Times x 0 25 EU ml V Troubleshooting Problem Possible Cause Suggestions Pay more attention to Negative control produces The materials e g tips vials y ti dk th a gel using L00351 etc may be contaminated SPETEn OM BNU SERP le aseay under laminar flow cabinets Positive control does not The standard of endotoxin is Me Sandara SNUG DE form gel notmixediwell vigorously vortexed for 15 minutes prior to use re Customer Refere
9. in in a licensed reagent clinical Cat No Size Test Methods Sensitivity samples or the diagnosis of human disease A measurable L00350C 16 rxns Quantitative Endpoint 0 005 to 1 EU mi endotoxin concentration range of 0 005 to 1 EU mI can be L00350 32 rxns Chromogenic achieved L00351 40 rxns Semi quantitative Gel clot gt 0 25 EU ml Warning For In Vitro Diagnostic Use Only Not intended to detect endotoxemia in man or animals or for use in clinical diagnosis patient management cell bacterial culture medium serum blood or blood products Background For biopharmaceutical companies endotoxin detection is the most critical quality control test to ensure that manufacturing of pharmaceutical products are free of endotoxin contaminations There are three LAL test methods and the following is a general selection guide to help you decide which method to use Methods Maximum Sensitivity Regulatory Requirements Gel clot 0 03 EU ml Non circulating water bath or dry bath incubator Chromogenic 0 005 EU ml A microplate reader an incubating reader is required for the kinetic method Turbidimetric 0 001 EU ml An incubating microplate reader ToxinSensor Endotoxin Detection System www genscript com Product Overview ToxinSensor Chromogenic LAL Endotoxin Assay Kit Kit Contents This kit is designed as a qualitative test that is simple and sensitive for detection of the presence of lipopolysaccharides in samples I
10. interference Consequently samples should be diluted with LAL reagent water before proceeding In addition it is necessary to calculate the MVD to ensure a margin of safety we recommend not exceeding the MVD of your sample MVD Maximum Valid Dilutionis a dilution factor showing endotoxin limit in EU ml divided by lambda The labeled lysate reagent sensitivity in the gel clot methods of our kit is 0 25 EU ml Note Our kit is used for samples certified free of Beta Glucans contaminant This contaminant can come from yeast and cellulosic materials such as blood products BET ToxinSensor Endotoxin Detection System www genscript com ToxinSensor Endotoxin Detection System www genscript com Protocols continued Semi Quantitative Detection Protocols continued ToxinSensor Gel Clot II Reagent preparation Endotoxin Assay Kit Limulus Amebocyte Lysate LAL continued Reconstitute lyophilized lysate by adding 2 ml LAL reagent water before proceeding Each reconstitution should be vortexed for 30 60 seconds with a vortex mixer or mixed gently by swirling Do not shake or invert vortex to avoid foaming Reconstituted lysate can remain stable for one week if stored at 20 C or for long term use if frozen at 80 C immediately after reconstitution Avoid repeated freeze and thaw cycles Positive controls Reconstitute E coli endotoxin Standard by adding 1 ml LAL reagent water to a concentration of 0 5 EU ml
11. nces Wu JW etc Stimulation of the sacral nerve reduces gut bacterial translocation and endotoxemia caused by acute spinal cord injury in rabbits Spinal Cord Apr 6 2010 PMID 20368712 Sharon Altmann etc A Quantitative Rabbit Model of Vaccinia Keratitis Invest Ophthalmol Vis Sci Apr 7 2010 PMID 20375331 Stefan Tukaj etc Hsp40 proteins modulate humoral and cellular immune response in rheumatoid arthritis patients Cell Stress Chaperones Feb 2 2010 PMID 20127215 Suree Lekawanvijit etc Does indoxyl sulfate a uraemic toxin have direct effects on cardiac fibroblasts and