Mouse GM-CSF ELISA Assay

Additional Information

Assay Procedure

1. Prepare all reagents and working standards as directed in the previous sections.
2. Determine the number of microwell strips required to test the desired number of samples plus appropriate number of wells needed for running blanks and standards. Remove extra microwell strips from holder and store in foil bag with the desiccant provided at 2-8°C sealed tightly.
3. Add 100 µL of Standard, control, or sample, per well. Cover with the adhesive strip provided. Incubate for 1.5 hours at 37°C.
4. Aspirate each well and wash, repeating the process three times for a total of four washes. Wash by filling each well with Wash Buffer (350 µL) using a squirt bottle, manifold dispenser or auto-washer. Complete removal of liquid at each step is essential to good performance. After the last wash, remove any remaining Wash Buffer by aspirating or decanting. Invert the plate and blot it against clean paper towels.
5. Add 100 µL of the working solution of Biotin-Conjugate to each well. Cover with a new adhesive strip and incubate 1 hour at 37°C.
6. Repeat the aspiration/wash.
7. Add 100 µL of the working solution of Streptavidin-HRP to each well. Cover with a new adhesive strip and incubate for 30 minutes at 37°C.   Avoid placing the plate in direct light.
8. Repeat the aspiration/wash.
9. Add 100 µL of Substrate Solution to each well. Incubate for 10-20 minutes at 37°C. Avoid placing the plate in direct light.
10. Add 100 µL of Stop Solution to each well. Gently tap the plate to ensure thorough mixing.
11. Determine the optical density of each well immediately, using a microplate reader set to 450 nm.(optionally 630nm as the reference wave length; 610-650nm is acceptable)

Assay Background

Granulocyte macrophage colony-stimulating factor (GM-CSF), a 22 kDa glycosylated protein, was initially characterized as a factor that can support the in vitro colony formation of granulocyte macrophage progenitors. It is also a growth factor for erythroid, megakaryocyte, and eosinophil progenitors. GM-CSF is produced by a number of different cell types (including T cells, B cells, macrophages, mast cells, endothelial cells, fibroblasts, and adipocytes) in response to cytokine or inflammatory stimuli. On mature hematopoietic cells, GM-CSF is a survival factor for and activates the effector functions of granulocytes, monocytes/macrophages, and eosinophils (1, 2). GM-CSF promotes a Th1 biased immune response, angiogenesis, allergic inflammation, and the development of autoimmunity (3 5). It shows clinical effectiveness in ameliorating chemotherapy induced neutropenia, and GM-CSF transfected tumor cells are utilized as cancer vaccines (6, 7). Mature mouse GM-CSF shares 49% -54% amino acid sequence identity with canine, feline, human, and porcine GM-CSF and 69% with rat GM-CSF. GM-CSF exerts its biological effects through a heterodimeric receptor complex composed of GM-CSF Rα/CD116 and the signal transducing common β chain (CD131) which is also a component of the high affinity receptors for IL-3 and IL-5 (8,9). In addition, GM-CSF binds a naturally occurring soluble form of GM-CSF Rα (10). The activity of GM-CSF is species specific between human and mouse. Mouse GM-CSF is only weakly active on rat cells, although rat GM-CSF is fully active on mouse cells (11).