Mouse TNF-alpha Product Information Sheet
Contents
1. Reader was at wrong wavelength or reader was malfunctioning Make sure that the wavelength is set to 450 nm and read the plate again Verify reader calibration and lamp alignment Excessive kit stress has occurred Check records to see how many times the kit has cycled from the refrigerator Check to see if the kit was left at extreme temperatures for too long Assay plates were compromised Always refrigerate plates in sealed bags with a desiccant to maintain stability Prevent condensation from forming on plates by allowing them to equilibrate to room temperature 20 25 C 68 77 F while in the packaging High Background or High Optical Density OD Readings Possible Causes Recommended Action Poor quality water was used in wash solution If water quality is questionable try substituting an alternate source of distilled deionized water to prepare the wash solution Substrate solution has deteriorated Make sure that the substrate is colorless prior to addition to the plate There was insufficient washing or poor liquid handling technique Use the number of washes per the protocol instruction Make sure that 250 uL of wash solution is dispensed per well per wash If you use a multichannel pipette or robotic liquid handling system verify its performance have the system repaired if any ports drip dispense or aspirate poorly Reader was malfunctioning or not blanked prope2. Human IL 20 lt 0 01 Mouse IL 1a lt 0 01 Mouse 11 16 lt 0 01 Mouse IL 2 lt 0 01 Mouse IL 4 lt 0 01 Mouse IL 5 lt 0 01 Mouse IL 6 lt 0 01 Mouse IL 13 lt 0 01 Mouse IL 15 lt 0 01 Mouse IL 17 lt 0 01 Mouse IL 23 lt 0 01 Mouse IL 12p70 lt 0 01 Rat IL 1B lt 0 01 Rat IL 6 lt 0 01 Return any Warnings and Precautions Neogen strongly recommends that you read the following warnings and precautions to ensure your full awareness of ELISA techniques and other details you should pay close attention to when running the assays More information can also be found in the Troubleshooting section Periodically optimizations and revisions are made to the kit and manual Therefore it is important to follow the version of the protocol included with the kit If you need further assistance please contact a Neogen technical services representative at techservice lifesciences neogen com standard contains mouseTNF a Handle with particular care Do not use the kit past the expiration date Do not intermix reagents from different kits or different lots ANTIBODIES AND PLATES ARE KIT AND LOT SPECIFIC Make sure that the standards detection antibody avidin HRP and diluent are mixed in correct volumes Try to maintain a laboratory temperature of 20 25 C 68 77 F Avoid running assays un
3. Standards Possible Causes Recommended Action Reagents were used in the wrong order or a step was skipped Follow the protocol carefully and repeat the assay Wrong antibodies were used Either Detection Antibody or Avidin HRP was prepared incorrectly or has deteriorated Make sure that the antibodies used are the ones that came with the kit All antibodies are kit and lot specific Make sure that the Detection Antibody Avidin HRP and diluent are mixed in correct volumes TMB Substrate has deteriorated Use a new set of TMB substrate Note if TMB substrate shows any color before use it should not be used for the assay Low Optical Density OD Readings Possible Causes Recommended Action Reagents were expired or mixed with a different lot number Verify the expiration dates and lot numbers Wash solution was prepared incorrectly Verify that the solution was prepared as described in the protocol Too many wash cycles were used Make sure to use the number of washes per the protocol instruction Incubation times were too short Follow protocol and ensure accurate incubation time Lab temperature was too low Maintain the lab room temperature within 20 25 C 68 77 F Do not run assays under air conditioning vents or near cold windows Reagents and plates were too cold Make sure that the plates and reagents are brought up to room temperature
4. from blue to yellow The wells are then read at 450 nm Kit Contents Storage and Shelf Life The Mouse TNF a ELISA Test Kit has the capacity for 96 determinations or testing of 40 samples in duplicate assuming 16 wells for standards and negative controls unused microwells to the foil bag and reseal them with the desiccant provided in the original package Store the kit components as recommended in the table below The shelf life is 12 months when the kit is properly stored Kit Contents Amount Storage Capture Ab coated Microtiter Plate 1 x 96 well plate 8 wells x 12 strips 2 8 C Mouse TNF a Standard 20 pL x 3 20 C 500X Detection Antibody 40 uL 20 C 500X Avidin HRP 40 uL 20 C 5X Assay Diluent 15 mL 2 8 C 20X Wash Solution 28 mL 2 8 C Stop Buffer 20 mL 2 8 C TMB Substrate 12 mL 2 8 C Components with the same part numbers are interchangeable among kits as long as they are used before their expiration dates Required Materials Not Provided With the Kit Sensitivit Specificit Microtiter plate reader 450 nm 10 20 100 and 1000 uL pipettes Multi channel pipette 50 300 uL Optional Distilled deionized water Detection Limit 15 pg mL Cross Reactivit Analytes Cross Reactivity Mouse TNF a 100 Human IL 1 lt 0 01 Human IL 2 lt 0 01 Human IL 4 lt 0 01 Human IL 6 lt 0 01 Human IL 8 lt 0 01
