Mouse CD40 Ligand (CD40LG) ELISA Kit, Lot 21AO-393 [Cancer Immune Checkpoint Assay Kit] (CAT#: IOK-05-P304)

Reagent (Quantity): Assay plate (1), Standard (2), Sample Diluent (1x20ml), Assay Diluent A (1x10ml), Assay Diluent B (1x10ml), Detection Reagent A 1x120µl Detection Reagent B 1x120µl Wash Buffer(25 x concentrate) (1x30ml), Substrate (1x10ml), Stop Solution (1x10ml)

100 μL

Pre-coated

Bring all reagents and samples to room temperature for 20 minutes before use.
Wash Buffer
Standards
Preparation of Biotin-labeled Antibody Working Solution
Preparation of HRP-Streptavidin Conjugate (SABC) Working Solution

1.Add 100µL of standard or sample per well. Cover with the adhesive strip provided. Incubate for 2 hours at 37°C.
2.Remove the liquid of each well, don't wash.
3.Add 100µL of Biotin-antibody (1×) to each well. Cover with a new adhesive strip. Incubate for 1 hour at 37°C.
4.Aspirate each well and wash, repeating the process two times for a total of three washes.After the last wash, remove any remaining wash Buffer by aspirating or decanting. Invert the plate and blot it against clean paper towels.
5.Add 100µL of HRP-avidin (1×) to each well. Cover the microtiter plate with a new adhesive strip. Incubate for 1 hour at 37°C.
6.Repeat the aspiration/wash process for five times as in step 6.
7.Add 90µL of TMB Substrate to each well. Incubate for 20 minutes at 37°C. Protect from light.
8.Add 50µL of Stop Solution to each well, gently tap the plate to ensure thorough mixing.
9.Determine the optical density of each well within 5 minutes using a microplate reader set to 450nm.

Average the duplicate readings for each standard, control, and sample and subtract the average zero standard optical density. Create a standard curve by reducing the data using computer software capable of generating a four parameter logistic (4-PL) curve-fit.

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

The Standard, Detection Reagent A, Detection Reagent B and the 96-well strip plate should be stored at -20 °C upon being received. The other reagents can be stored at 4 °C.

The Standard, Detection Reagent A, Detection Reagent B and the 96-well strip plate should be stored at -20 °C upon being received. The other reagents can be stored at 4 °C.

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

CD40 Ligand [CD40L, also known as CD154, gp39, TRAP (TNF-Related Activation Protein) or TBAM (T-cell B-cell Activating Molecule)], a member of the TNF superfamily (TNFSF), is a multi-functional ligand. Interaction between CD40 and CD40L is critical to the control of thymus-dependent humoral immunity and cell-mediated immune responses.

21947

P27548

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

A monoclonal 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. An enzyme-linked monoclonal antibody specific for SCD40L 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 SCD40L bound in the initial step.