AssayMax Human Lysozyme ELISA Kit
Contents
1. 12 ug of Human Lysozyme Standard with 4 ml of EIA Diluent to generate a 3 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 3 ug ml 1 2 with equal volume of EIA Diluent to produce 1 5 0 75 0 375 0 188 0 094 and 0 047 ug ml solutions EIA Diluent serves as the zero standard 0 ug ml Any remaining solution should be frozen at 20 C and used within 30 days Standard Point Dilution Lysozyme ug ml P1 1 part Standard 3 ug ml 3 000 1 part P1 1 part EIA Diluent 1 500 1 part P2 1 part EIA Diluent 0 750 P4 A part P3 1 part EIA Diluent 0 375 P6 tparPS ipattlADilunt 0 094 PBT HADiuent ooo e Biotinylated Human Lysozyme 1x Reconstitute Biotinylated Human Lysozyme with 4 ml EIA Diluent to produce a stock solution Allow the biotin to sit for 10 minutes with gentle agitation prior to use Any remaining solution should be frozen at 20 C and used within 30 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 EIA Diluent Any remaining solution should be frozen at 20 C Assay Pr2. Centrifuge samples at 800 x g for 10 minutes Dilute samples 1 200 into EIA Diluent or within the range of 1 20 to 1 2000 and assay The undiluted samples can be stored at 20 C or below for up to 30 days 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 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 Human Lysozyme Standard Reconstitute the
3. A assarPno AssayMax Human Lysozyme 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 10 minutes Step 4 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 Lysozyme ELISA Kit Catalog No EL3020 1 Sample insert for reference use only Introduction Lysozyme is one of the anti microbial agents found in human milk and is also present in spleen lung kidney white blood cells plasma saliva and tears Lysozyme has 130 amino acids is synthesized by granulocytes and macrophages and its natural substrate is the bacterial cell wall peptidoglycan 1 2 Principle of the Assay The AssayMax Human Lysozyme ELISA Enzyme Linked Immunosorbent Assay kit is designed for detection of lysozyme in human milk This assay employs a quantitative competitiv
4. ctively e This assay recognizes both natural and recombinant human lysozyme Linearity Average Percentage of Expected Value Sample Dilution Milk 1 100 10696 1 200 98 1 400 94 Recovery Standard Added Value 0 09 1 5 ug ml Recovery 86 114 Average Recovery 96 98 Cross Reactivity Species Cross Reactivity Canine None Bovine None Monkey None Mouse None Rat None Swine lt 10 Human 100 Troubleshooting Issue Causes Course of Action Use of expired e Check the expiration date listed before use c components e Do not interchange components from different lots 2 e Check that the correct wash buffer is being used is e Check that all wells are dry after aspiration Improper wash step e Check that the microplate washer is dispensing properly 3 e If washing by pipette check for proper pipetting 3 technique Splashing of reagents e Pipette properly in a controlled and careful manner while loading wells 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
5. ddition 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 Improper pipetting e Pipette properly in a controlled and careful manner e Check pipette calibration e Check pipette for proper performance Insufficient mixing of reagent dilutions References 1 2 e Thoroughly agitate the lyophilized components after reconstitution e Thoroughly mix dilutions Chung LP et al 1988 Proc Natl Acad Sci USA 85 6227 6231 Lollike K et al 1995 Leukemia 9 159 164 Version 2 5R Related Products EL3010 1 AssayMax Human Lysozyme ELISA Kit Plasma Serum Saliva Urine and Cell Culture samples www assaypro com e mail Support assaypro com
6. e enzyme immunoassay technique that measures lysozyme in less than 3 hours A polyclonal antibody specific for lysozyme has been pre coated onto a 96 well microplate with removable strips Lysozyme in standards and samples is competed with a biotinylated lysozyme 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 Lysozyme Microplate A 96 well polystyrene microplate 12 strips of 8 wells coated with a polyclonal antibody against human lysozyme 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 Lysozyme Standard Human lysozyme in a buffered prot
7. ein base 12 ug lyophilized e Biotinylated Human Lysozyme 1 vial lyophilized 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 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 days in a 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 Milk Collect milk using sample tube
8. les 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 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 Standard Curve e The curve is provided for illustration only A standard curve should be generated each time the assay is performed Human Lysozyme Standard Curve 1 0 OD459 nm 0 1 0 01 0 10 1 00 10 00 hLysozyme ug ml Performance Characteristics e The minimum detectable dose of lysozyme is typically 0 04 ug ml e Intra assay and inter assay coefficients of variation were 4 9 and 7 0 respe
9. ocedure 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 Add25 ul of Human Lysozyme Standard and or sample per well and immediately add 25 ul of Biotinylated Human Lysozyme 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 at room temperature 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 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 bubb
10. 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 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 e A new tip must be used for each a
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