Human CD40 Ligand (CD40LG) ELISA Kit, Lot 21AO-383 [Cancer Immune Checkpoint Assay Kit] (CAT#: IOK-05-P294)

Assay plate (12 × 8 coated Microwells)
Standard (freeze dried)
Biotin-antibody (100 × concentrate)
HRP-avidin (100 × concentrate)
Biotin-antibody Diluent
HRP-avidin Diluent
Sample Diluent
Wash Buffer (25 × concentrate)
TMB Substrate
Stop Solution
Adhesive Strip (for 96 wells)
Instruction manual

Microplate reader capable of measuring absorbance at 450nm, with the correction wavelength set at 540nm or 570nm.
An incubator which can provide stable incubation conditions up to 37°C ± 0.5°C.
Squirt bottle, manifold dispenser or automated microplate washer.
Absorbent paper for blotting the microtiter plate.
100mL and 500mL graduated cylinders.
Deionized or distilled water.
Pipettes and pipette tips.
Test tubes for dilution.

0.078 ng/mL

100 μL

1 - 4.5 h

Pre-coated

Biotin-antibody (1×) - Centrifuge the vial before opening.
HRP-avidin (1×) - Centrifuge the vial before opening.
Wash Buffer (1×) - If crystals have formed in the concentrate, warm up to room temperature and mix gently until the crystals have completely dissolved.
Standard - Centrifuge the standard vial at 6000-10000rpm for 30s.

1. Prepare all reagents, working standards and samples as directed in the respective sections.
2. Refer to the Assay Layout Sheet to determine the number of wells to be used and put any remaining wells and the desiccant back into the pouch and seal the ziploc, store unused wells at 4°C.
3. Add 100µL of standard or sample per well. Incubate for 2 hours at 37°C.
4. Remove the liquid of each well, don't wash.
5. Add 100µL of Biotin-antibody (1×) to each well. Incubate for 1 hour at 37°C.
6. Aspirate each well and wash, repeating the process two times for a total of three washes.
7. Add 100µL of HRP-avidin (1×) to each well. Incubate for 1 hour at 37°C.
8. Repeat the aspiration/wash process for five times as in step 6.
9. Add 90µL of TMB Substrate to each well. Incubate for 20 minutes at 37°C. Protect from light.
10. Add 50µL of Stop Solution to each well, gently tap the plate to ensure thorough mixing.
11. Determine the optical density of each well within 5 minutes using a microplate reader set to 450nm.

Average the duplicate readings for each standard and sample and subtract the average zero standard optical density.

Intra-assay precision (precision within an assay): Three samples of known concentration were tested twenty times on one plate to assess precision.
Inter-assay precision (precision between assays): Three samples of known concentration were tested in twenty assays to assess precision.
Intra-assay: CV% less than 8%
Inter-assay: CV% less than 10%

The Stop Solution provided with this kit is an acid solution. Wear eye, hand, face and clothing protection when using this material.

The kit should not be used beyond the expiration date on the kit label.
Do not mix or substitute reagents with those from other lots or sources.
If samples generate values higher than the highest standard, dilute the samples with Sample Diluent and repeat the assay.
Any variation in Sample Diluent, operator, pipetting technique, washing technique, incubation time/temperature and kit age can cause variation in binding.
This assay is designed to eliminate interference by soluble receptors, binding proteins and other factors present in biological samples. Until all factors have been tested in the Immunoassay, the possibility of interference cannot be excluded.

4 °C,-20 °C

May be stored at 2-8°C for up to 1 month. For long term storage, please store at -20°C. Try to keep assay plate in a sealed aluminium foil bag and avoid dampness.

6 months

May be stored at 2-8°C for up to 1 month. For long term storage, please store at -20°C. Try to keep assay plate in a sealed aluminium foil bag and avoid dampness.

For Research Use only

Cluster Of Differentiation 40 Ligand (CD40L)

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

Synonyms: RP3-527F8.3, CD154, CD40L, HIGM1, IGM, IMD3, T-BAM, TNFSF5, TRAP, gp39, hCD40L, CD40 antigen ligand|T-B cell-activating molecule|TNF-related activation protein|tumor necrosis factor (ligand) superfa

959

P29965

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

This assay employs the quantitative sandwich enzyme immunoassay technique. Antibody specific for sCD40L has been pre-coated onto a microplate. Standards and samples are pipetted into the wells and any sCD40L present is bound by the immobilized antibody. After removing any unbound substances, a biotin-conjugated antibody specific for sCD40L is added to the wells. After washing, avidin conjugated Horseradish Peroxidase (HRP) is added to the wells. Following a wash to remove any unbound avidin-enzyme reagent, a substrate solution is added to the wells and color develops in proportion to the amount of sCD40L bound in the initial step. The color development is stopped and the intensity of the color is measured.