Manual - RayBiotech, Inc.
Contents
1. 2015 RayBiotech Inc2. Invert the plate and blot it against clean paper towels 4 Add 100 ul of each standard see Reagent Preparation Section C Positive Control see Reagent Preparation Section D and sample see Reagent Preparation Section E in 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 overnight or at 4 C 5 Discard the solution and wash 4 times as directed in Step 3 Add 100 ul of prepared HRP Streptavidin solution see Reagent Preparation step 7 to each well Incubate for 45 minutes at room temperature with gentle shaking It is recommended that incubation time should not be shorter or longer than 45 minutes Discard the solution and wash 4 times as directed 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 1 2 cycles sec Add 50 ul of Stop Solution Item to each well Read at 450 nm immediately Assay Procedure Summary Prepare all reagents samples and standards as instructed Add 100 ul anti Amylin to each well Incubate 1 5 hours at room temperature or overnight at 4 C Add 100 ul standard or sample to each well Incubate 2 5 hours at room temperature or overnight at 4 C Add 100 ul prepared Streptavidin solution Incubate 45 minutes at room temperature Add 100 ul TMB One Ste
3. a dilution series as shown in the illustration below Each time use 450 ul of biotinylated Amylin and 50 ul of the prior concentration until the 0 1 pg ml is reached Mix each tube thoroughly before the next transfer 50 PAN 50 ul 90 ul 50 ul OS AS OS Is SAE 1000 100 10 1 100 0 ng ml ng ml ng ml ng ml pg ml pg ml D Positive Control Preparation 11 Briefly centrifuge the Positive Control vial Item M 12 Refer to step 6b This is a 2 fold dilution of the Positive Control The final concentration of biotinylated Amylin should still be 10 ng ml The Positive Control is a cell culture media sample that serves as a system control to verify that the kit components are working The resulting OD will not be used in any calculations if no positive competition is observed please contact RayBiotech Technical Support The Positive Control may be diluted further if desired but be sure the final concentration of biotinylated Amylin is 10 ng ml E Sample Preparation 13 If you wish to perform a 2 fold dilution of your sample proceed to step 6c If you wish to perform a higher dilution of your sample dilute your sample with 1X Assay Diluent B before performing step 6c EXAMPLE to make a 4 fold dilution of sample a Dilute sample 2 fold 62 5 ul of sample 62 5 ul of 1X Assay Diluent B b Perform step 6c 125 ul of working solution Item F 125 ul of sample prepared above The total volume is 250 ul enough for duplicate wells o
4. reduced by inhibiting secretion of the gluconeogenic hormone glucagon These actions which are mostly carried out via a glucose sensitive part of the brain stem the area postrema may be over ridden during hypoglycemia They collectively reduce the total insulin demand ll General Description The RayBio Amylin Enzyme Immunoassay EIA Kit is an in vitro quantitative assay for detecting Amylin peptide based on the competitive enzyme immunoassay principle In this assay a biotinylated Amylin peptide is spiked into the samples and standards The samples and standards are then added to the plate where the biotinylated Amylin peptide competes with endogenous unlabeled Amylin for binding to the anti Amylin antibody After a wash step any bound biotinylated Amylin then interacts with horseradish peroxidase HRP streptavidin which catalyzes a color development reaction The intensity of the colorimetric signal is directly proportional to the amount of captured biotinylated Amylin peptide and inversely proportional to the amount of endogenous Amylin in the standard or samples A standard curve of known concentration of Amylin peptide can be established and the concentration of Amylin peptide in the samples can be calculated accordingly Ill How It Works Y Y ace yi yi Target molecule Biotinylated tt in sample Peptide bed tk gt Capture antibody yi yi pn is added to the wells Biotin peptide Standard w 5 Sample interact comp
