ELIS method and kit for panoramic identification of intra-genus broad spectrum of human norovirus antibody
A technology for identifying people and kits, which is applied in ELISA methods and kits to comprehensively identify the broad-spectrum of human Norovirus antibodies, and can solve the problems of measuring the broad-spectrum of NoV serum antibodies
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Embodiment 1
[0035] Example 1: Preparation of a broad-spectrum kit for panoptic identification of human norovirus antibodies
[0036] 1. Dilute the NoV capsid protein of 28 genotypes (9 GI types and 19 GII types, each genotype is shown in Table 1) to 2 μg / μl, take 100 μl and coat it in a 96-well microtiter plate, Each protein was coated in triplicate wells. Glutathione S-transferase (GST)-tagged protein was used as a positive control. Place overnight at 4°C.
[0037] 2. Take out the 96-well plate and wash it 3 times with PBST. Add 5% skimmed milk powder solution and incubate at 37°C for 2h.
[0038] 3. After the incubation, take out the 96-well plate, wash it with PBST for 3 times, control the dry water and place it at 4°C for later use, and obtain a broad-spectrum kit for the panorama identification of human norovirus antibody ( figure 2 ).
[0039] Table 1
[0040]
[0041]
Embodiment 2
[0042] Example 2 Determination of Norovirus Antibody Titer
[0043] Take 100 μl of 2 μg / μl GII.4 and GII.8 capsid proteins and coat them in a 96-well microtiter plate, overnight at 4°C. The next day, the 96-well plate was washed with PBST three times, and then 200 μl of 5% skimmed milk powder solution was added to each well to block at 37° C. for 2 hours. After blocking, wash the plate 4 times with PBST, dilute the GII.4 monoclonal antibody and GII.4+GII.8 antiserum to be tested from 1:2000 and 1:500 respectively, and add 100 μl to each well at 37°C Incubate for 1 hour. After incubation, wash the plate 4 times with PBST, add 100 μl of HRP-labeled goat anti-mouse secondary antibody serum diluted 1:3000, and incubate at 37°C for 0.5h. After that, the plate was washed 5 times with PBST, 100 μl of TMB single-component chromogenic solution was added, and incubated at 37° C. in the dark for 5 minutes. Immediately add 50 μl 2M H at the end 2 SO 4 The reaction was terminated, and...
Embodiment 3
[0045] Embodiment 3 Norovirus antibody is broad-spectrum determination in the genus
[0046] Take out the sealed 96-well plate ( figure 2 ), GII.4 monoclonal antibody and GII.4+GII.8 antiserum were diluted 1:5000 and 1:500 respectively, and 100 μl was added to each well and incubated at 37°C for 1 hour, and GST protein antibody for positive control was added at the same time. Wash the plate 4 times with PBST, add 100 μl of HRP-labeled goat anti-mouse secondary antibody at a dilution ratio of 1:3000, and incubate at 37°C for 0.5h. After that, the plate was washed 5 times with PBST, 100 μl of TMB single-component chromogenic solution was added, and incubated at 37° C. in the dark for 5 minutes. Finally join 2M H immediately 2 SO 4 The reaction was terminated, and the OD 450nm was measured with a microplate reader. An OD value below 0.2 was defined as no binding; an OD value between 0.2-0.6 was defined as moderately strong binding; an OD value greater than 0.6 was defined as...
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