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1. Dilution Buffer is pipetted into blank wells Typical Standard Curve of Mouse IGS15 Standard Curve A450 0 2 5 5 7 5 10 12 5 Mouse ISG15 conc ng ml C CY 8091 12 Version 120710 Mouse ISG15 ELISA Kit n TM CircuLex User s Manual For Research Use Only Not for use in diagnostic procedures 2 Precision Intra assay Precision Precision within an assay Three samples of known concentration were tested sixteen times on one plate to assess intrazass precision e Intra assay Within Run n 16 CV 2 6 3 9 O Mouse ISG15 conc ng mL Sampel Sample 2 Sample 3 0 7 50 8 C CY 8091 13 Version 120710 Mouse ISG15 ELISA Kit n TM CircuLex User s Manual For Research Use Only Not for use in diagnostic procedures Inter assay Precision Precision between assays Three samples of known concentration were tested in five separate assays to assess inter ass precision e Inter assay Run to Run n 5 CV 5 0 7 7 Mouse ISG15 conc ng mL O ample ample ample 2 1 59 3 19 7 69 3 1 59 3 04 7 56 4 178 321 830 1 56 3 21 3 Linearity To assess the linearity of the assay samples containin an spiked with high concentrations of mouse ISGI5 were serially diluted with the Dilutio e oduce samples with values within the dynamic range of the assay N Linearity Sample 1 e Sample 2 a amp Sample 3 z n un2. Mouse ISG15 ELISA Kit n TM CircuLex User s Manual For Research Use Only Not for use in diagnostic procedures ELISA Kit for Measuring Mouse ISG15 CircuLex Mouse ISG15 ELISA Kit cA Cat CY 8091 O Intended USE cavsssinncssteieeceussatactenasastioeainin 1 PA aes creer EEE 1 DC Oi eesis 2 Principle of the Assay 2 3 Materials Provided ys ccssccassasccaeoscasscencsvava neds 3 Materials Required but not Provided 4 Precautions and Recommendations 5 Sample Collection and Storage 0 6 Detailed Protocol scsictacidccictnnnsisnaneeniiectus 7 8 CaCl 35334 avevantevae capsaicin thease 9 Measurement Ratge c ssscsssscssecrseessteneees 9 Trou DleshO Ong isisisiiccsssssastecacsrseasedueevsevedevien 9 Reagent SCADILILY asad snccasecedrasssantavsaudiaseonsenaes 9 Sample PreparavtOtssic scsscaetscessered antenesecessnas 9 Assay Characteristics cccesccccssceeseeeeeeee Example of Test Results eeeeeeeeeeeeeeeee PRTG Saien rosne niis oriei Related PrOGUCTS sea sctsencssbeceeedacaenenoaniecanes Intended Use The CycLex Research Product Circ kex Mouse ISG15 ELISA Kit is used for the quantitative 1 measurement of mouse ISG15 in cell e d media and serum This assay kit is for research use only and not for use in diagnostic or therapeutic procedures Storage e Upon receipt store all e C CY 8091 1 Version 120710 Mouse ISG15 ELISA Kit n TM Circulex User s Man
3. Mouse ISG15 conc ng mL 0 0 0 0 2 0 4 0 6 0 8 1 Sample Dilution Ratio C CY 8091 14 Version 120710 Mouse ISG15 ELISA Kit m TM CircuLex User s Manual For Research Use Only Not for use in diagnostic procedures Example of Test Results Fig l Concentrations of total mouse ISG15 in cell lysates of three mouse cell lines after FNa treat for 24 hours Mouse ISG15 concentration of cell lysate 1x10 cells mL Treated with 1 KU ml mouse IFNa for 24hrs w N Wn N we Mouse ISG15 ng mL _ un o e a inal gleie ieee lei Ecie eieie inn ERIIER 5 Da se hide Sade gt ns nr an pb Side push aA zd Fa de ede le pte AA Side ede de yee de ie Side ist edo ieee Apap ip Ep ae 0 e IFNa IFNa IFNa IFNa IFNa IFNa NIH 3T3 lysate PT67 lysate Balb 3T3 lysate Fig 2 Concentrations of unconjugated free form ISGI5 in Balb 3T3 culture supernatant after IFNa treatment for indicated times Mouse ISG15 concentration in Balb 3T3 culture supernatant after IFNa treatment at indicated times E IFNo a gs e a iv Mouse ISG15 conc ng mL 12 24 36 48 72 Treatment time hrs C CY 8091 15 Version 120710 Mouse ISG15 ELISA Kit TM Circulex User s Manual For Research Use Only Not for use in diagnostic procedures Fig 3 Time course of mouse ISG15 induction in Bal
