TNF (Human) ELISA Kit
Contents
1. FIFTH STEP READING AND CALCULATION 9 Calculate the mean of reagent blank absorbance values and subtract it from all test well values standard and test samples Mean reagent blank absorbance value at 450 nm should be less than 0 200 10 Calculate your results against standard curve KA0648 9 15 Abnova 1 Prepare all reagents samples and standards Dilution of samples not required at initial screening Summary 2 Add 50 ul standard starting from 1000 pg ml test samples and sample diluent as a blank into the appropriate wells of the strips Incubate 1 hour at room temperature Wash 5x 3 Add 50 Ul ready for use biotin antibody promptly to each well Incubate 30 min at room temperature Wash 5x 4 Add 50 Ul ready for use HRP Streptavidin solution Incubate 30 minutes at room temperature Wash 5x Add 50 ul TMB Substrate Solution to each well Incubate 20 minutes at room temperature 6 Add 25 Ul Stop Solution to each well Read at 450 nm against 630 nm immediately Subtract blank values from values for standards and test samples Correcting for optical imperfections in the microplates by subtracting Asso nm is recommended but not an essential procedure KA0648 10 15 Q Abnova TI TNF a Antibody coated test well Add 50 uL of standards and test samples to test well and STEP 1 incubate 1 hr at Room Temperature STEP 2 Add 50 uL of Biotinylated TNF a antibody to test well and incubate2. 30 min at Room Temperatur C TNF a u 9 P docs TNF a Add 50 uL of HRP Streptavidin to test well and sTEP3 3 incubate 30 minutes at Room Temperature xy TNF a antibody Add 50 uL of TMB substrate to test well incubate 20 minutes at Room Temperature STEP 4 j KA0648 11 15 Abnova Data Analysis Calculation of Results The standard curve must be determined individually for each experiment Correct the absorbance values of all standards by subtracting from them the mean O D value of the reagent blank B1 only sample diluent Calculate the mean absorbance value for each standard from the duplicates The standard curve is used to determine the amount of TNF alpha in an unknown sample The standard curve is generated by plotting the average O D 450 nm obtained for each of the standard concentrations on the vertical Y axis versus the corresponding TNF alpha concentration pg mL on the horizontal X axis Construct the standard curve using graph paper or statistical software If samples generate values higher than the highest standard dilute the samples with sample diluent and repeat the assay Note that the concentration read from the standard curve must be multiplied by the dilution factor Performance Characteristics TNF a Assay range 0 1000 pg ml Standard curve points 1000 500 250 125 62 5 31 25 15 6 and 0 pg ml Intra Assay Precision lt 6 Inter Assay Precision lt 4 Inter Lo
3. 300 ul 30 ml 2 16 wells 1 ml 1 ml 1 ml 600 ul 55 ml 4 32 wells 2ml 2ml 2ml 1 2 ml 110 ml 6 48 wells 3 ml 3 ml 3 ml 1 8 ml 165 ml 8 64 wells 4 ml 4 ml 4 ml 2 4 ml 220 ml 12 96 wells 6 ml 6 ml 6 ml 4 ml 350 ml Sample Preparation Sample preparation and dilution Dilution of samples is not required for initial screening Samples that exceed the measuring range should be diluted in sample diluent serially 1 2 1 4 or further if necessary and measured again The dilution factor must be taken in account when calculating the results Dilute and store all samples in tubes or plates made of material with low binding surface such as polypropylene Sample collection and storage Serum EDTA heparin or citrate anti coagulated plasmas cerebrospinal fluid urine synovial fluid other body fluids and cell culture supernatants are suitable for use in the assay caution separate plasma serum and blood cells within 4 hours after collection non separated samples must be kept at 2 8 C Do not use grossly haemolysed or lipemic specimens If samples are to be run within 24 hours they may be stored at 2 8 C otherwise samples should be stored frozen at least between 18 to 32 C but preferably lt 70 C Up to 3 freeze thaw cycles have no effect on the TNF a levels of samples Nonetheless excessive freeze thaw cycles should be avoided Prior to the assay frozen samples should be thawed as quickly as possible in tap wat
