CY-8071 High-Sensitivity CRP ELISA Kit
Contents
1. Preparation of Working Solutions All reagents need to be brought to room temperature prior to the assay Assay r agents are supplied ready to use with the exception of 10X Wash Buffer and Human CRP Standard 1 Prepare a working solution of Wash Buffer by adding 100 mL of the 10X Wash Buffer to 900 mL of deionized distilled water Mix well Store at 4 C for two weeks or 20 C for long term storage 2 Reconstitute Human CRP Standard with 1 25 mL of Dilution Buffer The concentration of the human CRP in vial should be 15 2 ng mL which is referred as a Master Standard of human CRP Prepare Standard Solutions as follows Use the Master Standard to produce a dilution series below Mix each tube thoroughly before the next transfer The 3 800 pg mL standard Std 1 s nves as the high standard The Dilution Buffer serves as the zero standard Blank Volume of Standard Dilution Buffer Concentration Std 1 150 uL of Master Standard 450 uL 3 800 pg mL Std 2 300 uL of Std 1 3 800 pg mL 300 uL 1 900 pg mL Std 3 300 uL of Std 2 1 900 pg mL 300 uL 950 pg mL Std 4 300 uL of Std 3 950 pg mL 300 uL 475 pg mL Std 5 300 uL of Std 4 475 pg mb 300 uL 237 5 pg mL Std 6 300 uL of Std 5 237 Spg mL 300 uL 118 8 pg mL Std 7 300 uL of Std 6 1 8 8ypg mL 300 uL 59 4 pg mL Blank 4 300 uL 0 pg mL Note Do not use a Repeating pipette Change tips for every dilution Wet tip with Dilution Buffer befo2. Substrate Reagent One botthe containing 20 mL of the chromogenic substrate tetra methylbenzidine TMB Ready to use Stop Solution One bottle Containing 20 mL of 1 N H2SO4 Ready to use Tt containsfhuman s ra Handle as if capable of transmitting infectious agents The sera were tested and foundsto benegative for HIV HBV and HCV However because no test method can offer complete assurance that HIV HBV HCV or other infectious agents are absent this component should be handled at the Bio saf typLevel 2 BSL 2 as recommended for any potentially infectious human serum or blood specimen in the CDC NIH manual Biosafety in microbiological and Biomedical Laboratories Cat CY 8071 3 Version 140318 High Sensitivity CRP ELISA Kit C ircuLex User s Manual 4 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 e Precision repeating pipettor e Orbital microplate shaker e Microcentrifuge and tubes for sample preparation e Plate reader capable of measuring absorbance in 96 well plates at dual wa s of 450 nm 540 nm Dual wavelengths of 450 550 or 450 595 nm can also be used n also be read at a single wavelength of 450 nm which will give a somewhat higher readi e Vortex mixer Qy e Microplate washer optional Manual washing is possible but not preferable f Software package fa
3. a High Sensitivity CRP ELISA Kit CircuLex Usei Manil For Research Use Only Not for use in diagnostic procedures ELISA Kit for Measuring Human High Sensitivity CRP CircuLex High Sensitivity CRP ELISA Ki Cat CY 8071 Intended Use cisssiesatstassntcssaasaaaiaaabalonatonatoons 1 AOI aT EEEE 1 TREOOU CUI OM a iesscascsnceadatacatantonatabotectacincevedeads 2 Qy Principle of the Assay 2 3 Materials Provided jissiccsessssanconcecsasndecancesuaneds 3 Materials Required but not Provided 4 Precautions and Recommendation 5 Sample Collection and Storage 0 6 Detailed Protocol suscscctsasssvasansassreeinasvseancens 7 9 CACM LAGOS cecancasaacestesassacnavessconeinwedseriasaans 9 Measurement Rative ccssecsectcarsnnvsaacenss 9 TroubleshoOotin iccaccsssenccncactssnsssarsenssensvene 9 Reagent SCADIIY vsscccsancrs dcasavnrvecaccaronsasanasoas 10 Assay CHaraCteriStics ccccsasnccacecersnnvvnancnsonsne 10 Example of Test Results 0 eeeeeeeeees 1 RGLELCHCES sasctssscnisvsinccedaqantebosaainanad tates 1 Related PROGUCtS crisicnscsncassssatsciectaaneonsnatosents 15 Intended Use The CycLex Research Product CircuL igh Sensitivity CRP ELISA Kit is used for the o quantitative measurement of Human iv tein CRP in serum plasma and other biological media This assay kit is for research use o d not for use in diagnostic or therapeutic procedures Storage e Upon rec
