PyroGeneTM Recombinant Factor C Endotoxin Detection Assay
Contents
1. PyroGene Recombinant Factor C Endotoxin Detection Assay Catalog Number 50 658U 50 658NV Certificate of Analysis at www lonza com coa TABLE OF CONTENTS TITLE PAGE AAA veneer A 3 sn A EEE Saunas ds Sneaks 3 Safety Precautions 3 BACKOFOUNG een nee ren ecos 3 PIIDEIDIG See te ame ne ae soars ere eet Be sa ne ae ee 5 Storage 5 Reagents Supplied and Kit Configurations 5 Materials and Equipment Required but not Supplied 6 Specimen Collection and Preparation 7 Sensitivity Setting for Fluorescent T Preparation of Endotoxin Standards 9 Testing Procedure 24 224 10 Types OF PYrDGee ASSAYS crasas na 13 Pertormance Ghataetetisiies Rene nannten 15 Product Inhibition un acer ra rra da a td det des Bed alk hama 15 All des 17 Parent WO ACO an ra ee 18 Other Technical 155 5 18 Trademark NWO A ee an ae aca 18 INTENDED U2. Navas M A A Ho B Two forms of Factor C from the amoebocytes of Carcinoscorpius rotundicauda purification and characterization Biochim Biophys Acta 1202 149 1993 Ding J L Navas M A A Ho B Molecular cloning and sequence analysis of Factor C cDNA from the Singapore horseshoe crab Carcinoscorpius rotundicauda Mol Mar Biol Biotechnol 4 90 1995 Ding J L and Ho New era in pyrogen testing Trends in Biotechnology 19 277 2001 U S Department of Health and Human Service Food and Drug Administration Guideline on Validation of the Limulus Amebocyte Lysate Test as an End Product Endotoxin Test for Human and Animal Parenteral Drugs Biological Products and Medical Devices 1987 Chapter lt 85 gt Bacterial Endotoxins Test Rockville MD United States Pharmacopeia 17 PATENT INFORMATION Various components of PyroGene are protected under the following patents US 5 716 834 US 5 712 144 Australia 581 174 Canada 1 222 213 Columbia 24 556 Denmark 172 401 127 839 Ireland 58 011 Israel 71 906 Japan 2 129 487 2 644 447 Korea 51 077 Mexico 164 250 Norway 173 944 New Zealand 208 259 Philippines 25 395 South Africa 843 914 Spain 532 825 Taiwan 50 740 Additional Patents Pending OTHER TECHNICAL ISSUES Improving spike recovery By using a different data treatment procedure for example a polynomial curve fit using POWERCURVE calculation of the recovery of the positive product con
3. Prior to routine use microplates should be pre qualified 25 340 or equivalent Reagent reservoirs 25 364 or equivalent Eight channel pipettor Fluorescence microplate reader 25 344 FLx800 TBIE reader with Ex 380 20 and Em 440 30 Software WinKQCL V 4 0 2 or higher recommended Timer Vortex Mixer For 50 658NV LAL Reagent Water W50 640 or equivalent LAL Reagent Water is equivalent to Water for Bacterial Endotoxins Test BET SPECIMEN COLLECTION AND PREPARATION Careful technique must be used to avoid microbiological or endotoxin contamination All materials coming in contact with the specimen or test reagents must be endotoxin free Clean glassware and materials may be rendered endotoxin free by heating at 250 C for 30 minutes Appropriate precautions should be taken to protect depyrogenated materials from subsequent environmental contamination From experience most sterile individually wrapped plastic pipettes and pipette tips are endotoxin free However these materials should be tested before regular use It may be necessary to adjust the pH of the sample to within the range 6 0 8 0 using endotoxin free sodium hydroxide or hydrochloric acid Always measure the pH of an aliquot of the bulk sample to avoid contamination by the pH electrode Do not adjust unbuffered solutions Samples to be tested must be stored in such a way that all bacteriological activity is Stopped or the endotoxin level may increa
4. ml standard The RFU range from 1000 to 10000 corresponds to log range from 3 to 4 with a log mid point of 3 5 Thus the target absolute net RFU for the 1 EU ml is approximately 3000 RFU To maintain adequate separation between the blanks and the lowest standard in a routine assay this value should be considered the minimum of a desirable range Determination of appropriate sensitivity setting NOTE This procedure should be conducted as part of the performance qualification for any new lot of reagents after major equipment maintenance such as lamp replacement or when reader performance is questioned A periodic re verification is recommended as part of any on going equipment maintenance program 1 Prepare a 1 EU ml endotoxin solution see Preparation of Endotoxin Standards Allow reagents to equilibrate to ambient temperature before mixing See WinKQCL Software Manual for prompts to the Sensitivity Test Enter appropriate information in blank fields and confirm settings for the test Endotoxin Concentration Unitage EU Time Between Reads hh mm ss 01 00 00 Excitation Filter nm 380 20 Emission Filter nm 440 30 Scans per Well 4 Delay before scanning millisec 150 Delay between scans millisec 1 Optics Position bottom Wells to Use Pre defined 06 7 E6 7 F6 7 Sensitivity select by checking 30 35 40 45 50 55 Add 100 ul of the 1 EU ml solution to wells 06 7 E6 7 F6 7 Pre incubate covered pla
