Manual - RayBiotech, Inc.
Contents
1. Final concentration 10 pg 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 BNP 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 BNP is 10 pg ml in all standards 8 Briefly centrifuge the vial of BNP 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 BNP 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 BNP standard 10 pg ml biotinylated BNP 9 To make the 100 pg ml standard pipette 50 ul of the 1000 pg ml BNP standard into the tube labeled 100 pg ml Mix thoroughly 10 Repeat this step with each successive concentration preparing a dilution series as shown in the illustration below Each time use 450 ul of biotinylated BNP 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 pl SO OO py oS os Ke a SS 0 1 0 1000 100 10 1 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 wi2. extracts of porcine brain but in humans it is produced mainly in the cardiac ventricles Its counterpart in rats is a 45 amino acid peptide hormone At the time of release a co secreted 76 amino acid N terminal fragment NT proBNP is also released with BNP BNP binds to and activates NPRA in a similar fashion to atrial natriuretic peptide ANP but with 10 fold lower affinity The biological half life of BNP however is twice as long as that of ANP Both ANP and BNP have limited ability to bind and activate NPRB Physiologic actions of BNP include decrease in systemic vascular resistance and central venous pressure as well as an increase in natriuresis Thus the resulting effect of BNP is a decrease in cardiac output and a decrease in blood volume Tests showing elevated levels of BNP or NT proBNP in blood are used as a diagnosis of heart failure and may be useful to establish prognosis in heart failure as both markers are typically higher in patients with poorer outcome Both BNP and NT proBNP have been approved as a marker for acute congestive heart failure CHF The plasma concentrations of both BNP are increased in patients with asymptomatic and symptomatic left ventricular dysfunction There is no level of BNP that perfectly separates patients with and without heart failure ll General Description The RayBio BNP Enzyme Immunoassay EIA Kit is an in vitro quantitative assay for detecting BNP peptide based on the competitive en
3. 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 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 200 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 BNP 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 o
4. RayBio Human Mouse Rat BNP Enzyme Immunoassay Kit Catalog EIA BNP EIAM BNP EIAR BNP 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 BNP 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 Brain natriuretic peptide BNP aka B type natriuretic peptide is a 32 amino acid polypeptide secreted by the ventricles of the heart in response to excessive stretching of myocytes in the ventricles BNP was originally identified in
5. ble 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 BNP 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 BNP 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 BNP 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 BNP Item F 5 Briefly centrifuge the vial of Biotinylated BNP Item F and reconstitute with 20 ul of ddH20 before use 6 See t
6. escription epee Stay Stability BNP BNP Mirplato tem A Item A BNP Microplate tem a wells 12 ee x 8 wells coated with Hronka month at aor a antibody Wesel eo 25 ml 25 mi of 20x concentrated soon 20X concentrated solution 1 month at month ata eo Item B oe BNP oer Item 2 vials of Te BNP Peptide 1 vial is ee not store and en ee to run each standard in duplicate ee Anti BNP fo ae vials of Lyopniized amene D natstoreand not store and fo ae Item N 2 vials of Lyophilized anti BNP avis 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 TOR BNP Peptide 2 vials of a Biotinylated BNP Peptide 1 P not store and TOR F vial is a to assay the whole plate P HRP oe 600 eee 200X concentrated HRP conjugated ee not store and oe Item G eee ee Poste Convoi em M Poste Convoi em M Item M 1 vial of Lyophilized Positive 1 vial of Lyophilized Postive Conta 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 capa
7. he 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 BNP 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 BNP will be 10 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 BNP 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 BNP will be 10 pg ml Reconstitute Item F in 20 ul ddH 0 1 vial is enough to run Item F the whole plate Working Stock Item F b Positive Control 100 ul of Working Stock Item F 100 ul Prepared Positive Control First Dilution 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 a Standards 2 ml of Working Stock Item F 2 ml of Assay Diluent
8. hooting 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 2015 RayBiotech Inc
9. nly 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 BNP 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 densit
10. s of Angiotensin II and Protect from Cardiac Hypertrophy PLoS ONE 2012 7 7 e41545 doi 10 1371 journal pone 0041545 Species Mouse Sample Type Serum 2 Ku HC et al 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 Martin R Cordova C San Roman JA Gutierrez B Cachofeiro V Nieto ML Oleanolic acid Modulates the Immune Inflammatory Response in Mice with Experimental Autoimmune Myocarditis and Protects from Cardiac Injury J Mol Cell Cardiol 2014 Apr 13 72C 250 262 Species Mouse Sample Type Serum 4 Martin R Cordova C San Roman JA Gutierrez B Cachofeiro V Nieto ML Oleanolic acid Modulates the Immune Inflammatory Response in Mice with Experimental Autoimmune Myocarditis and Protects from Cardiac Injury J Mol Cell Cardiol 2014 Apr 13 72C 250 262 Species Mouse Sample Type Conditioned Media 5 Liu L Aquirre SA Evering WE Hirakawa BP May JR Palacio K Wang J Zhang Y Stevens GJ mir 208a as a Biomarker of isoproterenol induced Cardiac Injury in Sod 2 and C57BL 6 Wild Type Mice Toxicol Pathol 2014 Apr 8 Epub ahead of print Species Mouse Sample Type Plasma For additional publications citing this product please contact technical support at 888 494 8555 or techsupport raybiotech com XIII Troubles
11. th 100 ul of ddH20 12 Refer to step 6b This is a 2 fold dilution of the Positive Control The final concentration of biotinylated BNP should still be 10 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 BNP 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 BNP 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 2X Mouse 2X Rat 2X If you have any questions regarding the recommendended dilutions you may contact technical support at 888 494 8555
12. y 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 BNP EIA 1 120 100 80 4 60 B BO 40 4 20 gt Si 0 01 0 1 1 10 100 1000 10000 BNP Peptide Concentration pg ml B Sensitivity The minimum detectable concentrations of BNP is 1 66 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 BNP 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 Cross Reactivity This EIA kit shows no cross reactivity with any of the adipokines tested Ghrelin Nesfatin Angiotensin Il NPY and APC XIV Publications Citing This Product 1 Martin R Miana M Jurado Lopez R Martinez Martinez E GOmez Hurtado N et al DIOL Triterpenes Block Profibrotic Effect
13. zyme immunoassay principle In this assay a biotinylated BNP peptide is spiked into the samples and standards The samples and standards are then added to the plate where the biotinylated BNP peptide competes with endogenous unlabeled BNP for binding to the anti BNP antibody After a wash step any bound biotinylated BNP 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 BNP peptide and inversely proportional to the amount of endogenous BNP in the standard or samples A standard curve of known concentration of BNP peptide can be established and the concentration of BNP 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 be 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 D
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