AssayMaxTM Rat ANP ELISA Kit
Contents
1. for proper performance e Thoroughly agitate the lyophilized components after reconstitution e Thoroughly mix dilutions e Check the microplate pouch for proper sealing Improperly sealed e Check that the microplate pouch has no punctures microplate e Check that three desiccants are inside the microplate pouch prior to sealing Inconsistent volumes loaded into wells Low Precision Insufficient mixing of reagent dilutions Microplate was left e Each step of the procedure should be performed T unattended between uninterrupted E steps a Omission of step e Consult the provided procedure for complete list of steps Steps performed in e Consult the provided procedure for the correct order zZ incorrect order 5 z Insufficient amount of e Check pipette calibration 2 3 reagents added to e Check pipette for proper performance 3 g wells zs Wash step was skipped e Consult the provided procedure for all wash steps 3 Improper wash buffer e Check that the correct wash buffer is being used E Improper reagent e Consult reagent preparation section for the correct Q preparation dilutions of all reagents a Insufficient or e Consult the provided procedure for correct incubation gt prolonged incubation time periods 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 ELISA2. 0 07 OD 450 nm 0 1 LO 10 0 rat ANP ng ml Performance Characteristics e The minimum detectable dose of rat ANP as calculated by 25D from the mean of a zero standard was established to be 0 09 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 3 1 3 ng ml Recovery 86 108 Average Recovery 101 Cross Reactivity Species Cross Reactivity Canine lt 40 Bovine None Monkey lt 40 Mouse lt 40 Rat 100 Swine 100 Human lt 40 Troubleshooting 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 Check that the microplate washer is dispensing properly e If washing by pipette check for proper pipetting technique Splashing of reagents e Pipette properly in a controlled and careful manner while loading wells e Pipette properly in a controlled and careful manner e Check pipette calibration e Check pipette
3. ANP Microplate A 96 well polystyrene microplate 12 strips of 8 wells coated with a polyclonal antibody against rat ANP 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 Rat ANP Standard Rat ANP in a buffered protein base 20 ng lyophilized e Biotinylated Rat ANP Antibody 70x A 70 fold biotinylated polyclonal antibody against rat ANP 105 ul e _ MIX Diluent Concentrate 10x A 10 fold concentrated buffered protein base 30 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 components 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
4. 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 Atlas SA et al 1992 p 1577 673 2 Leitman DC et al 1988 J Biol Chem 263 3720 8 Version 2 3R www assaypro com e e mail Support assaypro com
5. ater Store for up to 30 days at 2 8 C e Rat ANP Standard Reconstitute the 20 ng of Rat ANP Standard with 5 ml of MIX Diluent to generate a 4 ng ml standard stock solution Allow the standard to sit for 10 minutes with gentle agitation prior to making dilutions Prepare duplicate or triplicate standard points by serially diluting the standard stock solution 4 ng ml 1 2 with MIX Diluent to generate 2 1 0 5 and 0 25 ng ml solutions MIX Diluent serves as the zero standard 0 ng ml Any remaining solution should be frozen at 20 C and used within 30 days Standard Point Dilution Rat ANP ng ml P1 1 part Standard 4 ng ml 4 000 1 part P1 1 part MIX Diluent 2 000 1 part P2 1 part MIX Diluent 1 000 P4 1patP3 1part MIX Diluent 0 500 1 part P4 1 part MIX Diluent 0 250 re MIX Diluent 0 000 e Biotinylated Rat ANP Antibody 70x Spin down the biotinylated antibody briefly and dilute the desired amount of the antibody 1 70 with MIX 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 MIX Diluent Any remaining solution should be frozen at 20 C Assay Procedure e Prepare all reagents standard so
6. 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 a final concentration of 0 1 M sodium citrate as an anticoagulant Centrifuge samples at 3000 x g for 10 minutes and assay undiluted plasma for medium and high level of ANP Samples can be stored at 20 C or below for up to 3 months Avoid repeated freeze thaw cycles For low level of ANP please use ANP extraction protocol below Low Level ANP Extraction Protocol Buffer A 1 trifluoroacetic acid TFA HPLC Grade in H O Buffer B 60 acetonitrile HPLC Grade in 1 TFA 1 a WW Acidify the sample with equal amount of Buffer A 1 ml sample 1 ml Buffer A Mix and centrifuge samples at 6000 x g for 20 minutes at 4 C Pack an extraction column using 200 mg of C18 resin Pre equilibrate the column with 1 ml of Buffer B once and then with 3 ml of Buffer Athree times Load the acidified plasma solution onto the pre treated C18 column Slowly wash the column with 3 ml of Buffer A twice Elute the peptide slowly with 3 ml of Buffer B once and collect the eluant Evaporate and dry the eluant in a freeze dryer or use a suitable substitute method Keep the dried extract at 20 C a