4. C May also be stored at Room Temperature Until expiry date at room temperature Materials Required but Not Supplied Absorbent paper SN NN NN S S S S S Timer Precautions for Use Distilled or de ionized water Micro plate reader capable of measuring absorbance at 450 nm Precision pipettes to deliver 2 ul to 1 ml volumes Multi channel pipet 25 ul to 350 ul Adjustable 1 25 ml pipettes for reagent preparation 100 ml and 1 liter graduated cylinders Tubes to prepare standard or sample dilutions Log log graph paper or computer and software for ELISA data analysis Y This kit has been configured for research use only and is not for diagnostic and clinical use Y Caution TMB substrate Tetramethylbenzidine and the Stop solution H2SO are toxic or corrosive and should be handled with care Use gloves during handling e Procedure Note Yv When not in use kit components should be refrigerated All reagents should be warmed to room temperature before use Y Microtiter plates should be allowed to come to room temperature before opening the foil pouches Y Once the desired number of strips has been removed immediately reseal the pouch and store at 2 8 C to maintain plate integrity Protect from humidity Y Samples should be collected in pyrogen endotoxin free tubes v Samples should be frozen if not analyzed shortly after collection Avoid multiple freeze thaw cycles of frozen samples
5. Thaw completely and mix well prior to analysis KA0648 5 15 s SN SN S S Abnova When possible avoid use of badly hemolyzed or lipemic sera If large amounts of particulate matter are present centrifuge or filter prior to analysis It is recommended that all standards controls and samples be run in duplicate Samples that are gt 400 pg ml should be diluted with Sample Diluent When pipetting reagents maintain a consistent order of addition from well to well This ensures equal incubation times for all wells Cover or cap all reagents when not in use Do not use reagents after the kit expiration date Read absorbances within 20 minutes of assay completion In house controls should be run with every assay If control values fall outside pre established ranges the accuracy of the assay is suspect All residual wash liquid must be drained from the wells by efficient aspiration or by decantation followed by tapping the plate forcefully on absorbent paper Never insert absorbent paper directly into the wells Because TMB Chromogen is light sensitive avoid prolonged exposure to light Also avoid contact between Stabilized Chromogen and metal or color may develop KA0648 6 15 Abnova Assay Protocol Bring all reagents and samples to room temperature 18 25 C before use Reagent Preparation Antibody coated plate Before opening the plastic pouch determine the number of strips required to test the desired numbe
6. a coated onto a 96 well plate Standards samples and biotinylated anti human TNF a are pipetted into the wells TNF a present in a sample is captured by the antibody immobilized to the wells and by the biotinylated TNF a specific detection antibody After washing away unbound biotinylated antibody HRP conjugated streptavidin is pipetted to the wells The wells are again washed Following the second wash step TMB substrate solution is added to the wells resulting in color development proportional to the amount of TNF a bound The Stop Solution changes the color from blue to yellow and the intensity of the color is measured at 450 nm KA0648 3 15 General Information Materials Supplied List of component Abnova Component State Amount 96 Well Plate with 12 Strips Break apart microtiter test strips each with ready to use 1 frame TNF a antibody coated single wells TNF a Standard Lyophilized amp stabilized human TNF a reconstitute p Lyophilized 4 vials with Sample Diluent volume shown on the label Biotinylated TNF a antibody ready to use 10 ml HRP Conjugated Avidin ready to use 12 ml 20x Wash solution concentrate sufficient for 1000 ml Dilute 1 20 concentrated 50 ml Dilution buffer ready to use 100 ml Stop solution 0 9 N H2SO ready to use 8 ml TMB Substrate ready to use 8ml Storage Instruction Reagent Storage Stability TNF a antibody coated 96 well plates with 12 st
