CD40 Ligand ELISA Kit

Catalog No. ABIN924794

Product Details CD40 Ligand ELISA Kit

Target: CD40 Ligand (CD40LG)

Binding Specificity: Soluble

Reactivity: Human

Detection Method: Colorimetric

Method Type: Sandwich ELISA

Detection Range: 32-2000 pg/mL

Minimum Detection Limit: 32 pg/mL

Application: ELISA

Purpose: The OmniKine? Human sCD40 Ligand ELISA Kit contains the components necessary for quantitative determination of natural or recombinant Human sCD40 Ligand concentrations within any experimental sample including cell lysates, serum and plasma. This particular immunoassay utilizes the quantitative technique of a "Sandwich" Enzyme- Linked Immunosorbent Assay (ELISA) where the target protein (antigen) is bound in a "sandwich" format by the primary capture antibodies coated to each well-bottom and the secondary detection antibodies added subsequently by the investigator. The capture antibodies coated to the bottom of each well are specific for a particular epitope on Human sCD40 Ligand while the user-added detection antibodies bind to epitopes on the captured target protein. Amid each step of the procedure, a series of wash steps must be performed to ensure the elimination of non-specific binding between proteins to other proteins or to the solid phase. After incubation and "sandwiching" of the target antigen, a peroxidase enzyme is conjugated to the constant heavy chain of the secondary antibody (either covalently or via Avidin/Streptavidin-Biotin interactions), allowing for a colorimetric reaction to ensue upon substrate addition. When the substrate TMB (3, 3', 5, 5'-Tetramethylbenzidine) is added, the reaction catalyzed by peroxidase yields a blue color that is representative of the antigen concentration. Upon sufficient color development, the reaction can be terminated through addition of Stop Solution (2 N Sulfuric Acid) where the color of the solution will turn yellow. The absorbance of each well can then be read by a spectrophotometer, allowing for generation of a standard curve and subsequent determination of protein concentration.

Brand: OmniKine™

Sample Type: Cell Lysate, Serum, Plasma

Analytical Method: Quantitative

Specificity: The Human sCD40 Ligand ELISA Kit allows for the detection and quantification of endogenous levels of natural and/or recombinant Human sCD40 Ligand proteins.

Cross-Reactivity (Details): The Human sCD40 Ligand ELISA is capable of recognizing both recombinant and naturally produced Human sCD40 Ligand proteins. The antigens listed below were tested at 50 ng/mL and exhibited less than 1% cross reactivity. Murine: sCD40L The antigens listed below were tested at 50 ng/mL and did not exhibit significant cross reactivity or interference. Human: 4-1BBL, BAFF, sFasL, FasR, IL-1α, sRANKL, sRANKR, sTNF-RI, sTNF-RII, TNFα, TNFβ, sTRAIL, sTRAIL-RI, sTRAIL-RII, TWEAK Murine: sRANKL, TNFα Rat: TNFα

Characteristics: The Human sCD40 Ligand ELISA Kit allows for the detection and quantification of endogenous levels of natural and/or recombinant Human sCD40 Ligand proteins within the range of 32-2000 pg/mL.

Components:

• Microstrips Coated w / Capture Antibody: 12 x 8-Well Microstrips
• Protein Standard: Lyophilized (100 ng), Red container
• Biotinylated Detection Antibody: Lyophilized, Yellow container
• 400x Streptavidin-HRP: 30 μL, Blue container
• Wash Buffer (10x): 50 mL, Clear containter
• Assay Diluent: 50 mL, Clear container
• Ready-to-Use Substrate: 12 mL, Brown container
• Stop Solution: 12 mL, Clear container
• Adhesive Plate Sealers: 4 Sheets
• Technical Manual 1 Manual

Material not included: The following materials and/or equipment are NOT provided in this kit but are necessary to successfully conduct the experiment:
Microplate reader able to measure absorbance at 450 nm (with correction wavelength set to 540 nm or 570 nm)
Micropipettes with capability of measuring volumes ranging from 1 μl to 1 mL
Deionized or sterile water
Squirt bottle, manifold dispenser, multichannel pipette reservoir or automated microplate washer
Graph paper or computer software capable of generating or displaying logarithmic functions
Absorbent paper or vacuum aspirator
Test tubes or microfuge tubes capable of storing ≥1 mL
Bench
top centrifuge (optional)
Bench
top vortex (optional)
Orbital shaker (optional)

