Sabin strain poliovirus type II monoclonal antibody and application thereof
A polio, monoclonal antibody technology, applied in the field of immunology, can solve the problem that the test results cannot truly reflect the immunogenicity of the content of type II antigen, and achieve the effect of good virus specificity
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Embodiment 1
[0030] Example 1 Immunogen preparation and animal immunization
[0031](1) Take the Vero cell working cell bank and culture it at 36.5±0.5°C after recovery until the cell concentration is 0.1-10×10 6 cells / ml, the virus was inoculated.
[0032] (2) Inoculate Vero cells with working seeds prepared from sabin polio type Ⅱ virus derived from Intravacc at an MOI of 10-0.05, and culture at 32.5±0.5°C.
[0033] (3) The virus was cultured for 2-4 days, and the cell supernatant was harvested, which was the poliovirus Sabin strain type II virus harvest solution.
[0034] (4) Type II harvest liquid is clarified and concentrated more than 5 times with ultrafiltration membrane bag.
[0035] (5) Then carry out molecular sieve chromatography and ion exchange chromatography, the monitoring wavelength is 280nm, collect the eluate and flow-through respectively to obtain the purified solution, and obtain the type II vaccine stock solution after formaldehyde inactivation.
[0036] (6) Mix and...
Embodiment 2
[0038] Example 2 Cell fusion and strain establishment
[0039] (1) Recover and culture the SP2 / 0 cell line before cell fusion, expand the culture 3 days before fusion, remove RPMI 1640 cell culture medium (Gibco) 1 day before fusion, and add culture medium again to prepare SP2 / 0 cells.
[0040] (2) The immunized mice were sacrificed, and the mouse splenocyte suspension was prepared according to conventional methods.
[0041] (3) Add an appropriate amount of incomplete IMDM culture medium (Gibco) according to the counting results of splenocytes and SP2 / 0 cells, shake and mix the SP2 / 0 cells, and pipette the splenocytes evenly. Then the splenocytes and SP2 / 0 cells were mixed in a 50ml centrifuge tube at a ratio of 1:2 to 10:1, and mixed well.
[0042] (4) Add incomplete IMDM culture medium to 50ml, centrifuge for 5-10 minutes, and pour out the supernatant. Lightly tap the bottom of the fusion tube to loosen and evenly precipitate the cells, and place the centrifuge tube in a 3...
Embodiment 3
[0050] Example 3 Preparation of monoclonal antibody cell line ascites and detection of antibody titer
[0051] Resuscitate the frozen hybridoma cells obtained in Example 2 according to conventional methods, and cultivate them. When the cells cover more than 50% of the bottom of the 25ml cell culture bottle, BALB / c mice can be inoculated intraperitoneally according to conventional methods, and the ascites can be collected regularly. SIPV-II.
[0052] Dilute the polio type Ⅱ vaccine stock solution with 0.01M PBS 1:20, coat 100 μl / well on an enzyme-titer plate, and overnight at 2-8°C to detect the antibody titer of polio type Ⅱ ascites SIPV-Ⅱ, and the antibody titer is the highest up to 10 6 , the results are shown in Table 1.
[0053] Table 1 Antibody titer test results
[0054]
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