IFU - DRG International
Contents
1. 9 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 10 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 DRG International Inc USA Fax 908 233 0758 e mail corp drg international com 5 DIRE Co C lt Revised 1 June 2012 rm Vers 2 1 USA DRG Cartilage Oligomeric Matrix Protein Human EIA 5265 11 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 the plate during the incubation 12 Stop the colour development by adding 100 uL of Stop Solution 13 Determine 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 12 Note If some samples and standard s 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 COMP concentration of off scale standards and samples The readings at 405 nm should not replace the readings for sa2. DIRE Co DRG Cartilage Oligomeric Matrix Protein Human EIA 5265 C lt Revised 1 June 2012 rm Vers 2 1 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 Human Cartilage Oligomeric Matrix Protein ELISA is a sandwich enzyme immunoassay for measurement of human cartilage oligomeric matrix protein COMP Features The total assay time is less than 3 5 hours The kit measures COMP in serum and plasma EDTA citrate heparin Assay format is 96 wells Quality Controls are human serum based Standard is recombinant protein based Components of the kit are provided ready to use concentrated or lyophilized 2 STORAGE EXPIRATION Store the complete kit at 2 8 C Under these conditions the kit is stable until the expiration date see label on the box 3 TEST PRINCIPLE In the Human Cartilage Oligomeric Matrix Protein ELISA standards quality controls and samples are incubated in microplate wells pre coated with monoclonal anti human COMP antibody After 60 minutes incubation and washing biotin labelled second monoclonal anti human COMP antibody is added and incubated with captured COMP for 60 minutes After another washing streptavidin HRP conjugate is added After 30 minutes incubation and the last washing step the remaining conjugate is allowed to react with the substrate solution TMB The
3. A 5265 12 1 Effect of Freezing Thawing No decline was observed in concentration of human COMP in serum and plasma samples after repeated 5x freeze thaw cycles However it is recommended to avoid unnecessary repeated freezing thawing of the samples Sample Number of fit ae Plasma ng mL 716 614 128 woo pa a pae rs a e e s fa e n 13 DEFINITION OF THE STANDARD The recombinant human COMP is used as the Standard The recombinant human COMP is produced in cell line HEK293 13 1 Reference range The data quoted in these instructions should be used for guidance only It is recommended that each laboratory include its own panel of control sample in the assay Each laboratory should establish its own normal and pathological references ranges for COMP levels with the assay DRG International Inc USA Fax 908 233 0758 e mail corp drg international com 9 DIRE Co DRG Cartilage Oligomeric Matrix Protein Human EIA 5265 C lt Revised 1 June 2012 rm Vers 2 1 USA 14 TROUBLESHOOTING AND FAQS gt 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 absorbance gt High signal and background in all wells Possible explanations Improper or inadequate washing Overdeveloping incubation time with Substrate Solution should
4. be decreased before addition of Stop Solution Incubation temperature over 30 C gt High coefficient of variation CV Possible explanation Improper or inadequate washing Improper mixing Standards Quality Controls or samples 15 REFERENCES V Vilim M E Lenz R Vytasek K Masuda K Pavelka K E Kuettner and E J M Thonar Characterization of monoclonal antibodies recognizing different fragments of cartilage oligomeric matrix protein in human body fluid Arch Biochem Biophys 341 8 16 1997 V Vilim R Vytasek M Olejarova S Machacek J Gatterova B Prochazka V B Kraus and K Kavala Serum cartilage oligimeric matrix protein reflects the presence of clinically diagnosed synovitis in patients with knee osteoarthritis Osteoarthritis and Cartilage 9 612 618 2001 K Misumi V Vilim P D legg C C M Thompson and S D Carter Measurement of cartilage oligomeric matrix protein COMP in normal and diseased equine synovial fluids Osteoarthritis and Cartilage 9 119 127 2001 V Vilim M Olejarova S Machacek J Gatterova V B Kraus K Pavelka Serum levels of cartilage oligomeric matrix protein COMP correlate with radiographic progression of knee osteoarthritis Osteoarthr Cart 10 707 713 2002 A D Dragomir V B Kraus J B Rennef G Luta A Clark V Vilim M C Hochberg C G Helmick and J M Jordan Serum cartilage oligomeric matrix protein and clinical signs and symptoms of potential pre radiograp