myocytes Eur Heart J Jan 4 2010 PMID 20047993 Shi Y etc Endotoxin promotes adverse effects of amorphous silica nanoparticles on lung epithelial cells in vitro J Toxicol Environ Health A Jan 2010 73 11 748 56 PMID 20391117 Jana Ryckaert etc Heat shock proteins protect platyfish Xiphophorus maculatus from Yersinia ruckeri induced mortality Fish Shellfish Immunol Jan 2010 28 1 228 31 Epub Sep 12 2009 PMID 19751832 48 ToxinSensor Endotoxin Detection System www genscript com ToxinSensor Endotoxin Detection System www genscript com Related Products Cat No Products Quantity Price M01062 ToxinSensor Endotoxin free Vials 2 ml Clear 16 Vials 20 00 M01063 ToxinSensor Endotoxin free Pipette Tips 1 ml Blue 6 Tips 6 00 L00338 ToxinEraser Endotoxin Removal Kit 3 5 Ass
12. or invert vortex to avoid foaming Store prepared endotoxin standard solutions 6 EU ml at 20 C for less than 24 hours The solutions can remain stable for up to 15 days if frozen at 80 C Dilute 0 1 ml of the 6 EU ml endotoxin standard solution with 0 5 ml of LAL reagent water to make the 1 EU ml standard solution The 1 EU ml standard solutions will be used for making the standard curve ToxinSensor Endotoxin Detection System www genscript com ToxinSensor Endotoxin Detection System www genscript com Protocols continued Protocols continued Quantitative Detection Protocols continued Quantitative Detection Protocols continued ToxinSensor II Reagents Preparation continued ToxinSensor Chromogenic LAL III Test Procedure continued In the assay example below at least five serial dilutions of Chromogenic LAL 4 After proper incubation add 100 pl of reconstituted Endotoxin Assay Kit continued endotoxin solutions should be prepared to make a standard curve in each assay If the expected endotoxin concentration range of samples is 0 005 0 1 EU ml the recommended concentrated endotoxin solutions should be 0 1 0 04 0 02 0 01 and 0 005 EU ml respectively The serial dilution of endotoxin solutions can be made as outlined in following figure Each solution should be mixed thoroughly for 30 seconds with a vortex mixer LAL Reagent Water 0 3 el 0 2 0 2 a 0 2 l 0 1EU ml 0 04EU ml 0 02EU ml 0
13. otoxin free Tips 1000 ul 1 Incubation Rack Product Overview continued Materials and equipments not provided 1 Sodium hydroxide 0 1N or hydrochloric acid 0 1N dissolved in LAL Reagent Water for pH adjustment of samples if necessary 2 Oven or non circulating hot water bath 37 1 C 3 Test tube rack 4 Vortex Mixer Kit Storage The kit should be stored dry at room temperature for up to one month For longer storage the kit can be kept at 2 8 C for up to one year Do not freeze the kit or any of its components Ordering Information Cat No Product Quantity Price L00351 ToxinSensor Gel Clot Endotoxin Assay Kit 40 rxns 90 00 ToxinSensor Endotoxin Detection System www genscript com ToxinSensor Endotoxin Detection System www genscript com Protocols Quantitative Detection Protocols ToxinSensor Chromogenic LAL Endotoxin Assay Kit Proceed to the Reagent Preparation Area in a laminar flow cabinet at room temperature Wear disposable gloves and use endotoxin free materials in order to avoid contamination I Specimen Preparation pH The pH value of the sample should be at pH 6 8 to ensure good linearity Consequently we recommed adjusting pH value using sodium hydroxide 0 1 N dissolved in LAL reagent water or hydrochloric acid 0 1 N diluted in LAL reagent water if necessary II Reagents Preparation Limulus Amebocyte Lysate LAL Recon
