AAV8 neutralizing antibody detection method and detection kit
An antibody detection and carrier technology, applied in biochemical equipment and methods, microbial determination/inspection, analysis by chemical reaction of materials, etc., can solve the problems of long cycle time, poor data stability and high cost
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Embodiment 1
[0026] Working concentration of AAV8 viral vector
[0027] Take the cultured 293T (manufacturer ATCC, product number: CRL-3216) cells, use DMEM cell culture medium to plant 96-well white plate (manufacturer Corning, product number: 3917) at 20000 cells / well, 100ul / well, put in 37 ℃ 5%CO 2 The incubator cultures were grown overnight. On the second day, AAV8[PAAV-CMV-Luc] (manufacturer: Jinweizhi, customized) was diluted to 2E10 vg / ml using DMEM serum-free basal medium, and then 3-fold serially diluted 5 points to obtain a concentration of 6.6E9 AAV8 viral vector dilutions of vg / ml, 3.3E9 vg / ml, 1.1E9 vg / ml, 3.7E8 vg / ml, 1.2E8 vg / ml. Take the cell culture plate, draw 50ul / well supernatant, add 50ul / well gradient dilution of AAV8 virus, mix gently with a pipette gun, put in 37°C 5% CO 2 Cultured in an incubator for 24 hours. On the third day, take out the 96-well plate and add 50ul Bright-Lumi TM Firefly luciferase detection reagent (manufacturer: Biyuntian, product number: ...
Embodiment 2
[0030] Mixed human serum and individual serum AAV8 neutralizing antibody detection test
[0031] Take the cultured 293T (manufacturer ATCC, product number: CRL-3216) cells, use DMEM cell culture medium to plant 96-well white plate (manufacturer Corning, product number: 3917) at 20000 cells / well, 100ul / well, put in 37 ℃ 5%CO 2 The incubator cultures were grown overnight. On the second day, AAV8[PAAV-CMV-Luc] (manufacturer: Jinweizhi, customized) was diluted to 2.5E9vg / ml using DMEM serum-free basal medium, and individual serum and mixed human serum were mixed using DMEM serum-free basal medium 4-fold gradient dilution for 3 points, take AAV8 dilution and serum gradient dilution for 1:4 dilution, mix well, and use DMEM serum-free medium as a negative control, incubate at room temperature for 30min, as shown in the table below, follow the layout diagram 50ul The gradient dilution of AAV8 virus per well, mixed gently with a pipette gun, placed in 37 ° C 5% CO 2 Cultured in an i...
Embodiment 3
[0035] Screening of negative sera and preparation of sample diluents
[0036] Take the cultured 293T (manufacturer ATCC, product number: CRL-3216) cells, use DMEM cell culture medium to plant 96-well white plate (manufacturer Corning, product number: 3917) at 20000 cells / well, 100ul / well, put in 37 ℃ 5%CO 2 The incubator cultures were grown overnight. The next day, AAV8[PAAV-CMV-Luc] (manufacturer: Jinweizhi, customized) was diluted to 4E9vg / ml using DMEM serum-free basal medium, and individual serum was diluted 2.5 times and 5 times, take AAV8 dilution and serially diluted serum for 1:1 dilution, mix evenly, and use DMEM serum-free medium instead of human serum dilution as a negative control (NSC), incubate at room temperature for 30min, according to the layout in the table below Figure 50ul / well AAV8 virus gradient dilution, mix gently with a pipette gun, put in 37 ℃ 5% CO 2 Cultured in an incubator for 24 hours. On the third day, take out the 96-well plate and add 50ul ...
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