5. dwin H Shen F J Pellett S Shanmugarajah C F Rosen and D D Gladman Soluble biomarkers differentiate patients with psoriatic arthritis from those psoriasis without arthrithis Rheumatology 49 1399 1405 2010 DRG International Inc USA Fax 908 233 0758 e mail corp drg international com 11 DIRE Co DRG Cartilage Oligomeric Matrix Protein Human EIA 5265 C lt Revised 1 June 2012 rm Vers 2 1 USA 16 ASSAY PROCEDURE SUMMARY Antibody Coated Micrmtiter Strips Reconstitute GCs and Master Standard and prepare set of Dilute samples 62x standards Add Standards QCs and samples 100 wl Prepare Wash Solution Incubate at RT for 1 hour 300 rpm Add Biotin Labelled Antibody 100 ul l Incubate at RT for 1 hour 300rpm D Add Streptavidin HRP Conjugate 100 wl Incubate at RT for 30 min 300rpm Wash 2x Add Substrate Solution 100 ul incubate at RT for 10 min Add Stop Solution 100 ul Read absorbance and calculate results DRG International Inc USA Fax 908 233 0758 e mail corp drg international com 12 DRG C lt Revised 1 June 2012 rm Vers 2 1 USA DRG Cartilage Oligomeric Matrix Protein Human EIA 5265 Rev 5 31 12cc DRG International Inc USA Fax 908 233 0758 e mail corp drg international com 13
6. hic hip and knee pathology Osteoarthritis and Cartilage 10 687 691 2002 DRG International Inc USA Fax 908 233 0758 e mail corp drg international com 10 DIRE Co DRG Cartilage Oligomeric Matrix Protein Human EIA 5265 Revised 1 June 2012 rm Vers 2 1 USA V Vilim Z Voburka R Vytasek L Senolt I Tchetverikov V B Kraus and K Pavelka Monoclonal antibodies to human cartilage oligomeric matrix protein epitope mapping and characterization of sandwich ELISA Clinica Chimica Acta 328 59 69 2003 O Bruzere J H Collette O Ethgen L C Rovati G Giacovelli Y E Henrotin L Seidel and J Y L Reginster Biochemical Markers of bone and Cartilage remodelling in prediction of longterm progression of knee osteoarthritis The Journal of Rheumatilogy 30 1043 1050 2003 J M Jordan G Luta T Stabler J B Renner A D Dragomir V Vilim M C Hochberg C G Helmick and V B Kraus Ethnic and sex differences in serum levels of cartilage oligomeric matrix protein Arthritis amp Rheumatism 3 675 681 2003 M Skoumal G Haberhauer J Feyertag E M Kittl K Bauer and A Dunky Serum levels of cartilage oligomeric matrix protein are elevated in rheumatoid arthritis but not in inflammatory rheumatic diseases such as psoriatic arthritis Raynaund s syndrome scleroderma systemic lupus erythematosus vasculitis and SjOgren s syndrome Arthritis Res Ther 6 73 74 2004 V Chandran R J Cook J E
7. international com 3 DRG C lt Revised 1 June 2012 rm Vers 2 1 USA DRG Cartilage Oligomeric Matrix Protein Human EIA 5265 Refer to the Certificate of Analysis for current volume of Dilution Buffer needed for reconstitution of standard Reconstitute the lyophilized Master Standard with Dilution Buffer just prior to the assay Let it dissolve at least 15 minutes with occasional gentle shaking not to foam The resulting concentration of the COMP in the stock solution is 128 ng mL Prepare set of standards using Dilution Buffer as follows sa f fem 300 uL of 32 ng mL 300 uL 16 ng mL 300 uL of 16 ng mL 300 uL 8 ng mL 300 uL of 8 ng mL 300 uL 4 ng mL Prepared Standards are ready to use do not dilute them Stability and storage Do not store the Standard stock solution and set of standards Quality Controls HIGH LOW Refer to the Certificate of Analysis for current volume of Dilution Buffer needed for reconstitution and for current Quality Control concentration Reconstitute each Quality Control HIGH and LOW with Dilution Buffer just prior to the assay Let it dissolve at least 15 minutes with occasional gentle shaking not to foam Reconstituted Quality Controls are ready to use do not dilute them Stability and storage The reconstituted Quality Controls must be used immediately or stored frozen at 20 C for 1 month Avoid repeated freeze thaw cycles Note Concentration of analyte in Quality Contr
8. ity 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 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 human COMP Note It is recommended to use a precision pipette and a careful technique to perform the dilution in order to get precise results 10 ASSAY PROCEDURE 1 Pipet 100 uL of 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 1 hour 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 Biotin Labelled Antibody into each well 5 Incubate the plate at room temperature ca 25 C for 1 hour shaking at ca 300 rpm on an orbital microplate shaker 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 Streptavidin HRP Conjugate into each well 8 Incubate the plate at room temperature ca 25 C for 30 minutes shaking at ca 300 rpm on an orbital microplate shaker