5. RayBio Human Mouse Rat Amylin Enzyme Immunoassay Kit Catalog EIA AMY EIAM AMY EIAR AMY User Manual Last revised December 1 2015 Caution Extraordinarily useful information enclosed Re RayBiotech 1 ii The protein array pioneer ISO 13485 Certified 3607 Parkway Lane Suite 100 Norcross GA 30092 Tel 1 888 494 8555 Toll Free or 770 729 2992 Fax 770 206 2393 Web www RayBiotech com Email info raybiotech com Table of Contents ease SSSCS di i In oenemionsowin SSSCSCS d Cd m forne Cd ao paot ooo a ao Preparation A Preparation of Plate and Anti Amylin Antibody B Preparation of Biotinylated Peptide Item F C Preparation of Standards D Preparation of Positive Control E Preparation of Samples F Preparation of Wash Buffer and HRP Strep vm Assay Procedure lege fix Assay Procedure Summary O Assay Procedure Summary pe Calculation of Results A Typical Data B Sensitivity a See eee C Detection Range D Reproducibility E Assay Diagram x Specifcty OOOO O O Specificity Ce xu Select Publications OO O O Select Publications a Troubleshooting Guide Please read the entire manual carefully before starting your experiment I Introduction Amylin or Islet Amyloid Polypeptide IAPP is a 37 residue peptide hormone It is cosecreted with insulin from the pancreaticA A beta cells Amylin plays a role in glycemic regulation by slowing gastric emptying and promoting sat
6. Tschop M Smiley DL Heiman ML Ghrelin induces adiposity in rodents Nature 2002 407 6806 908 913 9 Kojima M Hosoda H Date Y Nakazato M Matsuo H Kangawa K Ghrelin is a growth hormone releasing acylated peptide from stomach Nature 1999 402 6762 656 60 XIII Troubleshooting Guide e Check pipettes Poor standard e Inaccurate pipetting Briefly centrifuge Item C and dissolve curve e Improper standard dilution the powder thoroughly by gently mixing Briefly spin down vials before e Improper preparation of opening Dissolve the powder standard and or thoroughly biotinylated antibody Ensure sufficient incubation time e Too brief incubation times assay procedure step 2 may be done e Inadequate reagent overnight volumes or improper dilution Check pipettes and ensure correct preparation Low signal e Inaccurate pipetting Check pipettes e Air bubbles in wells e Remove bubbles in wells Review the manual for proper wash If using a plate washer ensure that all ports are unobstructed Make fresh wash buffer e Plate is insufficiently washed e Contaminated wash buffer Follow storage recomendations in e Improper storage of the sections IV and V Keep substrate ELISA kit solution protected from light e Stop solution Add stop solution to each well before reading plate RayBio ELISA Kits Over 2 000 ELISA kits available visit www RayBiotech com ELISA Kits html for details This product is for research use only
7. e Control Item M Positive Control Item M Item M 1 1 vial of Positive Control of Positive 1 vial of Positive Control 2 3 re at 2 3 days at4ec TMB One Step Substrate 12 ml of 3 3 5 5 tetramethylbenzidine TMB i Reagent Item H buffer solution Stop Solution Item 1 8 8 mi of 0 2 M sulfuric acid of 0 2 M sulfuric acid laa m Return unused wells to the pouch containing desiccant pack reseal along entire edge Vi NOOR OWOD 8 9 10 11 Vil 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 SigmaPlot software or other software which can perform four parameter logistic regression models Tubes to prepare standard or sample dilutions Orbital shaker Aluminum foil Plastic wrap Reagent Preparation Keep kit reagents on ice during reagent preparation steps A Preparation of Plate and Anti Amylin Antibody 1 2 Equilibrate plate to room temperature before opening the sealed pouch Label removable 8 well strips as appropriate for your experiment 5X Assay Diluent B Item E should be diluted 5 fold with deionized or distilled water Briefly centrifuge the anti Amylin antibody vial Item N Then add 50 ul of 1X Assay Diluent B to
