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|Application / Reactivity||Human|
|ELISA||5 ELISA Kits|
|Antigen||Tumor Necrosis Factor Receptor Superfamily, Member 10c, Decoy Without An Intracellular Domain (TNFRSF10C) ELISA Kits|
Kits with alternative reactivity to:
|Methode Type||Sandwich ELISA|
|Minimum Detection Limit||147 pg/mL|
|Supplier||Log in to see|
Product Details TNFRSF10C ELISA KitTarget details Application Details Handling Images
|Purpose||The human TRAIL R3/DcR1 ELISA is a solid phase sandwich ELISA for the in-vitro qualitative and quantitative determination of TRAIL R3/DcR1 in cell culture supernatants, buffered solutions or human serum, plasma, or other body fluids. This assay will recognize both natural and recombinant human TRAIL R3/DcR1.|
|Sample Type||Cell Culture Supernatant, Serum, Plasma, Biological Samples|
|Specificity||The assay recognizes natural and recombinant human TRAIL R3. To define specificity of this ELISA, several proteins were tested for cross reactivity. There was no cross reactivity observed for any protein tested: TRAIL, CD117, IL-6R, IL-2R, CD116, TRAIL R2, TRAIL R1, TRAIL R4, CD178 and Granzyme B.|
|Sensitivity||The sensitivity, minimum detectable dose of human TRAIL R3 using this TRAIL R3 ELISA kit was found to be 147 pg/mL. This was determined by adding 3 standard deviations to the mean OD obtained when the zero standard was assayed 32 times.|
96 well microtitre strip plate Ready to use (Pre-coated)
Plastic plate covers
Standard: 10 000pg/ml, Reconstitute as directed on the vial
Standard Diluent (Buffer), 10x Concentrate, dilute in distilled water
Biotinylated anti-TRAIL R3, Dilute in Biotinylated Antibody Diluent
Biotinylated Antibody Diluent Ready to use
Streptavidin-HRP, add 0.5ml of HRP diluent prior to use
HRP Diluent Ready to use
Wash Buffer, 200x Concentrate dilute in distilled water
TMB Substrate Ready to use
H2SO4 stop reagent Ready to use
|Material not included||
Microtiter plate reader fitted with appropriate filters (450nm required with optional 630nm reference filter)
Microplate washer or wash bottle
10, 50, 100, 200 and 1,000 μL adjustable single channel micropipettes with disposable tips
50-300 μL multi-channel micropipette with disposable tips
Multichannel micropipette reagent reservoirs
Miscellaneous laboratory plastic and/or glass, if possible
Target detailsProduct Details TNFRSF10C ELISA Kit Application Details Handling Images back to top
|Alternative Name||TRAILR3_DcR1 (TNFRSF10C ELISA Kit Abstract)|
|Background||Human Trail R3, also called DcR1, CD263, LIT, or TRID is a glycosyl-phosphatidylinositol-linked membrane protein which binds TRAIL with high affinity. In the Trail receptor family, Trail R3 (DcR1) but also Trail R4 (DcR2) antagonize TRAIL-induced apoptosis whereas Trail R1 (DR4) and Trail R2 (DR5) transduce an apoptosis signal. Trail R3 has extracellular TRAIL-binding cystein-rich domain but lacks intracellular signalling domain. Expression of Trail R3 has been shown to protect cells bearing Trail R1 and/or Trail R2 from Trail-induced apoptosis. Many tumor cell lines don't express the decoy receptors and are therefore sensitive to TRAIL if they express Trail R1 and/or Trail R2.|
Application DetailsProduct Details TNFRSF10C ELISA Kit Target details Handling Images back to top
|Application Notes||Optimal working dilution should be determined by the investigator.|
Spike recovery: The spike recovery was evaluated by spiking concentrations of natural TRAIL R3 in human serum. Recoveries ranged from 103% to 122% with an overall mean recovery of 115%. t 12.6. Expected Serum Values TRAIL R3 is not detected in healthy donor sera (95 sera tested).
|Plate||Pre-coated,Strips (16 x 6)|
|Protocol||A capture Antibody highly specific for TRAIL R3 has been coated to the wells of the microtiter strip plate provided during manufacture. Binding of TRAIL R3 in samples and known standards to the capture antibodies is completed and then any excess unbound analyte is removed. During the next incubation period the binding of the biotinylated anti- TRAIL R3 secondary antibody to the analyte occurs. Any excess unbound secondary antibody is then removed. The HRP conjugate solution is then added to every well including the zero wells, following incubation excess conjugate is removed by careful washing. A chromogen substrate is added to the wells resulting in the progressive development of a blue coloured complex with the conjugate. The colour development is then stopped by the addition of acid turning the resultant final product yellow. The intensity of the produced coloured complex is directly proportional to the concentration of TRAIL R3 present in the samples and standards. The absorbance of the colour complex is then measured and the generated OD values for each standard are plotted against expected concentration forming a standard curve. This standard curve can then be used to accurately determine the concentration of TRAIL R3 in any sample tested|
Bring all reagents to room temperature before use
8.1.Assay Design Determine the number of microwell strips required to test the desired number of samples plus appropriate number of wells needed for running zeros and standards. Each sample, standard and zero should be tested in duplicate. Remove sufficient microwell strips for testing from the pouch immediately prior to use. Return any wells not required for this assay with desiccant to the pouch. Seal tightly and return to 2-8 °C storage.
