Mouse Decorin ELISA Kit（KT20451） User Manual
Contents
1. Cell culture media 111 1 68 130 D LINEARITY Sample Type Serum Plasma Cell Culture Media 1 2 Average of Expected 103 8 107 7 106 5 Range 96 112 100 116 101 112 1 4 Average of Expected 114 0 106 0 106 1 Range 106 122 97 114 101 111 E REPRODUCIBILITY Intra Assay CV lt 10 Inter Assay CV lt 12 IX SPECIFICITY Cross Reactivity This ELISA kit shows no cross reactivity with the following cytokines tested Mouse CD30 L CD30 T CD40 CRG 2 CTACK CXCL16 Eotaxin Eotaxin 2 Fas Ligand Fractalkine GCSF GM CFS IFN y IGFBP 3 IGFBP 5 IGFBP 6 IL 1 a IL 1f IL 2 IL 3 IL 3 Rb IL 4 IL 5 IL 9 IL 10 IL 12 p40 p70 IL 12 p70 IL 13 IL 17 KC Leptin R LEPTIN OB LIX L Selectin Lymphotactin MCP 1 MCP 5 M CSF MIG MIP 1a MIP 1y MIP 2 MIP 3B MIP 3a PF 4 P Selectin RANTES SCF SDF 1a TARC TCA 3 TECK TIMP 1 TNF RI TNF RII TPO VCAM 1 VEGF X TROUBLESHOOTING GUIDE Problem Cause Solution 1 Poor standard 1 Inaccurate pipetting 1 Check pipettes curve 2 Improper standard 2 Ensure briefly spin dilution the vial of Item C and dissolve the powder thoroughly by a gentle mix 2 Low signal 1 Too brief incubation times Ensure sufficient incubation time assay procedure step 2 change to over night 2 Inadequate reagent 2 Check pipettes and volumes or improper ensure correct dilution preparation 3 Large CV 1 Inaccurate pipetting2. Mouse Decorin ELISA Kit KT20451 User Manual For research use only Not intended for diagnostic testing AP waa noptec AS ARGENT IL MI IV VI VII VIII lt S TABLE OF CONTENTS Introduction re knra neni ngiseni 2 Reagents i aineta oaa a a E E eel 2 MOTA ii ae 3 Additional Materials Required 3 Reagent PreparatiOM ooooocccccccncccocncons 3 Assay Procedure oooooccoccoccononcncncninnnns 5 Assay Procedure Summary 7 Calculation of Results Typical Data searre dan coe sereeenin teks 7 SEMSIIVILY A re a eE a ig 8 RECO Vaio sak dees veer EA NRA 8 LAM CALICY 5 55 53 s Satya da 9 Reproducibility oooooooooccccnononco ro 9 POC 9 Troubleshooting Guide oooooocococ ooo 10 I INTRODUCTION The Mouse Decorin ELISA Enzyme Linked Immunosorbent Assay kit is an in vitro enzyme linked immunosorbent assay for the quantitative measurement of mouse Decorin in serum plasma collect plasma using EDTA and heparin as an anticoagulant Citrate is not recommended as anticoagulants for use in this assay This assay employs an antibody specific for mouse Decorin coated on a 96 well plate Standards and samples are pipetted into the wells and Decorin present in a sample is bound to the wells by the immobilized antibody The wells are washed and biotinylated anti mouse Decorin antibody is added After washing away unbound biotinylated antibody HRP conjugated streptavidin is pipetted t
3. 1 Check pipettes 4 High background 1 Plate is insufficiently 1 Review the manual washed for proper wash If using ana plate washer check that all ports are unobstructed 2 Contaminated wash 2 Make fresh wash buffer buffer 5 Low sensitivity 1 Improper storage of the ELISA kit 2 Stop solution Store your standard at lt 20 C after reconstitution others at 4 C Keep substrate solution protected from light Stop solution should be added to each well before measure Note This product is for research use only aR 2 USA Abgent Inc Toll Free 888 735 7227 Or 858 875 1900 info_us abgent wuxiapptec com CHINA Abgent Suzhou 86 512 69369088 sales abgent wuxiapptec com EUROPE Abgent Europe 44 0 1235 854042 eurosales abgent wuxiapptec com For other countries www abgent com
4. all reagents and samples to room temperature 18 25 C before use It is recommended that all standards and samples be run at least in duplicate 2 Add 100 pl of each standard see Reagent Preparation step 2 and sample into appropriate wells Cover well and incubate for 2 5 hours at room temperature or over night at 4 C with gentle shaking 3 Discard the solution and wash 4 times with 1x Wash Solution Wash by filling each well with Wash Buffer 300 ul using a multi channel Pipette or autowasher Complete removal of liquid at each step is essential to good performance After the last wash remove any remaining Wash Buffer by aspirating or decanting Invert the plate and blot it against clean paper towels Add 100 ul of 1x prepared biotinylated antibody Reagent Preparation step 6 to each well Incubate for 1 hour at room temperature with gentle shaking Discard the solution Repeat the wash as in step 3 Add 100 ul of prepared Streptavidin solution see Reagent Preparation step 7 to each well Incubate for 45 minutes at room temperature with gentle shaking Discard the solution Repeat the wash as in step 3 Add 100 ul of TMB One Step Substrate Reagent Item H to each well Incubate for 30 minutes at room temperature in the dark with gentle shaking Add 50 ul of Stop Solution Item I to each well Read at 450 nm immediately VII ASSAY PROCEDURE SUMMARY 1 Prepare all reagents samples and standards as in
