Mouse CD40 ELISA Kit（KT20435） User Manual
Contents
1. B SENSITIVITY The minimum detectable dose of CD40 is typically less than 3 pg ml C RECOVERY Recovery was determined by spiking mouse CD40 into mouse serum plasma and cell culture media Mean recoveries are as follows Sample Type Average Recovery Range Serum 77 26 71 87 Plasma 76 68 69 87 Cell culture media 84 23 74 93 D LINEARITY Sample Type Serum Plasma Cell Culture Media 1 2 Average of Expected 100 9 102 5 108 2 Range 91 110 93 110 97 117 1 4 Average of Expected 104 9 102 0 108 0 Range 92 112 90 109 96 17 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 Fractalkine GCSF GM CES IFN y IGFBP 3 IGFBP 5 IGFBP 6 IL 1 a IL 1B 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 MIG MIP la MIP 1y MIP 2 MIP 3f MIP 3a PF 4 P Selectin RANTES SCF SDF 1a TARC TCA 3 TECK TIMP 1 TNF a 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 a brief spin dilution of Item C and dissolve the powder thoroughly by a gentle mix2. 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 instructed 2 Add 100 ul standard or sample to each well Incubate 2 5 hours at room temperature or over night at 4 C y 3 Add 100 ul prepared biotin antibody to each well Incubate 1 hour at room temperature 4 Add 100 ul prepared Streptavidin solution Incubate 45 minutes at room temperature y 5 Add 100 ul TMB One Step Substrate Reagent to each well Incubate 30 minutes at room temperature 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 A Assay Diluent B 10 10 1 E 1 Q o a A o 01 O 0 1 0 01 0 01 1 10 100 1000 10000 4 10 100 1000 10000 Mouse CD40 concentration pg ml Mouse CD40 concentration pg ml
3. Mouse CD40 ELISA Kit KT20435 User Manual For research use only Not intended for diagnostic testing AP waa noptec AS ARGENT TABLE OF CONTENTS I IntroductiON ooooooccccncccnccncncnncncnncncnnnncncnnss 2 H AAA ana n E a 2 M Storage seenen i ea E E a E aa 3 IV Additional Materials Required ooo o 3 V Reagent Preparation ccceceeeeeee eee eees 3 VI Assay Procedure cccecece cece eee eee eneeneens 5 VII Assay Procedure Summaty 0 eee 7 VIII Calculation of Results As Typical Data esperen n E n E E levee 7 By SSI iii NE E EEA ide 8 C RECO lenio oT ne cd ee a ee hs 8 Di LM is 9 E Reproducibility oooooccoroccccnonconcnnonanns 9 TX Specificity dto at 9 X Troubleshooting GUId o oooooococconooconnnnnos 10 I INTRODUCTION The Mouse CD40 ELISA Enzyme Linked Immunosorbent Assay kit is an in vitro enzyme linked immunosorbent assay for the quantitative measurement of mouse CD40 TNFRSES in serum plasma and cell culture supernatants This assay employs an antibody specific for mouse CD40 coated on a 96 well plate Standards and samples are pipetted into the wells and CD40 present in a sample is bound to the wells by the immobilized antibody The wells are washed and biotinylated anti mouse CD40 antibody is added After washing away unbound biotinylated antibody HRP conjugated streptavidin is pipetted to the wells The wells are again wash
4. 2 Low signal 1 Too brief incubation times Ensure sufficient incubation time assay procedure step 2 may change to over night 2 Inadequate reagent 2 Check pipettes and volumes or improper ensure correct dilution preparation 3 Large CV 1 Inaccurate pipetting 1 Check pipettes 4 High background 1 Plate is insufficiently 1 Review the manual washed for proper wash If using a 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 a 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
5. gently Add 50 ul of HRP Streptavidin concentrate into a tube with 10 ml Ix Assay Diluent B to prepare a 200 fold diluted HRP Streptavidin solution don t store the diluted solution for next day use Mix well VI ASSAY PROCEDURE 1 Bring 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 ul 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
6. ard 50 ng ml from the vial of Item C into a tube with 485 ul Assay Diluent A or 1x Assay Diluent B to prepare a 1 500 pg ml standard solution Pipette 400 ul Assay Diluent A or 1x Assay Diluent B into each tube Use the 1 500 pg ml standard solution to produce a dilution series shown below Mix each tube thoroughly before the next transfer Assay Diluent A or 1x Assay Diluent B serves as the zero standard 0 pg ml 15 pl standard 200 pul 485 yl 20041 2001 200 pl 200 pl H 200 pul 1 500 500 166 7 55 56 18 52 6 17 2 06 0 pg ml pg ml pg ml pg ml pg ml pg ml pg ml pg ml 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 pl of 1x Assay Diluent B 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 B 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 200 fold with 1x Assay Diluent B For example Briefly spin the vial Item G and pipette up and down to mix
7. ed a TMB substrate solution is added to the wells and color develops in proportion to the amount of CD40 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 CD40 Microplate Item A 96 wells 12 strips x 8 wells coated with anti mouse CD40 2 Wash Buffer Concentrate 20x Item B 25 ml of 20x concentrated solution Standards Item C 2 vials of recombinant mouse CD40 4 Assay Diluent A Item D 30 ml diluent buffer 0 09 sodium azide as preservative For Standard Sample serum plasma diluent 5 Assay Diluent B Item E 15 ml of 5x concentrated buffer For Standard Sample cell culture medium diluent 6 Detection Antibody CD40 Item F 2 vial of biotinylated anti mouse CD40 each vial is enough to assay half microplate 7 HRP Streptavidin Concentrate Item G 200 ul 200x concentrated HRP conjugated streptavidin 8 TMB One Step Substrate Reagent Item H 12 ml of 3 3 5 5 tetramethylbenzidine TMB in buffer solution 9 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 stored for up to month at 2 to 8 C Return unused wells to the pouch containing desiccant pack resea
8. l along entire edge Note the kit can be used within one 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 Assay Diluent A Item D should be used for dilution of serum plasma samples 1x Assay Diluent B Item E should be used for dilution of cell culture supernatants Suggested dilution for normal serum plasma 3 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 3 Assay Diluent B should be diluted 5 fold with deionized or distilled water before use 4 Preparation of standard Briefly spin the vial of Item C and then add 400 ul Assay Diluent A for serum plasma samples or 1x Assay Diluent B for cell culture supernates into Item C vial to prepare a 50 ng ml standard Dissolve the powder thoroughly by a gentle mix Add 15 ul CD40 stand
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