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Human TNF-a

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1. EA 15 ead dd E ee ee 16 15 TROUBLESHOOTING 17 AviBion Human TNF a T 2207 2 08 JJyour customized diagnostic solutions 1 INTENDED USE Orgenium Laboratories TNF a ELISA is an enzyme linked immunosorbent assay for quantitative detection of human TNF a in cell culture supernatants human plasma EDTA heparin and citrate serum cerebrospinal fluid urine synovial fluid or other body fluids The assay will recognize both natural and recombinant Hu TNF a 2 INTRODUCTION 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
2. a see label for stock concentration Add 1 ml of Sample Diluent before use Biotinylated TNF a antibody Ready for use HRP Conjugated Avidin Ready for use 20x Washing solution concentrate sufficient for 1000ml Dilute 1 20 Dilution buffer Ready for use Stop solution 2 N H2SO Ready for use TMB Substrate Ready for use Quality Control Certificate AviBion Human TNF a ELISA 4 17 Rev 2 08 100 ml ml ml ar JJyour customized diagnostic solutions 4 STORAGE AND STABILITY Stability TNF a antibody coated 96 well plates with 12 strips aluminum bag with desiccant Store at 2 8 C in closed 3 months after opening Break apart microtiter test Strips which are not used strips each with 8 antibody must be stored in the re coated single wells sealable aluminum bag in humidity free and airtight conditions Store at 2 8 C Until date of kit expiry in TNF alpha Standard lyophilized format Lyophilized At least 3 weeks after dissolving with sample diluent Store at 2 8 C 3 months after opening Avoid contamination Use clean sterile tips Store at 2 8 C 3 months after opening Avoid contamination Use clean sterile tips Store at 2 8 C 3 months after opening Avoid contamination Use clean sterile tips or pipettes Store at 2 8 C Until expiry date To avoid crystal formation wash buffer concentrate may also be stored at Room Biotinylated antibody Ready for use HR
3. ml AviBion Human TNF a ELISA eu _ Rev 2 08 l Orgenium your customized diagnostic solutions 3 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 4 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 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 water 18 25 C do not use 37 C or 56 C water bath for this purpose 5 Preparation of reagents a Wash Buffer If the 20x concentrated Wash Buffer contain
4. samples to room temperature 18 25 C before use It 15 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 250 pg mL see page 7 into appropriate wells Pipette 50 ul of sample diluent to the wells which will be used as a blank Incubate 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 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 shaki
5. AviBion Human TNF a ELISA User Manual REF TNFa021 NE Regulatory Status For research use only RUO Please contact Orgenium s customer service representatives for inquiries feedback or about non conforming products uud Orgenium Laboratories Viikinkaari 6 FIN 00790 Helsinki FINLAND Tel 358 9 319 36450 Fax 358 20 781 8175 www orgenium com CustomerCare orgenium com AviBion Human TNF a ELISA 1 1 7 your customized diagnostic solutions Rev02 08 TABLE OF CONTENTS INIENDED ado ores gaa sp sce tongue 3 2e ENTRODUC PEION ius cicer 3 Se CON TEING CHE KIT i ive S 4 4 STORAGE AND STABILITY 6 i EPA REC 5 5 ADDITIONAL MATERIALS REQUIRED ee eee eee eee eee ooo eee e ooo sooo essc cce O 6 AMOUNT OF THE REAGENTS NEEDED TO PERFORM THE TEST 6 7 REAGENT AND SAMPLE PREPARATION eee eere neenon NI 8 TEST PROCEDURE SUMMARY 5e eere segre pore n eee enw 9 PROCEDURAL NOLES UNE ENET 10 I ASSAY PROCEDURE II CALCUEATION OE RESULTS eenPbstuPPESEQVMVERMESCPUF RR qET VER EU 13 TYP AGDA rc R 14 13 TEST PERPORNEAN CE
6. P Conjugated Avidin Ready for use Sample Diluent 20x Concentrated Wash Buffer Temperature Diluted Wash Buffer Ix working dilution week at room temperature or one month at 2 8 C Bottles used for the working dilution should be cleaned regularly discard cloudy solutions Store at 2 8 C protected from Until expiry date light Avoid contamination TMB Substrate Solution Use clean sterile tips Store at 2 8 C Until expiry date at room May also be stored at Room temperature Temperature Stop Solution AviBion Human TNF a m 1227 2 08 l Orgenium your customized diagnostic solutions 5 ADDITIONAL MATERIALS REQUIRED e Microplate reader capable of measuring absorbance at 450 nm Precision pipettes to deliver 2 ul to 1 ml volumes e Multi channel pipet 25 ul to 350 ul e Adjustable 1 25 ml pipettes for reagent preparation 100 ml and 1 liter graduated cylinders e Absorbent paper e Distilled or de ionized water Log log graph paper or computer and software for ELISA data analysis e Tubes to prepare standard or sample dilutions e Timer 6 AMOUNTS OF THE REAGENTS NEEDED TO PERFOM THE TEST Reagents No of strips used Biotinylated antibody Avidin HRP TMB substrate Stop Wash Buffer with 8 well each Solution 50 ul well 50ul well 50 ul well 300 ul well 25 ul well 1 8 wells 500 ul 500 ul 500 uL 300 ul AviBion Human TNF a ELISA 227 6 17 2 08 0 O
