CD40, Mouse, BioAssay(TM) ELISA Kit

CD40, also known as TNFRSF5, is a 45-50kD variably glycosylated type I transmembrane protein belonging to the TNF receptor (TNFR) superfamily (1, 2). It exhibits diverse activities in normal immune system development and function and participates in the control or progression of a variety of diseases. Mature mouse CD40 consists of a 174aa extracellular domain (ECD) with four TNFR repeats, a 22 aa transmembrane segment, and a 74 aa cytoplasmic domain (3). Within the ECD, mouse CD40 shares 58% and 83% aa sequence identity with human and rat CD40, respectively. Additional splice forms show substitutions and/or deletions within and following the fourth TNFR repeat (4). Soluble CD40 (27-28kD), which functions as an inhibitor of CD40 bioactivity, can be generated by alternate splicing or by proteolytic cleavage of transmembrane CD40 by TACE (4-6). CD40 is expressed on B cells, dendritic cells, monocytes, macrophages, T cells, neutrophils, platelets, fibroblasts, smooth muscle cells, epithelial cells, endothelial cells, neurons, and many hematopoietic and epithelial cancers (1, 7, 8). It is upregulated on these cell types during inflammation and notably on CD4+ T cells in autoimmune mice (9-11). CD40 homotrimerization is important for its ability to initiate signaling through TRAF family proteins (12). This is induced by the binding of CD40 to the 39kD transmembrane glycoprotein CD40 Ligand/CD154 (CD40L). CD40L itself is upregulated on T cells, B cells, dendritic cells, neutrophils, platelets, vascular endothelial cells, and smooth muscle cells during activation or inflammation (1, 7, 13, 14). CD40L is expressed on the cell surface in heteromultimers of different isoforms (15). Soluble CD40L is generated by intracellular proteolytic cleavage and is secreted as a homotrimer of 15-18kD subunits (15, 16). Both the membrane bound and soluble forms of CD40L induce signaling through CD40 (17, 18). Interactions between CD40 and CD40L are involved in multiple aspects of humoral immunity, cellular immunity, and inflammation. CD40L on activated CD4+ T cells provides a costimulatory signal that augments B cell proliferation, germinal center formation, immunoglobulin class switching, and antibody secretion (1, 11, 18). CD40 ligation likewise promotes the activation of the many other cell types which express it and additionally promotes the development of medullary thymic epithelial, Th17, and FoxP3+ Treg cells (14, 19-23). CD40-CD40L interactions regulate lymphocyte development and the balance between immune self-tolerance and autoimmunity (11, 24). CD40 signaling reinforces inflammatory responses but can also fulfill a tissue protective role by enhancing epithelial cell survival during oxidative stress (7, 23, 25-28). CD40-CD40L interactions in disease can be beneficial (contributing to the immune response to cancer) or detrimental (contributing to the progression of atherosclerosis and graft versus host disease) (1, 7, 8). CD40-mediated responses are modulated by TLR, RANK, and TNF receptor activity through intracellular signaling crosstalk or direct interaction of CD40 with those receptors (2, 20, 21). The Mouse CD40 immunoassay is a 4.5 hour solid phase ELISA designed to measure CD40 in cell culture supernates, cell lysates, serum, and plasma. It contains NS0-expressed recombinant mouse CD40 and has been shown to accurately quantitate the recombinant factor. Results obtained using natural CD40 showed linear curves that were parallel to the standard curves obtained using the kit standards. These results indicate that this kit can be used to determine relative mass values for naturally occurring mouse CD40. Sample Type: Serum/Plasma-Samples were evaluated for the presence of mouse CD40 in this assay. Sensitivity: Fifty-nine assays were evaluated and the minimum detectable dose (MDD) of mouse CD40 ranged from 0.31-3.70 pg/ml. The mean MDD was 1.49 pg/ml. The MDD was determined by adding two standard deviations to the mean optical density value of twenty zero standard replicates and calculating the corresponding concentration. Specificity: This assay recognizes recombinant and natural mouse CD40. The factors listed below were prepared at 50ng/ml in Calibrator Diluent and assayed for cross-reactivity. Preparations of the following factors at 50ng/ml in a mid-range recombinant mouse CD40 control were assayed for interference. No significant cross-reactivity or interference was observed. Test Principle: This assay employs the quantitative sandwich enzyme immunoassay technique. A monoclonal antibody specific for mouse CD40 has been pre-coated onto a microplate. Standards, control, and samples are pipetted into the wells and any mouse CD40 present is bound by the immobilized antibody. After washing away any unbound substances, an enzyme-linked polyclonal antibody specific for mouse CD40 is added to the wells. Following a wash to remove any unbound antibody-enzyme reagent, a substrate solution is added to the wells and color develops in proportion to the amount of mouse CD40 bound in the initial step. The color development is stopped and the intensity of the color is measured. Precision: Intra-assay:, Mean (ng/ml): 21.0, 67.2, 159, Standard Deviation: 1.5, 3.3, 5.2, CV (%): 7.1, 4.9, 3.3, Inter-assay:, Mean (ng/ml): 22.7, 70.9, 169, Standard Deviation: 1.7, 2.9, 7.8, CV (%): 7.5, 4.1, 4.6, Kit Components: Mouse CD40 Microplate 1x96 well polystyrene microplate (12 strips of 8 wells) coated with a rat monoclonal antibody against mouse CD40., Mouse CD40 Conjugate 12.5ml of a polyclonal antibody against mouse CD40 conjugated to horseradish peroxidase with preservatives., Mouse CD40 Standard 2.5ng of recombinant mouse CD40 in a buffered protein solution with preservatives, lyophilized., Mouse CD40 Control 1 vial of recombinant mouse CD40 in a buffered protein base with preservatives, lyophilized. The concentration range of mouse CD40 after reconstitution is shown on the vial label. The assay value of the Control should be within the range specified on the label., Assay Diluent RD1-21 12.5ml of a buffered protein solution with preservatives, Calibrator Diluent RD5-3 21ml of a buffered protein solution with preservatives., Wash Buffer Concentrate 50ml of a 25-fold concentrated solution of a buffered surfactant with preservatives., Color Reagent A 12.5ml of stabilized hydrogen peroxide. , Color Reagent B 12.5ml of stabilized chromogen (tetramethylbenzidine). , Stop Solution 23ml of a diluted hydrochloric acid solution. Plate Covers-4 adhesive strips. Storage and Stability: Store components at 4°C. Stable for at least 6 months. For maximum recovery of product, centrifuge the original vial after thawing and prior to removing the cap.