Human CD40 Ligand (CD40LG) ELISA Kit, Lot 21AO-387 [Cancer Immune Checkpoint Assay Kit] (CAT#: IOK-05-P298)

Plate, Standard, Diluent

Microplate reader capable of measuring absorbance at 450 nm, with correction wavelength set at 570 nm or 630 nm.
Pipettes and pipette tips.
50 μL to 300 μL adjustable multichannel micropipette with disposable tips.
Multichannel micropipette reservoir.
Beakers, flasks, cylinders necessary for preparation of reagents.
Deionized or distilled water. Polypropylene test tubes for dilution.

11.3 pg/mL

100 μL

3 - 4 h

Pre-coated

1.Add the Standard working solution to the first two columns: Each concentration of the solution is added in duplicate, to one well each, side by side (100 μL for each well).Cover the plate with the sealer provided in the kit. Incubate for 90 min at 37 °C.
2.Remove the liquid out of each well, do not wash. Immediately add 100 μL of Biotinylated Detection Ab working solution to each well. Cover with the Plate sealer. Gently mix up. Incubate for 1 hour at 37 °C.
3.Aspirate or decant the solution from each well, add 350 μL of wash buffer to each well. Soak for 1~2 min and aspirate or decant the solution from each well and pat it dry against clean absorbent paper. Repeat this wash step 3 times.
4.Add 100 μL of HRP Conjugate working solution to each well. Incubate for 30 min at 37 °C.
5.Aspirate or decant the solution from each well, repeat the wash process for five times as conducted in step 3.
6.Add 90 μL of Substrate Reagent to each well. Cover with a new plate sealer. Incubate for about 15 min at 37 °C. Protect the plate from light.
7.Add 50 μL of Stop Solution to each well.
8.Determine the optical density (OD value) of each well at once with a micro-plate reader set to 450 nm.

Average the duplicate optical density readings for each standards and sample, then subtract the average optical density value of the zero standard. Standard Concentration as horizontal axis, optical density (OD) Value as the vertical axis, regressing the data and create a standard curve using computer software.

Intra-assay precision (precision within an assay): Three serum-based and buffer-based samples of known concentration were tested twenty times on one plate to assess intra-assay precision.
Inter-assay precision (precision between assays): Three serum-based and buffer-based samples of known concentration were tested in six separate assays to assess inter-assay precision.

This product contains Sodium azide: a POISONOUS AND HAZARDOUS SUBSTANCE which should be handled by trained staff only.

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

Store kit reagents between 2 and 8 °C. Immediately after use remaining reagents should be returned to cold storage (2 to 8 °C).
Opened/Reconstituted Reagents: Up to 1 month at 2 - 8 °C.

Store kit reagents between 2 and 8 °C. Immediately after use remaining reagents should be returned to cold storage (2 to 8 °C).
Opened/Reconstituted Reagents: Up to 1 month at 2 - 8 °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, CD154) is a member of the TNF superfamily of molecules and is a CD40L is a 39 kDa glycoprotein that is primarily expressed on activated T cells. 15-18 kDa soluble forms of CD40L (sCD40L) is produced by proteolytic cleavage. CD40L binds to CD40 on antigen-presenting cells (APC), which leads to many effects depending on the target cell type. CD40L acts as a costimulatory molecule and is particularly important on a subset of T cells called T follicular helper cells (TFH cells).

959

P29965

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

1.Prepare all reagents and standards as directed.
2.Add 50 μL Assay Buffer to each well.
3.Add 50 μL Standard or sample per well within 15 minutes.
4.Add 50 μL Detect Antibody to each well.
5.Incubate for 2 hours at RT.
6.Aspirate and wash 6 times.
7.Add 100 μL Substrate Solution to each well. Incubate for 10 - 30 minutes at RT. Protect from light.
8.Add 100 μL Stop Solution to each well.
9.Read at 450 nm within 30 minutes. Correction 570 or 630 nm