Manual - RayBiotech, Inc.
Contents
1. The antibody concentrate should then be diluted 100 fold with 1X Assay Diluent B This is your anti PYY antibody working solution which will be used in step 2 of Assay Procedure Section VIII 6 Note The following steps may be done during the antibody incubation procedure step 2 of Assay Procedure B Preparation of Biotinylated PYY Item F 5 Briefly centrifuge the vial of Biotinylated PYY Item F and reconstitute with 20 ul of ddH20O 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 the appropriate Assay Diluent This is your Working Stock of Item F Pipette up and down to mix gently The final concentration of biotinylated PYY will be 80 pg ml a Second Dilution of Item F for Standards Add 2 ml of Working Stock Item F to 2 ml of the appropriate Assay Diluent The final concentration of biotinylated PYY will be 40 pg 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 PYY will be 40 pg 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 PYY will be 40 pg ml Reconstitute2. 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 noai Storage Stability PYY Microplate Item A 96 wells 12 strips x 8 wells coated with 1 month at 4 C secondary antibody Wash Buffer Concentrate K 20X Item B 25 ml of 20X concentrated solution 1 month at 4 C Standard PYY Peptide Item 2 vials of Lyophilized PYY Peptide 1 vial is Do not store and C enough to run each standard in duplicate reuse Anti PYY Polyclonal Antibody 2 vials of Lyophilized anti PYY Do not store and Item N reuse 30 ml contains 0 09 sodium azide as Assay Diluent A Item D preservative Diluent for standards and serumor N A plasma 15 ml of 5X concentrated buffer Diluent for Assay Diluent B Item E standards cell culture media or other sample 1 month at 4 C types and HRP Streptavidin Biotinylated PYY Peptide 2 vials of Lyophilized Biotinylated PYY Peptide 1 Do not store and Item F vial is enough to assay the whole plate reuse HRP Streptavidin 600 ul 60X concentrated HRP conjugated Do not store and Concentrate Item G streptavidin reuse 1 vial of Lyophilized Positive Control POT OLS AEAN reuse TMB One Step Substrate 12 ml of 3 3 5 5 tetramethylbenzidine TMB in Reagent Item H buffer solution Stop Solution Item 1 8 ml of 0 2 M sulfuric acid wa Return unused wells t
3. Item F in 20 ul ddH 0 1 vial is enough to run the whole plate First Dilution Add entire vial of Item F to SHS aes Item F 10 ml Assay Diluent Second Dilution Perform a 2 fold dilution of c Sample 125 ul b Positive Control a Standards 2 ml of Working Stock 100 ul of Working of Working Stock a Item F ie of Stock Item F 100 pl Item F 5 ul Final concentration Prepared Positive Assay Diluent Prepared Sample 40 pg ml Control C Preparation of Standards 7 Label 6 microtubes with the following concentrations 1 000 pg ml 250 pg ml 62 5 pg ml 15 6 pg ml 3 91 pg ml and 0 pg ml Pipette 300 ul of PYY Item F working solution prepapred in step 6a into each tube except the 1 000 pg ml It is very important to make sure the concentration of biotinylated PYY is 40 pg ml in all standards 8 Briefly centrifuge the vial of PYY Standard Item C Reconstitute with 10 ul of ddH20 and briefly vortex if desired Pipette 6 ul of Item C and 594 ul of 40 pg ml biotinylated PYY working solution prepapred in step 6a into the tube labeled 1 000 pg ml Mix thoroughly This solution serves as the first standard 1 000 pg ml PYY standard 40 pg ml biotinylated PYY 9 To make the 250 pg ml standard pipette 100 ul of the 1 000 pg ml PYY standard into the tube labeled 250 pg ml Mix thoroughly 10 Repeat this step with each successive concentration preparing a dilution series
4. as shown in the illustration below Each time use 300 ul of biotinylated PYY and 50 ul of the prior concentration until the 3 91 pg ml is reached Mix each tube thoroughly before the next transfer 100n1 100n1 100ul 100 yl 1000 250 62 5 15 63 3 91 0 pg ml pg ml pg ml pg ml pg ml pg ml D Positive Control Preparation 11 Briefly centrifuge the Positive Control vial Item M and reconstitute with 100 ul of ddH20 12 Refer to step 6b This is a 2 fold dilution of the Positive Control The final concentration of biotinylated PYY should still be 40 pg 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 PYY is 40 pg ml E Sample Preparation NOTE Not compatiable with plasma samples 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 the appropriate Assay Diluent 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 the appropriate Assay Diluent b Perform step 6c 125 ul of working solution Item F 125 ul of sample pre
5. obesity therapy in the future ll General Description The RayBio PYY Enzyme Immunoassay EIA Kit is an in vitro quantitative assay for detecting PYY peptide based on the competitive enzyme immunoassay principle In this assay a biotinylated PYY peptide is spiked into the samples and standards The samples and standards are then added to the plate where the biotinylated PYY peptide competes with endogenous unlabeled PYY for binding to the anti PYY antibody After a wash step any bound biotinylated PYY 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 PYY peptide and inversely proportional to the amount of endogenous PYY in the standard or samples A standard curve of known concentration of PYY peptide can be established and the concentration of PYY peptide in the samples can be calculated accordingly NOTE Not compatible with plasma ll How It Works Anti igG antibody Be Y Y pre coated on the plate Yy Yy Target molecule Biotinylated ry P in sample Peptide bes 3 gt Capture antibody yi a is added to the wells Biotin peptide Standard w Sample interact competitivly a i i for spots on the capture antibodies Y Y BNP EIA Kit ae Add HRP Streptavidin and Color Substrate Data Analysis IV Storage The entire kit may be stored at 20 C to 80
