AssayMaxTM Human Serum AA ELISA Kit
Contents
1. T c 29 a lt bo Im o 3 e 2 v o x gt E 7 c w 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 reagent preparation section for the correct dilutions of all reagents Insufficient or prolonged incubation periods e Consult the provided procedure for correct incubation time Deficient Standard Curve Non optimal sample dilution e Sandwich ELISA If samples generate OD values higher than the highest standard point P1 dilute samples further and repeat the assay e Competitive ELISA If samples generate OD values lower 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 reagents Contents of wells evaporate e A new tip must be used for each addition of dif2. SAA3 is a pseudogene that does not express protein and SAA4 is expressed constitutively in the liver 4 Principle of the Assay The AssayMax Human Serum Amyloid A ELISA Enzyme Linked Immunosorbent Assay kit is designed for detection of human SAA in plasma serum and cell culture samples This assay employs a quantitative sandwich enzyme immunoassay technique that measures total SAA in less than 4 hours A polyclonal antibody specific for SAA has been pre coated onto a 96 well microplate with removable strips SAA in standards and samples is sandwiched by the immobilized antibody and the biotinylated antibody specific for SAA 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 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 the biotinylated antibody vial and the standard vial before opening and using contents e The Stop Solution is an acidic soluti
3. Value Sample Dilution Plasma Serum 1 2 91 92 1 4 100 98 1 8 107 108 Cross Reactivity Species Cross Reactivity Canine lt 20 Bovine None Monkey None Mouse None Rat lt 10 Swine lt 20 Rabbit None Human 100 Notes The conversion of IU and mg ml is 1 International Unit 11U 1 04 mg Troubleshooting Causes Course of Action Low Precision Use of expired components e Check the expiration date listed before use e Do not interchange components from different lots Improper wash step e Check that the correct wash buffer is being used e Check that all wells are dry after aspiration e Check that the microplate washer is dispensing properly e If washing by pipette check for proper pipetting technique Splashing of reagents while loading wells e Pipette properly in a controlled and careful manner Inconsistent volumes loaded into wells e Pipette properly in a controlled and careful manner e Check pipette calibration 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
4. for up to 30 daysina vacuum desiccator Diluent 1x may be stored for up to 30 days at 2 8 C Other Supplies Required Microplate reader capable of measuring absorbance at 450 nm Pipettes 1 20 ul 20 200 ul 200 1000 ul and multiple channel 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 Centrifuge samples at 3000 x g for 10 minutes Dilute samples 1 4 into MIX Diluent and assay The undiluted samples can be stored at 20 C or below for up to 3 months Avoid repeated freeze thaw cycles EDTA can also be used as an anticoagulant e Serum Samples should be collected into a serum separator tube After clot formation centrifuge samples at 3000 x g for 10 minutes and remove serum Dilute samples 1 4 into MIX Diluent 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 Collect cell culture media and centrifuge at 3000 x g for 10 minutes at 4 C to remove debris 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 MIX Diluent Concentrate 10x If crystals have formed in the concentrate mix gently until the crystals have completely dissolved Dilute the MIX Diluent Concen
5. MA assarpro AssayMax Human Serum AA 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 15 minutes Step 5 Add 50 ul of Stop Solution per well Read at 450 nm immediately Symbol Key 3 Consult instructions for use Assay Template 12 11 10 Human Serum Amyloid A ELISA Kit Catalog No EA8001 1 Sample insert for reference use only Introduction Human serum amyloid A SAA is a major apolipoprotein of high density lipoprotein in plasma It is not only synthesized by the liver and adipose tissue but also produced extrahepatically 1 SAA is a 12 5 kDa protein containing 122 amino acids with polymorphic forms 2 3 Four SAA genes have been identified and three encode functional proteins in humans In response to inflammatory stimuli acute phase SAA1 and SAA2 are secreted and increased
6. agents 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 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 Human Serum AA 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 Serum AA Antibody to each well and incubate for 1 hour e Wash the microplate as described above e 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 e Wash the microplate as described above e Add 50 ul of Chromogen Substrate per well and incubate for 15 minutes or till the optimal blue color density develop Gently tap the plate to ensure thorough mixing and break the bubbles in the well with pipette tip e Add 50 ul of S
