Human Ghrelin ELISA Kit
Contents
1. 1155 2010 945056 PMC 2925405 PMID 20798855 3 Cummings DE et al 2002 The New England Journal of Medicine 346 21 1623 30 doi 10 1056 NEJMoa012908 PMID 12023994 4 Fonken LK Nelson RJ 2014 Endocrine Reviews 35 4 648 70 doi 10 1210 er 2013 1051 PMID 24673196 5 Garcia JM et al The Journal of Clinical Endocrinology and Metabolism 90 5 2920 6 doi 10 1210 jc 2004 1788 PMID 15713718 6 Ruperto M et al 2014 Nutrici n Hospitalaria 29 4 735 50 doi 10 3305 nh 2014 29 4 7222 inactive 2015 02 01 PMID 24679014 7 Berardi E et al 2014 Frontiers in Physiology 5 119 doi 10 3389 fphys 2014 00119 PMC 3986550 PMID 24782779 8 Clynen E et al 2014 Molecular Neurobiology 50 2 626 46 doi 10 1007 s12035 014 8669 x PMC 4182642 PMID 24705860 9 Hasler WL 2014 Expert Opinion on Emerging Drugs 19 2 261 79 doi 10 1517 14728214 2014 899353 PMID 24669936 Version 1 0 www assaypro com e e mail Support assaypro com April 20152. hormones ghrelin is released in a circadian fashion suggesting that ghrelin levels can indicate interruptions in circadian rhythm 4 Elevated ghrelin levels have been observed in eating disorders and cachexia associated with chronic heart failure liver cirrhosis and cancer 1 5 The administration of synthetic ghrelin is being investigated as a potential treatment for cachexia and hemodialysis patients 6 7 In animal models ghrelin has been shown to suppress seizures and it may also be useful in treating gastroparesis 8 9 Principle of the Assay The AssayMax Human Ghrelin ELISA Enzyme Linked Immunosorbent Assay kit is designed for detection of human ghrelin in plasma serum and cell culture samples This assay employs a quantitative sandwich enzyme immunoassay technique which measures ghrelin in 4 hours A polyclonal antibody specific for ghrelin has been pre coated onto a 96 well microplate with removable strips Ghrelin in standards and samples is sandwiched by the immobilized antibody and biotinylated polyclonal antibody specific for ghrelin which is recognized by a streptavidin peroxidase conjugate All unbound material is washed away and a peroxidase enzyme substrate is added The color development is stopped and the intensity of the color is measured Caution and Warning Prepare all reagents working diluent buffer wash buffer standard biotinylated antibody and SP conjugate as instructed prior to running the assa
3. Wyssaypro Human Ghrelin ELISA Kit Assaypro LLC 3400 Harry S Truman Blvd St Charles MO 63301 T 636 447 9175 F 636 395 7419 WWW assaypro com For any questions regarding troubleshooting or performing the assay please contact our support team at support assaypro com Thank you for choosing Assaypro Assay Summary Step 1 Add 50 ul of Standard or Sample per well Incubate 2 hours Step 2 Wash then add 50 ul of Biotinylated Antibody per well Incubate 1 hour Step 3 Wash then add 50 ul of SP Conjugate per well Incubate 30 minutes Step 4 Wash then add 50 ul of Chromogen Substrate per well Incubate 30 minutes Step 5 Add 50 ul of Stop Solution per well Read at 450 nm immediately Symbol Key ci Consult instructions for use Assay Template 12 11 10 AssayMax Human Ghrelin ELISA Kit Catalog No EG3780 1 Sample protocol for reference use only Introduction Ghrelin colloquially known as the hunger hormone is a peptide produced in the gastrointestinal tract 1 2 It functions as a neuropeptide regulating hunger and participating in the regulation of energy use and distribution Higher levels of ghrelin contribute to the increase of appetite and metabolic function Ghrelin suppression is related to weight loss and is a potential treatment of obesity via gastric bypass 3 Like other metabolically related
