AssayMaxTM Human Albumin ELISA Kit
Contents
1. efore 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 450 ug of Human Albumin Standard with 4 5 ml of MIX Diluent to generate a 100 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 100 ug ml 1 4 with MIX Diluent to generate 25 6 25 1 563 and 0 391 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 30 days Standard Point Dilution Albumin ug ml P1 1 part Standard 100 ug ml 100 0 1 part P1 3 parts MIX Diluent 25 00 1 part P2 3 parts MIX Diluent 6 250 P4 1partP3 3 parts MIX Diluent 1 563 1 part P4 3 parts MIX Diluent 0 391 0o PSS MIX Diluent 0 000 e Biotinylated Human Albumin 3x Reconstitute Biotinylated Human Albumin with 4 ml MIX Diluent to produce a 3 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 3 with MIX Diluent to generate a working solution Any remaining solution should be frozen at 20 C and used within 30 days e Wash Buf2. fer 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 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 25 ul of Human Albumin Standard or sample per well and immediately add 25 ul of Biotinylated Human Albumin 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 1 hour 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 Streptavidin Peroxida
3. for human albumin has been pre coated onto a 96 well microplate with removable strips Albumin in standards and samples is competed with a biotinylated albumin sandwiched by the immobilized antibody and 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 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 Albumin Microplate A 96 well polystyrene microplate 12 strips of 8 wells coated with a polyclonal antibody against human albumin 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 Albumin Standard Human albumin in a buffered protein base 450 ug Iyophilized e Biotinylated Human Albumin 1 vial lyophilized e _ MIX Diluent Concent
4. laboratory means may vary from the values listed Variations between laboratories may be caused by technique differences Standard Point P1 Average OD 100 0 P2 25 00 P3 6 250 P4 1 563 P5 0 391 P6 0 000 Sample Pool Normal Sodium Citrate Plasma 10000x Standard Curve e The curve is provided for illustration only A standard curve should be generated each time the assay is performed H Albumin Standard Curve OD 459 nm 0 1FP s l siul sl asa 0 1 1 10 100 1000 hAlbumin u g ml Reference Value e The normal human plasma levels of albumin are 30 50 mg ml e Human plasma and serum samples from healthy adults were tested n 40 On average albumin level was 38 3 mg ml Sample Average Value mg ml Human Pool Normal Plasma 37 3 Human Normal Plasma 36 0 Human Pool Normal Serum 41 8 Performance Characteristics e The minimum detectable dose of human albumin as calculated by 25D from the mean of a zero standard was established to be 0 2 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 Recover
5. ntamination 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 Gekle M 2004 Annu Rev Physiol Version 7 3R Related Products EA3201 1 AssayMax Human Albumin ELISA Kit Urine Saliva Milk and Cell Culture samples e EMA2201 1 AssayMax Mouse Albumin ELISA Kit Plasma and Serum samples e EMA3201 1 AssayMax Mouse Albumin ELISA Kit Urine and Cell Culture samples e _ERA2201 1 AssayMax Rat Albumin ELISA Kit Plasma and Serum samples e _ERA3201 1 AssayMax Rat Albumin ELISA Kit Urine and Cell Culture samples ETA2202 1 AssayMax Rabbit Albumin ELISA Kit Plasma Serum Urine and Cell Culture samples e _ EPA3201 1 AssayMax Swine Albumin ELISA Kit Urine and Cell Culture samples e _ EPA2201 1 AssayMax Swine Albumin ELISA Kit Plasma and Serum samples www assaypro com e e mail Support assaypro com
6. oplate 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 Unexpectedly Low or High Signal Intensity 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 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 Co
7. rate 10x A 10 fold concentrated buffered protein base 30 ml e Wash Buffer Concentrate 20x A 20 fold concentrated buffered surfactant 30 ml 1 bottle 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 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 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 minu
8. se 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 15 minutes or until 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 low 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
9. tes Dilute samples 1 10000 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 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 and remove serum Dilute samples 1 10000 into MIX Diluent and assay The undiluted serum can be stored at 20 C or 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 b
10. y Standard Added Value 1 5 25 ug ml Recovery 92 109 97 Average Recovery Linearity e Plasma and serum samples were serially diluted to test for linearity Average Percentage of Expected Value Sample Dilution Plasma 1 5000 103 1 10000 99 1 20000 96 Cross Reactivity Species Cross Reactivity Bovine None Mouse None Rat None Swine None Beagle None Monkey None Rabbit 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 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 8 while loading wells v 2 e Pipette properly in a controlled and careful manner a Inconsistent volumes A Fides i e Check pipette calibration 3 loaded into wells 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 micr
11. yssaypro AssayMax Human Albumin 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 1 hour 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 Cli Consult instructions for use Assay Template 12 11 10 Human Albumin ELISA Kit Catalog No EA2201 1 Sample insert for reference use only Introduction Albumin is serum hepatic protein the most abundant protein in serum and contributes to the maintenance of oncotic pressure as well as to the transport of hydrophobic molecules 1 Principle of the Assay The AssayMax Human Albumin ELISA Enzyme Linked Immunosorbent Assay kit is designed for detection of albumin in human plasma and serum samples This assay employs a quantitative competitive enzyme immunoassay technique that measures human albumin in less than 2 hours A polyclonal antibody specific
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