7. and clean 3 Wash buffer volume is less than advised Make a fresh wash buffer Use 300ul per well Low sensitivity 1 Improper storage of the ELISA Store test kit components as kit advised in this user manual Keep substrate solution protected from light 2 Stop solution Stop solution should be added to each well before measure Use clean sterile tips Discard 3 Contamination of reagents contaminated reagents AviBion Human TNF a ELISA 1227 17 17 2 08 l Orgenium your customized diagnostic solutions
8. dards and test samples Correcting for optical imperfections in the microplates by subtracting nm is recommended but not an essential procedure THE HEA 1227 2 08 l Orgenium JJyour customized diagnostic solutions TEST PRINCIPLE 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 2 Add 50 uL of Biotinylated TNF a antibody to test well and wa 30 min at Room Temperatur TNF a 9 hm TNF a Add 50 uL of HRP Streptavidin to test well and STEP 3 3 incubate 30 minutes at Room Temperature Y TNF a antibody HRP Streptavidin Add 50 uL of TMB substrate to test well incubate 20 minutes at Room Temperature STEP 4 TMB substrate AviBion Human TNF a ELISA 1227 10 17 2 08 l Orgenium your customized diagnostic solutions 9 PROCEDURAL QUALITY CONTROL When not in use kit components should be refrigerated reagents should be warmed to room temperature before use Microtiter plates should be allowed to come to room temperature before opening foil bags e Once the desired number of strips has been removed immediately return unused strips to the bag reseal the bag and store at 2 8 C to maintain plate integrity Protect from humidity e Samples should be collected in pyrogen endotoxin free tubes e Samples shou
9. is 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 Tracey K J et al 1987 Anti cachectin TNF monoclonal antibodies prevent septic shock during lethal bacteraemia Nature 330 662 664 Piguet P F et al 1987 Tumor necrosis factor cachectin 15 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 Waage A et al 1987 Association between tumor necrosis factor in serum and fatal outcome in patients with meningococcal disease Lancet 1 355 357 Noraz N et al 1997 Human cytomegalovirus associated immunosuppression is mediated through IFN a Blood 89 7 2443 2452 Lekutis C et al 1998 HIV 1 env DNA vaccine administered to rhesus monkey elicits MHC class II restricted CD4 T helper cells that secrete IFN y and TNF a J Immunol 158 4471 4477 Neuman et al 1998 Role of cytokines in ethanol induced cytotoxicity in HepG2 cells Gastroenterology 114 7 157 169 Dong Z et al 1998 Activation of cytokine production tumoricidal properties and tyrosine phosphoryla
10. ld be frozen if not analyzed shortly after collection Avoid multiple freeze thaw cycles of frozen samples Thaw completely and mix well prior to analysis e When possible avoid the use of badly hemolyzed or lipemic sera If large amounts of particulate matter are present centrifuge or filter prior to analysis 15 recommended that all standards controls and samples are run in duplicate e Samples that are above the detection limit should be diluted with sample diluent e When pipetting reagents maintain a consistent order of addition from well to well This ensures equal incubation times for all wells e Cover or cap all reagents when not in use Do not use reagents past their expiration date e Read absorbencies within 20 minutes of assay completion e In house controls should be run with every assay If control values fall outside pre established ranges the accuracy of the assay is suspect residual wash buffer 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 e Because TMB substrate solution is light sensitive avoid prolonged exposure to light Also avoid contact between TMB substrate solution and metal or color may develop AviBion Human TNF a ELISA 2207 11 17 2 08 0 Orgenium your customized diagnostic solutions 10 ASSAY PROCEDURE 1 Bring all reagents and
11. mg mL and triglycerides up to 5 0 mg mL Specificity Recognizes both natural and recombinant human TNF a Sensitivity Expected values in TNF alpha serum levels in healthy controls was found healthy individuals between 1 to 4 pg ml Expected values in infection suspected individuals The average amount of TNF a in patients with a respiratory tract infection with an unknown cause was with Orgenium s TNF a ELISA kit found to be 40 pg ml range between 6 to 380 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 AviBion Human TNF a ELISA 1227 15 17 2 08 l Orgenium JJyour customized diagnostic solutions 14 REFERENCES 1 Seriolo B Paolino S Sulli 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 3 10 11 12 13 14 15 Intiso D Zarrelli MM Lagioia G Di Rienzo Checchia De Ambrosio C Simone P Tonali P Cioffi Dagger RP 2004 Tumor necros
12. ng THIRD STEP HRP CONJUGATED AVIDIN 5 Wash 5 times 5x as described in Step 3 Add 50 ul of prepared HRP conjugated avidin solution ready to use to each well Incubate for 30 minutes at room temperature FOURTH STEP TMB SUBSTRATE 6 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 9 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 Asso Is recommended but not an essential procedure AviBion Human 227 2 08 0 Orgenium your customized diagnostic solutions H FIFTH STEP READING AND CALCULATION 10 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 11 Calculate your results against standard curve as outlined below 11 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 Bl 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 s
