Human alpha-1- Microglobulin ELISA Kit
Contents
1. 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 formed 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 160 ng of Human alpha 1 Microglobulin Standard with 4 ml of EIA Diluent to generate a 40 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 40 ng ml 1 2 with EIA Diluent to produce 20 10 5 2 5 1 25 0 625 and 0 313 ng ml solutions EIA Diluent serves as the zero standard 0 ng ml Any remaining solution should be frozen at 20 C and used within 30 days Standard Dilution 1M2. 00 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 alpha 1 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 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 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 o
3. Point ng ml P1 1 part Standard 40 ng ml 1 part EIA Diluent 20 00 1 part P1 1 part EIA Diluent 10 00 1 part P2 1 part ElA Diluent 5 000 P4 1IpartP3 1partFlADiluent 2500 Ps 1ipatP5 lpattlADiuent 0 625 08 J ElADiluent 0000 e Biotinylated Human alpha 1 Microglobulin 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 Conjugate 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 alpha 1 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 2
4. 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 alpha 1 Microglobulin Microplate A 96 well polystyrene microplate 12 strips of 8 wells coated with a polyclonal antibody against human alpha 1 microglobulin 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 alpha 1 Microglobulin Standard Human alpha 1 microglobulin in a buffered protein base 160 ng lyophilized e Biotinylated Human alpha 1 Microglobulin Antibody 50x A 50 fold concentrated biotinylated polyclonal antibody against alpha 1 microglobulin 120 tl 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 Micropl
5. ate 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 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 and use supernatants Dilute samples 1 10000 with EIA 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 EIA 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 Urine Collect urine using sample pot Centrifuge samples at 800 x g for 10 minutes Dilute samples 1 500
6. atory and detoxification processes caused by immune system activation and extracellular heme catabolism 4 5 Principle of the Assay The AssayMax Human alpha 1 Microglobulin ELISA Enzyme Linked Immunosorbent Assay kit is designed for detection of human alpha 1 microglobulin in plasma serum urine saliva milk CSF and cell culture samples This assay employs a quantitative sandwich enzyme immunoassay technique that measures human alpha 1 microglobulin in less than 4 hours A polyclonal antibody specific for human alpha 1 microglobulin has been pre coated onto a 96 well microplate with removable strips Alpha 1 microglobulin in standards and samples is sandwiched by the immobilized antibody and biotinylated polyclonal antibody specific for human alpha 1 microglobulin 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
7. d assarpro AssayMax Human alpha 1 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 15 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 Microglobulin ELISA Kit Catalog No EM5110 1 Sample insert for reference use only Introduction Alpha 1 microglobulin 1M also called protein HC is a tubular plasma and tissue protein that belongs to the lipocalin transport protein superfamily for small hydrophobic molecules It contains 184 amino acids and weighs 26 kDa 1 2 Mature 1M and bikunin urinary trypsin inhibitor result from a common precursor 3 1M is found in blood both in free form and complex bound to immunoglobulin A IgA It is involved in inflamm
8. es 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 sealing c oo o lt E I LJ I 2 a YU E o w E o o o x o c 2 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 Ins
9. into EIA Diluent or within the range of 1 200 1 2000 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 Dilute samples 1 4 with EIA 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 samples 1 100 with EIA 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 CSF Collect cerebrospinal fluid CSF using sample pot Centrifuge samples at 3000 x g for 10 minutes Dilute samples 1 100 into ElA 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 4 ul sample 396 ul buffer 100x 100 fold dilution Assuming the needed volume is less than or equal to 400 ul
10. m detectable dose of alpha 1 microglobulin as calculated by 2SD from the mean of a zero standard was established to be 0 2 ng 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 96 Average CV 96 Spiking Recovery e Recovery was determined by spiking two plasma samples with different alpha 1 microglobulin concentrations Unspiked Sample ng ml Spike ng ml Expected Observed Recovery 76 5 0 15 0 20 0 19 9 100 6 5 0 10 0 9 5 95 2 0 7 0 6 9 99 15 0 17 0 16 8 99 5 0 7 0 6 4 91 2 0 4 0 3 6 90 Linearity Average Recovery 96 e Plasma and serum samples were serially diluted to test for linearity Average Percentage of Expected Value Serum Sample Dilution Plasma 1 5000 96 93 1 10000 100 98 1 20000 Cross Reactivity Species 106 Cross Reactivity 105 Canine None Bovine None Monkey 80 Mouse None Rat None Swine None Rabbit None Troubleshooting Caus
11. r 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 on the y axis The best fit line can be determined by regression analysis using 4 parameter or log log 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 20 00 P2 10 00 P3 5 000 P4 2 500 P5 1 250 P6 0 625 P7 0 313 P8 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 Human alpha 1 Microglobulin Standard Curve E Lor o L uv Q o 0 1 u 0 1 1 0 10 0 100 0 H alphal Microglobulin ng ml Reference Value e Human plasma and serum samples from healthy adults were tested n 40 On average alpha 1 microglobulin level was 42 ug ml Sample Average Value ug ml Human Pooled Normal Plasma 42 Human Normal Plasma 41 Human Pooled Normal Serum 43 Performance Characteristics e The minimu
12. ufficient 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 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 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 Insufficient mixing of Pam reconstitution reagent dilutions e Thoroughl
13. y mix dilutions References 1 2 3 4 5 Grubb AO et al 1983 Biol Chem 258 14698 14707 Ekstr m B and Bergg rd I 1977 J Biol Chem 252 8048 8057 Vetr H and Gebhard W 1990 Biol Chem Hoppe Seyler 371 1185 11 M ndez E et al 1986 Proc Natl Acad Sci USA 83 1472 1475 Allhorn M et al 2002 Blood 99 1894 1901 e Thoroughly agitate the lyophilized components after 96 Version 1 9R www assaypro com e e mail Support assaypro com
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