7. citrate serum cerebrospinal fluid urine synovial fluid or other body fluids The assay will recognize both natural and recombinant Hu TNF a Background Tumor Necrosis Factor a TNF a is a non glycosylated 17 5 kDa 157 amino acid protein TNF a is a potent lymphoid factor and exerts cytotoxic effects on a wide range of tumor cells and other target cells It is secreted by macrophages monocytes neutrophils T cells and NK cells following their stimulation by bacterial lipopolysaccharides TNF a has been suggested to play a pro inflammatory role and has been detected in synovial fluid of patients with rheumatoid arthritis Various pathological conditions are associated with the production of high levels of TNF a These include septic shock cachexia e g HIV tuberculosis cancer autoimmune diseases hepatitis leukemia myocardialischaemia organ transplantation rejection multiple sclerosis rheumatoid arthritis and meningococcal septicemia Annually many people die from septic shock syndrome triggered by TNF a following complications from an infectious disease In many cases elevated TNF a serum levels predict a higher mortality Principle of the Assay The TNF a ELISA kit is an in vitro enzyme linked immunosorbent assay for the quantitative measurement of human TNF a in cell culture supernatants serum plasma cerebrospinal fluid urine synovial fluid and other body fluids This assay employs an antibody specific for human TNF
8. each well with 300 ul of diluted wash buffer then empty plate contents again Repeat procedure 4 additional times for a total of FIVE washes Gently blot plate onto paper towels or other absorbent material Never let reaction wells dry Continue to the next step without delay or interruption For automated washing Aspirate all wells and wash 5 times with 300 ul diluted wash buffer Blot plate onto paper towels or other absorbent material Never let reaction wells dry Continue to the next step without delay or interruption 4 Promptly add 50 ul of green colored Biotinylated TNF alpha detection antibody to all wells Tap the plate gently by hand to homogenize your mixture Avoid touching to the reaction wells with the pipette tip Incubate at room temperature for 30 minutes without shaking THIRD STEP HRP CONJUGATED AVIDIN 5 Wash 5 times 5x as described in Step 3 Add 50 yl of prepared HRP conjugated avidin solution ready to use to each well Incubate for 30 minutes at room temperature FOURTH STEP TMB SUBSTRATE Wash 5 times as described in Step 3 7 Using a multichannel pipette promptly add 50 ul of TMB ready to use substrate reagent to each well Incubate for 20 minutes at room temperature in the dark 8 Add 25 ul of Stop Solution to each well Read at 450 nm within 15 minutes Correcting for optical imperfections in the microplates by subtracting Ass nm is recommended but not an essential procedure
9. pipetting error Large CV Inaccurate pipetting and drying of Check pipettes Fill the wells promptly with wash buffer and reagents High background 1 Plate is insufficiently washed 2 Contaminated wash Buffer 3 Wash buffer volume is less than advised Review the manual for proper wash If using a plate washer check that all ports are unobstructed and clean Make a fresh wash buffer Use 300pl per well Low sensitivity 1 Improper storage of the ELISA kit 2 Stop solution 3 Contamination of reagents Store test kit components as advised in this user manual Keep substrate solution protected from light Stop solution should be added to each well before measure Discard Use clean sterile tips contaminated reagents KA0648 13 15 Abnova References 1 Seriolo B Paolino S Sulli A Fasciolo D Cutolo M 2006 Effects of anti TNF alpha treatment on lipid profile in patients with active rheumatoid arthritis Ann N Y Acad Sci 1069 414 9 2 Intiso D Zarrelli MM Lagioia G Di Rienzo F Checchia De Ambrosio C Simone P Tonali P Cioffi Dagger RP 2004 Tumor necrosis factor alpha serum levels and inflammatory response in acute ischemic stroke patients Neurol Sci 24 390 396 Beutler B et al 1987 Cachectin more than a tumor necrosis factor N Engl J Med 316 379 385 4 Tracey K J et al 1987 Anti cachectin TNF monoclonal antibodies prevent septic shock during lethal ba