14. samples if required 3 Oven set at 37 C 1 0 C 4 Spectrometer or filter photometer with a 545 nm filter 5 Vortex mixer Kit Storage The kit should be stored dry at room temperature for up to one month For longer storage the kit can be kept at 2 8 C for up to one year Do not freeze the kit or any of its components Ordering Information Cat No Product Quantity Price L00350C ToxinSensor Chromogenic LAL Endotoxin Assay Kit 16 rxns 80 00 L00350 ToxinSensor Chromogenic LAL Endotoxin Assay Kit 32 rxns 150 00 Ase ToxinSensor Endotoxin Detection System www genscript com Product Overview continued ToxinSensor Gel Clot Endotoxin Assay Kit This kit is designed to be the simplest semi qualitative test for gram negative bacterial endotoxin that conforms to FDA Guideline Similar performance requirements for gel clot ToxinSensor Endotoxin Detection System www genscript com assays have been published and are updated regularly in the United States Pharmacope Kit Contents Good reproducibility Competitive price Ready to use reagents and materials such as endotoxin free tips endotoxin free tubes etc PK Label Volume 4 bottles LAL Reagent Water 10 ml 2 vials Limulus Amebocyte Lysate LAL 2 ml 2 vials E coli Endotoxin Standard 0 5 EU 5 x 16 vials Endotoxin free Vials 1 box 96 tips Endotoxin free Tips 200 ul 2 bags 12 tips End
15. stitute lyophilized lysate by adding 1 7 ml LAL reagent water Each reconstitution should be vortexed for 30 seconds with a vortex mixer or mixed gently by swirling Do not shake or invert vortex to avoid foaming Reconstituted lysate can remain stable if stored at 20 C for one week or for long term use if frozen at 80 C immediately after reconstitution Avoid repeated freeze and thaw cycles Chromogenic Substrate Reconstitute the substrate by adding 1 7 ml of LAL reagent water to a concentration of 2 mM Once reconstituted the substrate solution can remain stable for one month if stored at 2 8 C protected from light Lyophilized chromogenic substrate can remain stable for one year if stored at 2 8 C Protocols continued Quantitative Detection Protocols continued ToxinSensor Chromogenic LAL Endotoxin Assay Kit continued Stop Solution Reconstitute the color stabilizer 1 Stop Solution with 10 ml of buffer S Reconsituted Stop Solution can remain stable for one week if stored at 2 8 C Color stabilizer 2 and 3 Reconstitute color stabilizer 2 and 3 by adding 10 ml of LAL water for each Each reconstitution can remain stable for one week at 2 8 C Standard endotoxin solutions Dissolve 6 EU lyophilized endotoxin standard in 1 ml LAL reagent water to yield a concentration of 6 EU ml The dissolution should be vortexed for 15 minutes with a vortex mixer or mixed gently by swirling Do not shake
16. t uses a colorimetric method in which endotoxin catalyzes the activation of a proenzyme in LAL which will cleave a colorless substrate to produce a colored end product The end product can be measured spectrophotmetrically and compared to a standard curve Good linearity and good reproducibility e High sensitivity and board application range Ready to use reagents and materials such as endotoxin free tips endotoxin free tubes etc PK Label Volume L00350 L00350C 2 bottles 1 bottle LAL Reagent Water 50 ml 2 vials 1 vial Limulus Amebocyte Lysate LAL 2 vials 1 vial E coli Endotoxin Standard 6 EU 2 vials 1 vial Chromogenic Substrate 1 bottle 50 ml 1 bottle 10 ml Buffer S for Color stabilizer 1 50 ml 2 vials 1 vial Color stabilizer 1 2 vials 1 vial Color stabilizer 2 2 vials 1 vial Color stabilizer 3 6 x 8 vials 3 x 8 vials Endotoxin free Vials 1 box 96 tips 1 box 96 tips Endotoxin free Tips 200 ul 2 bags 12 tips 2 bags 12 tips Endotoxin free Tips 1000 ul 1 1 Incubation Rack ToxinSensor Endotoxin Detection System www genscript com Product Overview continued Materials and equipments not provided 1 Sodium hydroxide 0 1 N dissolved in LAL reagent water The reagent is for adjustment of the pH of samples if required 2 Hydrochloric acid 0 1 N diluted in LAL reagent water The reagent is for adjustment of the pH of

ToxinSensorTM

Contents

Download Pdf Manuals

Related Search

Related Contents