4. CY 8070 CycLex Co Ltd 1063 103 Terasawaoka Ina Nagano 396 0002 Japan Fax 81 265 76 7618 e mail info cyclex co jp URL http www cyclex A N CycLex CircuLex pr components th f products witho such commerci e supplied for research use only CycLex CircuLex products and t be resold modified for resale or used to manufacture commercial ten approval from CycLex Co Ltd To inquire about licensing for ease contact us via email C CY 8071 15 Version 140318
5. cilitating data generation and analysis opti 500 or 1000 mL graduated cylinder e Reagent reservoirs e Deionized water of the highest quality e Disposable paper towels C CY 8071 4 Version 140318 Tii High Sensitivity CRP ELISA Kit C 1rcu Lex User s Manual 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 e All microplate strips that are not immediately required should be returned to the zip lock pouch which 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 residue from glassware e Use deionized water of the highest quality e Do not mix reagents from different kits e The buffers and reagents in this kit may contain preservatives or oth r hemicals Care should be taken to avoid direct contact with these reagents e Do not mouth pipette or ingest any of the reagents e Do not smoke eat or drink when performing the assay orimyare as where samples or reagents are handled e Dispose of tetra methylbenzidine TMB containing solutions in compliance with local regulations e Avoid contact with the acidic Stop Solution and Substrate Solution which contains hydrogen peroxide e Wear gloves and eye protection when handling ummunodiagnostic materials a
6. cuLex High Sensitivity CRP BEISA Kit is designed to measure the concentration of Human CRP from cell lysate of cultured human cell lines cell culture conditioned medium and human serum plasma Cat CY 8071 2 Version 140318 Tii High Sensitivity CRP ELISA Kit C 1rcu Lex User s Manual For Research Use Only Not for use in diagnostic procedures Summary of Procedure Add 100 uL of diluted sample to the wells 4 Incubate for 1 hour at room temp Wash the wells t Add 100 uL of HRP conjugated anti Human CRP antibody 4 Incubate for 1 hour at room temp Wash the wells t Add 100 uL of Substrate Reagent Add 100 uL of Stop Solution t Measure absorbance at 450 nm Materials Provided All samples and standards should be assayed in duplicate The following components are supplied and are sufficient for the one 96 well microplate kit Microplate One microplate supplied ready to use with 96 wells 12 strips of 8 wells in a foil zip lock bag with a desiccant pack Wells are coated with anti Human CRP antibody as a capture antibody 10X Wash Buffer One 100 mL bottle of OX buffer containing 2 Tween 20 Dilution Buffer One bottle containing 50 mb of 1X buffer use for sample dilution Ready to use Human CRP Standard One vialicontaining 19 ng of lyophilized Human CRP HRP conjugated Detection Antibody One vial containing 12 mL of HRP horseradish peroxidase conjugated anti Human CRP antibody Ready to use
7. d at below 70 C fore d periods of time Avoid repeated freeze thaw cycles Plasma Collect plasma using EDTA Na or heparin as the anticoagulant If possible colle sma into a mixture of EDTA Na and Futhan5 to stabilize the sample against spontaneous vitro immediately or store samples on ice for up to 6 hours before assaying Aliquots of may also be stored at below 70 C for extended periods of time Avoid repeated freeze thaw cycl Note Citrate plasma has not been validated for use in this assay 7 Other biological samples Remove any particulates by centrifugation an ediately or aliquot and store samples at below 70 C Avoid repeated freeze thaw cycles complement activation Immediately centrifuge samples at 4 C for 15 minutes at gt x g Assay C CY 8071 6 Version 140318 Tii High Sensitivity CRP ELISA Kit C 1rcu Lex User s Manual For Research Use Only Not for use in diagnostic procedures Detailed Protocol The CycLex Research Product CircuLex High Sensitivity CRP ELISA Kit is provided wath removable strips of wells so the assay can be carried out on separate occasions using only the number of strips required for the particular determination Since experimental conditions may vary an aliquot of the Human CRP Standard within the kit should be included in each assay as a calibrator Disposable pipette tips and reagent troughs should be used for all liquid transfers to avoid cross contamination of reagents or samples
