Manual - RayBiotech, Inc.
Contents
1. enough to 2 3 days at 4 C Item C run each standard in duplicate Additional dilutions Do not store Anti Gastrin 1 oe oe Item N 2 vials ofantiGastint of anti vials ofantiGastint 1 1 month ata at 4 month ata 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 Gastrin 1 2 vials of a a a Gastrin 1 Peptide 1 vial is 2 3 esdaysatae esdaysatae oe Item F a a a to assay the whole plate y HRP o a 600 eee 200X concentrated HRP conjugated oe not store and Concentrate es G eee 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 desiccant 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 preparati2. IA kit shows no cross reactivity with any of the cytokines tested Ghrelin Nesfatin Angiotensin Il NPY and APC XIV Publications Citing This Product 1 Singh PK Hota D Dutta P et al Pantoprazole improves glycemic control in type 2 diabetes a randomized double blind placebo controlled trial J Clin Endocrinol Metab 2012 Nov 97 11 E2105 8 doi 10 1210 jc 2012 1720 Species Human Sample Type Plasma 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 Ra
3. 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 11 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 Gastrin 1 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 eac
4. RayBio Human Mouse Rat Gastrin 1 Enzyme Immunoassay Kit Catalog EIA GAS EIAM GAS EIAR GAS 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 C Cd m fome SSCS e ee nopot ooo ao Preparation A Preparation of Plate and Anti Gastrin 1 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 alee oo lx Assay Procedure Summary O Assay Procedure Summary pe Calculation of Results A Typical Data B Sensitivity C Detection Range D Reproducibility E Assay Diagram x Specifcty OOOO O O Specificity TE xu Select Publications OO O O Select Publications a Troubleshooting Guide Please read the entire manual carefully before starting your experiment I Introduction Gastrin is a hormone that stimulates secretion of gastric acid by the parietal cells of the stomach and aids in gastric motility It is released by G cells in the stomach duodenum and the pancreas Gastrin is a linear peptide that is synthesized as a prep
5. Working Stock Item F 2 ml of Final concentration Assay Diluent 10 pg ml Perform a 2 fold dilution of Working Stock Item F 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 Gastrin 1 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 Gastrin 1 is 10 pg ml in all standards 8 Briefly centrifuge the vial of Gastrin 1 Standard Item C Pipette 8 ul of Item C and 792 ul of 10 pg ml biotinylated Gastrin 1 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 Gastrin 1 standard 10 pg ml biotinylated Gastrin i 9 To make the 100 pg ml standard pipette 50 ul of the 1000 pg ml Gastrin 1 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 Gastrin 1 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 Foes Pa BS oe ee 1000 100 10 1 0 1 0 pg ml pg ml pg ml pg ml pg ml pg ml D Positive Control Preparation 11 Br
6. commendended 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 200 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 Gastrin 1 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 it against clean paper towels Add 100 ul of each standard see Reagent Preparation Section C
7. directly proportional to the amount of captured biotinylated Gastrin 1 peptide and inversely proportional to the amount of endogenous Gastrin 1 in the standard or samples A standard curve of known concentration of Gastrin 1 peptide can be established and the concentration of Gastrin 1 peptide in the samples can be calculated accordingly ll How It Works Y Y Anti IgG antibody D a Y pre coated on the plate Target molecule Biotinylated 3 4 in sample Peptide x A vk gt Capture antibody yi ae is added to the wells Biotin peptide Standard w 3 Sample interact competitivly tea yi j for spots on the capture antibodies BNP EIA Kit a Add HRP Streptavidin ai and Color Substrate Competition amp Data Analysis DP Peptide Concentration pg ml 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 Stay Stability Gastrin 1 Bastin Microplate tom A Item A astn 1 Micropate tem a WEIS UA e Sele code win 1 month at month at aor eee antibody Wash Buffer ee ft monthatas ee Item B 25 ml 25 mi of 20x concentrated soluton 20X concentrated solution 1 month at 4 ft monthatas The first standard Standard Gastrin 1 Peptide 2 vials of Gastrin 1 Peptide 1 vial is
8. h 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 Gastrin EIA 120 400 4 a oeo 80 4 gt 60 4 40 4 B B0 20 4 20 4 40 7 T T 1 T 0 01 0 1 1 10 100 1000 10000 Peptide Concentration pg ml B Sensitivity The minimum detectable concentrations of Gastrin 1 is 9 92 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 Standard Standard Standards Pos Contra SAB SAB Key Blank Buffer Only Total Binding Biotin Gastrin 1 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 E
