AssayMaxTM Human ApoB ELISA Kit
Contents
1. Serum 91 98 108 Cross Reactivity Species Beagle Bovine Cross Reactivity None None Monkey lt 5 Mouse Rat None None Swine None Proteins Cross Reactivity ApoA I lt 2 ApoC l lt 10 No significant cross reactivity observed with ApoA Il ApoC Il ApoC Ill and ApoE Troubleshooting Causes Course of Action Low Precision Use of expired components e Check the expiration date listed before use e Do not interchange components from different lots Improper wash step e Check that the correct wash buffer is being used e Check that all wells are dry after aspiration e Check that the microplate washer is dispensing properly e If washing by pipette check for proper pipetting technique Splashing of reagents while loading wells e Pipette properly in a controlled and careful manner Inconsistent volumes loaded into wells e Pipette properly in a controlled and careful manner e Check pipette calibration 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 sealing2. a c oo n lt E e wn 2 a YU E mo w E o w Q x c 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 complete list of steps Steps performed in incorrect order e Consult the provided procedure for the correct order Insufficient amount of reagents added to wells e Check pipette calibration e Check pipette for proper performance Wash step was skipped e Consult the provided procedure for all wash steps Improper wash buffer e Check that the correct wash buffer is being used Improper reagent preparation e Consult reagent preparation section for the correct dilutions of all reagents Insufficient or prolonged incubation periods e Consult the provided procedure for correct incubation time Deficient Standard Curve Fit Non optimal sample dilution e Sandwich ELISA If samples generate OD values higher than the highest standard point P1 dilute samples further and repeat the assay e Competitive ELISA If samples generate OD values lower than the highest standard point P1 dilute samples further and repeat the assay e User should determine the optimal dilution factor for samples Contamination of reagents e A new tip must be used for each addition of different samples or reagents during the assay procedure
3. 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 Samples are recommended for use at 1 20000 into EIA Diluent or within the range of 1 10000 to 1 40000 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 EDTA or Heparin can also be used as an anticoagulant Serum Samples should be collected into a serum separator tube After clot formation centrifuge samples at 3000 x g for 10 minutes Samples are recommended for use at 1 20000 into EIA Diluent or within the range of 1 10000 to 1 40000 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 CSF Collect cerebrospinal fluid CSF using sample pot Centrifuge samples at 3000 x g for 10 minutes Dilute samples 1 5 into EIA Diluent and assay The undiluted samples can be stored at 80 C for up to 3 months Avoid repeated freeze thaw cycles Cell Culture Supernatants 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 tub
4. P6 0 0078 P7 0 0039 P8 0 0000 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 B Standard Curve 1 0F OD 450 nm 0 1 1 1 N 0 001 0 010 0 100 1 000 APO B ug ml Reference Value e Normal human ApoB plasma levels range from 0 66 to 1 33 mg ml e _ Human plasma and serum samples from healthy adults were tested n 30 On average ApoB level was 1005 pg ml Sample Average Value ug ml Human Pool Normal Plasma 924 Human Pool Normal Serum 1086 Performance Characteristics e The minimum detectable dose of ApoB as calculated by 25D from the mean of a zero standard was established to be 0 002 ug 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 0 01 0 1 ug ml Recovery 88 111 Average Recovery 96 Linearity Plasma and serum samples were serially diluted to test for linearity Average Percentage of Expected Value Sample Dilution Plasma 1 10000 90 1 20000 99 1 40000 102
5. in less than 4 hours A polyclonal antibody specific for human ApoB has been pre coated onto a 96 well microplate with removable strips ApoB in standards and samples is sandwiched by the immobilized antibody and biotinylated polyclonal antibody specific for ApoB 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 ApoB Microplate A 96 well polystyrene microplate 12 strips of 8 wells coated with a polyclonal antibody against human ApoB 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 ApoB Standard Human ApoB in a buffered
6. protein base 0 325 ug 2 plastic vials lyophilized e Biotinylated Human ApoB Antibody 50x A 50 fold concentrated biotinylated polyclonal antibody against ApoB 120 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 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 20 C before and after reconstituting with Diluent Other Supplies Required e Microplate reader capable of measuring absorbance at 450 nm e Pipettes 1 20 ul 20 200 ul 200 1000 ul and multiple channel e Deionized or distilled reagent grade water Sample Collection
7. Contents of wells evaporate e Verify that the sealing film is firmly in place before placing the assay in the incubator or at room temperature Improper pipetting e Pipette properly in a controlled and careful manner e Check pipette calibration e Check pipette for proper performance 10 e Thoroughly agitate the lyophilized components after reconstitution e Thoroughly mix dilutions Insufficient mixing of reagent dilutions Reference 1 Brodsky JL et al 2008 Trends Endocrinol Metab Sep 19 7 254 9 Version 3 8R www assaypro com e e mail Support assaypro com
8. Massarbro AssayMax Human ApoB 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 3 Consult instructions for use Assay Template 12 11 10 Human Apolipoprotein B ELISA Kit Catalog No EA7001 1 Sample insert for reference use only Introduction Apolipoprotein B ApoB is the dominant protein constituent of LDL The levels of secreted ApoB directly correlate with circulating serum cholesterol levels 1 Principle of the Assay The AssayMax Human Apolipoprotein B ELISA Enzyme Linked Immunosorbent Assay Kit is designed for detection of human ApoB in plasma serum CSF and cell culture samples This assay employs a quantitative sandwich enzyme immunoassay technique that measures human ApoB
9. e 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 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 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 A 4 ul sample 396 ul buffer 100x 100 fold dilution Assuming the needed volume is less than or equal to 400 ul 4 ul sample 396 ul buffer 100x 4 ul of A 396 ul buffer 100x 10000 fold dilution Assuming the needed volume is less than or equal to 400 ul 1 1000 1 100000 4 ul sample 396 ul buffer 100x 24 ul of A 216 ul buffer 10x 1000 fold dilution Assuming the needed volume is less than or equal to 240 ul 4 ul sample 396 ul buffer 100x 4 ul of A 396 ul buffer 100x 24 ul of B 216 ul buffer 10x 100000 fold dilution Assuming the needed volume is less than 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 for
10. es 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 means may vary from the values listed Variations between laboratories may be caused by technique differences Standard Point Average OD P1 0 2500 P2 0 1250 P3 0 0625 P4 0 0313 P5 0 0156
11. med 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 0 325 ug of Human ApoB Standard with 1 3 ml of EIA Diluent to generate a 0 25 ug 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 0 25 ug ml 1 2 with EIA Diluent to produce 0 125 0 0625 0 0313 0 0156 0 0078 and 0 0039 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 48 Standard Point Dilution hours ApoB ug ml P1 1 part Standard 0 25 ug ml 0 2500 1 part P1 1 part EIA Diluent 0 1250 1 part P2 1 part EIA Diluent 0 0625 a ae 1 part P3 1 part EIA Diluent 0 0313 Ps ipartPS ipartFlADiluent 0 0078 ls EIA Diluent o0000 e Biotinylated Human ApoB Antibody 50x Spin down the antibody briefly and dilute the desired amount of the antibody 1 50 with EIA 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 C
12. onjugate 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 ApoB 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 ApoB Antibody to each well and incubate for 1 hour e Wash the microplate as described above e 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 e Wash the microplate as described above e Add 50 ul of Chromogen Substrate per well and incubate for 10 minut
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