Ghrelin EIA - RayBiotech, Inc.
Contents
1. Plasma ghrelin levels after diet induced weight loss or gastric bypass surgery N Engl J Med 346 21 1623 30 6 M Smiley DL Heiman ML 2000 Ghrelin induces adiposity in rodents Nature 407 6806 908 913 7 Kojima M Hosoda H Date Y Nakazato M Matsuo H Kangawa 1999 Ghrelin is growth hormone releasing acylated peptide from stomach Nature 402 6762 656 60 RayBio Human Ghrelin EIA Kit Protocol 14 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 5 Low sensitivity 1 Plate is insufficiently washed 2 Contaminated wash buffer 1 Improper storage of the EIA kit 2 Stop solution 1 Review the manual for proper wash If using a plate washer check that all ports are unobstructed 2 Make fresh wash buffer 1 Store your standard at lt 20 C after receipt of the kit 2 Stop solution should be added to each well before measure RayBio2. EIA kits If you are interested in other EIA kits please visit www raybiotech com for details RayBio Human Ghrelin Kit Protocol 15 Notes RayBio Human Ghrelin EIA Kit Protocol 16 This is for research use only 2013 RayBiotech Inc 3607 Parkway Lane Suite 200 Norcross GA 30092 Tel 770 729 2992 1 888 494 8555 Fax 770 206 2393 Web www raybiotech com RayBio Human Ghrelin EIA Kit Protocol 17
3. to both the constitutionally thin and normal weight controls These findings suggest that ghrelin plays a role in both anorexia and obesity Ghrelin levels are also high in patients who have cancer induced cachexia RayBio Human Ghrelin EIA Kit Protocol 2 Il GENERAL DESCRIPTION The RayBio amp Ghrelin Enzyme Immunoassay EIA Kit is an in vitro quantitative assay for detecting Ghrelin peptide based on the principle of Competitive Enzyme Immunoassay The microplate in the kit is pre coated with anti rabbit secondary antibody After a blocking step and incubation of the plate with anti Ghrelin antibody both biotinylated Ghrelin peptide and peptide standard or targeted peptide in samples interacts competitively with the Ghrelin antibody Uncompeted bound biotinylated Ghrelin peptide then interacts with Streptavidin horseradish peroxidase SA HRP which catalyzes a color development reaction The intensity of colorimetric signal is directly proportional to the amount of biotinylated peptide SA HRP complex and inversely proportional to the amount of Ghrelin peptide in the standard or samples This is due to the competitive binding to Ghrelin antibody between biotinylated Ghrelin peptide and peptides in standard or samples A standard curve of known concentration of Ghrelin peptide can be established and the concentration of Ghrelin peptide in the samples can be calculated accordingly RayBio Human Ghrelin EIA Kit Protocol 3 Principl
4. RayBio Human Mouse Rat Ghrelin Enzyme Immunoassay Kit Please Read the Manual Carefully Before Starting your Experiment User Manual 3 2 Revised Jan 2 2014 RayBio Ghrelin Enzyme Immunoassay Kit Protocol Cat EIA GHR 1 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 RayBiotech Inc RayBio Human Mouse Rat Ghrelin Enzyme Immunoassay Kit Protocol TABLE OF CONTENTS MEM WTO CIC OMe Tc I General Tede da RERO Da EA aUe MEE i o0 V Additional Materials Required VI Reagent Assay VIII Assay Procedure IX Calculation of A s Typical D Al sp Tm MES ds MERITI C Detection D RejpfodUciDil lly i i xy k nn n atre Pape gto ds SDOCH lyuueueshana dde om0M MM MM eed eheu aoa Al fBelerolb OS XII Troubleshooting RayBio Human Ghrelin EIA Kit Protocol 1 Oo N 11 12 13 13 13 13 13 15 INTRODUCTION Obesit