5. the Reference Standard Endotoxin RSE The assay requires a single series of RSE dilutions and one or more sets of dilutions of the CSE Depending on the concentration units of the CSE the WinKQCL Software automatically computes mean potency values in terms of EU ml or EU ng The user also has the option to enter units other than EU or ng 4 INITIAL QUALIFICATION INT QUAL An INITIAL QUALIFICATION assay is designed according to the requirements described in the FDA s Guideline on Validation of the Limulus Amebocyte Lysate Test as an End Product Endotoxin Test for Human and Animal Parenteral Drugs Biological Products and Medical Devices This assay is required as part of the validation of the LAL assay and is also to be performed with each new kit lot testing evolved from the LAL test and Lonza suggests a similar approach be utilized The INITIAL QUALIFICATION assay performs a log log linear correlation of the individual Net ARFU values for each replicate of each endotoxin standard The other assays use the average Net ARFU of all the replicates of each standard 14 The INITIAL QUALIFICATION assay does not provide for the inclusion of any Samples PERFORMANCE CHARACTERISTICS Linearity The linearity of the standard curve within the concentration range used to determine endotoxin values should be verified Assay no less than 3 endotoxin standards Spanning the desired concentration range and a LAL Reag
6. 0 100 Endotoxin EU ml 11 Calculate endotoxin concentration of samples according to the standard curve When using FLx800 and WinKQCL Software The fluorescence reader FLx800 and WinKQCL Software can be used in this assay Turn on the reader and launch the software In the software create a template for the rFC assay Several types of assays may be programmed Consult the Software Software User Manual for more information 11 1 Save the template From the list of the templates an assay is initiated by selecting Run Follow on screen prompts to begin the test Consult Software User Manual for more details Place the microplate loaded with blank standards and samples in the reader and pre incubate the plate for a minimum of 10 minutes at 37 C 1 C During the 10 minute incubation prepare working reagent by mixing the fluorogenic Substrate the assay buffer and the rFC enzyme solution ina 5 4 1 ratio respectively To ensure enough working reagent is prepared for the assay and excess 5 minimal determine the number of wells requiring working reagent add 4 additional wells and calculate the appropriate amount for each component to make the working reagent Allow reagents to equilibrate to room temperature before mixing The order of mixing should remain consistent Add enzyme last to the buffered substrate Mix gently but thoroughly Do not vortex Refer to the following table NOTE Once prepared the working reagen
7. SE PyroGene is intended for use as an in vitro end product endotoxin test for human and animal parenteral drugs biological products and medical devices This product is not intended for use in the detection of endotoxin in clinical samples or the diagnosis of human disease The PyroGene test utilizes recombinant Factor C rFC an endotoxin sensitive protein rFC is used in combination with a fluorogenic substrate an incubating fluorescence microplate reader and appropriate software to measure endotoxin A minimum detection limit of 0 01 EU ml and a measurable endotoxin concentration range of 0 01 to 10 EU ml can be achieved WARNING For n Vitro Diagnostic Use Only PyroGene is not intended to detect endotoxemia in man or animals or for use in clinical diagnosis patient management or for the qualification of blood or blood products SAFETY PRECAUTIONS The toxicological properties of the reagents supplied have not been tested These reagents are not considered hazardous according to the OSHA Hazard Communication Standard 29 CFR 1910 1200 It is recommended that the NIH guidelines for recombinant DNA experiments be followed along with the use of standard laboratory precautions BACKGROUND A Gram negative bacterial infection of Limulus polyphemus the horseshoe crab may result in fatal intravascular coagulation At the molecular level it has been demonstrated that endotoxin activates a serine protease catalytic coagula
