Jul 11, 2022
Neutralizing antibody detection
- Shihong Fu1,
- Wenjing Liu2
- 1Department of Arbovirus, NHC Key Laboratory of Biosafety, National Institute for Viral Disease Control and Prevention, Chinese Center for Disease Control and Prevention, Beijing, People’s Republic of China;
- 2Department of Basic Medicine, School of Medicine, Qingdao University, Qingdao, People’s Republic of China
Protocol Citation: Shihong Fu, Wenjing Liu 2022. Neutralizing antibody detection. protocols.io https://dx.doi.org/10.17504/protocols.io.bdati2en
License: This is an open access protocol distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited
Protocol status: Working
We use this protocol and it’s working
Created: March 05, 2020
Last Modified: July 11, 2022
Protocol Integer ID: 33843
Abstract
Neutralizing antibodies of serum samples were detected by 90% plaque reduction neutralization test (PRNT90).
Materials
serum samples ,Japanese encephalitis virus P3 isolate,West Nile virus,BHK-21 cell.
Before start
the serum samples should be inactived (56℃，30min,in water)before the test.
Neutralizing antibody detection
Neutralizing antibody detection
1. Diluted serum: dilute the serum sample in a 96-well plate. Take 24 μl of inactivated serum and mix it with 96 μl of MEM culture medium to complete the 5-fold dilution of serum,the Serum samples were serially diluted two-fold beginning at a 1:5 dilution and ending at 1: 160 .
2. Dilute virus: use MEM culture medium to dilute virus to 200 pfu/100 μl.
3. The diluted serum (from 1:5-1:160) was mixed with an equal volume of 200 pfu of JE virus (strain P3). At the same time, different dilutions of virus (100 PFU, 10 PFU, 1 PFU) were set as controls, and the diluted virus solution was mixed with serum samples and incubated at 37 ° C and 5% CO2 for 1 h.
4 Incubation of infected cells with the neutralized mixture: inoculate 6-well plates of BHK-21 cells grown to 90% confluence in monolayer, and incubate at 37℃, 5% CO2 for 1 h.
5 Overlay methylcellulose medium culture: supplement 4 ml of 1.2% methylcellulose-MEM medium containing 2% FBS, culture at 37℃ and 5% CO2 for 3-5 days until obvious viral plaque is observed.
6. Stain and calculate the neutralization: remove the culture medium, stain with crystal violet for 1h, gently rinse with running water, dry and count the number of viral plaques, and calculate the neutralizing antibody of JE virus in the sample.