5. e of Competitive EIA TLE Anti rabbit IgG coated plate AR Add Capture N Antibody signal signal Add mixture of LA Biotin peptide Sample Add Streptavidin HRP and Color Substrate Wu B Biotinylated peptide competes with target MER HRP streptavidin 1 Calibration RayBio Human Ghrelin EIA Kit Protocol 4 Ill REAGENTS 1 aa 8 9 10 11 12 Ghrelin Microplate Item 96 wells 12 strips 8 wells coated with secondary antibody Wash Buffer Concentrate 20x Item B 25 ml Lyophilized standard Ghrelin peptide Item C 2 vials Lyophilized anti Ghrelin polyclonal antibody Item N 2 vials 1X Assay Diluent E Item R 2 vials 25 ml vial Diluent for both standards and samples including serum or plasma cell culture media or other sample types Lyophilized biotinylated Ghrelin peptide Item F 2 vials HRP Streptavidin concentrate 600 100x concentrated HRP conjugated Streptavidin Lyophilized positive control Item M 1 vial TMB One Step Substrate Reagent Item H 12 ml of 3 3 5 5 tetramethylbenzidine TMB in buffered solution Stop Solution Item 1 8 ml of 0 2 M sulfuric acid Assay Diagram J User Manual Item K IV STORAGE Standard Biotinylated Ghrelin peptide and Positive Control should be stored at 20 C after arrival Avoid multiple freeze thaws The remaining kit components may be sto
6. ep 3 to each well Incubate for 1 5 hours at room temperature with gentle shaking 1 2 cycles sec You may also incubate overnight at 4 C RayBio Human Ghrelin EIA Kit Protocol 9 3 Discard the solution and wash wells 4 times with 1x Wash Buffer 200 300 yl each Washing may be done with a multichannel pipette or an automated plate washer Complete removal of liquid at each step is essential to good assay performance After the last wash remove any remaining Wash Buffer by aspirating or decanting Invert the plate and blot it against clean paper towels 4 Add 100 ul of each standard see Reagent Preparation step 5 positive control see Reagent Preparation step 7 and sample see Reagent Preparation step 9 into appropriate wells Be sure to include a blank well Assay Diluent only Cover wells and incubate for 2 5 hours at room temperature with gentle shaking 1 2 cycles sec or overnight at 4 C 5 Discard the solution and wash 4 times as directed in Step 3 6 Add 100 yl of prepared HRP Streptavidin solution see Reagent Preparation step 10 to each well Incubate with gentle shaking for 45 minutes at room temperature It is recommended that incubation time should not be shorter or longer than 45 minutes 7 Discard the solution and wash 4 times as directed in Step 3 8 Add 100 ul of TMB One Step Substrate Reagent Item to each well Incubate for 30 minutes at room temperature in the dark with gentle shaking 1 2 cycles
7. ep with each successive concentration preparing a dilution series as shown in the illustration below Each time use 450 ul of biotinylated Ghrelin and 50 ul of the prior concentration until 100 pg ml is reached Mix each tube thoroughly before the next transfer d The final tube 0 pg ml Ghrelin 10 ng ml biotinylated Ghrelin serves as the zero standard or total binding RayBio Human Ghrelin EIA Kit Protocol 7 50u 5041 50 ul 50 ul 1000 100 10 1 100 0 ng ml ng ml ng ml ng ml pg ml pg ml 6 Prepare a 10 fold dilution of Item F To do this add 2 ul of Item F to 18 ul of 1X Assay Diluent E This solution will be used in steps 7 and 9 7 Positive Control Preparation Briefly centrifuge the positive control vial and reconstitute with 100 ul of ddH2O before use Item M To the tube of Item M add 101 ul 1x Assay Diluent E Also add 2 yl of 10 fold diluted Item F prepared in step 6 to the tube This is a 2 fold dilution of the positive control Mix thoroughly The positive control is a cell culture medium sample with an expected signal between 1096 and 309 of total binding 70 9096 competition if diluted as described above It may be diluted further if desired but be sure the final concentration of biotinylated Ghrelin is 10 ng ml 8 If Item B 20X Wash Concentrate 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 y
