Human fT3 (Free Triiodothyronine) ELISA Kit - Information
The Assay Genie Human fT3 (Free Triiodothyronine) ELISA Kit can assay for Human fT3 in the following samples: serum, blood, plasma, cell culture supernatant and other related supernatants and tissues.How do our Human fT3 (Free Triiodothyronine) ELISA Kits Work?
This Human fT3 (Free Triiodothyronine) ELISA Kit is based on Competitive-ELISA detection method. The microtiter plate provided in this kit has been pre-coated with analyte of interest. During the reaction, the analyte in the sample or standard competes with a fixed amount of target on the solid phase supporter for sites on the Biotinylated Detection Antibody specific to analyte. Excess conjugate and unbound sample or standard are washed from the plate, and HRP-Streptavidin (SABC) is added to each microplate well and incubated. TMB substrate solution is then added to each well. The enzyme-substrate reaction is terminated by the addition of a sulphuric acid solution and the colour change is measured spectrophotometrically at a wavelength of 450nm. The concentration of target in the samples is then determined by comparing the OD of the samples to the standard curve.
Human fT3 (Free Triiodothyronine) ELISA Kit Data
|Product Code|| |
fT3, Free Triiodothyronine
|Detection method|| |
Competitive ELISA, Coated with Antibody
This immunoassay kit allows for the in vitro quantitative determination of Human fT3 concentrations in serum plasma and other biological fluids.
4'C for 6 months
Matrices listed below were spiked with certain level of Human fT3 and the recovery rates were calculated by comparing the measured value to the expected amount of Human fT3 in samples.
The linearity of the kit was assayed by testing samples spiked with appropriate concentration of Human fT3 and their serial dilutions. The results were demonstrated by the percentage of calculated concentration to the expected.
For Research Use Only
Human fT3 (Free Triiodothyronine) ELISA Kit Protocol
The below protocol is a sample protocol for a Human fT3 (Free Triiodothyronine) ELISA Kit. Competitive ELISA kits allow for the detection and quantification of an analyte in a sample.This Human fT3 (Free Triiodothyronine) ELISA Kit allows the researcher to calculate the amount of Human fT3 intheir sample. Equilibrate the TMB substrate for at least 30 min at 37°C beforeuse. When diluting samples and reagents, they must be mixed completely andevenly. It is recommended to plot a standard curve for each test.
|1.||Set standard, test sample and control (zero) wells on the pre-coatedplate respectively, and then, record their positions. It isrecommended to measure each standard and sample in duplicate. Washplate 2 times before adding standard, sample and control (zero) wells!|
|2.||Add Sample and Biotin-detection antibody: Add 50µL of Standard, Blank or Sample per well. The blankwell is added with Sample Dilution Buffer. Immediately add 50 µL of biotin-labelled antibody workingsolution to each well. Cover with the plate sealer provided. Gently tap the plate to ensure thoroughmixing. Incubate for 45 minutes at 37°C. (Solutions are added to the bottom of micro-ELISA platewell, avoid touching plate walls and foaming).|
|3.||Wash: Aspirate each well and wash, repeating the process three timesWash by filling each well with Wash Buffer (approximately 350µL)using a squirt bottle, multi-channel pipette, manifold dispenser orautomated washer. Complete removal of liquid at each step is essentialto good performance. After the last wash, remove any remaining WashBuffer by aspirating or decanting. Invert the plate and pat it againstthick clean absorbent paper.|
|4.||HRP-Streptavidin Conjugate(SABC): Add 100µL of SABC workingsolution to each well. Cover with a new Plate sealer. Incubate for30minutes at 37°C.|
|5.||Wash: Repeat the aspiration/wash process for five times.|
|6.||TMB Substrate: Add 90µL of TMB Substrate to each well. Coverwith a new Plate sealer. Incubate for about 10-20 minutes at 37°C.Protect from light. The reaction time can be shortened or extendedaccording to the actual color change, but not more than 30minutes.When apparent gradient appeared in standard wells, you can terminatethe reaction.|
|7.||Stop: Add 50µL of Stop Solution to each well. Color turn toyellow immediately. The adding order of stop solution should be as thesame as the substrate solution.|
|8.||OD Measurement: Determine the optical density (OD Value) of each wellat once, using a microplate reader set to 450 nm. You should open themicroplate reader ahead, preheat the instrument, and set the testingparameters.|
Human fT3 (Free Triiodothyronine) ELISA Kit components
|ELISA Microplate(Dismountable)||8×12 strips||4°C for 6 months|
|Sample/Standard Dilution Buffer||20ml||4°C|
|Biotin-labeled Antibody(Concentrated)||60ul||4°C (Protect from light)|
|Antibody Dilution Buffer||10ml||4°C|
|HRP-Streptavidin Conjugate(SABC)||120ul||4°C (Protect from light)|
|SABC Dilution Buffer||10ml||4°C|
|TMB Substrate||10ml||4°C (Protect from light)|
Other materials and equipment required:
The Assay Genie Human fT3 (Free Triiodothyronine) ELISA Kit will require other equipment andmaterials to carry out the assay. Please see list below for further details.
