Manual - RayBiotech, Inc.
Contents
1. ANP 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 ANP is 10 pg ml in all standards 8 10 Briefly centrifuge the vial of ANP Standard Item C Reconstitute with 10 ul of ddH20 and briefly vortex if desired Pipette 8 ul of Item C and 792 ul of 10 pg ml biotinylated ANP 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 ANP standard 10 pg ml biotinylated ANP To make the 100 pg ml standard pipette 50 ul of the 1000 pg ml ANP standard into the tube labeled 100 pg ml Mix thoroughly Repeat this step with each successive concentration preparing a dilution series as shown in the illustration below Each time use 450 ul of biotinylated ANP and 50 ul of the prior concentration until the 0 1 pg ml is reached Mix each tube thoroughly before the next transfer 50 ul 50 ul 50 ul 50 ul AANA DOo0C 1000 100 10 1 0 1 0 pg ml pg ml pg ml pg ml pg ml pg ml ox D Positive Control Preparation 11 Briefly centrifuge the Positive Control vial Item M and reconstitute with 105 ul of ddH20 12 Refer to step 6b This is a 2 fold dilution of the Positive Control The final concentration of biotinylated ANP should still be 10 pg ml The Positive Control is a cell culture media sample that serves as a system2. 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 ANP is 10 pg 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 on the microplate It is very important to make sure the final concentration of the biotinylated ANP is 10 pg ml Note Optimal sample dilution factors should be determined empirically however you may reference below for recommended dilution factors for serum Human 4X Mouse 4X Rat 4X 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 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 i
3. ml 25 mi of 20x concentrated soon 20X concentrated solution 1 month at month ata eo Item B a ANP Item 2 vials of Te ANP Peptide 1 vial is ee not store and en S to run each standard in duplicate ee Anti ANP oa ae vias of Lyophiized anane pppoe not store and a ae Item N 2 vials of Lyophilized anti ANP vias or tyophizea anane ont store and 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 TOOR ANP Peptide 2 vials of S Biotinylated ANP Peptide 1 P not store and TOOR F vial is S to assay the whole plate P HRP oe 600 ee 50X concentrated HRP conjugated ee not store and oe Item G ee ee Poste Convoi em M Poste Convoi em M Item M 1 vial of Lyophilized Positive 1 vial of Lyophilized Postive Cono e SOIS oe TMB One Step Substrate 12 ml of 3 3 5 5 tetramethylbenzidine TMB i Reagent Item H buffer solution Stop Solution Item 1 8 ml of 0 2 M sulfuric acid TE mai ed 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 Absorben
4. DPP4 deficiency preserves cardiac function via GLP 1 signaling in rats subjected to myocardial ischemia reperfusion Naunyn Schmiedebergs Arch Pharmacol 2011 Aug 384 2 197 207 doi 10 1007 s00210 01 1 0665 3 Species Rat Sample Type Plasma 3 Muchir A et al Treatment with selumetinib preserves cardiac function and improves survival in cardiomyopathy caused by mutation in the lamin A C gene Cardiovasc Res 2012 Feb 1 93 2 311 9 doi 10 1093 cvr cvr301 Species Mouse Sample Type Serum 4 Samillan V et al Combination of erythropoietin and sildenafil can effectively attenuate hypoxia induced pulmonary hypertension in mice Pulm Circ 2013 Dec 3 4 898 907 doi 10 1086 674758 Species Mouse Sample Type Serum 5 Hamdani N et al Left ventricular diastolic dysfunction and myocardial stiffness in diabeticmice is attenuated by inhibition of dipeptidyl peptiase 4 Cardiovascular Research 2014 104 423A A 431 doi 10 1093 cvr cvu223 Species Mouse Sample Type Serum For additional publications citing this product please contact technical support at 888 494 8555 or techsupport raybiotech com 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 Ensu
5. RayBio Human Mouse Rat ANP Enzyme Immunoassay Kit Catalog EIA ANP EIAM ANP EIAR ANP User Manual Last revised November 30 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 ANP 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 Atrial natriuretic peptide ANP is a 28 amino acid peptide hormone secreted by cardiac myocytes of the atrium ANP plays an important role in the homeostatic regulation of body water sodium potassium and fat by acting to reduce the
6. d down to mix gently The final concentration of biotinylated ANP will be 20 pg 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 ANP will be 10 pg ml b Second Dilution of Item F for Positive Control Add 105 ul of Working Stock Item F to 105 ul of the prepared Positive Control Item M See section D for Positive Control preparation The final concentration of biotinylated ANP will be 10 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 ANP will be 10 pg ml Reconstitute Item F in 20 ul ddH 0 1 vial is enough to run Item F the whole plate Add entire vial of Item F to logon Stock First Dilution Item F 10 ml Assay Diluent Second Dilution gt c Sample 125 ul Performa 2 fold dilution of a Standards 2 ml Working Stock Item F of Working Stock 105 pl of Working of Working Stock 3 ItemF 2 ml of Stock en 105 yl Item F 125 aiiai Final concentration Assay Diluent Prepared Positive aiiai Sample 10 pg ml Control f b Positive Control i 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
