DESCRIPTION

OPTN (Optineurin) is a crucial autophagy receptor that facilitates the recognition of infected pathogens, protein aggregates, and mitochondria. The depletion of OPTN significantly increases the formation of protein aggregates and results in a loss of function of selective autophagy. In contrast, upregulating the expression of OPTN can induce autophagy. OPTN is associated with many human disorders that are closely related to autophagy, such as rheumatoid arthritis, osteoporosis, and nephropathy.

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 (ng/mL)	SD	CV%
1	8	5.09	0.07	1.38
2	8	1.39	0.03	2.16
3	8	0.79	0.03	3.80

Inter-assay Precision

Sample	n	Mean (ng/mL)	SD	CV%
1	16	5.14	0.10	1.95
2	16	1.40	0.03	2.14
3	16	0.82	0.05	6.10

RECOVERY

The recovery of human OPTN/Optineurin spiked to three different levels throughout the range of the assay in various matrices was evaluated.

Sample Type		Average% of Expected	Range (%)
Human plasma	1:2	117	112-126
Cell lysate	1:16	101	97-107

LINEARITY

To assess the linearity of the assay, human plasma samples were spiked with high concenthumanions of human OPTN/Optineurin and diluted with the appropriate Sample Diluent to produce samples with values within the dynamic range of the assay. Cell lysate samples were diluted with the appropriate Sample Diluent to produce samples with values within the dynamic range of the assay.

		Human plasma	Cell lysate
1:2	Average% of Expected	128	100
	Range (%)	128-129	-
1:4	Average% of Expected	111	103
	Range (%)	110-112	98-108
1:8	Average% of Expected	109	106
	Range (%)	106-111	98-114
1:16	Average% of Expected	104	108
	Range (%)	95-112	100-117

CALIBRATION

This immunoassay is calibrated against a highly purified recombinant human Human OPTN/Optineurin One-Step ELISA Kit produced at Sunlong Medical™.