DESCRIPTION

CD30, also known as TNFRSF8, is a member of the tumor necrosis factor receptor (TNF-R) superfamily. It plays a crucial role in immune regulation and signaling. CD30 interacts with its ligand, CD30L (CD153), which is expressed on activated T cells, leading to the activation of downstream signaling pathways such as NF-κB, MAPK, and JAK/STAT. These pathways are involved in cell proliferation, survival, and immune response modulation. In mice, soluble CD30 (sCD30) can be detected in serum and other body fluids. Studies in mice have shown that sCD30 can modulate immune responses by competing with membrane-bound CD30 for CD30L binding, thereby reducing the activation of downstream signaling pathways.

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	1,028.2	76.4	7.4
2	8	268.7	11.3	4.2
3	8	140.3	7.0	5.0

Inter-assay Precision

Sample	n	Mean (pg/mL)	SD	CV%
1	16	1,027.2	73.8	7.2
2	16	263.6	12.4	4.7
3	16	137.7	6.7	4.9

RECOVERY

The recovery of mouse CD30/TNFRSF8 spiked to three different levels throughout the range of the assay in various matrices was evaluated.

Sample Type		Average% of Expected	Range (%)
Mouse serum	1:4	91	84-102
Cell culture supernatant	1:4	100	97-105

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.

		Mouse serum	Cell culture supernatant
1:2	Average% of Expected	100	100
	Range (%)	-	-
1:4	Average% of Expected	105	89
	Range (%)	94-115	87-91
1:8	Average% of Expected	99	74
	Range (%)	90-107	72-75

CALIBRATION

This immunoassay is calibrated against a highly purified recombinant human Mouse CD30/TNFRSF8 One-Step ELISA Kit produced at Sunlong Medical™.