DESCRIPTION

CD40, also named as TNFRSF5 and Bp50, is a type I transmembrane protein that belongs to the tumor necrosis factor receptor (TNFR) family. CD40 is expressed on B cells, dendritic cells (DCs), monocytes, platelets, and macrophages as well as by nonhematopoietic cells such as myofibroblasts, fibroblasts, epithelial, and endothelial cells. It is a receptor for TNFSF5/CD40L. CD40L/CD40 interactions are essential for immunoglobulin (Ig) isotype switching, germinal center formation and the development of B cell memory.

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	650.6	31.5	4.8
2	8	165.6	3.6	2.2
3	8	87.5	3.3	3.8

Inter-assay Precision

Sample	n	Mean (pg/mL)	SD	CV%
1	16	623.4	37.1	6.0
2	16	159.1	7.6	4.8
3	16	88.5	6.1	6.9

RECOVERY

The recovery of mouse CD40 spiked to three different levels throughout the range of the assay in various matrices was evaluated.

Sample Type		Average% of Expected	Range (%)
Mouse serum	1:2	91	83-98
	1:4	117	112-125
Cell culture supernatant	1:2	92	88-98
	1:4	81	75-86
Tissue lysate	1:4	93	86-99
	1:8	84	81-87

LINEARITY

To assess the linearity of the assay, mouse serum samples were spiked with high concentrations of mouse CD40 and diluted with the appropriate Sample Diluent to produce samples with values within the dynamic range of the assay. Cell culture surpernatant and cell lysate 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 1:40.)

		Mouse serum(Sample Diluent PT 1)	Cell culturesupernatant (SampleDiluent PT 4B1)	Cell lysate(Sample Diluent PT 4B1)
1:2	Average% of Expected	90	100	100
	Range (%)	79-101	-	-
1:4	Average% of Expected	121	102	105
	Range (%)	112-130	99-106	104-106
1:8	Average% of Expected	121	91	108
	Range (%)	121-122	89-94	103-112
1:16	Average% of Expected	110	98	110
	Range (%)	105-115	97-99	105-116

CALIBRATION

This immunoassay is calibrated against a highly purified recombinant mouse CD40 produced at Sunlong Medical™.