DESCRIPTION

Thimet oligopeptidase (THOP1), also known as EP24.15, MP24.15, and EC 3.4.24.15, belongs to the M3 family of zinc-dependent endopeptidases. It moves between the cytoplasm and nucleus of neurons and glial cells and is secreted into extracellular space via multiple mechanisms. Its function in major histocompatibility class I antigen presentation has been well studied, and more recently discovered roles in hyperlipidic diet-induced obesity, non-alcoholic liver steatosis, and insulin resistance are under investigation.

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 (ng/mL)	SD	CV%
1	8	26.10	1.50	5.75
2	8	7.19	0.43	6.00
3	8	3.73	0.24	6.46

Inter-assay Precision

Sample	n	Mean (ng/mL)	SD	CV%
1	16	25.15	1.72	6.83
2	16	6.74	0.59	8.78
3	16	3.42	0.40	11.64

RECOVERY

The recovery of human THOP1 spiked to three different levels throughout the range of the assay in cell lysate was evaluated.

Sample Type		Average% of Expected	Range (%)
Lysate	1:8	76	72-81

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.

		Lysate
1:2	Average% of Expected	100
	Range (%)	-
1:4	Average% of Expected	87
	Range (%)	80-95
1:8	Average% of Expected	100
	Range (%)	89-111
1:16	Average% of Expected	109
	Range (%)	107-110

CALIBRATION

This immunoassay is calibrated against a highly purified recombinant human Human THOP1 Sandwich ELISA Kit produced at Sunlong Medical™.