sCD-40 ELISA Kit

Produktnummer ABIN955775

Produktdetails

Target: sCD-40

Reaktivität: Human

Nachweismethode: Colorimetric

Methodentyp: Sandwich ELISA

Applikation: ELISA

Verwendungszweck: The Human sCD40 ELISA is an enzyme-linked immunosorbent assay for the quantitative determination of human sCD40 in cell culture supernatant, plasma, amniotic fluid, and serum.

Proben: Cell Culture Supernatant, Plasma, Serum

Analytische Methode: Quantitative

Spezifität: The interference of circulating factors of the immune system was evaluated by spiking these proteins at physiologically relevant concentrations into a serum sample. There was no detectable cross reactivity.

Sensitivität: The limit of detection of sCD40 defined as the analyte concentration resulting in an absorption significantly higher than that of the dilution medium (mean plus 2 standard deviations) was determined to be 1.3 pg/mL (mean of 6 independent assays).

Produktmerkmale: An anti-sCD40 monoclonal coating antibody is adsorbed onto microwells. sCD40 present in the sample or calibrator binds to antibody adsorbed to the microwells, a biotin-conjugated monoclonal anti- sCD40 antibody is added and binds to sCD40 captured by the first antibody. Following incubation unbound biotin-conjugated anti-sCD40 is removed during a wash step. Streptavidin-HRP is added and binds to the biotin-conjugated anti-sCD40. Following incubation unbound Streptavidin-HRP is removed during a wash step, and substrate solution reactive with HRP is added to the wells. A colored product is formed in proportion to the amount of sCD40 present in the sample. The reaction is terminated by addition of acid and absorbance is measured at 450 nm. A calibration curve is prepared from seven sCD40 calibrator dilutions and sCD40 sample concentration determined.

Bestandteile: 1 aluminum pouch with Microwell Plate (12 x 8-well strips) coated with monoclonal antibody (mouse) to human sCD40
1 vial (100 µL) Biotin-Conjugate anti-sCD40 monoclonal (mouse) antibody
1 vial (150 µL) Streptavidin-HRP
2 vials sCD40 Calibrator, lyophilized, 1,000 pg/mL upon reconstitution
1 bottle (50 mL) Wash Buffer Concentrate 20X (PBS with 1% Tween 20)
1 vial (5 mL) Assay Buffer Concentrate 20X (PBS with 1% Tween 20 and protein stabilizer)
1 vial (12 mL) Sample Diluent
1 vial (15 mL) Substrate Solution
1 vial (12 mL) Stop Solution (1M Phosphoric acid)
1 vial (0.4 mL each), Blue Dye, Green Dye, Red Dye
4 adhesive Plate Covers.

Benötigtes Material: 5 mL and 10 mL graduated pipettes
5 µL to 1,000 µL adjustable single channel micropipettes with disposable tips
50 µL to 300 µL adjustable multichannel micropipette with disposable tips
Multichannel micropipette reservoir
Beakers, flasks, cylinders necessary for preparation of reagents
Device for delivery of wash solution (multichannel wash bottle or automatic wash system)
Microwell strip reader capable of reading at 450 nm (620 nm as optional reference wave length)
Glass-distilled or de-ionized water
Statistical calculator with program to perform regression analysis

Anwendungsinformationen

Plattentyp: Pre-coated

Aufbereitung der Reagenzien:

Buffer Concentrates should be brought to room temperature and should be diluted before starting the test procedure. If crystals have formed in the Buffer Concentrates, warm them gently until they have completely dissolved.

A. Wash Buffer (1X): Pour entire contents (50 mL) of the Wash Buffer Concentrate (20X) into a clean 1,000 mL graduated cylinder. Bring final volume to 1,000 mL with distilled or de-ionized water. Mix gently to avoid foaming. The pH of the final solution should adjust to 7.4. Transfer to a clean wash bottle and store at 2°to 25°C. Please note that the Wash Buffer (1X) is stable for 30 days.

B. Assay Buffer (1X:) Pour entire contents (5 mL) of the Assay Buffer Concentrate (20X) into a clean 100 mL graduated cylinder. Bring to final volume of 100 mL with distilled water. Mix gently to avoid foaming. Store at 4°C. Please note that the Assay Buffer (1X) is stable for 30 days.

C. Preparation of sCD40 Calibrator Reconstitute sCD40 Calibrator by addition of distilled water. Reconstitution volume is stated on the label of the Calibrator vial. Swirl or mix gently to insure complete and homogeneous solubilization (concentration of reconstituted calibrator = 1,000 pg/mL). Allow the reconstituted calibrator to sit for exactly 10 minutes. Mix well prior to making dilutions. After usage remaining calibrator cannot be stored and has to be discarded.

D. Preparation of Biotin-Conjugate Please note that the Biotin-Conjugate should be used within 30 minutes after dilution. Dilute the Biotin-Conjugate 1:100 just prior to use with Assay Buffer (1X) in a clean plastic tube. Mix the contents of the tube well.

E. Preparation of Streptavidin-HRP: Please note that the Streptavidin-HRP should be used within 30 minutes after dilution. Make a 1:100 dilution of the concentrated Streptavidin-HRP solution with Assay Buffer (1X) in a clean plastic tube as needed.