5. Diluent into each tube Use the 1 000 pg ml standard solution to produce a dilution series shown below Mix each tube thoroughly before the next transfer 1x Assay Diluent serves as the zero standard 0 pg ml 10 ul standard 490 ul 20041 200ul 200ul 2001 200 nS 200 ul 1 000 400 160 25 6 10 2 pg ml pg ml pg ml oa pg ml pg ml val aa 5 If the Wash Concentrate 20x Item B contains visible crystals warm to room temperature and mix gently until dissolved Dilute 20 ml of Wash Buffer Concentrate into deionized or distilled water to yield 400 ml of 1x Wash Buffer 6 Briefly spin the Detection Antibody vial Item F before use Add 100 ul of 1x Assay Diluent into the vial to prepare a detection antibody concentrate Pipette up and down to mix gently the concentrate can be stored at 4 C for 5 days The detection antibody concentrate should be diluted 80 fold with 1x Assay Diluent and used in step 4 of Part VI Assay Procedure 7 Briefly spin the HRP Streptavidin concentrate vial Item G and pipette up and down to mix gently before use HRP Streptavidin concentrate should be diluted 300 fold with 1x Assay Diluent For example Briefly spin the vial Item G and pipette up and down to mix gently Add 40 ul of HRP Streptavidin concentrate into a tube with 12 ml 1x Assay Diluent to prepare a 300 fold diluted HRP Streptavidin solution don t store the diluted solution for next day use Mix well VI ASSAY PROCEDURE 1 Bring
6. o the wells The wells are again washed a TMB substrate solution is added to the wells and color develops in proportion to the amount of Decorin bound The Stop Solution changes the color from blue to yellow and the intensity of the color is measured at 450 nm Il REAGENTS 1 Decorin Microplate Item A 96 wells 12 strips x 8 wells coated with anti mouse Decorin 2 Wash Buffer Concentrate 20x Item B 25 ml of 20x concentrated solution Standards Item C 2 vials recombinant mouse Decorin 4 Assay Diluent Item E 15 ml of 5x concentrated buffer For Standard Sample serum plasma samples cell culture medium diluent 5 Detection Antibody Decorin Item F 2 vial of biotinylated anti mouse Decorin each vial is enough to assay half microplate 6 HRP Streptavidin concentrate Item G 200 ul 300x concentrated HRP conjugated streptavidin 7 TMB One Step Substrate Reagent Item H 12 ml of 3 3 5 5 tetramethylbenzidine TMB in buffered solution 8 Stop Solution Item I 8 ml of 0 2 M sulfuric acid W IHI STORAGE May be stored for up to 6 months at 2 to 8 C from the date of shipment Standard recombinant protein should be stored at 20 C or 80 C recommended at 80 C after reconstitution Opened Microplate Wells or reagents may be store for up to 1 month at 2 to 8 C Return unused wells to the pouch containing desiccant pack reseal along entire edge Note the kit can be used within one
7. structed 2 Add 100 ul standard or sample to each well Incubate 2 5 hours at room temperature or over night at 4 C 3 Add 100 ul prepared biotin antibody to each well Incubate 1 hour at room temperature y 4 Add 100 ul prepared Streptavidin solution Incubate 45 minutes at room temperature I 5 Add 100 ul TMB One Step Substrate Reagent to each well Incubate 30 minutes at room temperature ul 6 Add 50 ul Stop Solution to each well Read at 450 nm immediately VIII CALCULATION OF RESULTS Calculate the mean absorbance for each set of duplicate standards controls and samples and subtract the average zero standard optical density Plot the standard curve on log log graph paper or using Sigma plot software with standard concentration on the x axis and absorbance on the y axis Draw the best fit straight line through the standard points A TYPICAL DATA These standard curves are for demonstration only A standard curve must be run with each assay Assay Diluent 0 1 4 OD 450 nm 0 001 r r r 1 10 100 1 000 10 000 Mouse Decorin concentration pg ml B SENSITIVITY The minimum detectable dose of Decorin is typically less than 5 pg ml C RECOVERY Recovery was determined by spiking various levels of Decorin into normal mouse serum plasma and cell culture media Mean recoveries are as follows Sample Type Average Recovery Range Serum 104 7 83 126 Plasma 93 91 74 119
8. year if the whole kit is stored at 20 C Avoid repeated freeze thaw cycles IV ADDITIONAL MATERIALS REQUIRED Microplate reader capable of measuring absorbance at 450 nm Precision pipettes to deliver 2 ul to 1 ml volumes Adjustable 1 25 ml pipettes for reagent preparation 100 ml and 1 liter graduated cylinders Absorbent paper Distilled or deionized water Log log graph paper or computer and software for ELISA data analysis Tubes to prepare standard or sample dilutions OAYDMABRWN Ke V REAGENT PREPARATION 1 Bring all reagents and samples to room temperature 18 25 C before use 2 Sample dilution If your samples need to be diluted 1x Assay Diluent Item E should be used for dilution of serum plasma culture supernatants Suggested dilution for normal serum plasma 200 2 000 fold Please note that levels of the target protein may vary between Different specimens Optimal dilution factors for each sample must be determined by the investigator Assay Diluent Item E should be diluted 5 fold with deionized or distilled water before use 4 Preparation of standard Briefly spin the vial of Item C Add 400 ul 1x Assay Diluent Item E into Item C vial to prepare a 50 ng ml standard solution Dissolve the powder thoroughly by a gentle mix Add 10 ul Decorin standard solution from the vial of Item C into a tube with 490 ul 1x Assay Diluent to prepare a 1 000 pg ml standard solution Pipette 300u1 1x Assay
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