9. iefly 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 Gastrin 1 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 Gastrin 1 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 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 Gastrin 1 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 re
10. ing pancreatic secretions and gallbladder emptying The role of gastrin in certain diseases has also been reported including Zollinger Ellison Syndrome autoimmune gastritis and mucolipidosis In Zollinger Ellison syndrome gastrin is produced at excessive levels often by a gastrinoma of the duodenum or the pancreas In autoimmune gastritis the immune system attacks the parietal cells leading to hypochlorhydria This results in an elevated gastrin level in an attempt to compensate for increased pH in the stomach Eventually all the parietal cells are lost and achlorhydria results leading to a loss of negative feedback on gastrin secretion Plasma gastrin concentration is elevated in virtually all individuals with mucolipidosis type IV secondary to a constitutive achlorhydria ll General Description The RayBio Gastrin 1 Enzyme Immunoassay EIA Kit is an in vitro quantitative assay for detecting Gastrin 1 peptide based on the competitive enzyme immunoassay principle In this assay a biotinylated Gastrin 1 peptide is spiked into the samples and standards The samples and standards are then added to the plate where the biotinylated Gastrin 1 peptide competes with endogenous unlabeled Gastrin 1 for binding to the anti Gastrin 1 antibody After a wash step any bound biotinylated Gastrin 1 then interacts with horseradish peroxidase HRP streptavidin which catalyzes a color development reaction The intensity of the colorimetric signal is
11. on 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 Gastrin 1 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 Gastrin 1 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 Diluent B This is your anti Gastrin 1 antibody working solution which will be used in step 2 of Assay Procedure Section VIII 7 Note The following steps may be done during the antibody incubation procedure step 2 of Assay Procedure B Preparation of Biotinylated Gas
12. rohormone and post translationally cleaved to form a family of peptides with identical carboxytermini The predominant circulating form is gastrin 34 a k a big gastrin but full biologic activity is present in the smaller peptides i e gastrin 17 a k a little gastrin and gastrin 14 a k a minigastrin In addition pentagastrin is an artificial 5 amino acid peptide identical to the carboxyterminus of gastrin It is worth noting that the five C terminal amino acids of gastrin and cholecystokinin are identical which explains their overlapping biological effects Preprogastrin es a Progastrin Co aa aa Gastrin 34 Gastrin 17 Gastrin 14 C Pentagastrin Gastrin is released in response to certain stimuli including stomach distension vagal stimulation hypercalcemia and the presence of partially digested proteins On the other hand gastrin release can be inhibited by the presence of gastric acid or inhibitory hormones such as secretin GIP VIP glucagon and calcitonin As a peptide hormone in GI system gastrin stimulates gastric acid secretion from parietal cells of the stomach Additional functions by gastrin include the following Stimulating parietal cell maturation and fundal growth Causing chief cells to secrete pepsinogen Increasing antral muscle mobility and promoting stomach contractions Strengthening antral contractions against the pylorus and constricting the pyloric sphincter to slow gastric emptying induc
13. trin 1 Item F 5 Briefly centrifuge the vial of Biotinylated Gastrin 1 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 Gastrin 1 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 the appropriate Assay Diluent The final concentration of biotinylated Gastrin 1 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 Gastrin 1 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 Gastrin 1 will be 10 pg ml 1 vial is enough to run the whole plate Item F First Dilution Working Stock Item F Add entire vial of Item F to 10 ml Assay Diluent Second Dilution A b Positive Control 100 ul of Working Stock Item F 100 ul Prepared Positive Control c Sample 125 ul of Working Stock Item F 125 ul Prepared Sample a Standards 2 ml of
14. yBio 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
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