The test principle applied in this kit is Sandwich enzyme immunoassay. The microtiter plate provided in this kit has been pre-coated with an antibody specific to Rat FN. Standards or samples are added to the appropriate microtiter plate wells then with a biotin-conjugated antibody specific to Rat FN. Next, Avidin conjugated to Horseradish Peroxidase (HRP) is added to each microplate well and incubated. After TMB substrate solution is added, only those wells that contain Rat FN, biotin-conjugated antibody and enzyme-conjugated Avidin will exhibit a change in color. 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 Rat FN 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
400.00
1.857
1.774
200.00
1.315
1.232
100.00
0.998
0.915
50.00
0.791
0.708
25.00
0.495
0.412
12.50
0.329
0.246
6.25
0.175
0.092
0.00
0.083
0.000
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 FN and the recovery rates were calculated by comparing the measured value to the expected amount of FN in samples.
Matrix
Recovery range
Average
serum(n=5)
85-97%
91%
EDTA plasma(n=5)
91-99%
95%
Heparin plasma(n=5)
80-92%
86%
線性
The linearity of the kit was assayed by testing samples spiked with appropriate concentration of FN and their serial dilutions. The results were demonstrated by the percentage of calculated concentration to the expected.