Ⅰ. envelope antigen

1.TDissolve the antigen with 50mm carbonate coated buffer (pH 9.6), make the concentration of the antigen 10-20 μ g / ml, add 100 μ L / well to 96 well enzyme plate, and place it overnight at 4°C.

2.After discarding the coating solution the next day, wash it with PBST for three times, and add 150 μl 1% BSA into each hole for one hour at 37°C.

3.After 3 times of PBST washing, 100 μl of serum with different times of dilution was added into each pore, and the control sample was added and incubated at 37°C for 2 hours.

4.After 5 times of PBST washing, add 100 μ l diluted HRP labeled secondary antibody and incubate at 37°C for 1 hour.

5.After 5 times of PBST washing and 20 minutes of color reagent development, the absorption value of A₄₀₅ was read on the enzyme reader.

Ⅱ. encapsulated cell

1.The number of cells inoculated on 96 well plate was 1 x 10⁴ cells/well and cultured overnight at 37℃.

2.The next day, the culture plates were washed with PBS 2-3 times.

3.Add 125 µl/well 10% formalin (1:10 dilution), and fix it at room temperature for 15 min.

4.Wash the culture plate with ddH₂O for three times, and dry it, and store it at 2-8°c for standby..

5.Wash 3 times with PBST, add 150 µl 1% BSA into each hole and seal it at 37°C for 1 hour.

6.After 3 times of PBST washing, 100 μl of serum with different times of dilution was added into each pore, and the control sample was added and incubated at 37°C for 2 hours.

7.After 5 times of PBST washing, add 100 μl diluted HRP labeled secondary antibody and incubate at 37°C for 1 hour.

8.After 5 times of PBST washing and 20 minutes of color reagent development, the absorption value of A₄₀₅ was read on the enzyme reader. 50mM carbonate coated buffer solution: 0.05mol/l ph9.6 carbonate buffer solution, stored at 4 ℃,Na₂CO₃ 0.15 g, NaHCO₃ 0.293 g, diluted to 100 ml with distilled water. ABTS as substrate for color reaction (10ml):
· 0.2M  Na₂HPO₄  2.4ml
· 0.1M  citric  acid  2.6ml
· ddH₂O  5ml · ABTS  5mg · H₂O₂(30%)  4ul(Join before use)

notice：
· In general, it is necessary to have the corresponding control serum for multiple dilution test.
· Different color systems correspond to different light absorption values.