DESCRIPTION

JAB1 (also known as COPS5/CSN5) is the catalytic subunit of the conserved COP9 signalosome complex. It possesses JAMM-motif deneddylase activity that removes NEDD8 from cullin E3 ligases, governing ubiquitin-mediated protein turnover. As a transcriptional coactivator for c-Jun/AP-1, JAB1 controls cell-cycle progression, DNA damage response and apoptosis by modulating p27, p53 and PD-L1 stability. JAB1 overexpression is widely detected in multiple malignancies, correlating with tumor invasion, drug resistance and poor patient prognosis, making it a promising oncogenic biomarker and therapeutic target.

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	3.61	0.06	1.66
2	8	1.08	0.02	1.85
3	8	0.57	0.02	3.51

Inter-assay Precision

Sample	n	Mean (ng/mL)	SD	CV%
1	16	3.75	0.16	4.27
2	16	1.14	0.09	7.89
3	16	0.59	0.03	5.08

RECOVERY

The recovery of human JAB1 spiked to three different levels throughout the range of the assay in cell lysate was evaluated.

Sample Type		Average% of Expected	Range (%)
Cell lysate	1:4	81	78-83
	1:8	83	82-84

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.

		Cell lysate
1:2	Average% of Expected	100
	Range (%)	-
1:4	Average% of Expected	100
	Range (%)	99-102
1:8	Average% of Expected	95
	Range (%)	81-109
1:16	Average% of Expected	86
	Range (%)	80-92

CALIBRATION

This immunoassay is calibrated against a highly purified recombinant human Human JAB1 One-Step ELISA Kit produced at Sunlong Medical™.