10. TNF Human ELISA Kit Catalog Number KA0648 96 assays Version 02 Intended for research use only www abnova com Table of Contents Soligor Uieira etm 3 WPS MOLE c 3 Background rr 3 Principle OF the ASSAY met Tm 3 General Information m 4 fzitzizi Ee ee 4 Storage neis e H 4 Materials Required but Not Supplied eeeeesssseeessneeeeeeeneeeren 5 Precautions for US e eenn 5 PSSAY ger c Q 7 Reagent Preparation saec esciepede etes ce eS AE Eden ise ect beum o eas e Dn SEENE EANTA D dee E ERE EI ERES 7 Sample PRED ar AM OM ass eissis a a aea 8 Assay POCO CUS is asinn innean aea E aA a a EAE RAA AEE A TA A 9 Dat An lySiS pe 12 Calculation of ROSUNG EET RT 12 Performance Characteristics eeeeeseessssssssesseseeseee eene nennen nennen 12 alnj plM easanere 13 TG UDISS HOO TG PRI RIED 13 REIGIENCOS araona e A T S 14 Plate Layout FEM 15 KA0648 2 15 Abnova Introduction Intended Use For quantitative detection of human TNF a in cell culture supernatants human plasma EDTA heparin and
11. cteraemia Nature 330 662 664 5 Piguet P F et al 1987 Tumor necrosis factor cachectin is an effector of skin and gut lesions of the acute phase of graft versus host disease J Exp Med 166 1280 1289 Aukrust P et al 1994 Serum levels of tumor necrosis factor a TNF a and soluble TNF receptors in human immunodeficiency virus type 1 infection correlations to clinical immunologic and virologic parameters J Inf Dis 169 420 424 8 Waage A et al 1987 Association between tumor necrosis factor in serum and fatal outcome in patients with meningococcal disease Lancet 1 355 357 9 Noraz N et al 1997 Human cytomegalovirus associated immunosuppression is mediated through IFN a Blood 89 7 2443 2452 10 Lekutis C et al 1998 HIV 1 env DNA vaccine administered to rhesus monkey elicits MHC class Il restricted CD4 T helper cells that secrete IFN y and TNF a J Immunol 158 4471 4477 11 Neuman M G et al 1998 Role of cytokines in ethanol induced cytotoxicity in HepG2 cells Gastroenterology 114 7 157 169 12 Dong Z et al 1998 Activation of cytokine production tumoricidal properties and tyrosine phosphorylation of MAPKs in human monocytes by a new synthetic lipopeptide JBT3002 J Leukocyte Biol 63 766 774 13 Murato PA et al 1997 Immunodominance of a low affinity major histocompatibility complex binding myelin basic protein epitope Residues 111 129 in HLA DR4 B1 0401 Subjects is associated with a
12. er 18 25 C do not use 37 C or 56 C water bath for this purpose Preparation of reagents v Wash Buffer If the 20x concentrated Wash Buffer contains visible crystals warm it at 37 C and mix gently until dissolved Dilute 1 20 with de ionized or distilled water e g 25 ml of Wash Buffer Concentrate and 475 ml distilled water to yield 500 ml of 1x Wash Buffer Check the pH of the diluted wash buffer and adjust to 7 4 if necessary Y Vortex mix Biotinylated antibody solution gently before use Y Vortex mix peroxidase HRP labeled avidin gently before use Caution TMB substrate Tetramethylbenzidine and the Stop solution H2SO are toxic or corrosive and should be handled with care Use gloves during handling KA0648 8 15 Abnova Assay Procedure 1 Bring all reagents and samples to room temperature 18 25 C before use It is recommended that all standards and samples be run at least in duplicate Leave some wells as a reagent blank 2 to 4 wells FIRST STEP STANDARD SAMPLES AND BLANK BIOTINYLATED ANTIBODY 2 Pipette 50 ul of sample and 50 ul of each diluted standard starting from 1000 pg ml into appropriate wells Pipette 50 ul of sample diluent to the wells which will be used as a blank Incubate 1 hr at room temperature without shaking SECOND STEP BIOTINYLATED ANTIBODY 3 Wash 5x with 1x Wash Solution 300 ul each To wash manually Empty plate contents Use a multi channel pipette to fill
