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Double antibody sandwich enzyme-linked immunosorbent assay method for detecting chimpanzee adenoviruses AdC68

An adenovirus and chimpanzee technology, applied in the field of immune analysis, can solve the problems of weakening immune response and hindering the application of vectors

Inactive Publication Date: 2018-11-16
天津津斯特生物技术有限责任公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, due to the widespread presence of neutralizing antibodies against common human serotypes of adenoviruses in the population, the immune response induced by the corresponding adenovirus vectors is weakened, hindering the clinical application of these vectors

Method used

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  • Double antibody sandwich enzyme-linked immunosorbent assay method for detecting chimpanzee adenoviruses AdC68
  • Double antibody sandwich enzyme-linked immunosorbent assay method for detecting chimpanzee adenoviruses AdC68
  • Double antibody sandwich enzyme-linked immunosorbent assay method for detecting chimpanzee adenoviruses AdC68

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0018] Embodiment 1 draws standard curve

[0019] 1) Operation

[0020] Dilute the purified antibody with coating buffer, the optimal coating concentration is 1:50, 100ul per well, overnight at 4°C; after washing, prepare 2% BSA with PBST (PBS containing 0.1% Tween 20), 200ul per well , blocked at 37°C for 2hr; the standard reference substance and the sample to be determined were diluted with PBST containing 2% goat serum, the sample volume was 100ul, and incubated at 37°C for 1hr; after washing, HRP-labeled goat anti-adenovirus HEXON was added at a concentration of 1 : 500, 100ul per well, incubate at 37°C for 1hr; after washing, use 100ul of chromogenic solution containing 0.01% TMB, and develop color at room temperature in the dark for 30min.

[0021] 2) Drawing

[0022] Serially dilute the purified virus standard to 1 / 5, 1 / 10, 1 / 20, 1 / 40, 1 / 80, 1 / 160, 1 / 320, 1 / 640, and the concentration of each diluted reference product is 7.76ug / ml , 3.88ug / ml, 1.94ug / ml, 0.97ug / ml, 0....

Embodiment 2

[0025] Example 2 Verification of accuracy and precision

[0026] Operated according to the method of the present invention, the AdC68 standard substance was diluted, and samples with high and low concentrations were prepared, and the protein content was respectively 0.776ug / ml and 0.388ug / ml, and the established ELISA method was used to repeat the measurement 9 times, and the recovery rate and variation of the samples were calculated. Coefficients, etc. (see table), the results show that the recovery rate of the determination is between 84.38% and 102.6%, and the coefficients of variation are 3.44% and 6.72%, respectively, indicating that the method has good accuracy and precision, and can accurately determine viral proteins content.

[0027] Table 1

[0028]

Embodiment 3

[0030] Experimental operation is carried out according to the present invention, and different samples in the research process of adenovirus AdC68 purification process are selected to measure the content of antigen virus protein. Simultaneous determination of titer, viral particles. To verify the applicability of the established double antibody ELISA method. The viral protein in Table 2 is the data measured by the method of the present invention. From the sample detection during the virus purification process, the established ELISA method can well detect the content of recombinant virus protein, which has a good correlation with the measured virus titer and virus particle number. Moreover, by comparing with the virus titer of the sample and calculating the specific activity, the influence of different purification steps on the purity of AdC68 virus can be roughly estimated, and the purification method 1 is obviously better than 2 and 3.

[0031] Table 2 Comparison of differe...

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Abstract

The invention discloses a double antibody sandwich enzyme-linked immunosorbent assay method for detecting chimpanzee adenoviruses AdC68 and belongs to the technical field of immunoassay. The method uses a purified rabbit anti-AdC68 antibody as a coated antibody, purified recombinant ADC68GFP as a standard product, a HRP enzyme-labeled goat anti-adenovirus HEXON antibody as an enzyme-labeled antibody, PBST containing 2% of sheep serum as a sample diluent and a double antibody sandwich method to detect the chimpanzee adenovirus AdC68 antigen, a viral protein, titer and virus particles. The method has high specificity, sensitivity and precision. Compared with the common UV method for measuring virus particles and the plaque method for measuring virus titer, the method has a wide use range andis suitable for on-line detection of the recombinant chimpanzee adenovirus in the purification process. The method can fast estimate the amount of viruses in the preparation of AdC68 and guide the upstream virus culture and downstream purification process.

Description

technical field [0001] The invention relates to a double-antibody sandwich ELISA method for detecting chimpanzee adenovirus AdC68, which belongs to the technical field of immune analysis. Background technique [0002] Due to the characteristics of adenovirus with wide host range and high transgene expression ability, up to 300 human therapeutic or prophylactic recombinant adenovirus drugs are currently being used in clinical trials. The widely used adenovirus vectors are human serotype adenovirus AdHu2 and AdHu5. However, the widespread presence of neutralizing antibodies against common human serotypes of adenoviruses in the population weakens the immune response induced by the corresponding adenovirus vectors, hindering the clinical application of these vectors. In order to avoid the impact of pre-existing antibodies in the population on the immune or therapeutic effects of adenovirus vector vaccines, a number of types derived from other populations are increasingly used i...

Claims

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Application Information

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IPC IPC(8): G01N33/535G01N33/569
CPCG01N33/535G01N33/56983
Inventor 王宁吴冠军石慧颖
Owner 天津津斯特生物技术有限责任公司
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