8. eipt store all co e Don t expose reagents A C CY 8071 1 Version 140318 Tii High Sensitivity CRP ELISA Kit C 1rcu Lex User s Manual For Research Use Only Not for use in diagnostic procedures Introduction C Reactive protein CRP is a pentameric acute phase reactant that is synthesized by the liverelts production is controlled primarily by interleukin 6 The serum CRP concentration may increase by up to 1000 fold with infection ischemia trauma surgery and other acute inflammatory events 1 Thus CRP can be used as an extremely sensitive systemic marker of inflammation Especially in situlattons where microbiological diagnosis is difficult or too slow in the clinical context CRP measurements can be used to infer the presence of bacterial infection 2 Ligand bound or aggregated CRP binds Clq and in so doing activates the classical complement pathway 3 Although primarily synthesized in hepatocytes there is evidence for local expression of CRP in macrophages of the lung and the brain 4 6 In atherosclerotic plaquesf it Has been found associated with complement proteins and within foam cells 7 8 A growing number of studies suggest that CRP is an independent risk faetor forfatherosclerotic vascular disease Plasma CRP concentrations in the highest quartile are associated jdepending on the subject group with 1 5 to 7 fold increases in relative risk of symptomatic atherosclerosis 1 9 The baseline plasma concentrati
9. hemistry 39 2 293 297 3 Du Clos TW Function of C reactive protein Ann Med 2000 32 274 278 4 Dong Q Wright JR Expression of C reactive protein by alveolar macrophages J Immunol 1996 156 4815 4820 5 Gould JM Weiser JN Expression of C reactive protein in the human respiratory tract Infeet Immun 2001 69 1747 1754 6 Yasojima K Schwab C McGeer EG et al Generation of C reactive protei amd complement components in atherosclerotic plaques Am J Pathol 2001 158 1039 1051 7 Torzewski M Rist C Mortensen RF et al C reactive protein in the arterial intima r le of C reactive protein receptor dependent monocyte recruitment in atherogenesis Arteridsclef Thromb Vasc Biol 2000 20 2094 2099 Tracy RP Inflammation markers and coronary heart disease Curr Opin Mapidol 4999 10 435 451 Mendall MA Patel P Ballam L et al C reactive protein and its relation to cardiovascular risk factors a population based cross sectional study Br Med J 1996 312 106L 1L065 10 Ridker PM Cushman M Stampfer MJ et al Inflammation aspirin and the risk of cardiovascular disease in apparently healthy men N Engl J Med 1997 336 973 979 11 Tomoda H Aoki N Prognostic value of C reactive protein levels within six hours after the onset of acute myocardial infarction Am Heart J 2000 140 324 328 12 Heeschen C Hamm CW Bruemmer J et al Predictive value ofC reactive protein and troponin T in patients with unstable angi
10. l pipette manifold dispenser or microplate washer 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 shaking at ca 300 rpm on an orbital microplate shaker 8 Wash 4 times by filling each well with Wash Buffer 50 uD using a squirt bottle multi channel pipette manifold dispenser or microplate washer 9 Add 100 uL of Substrate Reagent Avoid exposing the microtiter plate to direct sunlight Covering the plate with e g aluminum foil is recommended Return Substrate Reagent to 4 C immediately after the necessary volume is removed 10 Incubate the plate at room temperature ca 25 C for 10 20 minutes shaking at ca 300 rpm on an orbital microplate shaker The incubation timegmay be extended up to 30 minutes if the reaction temperature is below than 20 C 11 Add 100 uL of Stop Solution t each well in the same order as the previously added Substrate Reagent 12 Measure absorbance in eachfwell using a spectrophotometric microplate reader at dual wavelengths of 450 540 nm Dual wavelengths of 450 550 or 450 595 nm can also be used Read the microplate at 450 nm if only a singlegwavelength can be used Wells must be read within 30 minutes of adding the Stop Solution Note 1 Complete removal of liquid at each step is essential to good performance After the last wash remove any femaining Wash Buffer by aspirating or decanting Invert the pla