8. a suitable container and label 0 01 EU ml The solution should vigorously vortexed for approximately 1 minute before proceeding TESTING PROCEDURE Allow reagents to equilibrate to room temperature prior to use General testing procedure 1 PyroGene endotoxin assay uses a 96 well plate assay format Add 100 ul of the blank endotoxin standards and samples to appropriate wells of the microplate Duplicate or triplicate wells are generally used 2 spike the samples with endotoxin add 10 ul of the 1 EU ml solution to the designated Positive Product Control wells 3 Pre incubate the plate in the reader at 37 C 1 C for a minimum of 10 minutes 4 During the incubation period prepare the working reagent which consists of fluorogenic substrate assay buffer and rFC enzyme solution in a 5 4 1 ratio respectively 10 Carefully dispense 100 ul of the working reagent to each well Read fluorescence at time zero Incubate the reaction for one hour and read the plate at elapsed time one hour The difference of time one hour and time zero readings are corrected with the blank 9 log net ARFU is then plotted against log endotoxin concentration in a linear regression curve 10 example curve is shown below NoD Endotoxin Standard Curve in the rFC assay 1e 6 1 5 1 4 1 3 1 2 Fluorescence RFU Log y 0 946 Log x 3 588 1e 1 r 1 000 1e 0 0 001 0 01 0 1 1 1
9. ains Two vials of rFC Enzyme Solution R50 658 1 2 ml vial Two vials of the Fluorogenic Substrate 550 658 6 0 ml vial Two vials of the rFC Assay Buffer B50 658 5 0 ml vial Two vials of the coli Endotoxin 055 B5 E50 643 lyophilized The reconstitution volume of the vial is stated on the Certificate of Analysis and is calculated to yield a solution containing 20 EU ml Reconstituted endotoxin is stable for 4 weeks at 2 8 C Two vials of LAL Reagent Water W50 640 30 ml vial This water is used to rehyd rate E coliendotoxin and to prepare endotoxin standard and sample dilutions LAL Reagent Water is equivalent to Water for Bacterial Endotoxins Test BET Catalog No 50 658NV contains Thirty vials of rFC Enzyme Solution R50 658 1 2 ml vial Thirty vials of the Fluorogenic Substrate 550 658 6 0 ml vial Thirty vials of the rFC Assay Buffer B50 658 5 0 ml vial Ten vials of the coli Endotoxin 055 5 E50 643 lyophilized The reconstitution volume of the vial is stated on the Certificate of Analysis and is calculated to yield a solution containing 20 EU ml Reconstituted endotoxin is stable for 4 weeks at 2 8 C MATERIALS AND EQUIPMENT REQUIRED BUT NOT SUPPLIED ie Disposable endotoxin free glass dilution tubes 13 x 100 mm N207 or equivalent Individually wrapped sterile measuring pipettes Automatic hand held pipettes with sterile individually wrapped or racked tips Disposable sterile microplates Note
10. ent Water blank at least in triplicate Additional standards should be included to bracket each log interval over the range of the standard curve The value of the correlation coefficient r of the calculated standard curve should be 0 980 See Further data considerations under Other Technical Issues section PRODUCT INHIBITION Product inhibition occurs when substances in the test sample interfere with the enzyme reaction Inhibition results in a lower final ARFU in a sample reading indicating lower levels of endotoxin than what may actually be present in the test sample The lack of product inhibition should be determined for each specific sample either undilute or at an appropriate dilution To verify the lack of product inhibition an aliquot of a test sample or a dilution of a test sample is spiked with a known amount of endotoxin It is recommended that the endotoxin spike result in a final endotoxin concentration in the sample equal to 0 1 EU ml For samples which may contain a background endotoxin level gt 0 1 EU ml the endotoxin spike should result in a final endotoxin concentration of 1 0 EU ml The spiked solution is assayed along with the unspiked samples and their respective endotoxin concentrations are determined The differences between these two calculated endotoxin values should equal the known concentration of the spike within the range of 50 200 15 dilutions of the test sample A spiked aliquot of the
