RayBio® Human Eotaxin-2 ELISA Kit
Contents
1. 1997 3 Kitaura M et al Molecular cloning of human eotaxin an eosinophil selective cc chemokine and identification of a specific eosinophil eotaxin receptor cc chemokine receptor 3 Journal Biological Chemistry 13 7725 7730 1996 RayBio Human EOTAXIN 2 ELISA Kit Protocol 10 XI TROUBLESHOOTING GUIDE Problem Cause Solution 1 Poor standard curve 1 Inaccurate pipetting 2 Improper standard dilution 1 Check pipettes 2 Ensure briefly spin the vial of Item C and dissolve the powder thoroughly by a gentle mix 2 Low signal 1 Too brief incubation 1 Ensure sufficient times 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 washed 2 Contaminated wash buffer 1 Review the manual for proper wash If using anaplate washer check that all ports are unobstructed 2 Make fresh wash buffer 5 Low sensitivity 1 Improper storage of the ELISA kit 2 Stop solution 1 Store your standard at lt 20 C after reconstitution others at 4 C Keep substrate solution protected from light 2 Stop solution should be added to each well before measure RayBio Human EOTAXIN 2 ELISA Kit Protocol 11 RayBio ELISA kits Over 200 ELISA kits custom2. ELISA kit choose from over 300 list visit www raybiotech com for details RayBio Human EOTAXIN 2 ELISA Kit Protocol 12 This product is for research use only 02004 RayBiotech Inc RayBio Human EOTAXIN 2 ELISA Kit Protocol 13
3. VIL ASSAY PROCEDURE SUMMARY 1 Prepare all reagents samples and standards as instructed J 2 Add 100 ul standard or sample to each well Incubate 2 5 hours at room temperature or over night at 4 C U 3 Add 100 ul prepared biotin antibody to each well Incubate hour at room temperature 4 Add 100 ul prepared Streptavidin solution Incubate 45 minutes at room temperature J 5 Add 100 ul TMB One Step Substrate Reagent to each well Incubate 30 minutes at room temperature J 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 RayBio Human EOTAXIN 2 ELISA Kit Protocol 7 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 450 nm 450 nm OD OD 0 1 1 10 100 1000 10000 0 1 1 10 100 1000 10000 Eotaxin 2 concentration pg ml Eotaxin 2 concentration pg ml B SENSITIVITY The minimum detectable dose of Eotaxin 2 is typically less than 2 pg ml C RECOVERY Recovery was determined by
4. or deionized water Log log graph paper or computer and software for ELISA data analysis Tubes to prepare standard or sample dilutions RayBio Human EOTAXIN 2 ELISA Kit Protocol 3 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 Suggested dilution for normal serum plasma 2 20 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 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 medium and urine into Item C vial to prepare a 50 ng ml standard Dissolve the powder thoroughly by a gentle mix Add 20 ul Eotaxin 2 standard from the vial of Item C into a tube with 980 0 ul Assay Diluent A or 1x Assay Diluent B to prepare a 1000 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 As
5. RayBio Human Eotaxin 2 ELISA Kit User Manual Revised Mar 1 2012 RayBio Human Eotaxin 2 ELISA Kit Protocol Cat ELH Eotaxin2 001 2 RayBiotech Inc We Provide You With Excellent Protein Array System And Service Tel Toll Free 1 888 494 8555 or 770 729 2992 Fax 770 206 2393 web www raybiotech com Email info raybiotech com Ray Biotech Inc RayBio Human Fotaxin 2 ELISA Kit Protocol TABLE OF CONTENTS I ntrodectons urmvadvsarriostraramaker veni 2 Eh Rasens aaa 2 MI SUGTA GC isacaw sn cchredwsccndcmencucaate gus tiwensdwacenese 3 IV Additional Materials Required 4 3 V Reagent Preparation sso iccccis vvscecaeeaiextsocess 4 VI Assay Procedure ic cecscccccecksccoedeonscatddsenesvess 6 VII Assay Procedure Summary 00eee eee 7 VIII Calculation of Results A Typical PD vred e 8 B Sr 8 een 8 ID JD 81500 OE ERE EEE 9 E Reproducibility cccccccceccsccicceessnesstaiesieeeces 9 IX DET reset 9 Me RANE Saeed 10 XI Troubleshooting Guide raannannnnrnnnnnrnnnnr 11 RayBio Human EOTAXIN 2 ELISA Kit Protocol 1 I INTRODUCTION Eotaxin is composed of 73 amino acids and has been found in the bronchoalveolar lavage of guinea pigs used as a model of allergic Inflammation The factor belongs to the platelet factor 4 family of Chemokines It exhibits homology of 53 percent with human MCP 1 44 percent with guinea pig MCP 1 31 percent with human MIP 1 al