5. CORPORATION PRODUCT INFORMATION AND MANUAL Enzyme linked immunosorbent assay for quantitative detection of mouse TNF a For Research Use Only Not for diagnostic or therapeutic procedures 96 Wells Mouse TNF a Product 455210 944 Nandino Blvd Lexington KY 40511 1205 USA 859 254 1221 or 800 477 8201 USA CANADA Fax 859 255 5532 Email inform neogen com Web www neogen com LifeSciences Technical assistance is available Monday Friday between 8 00 a m and 6 00 p m EST COPYRIGHT All rights reserved worldwide No part of this publication may be reproduced transmitted transcribed or stored in any information retrieval system or translated into any human or computer language in any form or by any means manual electronic mechanical magnetic optical chemical or otherwise without expressed written permission WARRANTY Neogen Corporation makes no warranty of any kind either expressed or implied except that the material from which its products are made are of standard quality If any materials are defective Neogen Corporation will provide a replacement product Buyer assumes all risk and liability resulting from the use of this product and any of the predictive models There is no warranty of merchantability of this product or of the fitness of the product for any purpose Neogen Corporation shall not be liable for any damages including special or consequential damage or expense arising directly or indirectly from the
6. MOUSE TNF a ELISA TEST KIT PROTOCOL Reagent Preparation IMPORTANT All reagents should be brought up to room temperature before use 1 2 hours at 20 25 68 77 F Make sure you read the Warnings and Precautions section on page 3 Solutions should be prepared just prior to ELISA test All reagents should be mixed by gently inverting or swirling prior to use Prepare volumes that are needed for the number of wells being run Do not return the reagents to the original stock tubes bottles Disposable reservoirs are recommended to minimize the risk of contamination 1 Preparation of 1X Assay Diluent Mix 1 volume of 5X Assay Diluent with 4 volumes of distilled water In general 50 mL of 1X Assay Diluent is sufficient for one whole plate Preparation of Standards a Add 10 ul of Standard stock to 1 mL of 1X Assay Diluent to prepare 960 pg mL b Make serial 2X dilutions in the same diluent to make standards of 480 pg mL 240 pg mL 120 pg mL 60 pg mL 30 pg mL and 15 pg mL Discard the remaining unused standards c Use 1X Assay Diluent as negative control Preparation of 1X Wash Solution Mix 1 volume of the 20X Wash Solution with 19 volumes of distilled water Preparation of 1X Detection Antibody Mix 1 volume of 500X Detection Antibody with 499 volumes of 1X Assay Diluent When the 500X Detection Antibody is thawed it can be used for 2 weeks when stored at 4 C If you do not plan to use it for a long tim
7. andling system verify its performance have the system repaired if any ports drip dispense or aspirate poorly Inconsistent incubation times occurred from plate to plate Time each plate separately to ensure consistent incubation times Pipette was inaccurate Check the pipette calibration Verify that pipette tips are on tight before use and that all channels draw and dispense equal volumes Kit plates reagents standards and samples were at different temperatures Make sure to allow sufficient time for kit plates reagents standards and samples to come to room temperature 20 25 68 77 F Larger volumes will require longer equilibration time If using a water bath to hasten equilibration make sure that it is maintained at room temperature do not use a warm water bath to warm reagents samples and kit standards Reagents used were intermixed from different kit lots or the kits were of different expiration dates Carefully label each user prepared reagent to make sure that the reagents are not intermixed Kits with different expiration dates might generate different range of OD readings however the relative absorbance values will typically be comparable In general a value of less than 1 0 reading for the highest standard may indicate deterioration of reagents One or More of the Standard Curve Points Are Out of Range Possible Causes Recommended Action Standards were added i
8. ansformed cell lines and can exert cytotoxic effects against certain solid tumors In vivo TNF a serves as a primary mediator in protective immune responses against microbial and viral pathogens Interestingly TNF a has also been implicated as a central mediator in a number of pathologic responses including septic shock cachexia and autoimmune diseases The specifications of the Mouse TNF a ELISA Test Kit are as follows Format 96 well strip plate Assay range 15 960 pg mL Sensitivity 15 pg mL Total Assay Incubation Time 3 hours and 45 minutes Sample Size 100 uL well Sample types Serum Plasma and Cell Culture Supernatant Wavelength for plate reading 450 nm Procedure Overview The method is a solid phase sandwich ELISA Enzyme Linked Immunosorbent Assay It utilizes a monoclonal antibody capture antibody specific for mouse TNF a coated on a 96 well plate Standards and samples are added to the wells and any TNF a present binds to the immobilized antibody The wells are washed and biotinylated polyclonal anti mouse TNF a antibody detection antibody is added After a second wash avidin horseradish peroxidase avidin HRP is added producing an antibody antigen antibody sandwich The wells are again washed and a substrate solution is added which produces a blue color in direct proportion to the amount of TNF a present in the initial sample The stop buffer is then added to terminate the reaction This results a color change
