DESCRIPTION

RNF40 (Ring Finger Protein 40) is an E3 ubiquitin ligase, which is involved in the monoubiquitination of histone H2B at lysine 120 (H2Bub1), a modification that is crucial for transcriptional elongation and DNA repair. RNF20, RNF40 and H2Bub1 were also found to correlate with poor prognosis in hepatocellular carcinoma and to support the tumorigenic properties of prostate cancer and MLL-rearranged lymphoblastic leukemia.

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 (pg/mL)	SD	CV%
1	8	1,999.9	54.3	2.7
2	8	471.8	10.7	2.3
3	8	222.3	11.3	5.1

Inter-assay Precision

Sample	n	Mean (pg/mL)	SD	CV%
1	16	1,973.2	68.1	3.5
2	16	475.0	14.5	3.1
3	16	237.8	21.1	8.9

RECOVERY

The recovery of human RNF40 spiked to three different levels throughout the range of the assay in cell lysate was evaluated.

Sample Type		Average% of Expected	Range (%)
Cell lysate	1:512	114	109-124

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 cell lysate was initially diluted 1:128.)

		Cell lysate
1:2	Average% of Expected	100
	Range (%)	-
1:4	Average% of Expected	117
	Range (%)	117-118
1:8	Average% of Expected	128
	Range (%)	127-129
1:16	Average% of Expected	126
	Range (%)	119-133

CALIBRATION

This immunoassay is calibrated against a highly purified recombinant human Human RNF40 Sandwich ELISA Kit produced at Sunlong Medical™.