Creative Biolabs is dedicated to providing comprehensive support and services based on neutralizing antibodies for our clients. Here, we offer the protocol for Micro-neutralization (MN) Assay, aiming to provide clients with a detailed understanding of the experiment's procedures and workflow. This will assist clients in making informed experimental plans and decisions.
All-round Micro-Neutralization (MN) Assay Guidance
The Micro-neutralization (MN) assay is an imaging-based virology technique used to quantify neutralizing antibodies and antiviral activities. Unlike traditional assays, it counts infected cells on the cellular level, overcoming limitations related to plaque size. By employing imaging technology, including optical microscopy and high-throughput well-plate readers, the MN assay achieves accurate and sensitive results. This approach offers a cost-effective, high-throughput, and user-friendly method for antigenic characterization of viruses, as well as for measuring antiviral activities and neutralizing antibodies. The MN assay has undergone extensive testing with influenza viruses, making it a valuable tool in virology research for studying neutralizing antibodies and assessing antiviral potency.
| Reagents | Instruments and Consumables |
| Virus Growth Medium (VGM) | Washing Machine |
| Overlay Buffer | Eppendoff |
| Fixation Buffer | 96-well plate |
| Phosphate Buffered Saline (PBS) | Flatbed scanner |
| Permeabilization Buffer | Microplate Reader |
| Washing Buffer | |
| Detect Antibody | |
| HRP-Conjugated Secondary Antibody | |
| TMB Solution | |
| Receptor-Destroying Enzyme (RDE) |
Virus Titration
1. Inoculate cells (200 μL/well) into a 96-well plate. Incubate cells for 2-3 days to allow fusion.
2. Wash cells three times with VGM (200 μL/well). Add fresh VGM (50 μL/well).
3. Dilute the virus into a gradient of viral concentrations. Add different virus dilutions (50 μL/well) to the cells and incubate for 2-3 hours to allow virus infection.
4. Remove the virus solution. Add Overlay Buffer (200 μL/well) and incubate overnight at 37°C.
5. Remove the Overlay Buffer. Add pre-cooled Fixation Buffer (200 μL/well). Incubate at 4°C for 30 minutes.
6. Remove the Fixation Buffer and wash the plate twice with PBS (200 μL/well).
7. Add Permeabilization Buffer (100 μL/well) and incubate at room temperature for 30 minutes.
8. Wash the plate twice with PBS (200 μL/well).
9. Add the detection antibody (50 μL/well) and incubate on a shaker at room temperature for 1 hour or overnight at 4°C.
10. Wash the plate three times with wash buffer (200 μL/well).
11. Add HRP-conjugated Secondary Antibody (50 μL/well) and incubate on a shaker at room temperature for 1 hour.
12. Wash the plate three times with wash buffer (200 μL/well).
13. Add TMB Solution (50 μL/well) and incubate at room temperature until a clear blue color develops (approximately 30 minutes).
14. Wash the plate twice with distilled water (200 μL/well).
15. Air dry the plate and store it away from light.
Titration Quantitation
16. Image the infected cell populations using a flatbed scanner.
17. Read the OD values at 490nm wavelength for each well using a Microplate Reader to calculate the required virus concentration based on infected cell population measurements.
Virus Neutralization
18. Inoculate cells (200 μL/well) into a 96-well plate. Incubate cells for 2-3 days to allow fusion.
19. Wash cells three times with VGM (200 μL/well). Add fresh VGM (50 μL/well).
20. Dilute RDE-treated serum into a gradient of dilutions.
21. Add different dilutions of serum (50 μL/well).
22. Based on the results of Virus Titration, add the most suitable working virus solution (50 μL/well) and incubate for 2-3 hours to allow virus infection.
23. Repeat steps 4-15.
Neutralization Quantitation
24. Image the infected cell populations using a flatbed scanner.
25. Read the OD values at 490nm wavelength for each well using a Microplate Reader to calculate the required virus concentration based on infected cell population measurements.
Creative Biolabs is an exceptional choice for your needs! If you require purchasing or custom synthesizing neutralizing antibodies for subsequent MN assay, do not hesitate to contact us for unparalleled support. Their expertise and dedication will undoubtedly provide you with the best solutions for your research.