Application Details

Plate: Pre-coated

Protocol: This particular immunoassay utilizes the quantitative technique of a Sandwich Enzyme- Linked Immunosorbent Assay (ELISA) where the target protein (antigen) is bound in a sandwich format by the primary capture antibodies coated to each well-bottom and the secondary detection antibodies added subsequently by the investigator. The capture antibodies coated to the bottom of each well are specific for a particular epitope on the Human sCD40 Ligand cytokine while the user-added detection antibodies bind to epitopes on the captured target protein. Amid each step of the procedure, a series of wash steps must be performed to ensure the elimination of non- specific binding between proteins to other proteins or to the solid phase. After incubation and sandwiching of the target antigen, a peroxidase enzyme is conjugated to the constant heavy chain of the secondary antibody (either covalently or via Avidin/Streptavidin-Biotin interactions), allowing for a colorimetric reaction to ensue upon substrate addition. When the substrate TMB (3, 3’, 5, 5’-Tetramethylbenzidine) is added, the reaction catalyzed by peroxidase yields a blue color that is representative of the antigen concentration. Upon sufficient color development, the reaction can be terminated through addition of Stop Solution (2 N Sulfuric Acid) where the color of the solution will turn yellow. The absorbance of each well can then be read by a spectrophotometer, allowing for generation of a standard curve and subsequent determination of protein concentration.

Sample Preparation:

If samples are to be used within 24 hours, aliquot and store at 4 °C. If samples are to be used over a long period of time, aliquot and store between -20 °C and -80 °C, depending on the duration of storage.
Note: Samples containing a visible precipitate or pellet must be clarified prior to use in the assay.
Caution: Avoid repeated freeze/thaw cycles to prevent loss of biological activity of proteins in experimental samples.

• Cell Lysate and Supernatants:
  Remove large cell components via centrifugation and perform the assay. Cell lysates and supernatants require a dilution using Assay Diluent. A serial dilution may be performed to determine a suitable dilution factor for the sample. For future use of the sample, follow the sample storage guidelines stated above.
• Serum:
  Allow samples to clot in a serum separator tube (SST) for 30 minutes. After sufficient clotting, centrifuge at 1000 x g for 15 minutes and remove serum from SST in preparation for the assay. Serum samples require at least a 1:50 dilution using Assay Diluent. For future use of the sample, follow the storage guidelines above.
• Plasma:
  Use heparin, citrate or EDTA as an anticoagulant to gather plasma from original biological sample. After collection of the plasma, centrifuge for 15 minutes at 1000 x g. This step must be performed within 30 minutes of plasma collection. Plasma samples require at least a 1:50 dilution using Assay Diluent. Afterwards, perform the assay or for future use of the sample, follow the storage guidelines stated above.

Assay Procedure:

Note: If possible, all incubation steps should be performed on an orbital shaker to equilibrate solutions when added to the microplate wells. Also, all provided solutions should be at ambient temperature prior to use.
Note: Avoid adding solutions into wells at an angle, always keep pipette tip perpendicular to plate bottom.

Reconstitution of Provided Materials:

1. Reconstitute the Biotin-Conjugated Detection Antibody in 67 µL of ddH₂O for a concentration of 180 µg/ml.
2. Reconstitute the Protein Standard in 100 µL of ddH₂O for a concentration of 340 ng/ml.
3. Dilute the 50 mL of 10x Wash Buffer in 450 mL of ddH2O for 500 mL of 1x Wash Buffer.