9. der or near air vents as this may cause excessive cooling heating and or evaporation Also do not run assays in direct sunlight as this may cause excessive heat and evaporation Cold bench tops should be avoided by placing several layers of paper towel or some other insulation material under the assay plates during incubation sure you are using only distilled deionized water since water quality is very important Incubations of assay plates should be timed as precisely as possible Be consistent when adding standards to the assay plate Add your standards first and then your samples Add standards to plate only in the order from low concentration to high concentration as this will minimize the risk of compromising the standard curve Always refrigerate plates in sealed bags with a desiccant to maintain stability Prevent condensation from forming on plates by allowing them to equilibrate to room temperature 20 25 C 68 77 F before opening plates provided in packaging contain dessicant sure samples are properly stored In general samples should be refrigerated at 2 4 C for no more than 1 2 days Freeze samples to 20 C or colder if they need to be stored for a longer period Frozen samples can be thawed at room temperature 20 25 C 68 77 F or in a refrigerator before use Mouse TNF a in plasma sera and cell culture supernatant can be measured directly using this kit without extraction
10. e you should store it at a freezer Repeated freeze thaw cycles may result in reduced antibody activity Preparation of 1X Avidin HRP Mix 1 volume of 500X Avidin HRP with 499 volumes of 1X Assay Diluent When the 500X Avidin HRP is thawed it can be used for 2 weeks when stored at 4 C If you do not plan to use it for a long time you should store it at a freezer Repeated freeze thaw cycles may result in reduced HRP activity ELISA Testing Protocol Label the individual strips that will be used and prepare working solutions of reagents as shown in the following example Adjust the total amount as needed for number of wells that will be used Component Volume per Well 24 Wells 1X Detection Antibody 100 uL 24 mL 1X Avidin HRP 100 uL 2 4 mL 1X Wash Solution 3 0 mL 72 mL Stop Buffer 100 uL 24 mL TMB Substrate 100 pL 24 mL Add 100 ul of 1X Assay Diluent to duplicate wells to serve as negative controls Add 100 uL of each dilution of Mouse TNF a Standards in duplicate into different wells 0 Add standards to plate only in the order from low concentration to high concentration 2 Add 100 uL of each sample in duplicate into different sample wells 10 11 Incubate for 2 hours at room temperature 20 25 C 68 77 F Avoid direct sunlight and cold bench tops during the incubation Covering the plate with aluminum foil while incubating is recommended Aspirate all f
11. he first minute of incubation Do not put any substrate back into the original container which could lead to contamination Any substrate solution exhibiting coloration is indicative of deterioration and should be discarded Covering the plate with aluminum foil while incubating is recommended Add 100 uL of Stop Buffer in the order of adding TMB substrate to stop the enzyme reaction Read the plate as soon as possible following the addition of Stop Buffer Read plate on a 6 plate reader at 450 nm wavelength Before reading wipe the bottom of the plate with a lint free tissue to remove any moisture or fingerprints that could interfere with the reading Mouse TNF a Concentration Calculations A standard curve can be constructed by plotting the adjusted average absorbance for each reference standard against its concentration in pg mL on a logarithmic scale as shown in the figure below The adjusted average absorbance is obtained by subtracting the average of the negative control absorbance from the average of each observed absorbance To determine the corresponding concentration of Mouse TNF a Standard Curve mouse TNF a in pg mL in samples plot the adjusted absorbance value for each sample on the 0 standard curve The figure to the right shows a typical mouse TNF a standard curve m 0 5 Z 1 0 1 5 2 0 10 15 20 25 30 35 Log10 Concentration pg mL TROUBLESHOOTING No Color Development or No Signals with
12. luid from each well after the incubation and wash wells 3 times with 250 uL of 1X Wash Solution per wash It is not necessary to agitate the plate during the wash steps Allow Wash Solution to remain in wells for 1 2 min during each wash step After last wash invert the plate and gently tap the plate on paper towels to remove residual fluid Perform the next step immediately after plate washings Do not allow the plate to air dry between working steps Add 100 uL of 1X Detection Antibody into each well and incubate the plate for 1 hour at room temperature Aspirate and wash the plate 3 times with 250 uL of 1X Wash Solution per wash After the last wash invert the plate and gently tap the plate on paper towels Perform the next step immediately after the third wash Do not allow the plate to air dry between steps Add 100 uL of 1X Avidin HRP into each well Incubate the plate for 30 minutes at room temperature 20 25 C 68 77 F Wash the plate 3 times with 250 uL of 1X Wash Solution per wash After the last wash invert the plate and gently tap the plate dry on paper towels Perform the next step immediately after the third wash Do not allow the plate to air dry between steps Add 100 uL of TMB Substrate and incubate for 15 minutes at room temperature 20 25 68 77 F Start timing the reaction immediately after adding the substrate to the last well Mix the solution by gently rocking the plate manually during t