6. Y PROCEDURE 1 Bring all reagents and samples to room temperature 18 25 C before use t is recommended that all standards and samples be run at least in duplicate 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 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 RayBio Human EOTAXIN 2 ELISA Kit Protocol 6 9 Add 50 ul of Stop Solution Item I to each well Read at 450 nm immediately
7. man EOTAXIN 2 ELISA Kit Protocol 2 Assay Diluent A Item D 30 ml 0 09 sodium azide as preservative For Standard Sample serum plasma diluent Assay Diluent B Item E 15 ml of 5x concentrated buffer For Standard Sample cell culture medium urine diluent Detection Antibody Eotaxin 2 Item F 2 vial of biotinylated anti human Eotaxin 2 each vial is enough to assay half microplate HRP Streptavidin concentrate Item G 200 ul 600x concentrated HRP conjugated 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 HI 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 NAD WN Microplate reader capable of measuring absorbance at 450 nm Precision pipettes to deliver 2 ul to I ml volumes Adjustable 1 25 ml pipettes for reagent preparation 100 ml and I liter graduated cylinders Absorbent paper Distilled
8. pha and 26 percent with human RANTES Eotaxin 2 induces chemotaxis of eosinophils and basophils and the release of histamine and leukotriene C 4 from basophils primed with IL3 Eotaxin 2 activates chemotaxis related events such as actin polymerization and the respiratory burst almost as efficiently as Eotaxin The RayBio Human Eotaxin 2 ELISA Enzyme Linked Immunosorbent Assay kit is an in vitro enzyme linked immunosorbent assay for the quantitative measurement of human Eotaxin 2 in serum plasma cell culture supernatants and urine This assay employs an antibody specific for human Eotaxin 2 coated on a 96 well plate Standards and samples are pipetted into the wells and Eotaxin 2 present in a sample is bound to the wells by the immobilized antibody The wells are washed and biotinylated anti human Eotaxin 2 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 Eotaxin 2 bound The Stop Solution changes the color from blue to yellow and the intensity of the color is measured at 450 nm I REAGENTS 1 Eotaxin 2 Microplate Item A 96 wells 12 strips x 8 wells coated with anti human Eotaxin 2 2 Wash Buffer Concentrate 20x Item B 25 ml of 20x concentrated solution 3 Standards Item C 2 vials recombinant human Eotaxin 2 RayBio Hu
9. say Diluent A or 1x Assay Diluent B serves as the zero standard 0 pg ml RayBio Human EOTAXIN 2 ELISA Kit Protocol 4 20 ul standard 980 0 ul 200u1 200ul 200ul 200ul 200u1 200 ul QSOS OS IS IS OS Os z 1000 333 3 111 1 37 04 12 35 4 12 1 37 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 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 600 fold with 1x Assay Diluent B For example Briefly spin the vial Item G and pipette up and down to mix gently Add 25 ul of HRP Streptavidin concentrate into a tube with 15 ml Ix Assay Diluent B to prepare a final 600 fold diluted HRP Streptavidin solution don t store the diluted solution for next day use Mix well RayBio Human EOTAXIN 2 ELISA Kit Protocol 5 VI ASSA
10. spiking various levels of human Eotaxin 2 into human serum plasma and cell culture media Mean recoveries are as follows Sample Type Average Recovery Range Serum 94 86 83 103 Plasma 96 74 85 104 Cell culture media 95 77 82 103 RayBio Human EOTAXIN 2 ELISA Kit Protocol 8 D LINEARITY Sample Type 1 2 Average of Expected Range 1 4 Average of Expected Range E REPRODUCIBILITY Intra Assay CV lt 10 Inter Assay CV lt 12 IX SPECIFICITY Serum 98 85 104 97 84 103 Plasma Cell Culture Media 96 94 83 103 82 102 98 96 84 105 83 103 Cross Reactivity This ELISA kit shows no cross reactivity with any of the cytokines tested e g human Angiogenin BDNF BLC ENA 78 Eotaxin Eotaxin 3 FGF 4 IL la IL 1B IL 2 IL 3 IL 4 IL 5 IL 7 IL 8 IL 9 IL 10 IL 11 IL 12 p70 IL 12 p40 IL 13 IL 15 IL 309 IP 10 G CSF GM CSF IFN y MCP 1 MCP 2 MCP 3 MDC MIP 1a MIP 1 B MIP 15 PARC PDGF RANTES SCF TARC TGF B TIMP 1 TIMP 2 TNF a TNF B TPO VEGF RayBio Human EOTAXIN 2 ELISA Kit Protocol X REFERENCES 1 Bartels J et al Human dermal fibroblasts express eotaxin molecular cloning mRNA expression and identification of eotaxin sequence variants Biochemical and Biophysical Research Communications 22563 1045 51 1996 2 Yamada H et al Eotaxin is a potent chemotaxin for human basophils Biochemical and Biophysical Research Communications 231 2 365 368
Download Pdf Manuals
Related Search