8. etitivly m_a i for spots on the capture antibodies Y Y bii a Add HRP Streptavidin and Color Substrate Data Analysis IV Storage The entire kit may be stored at 20 C to 80 C for up to 6 months from the date of shipment For extended storage it is recommended to store at 80 C Avoid repeated freeze thaw cycles For prepared reagent storage see table below V Reagents Size Description Storage Sabiny Stability Amyin wcroplat tom A Microplate Item A myin microplate toma wells 12 ee x 8 wells coated with Geis tae month at aor eee antibody Wash Buffer el ft monthatas el Item B 25 ml 25 mi of 20x concentrated soluton 20X concentrated solution 1 month at 4 ft monthatas The first standard Standard Amylin Peptide 2 vials of Amylin Peptide 1 vial is enough to run 2 3 days at 4 C Item C each standard in duplicate Additional dilutions Do not store Anti oo Polyclonal oo Item N 2 vials of anti vials ofantramyin 1 month ato at 4 month ato 15 ml of 5X concentrated buffer Diluent for both 5X Assay Diluent B Item E standards and samples including serum plasma 1 month at 4 C cell culture media or other sample types ogee ane Amylin Peptide 2 vials of ee Amylin Peptide 1 vial is 2 3 podaysaao at 4 C ogee ane F ee to assay the whole plate HRP te 600 eee 1000X concentrated HRP conjugated Cel not store and Concentrate te G eee Cel Positiv
9. icity Cross Reactivity This EIA kit shows no cross reactivity with any of the cytokines tested Ghrelin Nesfatin Angiotensin Il NPY and APC XIV Select EIA Publications N Plum L Lin HV Dutia R Tanaka J Aizawa KS et al The Obesity Susceptibility Gene Carboxypeptidase E Links FoxO1 Signaling in Hypothalamic Pro opiomelanocortin Neurons with Regulation of Food Intake Nature Med 2009 15 10 1195 1201 Ghrelin EIA EIA GHR 1 Hug C Lodish HF Visfatin a new adipokine Science 2005 307 5708 366 7 Kim MK Crystal structure of visfatin pre B cell colony enhancing factor 1 nicotinamide phosphoribosyltransferase free and in complex with the anti cancer agent FK 866 J Mol Biol 2006 362 1 66 77 Revollo J R et al The NAD biosynthesis pathway mediated by nicotinamide phosphoribosyltransferase regulates Sir2 activity in mammalian cells J Biol Chem 2004 279 50754 50763 Oh I S Shimizu H Satoh T et al Identification of nesfatin 1 as a satiety molecule in the hypothalamus Nature 2006 443 7112 709 12 Zhang J Ren P Avsian Kretchmer O Luo C Rauch R Klein C Hsueh A Obestatin a peptide encoded by the ghrelin gene opposes ghrelin s effects on food intake Science 2005 310 5750 996 9 Cummings D Weigle D Frayo R Breen P Ma M Dellinger E Purnell J Plasma ghrelin levels after diet induced weight loss or gastric bypass surgery N Engl J Med 2002 346 21 1623 30
10. iety thereby preventingA A post prandialA A spikes in blood glucose levels APP is processed from an 89 residueA A coding sequence A A Proislet Amyloid PolypeptideA A is produced in the pancreatic beta cells beta cells as a 67 amino acid 7404 Dalton pro peptide and undergoesA A post translational modificationsA A including protease cleavage to produce amylin Insulin and APP are regulated by similar factors since they share a common regulatory promoter motif The APP promoter is also activated by stimuli which do not affect insulin such as tumor necrosis factor alpha and fatty acids One of the defining features of Type 2 diabetes is insulin resistance This is a condition wherein the body is unable to utilize insulin effectively resulting in increased insulin production since proinsulin and prolAPP are cosecreted this results in an increase in the production of prolAPP as well Amylin s metabolic function is well characterized as an inhibitor of the appearance of nutrient in the plasma It thus functions as a synergistic partner to insulin with which it is cosecreted from pancreatic beta cells in response to meals The overall effect is to slow the rate of appearance of glucose in the blood after eating this is accomplished via coordinate slowing down gastric emptying inhibition of digestive secretion gastric acid pancreatic enzymes and bile ejection and a resulting reduction in food intake Appearance of new glucose in the blood is
11. lin will be 10 ng ml 1 vialis enough to run the Item F whole plate First Dilution Working Stock Item F Add entire vial of Item F to 10 ml Assay Diluent Second Dilution Perform a 2 fold dilution of Working Stock Item F c Sample 125 ul of Working Stock Item F 125 ul Prepared Sample b Positive Control 100 ul of Working Stock Item F 100 ul Prepared Positive Control a Standards 2 ml of Working Stock Item F 2 ml of Assay Diluent Final concentration 10 ng ml C Preparation of Standards 7 Label 6 microtubes with the following concentrations 1000 pg ml 100 pg ml 10 pg ml 1 pg ml 0 1 pg ml and 0 pg ml Pipette 450 ul of biotinylated Amylin Item F working solution prepapred in step 6a into each tube except the 1 000 pg ml leave this one empty It is very important to make sure the concentration of biotinylated Amylin is 10 ng ml in all standards 8 Briefly centrifuge the vial of Amylin Standard Item C Pipette 8 ul of Item C and 792 ul of 10 ng ml biotinylated Amylin working solution prepared in step 6a into the tube labeled 1000 pg ml Mix thoroughly This solution serves as the first standard 1000 pg ml Amylin standard 10 ng ml biotinylated Amylin 9 To make the 100 pg ml standard pipette 50 ul of the 1000 pg ml Amylin standard into the tube labeled 100 pg ml Mix thoroughly 10 Repeat this step with each successive concentration preparing
