IFU - DRG International
Contents
1. Wash Solution Conc 10x concentrated 100 mL Substrate Solution ready to use 13 mL Stop Solution ready to use 13 mL Product Data Sheet Certificate of Analysis I pc 7 MATERIAL REQUIRED BUT NOT SUPPLIED Deionized distilled water Test tubes for diluting samples Glassware graduated cylinder and bottle for Wash Solution Dilution Buffer Precision pipettes to deliver 10 1000 uL with disposable tips Multichannel pipette to deliver 100 uL with disposable tips Absorbent material e g paper towels for blotting the microtitrate plate after washing Vortex mixer Orbital microplate shaker capable of approximately 300 rpm Microplate washer optional Manual washing is possible but not preferable Microplate reader with 450 10 nm filter preferably with reference wavelength 630 nm alternatively another one from the interval 550 650 nm Software package facilitating data generation and analysis optional 8 PREPARATION OF REAGENTS All reagents need to be brought to room temperature prior to use Always prepare only the appropriate quantity of reagents for your test Do not use components after the expiration date marked on their label DRG International Inc USA Fax 908 233 0758 e mail corp drg international com 3 DRE iS DRG Cystatin C human ELISA EIA 4394 REVISED 1 JUNE 2012 RM VERS 11 1 IN THE USA Assay reagents supplied ready to use Ant2. JH Rebeck GW Elevation of cystatin C in susceptible neurons in Alzheimer s disease Am J Pathol 159 1061 1068 2001 Dharnidharka VR Kwon C Stevens G Serum cystatin C is superior to serum creatinine as a marker of kidney function a meta analysis Am J Kidney Dis 40 221 226 2002 Mojiminiyi OA and Abdella N Evaluation of cystatin C and beta 2 microglobulin as markers of renal function in patients with type 2 diabetes mellitus J Diabetes Complications 17 160 168 2003 Perlemoine C Beauvieux MC Rigalleau V Baillet L Barthes N Derache P Gin H Interest of cystatin C in screening diabetic patients for early impairment of renal function Metabolism 52 1258 1264 2003 Mussap M and Plebani M Biochemistry and Clinical Role of Human Cystatin C Crit Rev Clin Lab Sci 41 467 550 2004 Johnston N Jernberg T Lindahl B Lindback J Stridsberg M Larsson A Venge P Wallentin L Biochemical indicators of cardiac and renal function in a healthy elderly population Clin Biochem 37 210 216 2004 Luc G Bard JM Lesueur C Arveiler D Evans A Amouyel P Ferrieres J Juhan Vague I Fruchart JC Ducimetiere P Plasma cystatin C and development of coronary heart disease The PRIME Study Atherosclerosis 2005 Macisaac RJ Tsalamandris C Thomas MC Premaratne E Panagiotopoulos S Smith TJ Poon A Jenkins MA Ratnaike SI Power DA Jerums G Estimating glomerular filtration rate in diabetes a comparison of cystatin C and creatinine bas
3. DRE iS DRG Cystatin C human ELISA EIA 4394 REVISED 1 JUNE 2012 RM VERS 11 1 IN THE USA 10 ASSAY PROCEDURE 1 Pipet 100 uL of diluted Standards Quality Controls Dilution Buffer Blank and samples preferably in duplicates into the appropriate wells See Figure 1 for example of work sheet 2 Incubate the plate at room temperature ca 25 C for 30 minutes shaking at ca 300 rpm on an orbital microplate shaker 3 Wash the wells 3 times with Wash Solution 0 35 mL per well After final wash invert and tap the plate strongly against paper towel 4 Add 100 uL of Conjugate Solution into each well 5 Incubate the plate at room temperature ca 25 C for 30 minutes shaking at ca 300 rpm on an orbital microplate shaker Incubation without shaking is an alternative that requires extended incubation with substrate see step 8 below 6 Wash the wells 3 times with Wash Solution 0 35 mL per well After final wash invert and tap the plate strongly against paper towel 7 Add 100 uL of Substrate Solution into each well Avoid exposing the microtiter plate to direct sunlight Covering the plate with e g aluminium foil is recommended 8 Incubate the plate for 10 minutes at room temperature The incubation time may be extended up to 20 minutes if the reaction temperature is below than 20 C Do not shake with the plate during the incubation 9 Stop the colour development by adding 100 pL of Stop Solution 10 Determ
