AssayMaxTM Human ApoA-I ELISA Kit
Contents
1. Average Percentage of Expected Value Sample Dilution Plasma Serum 1 100 92 91 1 200 97 99 1 400 105 106 Cross Reactivity Species Cross Reactivity Canine None Bovine None Monkey lt 5 Mouse None Rat None Swine None Rabbit None e _ No significant cross reactivity observed with ApoA Il ApoB ApoC I ApoC Il and ApoC Ill 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 technique Splashing of reagents e Pipette properly in a controlled and careful manner while loading wells e Pipette properly in a controlled and careful manner e Check pipette calibration e Check pipette for proper performance Inconsistent volumes loaded into wells Low Precision e Thoroughly agitate the lyophilized components after reconstitution e Thoroughly mix dilutions e Check the microplate pouch for proper sealing Improperly sealed e Check that the microplate pouch has no punctures microplate e Check that three desiccants are inside the microplate pouch prior to sealing Insufficient mixing of reagent d2. clot formation centrifuge samples at 3000 x g for 10 minutes and remove serum Dilute samples 1 200 into MIX Diluent or within the range of 1 100 1 800 and assay User should determine the optimal dilution factor The undiluted samples can be stored at 20 C and 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 suggested 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 gr
3. 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 g for 10 minutes Dilute samples 1 200 into MIX Diluent or within the range of 1 100 1 800 and assay User should determine the optimal dilution factor The undiluted samples can be stored at 20 C and 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
4. 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 Add 25 ul of Human ApoA I Standard or sample per well and immediately add 25 ul of Biotinlylated Human ApoA I Protein to each well on top of the standard or sample and tap plate to mix gently 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 15 minutes or till the optimal blue color density develops Gently tap plate to ensure thorough mi
5. transport and anti inflammation 2 Oxidation of specific amino acid residues in ApoA may contribute to atherogenesis by impairing cholesterol efflux from macrophages 3 A majority of HDL functionality is derived from the ability of ApoA I to sequester phospholipids and cholesterol as well as interact with plasma enzymes and cellular receptors 4 During reverse cholesterol transport HDL interacts with lecithin cholesteryl acyltransferase LCAT and cellular receptors including ATP binding cassette transporter protein A I ABCA1 and the scavenger receptor class B type in an ordered fashion that is reflected by HDL particle lipid composition The beta chain of ATP synathase found on the surface of hepatocytes contains a high affinity HDL receptor for ApoA 1 5 Principle of the Assay The AssayMax Human Apolipoprotein A I ELISA Enzyme Linked Immunosorbent Assay kit is designed for detection of human apoA I in plasma and serum samples This assay employs a quantitative competitive enzyme immunoassay technique that measures human apoA l in less than 3 hours A polyclonal antibody specific for human apoA I has been pre coated onto a 96 well microplate with removable strips ApoA l in standards and samples is competed with a biotinylated apoA I sandwiched by the immobilized antibody and streptavidin peroxidase conjugate All unbound material is then washed away and a peroxidase enzyme substrate is added The color development is stopped
6. DA assarbro AssayMax Human ApoA I 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 15 minutes Step 4 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 A I ELISA Kit Catalog No EA5201 1 Sample insert for reference use only Introduction Human Apolipoprotein A I ApoA I comprises about 70 of the high density lipoprotein s HDL protein mass while ApoA Il comprises 15 20 ApoA l a 243 amino acid molecule that contains a series of highly homologous amphipathic alpha helices is a 28 kDa single polypeptide that lacks glycosylation or disulfide linkages 1 About 5 10 of human plasma ApoA I exists in a lipoprotein unassociated state ApoA l appears to have effects on the atherosclerosis inhibition reverse cholesterol