12. n the microplate It is very important to make sure the final concentration of the biotinylated Amylin is 10 ng ml 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 factors for serum F Preparation of Wash Buffer and HRP 14 If Item B 20X Wash Concentrate contains visible crystals warm to room temperature and mix gently until dissolved 15 Dilute 20 ml of Wash Buffer Concentrate into deionized or distilled water to yield 400 ml of 1X Wash Buffer 16 Briefly centrifuge the HRP Streptavidin vial Item G before use 17 Dilute the HRP Streptavidin concentrate 1000 fold with 1X Assay Diluent B Vill 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 2 Add 100 ul of Anti Amylin Antibody Item N See Reagent Preparation step 3 to each well Incubate for 1 5 hours at room temperature with gentle shaking 1 2 cycle sec You may also incubate overnight at 4 C 3 Discard the solution and wash wells 4 times with 1X Wash Solution Buffer 200 300 ul 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
13. p Substrate Reagent to each well Incubate 30 minutes at room temperature Add 50 ul Stop Solution to each well Read at 450 nm immediately X Calculation of Results Calculate the mean absorbance for each set of duplicate stands 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 x axis and percentage of absorbance see calculation below on the y axis Draw the best fit curve through the standard points Percentage absorbance B blank OD Bo blank OD where B OD of sample or standard and Bo OD of zero standard total binding A Typical Data These standard curves are for demonstration only A standard curve must be run with each assay Amylin EIA 120 100 Joo 80 N 604 a i a 40 N 20 4 Ny T T T T T 0 01 0 1 1 10 100 1000 Amylin Peptide concentration ng ml B Sensitivity The minimum detectable concentrations of Amylin is 0 62 ng ml C Detection Range 0 1 1 000 ng ml D Reproducibility Intra Assay CV lt 10 Inter Assay CV lt 15 E Assay Diagram Recommended Plate Layout Key Blank Buffer Only Total Binding Biotin Amylin only Standard 1 1000 pg ml Standard 2 100 pg ml Standard 3 10 pg ml Standard 4 1 pg ml Standard 5 0 1 pg ml Pos Control Biotin with Item M XI Specif
14. the vial to prepare the antibody concentrate Pipette up and down to mix gently The antibody concentrate should then be diluted 100 fold with 1X Assay Diluent B This is your anti Amylin antibody working solution which will be used in step 2 of Assay Procedure Section VIII Note The following steps may be done during the antibody incubation procedure step 2 of Assay Procedure B Preparation of Biotinylated Amylin Item F 5 Briefly centrifuge the vial of Biotinylated Amylin Item F before use 6 See the image below for proper preparation of Item F Transfer the entire contents of the Item F vial into a tube containing 10 ml of 1X Assay Diluent B This is your Working Stock of Item F Pipette up and down to mix gently The final concentration of biotinylated Amylin will be 20 ng ml a Second Dilution of Item F for Standards Add 2 ml of Working Stock Item F to 2 ml of 1X Assay Diluent B The final concentration of biotinylated Amylin will be 10 ng ml b Second Dilution of Item F for Positive Control Add 100 ul of Working Stock Item F to 100 ul of the prepared Positive Control Item M See section D for Positive Control preparation The final concentration of biotinylated Amylin will be 10 ng ml c Second Dilution of Item F for samples Add 125 ul of Working Stock Item F to 125 ul of prepared sample see section E for sample preparation This is a 2 fold dilution of your sample The final concentration of biotinylated Amy
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