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LAG-3

The lymphocyte activation gene-3 (LAG-3, also called CD223), a member of the immunoglobulin superfamily (IgSF) related to CD4, binds to the major histocompatibility complex (MHC) class II molecules but with higher affinity than CD4 [1]. LAG-3 is expressed in vivo in activated CD4+ and CD8+ lymphocytes residing in inflamed secondary lymphoid organs or tissues, such as human tumors, but not in spleen, thymus or blood [2]. LAG-3 negatively regulates the expansion of activated T cells [3]. LAG-3 also activates and matures antigen presenting cells (APCs) through its specific binding to MHC class II molecules expressed on immature monocytes or dendritic cells. Ectopic expression of LAG-3 also blocks antigen-induced proliferation of autoantigen-specific T cells, a hallmark of TREGs. In vitro, LAG-3 expression is associated with interferon-g (IFN-g production and the most potent stimulus for LAG-3 expression on both activated T and natural killer (NK) cells is the IFN-g-inducer interleukin-12 (IL-12). Under inflammatory conditions (e.g. IFN-g stimulation), both MHC class II and its high affinity ligand LAG-3 are strongly upregulated.

Several alternative mRNA splice-variants of human LAG-3 have been described, two of them encoding potential secreted forms: LAG-3V1 (i.e. the D1-D2 domains of the protein, 36 kDa) and LAG-3V3 (D1-D3, 52 kDa) [1, 4]. The longer form was detected by ELISA in the serum of healthy individuals as well as of tuberculosis patients with a favorable outcome [5]. LAG-3 expression by T cell clones correlated with IFN-g production, and hence soluble LAG-3 has been suggested as a serological marker of Th1 responses [6]. In addition, LAG-3 was identified in the supernatants of LAG-3-expressing T cell hybridoma cultures and was detected in the serum of WT mice [7].

LAG-3, Soluble (human) Detection Set (APO-54N-017)

Summary of features

	For the quantitative determination of soluble human LAG-3 (human) from biological fluids (serum and cell culture supernatant)
	Specificity : The antibodies used in this detection Set are specific for measurement of natural and recombinant human LAG-3. They do not cross-react with mouse or rat LAG-3
	Monoclonal antibodies-based sandwich ELISA
	Detection limit: 50pg / ml
	Range: 0-4000 pg / ml
	Format: contains sufficient materials to run ELISAs on 2x 96-well plates

Principle of the Kit

This assay is a sandwich Enzyme Linked-Immuno-Sorbent Assay (ELISA) developed for the direct measurement of human LAG-3 (hLAG-3) in serum, plasma and cell culture supernatants. A monoclonal antibody specific for hLAG-3 (11E3) is coated onto the wells of the supplied microtiter strips. Samples and concentration standards of LAG-3 are pipetted into the wells for binding to the coated antibody. After extensive washing to remove unbound compounds, hLAG-3 is recognized by the addition of a biotinylated monoclonal antibody specific for hLAG-3 (17B4 (biotin). After removal of excess biotinylated antibody, streptavidine-peroxidase is added. Following a final washing, peroxidase activity is quantified using the substrate 3,3’,5,5’-tetramethylbenzidine (TMB). The intensity of the color reaction is measured at 450 nm after acidification and is directly proportional to the concentration of hLAG-3 in the samples.

LITERATURE OVERVIEW

[1] Triebel, F. (2003). LAG-3: a regulator of T-cell and DC responses and its use in therapeutic vaccination. Trends Immunol 24, 619-622.

[2] Huard, B., Gaulard, P., Faure, F., Hercend, T., and Triebel, F. (1994). Cellular expression and tissue distribution of the human LAG-3-encoded protein, an MHC class II ligand. Immunogenetics 39, 213-217.

[3] Huard, B., Prigent, P., Pages, F., Bruniquel, D., and Triebel, F. (1996). T cell major histocompatibility complex class II molecules down-regulate CD4+ T cell clone responses following LAG-3 binding. Eur J Immunol 26, 1180-1186.

[4] Triebel, F., Hacene, K., and Pichon, M.F. (2005). A soluble lymphocyte activation gene-3 (sLAG-3) protein as a prognostic factor in human breast cancer expressing estrogen or progesterone receptors. Cancer Lett.

[5] Lienhardt, C., Azzurri, A., Amedei, A., Fielding, K., Sillah, J., Sow, O.Y., Bah, B., Benagiano, M., Diallo, A., Manetti, R., Manneh, K., Gustafson, P., Bennett, S., D'Elios, M.M., McAdam, K., and Del Prete, G. (2002). Active tuberculosis in Africa is associated with reduced Th1 and increased Th2 activity in vivo. Eur J Immunol 32, 1605-1613.

[6] Annunziato, F., Manetti, R., Tomasevic, I., Guidizi, M.G., Biagiotti, R., Gianno, V., Germano, P., Mavilia, C., Maggi, E., and Romagnani, S. (1996). Expression and release of LAG-3-encoded protein by human CD4+ T cells are associated with IFN-gamma production. Faseb J 10, 769-776.

[7] Li, N., Workman, C.J., Martin, S.M., and Vignali, D.A. (2004). Biochemical analysis of the regulatory T cell protein lymphocyte activation gene-3 (LAG-3; CD223). J Immunol 173, 6806-6812.