DESCRIPTION

CEBPA and its isoforms play important roles in lineage determination and gene activation in a variety of cell types by activating transcription from lineage-specific promoters. CEBPA is a DNA-binding protein that recognizes two different motifs: the CCAAT homology common to many promoters and the enhanced core homology common to many enhancers. In hematopoiesis, C/EBPa is a key factor in driving the development of myeloid cells interacting with a variety of factors, including c-Myc, PU.1, and microRNAs. It can also form heterodimers with the related proteins CEBP-beta and CEBP-gamma. The encoded protein has been shown to bind to the promoter and modulate the expression of the gene encoding leptin which plays an important role in body weight homeostasis. CEBPA can interact with CDK2 and CDK4, thereby inhibiting these kinases and causing growth arrest in cultured cells. Several pathways have been implicated as the means by which CEBPA mediates cell cycle arrest and proliferation, including p21, cyclin-dependent kinases and the E2F complex via c-Myc.

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 (ng/mL)	SD	CV%
1	8	21.20	1.04	4.92
2	8	5.51	0.27	4.92
3	8	2.85	0.15	5.36

Inter-assay Precision

Sample	n	Mean (ng/mL)	SD	CV%
1	16	21.87	1.15	5.27
2	16	5.40	0.22	4.13
3	16	2.97	0.17	5.75

RECOVERY

The recovery of human CEBP Alpha/CEBPA spiked to three different levels throughout the range of the assay in cell lysate was evaluated.

Sample Type		Average% of Expected	Range (%)
Cell lysate	1:16	82	71-88

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.

		Cell lysate
1:2	Average% of Expected	100
	Range (%)	-
1:4	Average% of Expected	86
	Range (%)	79-93
1:8	Average% of Expected	79
	Range (%)	74-85
1:16	Average% of Expected	84
	Range (%)	83-84

CALIBRATION

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