13. n wrong order or recorded in wrong position Make sure that the standards are applied and recorded correctly Standards were contaminated or intermixed with other standards Prepare a new set of standards Always add standards to plate in the order from low concentration to high concentration There was inconsistent washing or poor liquid handling technique Use the number of washes per the protocol instruction Make sure that 250 uL of wash solution is dispensed per well per wash If you use a multichannel pipette or robotic liquid handling system verify its performance have the system repaired if any ports drip dispense or aspirate poorly Inconsistent time was taken to add standards and reagents to plate Make sure all materials are set up and ready to use Add standards to plate only in the order from low concentration to high concentration at undisrupted constant pace Use a multichannel pipette to add reagents to multiple wells simultaneously to increase consistency Multichannel pipette was not functioning properly Verify pipette calibration and check that tips are on tight Be sure all channels of the pipette draw and dispense equal volumes Neogen Corporation 2012 Neogen is a registered trademark of Neogen Corp Lansing MI D455210 3 12 12
14. of mouse tumor necrosis factor alpha TNF a in serum plasma and cell culture supernatant TNF a is a potent mediator of immune and inflammatory responses It is produced by many activated cell types including monocytes astrocytes granulocytes T and B lymphocytes NK cells keratinocytes fibroblasts and certain tumor cells Activated cells initially express TNF a as a transmembrane protein that associates to form homotrimeric complexes After proteolytic cleavage the extracellular region of membrane TNF a sheds as a soluble homotrimer Membrane and soluble TNF a homotrimers are biologically active whereas monomeric TNF a is not The mature mouse TNF a monomer contains 156 residues has a predicted size of 17 kDa and contains one potential N glycosylation site TNF a exerts its biological activities by binding and signaling through membrane type and II TNF a Receptors TNFR and TNFR Il Interestingly TNF a binds to soluble forms of TNFRI and TNFRII which naturally shed by activated cells and can modulate biological activities of TNF a TNF a exerts many regulatory influences on the activation growth and differentiation of leukocytes and other cells For example TNF a can co stimulate differentiation of activated T and B lymphocytes upregulate the expressed levels of MHC class and class II molecules by various cell types and induce the expression of adhesion molecules by endothelial cells TNF a is selectively cytotoxic for some tr
15. rly This is a high possibility if the OD readings were high and the color was light Verify the reader s performance using a calibration plate and check the lamp alignment Verify the blanking procedure if applicable and reblank Lab temperature was too high Maintain the room temperature within 20 25 C 68 77 F Avoid running assays near heat sources or in direct sunlight Reagents were intermixed contaminated or prepared incorrectly Ensure that the correct reagents were used that working solutions were prepared correctly and that contamination has not occurred High Intra Plate or Inter Plate Variance Possible Causes Recommended Action Inconsistent time was taken when adding standards reagents or samples within and or between plates Make sure that all materials are set up and ready to use Use a multichannel pipette to add reagents to multiple wells whenever possible Do not interrupt procedure while adding standards reagents and samples Multichannel pipette was not functioning properly Verify pipette calibration and check that tips are on tight Be sure all channels of the pipette draw and dispense equal volumes There was inconsistent washing or poor liquid handling technique Use the number of washes per the protocol instruction Make sure that 250 uL of wash solution is dispensed per well per wash If you use a multichannel pipette or robotic liquid h
16. use of this product TABLE OF CONTENTS GENERAL INFORMATION Product a ia Procedure OV CV VLC Sikes sil Kit Contents Storage Shelf Life ss Required Materials Not Provided With the Sensitivity Detection Limit ersccccccciccscsvisessrcisveissrisrsecsiiesssesssestesaivsssssassasiseesiesssvesiasssaais Specificity Cross Reactivity Warnings and Precautions MOUSE TNF a ELISA TEST PROTOCOL eseeseeseseosseseesossesossossesoosoesossossesossossee Re pent a ELISA Testing Protocol Mouse TNF a Concentration Calculations n TROUBLESHOOTING sessssesesseseseseesesosorseseseecesororseseroeeesororseseroreesororseseroeeesoroesesereeeesoreee No Color Development or No Signals with 5 Low Optical Density OD High Background or High Optical Density OD Readings High Intra Plate or Inter Plate Variance s One or More of the Standard Curve Points Are Out of GENERAL INFORMATION Product Description The Mouse TNF a ELISA Test Kit is designed for quantitative determination of the concentration
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