8. ield 400 ml of 1X Wash Buffer RayBio Human Ghrelin EIA Kit Protocol 8 9 Sample Preparation Use 1X Assay Diluent biotinylated GHR to dilute samples including serum plasma cell culture medium and other sample types It is very important to make sure the final concentration of the biotinylated Ghrelin is 10 ng ml in every sample EXAMPLE to make a 4 fold dilution of sample mix together 2 5 ul of 10 fold diluted Item prepared in step 6 185 ul of 1X Assay Diluent and 62 5 ul of your sample mix gently The total volume is 250 ul enough for duplicate wells on the microplate Do not use ltem F diluent from Step 4 for sample preparation If you plan to use undiluted samples you must still add biotinylated Ghrelin to a final concentration of 10 ng ml EXAMPLE 2 5 ul of 10 fold diluted Item to 247 5 ul of sample NOTE Optimal sample dilution factors should be determined empirically however you may contact technical support 888 494 8555 techsupport raybiotech com to obtain recommended dilution ranges for serum or plasma Briefly centrifuge the HRP Streptavidin vial Item G before use The HRP Streptavidin concentrate should be diluted 100 fold with 1X Assay Diluent E ASSAY PROCEDURE 1 Keep kit reagents on ice during reagent preparation steps It is recommended that all standards and samples be run at least in duplicate Add 100 yl anti Ghrelin antibody see Reagent Preparation st
9. ing solution which will be used in step 2 of the Assay Procedure NOTE the following steps may be done during the antibody incubation procedure step 2 of Assay Procedure RayBio Human Ghrelin EIA Kit Protocol 6 4 Briefly centrifuge the vial of biotinylated Ghrelin peptide Item F and reconstitute with 20 ul of ddH20 before use Add 5 ul of Item F to 5 ml 1X Assay Diluent E Pipette up and down to mix gently The final concentration of biotinylated Ghrelin will be 10 ng ml This solution will only be used as the diluent in step 5 of Reagent Preparation 5 Preparation of Standards Label 6 microtubes with the following concentrations 1000 ng ml 100 ng ml 10 ng ml 1 ng ml 100 pg ml and 0 pg ml Pipette 450 pl of biotinylated Ghrelin solution into each tube except for the 1000 ng ml leave this one empty t is very important to make sure the concentration of biotinylated Ghrelin is 10 ng ml in all standards a Briefly centrifuge the vial of standard Ghrelin peptide Item C and reconstitute with 10 pl of ddH2O In the tube labeled 1000 ng ml pipette 8 ul of Item C and 792 ul of 10 ng ml biotinylated Ghrelin solution prepared in step 4 above This is your Ghrelin stock solution 1000 ng ml Ghrelin 10 ng ml biotinylated Ghrelin Mix thoroughly This solution serves as the first standard b To make the 100 ng ml standard pipette 50 ul of Ghrelin stock solution the tube labeled 100 ng ml Mix thoroughly c Repeat this st
10. red at 4 C Opened Microplate Wells and antibody Item N may be stored for up to 1 month at 2 to 8 Return unused wells to the pouch containing desiccant pack and reseal along entire edge If stored in this manner RayBiotech warranties this kit for 6 months from the date of shipment RayBio Human Ghrelin EIA Kit Protocol 5 V ADDITIONAL MATERIALS REQUIRED R L pa a 8 9 10 11 VI Microplate reader capable of measuring absorbance at 450nm 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 SigmaPlot software or other software which can perform four parameter logistic regression models Tubes to prepare standard or sample dilutions Orbital shaker Aluminum foil Saran Wrap REAGENT PREPARATION For sample and positive control dilutions refer to steps 5 6 7 and 9 of Reagent Preparation 1 Keep kit reagents on ice during reagent preparation steps Equilibrate plate to room temperature before opening the sealed pouch Briefly centrifuge the GHR Antibody vial N and reconstitute with 5 ul of ddH2O before use Add 50 yl of 1x Assay Diluent E into the vial to prepare a detection antibody concentrate Pipette up and down to mix gently The antibody concentrate should then be diluted 100 fold with 1x Assay Diluent E This is your anti Ghrelin antibody work
