DESCRIPTION

PDE5A (cGMP-binding cGMP-specific phosphodiesterase) plays a role in signal transduction by regulating the intracellular concentration of cyclic nucleotides. This phosphodiesterase catalyzes the specific hydrolysis of cGMP to 5'-GMP. Through alternative splicing, the PDE5A gene produces three proteins (PDE5A1, PDE5A2, and PDE5A3) that differ in their N termini and range in mass from 89 to 105 kDa. A second 70 kDa band was consistently observed in heart tissue that either reflected a splice variant or a proteolytic fragment of PDE5A.

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	4.70	0.20	4.22
2	8	1.25	0.04	3.22
3	8	0.65	0.03	3.90

Inter-assay Precision

Sample	n	Mean (ng/mL)	SD	CV%
1	16	4.69	0.17	3.59
2	16	1.27	0.04	3.46
3	16	0.68	0.04	6.49

RECOVERY

The recovery of human PDE5A spiked to three different levels throughout the range of the assay in cell lysate was evaluated.

Sample Type		Average% of Expected	Range (%)
Cell lysate	1:64	108	105-110
	1:128	106	99-112

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 1:8.)

		Cell lysate
1:2	Average% of Expected	100
	Range (%)	-
1:4	Average% of Expected	92
	Range (%)	85-99
1:8	Average% of Expected	93
	Range (%)	87-100
1:16	Average% of Expected	122
	Range (%)	117-126

CALIBRATION

This immunoassay is calibrated against a highly purified recombinant human Human PDE5A Sandwich ELISA Kit produced at Sunlong Medical™.

Ordering & shipping

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