DESCRIPTION

CCAAT/enhancer binding protein β (C/EBPβ), originally identified as a nuclear factor interleukin-6 (NF-IL6), plays a role in regulating inflammatory responses. C/EBPβ binds to the IL-1 response element within the promoter region of IL-6 and promotes the transcription of IL-6 and IL-8. C/EBPβ can directly interact with the Rel homologous domain situated between the nuclear factor κB (NF-κB) subunits p50, p65, and C-REL, thereby stabilizing NF-κB and synergistically activating the expression of IL-6 and IL-8. C/EBPβ can also regulate NF-κB by directly inactivating the inhibitor IκBα. C/EBPβ mediates the balance between antiinflammatory and pro-inflammatory signals during tissue repair.

PRINCIPLE OF THE ASSAY

Sandwich ELISA structure (Detection antibody labeled with HRP) A capture antibody is pre-coated onto the bottom of wells which binds to analyte of interest. A detection antibody labeled with HRP also binds to the analyte. TMB acts as the HRP substrate and the solution color will change from colorless to blue. A stop solution containing sulfuric acid turns solution yellow. The color intensity is proportional to the quantity of bound protein which is measurable at 450 nm with the correction wavelength set at 630 nm.

PRECISION

Intra-assay Precision (Precision within an assay) Three samples of known concentration were tested 8 times on one plate to assess intra-assay precision. Inter-assay Precision (Precision between assays) Three samples of known concentration were tested in 16 separate assays to assess inter-assay precision.

Intra-assay Precision

Sample	n	Mean (pg/mL)	SD	CV%
1	8	984.6	51.2	5.2
2	8	254.7	2.6	1.0
3	8	123.3	2.9	2.3

Inter-assay Precision

Sample	n	Mean (pg/mL)	SD	CV%
1	16	986.5	44.5	4.5
2	16	244.7	13.7	5.6
3	16	120.0	6.6	5.5

RECOVERY

The recovery of human CEBPB spiked to three different levels throughout the range of the assay in cell lysate was evaluated.

Sample Type		Average% of Expected	Range (%)
Cell lysate	1:160	102	95-109

LINEARITY

To assess the linearity of the assay, samples were diluted with the appropriate Sample Diluent to produce samples with values within the dynamic range of the assay. (The cell lysate samples were initially diluted 1:20.)

		Cell lysate
1:2	Average% of Expected	100
	Range (%)	-
1:4	Average% of Expected	110
	Range (%)	102-115
1:8	Average% of Expected	114
	Range (%)	101-125
1:16	Average% of Expected	111
	Range (%)	95-126

CALIBRATION

This immunoassay is calibrated against a highly purified recombinant human Human CEBPB Sandwich ELISA Kit produced at Sunlong Medical™.