DESCRIPTION

Cyclin-dependent kinase 2 (CDK2) is also named as CDKN2. CDK2 is a core cell-cycle kinase that phosphorylates many substrates to drive progression through the cell cycle. CDK2 is hyperactivated in multiple cancers and is therefore an attractive therapeutic target. CDK4/6-cyclin D complexes phosphorylate and inactivate the retinoblastoma protein (Rb1), liberating E2F to promote transcription of cyclins E and A, which activate CDK2. Activated CDK2 hyperphosphorylates Rb1, establishing a potent positive feedback loop that ensures the continued expression of cyclins E and A, as well as other proteins that are essential for S phase.

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	4,751.7	331.1	7.0
2	8	1,518.2	45.5	3.0
3	8	424.1	29.9	7.0

Inter-assay Precision

Sample	n	Mean (pg/mL)	SD	CV%
1	16	4,950.2	393.3	7.9
2	16	1,483.6	60.6	4.1
3	16	413.0	34.5	8.3

RECOVERY

The recovery of human CDK2 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	81	76-85

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 was initially diluted 1:20. )

		Cell lysate
1:2	Average% of Expected	100
	Range (%)	-
1:4	Average% of Expected	93
	Range (%)	93-94
1:8	Average% of Expected	100
	Range (%)	99-101
1:16	Average% of Expected	123
	Range (%)	122-125

CALIBRATION

This immunoassay is calibrated against a highly purified recombinant human Human CDK2 Sandwich ELISA Kit produced at Sunlong Medical™.