DESCRIPTION

ATP1A2 (Na+/K+-ATPase α-2 subunit) is the catalytic component of the active enzyme Na+/K+-ATPase, which catalyzes the hydrolysis of ATP coupled with the exchange of sodium and potassium ions across the plasma membrane. The Na+/K+-ATPase is composed of a larger catalytic α-subunit (~110 kDa) and a small β-subunit (~55 kDa). The α subunit has four isoforms identified to date: α1, α2, α3, and α4. The α1 isoform is expressed ubiquitously, but the α2 isoform is present largely in skeletal muscle, heart, and vascular smooth muscle. The α3 isoform is found almost exclusively in neurons and ovaries. The α4 isoform is expressed in sperm.

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	12.86	0.38	2.95
2	8	3.02	0.04	1.36
3	8	1.56	0.04	2.24

Inter-assay Precision

Sample	n	Mean (ng/mL)	SD	CV%
1	16	12.85	0.45	3.51
2	16	3.01	0.10	3.17
3	16	1.55	0.04	2.70

RECOVERY

The recovery of human/mouse ATP1A2 spiked to three different levels throughout the range of the assay in tissue lysate was evaluated.

Sample Type		Average% of Expected	Range (%)
Tissue lysate	1:320	103	98-109

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 tissue lysate was initially diluted 1:160.)

		Tissue lysate
1:2	Average% of Expected	100
	Range (%)	-
1:4	Average% of Expected	119
	Range (%)	117-121
1:8	Average% of Expected	132
	Range (%)	125-139
1:16	Average% of Expected	112
	Range (%)	109-114

CALIBRATION

This immunoassay is calibrated against a highly purified recombinant human Human/Mouse ATP1A2 Sandwich ELISA Kit produced at Sunlong Medical™.