Swine IL-2 ELISA Assay

Additional Information

Assay Procedure

1. Prepare all reagents and working standards as directed in the previous sections.
2. Determine the number of microwell strips required to test the desired number of samples plus appropriate number of wells needed for running blanks and standards. Remove extra microwell strips from holder and store in foil bag with the desiccant provided at 2-8°C sealed tightly.
3. Add 100 µl of Standard, control, or sample, per well, then add 50 µl of the working solution of Biotin-Conjugate to each well. Cover with the adhesive strip provided and incubate 2 hours at RT. Adequate mixing is very important for good result. Use a mini-vortexer at the lowest frequency.
4. Aspirate each well and wash, repeating the process three times for a total of four washes. Wash by filling each well with Wash Buffer (350 µl) using a squirt bottle, manifold dispenser or auto-washer. Complete removal of liquid at each step is essential to good performance. 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 100 µl of the working solution of Streptavidin-HRP to each well. Cover with a new adhesive strip and incubate for 30 minutes at RT Avoid placing the plate in direct light.
6. Repeat the aspiration/wash.
7. Add 100 µl of Substrate Solution to each well. Incubate for 10-20 minutes at RT. Avoid placing the plate in direct light.
8. Add 100 µl of Stop Solution to each well. Gently tap the plate to ensure thorough mixing.
9. Determine the optical density of each well immediately, using a microplate reader set to 450 nm.(optionally 630nm as the reference wave length;610-650nm is acceptable)

Assay Background

Interleukin 2 (IL-2) is a pleiotropic cytokine produced primarily by mitogen- or antigen-activated T lymphocytes (1, 2). Human IL-2 (also known as T-cell growth factor) is produced by T-cells in response to antigenic or mitogenic stimulation. IL-2 is a potent lymphoid cell growth factor which exerts its biological activity primarily on T cells promoting proliferation and maturation.

IL-2 has been found to stimulate growth and differentiation of B cells, NK cells, LAK cells, monocytes, and oligodendocytes. IL-2 is involved in treatment of cancers such as melanoma and renal cell cancer. It plays a key role in promoting the clonal expansion of antigen-specific T cells. In addition, IL-2 has also been shown to mediate multiple immune responses on a variety of cell types. At the amino acid sequence level, there is approximately 72% similarity between mature porcine and human IL-2. The biological effects of IL-2 are mediated by specific cell surface receptor complexes. The functional high-affinity receptor for IL-2 is composed of three distinct polypeptide chains (3, 4). IL-2 stimulates the proliferation of thymocytes; stimulates the proliferation and differentiation of activated B cells; promotes the growth, differentiation and cytocidal activity of monocytes; induces the growth of natural killer cells and stimulates cytokine production by these cells as well as the cytolytic activity of these cells; enhances the production of lymphocyte-activated killer (LAK) cells; and induces the proliferation and differentiation of oligodendrocytes (1, 2).