4. amples with Dilution Buffer See Sample Preparation above amp Pipette 100 uL of Standard Solutions Std1 Std7 Blank and diluted samples in d eS into the appropriate wells 4 Incubate the plate at room temperature ca 25 C for 1 hour shaking at ca 3 on an orbital microplate shaker 5 Wash 4 times by filling each well with Wash Buffer 350 uL using a sq i le multi channel pipette manifold dispenser or microplate washer N w 6 Add 100 uL of HRP conjugated Detection Antibody into each well 7 Incubate the plate at room temperature ca 25 C for 1 hour 4 microplate shaker at ca 300 rpm on an orbital 8 Wash 4 times by filling each well with Wash Buffer 35 singa squirt bottle multi channel pipette manifold dispenser or microplate washer 9 Add 100 uL of Substrate Reagent Avoid expo titer plate to direct sunlight Covering the plate with e g aluminum foil is recomme urn Substrate Reagent to 2 8 C immediately after the necessary volume is removed 10 Incubate the plate at room temperature for 10 20 minutes shaking at ca 300 rpm on an orbital microplate shaker The incubation time m ended up to 30 minutes if the reaction temperature is below than 20 C 11 Add 100 uL of Stop Solution to e in the same order as the previously added Substrate Reagent 12 Measure absorbance in each wellUsing a spectrophotometric microplate reader at dual wavelengths of 450 540 nm Dual w
5. avelen 550 or 450 595 nm can also be used Read the microplate at 450 nm if only a single wa can be used Wells must be read within 30 minutes of adding the Stop Solution Note 1 Complete r liquid at each step is essential to good performance After tNIH3T3st wash remov ining Wash Buffer by aspirating or decanting Invert the plate and blot it against er towels Note 2 Reliab curves are obtained when either O D values do not exceed 0 25 units for the blank entration or 3 0 units for the highest standard concentration The plate should be m at 5 minute intervals for approximately 30 minutes Note t icfoplate reader is not capable of reading absorbance greater than the absorbance of ig standard perform a second reading at 405 nm A new standard curve constructed values measured at 405 nm is used to determine ISG15 concentration of off scale C CY 8091 8 Version 120710 Mouse ISG15 ELISA Kit n TM CircuLex User s Manual For Research Use Only Not for use in diagnostic procedures Calculations Average the duplicate readings for each standard control and sample and subtract the average standard optical density Plot the optical density for the standards versus the concentration th standards and draw the best curve The data can be linearized by using log log paper and n analysis may be applied to the log transformation To determine the mouse ISG15 concent ea sample first find the absorbance value on the
6. b 3T3 cell lysate by IFNa treatment Mouse ISG15 concentration in Balb 3T3 cell lysate after IFNa treatment for indicated times E IFNa D IFNa AQ pp et enesee 30 pece ceedeeceeeoaeeceneers Os Weedadedadededdanede 1 0 _ 0 9 SL 4 6 8 12 24 36 48 72 Treatment time hrs Mouse ISG15 conc ng mL Fig 4 IFNa dose dependent induction of mouse V of Balb 3T3 cultured in 12 well plate for 24 hrs IFNa dose dependent induction of mouse ISG15 in Balb 3T3 cell lysate ith a 5 OO Fg ee eS amp z S 3 0 r Nn z N 2 0 E ee ee 2 ET E E l ADA a 0 0 0 013 0 064 0 32 1 6 40 200 1000 5000 IFNa conc U mL C CY 8091 16 Version 120710 CirculLex Mouse ISGI5 ELISA Kit User s Manual For Research Use Only Not for use in diagnostic procedures A 4 Fig 5 IFNa dose dependent induction of mouse ISG15 in cell lysate of Balb 3T3 cultured in 96 plate for 24 hrs IFNa dose dependent induction of mouse ISG15 in Balb 3T3 cell lysate 4 e a a El JO eats E E E a e a e 5 3 S n 2 ARATA ARAN ASA TAAA ARAN ARA DAARAAN mH x amp 7 lt 2 B RAT ARST nn AREN n ENEN EE eag EEA i n Le E 0 0 32 1 6 8 40 200 1000 5000 IFNa conc U mL 2 C CY 8091 17 Version 120710 Mouse ISG15 ELISA Kit n TM Circulex User s Manual For Research Use Only Not for use in diagnos
