When an animal is infected by a virus, specific neutralizing antibodies are produced in the body and bind specifically to the corresponding virus particles, thus preventing the virus from adsorbing to sensitive cells or inhibiting its invasion and rendering the virus incapable of infection. Neutralization Test (Neutralization Test) is based on the determination of the infectivity of the virus and the comparison of the residual infectivity of the virus after neutralization by the immune serum to determine the ability of the immune serum to neutralize the virus.
Operation method
Cross-protection neutralization experiments
Materials and Instruments
Strain Standard Serum Move I. Purpose of the experiment For more product details, please visit Aladdin Scientific website.
Bovine serum Hanks' solution Agarose
Water bath 24-well plate Incubator
Serotyping.
II. Preparation of experimental materials
1. Specimens
Serum from patients recovering from hemorrhagic fever
2. Materials
(1) Virus strains Hanban virus standard strain 76-118, Seoul virus standard strain;
(2) Standard serum Rabbit anti-Hantaan virus, Seoul virus serum;
(3) Reagents commonly used in the empty spot reduction neutralization test.
III. Steps
1. Dilute the serum to be examined 1:10 with bovine serum Hanks' solution and inactivate at 56°C for 30 minutes;
2. Further 2-fold dilution of serum to 1:20, 1:40, 1:80 ...... and 1:10 dilution of control serum;
3. Dilute both viruses (do this in an ice bath) to contain 200 pfu/ml;
4. Mix each serial dilution of serum with an equal amount of 200 pfu/ml of each of the two viral fluids (0.3 ml each) and place at 37°C for 1 hour (shaking every 15 minutes);
5. Aspirate the maintenance solution from each cell culture well;
6. Inoculate monolayers of Vero-E6 cells (24-well plates) with serovar mixture, standard serum control and two viral controls per well, two wells per dilution, 100 ul/well. adsorb at 37°C for 1 hr, shaking every 15 min;
7. Add the first layer of agarose covering solution (need to be cold at about 42 ℃), 1 ml per well, wait for solidification at room temperature, with the cell side facing up, and incubate at 37 ℃ in 5% CO2 incubator for 7-9 days;
8. Add the second layer containing neutral red agarose covering solution, 1 ml/well, and wait for solidification at room temperature, with the cell surface facing upwards, and incubate at 37℃ in 5% CO2 incubator for 2-5 days, and observe the number of empty spots from the second day onwards;
9. Antibody titers are determined by the reciprocal of the highest dilution of serum that is neutralized or reduced by 50% from the number of empty spots of the virus control as the neutralizing antibody titer of the serum to be tested;
10. Type determination: Differentiation is based on the different titers of the same serum reacting with the two viruses. If the titer of the antibody of a serum reacting with the Hanban virus (or Seoul virus) is 4 times or more higher than that of the antibody reacting with the Seoul virus, the serum is classified as Hanban virus, and vice versa for the Seoul virus. If the difference between the two titers is very small, it is not possible to determine the type. If the titer is less than 4-fold, it cannot be typed.
IV. Formulation
1. First layer of agarose cover solution
2. Second layer of agarose overlay solution Basically the same as the first layer of agarose overlay solution, except that the inactivated fetal bovine serum was changed to 5 ml, and 1 ml of Neutral Red solution (333.0 ug/L Neutral Red Sodium Salt Gibco) was added.