This assay employs the competitive inhibition enzyme immunoassay technique. The microtiter plate provided in this kit has been pre-coated with Human T4 antigen, and the Human T4 standard plate wells that pre-coated using protein-related techniques are provided separately. Standard/Sample Diluent Buffer or samples are added to the appropriate microtiter plate wells ,then added a HRP-conjugated antibody specific to Human T4 to each microplate well and incubated . After TMB substrate solution is added, the enzyme-substrate reaction is terminated by the addition of sulphuric acid solution and the color change is measured spectrophotometrically at a wavelength of 450nm ±10nm. The concentration of Human T4 in the samples is then determined by comparing the OD of the samples to the standard curve.
標(biāo)準(zhǔn)曲線
Concentration (ng/mL)
OD
Corrected OD
600.00
0.199
300.00
0.385
150.00
0.575
75.00
0.878
37.50
1.139
18.75
1.585
9.38
1.624
0.00
2.112
精密度
Intra-assay Precision (Precision within an assay):CV%<8%
Three samples of known concentration were tested twenty times on one plate to assess intra-assay precision.
Inter-assay Precision (Precision between assays):CV%<10%
Three samples of known concentration were tested in forty separate assays to assess inter-assay precision.
回收率
Matrices listed below were spiked with certain level of recombinant T4 and the recovery rates were calculated by comparing the measured value to the expected amount of T4 in samples.
Matrix
Recovery range
Average
serum(n=5)
82-97%
89%
EDTA plasma(n=5)
80-95%
87%
Heparin plasma(n=5)
87-95%
91%
線性
The linearity of the kit was assayed by testing samples spiked with appropriate concentration of T4 and their serial dilutions. The results were demonstrated by the percentage of calculated concentration to the expected.