7. e 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 optical 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 log log or four parameter 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 4 000 P2 2 000 P3 1 000 P4 0 500 PS 0 250 P6 0 000 Sample Normal Rat 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 Rat ANP Standard Curve 1
8. lular cGMP levels 2 Principle of the Assay The AssayMax Rat ANP ELISA Enzyme Linked Immunosorbent Assay kit is designed for detection of rat ANP in plasma serum tissue extract and cell culture samples This assay employs a quantitative sandwich enzyme immunoassay technique that measures rat ANP in less than 5 hours A polyclonal antibody specific for rat ANP has been pre coated onto a 96 well microplate with removable strips The rat ANP in standards and samples is sandwiched by the immobilized antibody and biotinylated polyclonal antibody specific for ANP which is recognized by a streptavidin peroxidase conjugate All unbound material is then 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 e This product is for Research Use Only and is Not For Use In Diagnostic Procedures Prepare all reagents working diluent buffer wash buffer standard biotinylated antibody and SP conjugate as instructed prior to running the assay e Prepare all samples prior to running the assay The dilution factors for the samples are suggested in this insert 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 The kit should not be used beyond the expiration date Reagents e Rat
9. lutions 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 immediately 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 Rat ANP Standard or sample per well Cover wells with a sealing tape and incubate for 2 hours Start the timer after the last addition 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 Add 50 ul of Biotinylated Rat ANP Antibody to each well and incubate for 2 hours 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 10 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 blu
10. nd perform the assay as early as possible Reconstitute the dried extract with 200 ul of MIX Diluent before the assay Check sample pH with pH papers If sample pH is below 6 5 neutralize the sample with 20 ul of 1M NaH PO If the peptide value exceeds or does not fall in the range of detection dilute or concentrate the sample accordingly Serum Samples should be collected into a serum separator tube After clot formation centrifuge samples at 3000 x g for 10 minutes Remove serum and perform the assay for medium and high level of ANP Samples can be stored at 20 C or below for upto 3 months Avoid repeated freeze thaw cycles For low level of ANP please use the extraction protocol as above Tissue Extract tissue samples with 0 1 M phosphate buffered saline pH7 4 containing 1 Triton X 100 and centrifuge at 14000 x g for 20 minutes Collect the supernatant and measure the protein concentration Freeze remaining extract at 20 C or below Cell Culture Supernatants Centrifuge cell culture media at 3000 x g for 10 minutes to remove debris Collect supernatants and assay Store samples at 20 C or below Avoid repeated freeze thaw cycles Reagent Preparation Freshly dilute all reagents and bring all reagents to room temperature before use e _ MIX Diluent Concentrate 10x If crystals have formed in the concentrate mix gently until the crystals have completely dissolved Dilute the MIX Diluent Concentrate 1 10 with reagent grade w
11. yssarpro AssayMax Rat ANP 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 2 hours 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 10 minutes Step 5 Add 50 ul of Stop Solution per well Read at 450 nm immediately Symbol Key BE Consult instructions for use Assay Template 12 11 10 Rat ANP ELISA Kit Catalog No ERA7010 1 Sample insert for reference use only Introduction Atrial natriuretic peptide ANP a 28 amino acid polypeptide is mainly secreted from the atrium of the heart where it is stored in secretory granules as a 136 amino acid pro hormone 1 Upon its secretion which is induced by increases in atrial pressure and stretch the pro hormone is processed by a serine protease to the active 28 amino acids peptide The peptide binds with high affinity to the membrane receptor guanylate cyclase GC A leading to increased intracel
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