8.2.Preparation of Wash Buffer Dilute the (200x) wash buffer concentrate 200 fold with distilled water to give a 1x working solution. Pour entire contents (10 mL) of the Washing Buffer Concentrate into a clean 2,000 mLgraduated cylinder. Bring final volume to 2,000 mLwith glass-distilled or deionized water. Mix gently to avoid foaming. Transfer to a clean wash bottle and store at 2°-8 °C for up to 1 week.
8.3.Preparation of Standard Diluent Buffer Add the contents of the vial (10x concentrate) to 225 mL of distilled water before use. This solution can be stored at 2-8 °C for up to 1 week.
8.4.Preparation of Standard Standard vials must be reconstituted with the volume of standard diluent shown on the vial immediately prior to use. This reconstitution gives a stock solution of 10 000 pg/mL of TRAIL R3. Mix the reconstituted standard gently by inversion only. Serial dilutions of the standard are made directly in the assay plate to provide the concentration range from 10 000 to 312. 5pg/mL. A fresh standard curve should be produced for each new assay. Immediately after reconstitution add 200 μL of the reconstituted standard to wells A1 and A2, which provides the highest concentration standard at 10 000 pg/mL Add 100 μL of Standard Diluent to the remaining standard wells B1 and B2 to F1 and F2 Transfer 100 μL from wells A1 and A2 to B1 and B2. Mix the well contents by repeated aspirations and ejections taking care not to scratch the inner surface of the wells Continue this 1:1 dilution using 100 μL from wells B1 and B2 through to wells F1 and F2 providing a serial diluted standard curve ranging from 10 000pg/mL to 312.5pg/mL Discard 100 μL from the final wells of the standard curve (F1 and F2) Alternatively these dilutions can be performed in separate clean tubes and immediately transferred directly into the relevant wells.
8.5.Preparation of Samples Before testing, human serum or plasmas samples have to be diluted 1:2 in standard buffer diluent.
8.6.Preparation of Biotinylated Anti TRAIL R3 It is recommended this reagent is prepared immediately before use. Dilute the biotinylated anti TRAIL R3 with the biotinylated antibody diluent in an appropriate clean glass vial using volumes appropriate to the number of required wells.
8.7.Preparation of HRP-Conjugate It is recommended to centrifuge vial for a few seconds in a microcentrifuge to collect all the volume at the bottom. Dilute the 5 μL vial with 0.5 mL of HRP diluent immediately before use. Do-not keep this diluted vial for future experiments. Further dilute the HRP solution to volumes appropriate for the number of required wells in a clean glass vial.
Cell culture supernatants, serum, plasma or other biological samples will be suitable for use in the assay
Remove serum from the clot or red cells, respectively, as soon as possible after clotting and separation. Cell culture supernatants: Remove particulates and aggregates by spinning at approximately 1000 x g for 10 min
Serum: Use pyrogen/endotoxin free collecting tubes
Serum should be removed rapidly and carefully from the red cells after clotting. Following clotting, centrifuge at approximately 1000 x g for 10 min and remove serum. Plasma: EDTA, citrate and heparin plasma can be assayed
Spin samples at 1000 x g for 30 min to remove Storage: If not analyzed shortly after collection, samples should be aliquoted (250-500 μL) to avoid repeated freeze-thaw cycles and stored frozen at -70 °C
Avoid multiple freeze-thaw cycles of frozen specimens. Recommendation: Do not thaw by heating at 37 °C or 56 °C. Thaw at room temperature and make sure that sample is completely thawed and homogeneous before use. When possible avoid use of badly haemolysed or lipemic sera. If large amounts of particles are present these should be removed prior to use by centrifugation or filtration. 7
We strongly recommend that every vial is mixed thoroughly without foaming prior to use except the standard vial which must be mixed gently by inversion only. Prepare all reagents as shown in section
8.Note: Final preparation of Biotinylated anti TRAIL R3 and Streptavidin-HRP should occur immediately before use.
1.Addition Prepare Standard curve as shown in section 8.4
2.Addition Add 100 μL of Standard Diluent to zero wells and 100 μL of Sample to sample wells
3.Incubation Cover with a plastic plate cover and incubate at room temperature (18 to 25 °C) for 2 hour(s)
4.Wash Remove the cover and wash the plate as follows: a) Aspirate the liquid from each well b) Dispense 0.3 mLof 1x washing solution into each well c) Aspirate the contents of each well d) Repeat step b and c another two times
5.Addition Add 50 μL of diluted biotinylated anti TRAIL R3 to all wells
6.Incubation Cover with a plastic plate cover and incubate at room temperature (18 to 25 °C) for 1 hour
7.Wash Repeat wash step
4.8. Addition Add 100 μL of Streptavidin-HRP solution into all wells
9.Incubation Cover with a plastic plate cover and incubate at room temperature (18 to 25 °C) for 30 min
10.Wash Repeat wash step
4.11. Addition Add 100 μL of ready-to-use TMB Substrate Solution into all wells
12.Incubation Incubate in the dark for 10-20 minutes* at room temperature. Avoid direct exposure to light by wrapping the plate in aluminium foil.
