For the quantitative determination of human interleukin 12 IL-12) concentrations in cell culture supernates, serum, plasma, and urine. Recognizes native and recombinant human Il-12
Interleukin 12 (IL-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 cultured EBV-transformed RPMI-8866 cells (1-3). IL-12 is a 75ka glycoprotein heterodimer composed of two genetically unrelated subunits linked by a disulfide bond. The smaller subunit (p35) has homology to IL-6 and G-CSF while the larger subunit (p40) demonstrates similarity to the soluble receptor for IL-6, leading to the suggestion that IL-12 might have evolved from a cytokine/soluble receptor complex (2-6). IL-12 apparently shows
species specificity, with human IL-12 showing minimal activity in the mouse system (3, 7). For reviews on IL-12, see references 8-12.
Each subunit of IL-12 apparently arises from a single copy gene. The mRNA transcription of the subunits is closely coordinated, although an excess of the larger subunit has been shown to be produced by B cells in addition to active IL-12 (1, 3). Expression of p35 is reported to be enhanced by simultaneous expression of p40. IL-12 activity cannot be demonstrated in the absence of either chain (3, 4). As suggested by their names, p35 has a native molecular weight of 35ka while p40 has a native molecular weight of 40kD. In humans, p35 is 197 amino acid residues in length with a predicted molecular weight of 22.5kD. This subunit possesses seven cysteines plus three potential N-linked glycosylation sites and the molecule is believed to be heavily glycosylated. The p40 subunit is 306 amino acid residues in length with a predicted molecular weight of 34.7kD. The molecule contains ten cysteine residues and four potential N-linked glycosylation sites (3). The mouse p35 subunit shows 60% sequence
identity with the corresponding human subunit and is 193 amino acid residues in length with seven conserved cysteines and one possible N-linked glycosylation site. Mouse p40 shows 70% sequence identity to human p40 and is 313 amino acid residues in length with eleven
conserved cysteines and three potential N-linked glycosylation sites (7). It is not clear what separate functions can be attributed to p35 and p40. Preliminary evidence suggests, however, that p40 is involved in receptor binding and p35 is important for signal transduction (13).
A unique, high affinity receptor for IL-12 (IL-12 R) has been characterized from PHA-stimulated human peripheral blood mononuclear cells (14). Approximately 110kD as determined by crosslinking studies, it has a Kdin the range of 100-600 pM (14). Cross-linking studies also suggested an association with a second protein of approximately 85kD. IL-12 receptor has also been reported to be present on PHA- or IL-2-stimulated CD4+, CD8+, and CD56+cells and on one T cell and one NK cell line (14, 15).
IL-12 is produced by macrophages and B lymphocytes and has been shown to have multiple effects on T cells and natural killer (NK) cells (16, 17). These include inducing production of IFN-and TNF by resting and activated T and NK cells, synergizing with other IFN-inducers at both the transcriptional and post-transcriptional levels to induce IFN-gene expression,
enhancing the cytotoxic activity of resting NK and T cells, inducing and synergizing with IL-2 in the generation of lymphokine-activated killer (LAK) cells, acting as a comitogen to stimulate proliferation of resting T cells, and inducing proliferation of activated T and NK cells (16).
Evidence indicates that IL-12, produced by macrophages in response to infectious agents, is a central mediator of the cell-mediated immune response by its actions on the development, proliferation, and activities of TH1 cells (8, 9, 18, 19). These activities of IL-12 are antagonized by IL-4 and IL-10, factors associated with the development of uncommitted T helper cells into TH2 cells and mediation of the humoral immune response (8, 9, 18, 19). In its role as the 2 initiator of cell-mediated immunity, it has been suggested that IL-12 has therapeutic potential as a stimulator of cell-mediated immune responses to microbial pathogens, metastatic
cancers, and viral infections such as AIDS (8, 9, 18-20).
Bioassays for human IL-12 are based on measurement of its ability to stimulate the proliferation of PHA-stimulated human lymphoblasts (2). This assay is time-consuming and not completely specific for IL-12. The IL-12 Immunoassay is a 3.5-4.5 hour solid phase ELISA designed to measure IL-12 in cell culture supernates, serum, plasma, and urine. It containsSf21 expressed recombinant human IL-12 and antibodies raised against the recombinant factor. This immunoassay has been shown to quantitate recombinant human IL-12 accurately. Results obtained using natural human IL-12 showed linear curves that were parallel to the standard curves obtained using the recombinant kit standards. These results indicate that the kit can be used to determine relative mass values for natural IL-12.
The presence of soluble IL-12 receptors in biological fluids would represent a potential source for interference in ELISAs. There is currently no evidence, however, for the existence of soluble receptors for IL-12 in biological fluids. The IL-12 Immunoassay was developed with a capture antibody that recognizes only the IL-12 heterodimer and not the individual subunits of the dimer, thus eliminating the potential for interference by these subunits.
Principle of the Assay:
This assay employs the quantitative sandwich enzyme immunoassay technique. A monoclonal antibody specific for IL-12 has been pre-coated onto a microplate. Standards and samples are pipetted into the wells and any IL- 12 present is bound by the immobilized antibody. After washing away any unbound substances, an enzyme-linked polyclonal antibody specific for IL-12 is added to the wells. Following a wash to remove any unbound antibody-enzyme reagent, a substrate solution is added to the wells and color develops in proportion to the amount of IL-12 bound in the initial step. The color development is stopped and the intensity of the color is measured.
IL-12 Microplate: 96 well polystyrene microplate (12 strips of 8 wells) coated with a murine monoclonal antibody against IL-12.
IL-12 Conjugate: 21ml of polyclonal antibody against IL-12 conjugated to horseradish peroxidase, with preservative.
IL-12 Standard: 2.5ng of recombinant human IL-12 in a buffered protein base with preservative, lyophilized.
Assay Diluent RD1F: 6ml of a buffered protein base with preservative. For use with cell culture supernate/serum/plasma samples.
Assay Diluent RD1U: 6ml of a buffered protein base with preservative. For use with urine samples.
Calibrator Diluent RD5C Concentrate (5X): 21ml of a buffered protein base with preservative.
Wash Buffer Concentrate: 21ml of a 25-fold concentrated solution of buffered surfactant with preservative.
Color Reagent A: 12.5ml of stabilized hydrogen peroxide.
Color Reagent B: 12.5ml of stabilized chromogen (tetramethylbenzidine).
Stop Solution: 6ml of 2 N sulfuric acid.
Materials required but not supplied:
Microplate reader capable of measuring absorbance at 450nm, with the correction wavelength set at 540nm or 570nm.
Pipettes and pipette tips.
Deionized or distilled water.
Multi-channel pipette, squirt bottle, manifold dispenser, or automated microplate washer.
100ml and 500ml graduated cylinders.
Human IL-12 Controls (optional; available from United States Biological).
Storage and Stability:
All components may be stored for up to 1 month at 4 degrees C. Aliquot and store the IL-12 standard and store up to 1 month at 4 degrees C.