Human ApoH ELISA Kit
Contents
1. ApoH Microplate A 96 well polystyrene microplate 12 strips of 8 wells coated with a polyclonal antibody against human ApoH 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 Human ApoH Standard Human ApoH in a buffered protein base 320 ng lyophilized e Biotinylated Human ApoH Antibody 100x A 100 fold concentrated biotinylated polyclonal antibody against ApoH 80 pul 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 desiccants 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 Stor2. 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 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 Human APO H Standard Curve OD 450 nm 0 1 1 0 100 100 0 APO H ng ml Performance Characteristics e The minimum detectable dose of ApoH is typically 0 6 ng ml e Intra assay and inter assay coefficients of variation were 4 8 and 7 2 respectively Linearity Average Percentage of Expected Value Sample Dilution Plasma Serum 1 10000 91 94 1 20000 99 100 1 40000 104 105 Recovery Standard Added Value 1 10 ng ml Recovery 88 111 98 Average Rec
3. 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 80 ng ml 1 2 with MIX Diluent to produce 40 20 10 5 2 5 1 25 and 0 625 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 ApoH Point ng ml 1 part Standard 80 ng ml 1 part MIX Diluent 40 00 1 part P1 1 part MIX Diluent 20 00 1 part P2 1 part MIX Diluent 10 00 Pa tart P3 1partMixDiluent_ 5000 Ps 1ipartPS ipartMIXDiluent 1250 Pr mMiDilen ooo e Biotinylated Human ApoH Antibody 100x Spin down the antibody briefly and dilute the desired amount of the antibody 1 100 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 working standards 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 micr
4. DA assarbro AssayMax Human ApoH 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 BE Consult instructions for use Assay Template 12 11 10 Human Apolipoprotein H ELISA Kit Catalog No EA8821 1 Sample insert for reference use only Introduction Apolipoprotein H ApoH previously known as B gt glycoprotein I is a 50 kDa plasma glycoprotein with 326 amino acids and circulates in plasma at approximately 200 ug ml 1 4 ApoH inhibits the generation of factor Xa Xia and Xlla preventing activation of the intrinsic blood coagulation cascade 5 6 Binding of ApoH to anionic phospholipids such as phosphatidylserine and cardiolipin plays a key role in the formation of antiphospholipid antibodi
5. M NaCl 1 Triton X 100 protease inhibitor cocktail For every 1 x 10 cells add approximately 100 uL of ice cold Lysis Buffer Incubate on ice for 60 minutes Centrifuge at 13000 rpm for 30 minutes at 4 C and collect supernatant for assay e Urine Collect urine using sample pot Centrifuge sample at 800 x g for 10 minutes Dilute sample 1 16 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 Saliva Collect saliva using sample tube Centrifuge sample at 800 x g for 10 minutes Dilute sample 1 16 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 Milk Collect milk using sample tube Centrifuge sample at 800 x g for 10 minutes Dilute sample 1 400 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 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 320 ng of Human ApoH Standard with 4 ml of MIX Diluent to generate an 80 ng ml standard stock solution Allow
6. e 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 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 g for 10 minutes Dilute samples 1 20000 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 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 Dilute samples 1 20000 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 Media 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 e Cell Lysate Rinse cell with cold PBS and then scrape the cell into a tube with 5 ml cold PBS with 0 5 M EDTA Centrifuge suspension at 1500 rpm for 10 minutes at 4 C and aspirate supernatant Re suspend pellet in ice cold Lysis Buffer 10 mM Tris pH8 0 130 m
7. es 7 8 Principle of the Assay The AssayMax Human Apolipoprotein H ELISA Enzyme Linked Immunosorbent Assay kit is designed for detection of human ApoH in plasma serum urine saliva milk and cell culture samples This assay employs a quantitative sandwich enzyme immunoassay technique that measures ApoH in less than 4 hours A polyclonal antibody specific for human ApoH has been pre coated onto a 96 well microplate with removable strips ApoH in standards and samples is sandwiched by the immobilized antibody and biotinylated polyclonal antibody specific for ApoH 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 e Human
8. oplate 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 ApoH Standard or sample per well Cover wells with a sealing tape and incubate for 2 hours Start the timer after the last sample 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 Add 50 ul of Biotinylated Human ApoH Antibody to each well and incubate for 1 hour Wash the microplate as described above 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 10 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
9. overy Cross Reactivity Species Cross Reactivity Beagle None Bovine None Monkey lt 50 Mouse None Rat None Rabbit None Swine lt 10 No significant cross reactivity observed with ApoA l ApoA Il ApoB ApoC ApoC Il and ApoC Ill Reference Value Normal human ApoH plasma levels range from 200 to 400 ug ml References 1 2 3 4 5 6 7 8 Lozier J et al 1984 Proc Natl Acad Sci U S A 81 3640 3644 Steinkasserer A et al 1991 Biochem J 277 387 391 Kristensen T et al 1991 FEBS Lett 289 183 186 Polz E and Lostner GM 1979 FEBS Lett 102 183 186 Shi W et al 1993 Thromb Haemost 70 342 345 Schousboe and Rasmussen MS 1995 Thromb Haemost 73 798 804 McNeil HP et al 1990 Proc Natl Acad Sci U S A 87 4120 4124 Timothy A et al 1999 Biochem J 340 59 67 Version 2 7R1 www assaypro com e mail Support assaypro com
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