13.Addition Add 100 μL of H2SO4:Stop Reagent into all wells Read the absorbance value of each well (immediately after step 14.) on a spectrophotometer using 450 nm as the primary wavelength and optionally 620 nm as the reference wave length (610 nm to 650 nm is acceptable). *Incubation time of the substrate solution is usually determined by the ELISA reader performance. Many ELISA readers only record absorbance up to 2.0 O.D. Therefore the colour development within individual microwells must be observed by the analyst, and the substrate reaction stopped before positive wells are no longer within recordable range
|Calculation of Results||Calculate the average absorbance values for each set of duplicate standards and samples. Ideally duplicates should be within 20 % of the mean. Generate a linear standard curve by plotting the average absorbance of each standard on the vertical axis versus the corresponding human TRAIL R3 standard concentration on the horizontal axis. The amount of TRAIL R3 in each sample is determined by extrapolating OD values against TRAIL R3 standard concentrations using the standard curve.Every laboratory must produce a standard curve for each set of microwell strips assayed. Do not extrapolate the standard curve beyond the maximum standard curve point. The dose-response is non-linear in this region and good accuracy is difficult to obtain. Concentrated samples above the maximum standard concentration must be diluted with Standard diluent or with your own sample buffer to produce an OD value within the range of the standard curve. Following analysis of such samples always multiply results by the appropriate dilution factor to produce actual final concentration. The influence of various drugs on end results has not been investigated. Bacterial or fungal contamination and laboratory cross-contamination may also cause irregular results. Improper or insufficient washing at any stage of the procedure will result in either false positive or false negative results. Completely empty wells before dispensing fresh Washing Buffer, fill with Washing Buffer as indicated for each wash cycle and do not allow wells to sit uncovered or dry for extended periods. Disposable pipette tips, flasks or glassware are preferred, reusable glassware must be washed and thoroughly rinsed of all detergents before use. As with most biological assays conditions may vary from assay to assay therefore a fresh standard curve must be prepared and run for every assay.|
|Restrictions||For Research Use only|
HandlingProduct Details TNFRSF10C ELISA Kit Target details Application Details Images back to top
Handling of reagents, serum or plasma specimens should be in accordance with local safety procedures , e.g.CDC/NIH Health manual: " Biosafety in Microbiological and Biomedical Laboratories" 1984
Laboratory gloves should be worn at all times
Avoid any skin contact with H2SO4 and TMB. In case of contact, wash thoroughly with water
Do not eat, drink, smoke or apply cosmetics where kit reagents are used
Do not pipette by mouth
When not in use, kit components should be stored refrigerated or frozen as indicated on vials or bottles labels
All reagents should be warmed to room temperature before use. Lyophilized standards should be discarded after use
Once the desired number of strips has been removed, immediately reseal the bag to protect the remaining strips from deterioration
Cover or cap all reagents when not in use
Do not mix or interchange reagents between different lots
Do not use reagents beyond the expiration date of the kit
Use a clean disposable plastic pipette tip for each reagent, standard, or specimen addition in order to avoid cross contamination, for the dispensing of H2SO4 and substrate solution, avoid pipettes with metal parts
Use a clean plastic container to prepare the washing solution
Thoroughly mix the reagents and samples before use by agitation or swirling
All residual washing 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
The TMB solution is light sensitive. Avoid prolonged exposure to light. Also, avoid contact of the TMB solution with metal to prevent colour development. Warning TMB is toxic avoid direct contact with hands. Dispose off properly
If a dark blue colour develops within a few minutes after preparation, this indicates that the TMB solution has been contaminated and must be discarded. Read absorbance's within 1 hour after completion of the assay
When pipetting reagents, maintain a consistent order of addition from well-to-well. This will ensure equal incubation times for all wells
Follow incubation times described in the assay procedure
Dispense the TMB solution within 15 min of the washing of the microtitre plate Version 3 23/01/2013 5 8
Store kit reagents between 2 and 8 °C. Immediately after use remaining reagents should be returned to cold storage (2-8 °C). Expiry of the kit and reagents is stated on box front labels. The expiry of the kit components can only be guaranteed if the components are stored properly, and if, in case of repeated use of one component, the reagent is not contaminated by the first handling.
Wash Buffer: Once prepared store at 2-8 °C for up to 1 week
Standard Diluent Buffer: Once prepared store at 2-8 °C for up to 1 week
Standards: Once prepared use immediately and do not store
Biotinylated Secondary Antibody: Once prepared use immediately and do not store
Streptavidin-HRP: Once prepared use immediately and do not store