This assay employs the competitive inhibition enzyme immunoassay technique. The microtiter plate provided in this kit has been pre-coated with Human POMC protein. Standards or samples are added to the appropriate microtiter plate wells then with a biotin-conjugated antibody specific to Human POMC. Next, Avidin conjugated to Horseradish Peroxidase (HRP) is added 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 POMC in the samples is then determined by comparing the OD of the samples to the standard curve.
標準曲線
Concentration (pg/mL)
OD
Corrected OD
10000.00
0.189
5000.00
0.386
2500.00
0.596
1250.00
0.812
625.00
1.157
312.50
1.592
156.25
1.775
0.00
2.123
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 POMC and the recovery rates were calculated by comparing the measured value to the expected amount of POMC in samples.
Matrix
Recovery range
Average
serum(n=5)
87-99%
93%
EDTA plasma(n=5)
85-97%
91%
Heparin plasma(n=5)
90-103%
96%
線性
The linearity of the kit was assayed by testing samples spiked with appropriate concentration of POMC and their serial dilutions. The results were demonstrated by the percentage of calculated concentration to the expected.