This ELISA kit can be applied to the in vitro quantitative determination of fT4 concentrations in serum, plasma and other biological fluids.
This ELISA kit uses Competitive-ELISA as the method. The microtiter plate provided in this kit has been pre-coated with fT4. During the reaction, fT4 in the sample or standard competes with a fixed amount of fT4 on the solid phase supporter for sites on the Biotinylated Detection Ab specific to fT4. Excess conjugate and unbound sample or standard are washed from the plate, and Avidin conjugated to Horseradish Peroxidase (HRP) are added to each microplate well and incubated. Then a TMB substrate solution is added to each well. The enzyme-substrate reaction is terminated by adding Stop Solution and the color change is measured spectrophotometrically at a wavelength of 450 nm ± 2 nm. The concentration of fT4 in the samples is then determined by comparing the OD of the samples to the standard curve.
|Detection Range||1.56—100 pg/mL|
|Sample Type||Serum, plasma and other biological fluids|
This kit recognizes some recombinant and natural fT4. No significant cross-reactivity or interference was observed.
For convenience in result calculation, absorbance can be used as ordinate and standard concentrations as abscissa. The standard curve provided in the manual is only for reference, and experimenters should draw the standard curve based on data of themselves.
Intra-assay Precision (Precision within an assay): 3 samples with low, middle and high level fT4 were tested 20 times on one plate, respectively.
Inter-assay Precision (Precision between assays): 3 samples with low, middle and high level fT4 were tested on 3 different plates, 20 replicates in each plate.
|Intra-assay Precision||Inter-assay Precision|
|C V (%)||5.88||4.55||5.20||5.77||4.55||4.44|
The recovery of fT4 spiked to three different levels in samples throughout the range of the assay in various matrices was evaluated.
|Sample Type||Range (%)||Average Recovery (%)|
|EDTA plasma (n=5)||90-101||96|
|Cell culture media (n=5)||96-107||102|
Samples were spiked with high concentrations of Human ACTH and diluted with Reference Standard & Sample Diluent to produce samples with values within the range of the assay.
|Serum (n=5)||EDTA plasma (n=5)||Cell culture media (n=5)|
The unopened kit can be stored at 4℃ for 1 month. If the kit is not used within 1 month, store the items separately according to the following conditions since the kit is received.
|Micro ELISA Plate(Dismountable)||8 wells ×12 strips||-20℃, 6 months|
|Reference Standard||2 vials|
|Concentrated Biotinylated Detection Ab (100×)||1 vial, 120 μL|
|Concentrated HRP Conjugate (100×)||1 vial, 120 μL||-20℃(shading light), 6 months|
|Reference Standard & Sample Diluent||1 vial, 20 mL||4℃, 6 months|
|Biotinylated Detection Ab Diluent||1 vial, 14 mL|
|HRP Conjugate Diluent||1 vial, 14 mL|
|Concentrated Wash Buffer (25×)||1 vial, 30 mL|
|Substrate Reagent||1 vial, 10 mL||4℃(shading light)|
|Stop Solution||1 vial, 10 mL||4℃|
|Plate Sealer||5 pieces|
|Product Description||1 copy|
|Certificate of Analysis||1 copy|
Note: All reagent bottle caps must be tightened to prevent evaporation and microbial pollution.
The volume of reagents in partial shipments is a little more than the volume marked on the label, please use in measuring instead of directly pouring.
1.Add 50 μL of standard or sample to each well.
2.Immediately add 50 μL of Biotinylated Detection Ab to each well.
3.Incubate for 45 min at 37℃. Aspirate and wash 3 times.
4.Add 100 μL of HRP Conjugate to each well. Incubate for 30 min at 37℃. Aspirate and wash 5 times.
5.Add 90 μL of Substrate Reagent. Incubate for 15 min at 37℃.
6.Add 50 μL of Stop Solution.
7.Read the OD at 450 nm immediately. Calculation of the results.