13. r of samples plus 16 wells needed for running standards and blanks in duplicate Remove non used strips from the plate frame and return them to the foil pouch containing the desiccant for up to 3 month at 2 8 C Dilution of test standard Dissolve the lyophilised TNF a standard with Sample Diluent volume shown on the label TNF a standard is unstable after dissolving Use immediately or keep on ice if not used within 1 hr after dissolving To obtain a standard curve dilute it as follows v Add 300 ul of TNF a standard from kit standard tube containing 1000 pg ml of TNF a Standard tube 1 v Add 150 ul of Sample Diluent to all other 6 dilution tubes Take 150 ul from the first tube 1000 pg ml and start 2 fold serial dilutions in dilution tubes as described in the figure by mixing several times with the pipet in each tube Total of 7 dilution tubes Y 150ylof sample Diluent in tube 8 serves as zero standard 0 pg ml 300 ul of TNF a Standard from dissolved stock 1000 pg ml cH 150 ul 150 ul 130 ul 1000 pg ml 500 250 125 62 5 31 25 15 6 Only Sample pg ml pg ml pg ml pg ml pg ml pg ml Diluent as a blank KA0648 7 15 Abnova Amounts of the reagents needed to perfom the test Reagents No of strips used Biotinylated Avidin HRP TMB substrate Stop Solution Wash Buffer 8 well each antibody 50 pl well 50 ul well 25 ul well 300 ul well 50 ul well 1 8 wells 500 ul 500 ul 500 ul
14. restricted T cell receptor repertoire J Clin Invest 100 2 339 349 14 Ludviksson B R et al 1998 Active Wegener s granulomatosis is associated with HLA DR CD4 T cells exhibiting an unbalanced Th1 type T cell cytokine pattern Reversal with IL 10 J Immunol 160 3602 3609 KA0648 14 15 Plate Layout 12 11 10 KA0648 15 15
15. rips Break apart microtiter test strips each with 8 antibody coated Store at 2 8 C in closed aluminum bag with desiccant Strips which are not used must be stored in the re sealable aluminum bag in 3 months after opening single wells humidity free and airtight conditions TNF alpha Standard Store at 2 8 C Until date of kit expiry in lyophilized Lyophilized format Unstable Use immediately after dissolving Keep on ice if not used within 1 hr after dissolving Biotinylated antibody Ready for use Store at 2 8 C Avoid contamination Use clean sterile tips 3 months after opening HRP Conjugated Avidin Ready for use Store at 2 8 C Avoid contamination Use clean sterile tips 3 months after opening Sample Diluent Store at 2 8 C Avoid contamination Use clean sterile tips or pipettes 3 months after opening 20x Concentrated Wash Buffer Store at 2 8 C To avoid crystal formation wash buffer concentrate may also be stored at Room Temperature Until expiry date KA0648 4 15 Abnova Diluted Wash Buffer 1x working dilution Bottles used for the working dilution should be cleaned regularly discard cloudy solutions 1 week at room temperature or one month at 2 8 C TMB Substrate Solution Store at 2 8 C protected from light Avoid contamination Use clean sterile tips Until expiry date Stop Solution Store at 2 8
16. t Precision lt 8 Cross Reactivity No cross reactivity was observed with the following recombinant human proteins IL 18 IL 1a IL 2 IL 3 IL 4 IL 5 IL 6 IL 7 IL 8 IL 9 IL 10 IL 12 IL 13 TARC Interferences No interferences to bilirubin up to 0 3 mg mL haemoglobin up to 8 0 mg mL and triglycerides up to 5 0 mg mL Specificity Recognizes both natural and recombinant human TNF a Sensitivity lt 15 pg ml TNF a levels may vary greatly between different study groups and sample types such as serum samples cell culture supernatant cell extracts or other biological samples Each research study should include a proper control group age sex locality or geographical region matched to establish more precise TNF a values Disease status or the use of drugs or TNF a stimulating agents may interfere with the TNF a levels and should be taken into careful consideration in all studies KA0648 12 15 Resources Troubleshooting Abnova Problem Cause Solution Poor standard Curve 1 Inaccurate pipetting or pipetting error 2 Improper standard dilution Check pipettes and calibrate regularly Vortex the stock before use and dilute carefully in an eppendorf tube wells during test procedure Low signal 1 Shorter incubation than Ensure sufficient incubation time recommended Check pipettes and ensure correct 2 Inadequate reagent volumes or performance improper dilution or
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