4. an anticoagulant e Serum Samples should be collected into a serum separator tube Samples collection and processing should be performed as quickly as possible Keep on ice when not in use It is recommended that protease inhibitor cocktail is added to samples for example o phenanthroline 0 44 mM EDTA 25 mM p hydroxy mercuribenzoic acid 1mM and pepstatin A 0 12 mM The user may need to optimize concentration of above reagents After clot formation centrifuge samples at 3000 x g for 10 minutes remove serum and assay The undiluted samples can be stored at 20 C or below for up to 3 months Avoid repeated freeze thaw cycles e Cell Culture Supernatants Centrifuge cell culture media at 3000 x g for 10 minutes to remove debris collect supernatants and assay The undiluted samples can be stored at 20 C or below Avoid repeated freeze thaw cycles Reagent Preparation e Freshly dilute all reagents and bring all reagents to room temperature before use e EIA Diluent Concentrate 10x If crystals have formed in the concentrate mix gently until the crystals have completely dissolved Dilute the EIA Diluent Concentrate 1 10 with reagent grade water Store for up to 30 days at 2 8 C e Human Ghrelin Standard Reconstitute the 12 8 ng of Human Ghrelin Standard with 0 8 ml of EIA Diluent to generate a 16 ng ml standard stock solution Allow the standard to sit for 10 minutes with gentle agitation prior to making dilutions Aliquot standard t
5. ck that the microplate washer is dispensing properly e If washing by pipette check for proper pipetting c technique 2 Splashing of reagents e Pipette properly in a controlled and careful manner 8 while loading wells v e Pipette properly in a controlled and careful manner a Inconsistent volumes A Pon A e Check pipette calibration 3 loaded into wells o e Check pipette for proper performance Insufficient mixing of reagent dilutions e Thoroughly agitate the lyophilized components after reconstitution e Thoroughly mix dilutions Improperly sealed microplate e Check the microplate pouch for proper sealing e Check that the microplate pouch has no punctures e Check that three desiccants are inside the microplate pouch prior to sealing Unexpectedly Low or High Signal Intensity Microplate was left unattended between steps e Each step of the procedure should be performed uninterrupted Omission of step e Consult the provided procedure for complete list of steps Steps performed in incorrect order e Consult the provided procedure for the correct order Insufficient amount of reagents added to wells e Check pipette calibration e Check pipette for proper performance Wash step was skipped e Consult the provided procedure for all wash steps Improper wash buffer e Check that the correct wash buffer is being used Improper reagent preparation e Consult rea
6. gent preparation section for the correct dilutions of all reagents Insufficient or prolonged incubation periods e Consult the provided procedure for correct incubation time e Sandwich ELISA If samples generate OD values higher than the highest standard point P1 dilute samples further and repeat the assay Non optimal sample e Competitive ELISA If samples generate OD values lower dilution than the highest standard point P1 dilute samples further and repeat the assay e User should determine the optimal dilution factor for samples Contamination of e A new tip must be used for each addition of different reagents samples or reagents during the assay procedure Contents of wells e Verify that the sealing film is firmly in place before placing evaporate the assay in the incubator or at room temperature e Pipette properly in a controlled and careful manner Improper pipetting e Check pipette calibration Deficient Standard Curve Fit e Check pipette for proper performance e Thoroughly agitate the lyophilized components after reconstitution e Thoroughly mix dilutions Insufficient mixing of reagent dilutions References 1 Inui A et al 2004 FASEB Journal Official Publication of the Federation of American Societies for Experimental Biology 18 3 439 56 doi 10 1096 fj 03 0641 rev PMID 15003990 2 Sakata Sakai T 2010 International Journal of Peptides 2010 1 doi 10