7. e ISG15 is pre coated onto a microplate Standards and samples are pipetted into the wells the immobilized antibody binds any mouse ISGI5 present After washing away any u bstances an HRP conjugated antibody it employs the quantitative specific for mouse ISGI5 is added to the wells Fo to remove any unbound antibody HRP conjugate the remaining conjugate is allowed to ith the substrate H O tetramethylbenzidine The reaction is stopped by addition of acidic solutio absorbance of the resulting yellow product is measured at 450 nm The absorbance is proportional tothe concentration of mouse ISG15 A standard curve is constructed by plotting absorbance versus mouse ISGI5 concentrations of calibrators and concentrations of unknown samples are di ied using this standard curve C CY 8091 2 Version 120710 Mouse ISG15 ELISA Kit n TM CircuLex User s Manual For Research Use Only Not for use in diagnostic procedures Summary of Procedure Add 100 uL of diluted samples to the wells Yy Incubate for 1 hour at room temp Wash the wells O l Add 100 uL of HRP conjugated anti mouse ISG15 antibody v Incubate for 1 hour at room temp Wash the wells Add 100 uL of Substrate Reagent a 4 Add 100 uL of Stop Solution v Measure absorbance at 450 nm Materials Provided All samples and standards should be assayed in e The following components are supplied and are sufficient for the one 96 well microplate kit Microplate One micr
8. er reading engths of 450 540 nm also be read at a single e Software package facilitating data generation and analysis opti e 500 or 1000 mL graduated cylinder e Reagent reservoirs e Deionized water of the highest quality Disposable paper towels C CY 8091 4 Version 120710 Mouse ISG15 ELISA Kit n TM CircuLex User s Manual A For Research Use Only Not for use in diagnostic procedures Precautions and Recommendations e Allow all the components to come to room temperature before use All microplate strips that are not immediately required should be returned to the zip lock W must be carefully resealed to avoid moisture absorption e Do not use kit components beyond the indicated kit expiration date e Use only the microtiter wells provided with the kit e Rinse all detergent residues from glassware Qy e Use deionized water of the highest quality A e Do not mix reagents from different kits O e The buffers and reagents used in this kit contain NaN as preserva e should be taken to avoid direct contact with these reagents Do not mouth pipette or ingest any of the reagents e Do not smoke eat or drink when performing the assay or eas where samples or reagents are handled e Dispose of tetra methylbenzidine TMB contain tions in compliance with local regulations e Avoid contact with the acidic Stop Solution and Substrate Solution which contains hydrogen peroxide e Wear gloves a
9. ficaftly different from those specified may give erroneous results 2 Poor duplicates accompanied by elev values for wells containing no sample indicate insufficient washing If all instructions i iled Protocol were followed accurately such results indicate a need for washer maintenan 3 Overall low signal may mdic at desiccation of the plate has occurred between the final wash and addition of Substrate Re t Do not allow the plate to dry out Add Substrate Reagent immediately after wash Reagent included in the CycLex Research Product CircuLex Mouse ISG15 ELISA Kit stability Reagents should not be used beyond the stated expiration date Upon CY 8091 9 Version 120710 Mouse ISG15 ELISA Kit n TM Circulex User s Manual For Research Use Only Not for use in diagnostic procedures Sample Preparation Several extraction methods can be used for measurement cellular total ISG15 The follo protocols have been shown to work with a number of different cell lines and are provided as ex suitable methods It is strongly advised that the user always perform an initial experiment te sufficient for this initial experiment All cell lysate preparation should be performed recovered cell lysate should be kept at below 70 C Preparation of Cell Lysate Qy A Preparation of Cell Lysis Buffer 20 mM Tris HCI pH 7 5 250 mM NaCl 10 glycerol 0 5 NP 40 1 A 1 mM EGTA 0 2 mM PMSF 1 ug mL pepstatin 0 5 ug mL le