4. DRE iS DRG Cystatin C human ELISA EIA 4394 REVISED 1 JUNE 2012 RM VERS 11 1 IN THE USA This kit is intended for Research Use Only Not for use in diagnostic procedures Please use only the valid version of the package insert provided with the kit 1 INTENDED USE The Cystatin C human ELISA is a sandwich enzyme immunoassay for easurement of human cystatin C Features The total assay time is less than 2 hours The kit measures total cystatin C in serum plasma EDTA citrate heparin urine and cerebrospinal fluid Assay format is 96 wells Quality Controls are human serum or human urine native protein based No animal sera are used Standard is purified native protein based Components of the kit are provided ready to use or concentrated Convenient for automatization 2 STORAGE EXPIRATION Store the complete kit at 2 C 8 C Under these conditions the kit is stable until the expiration date see label on the box 3 TEST PRINCIPLE In the Human Cystatin C ELISA standards quality controls and samples are incubated in microtitrate plate wells pre coated with polyclonal anti human cystatin C antibody After 30 minutes incubation and washing polyclonal anti human cystatin C antibody conjugated with horseradish peroxidase HRP is added to the wells and incubated for 30 minutes with captured cystatin C Following another washing step the remaining HRP conjugate is allowed to r
5. RMINATION For the determination of cystatin C in urine use the serum plasma protocol only with the following modifications 13 1 Sample collection and storage It is recommended to freeze down untreated urine although no significant decline was observed in concentration of human cystatin C in samples stored at 4 C for 14 days 13 2 Sample preparation Dilute urine samples 20x with Dilution Buffer just prior to use in the assay e g 20 uL of sample 380 uL of Dilution Buffer Stability and storage Untreated urine samples are stable for 3 months if stored at 20 C 70 C Do not store the diluted samples 13 3 Calculations of results Standard curve is plotted using values of undiluted Standards 10 000 4 000 2 000 1 000 400 and 200 ng mL As urine samples are diluted only 20x whereas Standards are diluted 400x the result read off the Standard curve has to be divided by dilution factor 20 in order to obtain the real concentration in the original undiluted sample DRG International Inc USA Fax 908 233 0758 e mail corp drg international com 10 DRE iS DRG Cystatin C human ELISA EIA 4394 REVISED 1 JUNE 2012 RM VERS 11 1 IN THE USA 14 TROUBLESHOOTING AND FAQS Weak signal in all wells Possible explanations Omission of a reagent or a step Improper preparation or storage of a reagent Assay performed before reagents were allowed to come to room temperature Improper wavelength when reading absorbanc
6. al attention when necessary The materials must not be pipetted by mouth TECHNICAL HINTS Reagents with different lot numbers should not be mixed Use thoroughly clean glassware Use deionized distilled water stored in clean containers Avoid any contamination among samples and reagents For this purpose disposable tips should be used for each sample and reagent Substrate Solution should remain colourless until added to the plate Keep Substrate Solution protected from light Stop Solution should remain colourless until added to the plate The colour developed in the wells will turn from blue to yellow immediately after the addition of the Stop Solution Wells that are green in colour indicate that the Stop Solution has not mixed thoroughly with the Substrate Solution Dispose of consumable materials and unused contents in accordance with applicable national regulatory requirements DRG International Inc USA Fax 908 233 0758 e mail corp drg international com 2 DRE iS DRG Cystatin C human ELISA EIA 4394 REVISED 1 JUNE 2012 RM VERS 11 1 IN THE USA 6 REAGENT SUPPLIED Kit Components State Quantity Antibody Coated Microtiter Strips ready to use 96 wells Conjugate Solution Conc 50x concentrated 0 26 mL Conjugate Diluent ready to use 13 mL Set of Standards concentrated 6x 0 1 mL Quality Control HIGH concentrated 0 1 mL Quality Control LOW concentrated 0 1 mL Dilution Buffer Conc 10x concentrated 10 mL