13. rgenium your customized diagnostic solutions 7 REAGENT AND SAMPLE PREPARATION Note All reagents and samples must be allowed to equilibrate to room temperature 18 25 C before use 1 Antibody coated plate Before opening the foil pouch determine the number of strips required to test the desired number 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 months at 2 8 C 2 Dilution of test standard Dissolve lyophilised TNF a standard with 1 ml of Sample Diluent TNF a standard is stable for at least 3 weeks after dissolving To obtain a standard curve dilute it as follows 1 Take 300 ul of TNF a standard from kit standard tube containing 250 pg ml of TNF a and pipette into Standard tube 1 2 Add 150 ul of Sample Diluent to all other 6 dilution tubes Take 150 ul from first tube 250 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 c 150 ul of sample Diluent serves as zero standard 0 ng ml in tube 8 300 ul of TNF a Standard 250 pg ml Only 150 pl of i dn Sample Diluent as a blank 150ul PE 150 1500 150 1504 150ul ze L d 250 125 62 5 31 25 15 6 7 8 3 9 0 pg ml pg ml pg ml pg ml pg ml pg ml pg ml pg
14. s 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 1f necessary b Vortex mix Biotinylated antibody solution gently before use c Vortex mix peroxidase HRP labeled avidin gently before use Caution TMB substrate Tetramethylbenzidine and Stop solution 5 4 are toxic or corrosive and should be handled with care Use gloves during handling AviBion Human TNF a ELISA 1227 8 17 Rev 2 08 l Orgenium your customized diagnostic solutions 8 TEST PROCEDURE SUMMARY 1 Prepare all reagents samples and standards Dilution of samples not required at initial screening 2 Add 50 ul standard starting from 250 pg mL test samples and sample diluent as a blank into the appropriate wells of the strips Incubate 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 5 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 stan
15. shock syndrome triggered by TNF a following complications from an infectious disease In many cases elevated TNF a serum levels predict a higher mortality Orgenium Laboratories human 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 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 1s measured at 450 nm AviBion Human TNF a ELISA 1227 3 17 2 08 Orgenium your customized diagnostic solutions 3 CONTENTS OF THE KIT Test components Amount Volume 96 Well Plate with 12 Strips Break apart microtiter test strips each with TNF a antibody coated single wells Ready for use TNF a Standard Lyophilized amp Stabilized Recombinant Human TNF
16. tandard curve 15 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 AviBion Human TNF a ELISA V 13 17 Rev 2 08 0 Orgenium JJyour customized diagnostic solutions 12 TYPICAL DATA The following standard curve is obtained for various concentrations of TNF a standard over the range of 0 to 250 pg mL Please note The curve is provided for illustration only A standard curve should be generated each time the assay is performed Absorbance 0450 0 50 100 150 200 250 300 TNF alpha Standard pg ml AviBion Human TNF a ELISA N 14 17 Rev 2 08 Orgenium your customized diagnostic solutions 13 TEST PERFORMANCE Standard curve points 250 125 62 5 31 25 15 62 7 8 3 9 and 0 pg ml Intra Assay Precision No cross reactivity was observed with the following Cross Reactivity recombinant human proteins IL 18 IL 1o IL 2 IL 3 IL 4 IL 5 IL 6 IL 7 IL 8 IL 9 IL 10 12 13 c No interferences to bilirubin up to 0 3 mg mL haemoglobin up to 8 0
17. tion of MAPKs in human monocytes by a new synthetic lipopeptide JBT3002 J Leukocyte Biol 63 766 774 Murato 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 restricted T cell receptor repertoire J Clin Invest 100 2 339 349 Ludviksson B R et al 1998 Active Wegener s granulomatosis 15 associated with HLA DR CD4 T cells exhibiting an unbalanced Thl type T cell cytokine pattern Reversal with IL 10 J Immunol 160 3602 3609 AviBion Human TNF a ELISA 1227 16 17 2 08 l Orgenium your customized diagnostic solutions 15 TROUBLE SHOOTING Poor standard Curve 1 Inaccurate pipetting or Check pipettes and calibrate pipetting error regularly 2 Improper standard dilution Vortex the stock before use and dilute carefully in an eppendorf tube Low signal 1 Shorter incubation than Ensure sufficient incubation recommended time 2 Inadequate reagent Check pipettes and ensure volumes or improper correct performance dilution or pipetting error Large CV Inaccurate pipetting and drying of Check pipettes wells during test procedure Fill the wells promptly with wash buffer and reagents High background 1 Plate 15 insufficiently Review the manual for proper washed wash If using a plate washer 2 Contaminated wash check that all ports are Buffer unobstructed

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