10. haw cycles Note Citrate plasma has not been validated for use in this assay Other biological samples Remove any particulates by centrifugation a ssi immediately or aliquot and store samples at below 70 C Avoid repeated freeze thaw cycles C CY 8091 6 Version 120710 aa Mouse ISGI5 ELISA Kit CircuLex User s Manual For Research Use Only Not for use in diagnostic procedures L Detailed Protocol The CycLex Research Product CircuLex Mouse ISG15 ELISA Kit is provided with remo mouse ISG15 Standard within the kit should be included in each assay as a calibrat pipette tips and reagent troughs should be used for all liquid transfers to avoid cross co reagents or samples Preparation of Working Solutions All reagents need to be brought to room temperature prior to the assay Assay r are supplied ready to use with the exception of 10X Wash Buffer and Mouse ISG15 Stand 1 Prepare a working solution of Wash Buffer by adding 100 mL of the 10X deionized distilled water ddH O Mix well Store at 4 C for two storage fer to 900 mL of 20 C for long term 2 Reconstitute Mouse ISG15 Standard with 0 46 mL of ddH O dissolved by gently mixing immediately dispense in small aliquots e g 100 uL to micro ge tubes and store at below 70 C to avoid repeated freezing and thawing The concentration of the mouse ISG15 in vial should be 120 ng mL which is referred as a Master Standard of mo Prepa
11. nd eye protection when hai iMmunodiagnostic materials and samples of human origin and these reagents In case of co ith the Stop Solution and the Substrate Solution wash skin thoroughly with water and seek i when necessary e Biological samples may be cont a ith infectious agents Do not ingest expose to open wounds or breathe aerosols Wear protective gloves and dispose of biological samples properly e CAUTION Sulfuric Aci ng acid Wear disposable gloves and eye protection when K amp Y C CY 8091 5 Version 120710 Mouse ISG15 ELISA Kit n TM CircuLex User s Manual For Research Use Only Not for use in diagnostic procedures Sample Collection and Storage Serum Use a serum separator tube and allow samples to clot for 60 30 minutes Centrifu samples at 4 C for 10 minutes at 1 000 x g Remove serum and assay immediately or store sam on ice for up to 6 hours before assaying Aliquots of serum may also be stored at below 70 periods of time Avoid repeated freeze thaw cycles Plasma Collect plasma using EDTA Na as the anticoagulant If possible collect the p toa mixture of EDTA Na and Futhan5 to stabilize the sample against spontaneous in vitro complement activation Immediately centrifuge samples at 4 C for 15 minutes at 1 000 x g Assa mummediately or store samples on ice for up to 6 hours before assaying Aliquots of plasma may als ba red at below 70 C for extended periods of time Avoid repeated freeze t
12. ommercial ior written approval from CycLex Co Ltd To inquire about licensing for e please contact us via email CY 8091 Version 120710
13. oplate supplied ready to with 96 wells 12 strips of 8 wells in a foil zip lock bag with a desiccant pack Wells are coated ti mouse ISG15 polyclonal antibody as a capture antibody 10X Wash Buffer One bottle containing 100 ML of 10X buffer containing 2 Tween 20 Dilution Buffer One bottle containing L of 1X buffer use for sample dilution Ready to use Mouse ISG15 Standard One ining 55 2 ng of lyophilized recombinant mouse ISG15 HRP conjugated Detection A y One bottle containing 12 mL of HRP horseradish peroxidase conjugated anti mouse IS antibody Ready to use Substrate Reag tle containing 20 mL of the chromogenic substrate tetra methylbenzidine TMB Ready t Stop ad e containing 20 mL of 1 N H SO Ready to use C CY 8091 3 Version 120710 Mouse ISG15 ELISA Kit TM Circulex User s Manual amp For Research Use Only Not for use in diagnostic procedures Materials Required but not Provided e Pipettors 2 20 uL 20 200 uL and 200 1000 uL precision pipettors with disposable tips Precision repeating pipettor e Orbital microplate shaker e Microcentrifuge and tubes for sample preparation e Vortex mixer of e Microplate washer optional Manual washing is possible but not preferable ca e Plate reader capable of measuring absorbance in 96 well plates at dug Dual wavelengths of 450 550 or 450 595 nm can also be used The ple wavelength of 450 nm which will give a somewhat high
