Human IL-12p40 ELISA Assay

Additional Information

Assay Background

Interleukin-12, also known as natural killer cell stimulatory factor (NKSF) or cytotoxic lymphocyte maturation factor (CLMF), is a pleiotropic cytokine originally identified in the medium of activated human B lymphoblastoid cell lines. IL-12 is produced by macrophages and B lymphocytes and has multiple effects on T-cells and NK cells, including stimulation of cytotoxic activity, proliferation, and promotion of Th1 development as well as IFNγ and TNF production. IL-12 is a disulfide linked, 70 kDa (p70) heterodimeric glycoprotein composed of a 40 kDa (p40) subunit and a 35 kDa (p35) subunit. The p40 and p35 subunits by themselves have no IL-12 activity, the p40 dimer has been shown to bind the IL-12 receptor and to be an IL-12 antagonist. Free p35 has not been detected in supernatant solutions of cultured cells expressing only p35 or both p35 and p40 mRNAs. In contrast, p40 is secreted in excess of IL-12 in cells expressing both p35 and p40 mRNAs. The p40 subunit of IL-12 has been shown to have extensive amino acid sequence homology to the extracellular domain of the human IL-6 receptor while the p35 subunit shows distant but significant sequence similarity to IL-6, G-CSF, and chicken MGF. These observations have led to the suggestion that IL-12 might have evolved from a cytokine/soluble receptor complex. Human and mouse IL-12 share 70% and 60% amino acid sequence homology in their p40 and p35 subunits, respectively. IL-12 apparently shows species specificity with human IL-12 reportedly showing minimal activity in the murine system.

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. Cover with the adhesive strip provided. Incubate for 1.5 hours at 37°C.
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 Biotin-Conjugate to each well. Cover with a new adhesive strip and incubate 1 hour at 37°C.
6. Repeat the aspiration/wash.
7. 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 37°C Avoid placing the plate in direct light.
8. Repeat the aspiration/wash.
9. Add 100 µl of Substrate Solution to each well. Incubate for 10-20 minutes at 37°C. Avoid placing the plate in direct light.
10. Add 100 µl of Stop Solution to each well. Gently tap the plate to ensure thorough mixing.
11. 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)