F. Addition of color-giving reagents: Blue Dye, Green Dye, Red Dye: In order to help our customers to avoid any mistakes in pipetting the Human sCD40 ELISA, we now offer a new tool that helps to monitor the addition of even very small volumes of a solution to the reaction well by giving distinctive colors to each step of the ELISA procedure. This procedure is optional, does not in any way interfere with the assay results, and is designed to help the customer with the performance of the assay, but can also be omitted, just following the package insert.

Aufbereitung der Proben:

Cell culture supernatants, human serum, EDTA or heparinized plasma, amniotic fluid, or other body fluids are suitable for use in the assay. Remove the serum or plasma from the clot or red cells, respectively, as soon as possible after clotting and separation. Samples containing a visible precipitate must be clarified prior to use in the assay. Do not use grossly hemolyzed or lipemic specimens. Samples should be aliquoted and must be stored frozen at -20°C to avoid loss of bioactive sCD40. If samples are to be run within 24 hours, they may be stored at 4°C. Avoid repeated freeze-thaw cycles. Prior to assay, frozen samples should be brought to room temperature slowly and mixed gently.

Ergebnisberechnung:

Calculate the mean absorbance values for each set of duplicate calibrators and samples. Duplicates should be within 20 percent of the mean.
Create a calibration curve by plotting the mean absorbance for each calibrator concentration on the ordinate against the sCD40 concentration on the abscissa. Draw a best-fit curve through the points of the graph (a 5-parameter curve fit is recommended).
To determine the concentration of circulating sCD40 for each sample, first find the mean absorbance value on the ordinate and extend a horizontal line to the calibration curve. At the point of intersection, extend a vertical line to the abscissa and read the corresponding sCD40 concentration.
For samples which have been diluted (1:2) according to the instructions given in this package insert, the concentration read from the calibration curve must be multiplied by the dilution factor (x2).
Calculation of samples with a concentration exceeding Calibrator 1 may result in incorrect, low human sCD40 levels. Such samples require further external predilution according to expected human sCD40 values with Sample Diluent in order to precisely quantitate the actual human sCD40 level.
It is suggested that each testing facility establishes a control sample of known sCD40 concentration and runs this additional control with each assay. If the values obtained are not within the expected range of this control, the assay results may be invalid.

Beschränkungen: Nur für Forschungszwecke einsetzbar

Handhabung

Lagerung: 4 °C

Informationen zur Lagerung: Store kit reagents at 4°C as indicated. Immediately after use remaining reagents should be returned to cold storage (4°C). Expiration date of the kit and reagents is stated on labels. The expiration date of the kit components can only be guaranteed if the components are stored properly, and if, in case of repeated use of one component, the reagent is not contaminated by the first handling.

Haltbarkeit: The expiry date is stated on the label.

Antigendetails

Target: sCD-40

Hintergrund: CD40 is a 50 kDa membrane-bound type I glycoprotein expressed by numerous cells, most notably B lymphocytes and monocytes and antigen presenting cells (APC) such as macrophages, dendritic cells and fibroblasts. It is furthermore highly expressed on various malignant cells. The expression of CD40 regulates T-cell-APC interaction and has been shown to be centrally involved in a wide array of inflammatory events. The function of CD40 is very broad. CD40 belongs to the TNF-receptor family. The ligand for CD40 (CD154) is a 33 kDa type II transmembrane protein mainly expressed by activated T-cells, and is a member of the TNF superfamily. CD40/CD40L interactions are essential for T- cell-dependent B cell proliferation and differentiation, for activation of antigen presenting cells and for cytokine production on numerous other cells. It is presently accepted that CD40 plays a critical role in the regulation of immune responses. CD40 expression has also been found on non-lymphoid cells such as fibroblasts, endothelia, and epithelial cells. Its critical role in T-cell-dependent humoral immune responses was demonstrated by patients with the hyper-IgM Syndrome. CD40 is also involved in the apoptotic pathway of cells. While it has been shown that the programmed cell death can be inhibited by the survival signals mediated from the binding of the CD40 receptor to the CD40 Ligand, very recently a novel proapoptotic mechanism induced by CD40 in carcinoma cells has been described. This mechanism is dependent on the endogenous production of cytotoxic cytokines. The interaction of CD40 and its ligand, CD154 (CD40L) was found to play a crucial role in many aspects of immune response and the development and progress of various diseases. It was found to be centrally involved in transplant rejection. The interaction of CD40 on synovial fibroblasts and CD40L expressed on activated T lymphocytes is directly involved in the neovascularization in rheumatoid synovitis. CD40 expression in thyroid tissue suggests a new pathway of pathogenesis of thyroid diseases. Functional expression of CD40 on human melanoma cells mediates T-cell co-stimulation and tumor cell growth. The interaction of CD40 and CD154 is centrally involved in a wide array of inflammatory events such as multiple sclerosis, atherosclerosis and asthma-associated airway inflammation. The expression of CD40 on various B cell malignancies such as leukemias, non-Hodgkin's lymphoma and multiple myeloma has been described. CD40 was found to regulate cell growth in squamous cell cancer of the head and neck. Its expression in hepatocellular carcinomas plays an important role in tumor biology. The expression of CD40 on human lung cancer correlates with metastatic spread and may serve as a prognostic marker and an indicator of advanced disease. An increased CD40 expression on muscle cells of polymyositis and dermatomyositis has been described.