7. e High signal and background in all wells Possible explanations e Improper or inadequate washing e Overdeveloping incubation time with Substrate Solution should be decreased before addition of Stop Solution e Incubation temperature over 30 C High coefficient of variation CV Possible explanation e Improper or inadequate washing e Improper mixing Standards Quality Controls or samples DRG International Inc USA Fax 908 233 0758 e mail corp drg international com 11 DRG iS C REVISED 1 JUNE 2012 RM VERS 11 1 IN THE USA DRG Cystatin C human ELISA EIA 4394 15 REFERENCES Ekiel I Abrahamson M Fulton DB Lindahl P Storer AC Levadoux W Lafrance M Labelle S Pomerleau Y Groleau D LeSauteur L Gehring K NMR structural studies of human cystatin C dimmers and monomers J Mol Biol 271 266 277 1997 Tian S Kusano E Ohara T Tabei K Itoh Y Kawai T Asano Y Cystatin C measurement and its practical use in patients with various renal diseases Clin Nephrol 48 104 108 1997 Bokenkamp A Domanetzki M Zinck R Schumann G Byrd D Brodehl J Cystatin C a new marker of glomerular filtration rate in children independent of age and height Pediatrics 101 875 881 1998 Risch L Blumberg A Huber A Rapid and accurate assessment of glomerular filtration rate in patients with renal transplants using serum cystatin C Nephrol Dial Transplant 14 1991 1996 1999 Deng A Irizarry MC Nitsch RM Growdon
8. e 3 months when stored at 2 C 8 C Do not store the diluted set of Standards Quality Controls High Low Refer to the Certificate of Analysis for current Quality Control concentration Dilute each Quality Control QC 400x with the Dilution Buffer just prior to the assay in two steps as follows DRG International Inc USA Fax 908 233 0758 e mail corp drg international com 4 DRE iS DRG Cystatin C human ELISA EIA 4394 REVISED 1 JUNE 2012 RM VERS 11 1 IN THE USA Dilution A 10x Add 10 uL of QC into 90 uL of Dilution Buffer Mix well not to foam Vortex is recommended Dilution B 40x Add 10 uL of Dilution A into 390 uL of Dilution Buffer to prepare final dilution 400x Mix well not to foam Vortex is recommended Stability and storage Opened Quality Controls are stable 3 months when stored at 2 C 8 C Do not store the diluted Quality Controls Note Concentration of analyte in Quality Controls need not be anyhow associated with normal and or pathological concentrations in serum or another body fluid Quality Controls serve just for control that the kit works in accordance with the user s manual and CoA and that ELISA test was carried out properly It is recommended to supplement two or three negative sample controls of customer s own in addition to those provided with this kit They can serve as evidence of the difference between positive and negative samples see Figure 5 and Fig
9. eact with the substrate solution TMB The reaction is stopped by addition of acidic solution and absorbance of the resulting yellow product is measured The absorbance is proportional to the concentration of cystatin C A standard curve is constructed by plotting absorbance values against concentrations of cystatin C standards and concentrations of unknown samples are determined using this standard curve 4 PRECAUTIONS For professional use only Wear gloves and laboratory coats when handling kit materials Do not drink eat or smoke in the areas where kit materials are being handled DRG International Inc USA Fax 908 233 0758 e mail corp drg international com 1 DRG iS C REVISED 1 JUNE 2012 RM VERS 11 1 IN THE USA DRG Cystatin C human ELISA EIA 4394 This kit contains components of human origin These materials were found non reactive for HBsAg HCV antibody and for HIV 1 2 antigen and antibody However these materials should be handled as potentially infectious as no test can guarantee the complete absence of infectious agents Avoid contact with the acidic Stop Solution and Substrate Solution which contains hydrogen peroxide and tetramethylbenzidine TMB Wear gloves and eye and clothing protection when handling these reagents Stop and or Substrate Solutions may cause skin eyes irritation In case of contact with the Stop Solution and the Substrate Solution wash skin eyes thoroughly with water and seek medic