- Microplate reader with 450 nm wavelength filter
- Multichannel Pipette, Pipette, microcentrifuge tubes and disposable pipettetips
- Deionized or distilled water
- Absorbent paper
- Buffer resevoir
When carrying out an ELISA assay it is important to prepare your samples inorder to achieve the best possible results. Below we have a list of proceduresfor the preparation of samples for different sample types.
If using serum separator tubes, allow samples to clot for 30 minutesat room temperature. Centrifuge for 10 minutes at 1,000x g. Collectthe serum fraction and assay promptly or aliquot and store the samplesat -80°C. Avoid multiple freeze-thaw cycles.
If serum separator tubes are not being used, allow samples to clotovernight at 2-8°C. Centrifuge for 10 minutes at 1,000x g. Removeserum and assay promptly or aliquot and store the samples at-80°C. Avoid multiple freeze-thaw cycles.
Collect plasma using EDTA or heparin as an anticoagulant. Centrifugesamples at 4°C for 15 mins at 1000 × g within 30 mins ofcollection. Collect the plasma fraction and assay promptly or aliquotand store the samples at -80°C. Avoid multiple freeze-thaw cycles.Note: Over haemolysed samples are not suitable foruse with this kit.
Urine & Cerebrospinal Fluid
Collect the urine (mid-stream) in a sterile container, centrifuge for20 mins at 2000-3000 rpm. Remove supernatant and assay immediately. Ifany precipitation is detected, repeat the centrifugation step. Asimilar protocol can be used for cerebrospinal fluid.
Cell culture supernatant
Collect the cell culture media by pipette, followed by centrifugationat 4°C for 20 mins at 1500 rpm. Collect the clear supernatant andassay immediately.
Solubilize cells in lysis buffer and allow to sit on ice for 30minutes. Centrifuge tubes at 14,000 x g for 5 minutes to removeinsoluble material. Aliquot the supernatant into a new tube anddiscard the remaining whole cell extract. Quantify total proteinconcentration using a total protein assay. Assay immediately oraliquot and store at ≤ -20 °C.
The preparation of tissue homogenates will vary depending upon tissuetype. Rinse tissue with 1X PBS to remove excess blood & homogenizein 20ml of 1X PBS (including protease inhibitors) and store overnightat ≤ -20°C. Two freeze-thaw cycles are required to break thecell membranes. To further disrupt the cell membranes you can sonicatethe samples. Centrifuge homogenates for 5 mins at 5000xg. Remove thesupernatant and assay immediately or aliquot and store at -20°C or-80°C.
Rinse tissue with PBS, cut into 1-2 mm pieces, and homogenize with atissue homogenizer in PBS. Add an equal volume of RIPA buffercontaining protease inhibitors and lyse tissues at room temperaturefor 30 minutes with gentle agitation. Centrifuge to remove debris.Quantify total protein concentration using a total protein assay.Assay immediately or aliquot and store at ≤ -20 °C.
Collect milk samples and centrifuge at 10,000 x g for 60 min at4°C. Aliquot the supernatant and assay. For long term use, storesamples at -80°C. Minimize freeze/thaw cycles.