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AssayMax Swine Haptoglobin ELISA Kit

1. ml Any remaining solution should be frozen at 20 C and used within 30 days Standard Dilution Swine Haptoglobin Point ng ml P1 1 part Standard 120 ng ml 120 0 1 part P1 1 part MIX Diluent 60 00 1 part P2 1 part MIX Diluent 30 00 Pa 1partP3 1 part MIX Diluent 15 00 Ps 1partP5 1 part MIX Diluent 3750 lr O MixDiluent 0000 e Biotinylated Swine Haptoglobin 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 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 Swine Haptoglobin Standard or sample per well Cover wells and incubate for 2 h
2. 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 OD 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 Standard Curve The curve is provided for illustration only A standard curve should be generated each time the assay is performed Swine Haptoglobin Standard Curve T OD 450 nm 0 1 1 1 1 1 0 10 0 100 0 Haptoglobin ng ml Performance Characteristics e The minimum detectable dose of swine haptoglobin is typically 1 5 ng ml Intra assay and inter assay coefficients of variation were 5 0 and 7 3 respectively Linearity Average Percentage of Expected Value Sample Dilution Plasma Serum 1 20000 88 91 1 40000 98 100 1 80000 104 105 Average Percentage of Expected Value Sample Dilution Urine 1 25 89 1 50 97 1 100 106 Recovery Standard Added Value 4 60 ng ml Recovery 85 111 Average Recovery 97 Cross Reactivity Species Cross Reactivity Beagle None Bovine None Monkey None Mouse None Rat None Human 1 Rabbit No
3. Wyssaypro AssayMax Swine Haptoglobin 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 20 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 Swine Haptoglobin ELISA Kit Catalog No EPH2003 1 Sample insert for reference use only Introduction Haptoglobin Hpt is a plasma protein with hemoglobin binding capacity and a plasma glycoprotein that forms a stable complex with hemoglobin to aid the recycling of heme iron 1 Principle of the Assay The AssayMax Swine Haptoglobin ELISA Enzyme Linked Immunosorbent Assay kit is designed for detection of swine haptoglobin in plasma serum urine and cell culture samples This assay employs a quantitative sandwich enzyme immunoassay techniqu
4. d 1 100 1 10000 4 ul sample 396 ul buffer 100x 4 ul sample 396 ul buffer 100x 100 fold dilution 4 ul of A 396 ul buffer 100x 10000 fold dilution Assuming the needed volume is less than Assuming the needed volume is less than or equal to 400 ul or equal to 400 ul 1 1000 1 100000 4 ul sample 396 ul buffer 100x 4 ul sample 396 ul buffer 100x 24 ul of A 216 ul buffer 10x 4 ul of A 396 ul buffer 100x 1000 fold dilution 24 ul of B 216 ul buffer 10x 100000 fold dilution Assuming the needed volume is less than Assuming the needed volume is less than or equal to 240 ul or equal to 240 ul 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 240 ng of Swine Haptoglobin Standard with 2 ml of MIX Diluent to generate a 120 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 120 ng ml 1 2 with MIX Diluent to produce 60 30 15 7 5 3 75 and 1 875 ng ml solutions MIX Diluent serves as the zero standard 0 ng
5. e that measures swine haptoglobin in less than 4 hours A polyclonal antibody specific for swine haptoglobin has been pre coated onto a 96 well microplate with removable strips Swine haptoglobin in standards and samples is sandwiched by the immobilized antibody and a biotinylated polyclonal antibody specific for swine haptoglobin 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 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 Swine Haptoglobin Microplate A 96 well polystyrene microplate 12 strips of 8 wells coated with a polyclonal antibody against swine haptoglobin Sealing Tapes Each kit contains 3 precut pressure sensitive sealing tapes that can be cut to fit the forma
6. ne Swine 100 10 FBS in culture media will not affect the assay Reference 1 Van Vlierberghe H et al 2004 Clin Chim Acta 345 1 2 35 42 Version 1 9R Related Products EH1003 1 AssayMax Human Haptoglobin ELISA Kit Plasma and Serum samples EH2003 1 AssayMax Human Haptoglobin ELISA Kit Urine Saliva Milk Cell Culture samples ERH1003 1 AssayMax Rat Haptoglobin ELISA Kit Plasma and Serum samples ERH2003 1 AssayMax Rat Haptoglobin ELISA Kit Urine and Cell Culture samples EBH2003 1 AssayMax Bovine Haptoglobin ELISA Kit Urine and Cell Culture samples ECH2003 1 AssayMax Canine Haptoglobin ELISA Kit Plasma and Cell Culture samples www assaypro com e e mail Support assaypro com
7. ours 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 Swine Haptoglobin Antibody to each well and incubate for 1 hour 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 20 minutes or till the optimal blue 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 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
8. t of the individual assay Swine Haptoglobin Standard Swine haptoglobin in a buffered protein base 240 ng lyophilized Biotinylated Swine Haptoglobin Antibody 50x A 50 fold concentrated biotinylated polyclonal antibody against swine haptoglobin 140 ul 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 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 for up to 30 daysina vacuum desiccator Diluent 1x may be stored for up to 30 days at 2 8 C Store Standard at 2 8 C before reconstituting with Diluent and at 20 C after reconstituting with Diluent Other Supplies Required Microplate reader capable of measuring absorbance at 450 nm Pipettes 1 20 ul 20 200 ul 200 1000
9. ul and multiple channel Deionized or distilled reagent grade water Sample Collection Preparation and Storage e Cell Culture Supernatants Centrifuge cell culture media at 3000 x g for 10 minutes to remove debris Collect supernatants and assay Store the remaining samples at 20 C or below Avoid repeated freeze thaw cycles e Urine Collect urine using sample pot Centrifuge samples at 800 x g for 10 minutes Urine dilution is suggested at 1 50 in MIX Diluent and assay Depending on application needs user should determine proper dilutions The undiluted samples can be stored at 20 C or below for up to 3 months Avoid repeated freeze thaw cycles 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 40000 with 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 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 40000 into MIX Diluent and assay The undiluted serum can be stored at 20 C or below for up to 3 months Avoid repeated freeze thaw cycles Refer to Sample Dilution Guidelines below for further instruction Guidelines for Dilutions of 1 100 or Greater for reference only please follow the insert for specific dilution suggeste

AssayMax Swine Haptoglobin ELISA Kit

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