7. lated ANP peptide competes with endogenous unlabeled ANP for binding to the anti ANP antibody After a wash step any bound biotinylated ANP 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 ANP peptide and inversely proportional to the amount of endogenous ANP in the standard or samples A standard curve of known concentration of ANP peptide can be established and the concentration of ANP peptide in the samples can be calculated accordingly ll How It Works Anti lgG antibody Bi ae Y Y pre coated on the plate Y Y Target molecule Biotinylated ry in sample Peptide D 4 e tk gt Capture antibody y is added to the wells Biotin peptide Standard w A Sample interact competitivly Ma a i for spots on the capture antibodies T Y BNP EIA Kit il 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 epee Stay Stability ANP ANP Microplate tem A Item A ANP microplate tem a wells 12 ee x 8 wells coated with Hronka month at aor eee antibody Washi Burer o 25
8. nto 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 50 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 ANP 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 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
9. raw 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 ANP EIA 1 B BO 0 01 0 1 1 10 100 1000 10000 ANP Peptide Concentration pg ml B Sensitivity The minimum detectable concentrations of ANP is 1 02 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 Blank Buffer Only Total Binding Biotin ANP 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 Specificity This kit targets the common sequence of human mouse and rat and thus may be used to detect ANP expression in all these species with high specificity and sensitivity Cross Reactivity This EIA kit shows no cross reactivity with any of the cytokines tested Ghrelin Nesfatin Angiotensin Il NPY and APC XIV Publications Citing This Product 1 Kim M Platt MJ Shibasaki T Quaggin SE Backx PH et al GLP 1 receptor activation and Epac2 link atrial natriuretic peptide secretion to control of blood pressure Nat Med 2013 19 567A A 575 Species Mouse Sample Type Serum 2 Ku HC et al
10. re 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. 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 ANP 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 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 D
12. t 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 ANP 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 ANP 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 The antibody concentrate should then be diluted 100 fold with 1X Assay Diluent B This is your anti ANP 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 ANP Item F 5 Briefly centrifuge the vial of Biotinylated ANP Item F and reconstitute with 20 ul of ddH20 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 an
13. water sodium and adipose loads on the circulatory system thus reducing blood pressure ANP peptide contains a 17 amino acid ring which is formed by a disulfide bond between two cysteine residues at positions 7 and 23 ANP is closely related to BNP brain natriuretic peptide and CNP C type natriuretic peptide which all share the same amino acid ring The mechanism of ANP induced vasodilatation is through binding to a specific set of ANP receptors Receptor agonist binding causes a reduction in blood volume and therefore a reduction in cardiac output and systemic blood pressure Lipolysis is increased and renal sodium reabsorption is decreased The overall effect of ANP on the body is to counter increases in blood pressure and volume caused by the renin angiotensin system In addition to its vasodilatation effect ANP also serves as a adipokine Studies have shown that ANP Increases the release of free fatty acids from adipose tissue activates adipocyte plasma membrane NPR A receptors and increases intracellular CGMP levels that induce the phosphorylation of a hormone sensitive lipase and perilipin A ll General Description The RayBio ANP Enzyme Immunoassay EIA Kit is an in vitro quantitative assay for detecting ANP peptide based on the competitive enzyme immunoassay principle In this assay a biotinylated ANP peptide is spiked into the samples and standards The samples and standards are then added to the plate where the biotiny
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