7. ade water Store for up to 30 days at 2 8 C e Standard Curve Reconstitute the 8 ug of Human ApoA I Standard with 0 4 ml of MIX Diluent to generate a 20 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 20 ug ml 1 2 with MIX Diluent to produce 10 5 2 5 and 1 25 ug ml solutions MIX Diluent serves as the zero standard 0 pg ml Any remaining solution should be frozen at 20 C and used within 10 days Standard Point Dilution ApoA 1 ug ml P1 1 part Standard 20 ug ml 20 0 1 part P1 1 part MIX Diluent Pa 1partP3 1 part MIX Diluent 250 f Pre o MiXDilvet ooo i e Biotinylated Human ApoA I Protein 2x Reconstitute Biotinylated ApoA I Protein with 4 ml MIX Diluent to produce a 2 fold stock solution Allow the biotin to sit for 10 minutes with gentle agitation prior to making dilutions The stock solution should be further diluted 1 2 with MIX Diluent Any remaining solution should be frozen at 20 C and used within 10 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 e SP Conjugate 100x Spin down the SP conjugate briefly and dilute the desired amount of the conjugate 1 100 with MIX Diluent Any
8. 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 Human ApoA I Microplate A 96 well polystyrene microplate 12 strips coated with a polyclonal antibody against human apoA l 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 I Standard Human apoA I in a buffered protein base 8 ug lyophilized e Biotinylated ApoA l Protein 1 vial lyophilized 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 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
9. e assay in the incubator or at room temperature e Pipette properly in a controlled and careful manner Improper pipetting e Check pipette calibration Deficient Standard Curve Fit e Check pipette for proper performance e Thoroughly agitate the lyophilized components after reconstitution e Thoroughly mix dilutions Insufficient mixing of reagent dilutions References 1 Davidson WS and Thompson TB 2007 J Biol Chem 282 31 22249 22253 2 Nessen SE et al 2003 J Am Med Assoc 290 2292 2300 3 Shao B etal 2006 Curr Opin Mol Ther 8 3 198 205 4 Forte T etal 1971 Biochim Biophys Acta 248 381 386 5 Martinez LO et al 2003 Nature 421 6918 75 79 Version 3 7R Related products e EA5301 1 AssayMax Human Apolipoprotein A I ELISA Kit Urine Milk Saliva and Cell Culture samples www assaypro com e mail Support assaypro com
10. ilutions Microplate was left e Each step of the procedure should be performed T unattended between uninterrupted D steps a Omission of step e Consult the provided procedure for complete list of steps El Steps performed in e Consult the provided procedure for the correct order incorrect order 5 z Insufficient amount of e Check pipette calibration 2 Fa reagents added to e Check pipette for proper performance 3 g wells 2s 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 gt 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 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 th
11. ovided for illustration only A standard curve should be generated each time the assay is performed H Apo A I Standard Curve 1 0 OD 450 nm 0 1 10 10 10 hAPO A I ug ml Reference Value e The normal human plasma levels of ApoA I are 0 7 1 5 mg ml e Human plasma and serum samples from healthy adults were tested n 20 On average ApoA I level was 1098 ug ml Sample Average Value ug ml Human Pool Normal Plasma 1027 Human Pool Normal Serum 1169 Performance Characteristics e The minimum detectable dose of ApoA l as calculated by 2SD from the mean of a zero standard was established to be 0 7 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 Spiking Recovery e Recovery was determined by spiking two plasma samples with different ApoA concentrations Unspiked Sample ng ml Spike ng ml 2 5 10 3 10 0 97 7 8 5 0 12 8 12 4 97 Recovery Expected Observed 10 0 17 8 18 0 101 2 5 12 7 12 3 97 5 0 15 2 15 0 99 10 0 20 2 19 4 96 Average Recovery 98 Linearity e Plasma and serum samples were serially diluted to test for linearity
12. xing 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 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 four parameter or log log logistic curve fit Determine the unknown sample concentration from the Standard Curve and multiply the value by the dilution factor Typical Data 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 20 0 P2 10 0 P3 5 00 P4 2 50 P5 1 25 P6 0 00 Sample Human Pool Normal Sodium Citrate Plasma 200x Standard Curve e The curve is pr
Download Pdf Manuals
Related Search