Background: T helper cells (Th cells) are a diversified group of CD4+ T cells that play an essential role in immune-response through cytokine secretion and cell to cell contact (adhesion molecule expression). The importance of Th cells is manifested by the acquired immunodeficiency in HIV patients whose CD4+ T cells are selectively infected and destroyed by HIV virus. The function of Th cells is multifaceted, including stimulation of CD8+ cytotoxic cells, promoting B cell proliferation, regulating antibody subclass switching, and activation of macrophages and other cells in innate immune system. T helper cells are originated from Naïve CD4+ T cells, which are generated in bone marrow and differentiate into effector Th cells in peripheral lymphoid organs and blood upon contact with cognate antigen that matches with the complementarity-determining region on T cell receptor. Th cells were classified into T helper 1 cells (Th1) and T helper 2 cells (Th2) in the mid-1980s based on their unique cytokine secretion patterns and their roles in cellular and humoral immunity. Th1 cells secrete IFN-gamma as their signature cytokine, and are also the primary producer of IL-2. Th2 cells fail to make IFN-gamma, but produce IL-4, IL-5, and IL-13 as their signature cytokines. Th1 cells are needed in host defense against intracellular bacteria and virus, while Th2 cells are essential for humoral responses against extracellular parasites. Cytokines play important roles in the development and maintenance of T helper cell lineage. In the presence of IL-12, Naïve CD4+ cells tend to differentiate into Th1, whereas IL-4 stimulates Th2 development. Interestingly, cytokines produced by one Th subset tends to inhibit the development of other Th subset(s). For example, IL-10 secreted by Th2 can counteract the effect of IL-12 through suppressing Th1 and stimulating Th2 differentiation. In 2005, Harrington et al described a new subset of IL-17 secreting CD4+ T cells as T helper 17 (Th17) cells, which is involved with autoimmunity and clearance of extracellular bacteria and fungi. It was observed that Th17 cells can be induced from Naïve CD4+ T cells by IL-23 in the absence of IFN-r and IL-4. More recently, T follicular helper cells (Tfh), regulatory T cells (Treg), T helper 9 cells (Th9) and T helper 22 cells (Th12) were proposed. IL-2 is one of the key cytokines that drive adaptive immunity. It is secreted by Naïve CD4+ cells immediately after T cell receptor is activated through binding with antigens presented on MHC by antigen-presenting cells. IL-2 stimulates Th1 differentiation, clone expansion and acts as autocrine to stimulate its own production. It also promotes the survival of antigen specific memory T cell, and stimulates the maturation of regulatory T-cells. The cytokine also influences the activity of B cells, CD8+ T cells, gamma delta T cells, NK cells and LAK cells. IL-10 is expressed by a variety of cell types and plays an important role in inflammatory and immune responses. It down-regulates the expression of Th1 cytokines. IL-10 also suppresses the production of pro-inflammatory cytokines by monocytes and neutrophils, down-regulates the expression of activating and co-stimulatory molecules on monocytes and dendritic cells, and stimulates the growth of B cells and mast cells. Produced by Th1, CD8+ T cells and natural killer (NK) cells, IFN-gamma is known to stimulate Naïve CD4+ T cells to differentiate into Th1 cells. It is both an inhibitor of viral replication and a regulator of numerous immunological functions. IFN-gamma has been reported to induce its own expression. IFN-gamma has documented antiviral, antiprotozoal and immunomodulatory activities. The immunomodulatory effects of IFN-gamma are extensive and diverse. In monocyte/ macrophages, IFN-gamma increases expression of class 1 MHC antigens; increases the production of IL-1, platelet-activating factor, H2O2, and pterin, protects monocytes against LAK cell-mediated lysis; and down-regulates IL-8 mRNA expression. IFN-gamma selectively enhances