sCD40L Assay Kit

Catalog No. ABIN955776

Product Details

Target: CD40 Ligand (CD40LG)

Detection Method: Colorimetric

Sample Type: Cell Culture Supernatant, Serum

Sensitivity: The limit of detection of sCD40L defined as the analyte concentration resulting in an absorption significantly higher than that of the dilution medium (mean plus three standard deviations) was determined to be 0.095 ng/mL (mean of 6 independent assays).

Components: 1 aluminum pouche with a Microwell Plate coated with Monoclonal Antibody (murine) to human sCD40L
1 vial (0.2 mL) HRP-Conjugate anti-sCD40L monoclonal (murine) antibody - contains preservative.
2 vials (20 ng/mL upon reconstitution) sCD40L Calibrator, lyophilized - contains preservative.
1 bottle (50 mL) Wash Buffer Concentrate 20x - contains preservative.
1 vial (12 mL) Sample Diluent (protein matrix). Please Note: In some cases the Sample Diluent contains insoluble white precipitations, which do not interfere with the test performance. Use according to protocol.
1 vial (5 mL) Assay Buffer Concentrate 20x - contains preservative.
1 vial (7 mL) Substrate Solution I (tetramethyl-benzidine)
1 vial (7 mL) Substrate Solution II (0.02 % buffered hydrogen peroxide)
1 vial (12 mL) Stop Solution (1M Phosphoric acid)
1 vial (0.4 mL each) Blue-Dye, Green-Dye
2 Dilution plates
2 adhesive Plate Covers

Material not included: 5 mL and 10 mL graduated pipettes
10 µL to 1,000 µL adjustable single-channel micropipettes with disposable tips
50 µL to 300 µL adjustable multi-channel micropipette with disposable tips
Multi-channel micropipette reservoir
Beakers, flasks, cylinders necessary for preparation of reagents
Device for delivery of Wash Solution (multi-channel wash bottle or automatic wash system)
Microwell strip reader capable of reading at 450 nm (620 nm as optional reference wavelength)
Glass-distilled or de-ionized water
Statistical calculator with program to perform linear regression analysis.

Application Details

Plate: Pre-coated

Reagent Preparation:

Except for the HRP-Conjugate, the sCD40L Calibrator, and the TMB Substrate Solution the reagents should be prepared before starting with the test procedure.

A. Wash Buffer: If crystals have formed in the Wash Buffer Concentrate, warm it gently until they have completely dissolved. Pour entire contents (50 mL) of the Wash Buffer Concentrate into a clean 1,000 mL graduated cylinder. Bring final volume to 1,000 mL with glass-distilled or de-ionized water. Mix gently to avoid foaming. The pH of the final solution should be adjusted to 7.4. Transfer to a clean wash bottle and store at 2°to 25°C. Please note that the Wash Buffer is stable for 30 days.

B. Assay Buffer: Mix the contents of the bottle well. Add contents of Assay Buffer Concentrate (5.0 mL) to 95 mL distilled or de-ionized water and mix gently to avoid foaming. Store at 2°to 8°C. Please note that the Assay Buffer is stable for 30 days.

C. Preparation of HRP-Conjugate: The HRP-Conjugate must be diluted 1:200 with Assay Buffer just prior to use in a clean plastic test tube. Please note that the HRP-Conjugate should be used within 30 minutes after dilution.

D. Preparation of sCD40L Calibrator:Reconstitute sCD40L Calibrator by addition of distilled water. Reconstitution volume is stated on the label of the calibrator vial. Mix gently to insure complete reconstitution. Prepare Calibrator shortly before use. Use immediately after reconstitution. Do not store reconstituted Calibrator.

E. TMB Substrate Solution: Using clean pipettes and containers known to be metal free, dispense an equal volume of Substrate Solution I into Substrate Solution II and swirl gently to mix. The TMB Substrate Solution may develop a yellow tinge over time. This does not seem to affect product performance. A blue color present in the TMB Substrate Solution, however, indicates that it has been contaminated and must be discarded. The TMB Substrate Solution must be used within a few minutes after mixing. Warm to room temperature before use. Avoid direct exposure of TMB reagents to intense light and oxidizing agents during storage or incubation.

Sample Preparation:

Cell culture supernatants and human serum will be suitable for use in the assay. Remove serum from the clot as soon as possible after clotting and separation. Do not use plasma preparations instead of sera for measurement. Samples containing a visible precipitate must be clarified prior to use in the assay. Do not use grossly hemolyzed or lipemic specimens. Clinical samples should be kept at 2°to 8°C and separated rapidly before storing at -20°C to avoid loss of bioactive sCD40L. Avoid repeated freeze-thaw cycles.

