DESCRIPTION

Phosphatidylinositol 4-phosphate 5-kinases (PIP5Ks) play diverse roles in the cellular biology of many organisms, including signal transduction, secretion and vesicular trafficking, and regulation of cytoskeleton assembly.

PRINCIPLE OF THE ASSAY

A capture antibody is pre-coated onto the bottom of wells which binds to analyte of interest. A detection antibody also binds to the analyte. Horseradish peroxidase (HRP)-conjugated secondary antibody binds to the detection antibody. 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 20 times on one plate to assess intra-assay precision. Inter-assay Precision (Precision between assays) Three samples of known concentration were tested in 24 separate assays to assess inter-assay precision.

Intra-assay Precision

Sample	n	Mean (ng/mL)	SD	CV%
1	20	0.30	0.02	8.4
2	20	1.17	0.03	2.6
3	20	4.92	0.14	2.9

Inter-assay Precision

Sample	n	Mean (ng/mL)	SD	CV%
1	24	0.28	0.02	6.5
2	24	1.20	0.08	7.0
3	24	5.57	0.38	6.8

RECOVERY

The recovery of PIP5K1A spiked to three different levels throughout the range of the assay in various matrices was evaluated.

Sample Type		Range (%)	Average% of Expected
Human plasma	1:2	81-91	85
	1:4	80-98	88
Mouse serum	1:50	76-112	98
	1:100	79-120	106
Rat serum	1:2	78-87	82
	1:4	81-97	89
Cell lysate	1:60	75-95	83
	1:120	83-99	88

LINEARITY

To assess the linearity of the assay, human plasma and rat serum were spiked with high concentrations of PIP5K1A in various matrices and diluted with the appropriate Sample Diluent to produce samples with values within the dynamic range of the assay. Mouse serum and cell lysate were diluted with the appropriate Sample Diluent to produce samples with values within the dynamic range of the assay.

Sample Type		Range (%)	Average% of Expected
Human plasma(Sample Diluent PT 4-af )	1:2	105-111	108
	1:4	119-126	122
	1:8	112-117	114
	1:16	101-103	102
Mouse serum(Sample Diluent PT 1-ef )	1:25	-	100
	1:50	115-126	121
	1:100	95-111	103
	1:200	70-75	73
Rat serum(Sample Diluent PT 3-ec )	1:2	78-89	84
	1:4	87-104	96
	1:8	99-108	104
	1:16	102-106	104
Cell lysate(Sample Diluent PT 3-ec )	1:8	-	100
	1:16	97-108	104
	1:32	97-109	105
	1:64	106-110	108

CALIBRATION

This immunoassay is calibrated against a highly purified recombinant human/mouse/rat PIP5K1A produced at Sunlong Medical™.

Ordering & shipping

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