Human TGF-β2 ELISA Kit（KT20369） User Manual
Contents
1. Human TGF B2 ELISA Kit KT20369 User Manual For research use only Not intended for diagnostic testing AP waa noptec AS ARGENT IL MI IV VI VII VIII xe MOO Pp TABLE OF CONTENTS Introduction 0 0 0 ccc eceeeneeeeneneeee 2 ReAaSentss oeeie oxtail eaS a 2 SOTA DE n a aeaa a IE S 3 Additional Materials Required 3 Reagent Preparation ccceecene ene eeeees 4 Assay Procedure s icccscticcavecdetetd a ii 6 Assay Procedure Summary 8 Calculation of Results Typical Data secca ea eee eee en 8 Sensitivity 0 soos oS a abs E led oa 9 Recovery ses toh eee eee eee 9 Linearity setae ha R AE E dex 10 Reproducibility ccceeceeeeee eee es 10 Specificity on nren onn RE TEE NRN 10 Troubleshooting Guide 0008 11 I INTRODUCTION TGF 6 exists in at least five isoforms known as TGF B1 TGF B2 TGF B3 TGF B4 TGF B5 Their amino acid sequences display homologies on the order of 70 80 The various TGF f isotypes share many biological activities and their actions on cells are qualitatively similar in most cases although there are a few examples of distinct activities TGF B2 is the only variant that does not inhibit the growth of endothelial cells TGF B2 and TGF B3 inhibit the survival of cultured embryonic chick ciliary ganglionic neurons The Human TGF B2 ELISA Enzyme Linked Immunosorbent Assay kit is an in vitro enzyme linked immunosorbent2. assay for the quantitative measurement of human TGF 2 in serum plasma cell culture supernatants and urine This assay employs an antibody specific for human TGF B2 coated on a 96 well plate Standards and samples are pipetted into the wells and TGF B2 present in a sample is bound to the wells by the immobilized antibody The wells are washed and biotinylated anti human TGF B2 antibody is added After washing away unbound biotinylated antibody HRP conjugated streptavidin is pipetted to 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 TGF B2 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 TGF B2 Microplate Item A 96 wells 12 strips x 8 wells coated with anti human TGF B2 2 Wash Buffer Concentrate 20x Item B 25 ml of 20x concentrated solution Standards Item C 2 vials recombinant human TGF B2 4 Assay Diluent A Item D 30 ml 0 09 sodium azide as preservative For Standard Sample serum plasma diluent w HI Assay Diluent B Item E 15 ml of 5x concentrated buffer For Standard Sample cell culture supernatants urine diluent Detection Antibody TGF B2 Item F 2 vial of biotinylated anti human TGF B2 each vial is enough to assay half microplate HRP Streptavidin concentrate Item G 200 pl of 500x concentrated HRP conjugat
3. bate 10 minutes at room temperature 3 Add 25 ul 1 2 N NaOH 0 5 M HEPES Mix through 4 Add 800 ul Assay Diluent A for serum plasma or 1x Assay Diluent B for cell culture supernatants urine Mix through and assay within 2 hours Note The concentration read off the standard curve must be multiplied by the dilution factor 7 8 If samples generate values higher than the highest standard further dilute the samples after activation with the 1x Assay diluent B 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 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 Str
4. e diluted 5 fold with deionized or distilled water 4 Preparation of standard Briefly spin the vial of Item C Add 400 ul Assay Diluent A for serum plasma samples or 1x Assay Diluent B for cell culture medium and urine into Item C vial to prepare a 50 ng ml standard Dissolve the powder thoroughly by a gentle mix Add 80 ul TGF B2 standard from the vial of Item C into a tube with 586 7 ul Assay Diluent A or 1x Assay Diluent B to prepare a 6 000 pg ml stock standard solution Pipette 400 ul Assay Diluent A or 1x Assay Diluent B into each tube Use the stock 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 80 ul standard 586 7 ul 200ul 200ul 200ul 200nl 200ul 200 ul eeesee 6000 2000 666 6 222 2 74 07 24 69 8 23 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 ul 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 f