IgG2a secretion by LPS-stimulated B cell activation. IFN-gamma has been shown to up-regulate ICAM-1, but not E-selectin or VCAM-1 expression on endothelial cells. IFN-gamma has also been reported to implicate in the development of a cholinergic phenotype in embryonic septal neurons. IL-4 was initially characterized as a B cell stimulatory factor (BSF-1) for its crucial role in Th2 cell differentiation, B cell expansion and differentiation into plasma cells. It was subsequently revealed that IL-4 is a pleiotropic cytokine with multiple immune response modulating functions on diverse cell types. IL-4 induces antibody class switching to IgE, up-regulates MHC class II production and plays a role in airway allergy. IL-4 stimulates protective immunity against parasites. It is also reported that IL-4 is implicated with metastasis. IL- 4 is produced by naïve CD4+ T cells, Th2 cells, fetal thymocytes, CD8+ T cells, mast cells and basophils. IL- 4 exhibits approximately 25% amino acid sequence homology to IL-13 that shares a number of biological functions with IL-4. Human IL-4 gene, composed of four exons and three introns, have been localized to a chromosome that contains tandem-arranged genes for IL-3, IL-5, IL-9, IL-13 and GM-CSF. IL-13 is secreted mainly by activated T helper 2 cells. IL-13 binds to IL-4Ralpha in addition to IL-13Ralpha1 and IL-13Ralpha2. The function of IL-13 partially overlaps with IL-4. Both IL-4 and IL-13 link to signal transducer and activator of transcription 6 (STAT6) through the receptor activation. IL-13 can induce B-cell proliferation and IgE classs switching. However, IL-13 is less competent in this function comparing with IL-4. Unlike IL-4, IL-13 does not play an important role in hematopoietic cell shift, such as naïve T helper differentiation to T helper 2. IL-13 is also associated with many features of the allergic airway diseases, including mucus hypersecretion, goblet metaplasia, chemokine induction and recruitment of effector cells. All of them contribute to airway obstruction. Polymorphisms in IL-13 gene have been found to link to increased eosinophil count, serum total IgE and high risk of asthma. It is reported that IL-I3 signaling is significant in inducing physiological changes to non-immuno cells in parasitized organs. IL-17A is the proto-type of a newly discovered pro-inflammatory cytokine family which consists of IL-17A, IL-17B, IL-17C, IL-17D, IL-17E (IL-25) and IL-17F. All the IL-17s have similar structure with four highly conserved cysteine residues and same 3 dimensional structures, which distinguish this family from other cytokines. IL-17 is produced by a group of CD4+ T help cells termed Th17 cells. In addition to previous discovered Th1 and Th2 cells, the Th17 cells consist in the third group of T helper cells. IL-23, a growth and stabilization factor was the first cytokine that was found to involve with the development of Th 17 cells. Later on, other cytokines including TGF-beta, IL-6, and IL-21, were also found to contribute to the procedure. The transcription factors implicated in the procedure were identified as STAT3, RORgt, and RORa. IL-17, like IFN-gamma, is a potent mediator of delayed-type reaction. IL-17 induces the secretion of many other pro-inflammatory cytokines and chemokines such as IL-6, IL-8, GM-CSF, G-CSF, TGF-beta, TNF-alpha, GRO-alpha, MCP-1, and stimulates the expression of NF-kappaB, mitogen activated protein kinase, and prostaglandings from many cell types. By stimulating cytokine production and recruiting monocytes neutrophils to site of inflammation, the cytokine exerts its effect in response to tissue damage and invasion.IL-22 is produced by dendritic cells, T-cells and natural killer cells during bacterial infection, auto-immunity and tissue inflammation. IL-22 acts upon innate immunity cells through its receptors expressed exclusively on these cells. In CD4+ T helper cells, IL-22 expression has been found to be associated with Th17 and Th1. Recently, an IL-22 expressing T helper cell subset (Th22) was characterized