Intended Use:Human MCAF enzyme-linked immunospot (ELISpot) whole kit with pre-coated PVDF-bottom Immunospot plates for the quantitation of single cells releasing human MCAF.
Background:Monocyte chemotactic and activating factor (MCAF), also known as monocyte chemotactic protein 1 (MCP-1), lymphocyte–derived chemotactic factor (LDCF), and glioma-derived chemotactic factor (GDF), is a recently-identified chemotactic cytokine for monocytes. cDNA cloning and structural analysis has revealed that this 76-amino acid polypeptide with a predicted molecular mass of 8,700 daltons belongs to a family of structurally-related low molecular weight proteins characterized by four conserved cysteine residues designated C-C family or intercrine Beta family (1,2,3).
MCAF is expressed by various types of cultured cells including monocytes, lymphocytes, fibroblasts, endothelial cells, smooth muscle cells and transformed cell lines upon stimulation with LPS or cytokines such as IL-1, TNF-alpha, and IFN-gamma. Although there exist some minor differences in expression patterns that are observed in some types of cells, almost all agents that induce IL-8 mRNA expression also induce MCAF mRNA expression. Platelet-derived growth factor is a strong inducer of MCAF mRNA in human fibroblasts whereas it failed to induce IL-8 mRNA in human fibroblasts (4). These results suggest that the regulatory mechanism of MCAF gene expression differs from that of the IL-8 gene. In addition to being chemotactic for monocytes, MCAF also activates human monocytes to become cytostatic for several human tumor cell lines (5), release lysosomal enzymes (6), and generate superoxide (6).
MCAF is also expressed in vivo by lung epithelial cells in patients with idiopathic pulmonary fibrosis (7), synovial tissues of rheumatoid arthritis (8), or in atheromatous plaques in atherosclerotic lesion (9), suggesting the participation of MCAF in the pathogenesis of these disorders. Furthermore, MCAF has a potent histamine-releasing activity on basophils (10) that indicates an associated effect in allergic inflammations. MCAF expression in vivo has been investigated by qualitative methods such as in situ hybridization and immunohistochemistry. In order to further clarify and elucidate its participation and relation with various disorders, quantitative analysis of its in vivo level is necessary (11, 12). This 2.5 hours ELISA kit is developed to detect and visualize of single cells secreting human MCAF.
Kit Components:1. ELISA Plate, 1x96 wellsPVDF-bottom Immunospot plates pre-coated with mouse anti-human MCAF monoclonalantibody. Unpacked before use
2. Positive Control, 1x1 vial Lyophilized recombinant human MCAF (2ng/vial). Reconstitute 1 vial in 250ul Cell Culture Media before use. Use in 1 hour. The final concentration is 8ng/ml
3. 20X Wash Buffer Concentrated, 1x60mlAdd 1 volume of 20X Wash Buffer Concentrated to 19 volume of deionized water/distilled water. Use in 1 week. Stored at room temperature
4. Concentrated Human MCAF Detection Antibody, 1x1 vial120ul 100X Concentrated Biotinylated mouse anti-human MCAF monoclonal antibody. Add 1 volume of Human MCAF Concentrated Detection Antibody to 100 volumes of Detection Antibody Diluent before use. Use in 1 month. Stored at 4 °C. 5. Detection Antibody Diluent, 1x11mlProtein with buffer and preservative. Ready to use. 6. Concentrated Streptavidin-AP, 1x1 vial 120ul 100X Concentrated Alkaline Phosphatase labeled Streptavidin. Add 1 volume of Concentrated Streptavidin-AP to 100 volumes of Streptavidin – AP Diluent before use. Use in 1 month. Store at 4°C. 7. Streptavidin – AP Diluent, 1x11mlProtein with buffer and preservative. Ready to use. 8. Substrate Solution, 1x11mlBCIP/NBT Substrate Solution. Ready to use.
Storage and Stability:Store other components at 4°C. Stable for 6 months. For maximum recovery of product, centrifuge the original vial after thawing and prior to removing the cap.
