DESCRIPTION

CA9 (Carbonic anhydrase 9) may be involved in the control of cell proliferation and transformation and appears to be a novel specific biomarker for a cervical neoplasia. It is a tumor-associated antigen that has been shown to have diagnostic utility in identifying cervical dysplasia and carcinoma. The protein is presentboth on the plasma membrane and in the nucleus of cells and has the molecular. Under hypoxic conditions, CA9 expression increases, helping cells adapt to the acidic environment.

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 (pg/mL)	SD	CV%
1	8	1,133.5	25.4	2.2
2	8	283.4	9.2	3.2
3	8	145.5	7.1	4.9

Inter-assay Precision

Sample	n	Mean (pg/mL)	SD	CV%
1	16	1,096.2	52.1	4.8
2	16	278.8	10.9	3.9
3	16	140.6	8.5	6.0

RECOVERY

The recovery of human Carbonic Anhydrase IX/CA9 spiked to three different levels throughout the range of the assay in various matrices was evaluated.

Sample Type		Average% of Expected	Range (%)
Human serum	1:8	82	79-86
	1:16	93	85-98
Urine	1:2	109	102-116
Cell culture supernatant	1:320	90	85-97

LINEARITY

To assess the linearity of the assay, urine samples were spiked with high concenthumanions of human Carbonic Anhydrase IX/CA9 in various matrices and diluted with the appropriate Sample Diluent to produce samples with values within the dynamic range of the assay. To assess the linearity of the assay, human serum and cell culture supernatant samples were diluted with the appropriate Sample Diluent to produce samples with values within the dynamic range of the assay. (The cell culture supernatant was initially diluted 1:20.)

		Human serum(Sample Diluent PT4B1)	Urine(Sample Diluent PT 6)	Cell culture supernatant(Sample Diluent PT 6)
1:2	Average% of Expected	100	104	100
	Range (%)	-	104-105	-
1:4	Average% of Expected	106	98	118
	Range (%)	96-117	98-98	116-119
1:8	Average% of Expected	100	92	77
	Range (%)	75-126	92-93	70-83
1:16	Average% of Expected	95	79	101
	Range (%)	76-114	77-80	98-104

CALIBRATION

This immunoassay is calibrated against a highly purified recombinant human Human Carbonic Anhydrase IX/CA9 One-Step ELISA Kit produced at Sunlong Medical™.