14. re Standard Solutions as follows Use the Master Standard to produce a diluti je w Mix each tube thoroughly before the next transfer The 12 ng mL standard erves as the highest standard The Dilution Buffer serves as the zero standard Blank Volume of Standar ilution Buffer Concentration Std 1 40 uL of Master St 360 uL 12 0 ng mL Std 2 300 uL of Std 1 1 300 uL 6 0 ng mL Std 3 300 uL of Std 2 6 0 ngfnl 300 uL 3 0ng mL Std 4 300 uL of Std ml 300 pL 1 5 ng mL Std 5 300 uL of Sadi 4 ng ml 300 uL 0 75 ng mL Std 6 300 uL of St 0 75 ng ml 300 uL 0 375 ng mL Std 7 300 u 0 375 ng ml 300 uL 0 188 ng mL Blank cts 300 uL 0 ng mL Note Do not use a Repeating pipette Change tips for every dilution Wet tip with Dilution Buffer before dispensin n sed ons of Master Standard should be aliquoted and stored at below 70 C immediat todultiple freeze and thaw cycles Sample Pre i C cell conditioned medium for measurement of ISG15 depends on cell number and require neat to appropriate dilution CY 8091 Version 120710 Mouse ISG15 ELISA Kit n TM Circulex User s Manual For Research Use Only Not for use in diagnostic procedures Standard Assay Procedure for Mouse ISG15 1 Remove the appropriate number of microtiter wells from the foil pouch and place them into the holder Return any unused wells to the foil pouch refold seal with tape and store at 4 C Dilute s
15. tic procedures References Korant BD Blomstrom DC Jonak GJ Knight E Jr 1984 J Biol Chem 259 14835 14839 2 Blomstrom DC Fahey D Kutny R Korant BD Knight E Jr 1986 J Biol Chem 261 881 w Haas AL Ahrens P Bright PM Ankel H 1987 J Biol Chem 1987 262 11315 11323 4 Feltham N Hillman M Jr Cordova B Fahey D Larsen B Blomstrom DC amp Knight E Jr 1989 J Interferon Res 9 493 507 Knight E Jr amp B Cordova 1991 J Immunol 146 2280 2284 Qy 6 Loeb K L amp Haas A L 1992 J Biol Chem 267 7806 7813 nn N D Cunha J Knight E Jr Haas A L Truitt R L amp Borden EC 1996 atl Acad Sci USA 93 211 215 oo D Cunha J Ramanujam S Wagner R J Witt PL Knight E den EC 1996 J Immunol 157 4100 4108 O Austin K J S K Ward M G Teixeira Dean VC Moore 600 606 n TR 1996 Biol Reprod 54 10 Kelso A 1998 Immunol Cell Biol 76 300 317 11 Durfee Larissa A Lyon Nancy Seo Kyungw and Huibregtse Jon M 2010 Mol Cell 38 722 732 Related Products CircuLex Human ISG15 ELIS Cat CY 8085 CircuLex Human ISG15 Cat 272 PRODUCED BY CycLex Co Ltd 1063 103 Terasawaoka Ina Nagano 396 0002 Japan Fax 81 265 76 7618 e mail info cyclex CycLex CircuL components ucts are supplied for research use only CycLex CircuLex products and y not be resold modified for resale or used to manufacture c
16. uLex User s Manual For Research Use Only Not for use in diagnostic procedures 3 Centrifuge at 3 500 rpm for 15 minutes at 4 C using a microplate bucket or transfer the cell lys to microcentrifuge tubes and centrifuge at 15 000 rpm for 5 minutes at 4 C 4 Transfer the clear cell lysates to new 96 well microplate or clean microcentrifuge tubes Dilute the cell lysates 10 times with Dilution Buffer 100 uL of these diluted cell lysates are ready forass o to the section Standard Assay Procedure for Mouse ISG15 at page 8 The cell lysates can be stored at below 70 C Avoid multiple freeze thaw cycles After cell lysates Centrifuge at 15 000 rpm for 5 minutes at 4 C again since the cell lysates should be f any sediments or particulate matter NOTE THE ABOVE PROCEDURES ARE INTENDED ONLY AS A INE THE OPTIMAL EXPERIMENTAL CONDITIONS WILL VARY D ON THE PARAMETERS BEING INVESTIGATED AND MUST BE D INED BY THE INDIVIDUAL USER NO WARRANTY OR GUARANTEE RFORMANCE USING THESE PROCEDURES IS MADE OR IMPLIED O C CY 8091 11 Version 120710 Mouse ISG15 ELISA Kit TM CircuLex User s Manual For Research Use Only Not for use in diagnostic procedures Assay Characteristics 1 Sensitivity The limit of detection defined as such a concentration of mouse ISG15 giving absorbance high a mean absorbance of blank plus three standard deviations of the absorbance of blank A blank is better than 48 3 pg ml of sample
