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.