11. lution factor 1 The results of unknown samples can be calculated with any computer program haying a 5 parameter logistic function It is important to make an appropriate mathematical adjustment to accommodate for the dilution factor 2 Most microtiter plate readers perform automatic calculations of analyte Concentration The calibration curve is constructed by plotting the absorbance Y of calibrators versus log of the known concentration X of calibrators using the four parametergfunction Alternatively the logit log function can be used to linearize the calibration curve i e logit of absorbance Y is plotted versus log of the known concentration X of calibrators Measurement Range The measurement range is 59 4 ug mL to 3 800 pg mL Any sample reading higher than the highest standard should be diluted with Dilution Buffer in highergdilution and re assayed Dilution factors need to be taken into consideration in calculating the human CPR concentration Troubleshooting 1 The Human CRP Standard should be f n in duplicate using the protocol described in the Detailed Protocol Incubation times or tefiperatures significantly different from those specified may give erroneous results 2 Poor duplicates accompanied byjeleyated values for wells containing no sample indicate insufficient washing If all instructions inthe Detailed Protocol were followed accurately such results indicate a need for washer maintenance 3 Overall l
12. na a comparative analysis CAPTURE Investigators Chimeric c7E3 AntiPlatelet Therapy in Unstable angina REfractory to standard treatment trial J Am Coll Cardiol 2000 35 1535 1542 13 Liuzzo G Biasucci LM Gallimore JR et al The prognostic value of C reactive protein and serum amyloid a protein in severe unstable angina N Engl Med 1994 331 417 424 14 Festa A D Agostino R Howard G et al Chronic subclinical inflammation as part of the insulin resistance syndrome The Insulin Resistan e Atherosclerosis Study IRAS Circulation 2000 102 42 47 15 Visser M Bouter LM McQuillan GMgget allElevated C reactive protein levels in overweight and obese adults JAMA 1999 282 2134 2135 16 Pradhan AD Manson JE Rifai N et alyC reactive protein interleukin 6 and risk of developing type 2 diabetes mellitus JAMA 2001 286 327 334 17 Tchernof A Nolan A Sites CK et al Weight loss reduces C reactive protein levels in obese postmenopausal women Circulation 2002 105 564 569 2 0 Cat CY 8071 14 Version 140318 High Sensitivity CRP ELISA Kit C ircuLex User s Manual A 4 For Research Use Only Not for use in diagnostic procedures Related Products CircuLex Human Adiponectin ELISA Kit Cat CY 8050 CircuLex 100A12 EN RAGE ELISA Kit Cat CY 8058 CircuLex S100A4 ELISA Kit Cat CY 8059 CircuLex CML N carboxymethyl lysine ELISA Kit Cat CY 8066 CircuLex Human NGAL Lipocalin 2 ELISA Kit Cat
13. nd samples of human origin and these reagents In case of contact with the Stop Solution and the Substrate Solution wash skin thoroughly with water and seek medieal attention when necessary Human CRP Standard containsphuman sera Handle as if capable of transmitting infectious agents The sera were tested and found to be negative for HIV HBV and HCV However because no test method can offer complete assurance that HIV HBV HCV or other infectious agents are absent this component should be handled at the Bio safety Level 2 BSL 2 as recommended for any potentially infectious human serum or blood specimen in the CDC NIH manual Biosafety in microbiological and Biomedical Laboratories Biological samples may be contaminated with infectious agents Do not ingest expose to open wounds or breathe aerosols Wear protective gloves and dispose of biological samples properly e Sulfuric Acid is ajstrong acid Wear disposable gloves and eye protection when handling Stop Solution Cat CY 8071 5 Version 140318 High Sensitivity CRP ELISA Kit C ircuLex User s Manual 4 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 Centrifug samples at 4 C for 10 minutes at 1 000 x g Remove serum and assay immediately or store sam 0 ice for up to 6 hours before assaying Aliquots of serum may also be store