10. ed methods Diabetologia 49 1686 1689 2006 Nakashima I Fujihara K Fujinoki M Kawamura T Nishimura T Nakamura M Itoyama Y Alteration of cystatin C in the cerebrospinal fluid of multiple sclerosis Ann Neurol 2006 Delanaye P Cavalier E Krzesinski JM Cystatin C renal function and cardiovascular risk Ann Intern Med Jul 33147 1 19 27 2007 Parikh NI Hwang SJ Yang Q Larson MG Guo CY Robins SJ Sutherland P Benjamin EJ Levy D Fox CS Clinical correlates and heritability of cystatin C from the Framingham Offspring Study Am J Cardiol Nov 1 102 9 1194 8 2008 Sundel f J Arnl v J Ingelsson E Sundstr m J Basu S Zethelius B Larsson A Irizarry MC Giedraitis V R nnemaa E Degerman Gunnarsson M Hyman BT Basun H Kilander L Lannfelt L Serum cystatin C and the risk of Alzheimer disease in elderly men Neurology Sep 30 71 14 1072 9 2008 Ortiz F Harmoinen A Paavonen T Koskinen P Gr nhagen Riska C Honkanen E Is Cystatin C more sensitive than creatinine in detecting early chronic allograft nephropathy Clin Nephrol 70 1 18 25 2008 Samouilidou EC Grapsa E Relationship of serum cystatin C with C reactive protein and apolipoprotein Al in patients on hemodialysis Ren Fail 30 7 711 5 2008 DRG International Inc USA Fax 908 233 0758 e mail corp drg international com 12 DRG iS C REVISED 1 JUNE 2012 RM VERS 11 1 IN THE USA DRG Cystatin C human ELISA EIA 4394 Muntner P Ma
11. ibody Coated Microtiter Strips Stability and storage Return the unused strips to the provided aluminium zip sealed bag with desiccant and seal carefully Remaining Microtiter Strips are stable 3 months stored at 2 C 8 C and protected from the moisture Conjugate Diluent Substrate Solution Stop Solution Stability and storage Opened reagents are stable 3 months when stored at 2 C 8 C Assay reagents supplied concentrated Dilution Buffer Conc 10x Dilute Dilution Buffer Concentrate 10x ten fold in 90 mL distilled water to prepare a 1x working solution e g 10 mL of Dilution Buffer Concentrate 10x 90 mL of distilled water for use of all 96 wells It is recommended to dilute only such a volume of Dilution Buffer Concentrate 10x to be used up in the one run of the test Stability and storage The diluted Dilution Buffer is stable 1 week when stored at 2 C 8 C Opened Dilution Buffer Concentrate 10x is stable 3 months when stored at 2 C 8 C Set of Standards Dilute each concentration of Standard 400x with the Dilution Buffer just prior to the assay in two steps as follows Dilution A 10x Add 10 uL of Standard into 90 uL of Dilution Buffer Mix well not to foam Vortex is recommended Dilution B 40x Add 10 uL of Dilution A into 390 uL of Dilution Buffer to prepare final dilution 400x Mix well not to foam Vortex is recommended Stability and storage Opened Standards are stabl
12. ine the absorbance of each well using a microplate reader set to 450 nm preferably with the reference wavelength set to 630 nm acceptable range 550 650 nm Subtract readings at 630 nm 550 650 nm from the readings at 450 nm The absorbance should be read within 5 minutes following step 9 Note If some samples and standards have absorbances above the upper limit of your microplate reader perform a second reading at 405 nm A new standard curve constructed using the values measured at 405 nm is used to determine cystatin C concentration of off scale standards and samples The readings at 405 nm should not replace the readings for samples that were in range at 450 nm Note 2 Manual washing Aspirate wells and pipet 0 35 mL Wash Solution into each well Aspirate wells and repeat twice After final wash invert and tap the plate strongly against paper towel Make certain that Wash Solution has been removed entirely DRG International Inc USA Fax 908 233 0758 e mail corp drg international com 7 DRE iS DRG Cystatin C human ELISA EIA 4394 REVISED 1 JUNE 2012 RM VERS 11 1 IN THE USA Figure 1 Example of a work sheet 11 CALCULATIONS Most microplate readers perform automatic calculations of analyte concentration The standard curve is constructed by plotting the mean absorbance Y of Standards against the known concentration X of Standards in logarithmic scale using the four parameter algorithm Result
