DESCRIPTION

CA6 (Carbonic anhydrase VI) is a member of CA family proteins that play a central role in pH regulation and electrolyte balance. CA6 is also known as gustin, is a zinc-containing secreted protein which catalyzes the hydration of carbon hydroxide in saliva. CA6 is specifically expressed in the salivary gland of a number of mammalian species. The amino acid sequences are highly conserved across the species. And it was reported that decreasing of CA6 protein was associated with loss of taste and pathological morphology of taste buds.

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	1,074.2	20.5	1.9
2	8	253.2	5.8	2.3
3	8	121.9	2.8	2.3

Inter-assay Precision

Sample	n	Mean (pg/mL)	SD	CV%
1	16	1,070.1	22.7	2.1
2	16	254.2	5.1	2.0
3	16	124.9	4.2	3.4

RECOVERY

The recovery of human Carbonic Anhydrase 6/CA6 spiked to three different levels throughout the range of the assay in various matrices was evaluated.

Sample Type		Average% of Expected	Range (%)
Human plasma	1:640	97	87-103
	1:1,280	106	101-108
Saliva	1:32,000	88	83-97
Human milk	1:16,000	98	92-102
	1:32,000	100	89-110

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 human plasma was initially diluted 1:80. The saliva was initially diluted 1:2,000. The human milk was initially diluted 1:2,000.)

		Human plasma	Saliva	Human milk
1:2	Average% of Expected	100	100	100
	Range (%)	-	-	-
1:4	Average% of Expected	106	96	101
	Range (%)	100-111	84-109	99-103
1:8	Average% of Expected	106	115	109
	Range (%)	105-107	109-120	105-113
1:16	Average% of Expected	116	116	120
	Range (%)	115-116	104-129	114-126

CALIBRATION

This immunoassay is calibrated against a highly purified recombinant human Human Carbonic Anhydrase 6/CA6 One-Step ELISA Kit produced at Sunlong Medical™.