As the OD values of the standard curve may vary according to the conditions of the actual assay performance (e.g. operator, pipetting technique, washing technique or temperature effects), the operator should establish a standard curve for each test. Typical standard curve and data is provided below for reference only.

(pg/mL)	OD	Corrected
1000.00	2.481	2.430
500.00	1.601	1.550
250.00	0.908	0.857
125.00	0.473	0.422
62.50	0.264	0.213
31.25	0.158	0.107
15.63	0.107	0.056
0.00	0.051	-

Intra-assay Precision (Precision within an assay): 3 samples with low, mid range and high level were tested 20 times on one plate, respectively.

Inter-assay Precision (Precision between assays): 3 samples with low, mid range and high level were tested on 3 different plates, 20 replicates in each plate, respectively.

Samples were spiked with high concentrations of target proteins and diluted with Reference Standard & Sample Diluent to produce samples with values within the range of the assay.

This ELISA kit uses the Sandwich-ELISA principle. The micro ELISA plate provided in this kit has been pre-coated with an antibody specific to Rat IL-1α. Standards or samples are added to the micro ELISA plate wells and combined with the specific antibody. Then a biotinylated detection antibody specific for Rat IL-1α and Avidin-Horseradish Peroxidase (HRP) conjugate are added successively to each micro plate well and incubated. Free components are washed away. The substrate solution is added to each well. Only those wells that contain Rat IL-1α, biotinylated detection antibody and Avidin-HRP conjugate will appear blue in color. The enzyme-substrate reaction is terminated by the addition of stop solution and the color turns yellow. The optical density (OD) is measured spectrophotometrically at a wavelength of 450 nm ± 2 nm. The OD value is proportional to the concentration of Rat IL-1α. You can calculate the concentration of Rat IL-1α in the samples by comparing the OD of the samples to the standard curve.