5. ed streptavidin TMB One Step Substrate Reagent Item H 12 ml of 3 3 5 5 tetramethylbenzidine TMB in buffered solution Stop Solution Item I 8 ml of 0 2 M sulfuric acid 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 year if the whole kit is stored at 20 C Avoid repeated freeze thaw cycles IV ADDITIONAL MATERIALS REQUIRED OAAIYDNABWN 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 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 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 culture supernatants and urine 3 Assay Diluent B should b
6. eptavidin 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 instructed 2 Add 100 ul standard or sample to each well Incubate 2 5 hours at room temperature or over night at 4 C p 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 5 Add 100 ul TMB One Step Substrate Reagent to each well Incubate 30 minutes at room temperature g 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 cur
7. old with 1x Assay Diluent B and used in step 4 of Part VI Assay Procedure N Briefly spin the HRP Streptavidin concentrate vial Item G before use HRP Streptavidin concentrate should be diluted 500 fold with 1x Assay Diluent B For example Briefly spin the vial Item G and pipette up and down to mix gently Add 20 ul of HRP Streptavidin concentrate into a tube with 10 ml Ix Assay Diluent B to prepare a 500 fold diluted HRP Streptavidin solution don t store the diluted solution for next day use Mix well ACTIVATION REAGENT PREPARATION To activate latent TGF 2 to the immunoreactive form prepare the following solutions for activation and neutralization The solutions may be stored in polypropylene bottles at room temperature for up to one month Use polypropylene test tubes Notes Do not activate the kit standards The kit standards contain active rhTGF 2 1 N HCI 100 ml Slowly add 8 33 mL of 12 N HCl into 91 67 ml deionized water Mix bottle 1 2 N NaOH 0 5 M HEPES 100 ml Slowly add 12 ml of 10 N NaOH into 75 mL deionized water Mix bottle Add 11 9 g HEPES Mix through Bring final volume to 100 mL with deionized water ACTIVATION PROCEDURE To activate latent TGF B2 to the immunoreactive form follow the activation procedure Use polypropylene test tubes Notes Do not activate the kit standards The kit standards contain active rhTGF B2 1 Add 25 ul 1 N HCI into 125 ul sample Mix through 2 Incu
8. petting 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 pipetting 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 A 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
9. ve must be run with each assay Assay Diluent A Assay Diluent B 10 io 1 E E 1 8 o ER D A ol i Q fe 0 1 0 01 r i 0 01 0 10 100 1 000 10 000 0 10 100 1 000 10 000 Human TGF beta 2 concentration pg ml Human TGF beta 2 concentration pg ml B SENSITIVITY The minimum detectable dose of TGF 2 is typically less than 15 pg ml C RECOVERY Recovery was determined by spiking various levels of human TGF 2 into human serum plasma and cell culture media Mean recoveries are as follows Sample Type Average Recovery Range Serum 94 74 82 104 Plasma 95 53 83 103 Cell culture media 96 14 84 105 D LINEARITY Sample Type Serum Plasma Cell culture media 1 2 Average of Expected 93 94 92 Range 84 103 83 103 84 103 1 4 Average of Expected 96 97 95 Range 85 104 84 102 85 103 E REPRODUCIBILITY Intra Assay CV lt 10 Inter Assay CV lt 12 IX SPECIFICITY Cross Reactivity This ELISA kit shows no cross reactivity with any of the cytokines tested e g human BDNF BLC ENA 78 FGF 4 IL la IL 1 IL 2 IL 3 IL 4 IL 5 IL 7 IL 8 IL 9 IL 11 IL 12 p70 IL 12 p40 IL 13 IL 15 IL 309 IP 10 G CSF GM CSF IFN y Leptin OB MCP 1 MCP 2 MCP 3 MDC MIP la MIP 1 B MIP 16 PARC PDGF RANTES SCF TARC TGF B1 TGF B3 TIMP 1 TIMP 2 TNF a TNF 8 TPO VEGF X TROUBLESHOOTING GUIDE Problem Cause Solution 1 Poor standard 1 Inaccurate pi
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