9. mples 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 D standardite Samples Sampie 11 Sampe 19 Sampie 27 Sample 35 E Standarda Sample Sampie 12 Sampe 20 Sampie 28 Sample 36 F f Standard Samples Sample 13 Sampe 21 Sampie 23 Sample 37 e acmon Samples Sampie 14 Sample 22 Sampie 30 Sample 38 m faciow Sample7 Sampe 15 Sampe 23 Sampe 31 Sample 35 DRG International Inc USA Fax 908 233 0758 e mail corp drg international com 6 DRG C lt Revised 1 June 2012 rm Vers 2 1 USA DRG Cartilage Oligomeric Matrix Protein Human EIA 5265 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 Results are reported as concentration of COMP ng mL in samples Alternatively the logit log function can be used to linearize the standard curve 1 e logit of the mean absorbance Y is plotted against log of the known concentration X of Standards The measured concentration of samples calculated from the standard curve m
10. ols 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 Certificate of Analysis and that ELISA test was carried out properly Wash Solution Conc 10x Dilute Wash Solution Concentrate 10x ten fold in distilled water to prepare a 1x working solution Example 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 1 month when stored at 2 C 8 C Opened Wash Solution Concentrate 10x is stable 3 months when stored at 2 C 8 C DRG International Inc USA Fax 908 233 0758 e mail corp drg international com 4 DIRE Co DRG Cartilage Oligomeric Matrix Protein Human EIA 5265 C lt Revised 1 June 2012 rm Vers 2 1 USA 9 PREPARATION OF SAMPLES The kit measures human COMP in serum and plasma EDTA citrate heparin 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 Dilute samples 50x with Dilution Buffer just prior to the assay e g 5 uL of sample 245 uL of Dilution Buffer for singlets or duplicates Mix well not to foam Vortex is recommended Stabil
11. on pipettes to deliver 5 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 Assay reagents supplied ready to use Antibody 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 when stored at 2 C 8 C and protected from the moisture Biotin Labelled Antibody Streptavidin HRP Conjugate Dilution Buffer Substrate Solution Stop Solution Stability and storage Opened reagents are stable 3 months when stored at 2 C 8 C Assay reagents supplied concentrated or lyophilized Human COMP Master Standard DRG International Inc USA Fax 908 233 0758 e mail corp drg
12. reaction is stopped by addition of acidic solution and absorbance of the resulting yellow product is measured The absorbance 1s proportional to the concentration of COMP A standard curve is constructed by plotting absorbance values against concentrations of 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 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 This kit contains components of animal origin These materials should be handled as potentially infectious DRG International Inc USA Fax 908 233 0758 e mail corp drg international com 1 DRG C lt Revised 1 June 2012 rm Vers 2 1 USA DRG Cartilage Oligomeric Matrix Protein Human EIA 5265 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
13. ust be multiplied by their respective dilution factor because samples have been diluted prior to the assay e g 20 ng mL from standard curve x 50 dilution factor 1 000 ng mL Human Cartilage Oligomeric Matrix Protein ELISA Standard Curve a m lt e L E Q D Q O Cc Q O 72 Q lt 10 100 Concentration of Hu COMP ng ml Figure 2 Typical Standard Curve for Human Cartilage Oligomeric Matrix Protein ELISA DRG International Inc USA Fax 908 233 0758 e mail corp drg international com 7 DRG C lt Revised 1 June 2012 rm Vers 2 1 USA DRG Cartilage Oligomeric Matrix Protein Human EIA 5265 12 STABILITY OF SAMPLES STORED AT 2 C 8 C Samples should be stored at 20 C However no decline in concentration of COMP was observed in serum and plasma samples after 7 days when stored at 2 C 8 C To avoid microbial contamination samples were treated with s aminocaproic acid and sodium azide resulting in the final concentration of 0 03 and 0 1 respectively Plasma ng mL serum ng mL na a EME EE 2 C 8 C 1 day 2 C 8 C 7 days 2 C 8 C 7 days 2 C 8 C 1 day 2 C 8 C 7 days 2 EM E Recall ee i al EOR C EE DRG International Inc USA Fax 908 233 0758 e mail corp drg international com 8 DRG C lt Revised 1 June 2012 rm Vers 2 1 USA DRG Cartilage Oligomeric Matrix Protein Human EI
14. wash skin eyes thoroughly with water and seek medical attention when necessary The materials must not be pipetted by mouth 5 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 6 REAGENT SUPPLIED Antibody Coated Microtiter Strips Product Data Sheet Certificate of l pe Analysis DRG International Inc USA Fax 908 233 0758 e mail corp drg international com 2 DIRE Co DRG Cartilage Oligomeric Matrix Protein Human EIA 5265 C lt Revised 1 June 2012 rm Vers 2 1 USA 7 MATERIAL REQUIRED BUT NOT SUPPLIED Deionized distilled water Test tubes for diluting samples Glassware graduated cylinder and bottle for Wash Solution Dilution Buffer Precisi
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