The multiplex human cytokine is a complete quantitative ELISA-based chemiluminescent assay that allows concurrent measurement of biomarkers or analytes. This Multiplex Elisa kit for pro-inflammatory cytokines is designed for semi-quantitative and simultaneous determination of pro-inflammatory cytokines. And this includes interleukin-1α (IL-1α), interleukin 1β (IL-1β), Interleukin-2 (IL-2), Interleukin-4 (IL-4), interleukin-5 (IL-5), Interleukin-6 (IL-6), interleukin-8 (IL-8), Interleukin-10 (IL-10), IL-12p70, IL-13, IL-15, IL-17, IL-23, granulocyte-macrophage colony-stimulating factor (GM-CSF), monocyte chemotactic and activating factor (MCAF), IFNγ, TNFβ and tumor necrosis factor-α (TNF-α) in cell culture supernatant and other biological samples.

What is Multiplex Assay

The multiplex assay is an immunoassay that measures multiple analytes in a single experiment simultaneously using magnetic beads. It is derived from Elisa like IL-1 Beta Elisa and uses beads to bind the captured antibody. The microsphere of designated colours is coated with antibodies in a multiplex assay in the defined binding specificities. Flow cytometry is the method used to read the results of the multiplex assay. And this is because the beads are distinguished by fluorescent signature. The number of various bead colours is dependent on the measured analytes number. The multiplex assay within a specified application area can be stratified based on the number of analytes measurable per assay, which is the highest.

What is Elisa kit

Elisa, which is an Enzyme-linked immunosorbent assay, is a plate-based assay technique. It is meant for detecting and quantifying peptides, proteins, antibodies, and hormones. To get the assay result using ELISA, an antigen must be immobilized to a solid surface and complexed with antibodies that have been linked to enzymes. ELISAs are commonly performed in 96-well or 384-well polystyrene plates, passively binding antibodies and proteins.

Elisa kits are ready-to-use immunoassay kits with all the reagents required to perform the ELISA contained in them, such as CXCL 10 Elisa. This tool makes the research quick, convenient, and accurate for detecting and quantitating targets of interest in cultures and samples. Elisa kits entail the attachment of a capture antibody to a solid state. ELISA is a plate-based experiment designed to detect and quantify molecules such as proteins, antibodies, hormones, peptides, and small molecules like vitamins and coenzymes. 

Multiplex Cytokine assays analysis 

In analyzing multiplex cytokine, there are different application measurements to be used. And this allows for evaluating the complexity and dynamic attributes of inflammatory responses in humans during clinical trials. This can be used to support the mechanism of action of immune-modulatory compounds. Alternatively, it can investigate the impact of therapy on the immune response in humans.

Contents of multiplex Elisa kit for human cytokine

1. Antibody coated microtiter plate 96 wells: Twelve 8-well Elisa strips are contained in the plate. Each of these 8-wells is coated with different monoclonal antibodies specific to one of the 8 cytokines.
2. Biotin conjugate mixture: this is a mixture of biotin, a conjugated anti-human cytokine antibody of 6ml.
3. HRP conjugate mixture: this contains a mixture of Horseradish Peroxidase Conjugates of 11ml.
4. Standard mixture: This contains 2 vials of the standard lyophilized mixture. There is a buffered protein base in each vial and 8 pro-inflammatory cytokines at different concentrations. The 8 pro-inflammatory cytokines are, IL-1α 1600pg, IL1β 2000pg, IL-6 700pg, IL-8 2000pg, GM-CSF 1800pg, IFN-γ 1300pg, MCAF 1600pg, TNFα 1800pg.
5. CALIBRATOR DILUENT I: 25mL, PBS buffer with bovine serum albumin and preservatives. It is suitable for plasma/serum testing. 
6. Calibrator diluent II: it contains 25mL, cell culture medium RPMI 1640 with newborn calf serum and preservative. It is suitable for testing of cell culture supernatant. 
7. Wash Buffer (20x): it contains a 20mL and 20-fold concentrated solution of buffered surfactant.
8. Substrate A: it has a 10mL buffered Solution with urea hydrogen peroxide.
9. Substrate B: this is a 10mL and buffered solution TMB.
10. Stop Solution: is in 14mL and 2N Sulphuric acid (H₂SO₄). Caution should be taken with this as it is a caustic material.

Validation of the Assay

• Before analyzing the clinical samples with multiplex immunoassay, pre-analytical sample handling and multiplex immunoassay must be validated. 
• A fit-for-purpose validation plan is designed to produce a robust, reliable, and precise data set. And this is based on the trial logistics and the intended purpose of the data.
• To run on a single multiplex cytokine assay plate to minimize the impact of inter-assay variation on cytokine concentrations, the preferable batches will be sampled from a single patient. Before carrying out the analysis, samples can be stored for some time
• Upon completion of the validation of multiplex cytokine assay, cytokine concentrations can be assessed for clinical samples. Comparison can then be carried out for pre-and post-dosing concentrations of cytokine with the use of IL-8 Elisa. Information around the effect of the therapy on the immune system will be provided with valuable information that will support the decision-making process for progress to the next phase of the analysis.