DESCRIPTION

CDKN2A encodes two transcripts with alternative transcriptional start sites. Both transcripts share exons 2 and 3, but are translated in different open reading frames to yield two distinct proteins: p16INK4a and ARF (p14ARF in humans, p19ARF in mice). The tumor suppressor protein p16Ink4a (encoded from the CDKN2A locus) is often transcriptionally activated in cells undergoing senescence and is one of the main regulators of this program, and it is upregulated in multiple tissues during aging. p16-Ink4a is the principal member of the Ink4 family of CDK inhibitors. p16-Ink4a contributes to the regulation of cell cycle progression by inhibiting the S phase. p16Ink4a binds to CDK4/6, inhibiting cyclin D-CDK4/6 complex formation and CDK4/6-mediated phosphorylation of Rb family members. Expression of p16-Ink4a maintains the Rb family members in a hypophosphorylated state, which promotes binding to E2F1 and leads to G1 cell cycle arrest.

PRINCIPLE OF THE ASSAY

An anti-tag antibody is pre-coated onto the bottom of wells. After adding antigen or samples, Tagged-Capture antibody and HRP-conjugated detection antibody, a sandwich complex is formed in the solution. TMB acts as a HRP substrate, and the solution color will change from colorless to blue. A stop solution containing sulfuric acid turns the 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	16.37	0.38	2.32
2	8	3.98	0.14	3.52
3	8	2.01	0.10	4.98

Inter-assay Precision

Sample	n	Mean (ng/mL)	SD	CV%
1	16	16.35	0.46	2.81
2	16	4.05	0.13	3.21
3	16	2.10	0.13	6.19

RECOVERY

The recovery of human P16-INK4A spiked to three different levels throughout the range of the assay in cell lysate was evaluated.

Sample Type		Average% of Expected	Range (%)
Cell lysate	1:80	103	102-104
	1:160	104	101-109

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 was initially diluted 1:10. )

		Cell lysate
1:2	Average% of Expected	100
	Range (%)	-
1:4	Average% of Expected	100
	Range (%)	98-101
1:8	Average% of Expected	91
	Range (%)	88-94
1:16	Average% of Expected	77
	Range (%)	74-79

CALIBRATION

This immunoassay is calibrated against a highly purified recombinant human Human P16-INK4A One-Step ELISA Kit produced at Sunlong Medical™.