DESCRIPTION

PAX6, a paired domain and homeodomain-containing transcription factor. Interaction with TRIM11 leads to ubiquitination of PAX6 and its proteasomal degradation. PAX6 is one of the earliest genes expressed in the eye field and considered a master control gene for retinal and eye development. PAX6 also regulates the development of the olfactory, central nervous systems, pituitary, and pancreas. PAX6 mutations can cause complex ocular disorders such as aniridia and Peter's anomaly. Pax6 facilitates important regulatory roles in breast cancer cell proliferation and tumor progression, and could serve as a diagnostic marker for clinical investigation. Moreover, tumor cells were arrested at G0/G1 phase after Pax6 was knocked down.

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	2,464.5	145.2	5.9
2	8	599.1	48.6	8.1
3	8	315.3	28.5	9.0

Inter-assay Precision

Sample	n	Mean (pg/mL)	SD	CV%
1	16	2,455.4	202.3	8.2
2	16	608.6	44.6	7.3
3	16	307.5	28.4	9.2

RECOVERY

The recovery of human/rat PAX6 spiked to three different levels throughout the range of the assay in cell lysate was evaluated.

Sample Type		Average% of Expected	Range (%)
Cell lysate	1:32	99	92-108
	1:64	88	82-92

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 1:4. )

		Cell lysate
1:2	Average% of Expected	100
	Range (%)	-
1:4	Average% of Expected	98
	Range (%)	95-101
1:8	Average% of Expected	106
	Range (%)	99-112
1:16	Average% of Expected	113
	Range (%)	105-121

CALIBRATION

This immunoassay is calibrated against a highly purified recombinant human/rat PAX6 produced at Sunlong Medical™.