Mouse Serum Amyloid P Component ELISA Kit
Contents
1. 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 Mouse Serum Amyloid P 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 Mouse Serum Amyloid P 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 10 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 Stop 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 nm2. tapes that can be cut to fit the format of the individual assay e Mouse Serum Amyloid P Standard Mouse SAP in a buffered protein base 32 ng lyophilized e Biotinylated Mouse Serum Amyloid P Antibody 50x A 50 fold concentrated biotinylated polyclonal antibody against mouse SAP 140 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 daysina 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 measurin
3. MA assarbro AssayMax Mouse Serum Amyloid P Component 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 10 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 Mouse Serum Amyloid P Component ELISA Kit Catalog No EMA8201 1 Sample insert for reference use only Introduction Serum amyloid P component SAP Serum AP APCS a 25kDa pentameric protein is a normal plasma protein and a universal non fibrillar constituent of amyloid deposits 1 SAP is a pentraxin similar to C reactive protein 2 3 Principle of the Assay The AssayMax Mouse Serum Amyloid P Component ELISA Enzyme Linked Immunosorbent Assay kit is designed for detection of mouse SAP in plasma serum and cell culture samp
4. e a 32 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 32 ng ml 1 2 with MIX Diluent to produce 16 8 4 2 1 and 0 5 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 Mouse SAP ng ml P1 1 part Standard 32 ng ml 32 0 1 part P1 1 part MIX Diluent 1 part P2 1 part MIX Diluent P4 1partP3 1 part MIX Diluent 400 Ps 1partP5 1 part MIX Diluent 100 ls MiXDiuent ooo e Biotinylated Mouse Serum Amyloid P 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 reagents standard solutions and samples as instructed Bring all reagents to room temperature before use The assay is performed at room temperature 20 25
5. ery Standard Added Value 1 0 20 ng ml Recovery 84 110 Average Recovery 97 Cross Reactivity Species Cross Reactivity Canine None Bovine None Monkey None Mouse 100 Rat None Swine None Rabbit None Human None e 10 FBS in culture media will not affect the assay References 1 Cathcart ES Shirahama T Cohen AS 1967 Biochim Biophy Acta 147 392 393 2 Jenny NS et al 2007 Arteriosclerosis Thrombosis and Vascular Biology 2007 27 352 3 Koenig W 2007 Arterioscler Thromb Vasc Biol 2007 27 698 700 Version 1 1R Related Products EA8201 1 AssayMax Human Serum Amyloid P Component ELISA Kit Plasma Serum Urine CSF and Cell Culture samples e ERA8201 1 AssayMax Rat Serum Amyloid P Component ELISA Kit Plasma Serum Urine and Cell Culture samples www assaypro com e mail Support assaypro com
6. g 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 Centrifuge samples at 3000 x g for 10 minutes Dilute samples 1 6000 into MIX Diluent and assay The undiluted samples can be stored at 20 C or below for upto 3 months Avoid repeated freeze thaw cycles 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 6000 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 and assay The 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 Concentrate 1 10 with reagent grade water Store for up to 30 days at 2 8 C e Standard Curve Reconstitute the 32 ng of Mouse Serum Amyloid P Standard with 1 ml of MIX Diluent to generat
7. 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 rea readings Data Analysis ction for about 10 minutes which will reduce the 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 OD ont he y axis The best fit line can be determined by regression analysis using log log or four parameter logistic curve fit Determine the unkno wn sample concentration from the Standard Curve and multiply the value by the dilution factor Standard Curve e The curve is used for generated each time OD 450 nm 0 1 illustration only A standard curve should be the assay is performed Mouse APCS Serum AP Standard Curve i 10 100 m APCS ng ml Performance Characteristics e The minimum detectable dose of mouse SAP is typically 0 5 ng ml Intra assay and inter respectively assay coefficients of variation were 4 9 and 7 0 Linearity Average Percentage of Expected Value Sample Dilution Plasma Serum 1 2000 88 87 1 6000 98 99 1 8000 107 104 Recov
8. les This assay employs a quantitative sandwich enzyme immunoassay technique that measures mouse SAP in less than 4 hours A polyclonal antibody specific for mouse SAP has been pre coated onto a 96 well microplate with removable strips SAP in standards and samples is sandwiched by the immobilized antibody and the biotinylated polyclonal antibody specific for mouse SAP 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 ofthe 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 Mouse Serum Amyloid P Microplate A 96 well polystyrene microplate 12 strips of 8 wells coated with a polyclonal antibody against mouse SAP e Sealing Tapes Each kit contains 3 precut pressure sensitive sealing
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