7. iately to the foil pouch with desiccants inside Reseal the pouch securely to minimize exposure to water vapor and store in a vacuum desiccator e Add 50 ul of Human Ghrelin Standard or sample per well Cover wells with a sealing tape and incubate for 2 hours Start the timer after the last addition e Wash five times with 200 ul of Wash Buffer manually Invert the plate each time and decant the contents hit 4 5 times on absorbent material to completely remove the liquid If using a machine wash six times with 300 ul of Wash Buffer and then invert the plate decanting the contents hit 4 5 times on absorbent material to completely remove the liquid e Add 50 ul of Biotinylated Human Ghrelin Antibody to each well and incubate for 1 hour e Wash the microplate as described above Add 50 ul of Streptavidin Peroxidase Conjugate per well and incubate for 30 minutes Turn on the microplate reader and set up the program in advance Wash the microplate as described above Add 50 ul of Chromogen Substrate per well and incubate for 30 minutes or till the optimal color density develops Gently tap the plate to ensure thorough mixing and break the bubbles in the well with pipette tip Add 50 ul of Stop Solution to each well The color will change from blue to yellow Read the absorbance on a microplate reader at a wavelength of 450 nm immediately If wavelength correction is available subtract readings at 570 nm from those at 450 nm to correct optica
8. l imperfections Otherwise read the plate at 450 nm only Please note that some unstable black particles may be generated at high concentration points after stopping the reaction for about 10 minutes which will reduce the readings Data Analysis Calculate the mean value of the duplicate or triplicate readings for each standard and sample To generate a standard curve plot the graph using the standard concentrations on the x axis and the corresponding mean 450 nm absorbance on the y axis The best fit line can be determined by regression analysis using four parameter or log log logistic curve fit Determine the unknown sample concentration from the Standard Curve and multiply the value by the dilution factor Typical Data The typical data is provided for reference only Individual laboratory means may vary from the values listed Variations between laboratories may be caused by technique differences Standard Point Average OD P1 16 0 P2 8 00 P3 4 00 P4 2 00 PS 1 00 P6 0 50 P7 0 25 P8 0 00 Sample Normal Sodium Citrate Plasma 1x Standard Curve e The curve is provided for illustration only A standard curve should be generated each time the assay is performed H Ghrelin Standard Curve OD 450 nm 0 1 ul loo sii 0 1 1 0 10 0 100 0 hGhrelin ng ml Reference Value e Normal human ghrelin plasma levels range from 200 to 1300 pg ml e Human plasma and se
9. o limit repeated freezing and thawing Prepare duplicate or triplicate standard points by serially diluting the standard stock solution 16 ng ml twofold with equal volume of EIA Diluent to produce 8 4 2 1 0 5 and 0 25 ng ml solutions EIA Diluent serves as the zero standard 0 ng ml Any remaining solution should be frozen at 20 C and use within 10 days Standard Point Dilution Ghrelin ng ml P1 1 part Standard 16 ng ml 16 0 1 part P1 1 part EIA Diluent 1 part P2 1 part EIA Diluent 400 Pa 1partP3 1partElADiluent Pe ipartPS ipartElADiluent 050 Ll Ps ADe ooo e Biotinylated Human Ghrelin Antibody 50x Spin down the antibody briefly and dilute the desired amount of the antibody 1 50 with ElA Diluent Any remaining solution should be frozen at 20 C e Wash Buffer Concentrate 20x If crystals have formed in the concentrate mix gently until the crystals have completely dissolved Dilute the Wash Buffer Concentrate 1 20 with reagent grade water e SP Conjugate 100x Spin down the SP Conjugate briefly and dilute the desired amount of the conjugate 1 100 with ElA Diluent Any remaining solution should be frozen at 20 C Assay Procedure e Prepare all reagents standard solutions and samples as instructed Bring all reagents to room temperature before use The assay is performed at room temperature 20 25 C e Remove excess microplate strips from the plate frame and return them immed
