Manual - RayBiotech, Inc.
Contents
1. S amp S OS Os 1000 100 10 1 0 1 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 12 Refer to step 6b This is a 2 fold dilution of the Positive Control The final concentration of biotinylated PTH should still be 20 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 PTH is 20 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 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 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 PTH is 20 pg ml Note Optimal sample dilution factors should be determined empirically however you may reference below for recomm2. 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 2015 RayBiotech Inc
3. 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 PTH EIA 120 100 rn ia 60 B BO 40 4 0 001 0 01 0 1 1 10 100 1000 10000 PTH Concentration pg ml B Sensitivity The minimum detectable concentrations of PTH is 1 27 pg ml C Detection Range 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 PTH 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 react
4. RayBio Human Mouse Rat PTH Enzyme Immunoassay Kit Catalog EIA PTH EIAM PTH EIAR PTH 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 PTH 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 Parathyroid hormone PTH is secreted by the chief cells of the parathyroid glands as a polypeptide containing 84 amino acids with molecular weight of 9 4KDa PTH acts to increase the concentration of calcium Ca2 in the blood by act
5. ended 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 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 1000 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 PTH 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 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 i
6. ing upon parathyroid hormone receptor in three parts of the body bone kidney and intestine In addition to regulation of serum calcium levels PTH also plays role in regulating serum phosphate and vitamin D synthesis PTH reduces the reabsorption of phosphate from the proximal tubule of the kidney resulting in increased phosphate excretion through the urine However PTH enhances the uptake of phosphate from the intestine and bones into the blood In the bone slightly more calcium than phosphate is released from the breakdown of bone In the intestines which are mediated by an increase in activated vitamin D the absorption of phosphate is not as dependent on vitamin D as is that of calcium The end result is a small net drop in the serum concentration of phosphate PTH increases the activity of 1 alpha hydroxylase enzyme which converts 25 hydroxycholecalciferol to 1 25 dihydroxycholecalciferol the active form of vitamin D PTH has shown significant clinical applications A high level of PTH in the blood is known as hyperparathyroidism The causes of primary hyperparathyroidism are parathyroid adenoma parathyroid hyperplasia and parathyroid cancer The cause for secondary hyperparathyroidism is often due to in chronic renal failure ll General Description The RayBio PTH Enzyme Immunoassay EIA Kit is an in vitro quantitative assay for detecting PTH peptide based on the competitive enzyme immunoassay principle In this assay a biotiny
7. ivity 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 Tschop M Smiley DL Heiman ML Ghrelin induces adiposity in
8. lated PTH peptide is spiked into the samples and standards The samples and standards are then added to the plate where the biotinylated PTH peptide competes with endogenous unlabeled PTH for binding to the anti PTH antibody After a wash step any bound biotinylated PTH 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 PTH peptide and inversely proportional to the amount of endogenous PTH in the standard or samples A standard curve of known concentration of PTH peptide can be established and the concentration of PTH 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 Description Storage Sabiny Stability PTH PTH micr
9. ml Assay Diluent Pee QUE 12 b Positive Control Perform a 2 fold dilution of n iada a 100 ul of Working Working Stock Item F oTo rng Babson Stock Item F 100 pl Final concentration oiana i died Sale ontro 20 pg ml c Sample 125 ul of Working Stock Item F 125 ul Prepared Sample 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 PTH 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 PTH is 20 pg ml in all standards 8 Briefly centrifuge the vial of PTH Standard Item C Pipette 8 ul of Item C and 792 ul of 20 pg ml biotinylated PTH 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 PTH standard 20 pg ml biotinylated PTH 9 To make the 100 pg ml standard pipette 50 ul of the 1000 pg ml PTH 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 PTH and 50 ul of the prior concentration until the 0 1 pg ml is reached Mix each tube thoroughly before the next transfer sOul SOul 50 ul 50 ul
10. oplate tema Item A PTH microplate tema wells 12 ee x 8 wells coated with Geis tae month at aor eee antibody Wash Buffer oo ft monthatas oo Item B 25 ml 25 mi of 20x concentrated soon 20X concentrated solution 1 month at 4 ft monthatas The first standard Standard PTH Peptide Item 2 vials of PTH Peptide 1 vial is enough to run 2 3 days at 4 C C each standard in duplicate Additional dilutions Do not store ALE alyeional AANDOAY ll aviais orante PTE 1 month at 4 C Item N 30 ml contains 0 09 sodium azide as Assay Diluent A Item D preservative Diluent for standards and serum or 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 oe ee PTH Peptide 2 vials of ee PTH Peptide 1 vial is 2 3 es daysatae at es daysatae oe ee F ee to assay the whole plate HRP ee 600 a eee ee 1000X concentrated HRP conjugated oe not store and Concentrate ee G a eee ee oe Positive Control Item M Positive Control Item M Item M 1 1 vial of Positive Control of Positive 1 vial of Positive Control 2 3 at 2 3daysat4C 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 ml of 0 2 M sulfuric acid of 0 2 M sulfuric acid E daea Return unused wells to the pouch containing desiccan
11. t B This is your anti PTH 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 PTH Item F 5 Briefly centrifuge the vial of Biotinylated PTH 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 the appropriate Assay Diluent This is your Working Stock of Item F Pipette up and down to mix gently The final concentration of biotinylated PTH will be 40 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 PTH will be 20 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 PTH will be 20 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 PTH will be 20 pg ml 1 vial is enough to run the Item F whole plate First Dilution Working Stock Item F Add entire vial of Item F to 10
12. t 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 PTH 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
13. t pack reseal along entire edge 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 PTH 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 PTH antibody vial Item N Then add 50 ul of 1X Assay Diluent B to 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 Diluen
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