AssayMax Human beta-2-Microglobulin ELISA Kit
Contents
1. 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 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 1000 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 1000 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 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 e Urine Collect urine using sample pot Centrifuge samples at 800 x g for 10 minutes Dilute samples 1 100 into MIX Diluent and assay The undiluted samples can be stored at 20 C or below for up2. 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 _ 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 Human beta 2 Microglobulin Standard Reconstitute the 75 ng 5376 mU of Human beta 2 Microglobulin Standard with 1 5 ml of MIX Diluent to generate a 50 ng ml 3584 mU 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 50 ng ml 1 4 with MIX Diluent to produce 12 5 3 125 0 781 0 195 and 0 049 ng ml solutions MIX Diluent serves as the zero standard 0 ng ml Any remaining solution should be frozen at 20 C and use within 30 days Standard rae B2M Point piluton ng ml P1 1 part Standard 50 ng ml 50 00 1 part P1 3 parts MIX Diluent 12 50 896 1 part P2 3 parts MIX Diluent 3 125 224 pa 1partP3 3partsMiXDiluent 0781 56 Ps 1partP5 3partsMiXDiluent 0 09 35 Al MiXDiluent 0 000 00 e Biotinylated Hu
3. DA assarbro AssayMax Human beta 2 Microglobulin 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 ci Consult instructions for use Assay Template 12 11 10 Human beta 2 Microglobulin B2M ELISA Kit Catalog No EM5001 1 Sample insert for reference use only Introduction Beta 2 microglobulin B2M is a small serum protein that constitutes the light chain of the major histocompatibility class human leukocyte antigen HLA class an integral membrane protein involved in the immune response The protein is 99 amino acid residues in length and has a molecular mass of 12 kDa 1 4 B2M is released from the cell surface of HLA class into the serum and carried to the kidneys for degradation an
4. anner a Inconsistent volumes A Pon 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 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 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 10 Deficient Standard Curve Fit e Sandwich ELISA If samples generat
5. d secretion 5 Principle of the Assay The AssayMax Human beta 2 Microglobulin ELISA Enzyme Linked Immunosorbent Assay kit is designed for detection of human B2M in plasma serum milk saliva urine CSF and cell culture samples This assay employs a quantitative sandwich enzyme immunoassay technique that measures B2M in less than 4 hours A polyclonal antibody specific for B2M has been pre coated onto a 96 well microplate with removable strips B2M in standards and samples is sandwiched by the immobilized antibody and biotinylated polyclonal antibody specific for B2M 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 H
6. e 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 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 Gussow D et al 1987 J Immunol 139 3132 3138 2 Krangel MS et al 1979 Cell 18 979 991 3 Cattoni Celli S et al 1992 Cancer Res 1 1201 1204 4 Saper MA et al 1991 J Mol Biol 219 277 319 5 Trinh CH et al 2002 Proc Natl Acad Sci USA 99 9771 9776 Version 2 5R www assaypro com e e mail Support assaypro com
7. ermined 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 19 12 5 ng ml Recovery 92 110 Average Recovery 96 Linearity e Plasma serum and milk samples were serially diluted to test for linearity Average Percentage of Expected Value Sample Dilution Plasma Serum 1 1000 95 93 1 2000 100 99 1 4000 104 104 1 8000 Cross Reactivity Species Cross Reactivity Canine None Monkey 50 Mouse None Rat None Swine None Bovine 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 2 e Pipette properly in a controlled and careful m
8. g 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 50 000 P2 12 500 P3 3 1250 P4 0 7810 P5 0 1950 P6 0 0488 P7 0 0000 Sample Normal Sodium Citrate Plasma 1000x Standard Curve e The curve is provided for illustration only A standard curve should be generated each time the assay is performed H Beta 2 Microglobulin Standard Curve 1 0 va 0 1 OD 450 nm 01 10 10 0 100 0 H b2 Microglobulin ng ml H Beta 2 Microglobulin Standard Curve 1 0 va 0 1 OD 450 nm 1 1 j 1 10 100 1000 10000 H b2 Microglobulin mU ml Reference Value Human plasma and serum samples from healthy adults were tested n 40 Average Value ng ml Sample 1086 1248 Human Pool Normal Plasma Human Normal Plasma 1305 Human Pool Normal Serum Performance Characteristics e Kit standard has been calibrated against WHO International Standard e The minimum detectable dose of beta 2 Microglobulin as calculated by 25D from the mean of a zero standard was established to be 0 04 ng ml e Intra assay precision was det
9. man beta 2 Microglobulin Antibody 50x Spin down the biotinylated 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 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 beta 2 Microglobulin 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
10. times on absorbent material to completely remove the liquid e Add 50 ul of Biotinylated Human beta 2 Microglobulin 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 10 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 usin
11. to 3 months Avoid repeated freeze thaw cycles e Saliva Collect saliva using sample tube Centrifuge samples at 800 x g for 10 minutes Dilute saliva 1 200 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 Milk Collect milk using sample tube Centrifuge samples at 800 x g for 10 minutes Dilute milk 1 4000 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 CSF Collect cerebrospinal fluid CSF using sample tube Centrifuge samples at 3000 x g for 10 minutes Dilute samples 1 1000 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 A 4ulsample 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
12. uman beta 2 Microglobulin Microplate A 96 well polystyrene microplate 12 strips of 8 wells coated with a polyclonal antibody against human B2M e Sealing Tapes Each kit contains 3 precut pressure sensitive sealing tapes which can be cut to fit the format of the individual assay e Human beta 2 Microglobulin Standard Human B2M in a buffered protein base 75 ng lyophilized e Biotinylated Human beta 2 Microglobulin Antibody 50x A 50 fold biotinylated polyclonal antibody against B2M 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
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