6. RayBio Human Mouse Rat PYY Enzyme Immunoassay Kit Catalog EIA PYY EIAM PYY EIAR PYY 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 amor SSSCS di i In oenemionewwin SSSCSCS d Cd m forne Cd ao paot ooo a ao Preparation A Preparation of Plate and Anti PYY 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 Peptide YY is a 36 amino acid peptide released by cells in the ileum and colon in response to feeding It is also known as PYY Peptide Tyrosine Tyrosine or Pancreatic Peptide YY3 36 There are two major forms of Peptide YY PYY1 36
7. and PYY3 36 which is the most common form of circulating PYY Peptide YY3 36 PYY is a linear polypeptide consisting of 36 amino acids with structural homology to NPY and pancreatic polypeptide Circulating PYY concentration increases postprandially and decreases by fasting PYY exerts its action through NPY receptors inhibits gastric motility and increases water and electrolyte absorption in the colon PYY may also suppress pancreatic secretion It is secreted by the neuroendocrine cells in the ileum and colon in response to a meal and has been shown to reduce appetite PYY works by slowing the gastric emptying hence it increases efficiency of digestion and nutrient absorption after meal PYY has been shown to play an important role in obesity Animal studies have shown that acute peripheral administration of PYY3 36 inhibits feeding of rodents and primates Studies on Y2R knockout mice have revealed that there is no anorectic effect on Y2R knockot mice Y2R is the receptor for PYY These findings indicate that PYY3 36 has anorectic effect which is suggested to be mediated by Y2R Studies on PYY knockout mice have shown that they have higher fat mass and lower glucose tolerance when compared to control mice indicating that PYY also plays very important role in energy homeostasis by balancing the food intake Studies have also shown that obese people secrete less PYY than non obese people The anorectic effect of PYY represents a possible anti
8. ank Buffer Only Total Binding Biotin PYY only Standard 1 1000 pg ml Standard 2 250 pg ml Standard 3 62 5 pg ml Standard 4 15 6 pg ml Standard 5 3 91 pg ml Pos Control Biotin with Item M XI Specificity Cross Reactivity This EIA kit shows no cross reactivity with any of the cytokines tested Ghrelin Nesfatin Angiotensin Il NPY and APC This kit detects the 1 36 form The 3 36 form may also be detected but this has not been conclusively tested XIV Publications Citing This Product 1 Bar F et al Carboxypeptidase E Modulates Intestinal Immune Homeostasis and Protects against Experimental Colitis in Mice PLOS One Published July 22 2014 DOI 10 1371 journal pone 0102347 Species Mouse Sample Type Conditioned Media 2 Hand K et al Hormone profiling in a novel enteroendocrine cell line pGIP neo STC 1 METABOLISM CLINICAL AND EXPERIMENTAL 61 2012 1683A A 1686 Species Mouse Sample Type Conditioned Media 3 Lin N Cai DL Jin D Chen Y Shi JJ Ginseng Panaxoside Rb1 Reduces Body Weight in Diet Induced Obese Mice Cell Biochem Biophys Cell Biochem Biophys 2014 68 189A A 194 DOI 10 1007 s12013 013 9688 3 Species Mouse Sample Type Serum 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 op
9. e 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 Step 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 PYY EIA 140 B BO 20 S C 0 1 1 10 100 1000 10000 Peptide Concentration pg ml B Sensitivity The minimum detectable concentrations of PYY is 2 84 pg ml C Detection Range 0 1 1 000 pg ml D Reproducibility Intra Assay CV lt 10 Inter Assay CV lt 15 E Assay Diagram Recommended Plate Layout Key Bl
10. ening 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 2015 RayBiotech Inc
11. o the pouch containing desiccant pack reseal along entire edge Positive Control Item M N A Vi NOOR WD 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 Note Assay Diluent A should be used for dilution of samples Item F and Item C when testing plasma or serum samples 1X Assay Diluent B should be used for dilution of samples Item F and Item C when testing cell culture media or other sample types A Preparation of Plate and Anti PYY 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 PYY antibody vial Item N and reconsititute with 55 ul of 1X Assay Diluent B to prepare the antibody concentrate Pipette up and down to mix gently
12. olution 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 Invert the plate and blot it against clean paper towels 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 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 10 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 PYY to each well Incubat
13. pared above The total volume is 250 ul enough for duplicate wells on the microplate It is very important to make sure the final concentration of the biotinylated PYY is 40 pg ml Note Optimal sample dilution factors should be determined empirically however you may reference below for recommended dilution factors for serum Human 8X Mouse 32X Rat 32X If you have any questions regarding the recommendended dilutions you may contact technical support at 888 494 8555 or techsupport raybiotech com F Preparation of Wash Buffer and HRP 14 15 16 17 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 yield 400 ml of 1X Wash Buffer Briefly centrifuge the HRP Streptavidin vial Item G before use Dilute the HRP Streptavidin concentrate 60 fold with 1X Assay Diluent B Note do not use Assay Diluent A for HRP Streptavidin preparation in step 17 VIII Assay Procedure 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 ul of Anti PYY 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 Discard the solution and wash wells 4 times with 1X Wash S
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