GM-CSF ELISA Kit | BioSupply UK

GM-CSF ELISA KIT

INTENDED USE

This Human GM-CSF ELISA Kit is to be used for the in vitro quantitative determination of human granulocyte macrophage colony stimulating factor (GM-CSF) concentrations in serum, plasma, cell culture supernatant, and other biological fluids. This GM-CSF ELISA Kit is intended for LABORATORY RESEARCH ONLY and is not for use in diagnostic or therapeutic procedures.

INRODUCTION

Granulocyte-Macrophage Colony Stimulating Factor (GM-CSF) is a member of the hematopoietic cytokine family, which includes interleukin-3 (IL-3) and interleukin-5 (IL-5). It is a pleiotropic cytokine that was one of the first growth factors characterized and shown to be necessary for the proliferation, differentiation, activation, and survival of hematopoietic cells. Human GM-CSF precursor (144 a.a.) is cleaved at the amino-terminal end to form a mature polypeptide ( 23 kDa, 127 a.a) that contains two intramolecular disulfide bonds, which are important for biological activity and two potential N-glycosylation sites. A single gene on chromosome 5 codes for the human GM-CSF protein. Human GM-CSF shows 56-60% amino acid (a.a) homology to murine GM-CSF but does not exhibit cross-species biological activity or receptor binding.Glycosylation does not appear to be essential for biological activity, since recombinant GM-CSF unlike native GM-CSF is non-glycosylated and it still retains high biologic activity. However, this glycoprotein does show a decrease in affinity for its receptor as a result of non-glycosylation.

Human GM-CSF is different from other family members in that it can be produced and act upon a much wider range of cell types. T-lymphocytes, B-lymphocytes, monocytes/macrophages, endothelial cells, fibroblasts, stromal cells, mesothelial cells, keratinocytes, osteoblasts, uterine epithelial cells, synoviocytes, mast cells, and various solid tumours produce GM-CSF. Usually a cytokine, inflammatory agent, or antigen is needed to stimulate the above cells to synthesize GM-CSF.For human GM-CSF to exert its biologic effects it will bind to a single class of cell surface receptors on hematopoietic and non-hematopoietic cells.The GM-CSF receptor has been clonedand, the α and β chains (80 kDa and 130 kDa) were found to members of the hematopoietin receptor family.

Numerous studies have shown diverse in vitro biological effects of GM-CSF on various cell types. GM-CSF can bind to pluripotent hematopoietic stem cells causing the proliferation and differentiation of various progenitor cells such as granulocyte and macrophage, whereas eosinophil, erythroid and megakaryocyte colony formation is stimulated at much higher concentrations. GM-CSF is also required for growth and differentiation of typical dendritic cells from human bone marrow, causes activation and prolonged survival of mature hematopoietic cells, and activates mature neutrophils and eosinophils causing antibody dependent cellular cytotoxicity, phagocytosis, superoxide generation. Also, GM-CSF stimulates macrophage production of TNF, M-CSF, G-CSF, and IL-1, intensifies killing by granulocytes and macrophages, and increases HIV-1 replication at the post-transcriptional level.GM-CSF binds to non-hematopoietic cells causing the proliferation and/or migration of fibroblast, endothelial, and various tumour cell lines.The significance of GM-CSF receptor expression on these non-hematopoietic cell types is unknown. Very little is known about the in vivo biological effects of GM-CSF in various pathological states. However in vivo studies showed a significant eosinophilic response and macrophage granuloma formation accompanied with tissue damage when GM-CSF was overexpressed in the rat lung. Thus role GM-CSF may play a role in the development of fibrotic reactions.In vivo, GM-CSF induces the upregulation of CD11b on neutrophils, induces temporary neutrophil sequestration in the lung, followed by specific granule release, and enhanced ex vivo production of superoxide anion on neutrophils.

Various pathological conditions are associated with increased GM-CSF levels. These include: lung cancer,acute mylogenous leukemia,tumour related thrombocytosis,myelodysplastic syndrome (MDS),thrombocytopenia, and psoriasis.GM-CSF expression is increased in bronchial asthma and lung inflammatory diseases;non-allergic respiratory diseases such as eosinophil pneumonia, hypersensitivity pneumonitis, iodiopathic pulmonary fibrosis, sarcoidosis, cryptogenic organizing pneumonia, HIV infection,rheumatoid arthritis, and systemic lupus erythmatosus.GM-CSF shows therapeutic value by accelerating neutrophil recovery in disease induced myelosuppression such as bone marrow transplantation, chemotherapy, and infectious disease.It is suggested that a GM-CSF may be useful in autologous bone marrow transplantation to detect GM-CSF toxicity for the diagnosis of post-transplant liver diseaseand in gestational trophoblastic disease (GTD) for the early identification of high risk choriocarcinoma cases.

PRINCIPLE OF THE ASSAY

GM-CSF ELISA Kit

GM-CSF ELISA Kit is to be used for the in vitro quantitative determination of human granulocyte macrophage colony stimulating factor (GM-CSF) concentrations in serum, plasma, cell culture supernatant, and other biological fluids. This GM-CSF ELISA Kit is intended for LABORATORY RESEARCH ONLY and is not for use in diagnostic or therapeutic procedures. This GM-CSF ELISA is a 3.5-hour solid phase immunoassay readily applicable to measure GM-CSF in serum, plasma, cell culture supernatant, and other biological fluids in the range of 0 to 500 pg/mL. It showed no cross reactivity with other cytokines tested. This GM-CSF ELISA is expected to be effectively used for further investigations into the relationship between GM-CSF and the various conditions mentioned above.

The microtiter plate provided in this kit has been pre-coated with a monoclonal antibody specific for GM-CSF. Standards or samples are then added to the appropriate microtiter plate wells and incubated. GM-CSF if present, will bind and become immobilized by the antibody pre-coated on the wells. The microtiter plate wells are thoroughly washed to remove unbound GM-CSF and other components of the sample. In order to quantitatively determine the amount of GM-CSF present in the sample, a standardized preparation of horseradish peroxidase (HRP)-conjugated polyclonal antibody specific for GM-CSF is added to each well to “sandwich” the GM-CSF immobilized during the first incubation. The microtiter plate then undergoes a second incubation. The wells are thoroughly washed to remove all unbound HRP-conjugated antibodies and 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 GM-CSF and enzyme-conjugated antibody will exhibit a change in colour. The enzyme-substrate reaction is terminated by the addition of a sulphuric acid solution and the colour change is measured spectrophotometrically at a wavelength of 450 ± 2 nm.

In order to measure the concentration of GM-CSF in the samples, this kit includes two diluents (Calibrator Diluent I for serum/plasma testing and Calibrator Diluent II for cell culture supernatant testing). According to the testing system, the provided standard is diluted (2-fold dilution series) with the appropriate Calibrator Diluent and assayed at the same time as the samples. This allows the operator to produce a standard curve of Optical Density (O.D.) versus GM-CSF concentration (pg/mL). The concentration of GM-CSF in the samples is then determined by comparing the O.D. of the samples to the standard curve.