Neutralization experiment protection steps - Database & Sql Blog Articles

First, the serotype of the test purpose. 2. Preparation of experimental materials 1. Samples of patients with hemorrhagic fever recovery period 2. Materials (1) Hantavirus standard strain 76-118, Seoul virus standard strain Seoul; (2) Standard serum rabbit anti-Hantan virus, Seoul virus Serum; (3) plaque reduction neutralization test commonly used reagents. 3. Step 1. Dilute the serum to be tested with bovine serum Hanks's solution to 1:10, inactivate at 30 °C for 30 minutes; 2. Further dilute the serum to 1:20, 1:40, 1:80... Control serum 1:10 dilution; 3. Dilute two viruses (in ice bath) to 200 pfu/ml; 4. Each serial dilution serum was mixed with 200 pfu/ml of two virus solutions respectively (each 0.3 ml), set at 37 ° C for 1 hour (oscillation every 15 minutes); 5. Aspirate the maintenance solution in each cell culture well; 6. Inoculate Vero-E6 cells (24-well plate) over a single layer, each well Serum virus mixture, standard serum control and two virus controls were inoculated, two wells per dilution, 100 ul/well. Adsorption for 1 hour at 37 ° C, shaking once every 15 minutes; 7. Add the first layer of agarose coating (about 42 ° C cold), 1 ml per well, to be solidified at room temperature, the cells face up, set 37 ° C Incubate in a 5% CO2 incubator for 7-9 days; 8. Add a second layer containing neutral red agarose overlay, 1 ml/well, to be coagulated at room temperature, with the cells facing up and placed in a 37 ° C 5% CO 2 incubator 2-5 days, observe the number of plaques from the next day; 9. Determine the antibody titer, judge the serum neutralization to be tested with the reciprocal of the highest dilution of the serum that is 50% or less than the number of plaques in the virus control. Antibody titer; 10. Type determination: According to the difference between the same serum and the reaction titer of the two viruses, if a serum reacts with Hantaan virus (or Seoul virus), the antibody titer is higher than that of Seoul virus ( Hantavirus has an antibody titer of 4 times or more, which is judged as Hantaan virus type, and vice versa. When the titer of the two is almost the same, it is not easy to type. If it is less than 4 times, it cannot be shaped. 4. Formulation 1. The first layer of agarose coating solution 2. The second layer of agarose coating is basically the same as the first layer of agarose coating, only the inactivated fetal bovine serum is changed to 5 ml, and a neutral red solution is added. 1 ml (333.0 ug/L neutral red sodium salt Gibco).