Human ApoA-II ELISA Kit
Contents
1. Mouse None Rat None Swine None Rabbit None No significant cross reactivity observed with ApoA l ApoB ApoC l ApoC 11 ApoC lll ApoE ApoH and ApoM Troubleshooting Issue Causes Course of Action Use of expired e Check the expiration date listed before use components e Do not interchange components from different lots e Check that the correct wash buffer is being used e Check that all wells are dry after aspiration Improper wash step e Check that the microplate washer is dispensing properly e If washing by pipette check for proper pipetting c technique 2 Splashing of reagents e Pipette properly in a controlled and careful manner 5 while loading wells 2 A e Pipette properly in a controlled and careful manner a Inconsistent volumes E AAT x e Check pipette calibration 3 loaded into wells t o 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 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 comple2. calibration e Check pipette for proper performance e Thoroughly agitate the lyophilized components after Insufficient mixing of YA reconstitution reagent dilutions e Thoroughly mix dilutions References 1 Tsao YK etal 1985 J Biol Chem 260 15222 15231 2 Gordon JI et al 1983 J Biol Chem 258 14054 14059 3 Brewer HB et al 1972 Proc Natl Acad Sci USA 69 1304 1308 4 Weisgraber KH and Mahley RW 1978 J Biol Chem 253 6281 6288 5 Blanco Vaca F et al 1992 J Lipid Res 33 1785 1796 6 Gillard BK et al 2005 Biochemistry 44 471 479 7 Durbin DM and Jonas A 1999 J Lipid Res 40 2293 2302 8 Forte TM et al 1995 J Lipid Res 36 148 157 9 Fidge NH 1999 J Lipid Res 40 187 201 10 Warden CH et al 1993 Science 261 469 471 11 Schultz JR et al 1993 Nature 365 762 764 12 Weng W and Breslow JL 1996 Proc Natl Acad Sci USA 93 14788 14794 Version 1 2R3 www assaypro com E mail Support assaypro com
3. to each 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 develops Gently tap 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 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 OD 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
4. DA 155AYPRO AssayMax Human ApoA ll 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 Apolipoprotein A II ELISA Kit Catalog No EA5333 1 Sample insert for reference use only Introduction Apolipoprotein A Il ApoA Il is the second most abundant apolipoprotein in human plasma HDL comprising about 25 of the protein mass After being synthesized by the liver and intestine as a preprotein containing 100 amino acids apoA ll is processed to 77 amino acids in the mature plasma protein 1 3 ApoA ll is found in plasma as a monomer homodimer of 17 4 kDa or heterodimer with ApoE and ApoD 4 7 It has been reported that apoA Il play
5. ith 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 mM 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 CSF Collect cerebrospinal fluid CSF using sample pot Centrifuge samples at 3000 x g for 10 minutes Dilute samples 1 10 into EIA Diluent and assay The undiluted samples can be stored at 80 C 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 suggested 1 100 1 10000 4 ul sample 396 ul buffer 100x A 4ulsample 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 i
6. means may vary from the values listed Variations between laboratories may be caused by technique differences Standard Point Average OD P1 120 0 P2 60 00 P3 30 00 P4 15 00 PS 7 500 P6 3 750 P7 1 875 P8 0 000 Sample Pool Normal Sodium Citrate Plasma 20000x Standard Curve e The curve is provided for illustration only A standard curve should be generated each time the assay is performed Human APO All Standard Curve OD 450 nm 0 1 100 101 102 APO AI ng ml Performance Characteristics e The minimum detectable dose of ApoA ll as calculated by 2SD from the mean of a zero standard was established to be 1 3 ng 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 Recovery Standard Added Value 3 30 ng ml Recovery 88 109 Average Recovery 96 Linearity e Plasma and serum samples were serially diluted to test for linearity Average Percentage of Expected Value Sample Dilution Plasma Serum 1 10000 83 86 1 20000 97 99 1 40000 110 112 Cross Reactivity Species Cross Reactivity Canine None Bovine None Monkey 10
7. s 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 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 180 ng of Human ApoA ll Standard with 1 5 ml of EIA 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 equal volume of EIA Diluent to produce 60 30 15 7 5 3 75 and 1 875 ng ml solutions EIA 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 ApoA I1 ng ml P1 1 part Standard 120 ng ml 120 0 1 part P1 1 part EIA Diluent 60 00 1 part P2 1 part EIA Diluent 30 00 P4 1partP3 1partElA Diluent 15 00 Pe 1partP5 1 part EIA Diluent 3750 Ll Pe ElADiluent 0000 e Biotinylated Human ApoA II Antibody 70x Spin down the antibody briefly and dilute the desired amount of the antibody 1 70 with EIA Diluent Any remaining solution should be frozen at 20 C e Wash Buffer Concentrate 20
8. s roles in HDL remodeling cholesterol efflux modulating HDL interaction with enzymes and receptors and triglyceride metabolism 7 12 Principle of the Assay The AssayMax Human Apolipoprotein A II ELISA Enzyme Linked Immunosorbent Assay kit is designed for detection of human apoA ll in plasma serum CSF and cell culture samples This assay employs a quantitative sandwich enzyme immunoassay technique that measures human apoA ll in less than 4 hours A polyclonal antibody specific for human apoA ll has been pre coated onto a 96 well microplate with removable strips ApoA ll in standards and samples is sandwiched by the immobilized antibody and biotinylated polyclonal antibody specific for human apoA ll 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 u
9. sing contents e The Stop Solution is an acidic solution e The kit should not be used beyond the expiration date Reagents e Human ApoA Il Microplate A 96 well polystyrene microplate 12 strips of 8 wells coated with a polyclonal antibody against human apoA Il 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 ApoA Il Standard Human apoA ll in a buffered protein base 180 ng lyophilized e Biotinylated Human ApoA II Antibody 70x A 70 fold concentrated biotinylated polyclonal antibody against apoA ll 105 ul 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 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 wi
10. te list of steps Steps performed in incorrect order e Consult the provided procedure for the correct order Deficient Standard Curve 5 c A wn lt E 5 z Insufficient amount of e Check pipette calibration 32 reagents added to e Check pipette for proper performance 3 g wells zs Wash step was skipped e Consult the provided procedure for all wash steps 3 Improper wash buffer e Check that the correct wash buffer is being used E Improper reagent e Consult reagent preparation section for the correct Q preparation dilutions of all reagents a Insufficient or e Consult the provided procedure for correct incubation gt prolonged incubation time periods e Sandwich ELISA If samples generate OD values higher than the highest standard point P1 dilute samples further and repeat the assay Non optimal sample e Competitive ELISA If samples generate OD values lower dilution than the highest standard point P1 dilute samples further and repeat the assay LL e User should determine the optimal dilution factor for samples Contamination of e A new tip must be used for each addition of different reagents samples or reagents during the assay procedure Contents of wells e Verify that the sealing film is firmly in place before placing evaporate the assay in the incubator or at room temperature e Pipette properly in a controlled and careful manner Improper pipetting e Check pipette
11. th 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 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 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 and use supernatants Dilute samples 1 20000 with 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 anticoagulant 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 20000 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 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 Cell Lysate Rinse cell with cold PBS and then scrape the cell into a tube w
12. x 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 EIA 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 Human Apo A II 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 ApoA Il Antibody to each well and incubate for 1 hour e Wash the microplate as described above e Add 50 ul of Streptavidin Peroxidase Conjugate
Download Pdf Manuals
Related Search