Human GM-CSF Matched Antibody Pair for ELISpot

INTENDED USE

Pre-titered capture antibody and biotinylated detection antibody matched pair for the development of enzyme-linked immunospot (ELISpot) assays for the quantitation of single cells releasing human GM-CSF. 

A recommended assay protocol is provided. The dilutions of capture antibody and detection antibody is determined according to this protocol. The researcher can optimize the dilutions if it is necessary. 

For laboratory research use only. Not for use in diagnostic procedures. 

INTRODUCTION 

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.^1,2,3 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.²  

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.^2,3  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.⁴  The GM-CSF receptor has been cloned³ and, the a  and b 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 macrophage³, whereas eosinophil, erythroid and megakaryocyte colony formation is stimulated at much higher concentrations.^2,3,5  GM-CSF is also required for growth and differentiation of typical dendritic cells from human bone marrow^2,6, causes activation and prolonged survival of mature hematopoietic cells^2,3, 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 macrophages³, and increases HIV-1 replication at the post-transcriptional level.⁷  GM-CSF binds to non-hematopoietic cells causing the proliferation and/or migration of fibroblast, endothelial, and various tumour cell lines.^8,9  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.¹⁰  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.¹¹ 

Various pathological conditions are associated with increased GM-CSF levels.  These include: lung cancer,¹²  acute mylogenous leukemia,¹³ tumour related thrombocytosis,¹⁴ myelodysplastic syndrome (MDS),¹⁵ thrombocytopenia,¹⁶and psoriasis.¹⁷  GM-CSF expression is increased in bronchial asthma and lung inflammatory diseases;^9,18 non-allergic respiratory diseases such as eosinophil pneumonia, hypersensitivity pneumonitis, iodiopathic pulmonary fibrosis, sarcoidosis, cryptogenic organizing pneumonia, HIV infection,⁹ rheumatoid arthritis, and systemic lupus erythmatosus.¹⁹  GM-CSF shows therapeutic value by accelerating neutrophil recovery in disease induced myelosuppression such as bone marrow transplantation, chemotherapy, and infectious disease.^2,3  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 disease²⁰ and in gestational trophoblastic disease (GTD) for the early identification of high risk choriocarcinoma cases.²¹  

This ELISpot kit is developed to detect and visualize of single cells secreting human GM-CSF.