Addition of Known Standard and Unknown Sample to Immunoassay:

The OmniKine™ Human CD163 ELISA Kit allows for the detection and quantification of endogenous levels of natural and/or recombinant Human CD163 proteins

Calculation of Results:

Generation of Standard Curve and Interpretation of Data
1. Average the duplicate or triplicate readings for each standard, control and sample and subtract the average zero standard optical density.
2. Generate a standard curve by using Microsoft Excel or other computer software capable of establishing a 4- Parameter Logistic (4-PL) curve fit. If using Excel or an alternative graphing tool, plot the average optical density values in absorbance units (y-axis) against the known standard concentrations in pg/ml (x-axis). Note: Only use the values in which a noticeable gradient can be established. Afterwards, generate a best fit curve or trend-line through the plotted points via regression analysis.

Restrictions: For Research Use only

Handling

Precaution of Use: Reagents provided in this kit may be harmful if ingested, inhaled or absorbed through the skin. Please carefully review the MSDS for each reagent before conducting the experiment.
Stop Solution contains 2 N Sulfuric Acid (H2SO4) and is an extremely corrosive agent. Please wear proper eye, hand and face protection when handling this material. When the experiment is finished, be sure to rinse the plate with copious amounts of running water to dilute the Stop Solution prior to disposing the plate.

Handling Advice: This ELISA kit is intended for research purposes only, NOT diagnostic or clinical procedures of any kind.
Materials included in this kit should NOT be used past the expiration date on the kit label.
Reagents or substrates included in this kit should NOT be mixed or substituted with reagents or substrates from any other kits.
Variations in pipetting technique, washing technique, operator laboratory technique, kit age, incubation time or temperature may cause differences in binding affinity of the materials provided.
The assay is designed to eliminate interference and background by other cellular macromolecules or factors present within any biological samples. However, the possibility of background noise cannot be fully excluded until all factors have been tested using the assay kit.

Reagents provided in this kit may be harmful if ingested, inhaled or absorbed through the skin. Please carefully review the MSDS for each reagent before conducting the experiment.
Stop Solution contains 2 N Sulfuric Acid (H2SO4) and is an extremely corrosive agent. Please wear proper eye, hand and face protection when handling this material. When the experiment is finished, be sure to rinse the plate with copious amounts of running water to dilute the Stop Solution prior to disposing the plate.

Storage: 4 °C

Storage Comment: Note: If used frequently, reagents may be stored at 4 °C.

• Unopened Kits: Store at 4 °C for 6 months.
• Microstrips Coated w/ Capture Antibody, 400x Streptavidin-HRP Wash Buffer (10x), Assay Diluent Ready-to-Use Substrate, Stop Solution: 6 Months at 4 °C
• Protein Standard, Biotinylated Detection Antibody: Lyophilized: 6 Months (if Reconstituted: 1 Month) at 4 °C

Target Details for CD40 Ligand

Target: CD40 Ligand (CD40LG)

Alternative Name: CD40 Ligand (CD40LG Products)

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

Background: Human sCD40 Ligand, also known as Tumor Necrosis Factor Ligand Superfamily Member 5, is a 149 amino acid cytokine protein encoded by the CD40LG gene located at locus Xq26 on the X chromosome. The soluble CD40 ligand is generated when the membrane form of the CD40 ligand undergoes proteolytic processing to remove the first 112 residues from the N-terminal region. The homotrimeric soluble CD40 ligand Mediates B-cell proliferation in the absence of co-stimulus as well as IgE production in the presence of IL-4, it is involved in immunoglobulin class switching. Moreover, release of soluble CD40L from platelets is partially regulated by GP IIb/IIIa, actin polymerization, and the matrix metalloproteinases (MMP) inhibitor-sensitive pathway. Defects in CD40LG are the cause of X-linked immunodeficiency with hyper-IgM type 1 (HIGM1), also known as X-linked hyper IgM syndrome (XHIM). HIGM1 is an immunoglobulin isoform switch defect characterized by elevated concentrations of serum IgM and decreased amounts of all other isoforms. Affected males present at an early age (usually within the first year of life) recurrent bacterial and opportunistic infections, including Pneumocystis carinii pneumonia and intractable diarrhea due to cryptosporidium infection. Source: Entrez Gene: CD40LG CD40 ligand [Homo sapiens], Swiss-Prot: P29965

Gene ID: 959

UniProt: P29965

Pathways: NF-kappaB Signaling, Production of Molecular Mediator of Immune Response, Cancer Immune Checkpoints