13. n ELISA EIA 4394 REVISED 1 JUNE 2012 RM VERS 11 1 IN THE USA Dilute samples serum plasma 400x with the Dilution Buffer just prior to the assay in two steps as follows Dilution A 10x Add 10 uL of sample into 90 uL of Dilution Buffer Mix well not to foam Vortex is recommended Dilution B 40x Add 10 uL of Dilution A into 390 uL of Dilution Buffer to prepare final dilution 400x Mix well not to foam Vortex is recommended Dilute samples CSF 1600x with the Dilution Buffer just prior to the assay as follows Dilution A 40x Add 10 ul of sample into 390 ul of Dilution Buffer Mix well not to foam Vortex is recommended Dilution B 40x Add 10 ul of Dilution A into 390 ul of Dilution Buffer to prepare final dilution 1600x Mix well not to foam Vortex is recommended Stability and storage Samples should be stored at 20 C or preferably at 70 C for long term storage Avoid repeated freeze thaw cycles Do not store the diluted samples For dilution of urine samples see Chapter 15 See Chapter 13 for stability of serum and plasma samples when stored at 2 C 8 C effect of freezing thawing and effect of sample matrix serum plasma on the concentration of cystatin C Note It is recommended to use a precision pipette and a careful technique to perform the dilution in order to get precise results DRG International Inc USA Fax 908 233 0758 e mail corp drg international com 6
14. nation in clinical diagnostics Biomed Papers 147 177 180 2003 Straface E Matarrese P Gambardella L Vona R Sgadari A Silveri MC Malorni W Oxidative imbalance and cathepsin D changes as peripheral blood biomarkers of Alzheimer disease A pilot study FEBS Lett 579 2759 2766 2005 Stejskal D Vavrouskova J Mares J Urbanek K Applications of new laboratory marker assays in neurological diagnoses A pilot study Biomed Pap Med Fac Univ Palacky Olomouc Czech Repub 149 265 266 2005 Taleb S Lacasa D Bastard JP Poitou C Cancello R Pelloux V Viguerie N Benis A Zucker JD Bouillot JL Coussieu C Basdevant A Langin D Clement K Cathepsin S a novel biomarker of adiposity relevance to atherogenesis FASEB J 19 1540 1542 2005 Barrientos LG Rollin PE Release of cellular proteases into the acidic extracellular milieu exacerbates Ebola virus induced cell damage Virology 2006 Liu XD Zeng BF Xu JG Zhu HB Xia QC Proteomic analysis of the cerebrospinal fluid of patients with lumbar disk herniation Proteomics 6 1019 1028 2006 Yang X Wang H Wang Z Dong M Alteration and significance of serum cardiac troponin I and cystatin C in preeclampsia Clin Chim Acta 374 168 169 2006 Taleb S Cancello R Poitou C Rouault C Sellam P Levy P Bouillot JL Coussieu C Basdevant A Guerre Millo M Lacasa D Clement K Weight loss reduces adipose tissue cathepsin S and its circulating levels in morbidly obese women J Clin End
15. nn D Winston J Bansilal S Farkouh ME Serum cystatin C and increased coronary heart disease prevalence in US adults without chronic kidney disease Am J Cardiol Jul 1 102 1 54 7 2008 Servais A Giral P Bernard M Bruckert E Deray G Isnard Bagnis C Is serum cystatin C a reliable marker for metabolic syndrome Am J Med May 121 5 426 32 2008 Muntner P Winston J Uribarri J Mann D Fox CS Overweight obesity and elevated serum cystatin C levels in adults in the United States Am J Med Apr 121 4 341 8 2008 Premaratne E MacIsaac RJ Finch S Panagiotopoulos S Ekinci E Jerums G Serial measurements of cystatin C are more accurate than creatinine based methods in detecting declining renal function in type I diabetes Diabetes Care May 31 5 971 3 2008 Moran A Katz R Smith NL Fried LF Sarnak MJ Seliger SL Psaty B Siscovick DS Gottdiener JS Shlipak MG Cystatin C concentration as a predictor of systolic and diastolic heart failure J Card Fail Feb 14 1 19 26 2008 Nedelkov D and Nelson RW Delineating protein protein interactions via biomolecular interaction analysis mass spectrometry J Mol Recognit 16 9 14 2003 Nedelkov D and Nelson RW Design and use of multi affinity surfaces in biomolecular interaction analysis mass spectrometry BLA MS a step toward the design of SPR MS arrays J Mol Recognit 16 15 19 2003 Mares J Stejskal D Vavrouskova J Urbanek K Herzig R Hlustik P Use of cystatin C determi
