What is Sandwich ELISA?
Sandwich ELISA (Enzyme-Linked Immunosorbent Assay) is an immunoassay technique used to detect and quantify a specific antigen in a sample. It works by sandwiching the target antigen between a capture antibody coated onto a solid surface and a detection antibody labeled with an enzyme. The enzyme generates a detectable signal, which is proportional to the amount of antigen in the sample.
Sandwich ELISA is highly sensitive and specific, making it a valuable tool in medical diagnosis, food safety testing, and environmental monitoring. It is particularly useful for detecting infectious agents such as viruses and bacteria, as well as in the diagnosis of autoimmune diseases and cancer.
Applications of Sandwich ELISA include the detection of viral and bacterial infections, the diagnosis of autoimmune diseases, the detection of cancer biomarkers, and food safety testing.
Materials:
- 96-well microtiter plates (flat bottom)
- Coating buffer (e.g., PBS)
- Capture antibody
- Blocking buffer (e.g., 3% BSA in PBS)
- Wash buffer (e.g., PBS with 0.05% Tween-20)
- Sample or standard protein
- Detection antibody
- Streptavidin-conjugated enzyme (e.g., HRP)
- Substrate solution (e.g., TMB)
- Stop solution (e.g., 2M H2SO4)
Procedure:
1. Dilute the capture antibody to a working concentration (usually 1-10 μg/mL) in coating buffer.
2. Add 100 μL of the capture antibody solution to each well of the microtiter plate. Cover the plate and incubate overnight at 4°C.
3. Discard the solution and wash the plate 3 times with wash buffer.
4. Block the wells with blocking buffer by adding 200 μL per well. Incubate for 1 hour at room temperature.
5. Discard the blocking buffer and wash the plate 3 times with wash buffer.
6. Add 100 μL of the sample or standard protein to each well. Cover the plate and incubate for 2-3 hours at room temperature or overnight at 4°C.
7. Discard the solution and wash the plate 3 times with wash buffer.
8. Dilute the detection antibody to a working concentration (usually 0.1-1 μg/mL) in blocking buffer.
9. Add 100 μL of the detection antibody solution to each well. Cover the plate and incubate for 1 hour at room temperature.
10. Discard the solution and wash the plate 3 times with wash buffer.
11. Add 100 μL of streptavidin-conjugated enzyme solution to each well. Cover the plate and incubate for 30-60 minutes at room temperature.
12. Discard the solution and wash the plate 3 times with wash buffer.
13. Add 100 μL of substrate solution to each well. Incubate for 15-30 minutes at room temperature in the dark.
14. Stop the reaction by adding 50 μL of stop solution to each well.
15. Read the absorbance of each well at 450 nm using a microplate reader.
Note: The dilution factors and incubation times may vary depending on the specific assay and reagents used. It is important to optimize the assay conditions for the best sensitivity and specificity.