14. on of C reactive protein predicts the risk of futire myocardial infarction and stroke 10 and is associated with a poor prognosis in unstable angina 11 13 Plasma CRP levels are also strongly associated with obesity and obesity r lated diseases including insulin resistance diabetes mellitus and hyperlipidemia 14 17 Althoughya recent report indicated that the plasma CRP level decreased during weight reduction 17 sthe precise interaction of CRP with obesity has not been fully elucidated Principle of the Assay The CircuLex High Sensitivity CRP ELISA Agit employs the quantitative sandwich enzyme immunoassay technique An antibody specific for Human CRP has been pre coated onto a microplate Standards and samples are pipetted into the wells and the immobilized antibody binds any Human CRP present After washing away any unbound substances anbHRP conjugated antibody specific for Human CRP is added to the wells Following a wash to remove any unbound antibody HRP conjugate the remaining conjugate is allowed to react with the substrate H O tetramethylbenzidine The reaction is stopped by addition of acidic solution and absorbance of the resulting yellow product is measured at 450 nm The absorbance is proportional to theoncentration of Human CRP A standard curve is constructed by plotting absorbance values versus Human CRP concentrations of calibrators and concentrations of unknown samples are determined using this standard curve The Cir
15. ow signal niayundicate that desiccation of the plate has occurred between the final wash and addition of Substrate Reagent Do not allow the plate to dry out Add Substrate Reagent immediately after wash Cat CY 8071 9 Version 140318 High Sensitivity CRP ELISA Kit C ircuLex User s Manual For Research Use Only Not for use in diagnostic procedures Reagent Stability All of the reagents included in the CycLex Research Product CircuLex High Sensitivity GRP ELISA Kit have been tested for stability Reagents should not be used beyond the stated expiration date Upon receipt kit reagents should be stored at 4 C except the reconstituted Human CRP Standard must be stored at below 70 C Coated assay plates should be stored in the original foil bag sealed by the zip lock and containing a desiccant pack For research use only not for use in diagnostic or therapeutic procedures Assay Characteristics 1 Sensitivity The limit of detection defined as such a concentration of Human CRP givingsabsorbance higher than mean absorbance of blank plus three standard deviations of the absorbance of blank A blank 3 SD blank is better than 28 6 pg mL of sample Dilution Buffer is pipetted into blank wells Eighty assays were evaluated and the minimum detectable dos MDD of Human CRP ranged from 21 5 34 3 pg mL The mean MDD was 28 6 pg mL The MDD was determined by adding three standard deviations to the mean optical density value of
16. rch Use Only Not for use in diagnostic procedures 4 Spiking Recover Serum samples were spiked with different amounts of Human CRP and assayed The recovery of Human CRP spiked to levels throughout the range of the assay was evaluated Sample Average Recovery Range Cell culture media n 4 e Recovery 104 96 112 5 Linearity To assess the linearity of the assay samples containing and or spiked with high concentrations of Human CRP were serially diluted with the Dilution Buffer to produce samples wi es within the dynamic range of the assay The linearity of the assay 4 0 Serum A A Serum B Serum C hu CRP conc ng ml 0 0 5 1 15 2 25 3 3 5 4 4 5 C CY 8071 12 Version 140318 High Sensitivity CRP ELISA Kit C ircuLex User s Manual A 4 For Research Use Only Not for use in diagnostic procedures Example of Test Results Fig 1 Human CRP level in sera from 63 healthy volunteers 40 30 20 E 2 10 i E lt 0 5 0 5 1 1 5 5 10 210 CRP conc ug ml Version 140318 C CY 8071 13 High Sensitivity CRP ELISA Kit C ircuLex User s Manual For Research Use Only Not for use in diagnostic procedures References j Westhuyzen J Healy H Biology and relevance of C reactive protein in cardiovascular and renal disease Ann Clin Lab Sci 2000 30 133 143 2 Nakayama T et al 1993 Clinical C