16. ocrinol Metab 91 1042 1047 2006 Hossain M A Emara M El moselhi H Shoker A Comparing measures of Cystatin C in human sera by three methods Am J Nephrology 29 5 381 391 2009 Wilson M E Boumaza I Lacomis D Bowser R Cystatin A candidate biomarker for amyotrophic lateral sclerosis PLoS ONE 5 12 1 9 2010 DRG International Inc USA Fax 908 233 0758 e mail corp drg international com 13 DRE iS DRG Cystatin C human ELISA EIA 4394 REVISED 1 JUNE 2012 RM VERS 11 1 IN THE USA e Tanindi A Olgun H Tuncel A Celik B Pasaoglu H Boyaci B Exercise electrocardiogrpahis responses and serum cystatin C levels among metabolic syndrome patients without overt diabetes mellitus Vascular Health and Risk Management 7 59 65 2011 DRG International Inc USA Fax 908 233 0758 e mail corp drg international com 14 DRE iS DRG Cystatin C human ELISA EIA 4394 REVISED 1 JUNE 2012 RM VERS 11 1 IN THE USA 16 PLATE LAYOUT N 11 10 DRG International Inc USA Fax 908 233 0758 e mail corp drg international com 15 DRE iS DRG Cystatin C human ELISA EIA 4394 REVISED 1 JUNE 2012 RM VERS 11 1 IN THE USA Rev 5 16 12cc DRG International Inc USA Fax 908 233 0758 e mail corp drg international com 16
17. s are reported as concentration of cystatin C ng mL in samples Alternatively the logit log function can be used to linearize the standard curve i e logit of the mean absorbance Y is plotted against log of the known concentration X of Standards Use values of undiluted standard range 10 000 4 000 2 000 1 000 400 200 ng mL Samples Quality Controls and Standards are all diluted 400x prior to analysis so there is no need to take this dilution factor into account Results are reported as total concentration of cystatin C ng mL in serum plasma samples For the determination of concentration in samples diluted differently use dilution factor for dividing multiplying results read off the standard curve DRG International Inc USA Fax 908 233 0758 e mail corp drg international com 8 DRG C DRG Cystatin C human ELISA EIA 4394 REVISED 1 JUNE 2012 RM VERS 11 1 IN THE USA Human Cystatin C ELISA Standard Curve ES o mm lt E fan LO st L 8 Cc 2 O wn 2 lt 1000 Concentration of Hu Cystatin C ng ml Figure 2 Typical Standard Curve for Human Cystatin C ELISA DRG International Inc USA Fax 908 233 0758 e mail corp drg international com DRE iS DRG Cystatin C human ELISA EIA 4394 REVISED 1 JUNE 2012 RM VERS 11 1 IN THE USA 12 DEFINITION OF THE STANDARD The Standard used in this kit is purified native protein based 13 URINE CYSTATIN C DETE
18. ure 6 Conjugate Solution Conc 50x Prepare the working Conjugate Solution by adding 1 part concentrated Conjugate Solution Concentrate 50x with 49 parts Conjugate Diluent Example 0 25 mL of Conjugate Solution Concentrate 50x 12 25 mL of Conjugate Diluent for use of all 96 wells Prepare only the volume needed for the test Mix well not to foam Stability and storage Opened Conjugate Solution Concentrate 50x is stable 3 months when stored at 2 C 8 C Do not store the diluted Conjugate Solution Wash Solution Conc 10x Dilute Wash Solution Concentrate 10x ten fold in 900 mL of distilled water to prepare a 1x working solution e g 100 mL of Wash Solution Concentrate 10x 900 mL of distilled water for use of all 96 wells Stability and storage The diluted Wash Solution is stable I month when stored at 2 C 8 C Opened Wash Solution Concentrate 10x is stable 3 months when stored at 2 C 8 C 9 PREPARATION OF SAMPLES The kit measures cystatin C in serum plasma EDTA citrate heparin urine and cerebrospinal fluid Samples should be assayed immediately after collection or should be stored at 20 C Mix thoroughly thawed samples just prior to the assay and avoid repeated freeze thaw cycles which may cause erroneous results Avoid using hemolyzed or lipemic samples DRG International Inc USA Fax 908 233 0758 e mail corp drg international com 5 DRE iS DRG Cystatin C huma
Download Pdf Manuals
Related Search