DESCRIPTION

CD40 is a member of the tumor necrosis factor receptor superfamily. It is expressed in various immune cells, including B cells, macrophages, and dendritic cells. CD40 functions as a co-stimulatory receptor that interacts with its ligand, CD40L (CD154), primarily expressed on activated CD4+ T cells. This interaction is essential for T-cell activation, immunoglobulin class switching, memory B cell development, and germinal center formation. CD40 signaling is mediated by recruitment of TRAFs (TNF receptorassociated factors) and NF-κB activation, supplemented by pathways such as PI3K/AKT, MAPKs, and JAK3/STATs. Additionally, circulating soluble CD40 levels are elevated in patients with chronic renal failure, chronic liver diseases, Alzheimer's disease, systemic sclerosis, and hematological malignancies.

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	2,690.8	41.0	1.5
2	8	662.4	5.8	0.9
3	8	330.7	6.1	1.8

Inter-assay Precision

Sample	n	Mean (pg/mL)	SD	CV%
1	16	2,689.1	81.8	3.0
2	16	664.2	12.5	1.9
3	16	327.5	9.0	2.7

RECOVERY

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

Sample Type		Average% of Expected	Range (%)
Human plasma	1:16	95	89-105
Cell culture supernatant	1:2	93	89-101
Urine	1:128	100	86-108
Saliva	1:4	89	75-98
Human milk	1:4	100	86-112

LINEARITY

To assess the linearity of the assay, cell culture supernatant samples were spiked with high concentrations of human CD40 and diluted with the appropriate Sample Diluent to produce samples with values within the dynamic range of the assay. Human plasma, urine, saliva and human milk samples were diluted with the appropriate Sample Diluent to produce samples with values within the dynamic range of the assay. (The urine was initially diluted 1:8.)

		Human plasma	Cell culturesupernatant	Urine	Saliva	Human milk
1:2	Average% ofExpected	100	117	100	100	100
	Range (%)	-	105-129	-	-	-
1:4	Average% ofExpected	109	110	88	119	98
	Range (%)	97-121	107-112	79-97	117-120	98-99
1:8	Average% ofExpected	111	93	97	110	99
	Range (%)	97-125	84-103	95-99	100-120	96-102
1:16	Average% ofExpected	111	105	93	-	98
	Range (%)	100-122	100-110	91-94	-	91-104

CALIBRATION

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