Calculation of Results:

Calculate the average 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 sCD4° Concentration on the abscissa. Draw a best fit curve through the points of the graph.
To determine the concentration of circulating sCD40L 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 sCD4° Concentration.
For samples which have been diluted 1:5 prior to testing, the concentration read from the calibration curve must be multiplied by the dilution factor (x 5).
Note: Calculation of samples with an O.D. exceeding 2.0 may result in incorrect, low sCD40L levels. Such samples require further dilution of 1:10
1:20 with Sample Diluent in order to precisely quantitative the actual sCD40L level.
It is suggested that each testing facility establishes a control sample of known sCD4° 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.

Restrictions: For Research Use only

Handling

Handling Advice: Since exact conditions may vary from assay to assay, a calibration curve must be established for every run.
Bacterial or fungal contamination of either screen samples or reagents or cross-contamination between reagents may cause erroneous results.

Storage: 4 °C

Storage Comment: Store all kit reagents between 2°and 8°C. Immediately after use remaining reagents should be returned to cold storage (2°to 8°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.

Expiry Date: The expiry date is stated on the label.

Target Details

Target: CD40 Ligand (CD40LG)

Alternative Name: sCD40L (CD40LG Products)

Synonyms: CD154 Kit, cd40l Kit, CD40L Kit, HIGM1 Kit, IGM Kit, IMD3 Kit, T-BAM Kit, TNFSF5 Kit, TRAP Kit, gp39 Kit, hCD40L Kit, CD40-L Kit, Cd40l Kit, Ly-62 Kit, Ly62 Kit, Tnfsf5 Kit, CD40 ligand Kit, TNF superfamily member 5 Kit, CD40LG Kit, cd40l Kit, Cd40lg Kit

Background: CD40 belongs to the TNF receptor superfamily. While the biological role of some of the ligand- receptor pairs in this family still remains obscure, CD40 has proven its importance. A key role of CD40/CD40L interactions in immune activation, particularly in T-cell dependent B cell responses is anticipated. This molecule as well as the other ligands of the family share the property of co-stimulation of T-cell proliferation and are all expressed by activated T-cells. The programmed cell death has been suggested to be involved in clonal elimination of self-reactive lymphocytes for the normal function of the immune system. Interaction with membrane bound self antigens may eliminate self-reactive nature B cells by apoptosis. Antigen receptor-mediated B cell apoptosis is blocked when a signal is transduced via the CD40 molecule on the B cell surface. Because CD40L is expressed on activated T helper cells, B cells may escape from apoptosis and are activated when the immune system interacts with foreign antigens, which are normally able to activate T-helper cells. Thus the CD40 - CD40L interaction plays a central role in the various phases of the B cell response to T-dependent antigens. Taken together, B cells can participate in regulating their own destruction. Protection against Fas- dependent apoptosis afforded by immunoglobulin-receptor engagement may constitute a fail-safe mechanism that eliminates bystander B cells activated by CD40L - expressing T cells, but ensures survival of antigen-specific B cells. CD40L is expressed on the surface of activated CD4+ T cells, basophils, and mast cells. Binding of CD40L to its receptor, CD40, on the surface of B cells stimulates B-cell proliferation, adhesion and differentiation. A soluble isoform of CD40L has been shown to exist in the circulation. This soluble molecule is a homotrimer of a 18kDa protein exhibiting full activity in B cell proliferation and differentiation assays, is able to rescue B cells from apoptosis and binds soluble CD40. CD40L is discussed in relation to a potential role in supporting B cell tumors and it has been discovered that the molecular defect in the X-linked Hyper-IgM-Syndrome is targeted to the CD40L gene. It is functionally involved in B cell hybridomas and chronic lymphocytic as well as several autoimmune diseases.An anti-sCD40L monoclonal coating antibody is adsorbed onto microwells. sCD40L present in the sample or calibrator binds to antibodies adsorbed to the microwells, a HRP- conjugated monoclonal anti- sCD40L is added and binds to sCD40L captured by the first antibody. Following incubation unbound enzyme conjugated anti- sCD40L is removed during a wash step and a substrate solution reactive with HRP is added to the wells. A colored product is formed in proportion to the amount of soluble sCD40L 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 sCD40L calibrator dilutions and sCD40L sample concentration determined.