11. sec 9 Add 50 ul of Stop Solution Item 1 to each well Read absorbances at 450 nm immediately RayBio Human Ghrelin Kit Protocol 10 VIII ASSAY PROCEDURE SUMMARY 1 Prepare all reagents samples and standards as instructed 100 ul anti Ghrelin antibody to each well Incubate 1 5 hours at room temperature or overnight at 4 C J Add 100 ul standard or sample to each well Incubate 2 5 hours at room temperature or overnight at 4 C J Add 100 ul prepared streptavidin solution Incubate 45 minutes at room temperature Add 100 ul TMB One Step Substrate Reagent to each well Incubate 30 minutes at room temperature l Add 50 ul Stop Solution to each well Read at 450 nm immediately IX CALCULATION OF RESULTS Calculate the mean absorbance for each set of duplicate standards controls and samples and subtract the blank optical density Plot the standard curve using SigmaPlot software or other software which can perform four parameter logistic regression models with standard concentration on the and percentage of absorbance see calculation below on the y axis Draw the best fit curve through the standard points RayBio Human Ghrelin EIA Kit Protocol 11 Percentage absorbance B blank OD B blank OD where B OD of sample or standard and OD of zero standard total binding A TYPICAL DATA These standard curves are for demonstration only A standard cur
12. ve must be run with each assay EIA GHRELIN 0 1 1 10 100 1000 10000 Peptide Concentration ng ml RayBio Human Ghrelin EIA Kit Protocol 12 SENSITIVITY The minimum detectable concentration of Ghrelin is 161 pg ml 12 46pM C DETECTION RANGE 0 1 1 000 ng ml D REPRODUCIBILITY Intra Assay CV lt 10 Inter Assay CV 1596 X SPECIFICITY Cross Reactivity This ELISA kit shows no cross reactivity with any of the cytokines tested Nesfatin Angiotensin Il NPY and XI REFERENCES Garcia J Garcia Touza M Hijazi R G Epner D Mann D Smith R Cunningham G Marcelli M 2005 Active ghrelin levels and active to total ghrelin ratio in cancer induced cachexia J Clin Endocrinol Metab 90 5 2920 6 Yildiz B Suchard M Wong M McCann 5 Licinio J 2004 Alterations in the dynamics of circulating ghrelin adiponectin and leptin in human obesity Proc Natl Acad Sci U S A 101 28 10434 9 Inui A Asakawa A Bowers CY et 2004 Ghrelin appetite and gastric motility the emerging role of the stomach as an endocrine organ FASEB J 18 3 439 56 Hewson AK Tung LY Connell DW Tookman L Dickson SL 2002 The rat arcuate nucleus integrates peripheral signals RayBio Human Ghrelin Kit Protocol 13 provided by leptin insulin and a ghrelin mimetic Diabetes 51 12 3412 3419 5 Cummings D Weigle D Frayo H Breen P Ma M Dellinger E Purnell J 2002
13. y which is characterized by excessive accumulation of adipose tissue in the body has become one of the greatest public health challenges Obesity is not only associated with health problems linked to increased weight dependent pressure overload on lung joints and bones but also a important risk factor for life threatening diseases such as cardiovascular diseases type 2 diabetes and certain cancers Ghrelin is synthesized as a preprohormone and then proteolytically processed to yield a 28 amino acid peptide Synthesis of ghrelin occurs predominantly in epithelial cells lining the fundus of the stomach with smaller amounts produced in the placenta kidney pituitary and hypothalamus Ghrelin has emerged as the first circulating hunger hormone Ghrelin increases food intake and thus fat mass by an action exerted at the level of the hypothalamus They activate cells in the arcuate nucleus that include the orexigenic neuropeptide Y NPY neurons Ghrelin responsiveness of these neurons is both leptin and insulin sensitive Ghrelin also activates the mesolimbic cholinergic dopaminergic reward link a circuit that communicates the hedonic and reinforcing aspects of natural rewards such as food Ghrelin levels in the plasma of obese individuals are lower than those in leaner individuals except in the case of Prader Willi syndrome induced obesity Those suffering from the eating disorder anorexia nervosa have high plasma levels of ghrelin compared
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