17. re dispensing Unused portions of Standards should be aliquoted and stored at below 70 C immediately Avoid multiple freeze and thaw cycles Sample Preparation e Serum and plasma samples require a 2 500 fold dilution e g First Make 50 fold dilution 2 uL of sample 98 uL of Dilution Buffer Second Make 2 500 fold dilution 5 uL of 50 fold diluted sample 245 uL Dilution Buffer Don tstor diluted samples e Other biological samples require 10 100 and 1 000 fold dilution Cat CY 8071 T Version 140318 Tii High Sensitivity CRP ELISA Kit C 1rcu Lex User s Manual For Research Use Only Not for use in diagnostic procedures Assay Procedure 1 Prepare the assay protocol assigning the appropriate wells for setting up Standard solutions diluted patient specimens and any internal laboratory controls in duplicate Remove the appropriate number of microtiter wells from the foil pouch and place them into the well holder Return any unused wells to the foil pouch refold seal with tape and store at 4 C 2 Dilute samples with Dilution Buffer See Sample Preparation above 3 Pipette 100 uL of Standard Solutions Std1 Std7 Blank and diluted samples in duplicates into the appropriate wells 4 Incubate the plate at room temperature ca 25 C for 1 hour shaking at cae300 mpm on an orbital microplate shaker 5 Wash 4 times by filling each well with Wash Buffer 350 uL using aisquirt bottle multi channe
18. te and blot it against Clean paper towels Note 2 Reliable standard curves are obtained when either O D values do not exceed 0 2 units for the blank Zero concentration or 2 5 units for the highest standard concentration The plate Should be monitored at 5 minute intervals for approximately 30 minutes Note 3 Ithe microplate reader is not capable of reading absorbance greater than the absorbance of the Cat CY 8071 8 Version 140318 High Sensitivity CRP ELISA Kit C ircuLex User s Manual For Research Use Only Not for use in diagnostic procedures highest standard perform a second reading at 405 nm A new standard curve constructed using the values measured at 405 nm is used to determine Human CRP concentration of off scale samples The readings at 405 nm should not replace the on scale readings at 450 nm Calculations Average the duplicate readings for each standard control and sample and subtract the average zero standard optical density Plot the optical density for the standards versus the concentration of the standards and draw the best curve To determine the Human CRP concentration of each sample first find the absorbance value on the y axis and extend a horizontal line to the standard curve At the point of intersection extend a vertical line to the x axis and read the corresponding Human CRP concentration If the samples have been diluted the concentration read from the standard curve must bednultiplied by the di
19. twenty zero standard replicates and calculating the corresponding concentration Typical standard curve 2 5 2 0 1 5 A450 1 0 0 5 0 0 0 0 1 0 2 0 3 0 4 0 Human CRP conc ng ml Cat CY 8071 10 Version 140318 Tii High Sensitivity CRP ELISA Kit C 1rcu Lex User s Manual For Research Use Only Not for use in diagnostic procedures 2 Specificity The antibodies in the CircuLex High Sensitivity CRP ELISA Kit are highly specific of Human CRP with no detectable cross reactivity to other serum protein and to other acute phase reactant including SAA 3 Precision Intra assay Precision Precision within an assay Three samples of known concentration were tested twelve times on one plate to assess imta assay precision e Intra assay Within Run n 12 CV 3 8 1 8 2 6 CRP conc in human Serum ug ml Inter assay Precision Precision between assays Three samples of known concentration yere tested in five separate assays to assess inter assay precision e Inter assay Run to Run n 5 CVi 5 2 5 2 2 6 CRP conc ug ml Day Serum 1 Serum 2 Serum 3 1 1 16 3 94 7 29 2 1 14 3 88 7 33 3 1 01 3 58 7 44 4 1 11 3 76 7 13 5 1 11 3 48 6 97 MAX 1 16 3 94 7 44 MIN 1 01 3 48 6 97 MEAN 1 10 3 73 7 23 S D 0 058 0 195 0 186 C V 5 2 5 2 2 6 Cat CY 8071 11 Version 140318 High Sensitivity CRP ELISA Kit C ircuLex User s Manual For Resea
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