14-8123

Contents: Recombinant Mouse IL-12/IL-23 p40 homodimer
Catalog Number: 14-8123
Formulation: Sterile liquid; 10 mM NaH2PO4, pH 6.0, 300 mM NaCl, with 1.0% BSA as a carrier. 0.22 uM filtered.
Storage Conditions: For greatest stability, keep concentration of primary stock at or above 10 µg/ml. For long term storage, aliquot into polypropylene vials (volumes of 20 µl or greater) and store at or below -80°C. Avoid repeated freeze/thaw cycles.
Handling Conditions: For best recovery, always quick-spin vial prior to opening. For dilution of current stock, always include carrier protein (1% BSA or 10% FBS) in the buffered saline diluent.
Source: Insect cells infected with baculovirus: mouse p40, amino acids met 1-ser 335, (accession # NM_008352).
Molecular Mass: P40, amino acids met 23-ser 335, has a predicted molecular mass of 35,791. On reducing SDS-PAGE the DTT-reduced protein migrates as a 40 kDa protein. The non-reduced homodimer migrates as a 78 kDa polypeptide.
Purity: Greater than 98% as determined by SDS-PAGE
Endotoxin Level: Less than 0.01 ng/ug cytokine as determined by the LAL assay.
Bioactivity: The recombinant mouse IL-12/IL-23 p40 homodimer at 60.0 ng/ml has been found to inhibit by 50% the biological effects of 1 ng/ml mouse IL-23 (14-8231) in an assay of IL-17 production by mouse splenocytes. The recombinant mouse IL-12/IL-23 p40 homodimer at 8.0 ng/ml has been found to inhibit by 50% the biological effects of 1 ng/ml mouse IL-12 p70 (14-8121) (with PMA as a costimulator) in an assay of IFN-γ production by mouse splenocytes.

Description

Interleukin-12 (IL-12) is a heterodimeric 70 kD (p70) cytokine composed of two covalently linked, glycosylated chains, 40kD (p40) and 35-kD (p35). IL-12 is mainly produced by monocytes, macrophages, and dendritic cells in response to bacterial products such as lipopolysaccharides (LPS), to intracellular pathogens or upon interaction with activated T cells. IL-12 was originally discovered because of its ability to induce interferon-γ production, cell proliferation, and cytotoxicity mediated by natural killer cells and T cells. It is now established that IL-12 also plays a key role in the development of Th1 responses, leading to IFN-γ and IL-2 production. These cytokines can in turn promote T-cell responses and macrophage activation. The p40 and p35 subunits by themselves have no IL-12 bioactivity, though the p40 homodimer has been shown to bind the IL-12 receptor and to be an antagonist of IL-12 p70. Free p40 is typically secreted in vast excess of IL-12 p70 by cells co-expressing both the p35 and p40 subunits. The p40 subunit of IL-12 has been shown to have extensive amino acid sequence homology to the extracellular domain of the IL-6 receptor, while the p35 subunit shows some homology to IL-6 and G-CSF.

Applications Reported

For research use only, not for diagnostic or therapeutic use. The recombinant mouse IL-12/IL-23 p40 homodimer has been reported for use as an ELISA standard and also for blocking IL-12 and IL-23 bioactivity.

Applications Tested

The recombinant mouse IL-12/IL-23 p40 homodimer has been tested as an ELISA standard in an ELISA measuring total IL-12/IL-23 p40 (monomer, dimer, heterodimer). For this assay, the biotinylated C17.8 antibody (13-7123) is used as the detection antibody in combination with the purified C15.6 antibody (14-7125) for capture and recombinant mouse IL-12/IL-23 p40 homodimer (14-8123) as the standard. A standard curve consisting of doubling dilutions of the recombinant standard over the range of 2,000 pg/ml - 15.0 pg/ml should be included in each ELISA plate.

The recombinant mouse IL-12/IL-23 p40 homodimer at 60.0 ng/ml has been found to inhibit by 50% the biological effects of 1 ng/ml mouse IL-23 (14-8231) in an assay of IL-17 production by mouse splenocytes.
The recombinant mouse IL-12/IL-23 p40 homodimer at 8.0 ng/ml has been found to inhibit by 50% the biological effects of 1 ng/ml mouse IL-12 p70 (14-8121) (with PMA as a costimulator) in an assay of IFN-γ production by mouse splenocytes. Detailed information and protocols about cytokine bioassays and in vitro cytokine neutralization using antibodies can be found in the BestProtocols

References

Chen, L., et al. 1997. Eradication of murine bladder carcinoma by intratumor injection of a bicistronic adenoviral vector carrying cDNAs for the IL-12 heterodimer and its inhibition by the IL-12 p40 subunit homodimer. J. Immunol. 159: 351-359.
Ling, P., et al. 1995. Human IL-12 p40 homodimer binds to the IL-12 receptor but does not mediate biologic activity. J. Immunol. 154: 116-127.
Russell, T., et al. 2003. IL-12 p40 homodimer-dependent macrophage chemotaxis and respiratory viral inflammation are mediated through IL-12 receptor beta1. J. Immunol. 171: 6866-6874.

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