11. exact dilution can be found by testing two fold dilutions around this 16 5 1 2 10 11 12 13 Levin J and Bang The Role of endotoxin in the extracellular coagulation of Limulus blood Bull Johns Hopkins Hosp 115 265 1964 Levin J and Bang A description of cellular coagulation in the Limulus Bull Johns Hopkins Hosp 115 337 1964 Levin J and Bang Clottable protein in Limulus its localization and kinetics of its coagulation by endotoxin Thromb Diath Haemorrh 19 186 1968 lwanaga S The Limulus clotting reaction Curr Opin Immunol 5 74 1993 Nakamura T Morita T Iwanaga Lipopolysaccharide sensitive serine protease zymogen factor found in Limulus hemocytes Eur J Biochem 154 511 1986 Muta T Miyata T Misumi Y et al Limulus Factor an endotoxin sensitive Serine protease zymogen with a mosaic structure of complement like epidermal growth factor like and lectin like domains J Biol Chem 266 6554 1991 Tokunaga F Nakajima H Iwanaga 5 Further studies on lipopolysaccharide Sensitive serine protease zymogen factor C its isolation from Limulus polyphemus hemocytes and identification as an intracellular zymogen activated by alpha chymotrypsin not by trypsin J Biochem 109 150 1991 Morita T et al Anew 1 3 beta D glucan mediated coagulation pathway found in Limulus amebocytes FEBS Letts 129 318 1981 Ding J L
12. igorously for approximately 15 minutes at high speed on a vortex mixer Prior to future use the solution must be warmed to room temperature and vigorously vortexed for approximately 15 minutes The following table suggests a dilution scheme for constructing a series of endotoxin dilutions from the endotoxin supplied in the kit Note Plastic tubes are not recommended for making endotoxin dilutions 9 Endotoxin Volume of Volume of Endotoxin Standard Concentration LAL Reagent added to EU ml Water LAL Reagent Water 0 5 ml of 20 EU ml solution 0 1 ml of 1 EU ml solution 0 1 ml of 10 EU ml solution 0 1 ml of 0 1 solution 1 Prepare a solution containing 10 EU ml endotoxin by adding 0 5 ml of the 20 EU ml endotoxin stock solution into 0 5 ml of LAL Reagent Water in a suitable container and label 10 EU ml This solution should be vigorously vortexed for approximately 1 minute before proceeding 2 Transfer 0 1 ml of the 10 EU ml endotoxin solution into 0 9 ml of LAL Reagent Water a suitable container and label 1 EU ml The solution should be vigorously vortexed for approximately 1 minute before proceeding 3 Transfer 0 1 ml of the 1 EU ml endotoxin solution into 0 9 ml of LAL Reagent Water in a suitable container and label 0 1 EU ml The solution should be vigorously vortexed for approximately 1 minute before proceeding 4 Transfer 0 1 ml of the 0 1 EU ml endotoxin solution into 0 9 ml of LAL Reagent Water in
13. n of endotoxin in unknowns by comparison to the performance of a series of endotoxin standards As part of a ROUTINE assay the user has the option to include a Positive Product Control PPC as a monitor for product inhibition or enhancement A PPC is a sample of product to which a known amount of endotoxin spike has been added The WinKQCL Software automatically calculates the amount of endotoxin recovered in the PPC allowing for a comparison to the known amount of endotoxin spike 2 INHIBITION ENHANCEMENT INH ENH The PyroGene reaction is enzyme mediated and as such has an optimal pH range and specific salt and divalent cation requirements Occasionally test samples may alter these optimal conditions to an extent that the rFC is rendered insensitive to endotoxin Negative results with samples which inhibit the PyroGene test do not necessarily indicate the absence of endotoxin An INHIBITION ENHANCEMENT assay is designed to determine what level of product dilution overcomes inhibition or enhancement Each product dilution must be accompanied by a Positive Product Control PPC The WinKQCL Software calculates the amount of endotoxin recovered in the PPC for comparison to the known amount of endotoxin spike In this manner it can be determined which product dilutions are non inhibitory 3 RSE CSE An RSE CSE assay is designed to determine the potency of a Control Standard Endotoxin CSE in terms of the concentration units of
14. se with time For example store samples at 2 8 C for less than 24 hours and frozen for periods greater than 24 hours It is the responsibility of the customer to validate the proper container and storage conditions for their samples SENSITIVITY SETTING FOR FLUORESCENT READER Fluorescence signals are normally recorded as Relative Fluorescence Units RFU Since the real fluorescence signal is converted to an electronic signal that can be tuned using the gain setting or sensitivity setting RFU is an arbitrary unit Depending on the strength of the detected signal the instrument can be adjusted to a higher gain sensitivity setting to boost a weak signal or tuned down to a lower gain sensitivity setting when the signal is too strong It is useful to consult the FLx800 Reader Operator s Manual Operation Section for the manufacturer s explanation In the PyroGene assay the 3 log endotoxin concentration range corresponds to the 3 log range in RFU If the sensitivity is tuned too low fluorescence at the lowest standard will be difficult to detect if the sensitivity is tuned too high fluorescence at the highest standard will be off scale Prior to conducting any assays it is important to determine the appropriate sensitivity for the PyroGene assay The FLx800 has fluorescence range from 0 to 99999 In order to establish the 3 log fluorescence range for the standard curve an RFU range from 1000 to 10000 was chosen for the 1 EU