which is distinct from other T cells by co-expression of the chemokine receptor CCR6 and the skin-homing receptors CCR4 and CCR10. IL-22 expression is elevated in psoriatic skin inflammation, atopic dermatitis, and inflammatory bowel disease. In cutaneous T-cell lymphoma, IL-22 dominates the tumor microenvironment and STAT3 phosphorylation was observed. IL-22 was also found to promote murine hepatocyte survival and ameliorate intestinal inflammation in mouse ulcerative colitis model. A natural antagonist of IL-22, IL-22 binding protein, was found to down-regulate IL-22 function. Tumor Necrosis Factor alpha (TNF-alpha), also known as cachectin was initially named for its remarkable ability to cause hemorrhagic necrosis of tumors in mice. The primary source of TNF-alpha is thought to be the monocyte/macrophage but various cell types are known to express this cytokine. TNF-alpha is produced upon stimulation with cytokines such as IL-1, 1L-2, GM-CSF, TNF-? itself and with bacterial lipopolysaccharide. Th1 cells secrete IFN-gamma, IL-2, IL-10, and TNF-alpha/beta. Various pathological conditions are associated with the production of high levels of TNF-alpha. These include septic shock syndrome, cachexia, autoimmune diseases, hepatitis, leukemia, myocardial ischaemia, organ transplantation rejection, multiple sclerosis, rheumatoid arthritis, and meningococcal septicemia. TNF-alpha is a pleiotropic cytokine that can induce disease through TNF-alpha toxicity (tissue injury, catabolic illness, and mediating septic shock) and improve host defense mechanisms (stimulating inflammation and increasing immune cell function). Since TNF-alpha has such a complex and important function in immune-response, studying TNF-alpha can help understanding its role in T cell differentiation.

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 IFN-gamma, IL-2, IL-4, IL-10, IL-13, IL-17, IL-22, 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.

Intended Uses: This multiplex ELISA kit for Th1/Th2/Th17 cytokines is designed for semi-quantitative and simultaneous determination of cytokines relevant to T helper cell differentiation. The kit simultaneously determines interferon-gamma(IFN-gamma), interleukin-2(IL-2), interleukin-4(IL-4), interleukin-10(IL-10), interleukin-13(IL-13), interleukin-17A(IL-17A), interleukin-22(IL-22), and tumor necrosis factor-alpha (TNF-alpha) in cell culture supernatant and other biological samples. In combination with other quantitative cytokine ELISA kits, the Th1/Th2/Th17 cytokine multiplex ELISA kit is expected to be useful for the investigation of the relationship of cytokine expression, T helper cell differentiation in various disease models. The kit is intended FOR LABORATORY RESEARCH USE ONLY and should not be used in any test or therapeutic procedures.

Small volumes of Th1/Th2/Th17 elisa kit vial(s) may occasionally become entrapped in the seal of the product vial during shipment and storage. If necessary, briefly centrifuge the vial on a tabletop centrifuge to dislodge any liquid in the container`s cap. Certain products may require to ship with dry ice and additional dry ice fee may apply.

MBS590071 is a ready-to-use microwell, strip-or-full plate ELISA (enzyme-linked immunosorbent assay) Kit for analyzing the presence of the Th1/Th2/Th17 Cytokines, ELISA Kit target analytes in biological samples. The concentration gradients of the kit standards or positive controls render a theoretical kit detection range in biological research samples containing Th1/Th2/Th17. The ELISA analytical biochemical technique of the MBS590071 kit is based on Th1/Th2/Th17 antibody-Th1/Th2/Th17 antigen interactions (immunosorbency) and an HRP colorimetric detection system to detect Th1/Th2/Th17 antigen targets in samples. The ELISA Kit is designed to detect native, not recombinant, Th1/Th2/Th17. Appropriate sample types may include undiluted body fluids and/or tissue homogenates, secretions. Quality control assays assessing reproducibility identified the intra-assay CV (%) and inter-assay CV(%).