DESCRIPTION

Uromodulin, also known as THP (Tamm-Horsfall glycoprotein), is the most abundant protein excreted in the urine under physiological conditions. Uromodulin is synthesized by the kidney and localizes in cells lining the ascending limb of Henle and distal convoluted tubule. Uromodulin has been linked to water/electrolyte balance and to kidney innate immunity. Also, studies in knockout mice demonstrated that it has a protective role against urinary tract infections and renal stone formation. Mutations of uromodulin lead to rare autosomal dominant diseases, collectively referred to as uromodulin-associated kidney diseases.

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	2,399.7	65.4	2.7
2	8	585.7	22.8	3.9
3	8	286.8	9.0	3.1

Inter-assay Precision

Sample	n	Mean (pg/mL)	SD	CV%
1	16	2,437.0	78.4	3.2
2	16	596.1	24.1	4.0
3	16	289.4	13.2	4.6

RECOVERY

The recovery of mouse Uromodulin spiked to three different levels throughout the range of the assay was evaluated.

Sample Type		Average% of Expected	Range (%)
Mouse serum	1:1,600	96	94-99
Urine	1:16,000	98	92-104
	1:32,000	99	95-101
Tissue lysate	1:640	93	91-95
	1:1,280	91	89-92

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 mouse serum was initially diluted 1:200. The urine was initially diluted 1:2,000. The tissue lysate was initially diluted 1:80.)

		Mouse serum	Urine	Tissue lysate
1:2	Average% of Expected	100	100	100
	Range (%)	-	-	-
1:4	Average% of Expected	106	97	94
	Range (%)	98-113	96-97	90-98
1:8	Average% of Expected	109	92	87
	Range (%)	102-117	84-101	84-89
1:16	Average% of Expected	116	104	96
	Range (%)	110-121	98-110	80-112

CALIBRATION

This immunoassay is calibrated against a highly purified recombinant mouse Uromodulin produced at Sunlong Medical™.