7. ated each time the assay is performed Human Serum AA Standard Curve 10r OD 450 nm fi fi J 0 01 0 1 1 10 H SAA g m1 Reference Value e Normal plasma SAA level is less than 6 ug ml e Human plasma and serum samples from healthy adults were tested n 40 On average serum AA level was 4 0 ug ml Sample Average Value ug ml Human Pool Normal Plasma 3 8 Human Normal Plasma 4 3 Human Pool Normal Serum 4 0 Performance Characteristics e The minimum detectable dose of serum AA as calculated by 25D from the mean of a zero standard was established to be 0 06 ug 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 Spiking Recovery e _ Recovery was determined by spiking two plasma samples with different serum AA concentrations Unspiked Sample ug ml Recovery Spike ug ml 0 15 0 70 0 80 114 0 55 0 30 0 85 0 89 105 1 00 1 55 1 49 96 0 15 1 35 1 40 104 0 30 1 50 1 45 97 1 00 2 20 2 16 98 Average Recovery 102 Expected Observed Linearity e Plasma and serum samples were serially diluted to test for linearity Average Percentage of Expected
8. ferent samples or reagents during the assay procedure e Verify that the sealing film is firmly in place before placing the assay in the incubator or at room temperature e Pipette properly in a controlled and careful manner Improper pipetting e Check pipette calibration e Check pipette for proper performance e Thoroughly agitate the lyophilized components after reconstitution Insufficient mixing of reagent dilutions e Thoroughly mix dilutions References 1 Malle E et al 2009 Cell Mol Life Sci 66 1 9 26 2 Kluve Beckerm B et al 1986 Biochem Genet 24 11 12 795 803 3 Sipe JD etal 1985 Biochemistry 24 12 2931 2936 4 Watson G et al 1992 Scand J Immunol 36 5 703 712 Version 2 8R1 www assaypro com e mail Support assaypro com
9. on The kit should not be used beyond the expiration date Reagents Human Serum AA Microplate A 96 well polystyrene microplate 12 strips of 8 wells coated with a polyclonal antibody against human SAA Sealing Tapes Each kit contains 3 precut pressure sensitive sealing tapes that can be cut to fit the format of the individual assay Human Serum AA Standard Human SAA in a buffered protein base 12 ug ml 0 6 ml Calibrated against WHO 1 International Standard Biotinylated Human Serum AA Antibody 50x A 50 fold concentrated biotinylated antibody against SAA 140 pul MIX Diluent Concentrate 10x A 10 fold concentrated buffered protein base 30 ml Wash Buffer Concentrate 20x A 20 fold concentrated buffered surfactant 30 ml 2 bottles Streptavidin Peroxidase Conjugate SP Conjugate A 100 fold concentrate 80 ul Chromogen Substrate A ready to use stabilized peroxidase chromogen substrate tetramethylbenzidine 8 ml Stop Solution A 0 5 N hydrochloric acid to stop the chromogen substrate reaction 12 ml Storage Condition Upon arrival immediately store components of the kit at recommended temperatures up to the expiration date Store Standard SP Conjugate and Biotinylated Antibody at 20 C Store Microplate Diluent Concentrate 10x Wash Buffer Stop Solution and Chromogen Substrate at 2 8 C Unused microplate wells may be returned to the foil pouch with the desiccant packs and resealed May be stored
10. top Solution to each well The color will change from blue to yellow e 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 e Calculate the mean value of the duplicate or triplicate readings for each standard and sample e 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 e Determine the unknown sample concentration from the Standard Curve and multiply the value by the dilution factor Typical Data e 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 2 000 P2 1 000 P3 0 500 P4 0 250 P5 0 125 P6 0 000 Sample Pool Normal Sodium Citrate Plasma 4x Standard Curve e The curve is used for illustration only A standard curve should be gener
11. trate 1 10 with reagent grade water Store for up to 30 days at 2 8 C e Standard Curve Briefly spin down the standard vial before opening and using contents Aliquot standard to limit repeated freezing and thawing Prepare duplicate or triplicate standard points by serially diluting the standard stock solution 12 ug ml 1 6 for the first point 2 ug ml then 1 2 with equal volume of MIX Diluent to produce 1 0 5 0 25 and 0 125 ug ml solutions MIX Diluent serves as the zero standard 0 ug ml Any remaining solution in the aliquot tube should be frozen at 20 C and used within 2 days Avoid repeated freeze thaw cycles Standard Dilution Point P1 1 part Standard 12 ug ml 5 parts MIX Diluent 1 part P1 1 part MIX Diluent 1 000 0 960 Pa IpartP3 1partMiXDiluent 0 250 0240 Pe MxDiluee 0 000 o000 e Biotinylated Human Serum AA Antibody 50x Spin down the antibody briefly and dilute the desired amount of the antibody 1 50 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 re
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