15. t cannot be stored 12 Volume ul of reagent required per total number of wells To assure slight excess assume volume prepared is sufficient for an additional 4 wells Total number of Fluorogenic assay rFC enzyme wells substrate buffer solution Total Volume 6 500 400 100 1000 12 800 640 160 1600 24 36 42 48 54 60 3200 56 640 6400 66 700 72 3800 760 7600 78 84 4400 52 880 8800 90 4700 3760 940 9400 96 5000 4000 1000 10000 4 When prompted by the software at the end of the incubation period dispense 100 ul of working reagent into the appropriate wells using an eight channel pipettor 5 Immediately follow screen prompt The software will initiate read one time zero reading followed by read two an hour later 6 After the assay is completed the data is automatically saved to the hard drive TYPES OF ASSAYS The incubating fluorescence microplate reader and WinKQCL Software are an integral part of the PyroGene assay It is important to become familiar with the operation of the incubating microplate reader and the features of the WinKQCL Software Please refer to the incubating microplate reader and WinKQCL Software Manuals for more detailed information There are four 4 basic types of PyroGene assays Each type is designed to perform a different aspect of endotoxin testing 1 ROUTINE A ROUTINE assay calculates the concentratio
16. te with standards at 37 C 1 C for 10 minutes Prepare working reagent by mixing the fluorogenic substrate rFC assay buffer and the rFC enzyme solution at 5 4 1 ratios respectively 500 ul 400 ul 100 ul is a sufficient quantity for the sensitivity test Refrigerate any unused reagents after opening Previously opened vials remain effective throughout the lifetime of the reconsti tuted endotoxin if returned to refrigeration Acceptable assay evaluation parameters will indicate proper reagent performance NOTE The order of mixing should remain consistent Add enzyme last to the buffered substrate Mix thoroughly but gently Do not vortex the working reagent mix NOTE Once prepared the working reagent cannot be stored At the appropriate software prompt add 100 ul of working reagent to all wells that contain the endotoxin sample Follow screen prompt to initiate test Note leave the plate uncovered for the duration of the test 8 At the completion of the test summary of the results with the calculated sensitivity will be displayed Using linear regression the sensitivity value correlated to log 3 5 is rounded to the nearest integer Successful assays Should result when using this setting as a minimum Both the Analyst and Reviewer should e sign then print the report Note If the predicted sensitivity result is outside of the range of the tested default parameters of 30 35 40 45 50 and 55 then the sensitivity assay sho
17. test sample or dilution may be prepared as in one of the following examples Tube Method Transfer 50 ul of the 10 EU ml solution into 4 95 ml of test sample or dilution This solution contains an endotoxin concentration of 0 1 EU ml in test sample or dilution This sample should be vigorously vortexed for one minute prior to use Plate Method 1 Transfer 10 ul of the 1 EU ml solution into each of the PPC wells in the 96 well plate as directed by the assay template To these wells add 100 ul of test sample or dilution Each well will now contain a 0 1 EU ml solution Mix gently Plate Method 2 Place 100 ul of test sample or dilution into the PPC wells in the 96 well plate as directed by the assay template To these wells add 10 ul of the 1 EU ml solution Each well will now contain a 0 1 EU ml solution Mix gently If the test sample or dilution is found to be inhibitory to the enzyme reaction the Sample may require further dilution until the inhibition is overcome Determination of a Non Inhibitory Dilution with 0 1 EU ml Endotoxin Spike Sample Raw Endotoxin EU ml Spike Sample Endotoxin Unspiked Spiked Difference Recovery Inhibition Raw x DF 0 025 25 Inhibito N A Inhibitory 0 105 0 080 80 Non Inhibitory 1 EU ml Initially Lonza recommends screening for product inhibition by testing ten fold Once the approximate non inhibitory dilution is determined the
