AssayMax Rat Haptoglobin ELISA Kit
Contents
1. 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 Standard Curve The curve is provided for illustration only A standard curve should be generated each time the assay is performed R Haptoglobulin Standard Curve 1 0 OD450 nm bI 10 100 1000 rHaptoglobulin g ml Performance Characteristics e The minimum detectable dose of rat haptoglobin is typically 0 9 ug ml Intra assay and inter assay coefficients of variation were 4 9 and 7 5 respectively Linearity Average Percentage of Expected Value Sample Dilution Plasma Serum 1 125 104 103 1 250 100 97 1 500 96 95 Recovery Standard Added Value 1 5 30 ug ml Recovery 83 119 Average Recovery 99 Cross Reactivity Species Cross Reactivity Beagle None Monkey lt 5 Mouse lt 10 Human None Swine None Rabbit None Reference 1 Van Vlierberghe H et al 2004 Clin Chim Acta 345 1 2 35 42 Version 1 3R Related Products EH2003 1 AssayMax2. e EIA 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 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 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 and Biotinylated Protein at 2 8 C 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 one tenth volume of 0 1 M sodium citrate as an anticoagulant Centrifuge samples at 3000 x
3. DA ssaYPRO AssayMax Rat 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 25 ul of Standard or Sample and 25 ul of Biotinylated Protein per well Incubate 2 hours Step 2 Wash then add 50 ul of SP Conjugate per well Incubate 30 minutes Step 3 Wash then add 50 ul of Chromogen Substrate per well Incubate 7 minutes Step 4 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 Rat Haptoglobin ELISA Kit Catalog No ERH1003 1 Sample insert for reference use only Introduction Haptoglobin Hpt is a plasma protein with hemoglobin binding capacity and plasma glycoproteins that form a stable complex with hemoglobin to aid the recycling of heme iron 1 Principle of the Assay The AssayMax Rat Haptoglobin ELISA Enzyme Linked Immunosorbent Assay kit is designed for detection of rat haptoglobin in plasma and serum samples This assay employs a quantitative competitive enzyme immunoassay technique that measures rat haptoglobin in less than 3 hours A polyclonal antibody sp
4. Human Haptoglobin ELISA Kit Urine Milk Saliva and Cell Culture samples e _ EH1003 1 AssayMax Human Haptoglobin ELISA Kit Plasma and Serum samples e ERH2003 1 AssayMax Rat Haptoglobin ELISA Kit Urine and Cell Culture samples e _ EBH2003 1 AssayMax Bovine Haptoglobin ELISA Kit Urine and Cell Culture samples e _ ECH2003 1 AssayMax Canine Haptoglobin ELISA Kit Plasma and Cell Culture samples EPH2003 1 AssayMax Swine Haptoglobin ELISA Kit Plasma Serum Urine and Cell Culture samples www assaypro com e E mail Support assaypro com
5. ecific for rat haptoglobin has been pre coated onto a 96 well microplate with removable strips Haptoglobin in standards and samples is competed with a biotinylated haptoglobin sandwiched by the immobilized antibody and 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 protein 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 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 Haptoglobin Microplate A 96 well polystyrene microplate 12 strips of 8 wells coated with a polyclonal antibody against rat haptoglobin 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 Haptoglobin Standard Rat haptoglobin in a buffered protein base 90 ug lyophilized e Biotinylated Rat Haptoglobin 1 vial lyophilized
6. g for 10 minutes Dilute samples 1 250 into EIA 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 or Heparin 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 1 250 into EIA Diluent and assay The undiluted samples can be stored at 20 C or below for up to 3 months Avoid repeated freeze thaw cycles Reagent Preparation e Freshly dilute all reagents and bring all reagents to room temperature before use e EIA Diluent Concentrate 10x If crystals have formed in the concentrate mix gently until the crystals have completely dissolved Dilute the EIA Diluent Concentrate 1 10 with reagent grade water Store for up to 30 days at 2 8 C e Standard Curve Reconstitute the 90 ug of Rat Haptoglobin Standard with 1 5 ml of EIA Diluent to generate a 60 ug ml standard 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 solution 60 ug ml 1 2 with EIA Diluent to produce 30 15 7 5 3 75 1 875 and 0 937 ug ml solutions EIA Diluent serves as the zero standard 0 ug ml Any remaining solution should be frozen at 20 C and used within 30 days Standard Rat Haptoglobin Point ug m
7. l Standard 60 ug ml 60 00 1 part P1 1 part EIA Diluent 30 00 1 part P2 1 part EIA Diluent 15 00 Pa 1partP3 1partElADiluenn 750 Pe ipartPS ipartElADiluent 1875 PB iluent_ 0000 e Biotinylated Rat Haptoglobin 4x Reconstitute Biotinylated Rat Haptoglobin with 4 ml ElA Diluent to produce a 4 fold stock solution Allow to sit for 10 minutes with gentle agitation prior to making dilutions The stock solution should be further diluted 1 4 with ElA Diluent Any remaining solution should be frozen at 20 C and used within 30 days 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 SP Conjugate 100x Spin down the SP Conjugate briefly and dilute the desired amount of the conjugate 1 100 with EIA Diluent Any remaining solution should be frozen at 20 C Assay Procedure 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 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 Add 25 ul of Rat Haptoglobin Standard or sample per well and immediately add 25 ul of Biotinylated Rat Ha
8. ptoglobin to each well on top of the standard or sample and gently tap plate to mix 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 Streptavidin Peroxidase Conjugate to each 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 7 minutes or till the optimal blue color density develops Gently tap 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 low concentration points after stopping the reaction for about 10 minutes which will reduce the readings Data Analysis
Download Pdf Manuals
Related Search