AssayMaxTM Human alpha-1-Antitrypsin ELISA Kit
Contents
1. microplate 12 strips of 8 wells coated with a polyclonal antibody against human A1AT 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 A1AT Standard Human A1AT in a buffered protein base 150 ng lyophilized e Biotinylated Human A1AT Antibody 50x A 50 fold biotinylated polyclonal antibody against human A1AT 120 ul 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 desiccant packs 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 Store Standard at 2 8 C before reconstitut2. 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 A1AT 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 A1AT Antibody to each well and incubate for 1 hour e Wash the microplate as described above e Add 50 ul of Streptavidin Peroxidase Conjugate per 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 20 minutes or till the optimal blue color density develops Gently tap the 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 wave
3. 6 3 Strange C etal 2006 Respriration 73 2 185 90 Version 3 2R Related Products e EA5001 1 AssayMax Human alpha 1 Antitripsin ELISA Kit Plasma and Serum samples www assaypro com e e mail Support assaypro com
4. A assarbro AssayMax Human alpha 1 Antitrypsin 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 20 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 alpha 1 Antitrypsin ELISA Kit Catalog No EA5101 1 Sample insert for reference use only Introduction Alpha 1 antitrypsin A1AT is a protein that protects the lungs The liver usually makes the protein and releases it into the bloodstream A1AT is a major protease inhibitor that controls tissue degradation A reduction of A1AT levels can cause a change in collagen metabolism 1 A1AT deficiency is an uncommon however 3 A1AT inhibits neutrophil elastase release into the lungs during inflammatory states 2 Principle of the Ass
5. F Collect cerebrospinal fluid CSF using sample pot Centrifuge samples at 3000 x g for 10 minutes Dilute samples 1 4000 into MIX 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 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 grade water Store for up to 30 days at 2 8 C e Standard Curve Reconstitut
6. The minimum detectable dose of A1AT as calculated by 25D from the mean of a zero standard was established to be 0 16 ng ml e Kit standard has been calibrated against WHO International Standard 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 A1AT concentrations Unspiked Sample ng ml Spike ng ml 1 5 4 9 5 4 110 3 4 5 0 8 4 9 2 109 10 0 13 4 13 3 99 1 5 7 7 8 0 104 5 0 11 2 11 1 99 10 0 16 2 15 9 98 Average Recovery 103 Recovery Expected Observed Linearity e Milk samples were serially diluted to test for linearity Average Percentage of Expected Value Sample Dilution Milk 1 1000 90 1 2000 97 1 4000 104 Cross Reactivity Species Cross Reactivity Bovine None Canine None Mouse None Monkey lt 5 Rat None Swine None Human Troubleshooting 100 Issue Causes Course of Action Use of expired e Check the expiration date listed before use components e Do not interchange components fro
7. ay The AssayMax Human alpha 1 Antitrypsin ELISA Enzyme Linked Immunosorbent Assay kit is designed for detection of A1AT in human urine milk saliva CSF and cell culture samples This assay employs a quantitative sandwich enzyme immunoassay technique that measures A1AT in less than 4 hours A polyclonal antibody specific for ALAT has been pre coated onto a 96 well microplate with removable strips Human A1AT in standards and samples is sandwiched by the immobilized antibody and a biotinylated polyclonal antibody specific for A1AT 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 A1AT Microplate A 96 well polystyrene
8. e the 150 ng of Human A1AT Standard with 1 5 ml of MIX Diluent to generate a 100 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 100 ng ml 1 4 with MIX Diluent to produce 25 6 25 1 563 and 0 391 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 Point Dilution A1AT ng ml P1 1 part Standard 100 ng ml 100 0 1 part P1 3 parts MIX Diluent 25 00 P4 1partP3 3 parts MIX Diluent 1563 LPS MiXDiluent 0o00 e Biotinylated Human A1AT Antibody 50x Spin down the antibody briefly and dilute the desired amount of the antibody 1 50 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 standard solutions and samples as instructed Bring all reagents to room temperature before use The assay is performed at room temperature 20
9. ing 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 Cell Culture Supernatants Centrifuge cell culture media at 3000 x g for 10 minutes to remove debris Collect supernatants and assay Store the remaining samples at 20 C or below Avoid repeated freeze thaw cycles e Urine Collect urine using sample tube Centrifuge samples at 800 x g for 10 minutes Samples are recommended for use at 1 20 into MIX Diluent or within the range of 1 2 to 1 200 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 e Milk Collect milk using sample tube Centrifuge samples at 800 x g for 10 minutes Dilute samples 1 2000 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 samples at 800 x g for 10 minutes Samples are recommended for use at 1 400 into MIX Diluent or within the range of 1 40 to 1 4000 and assay The undiluted samples can be stored at 20 C or below for up to 3 months Avoid repeated freeze thaw cycles e CS
10. length 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 100 0 P2 25 00 P3 6 250 P4 1 563 PS 0 391 P6 0 000 Sample Normal Human Milk 2000x Standard Curve e The curve is provided for illustration only A standard curve should be generated each time the assay is performed Human AlAT Standard Curve 1 0F OD 450 nm 0 1 1 0 10 0 100 0 ALAT ng ml Performance Characteristics e
11. m 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 Low Precision 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 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 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 buffe
12. r 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 Unexpectedly Low or High Signal Intensity e Consult the provided procedure for correct incubation time 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 the 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 Hauck EW et al 2004 Eur Urol 46 5 623 8 discussion 628 2 Chappell et al 2004 Hum Mutat 24 6 535
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