10. rum samples from healthy adults were tested n 30 On average ghrelin level was 429 pg ml Performance Characteristics e The minimum detectable dose of ghrelin as calculated by 2SD from the mean of a zero standard was established to be 0 1 ng ml e Intra assay precision was determined by testing replicates of three plasma samples in one assay e _ Inter assay precision was determined by testing three plasma samples in twenty assays Intra Assay Precision Inter Assay Precision Sample 1 2 3 1 2 3 n 20 20 20 20 20 20 CV Average CV Recovery Standard Added Value 0 5 5 ng ml Recovery 89 110 Average Recovery 96 Linearity e Plasma and serum samples were serially diluted to test for linearity Average Percentage of Expected Value Sample Dilution Plasma Serum No Dilution 94 91 1 2 98 99 1 4 103 107 Cross Reactivity Species Cross Reactivity Canine None Monkey gt 50 Mouse gt 50 Rat gt 50 Swine gt 90 Rabbit None Bovine None Human Troubleshooting 100 Issue Causes Course of Action Use of expired e Check the expiration date listed before use components e Do not interchange components from different lots e Check that the correct wash buffer is being used e Check that all wells are dry after aspiration Improper wash step e Che
11. ts of the kit at recommended temperatures up to the expiration date e Store SP Conjugate and Biotinylated Antibody at 20 C e Store Microplate Diluent Concentrate 10x Wash Buffer Stop Solution and Chromogen Substrate at 2 8 C e Unused microplate wells may be returned to the foil pouch with the desiccant packs and resealed May be stored for up to 30 days in a vacuum desiccator e Diluent 1x may be stored for up to 30 days at 2 8 C e Store Standard at 2 8 C before reconstituting with Diluent and at 20 C after reconstituting with Diluent Other Supplies Required e Microplate reader capable of measuring absorbance at 450 nm Pipettes 1 20 ul 20 200 ul 200 1000 ul and multiple channel e Deionized or distilled reagent grade water Sample Collection Preparation and Storage e Plasma Collect plasma using one tenth volume of 0 1 M sodium citrate as an anticoagulant Samples collection and processing should be performed as quickly as possible Keep on ice when not in use It is recommended that protease inhibitor cocktail is added to samples for example o phenanthroline 0 44 mM EDTA 25 mM p hydroxy mercuribenzoic acid 1mM and pepstatin A 0 12 mM The user may need to optimize concentration of above reagents Centrifuge samples at 3000 x g for 10 minutes and assay The undiluted samples can be stored at 20 C or below for up to 3 months Avoid repeated freeze thaw cycles EDTA or Heparin can also be used as
12. y e Prepare all samples prior to running the assay The dilution factors for the samples are suggested in this protocol However the user should determine the optimal dilution factor e Spin down the SP conjugate vial and the biotinylated antibody vial before opening and using contents e The Stop Solution is an acidic solution e This kit is for research use only e The kit should not be used beyond the expiration date Reagents e Human Ghrelin Microplate A 96 well polystyrene microplate 12 strips of 8 wells coated with a polyclonal antibody against human ghrelin e Sealing Tapes Each kit contains 3 precut pressure sensitive sealing tapes that can be cut to fit the format of the individual assay e Human Ghrelin Standard Human ghrelin in a buffered protein base 12 8 ng lyophilized e Biotinylated Human Ghrelin Antibody 50x A 50 fold biotinylated polyclonal antibody against ghrelin 120 ul e EIA Diluent Concentrate 10x A 10 fold concentrated buffered protein base 20 ml e Wash Buffer Concentrate 20x A 20 fold concentrated buffered surfactant 30 ml 2 bottles e Streptavidin Peroxidase Conjugate SP Conjugate A 100 fold concentrate 80 ul e Chromogen Substrate A ready to use stabilized peroxidase chromogen substrate tetramethylbenzidine 8 ml e Stop Solution A 0 5 N hydrochloric acid to stop the chromogen substrate reaction 12 ml Storage Condition e Upon arrival immediately store componen
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