17. ual For Research Use Only Not for use in diagnostic procedures Introduction ISG15 Interferon IFN Stimulated Gene 15 is a ubiquitin like protein containing two ubi homology domains and becomes conjugated to a variety of proteins when cells are treated wit interferon or lipopolysaccharide Therefore this modification so called SGylation involved in type I interferon signal transduction Unlike ubiquitylation SGylation does no degradation regulated by K48 linked ubiquitin ISG15 is expressed as a 165 amino acid is subsequently processed to expose the C terminal sequence LRLRGG Like ubiquitin diglycine residue is adenlylated and conjugated by a thiolester bond to sequential cystine residt El activating E2 carrier and E3 ligase enzymes before being transferred to lysine gesidues on target protein substrates ISG15 can also be found extracellularly in an unconjugated form Human lympho d monocytes were reported to release free ISG15 following treatment with IFN B More tha e total ISG15 could be detected in the culture medium after 24 hours following IFN stimula ddition ISG15 is highly elevated and extensively conjugated to cellular proteins in many tu an or cell lines The increased levels of ISGI5 in tumor cells were found to be associ decreased levels of polyubiquitinated proteins Principle of the Assay The CycLex Research Product CircuLex Mouse ISG15 sandwich enzyme immunoassay technique An antibody spe ous
18. upeptin 0 2 mM DTT B Preparation of poly I lysine coated plate Coat the plate with 25 ug mL poly l lysine PLL in PBS for 4 C Subsequently go to a washing step with PBS C Treatment of Cells 1 Plate adherent cells in PLL coated 96 well plate at around 2 ells well 2 Incubate the culture dish at 37 C for 7 12 hours i c r 3 Add appropriate amount of test compound or IFN vehicle for test compound to each well 4 Incubate the culture dish at 37 C for appropriate time D Cell Extraction Note This protocol has been successfully applied to Balb 3T3 cell line Users should optimize the cell extraction procedure for thei cations 1 Wash cells three times with mt Remove any remaining PBS by decanting Invert the plate and blot it against clean pa At this point the cells in the plate can be frozen at below 70 C and lysed at a later date 2 Lyse the cells by addin 300 rpm by an orbitalgmi 1 mL of Cell Lysis Buffer for 60 90 minutes at 4 C with rotating at ca te shaker could adjust the cell concentration to around 2 x 1 0 cells mL Cell Lysis rotein concentration of the Balb 3T3 cell lysate should be 0 03 0 04 mg mL using To get a ro j Buffer Resul this proce B Th volume of Cell Lysis Buffer depends on the cell line the cell number and the total ISG15 For example 2 x 10 Balb 3T3 cells can be lysed in 0 1 mL of Cell Lysis C CY 8091 10 Version 120710 Mouse ISG15 ELISA Kit n TM Circ
19. y axis and extend a horizontal line to the stay ve At the point of intersection extend a vertical line to the x axis and read the corresponding 15 concentration If the samples have been diluted the concentration read from the standard curv ust be multiplied by the dilution factor 1 The dose response curve of this assay fits best to a sigmoidal 4 parameter logistic equation The results of unknown samples can be calculated with any computer progr av a 4 parameter logistic function It is important to make an appropriate mathematical adjus accommodate for the dilution factor ration The calibration sus log of the known ior Alternatively the logit log Ofyabsorbance Y is plotted versus 2 Most microtiter plate readers perform automatic calculations of analyte curve is constructed by plotting the absorbance Y of calib concentration X of calibrators using the four parameter f function can be used to linearize the calibration curve i e lo log of the known concentration X of calibrators Measurement Range The measurement range is 0 188 ng mL to 12 standard should be diluted with Dilution Buffer in to be taken into consideration in calculating the mous ample reading higher than the highest dilution and re assayed Dilution factors need 15 concentration Troubleshooting 2 1 The ISG15 Standard should be run in duphi ing the protocol described in the Detailed Protocol Incubation times or temperatures signi
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