Interleukin 12 (IL-12) is a potent proinflammatory cytokine and multifunctional regulator of cell-mediated immune responses. Biologically active IL-12 is secreted by activated B lymphocytes and monocytes/macrophages as a 70-75 kD heterodimeric glycoprotein comprised of disulfide-bonded 35 kD (p35) and 40 kD (p40) subunits. IL-12 p70 - producing cells can also secrete high levels of IL-12 p40 monomers, much lower levels of IL-12 p40 homodimers, but no detectable free p35 subunits. Interestingly, IL-12 p40 homodimers have been shown to be capable of antagonizing IL-12 p70-mediated bioactivities. These findings underscore the importance of developing and applying immunoassays for research that can specifically measure the biologically active p70 species of IL-12 without interference by the competing high levels of p40 monomer.

PharMingen's Cytokine Product Development Group has developed research tools for characterizing mouse and human IL-12 expression at the mRNA, protein and cell level by three complementary approaches:

• Quantitation of secreted IL-12 p70 by sandwich ELISA
• Detection of IL-12-producing cell frequency by intracellular cytokine staining and multi-parameter flow cytomeric analysis
• Quantitation of IL-12 p35 and p40 mRNA levels by RNAse protection assay.

Cytokine Reagents for Immunoassay Development (Cytokine ELISA)

		ELISA Capture Antibody Product Catalog Number	ELISA Capture Antibody Product Catalog Number
Specificity	Standard	(Purified)	(Biotinylated)
Mouse IL-12 (p40, total)	19401W	18491D	18482D
Mouse IL-12 (p-70)	19361V	20011D	18482D
Human IL-12 (p40, monomer)	19931V	20711D	20512D
Human IL-12 (p70)	19721V	20501D	20512D

Cytokine Reagents for Immunofluorescence Staining and Flow Cytometric Analysis

(FITC and PE labeled anti-cytokine antibodies for staining intracellular cytokines. Purified anti-cytokine antibodies for pre-blocking)

			Catalog Number	Catalog Number	Catalog Number
Specificity	Clone	Isotype	FITC	PE	Purified
Mouse IL-12 (p40/p70)	C15.6	Rat IgG1	18494A	18495A	18491D
Human IL-12 (p40/p70)	C11.5.14	Mouse IgG1	18994A	18995A	18991A

Cytokine Reagents for Neutralization of Bioactivity and Capture Bioassay

Specificity	Clone	Isotype	Catalog Number	Applications
Mouse IL-12 (p40/p70)	C17.8	Rat IgG2a	18480D	Neutralization
Mouse IL-12 (p40/p70)	C15.1	Rat IgG1	20020D	Capture bioassay
Human IL-12 (p40/p70)	C8.6	Mouse IgG1	20510D	Neutralization
Human IL-12 (p40/p70)	C11.5	Mouse IgG1	18990D	Capture bioassay

RiboQuant™ Multi-Probe RNase Protection Assay System

RiboQuant™ Kit and Components	Catalog Number
RiboQuant™ In Vitro Transcription Kit	45004K
RiboQuant™ RNase Protection Assay Kit	45014K
mCK-1 RiboQuant™ Mouse Cytokine Multi-Probe Template Set	45001P
mCK-2 RiboQuant™ Mouse Cytokine Multi-Probe Template Set (Contains IL-12 p35 and p40)	45002P
mCK-3 RiboQuant™ Mouse Cytokine Multi-Probe Template Set	45003P

CD ROM Vol 2 was produced by staff at the Purdue University Cytometry Laboratories and distributed free of charge as an educational service to the cytometry community. If you have any comments please direct them to Dr. J. Paul Robinson, Professor & Director, PUCL, Purdue University, West Lafayette, IN 47907. Phone:(317) 494-0757; FAX (317) 494-0517; Web http://www.cyto.purdue.edu EMAIL robinson@flowcyt.cyto.purdue.edu