18. the assay mixture to produce a fluorescent signal in proportion to the endotoxin concentration in the sample Figure 1 Schematic drawing of the endotoxin detection mechanisms in the LAL system and the rFC system Endotoxin Detection LAL Endotoxin Recombinant Factor C Gl ucan FC FC y in FB FB FG FG FO Proclotting Clotting Enzyme Enzyme Fluorogenic Product Substrate Fluorescence Chromogenic Product KQCL Substrate Yellow Color Coagulin gel clot KT urb Coagulogen Turbidity PRINCIPLE Recombinant Factor C is activated by endotoxin binding and the active moiety created then acts to cleave a synthetic substrate resulting in the generation of a fluorogenic compound Figure 1 rFC pathway The assay is carried out in a 96 well plate Fluorescence is measured at time zero and after a one hour incubation at 37 C 1 C in a fluorescence microplate reader using excitation emission wavelengths of 380 440 nm The difference between the one hour reading and the time zero reading ARFU is corrected for blank ARFU fluorescence The log net fluorescence is proportional to the log endotoxin concentration and is linear in the 0 01 to 10 EU ml range Endotoxin in a Sample is calculated relative to a standard curve STORAGE CONDITIONS 1 The PyroGene kit catalog numbers 50 658U 50 658NV is stored at 2 8 C REAGENTS SUPPLIED AND KIT CONFIGURATIONS IS Catalog 50 6580 cont
19. tion cascade that results in the gelation of Limulus blood This cascade is used in the Limulus Amebocyte Lysate LAL endotoxin detection method The protease cascade and rationale of traditional LAL tests are illustrated in the LAL pathway in Figure 1 Factor C the first component in the cascade is a protease zymogen that is activated by endotoxin binding In this pathway Factor FB is activated by Factor C An alternative pathway the Factor G FG pathway can be activated by glucan binding Downstream Factor and Factor G pathways individually 3 activate proclotting enzyme into a clotting enzyme The chromogenic LAL assay Lonza s Kinetic QCL KQCL uses a synthetic chromogenic peptide substrate that can be cleaved by the clotting enzyme resulting in a product that exhibits a yellow color The turbidimetric assay Lonza s PYROGENT 5000 KTurb uses the native substrate coagulogen which can be cleaved into coagulin Coagulin then begins to self associate resulting in an increase in turbidity The densities of the yellow color OD 405 nm and turbidity OD 340 nm are correlated with endotoxin concentration Studies have demonstrated the ability of Factor C to selectively recognize endotoxin and activate the protease cascade To create an endotoxin specific assay Factor C has been purified and cloned When activated by endotoxin binding recombinant Factor C acts upon the fluorogenic substrate in
20. trol can be improved Truncation of standard curve In our experience a truncated range of endotoxin standards 0 01 to 1 EU ml or 0 1 to 10 EU ml can provide better linearity than the whole range of 0 01 to 10 EU ml Overcoming inhibition with a dispersing agent PYROSPERSE N190 a dispersing agent is not compatible with the PyroGene assay TRADEMARK INFORMATION The FLx800 is a trademark of BioTek Instruments Inc Unless otherwise noted all trademarks herein are marks of the Lonza Group or its affiliates 18 5 19 5 20 8830 Biggs Ford Road Walkersville MD 21793 301 898 7025 www lonza com 08299 P50 658U NV 8 01 11
21. uld be repeated utilizing a more appropriate range that will include that result It is not recommended to extrapolate beyond the range of the curve Sensitivity Setting Determined for Kit Lot PREPARATION OF ENDOTOXIN STANDARDS In order to calculate the endotoxin concentration in unknown samples each PyroGene test must be referenced to a valid standard curve Because of the large concentration range over which endotoxin values can be determined it is possible to adjust the quantitative range of any given test by adjusting the concentration of endotoxin standards used to generate the standard curve A minimum of three standards is required The PyroGene assay has been optimized to be linear from 0 01 EU ml to 10 EU ml However the individual user may choose to truncate the standard curve depending on Specific product testing requirements The coli endotoxin 055 B5 is provided for user convenience Other endotoxin preparations may be used to prepare the standards however their performance in the PyroGene assay relative to the current USP Reference Standard Endotoxin RSE must be determined The supplied lyophilized coli 055 B5 endotoxin is reconstituted with LAL Reagent Water to yield 20 EU ml stock solution The reconstitution volume of the vial is stated on the Certificate of Analysis and is calculated to yield a solution containing 20 EU ml Reconstitute with the specified volume of LAL Reagent Water Shake v
Download Pdf Manuals
Related Search