Multiplex Human Cytokine ELISA Kit (M1/M2/MDSC Cytokines)

Intended Uses: This multiplex ELISA kit for M1/M2/MDSC cytokines is designed for semi-quantitative and simultaneous determination of cytokines relevant to the proliferation of Myeloid Derived Suppressor Cells (MDSCs) and their differentiation toward M1 or M2 phenotype. The kit simultaneously determines granulocyte macrophage colony stimulating factor (GM-CSF), interferon-gamma?(IFN-gamma? interleukin-4 (IL-4), interleukin-6 (IL-6), interleukin-10 (IL-10), interleukin-12 (IL-12), monocyte chemotactic and activating factor (MCAF, also known as MCP-1), and tumor necrosis factor-alpha (TNF-alpha??in cell culture supernatant and other biological samples. In combination with other quantitative cytokine ELISA kits, the M1/M2/MDSC cytokine multiplexELISA kit is expected to be useful for the investigation of the relationship of cytokine expression and MDSC induced inmmunosuppression in various disease models. The kit is intended FOR LABORATORY RESEARCH USE ONLY and should not be used in any test or therapeutic procedures. Principle of the Assay: This enzyme linked immunosorbent assay (ELISA) applies a technique called a quantitative sandwich immunoassay. The microwells on the 8-well strips enclosed in the kit have been pre-coated with monoclonal antibodies specific to GM-CSF, IFN-gamma?? IL-4, IL-6, IL-10, IL-12, MCAF (also known as MCP-1), and TNF-alpha?respectively. Standards or samples are then added to the strips, and the biotin-conjugated detection antibody mixture will be added late on. The above cytokines, if present, will bind and become immobilized by the antibody pre-coated on the wells and then be "sandwiched" by biotin conjugate. The microtiter plate wells are thoroughly washed to remove unbound components of the sample. In order to quantitatively determine the amount of cytokine present in the sample, Avidin conjugated to Horseradish Peroxidase (HRP) is added to each microplate well and incubated. Avidin is a tetramer containing four identical subunits that each has a high affinity-binding site for biotin. The wells are thoroughly washed to remove all unbound HRP-conjugated Avidin. A TMB (3, 3' 5, 5' tetramethyl-benzidine) substrate solution is added to each well. The enzyme (HRP) and substrate are allowed to react over a short incubation period. Only those wells that contain coating antibody and the specific cytokine, biotin-conjugated antibody and enzyme-conjugated Avidin will develop a blue colour. The intensity of colour development is proportional to the concentration of the specific cytokine presented in the each wells. The enzyme-substrate reaction is terminated by the addition of a sulphuric acid solution and the colour will change to yellow. The intensity is measured spectrophotometrically at a wavelength of 450nm +/- 2 nm. Samples were tested together with standards diluted with a similar matrix, or one of the Calibrator Diluent provided with the kit. This allows the operator to produce Optical Density (O.D) versus cytokine concentration (pg/mL). The concentration of cytokines in the samples is then determined by comparing the O.D. of the samples to the standards. Background/Introduction: Myeloid derived suppressor cells (MDSCs) are myelogenous cells that are capable of negatively regulating T cell immunity. Increased numbers of MDSCs are found in pathological conditions such as malignancy, transplantation, chronic infection and inflammation. MDSCs cannot be classified by a standard leukocyte linage marker since MDSCs are comprised of various myeloid originated cells at immature status including myeloid progenitor cells, immature monocytes, dendritic cells, and granulocytes. MDSCs in human are broadly defined as lin(-/low)CD33(+) HLA-DR(-)CD11b(+) cells with altered enzyme and cytokine profile and immunosuppressive function. While CD33 (+) and CD11b (+) denote myeloid origin in human, Gr-1(+) and CD11b (+) define myeloid origin in MDSCs in mouse. In chronic inflammation caused by cancer, the interaction between tumor cells and MDSCs causes MDSCs to expand and increase its potential in T cell inhibition (Sevko et al. 2013). MDSCs have been recognized as one of the major mechanisms for tumor evasion from host immunity and are recently evaluated as target for cancer treatment (Sinha et al. 2005). Cytokines are believed to play a critical role in MDSC development and differentiation. GM-CSF and IL-6 have been shown to stimulate MDSC expansion in vivo and in vitro ((Lechner et al. 2010, Morales et al. 2010). T-helper 2 cytokines, IL-4 and IL-13, are the major polarization signals for MDSC to differentiate toward the more T-cell inhibitory M2 phenotype of MDSCs (Bronte et al. 2003, Sinha et al. 2005). Additionally, interleukin 4 receptor alpha (IL-4Ralpha), the common receptor for IL-4 and IL-13, has been found to be up-regulated in MDSCs (Mandruzzato et al. 2009). One of the main characteristics of M2 MDSCs is the up-regulation of IL-10 and down-regulation of IL-12 (Bunt et al. 2007). IL-10 inhibits cell immunity by decreasing the secretion of T helper 1 type cytokines and the expression of MHC class II antigens and co-stimulatory molecules. It has also been postulated that M2 MDSC-mediated T- cell inhibition is the consequence of increased production of arginases and reactive oxygen species by MDSCs (Zea et al. 2005, Rodriguez et al. 2006) and the enzymes secreted by MDSCs block the synthesis of zeta chain in T-cell receptor complex and sequester cystine and limit the availability of cysteine to T-cells. Signal transduction through calcium binding protein S100A8/A9 and signal transduction and activator of transcription (STAT) is likely implicated in MDSC activities (Zhao et al. 2012). M2 MDSCs inhibit effector T cells but promote Regulatory T cells. The increased expression of cytokines and chemokines such as VEGF, MCP-1 and MIF in the tumor microenvironment is believed to promote the infiltration of MDSCs and stimulate tumor angiogenesis and metastasis (Bellamy et al. 2001, Huang et al. 2007, and Simpson et al. 2013). IFN-gamma is a potent activator for MDSCs to develop into the more tumoricidal and virucidal M1 phenotype. IFN-? and other M1 polarizing signals up-regulate IL-12 and TNF-alpha in M1 MDSC. M1-polarized MDSCs express elevated signature markers such as inducible Nitric Oxide Synthase (iNOS), nitric oxide (NO), TNF-alpha, IFN-gamma (Yang et al. 2013). Nitric oxide (NO) and TNF-alpha play important roles in clearing bacterial, and certain fungal, viral, and parasitic invasions as well as in the necrosis of specific tumors and nitric oxide (NO)

Human

Quantitative Sandwich