Method for staining and determining titer of heterophilic mouse leukemia virus by using tissue half-infection method

A murine leukemia virus, half-infection technology, applied in the field of virus clearance/inactivation verification research, can solve the problems of time-consuming and labor-intensive plaque assay, it is not suitable to promote indicator cells, genetic stability cannot be guaranteed, etc., to achieve strong repeatability , reduce errors, and have strong sensitivity

Pending Publication Date: 2021-02-12
SUZHOU YAOMING KANGDE INSPECTION TESTING
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Benefits of technology

Inventors have developed an accurate way to measure how well mice develop tumor symptoms called xenosis. They found out when certain types of cancer were detected during their treatment but they didn't find it until later on. By combining these techniques together with immunological analysis, we could identify specific molecules associated with each type of disease without relying solely upon human judgment alone. These results would help us better study diseases related to bacterial growth such as syphilis.

Problems solved by technology

The technical problem addressed in this patented study relates to improving the efficiency and reliability of identifiable screenings during virus identification processes like measuring protein expression levels and evaluating virus severity index values. Current techniques involve either qualitative analysis or semi-quantitation of qNARISP-502 capsule fusion proteins containing nucleotides, followed by flow cytometry measurements. These current approaches require multiple steps and labor intensiveness due to their invasivity nature.

Method used

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  • Method for staining and determining titer of heterophilic mouse leukemia virus by using tissue half-infection method
  • Method for staining and determining titer of heterophilic mouse leukemia virus by using tissue half-infection method
  • Method for staining and determining titer of heterophilic mouse leukemia virus by using tissue half-infection method

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0044] Example 1 Tissue half infection method staining and determination of X-MuLV virus titer specific implementation method

[0045] After the X-MuLV virus was thawed, the virus was serially diluted with the virus diluent after ultrasonication and filtration, and a 3.2-fold serial dilution (0.5 log) was made according to the actual situation.

[0046] PG4 cells were seeded in a cell culture 96-well plate, and each cell well was seeded with 1.0×10 4 ~1.5×10 4 cells at 37°C, 5% CO 2 The cells are cultured under culture conditions, and the cells are observed on the second day, and subsequent operations can be performed when the cell confluency reaches 30%-50%. The number of seeded cells can also be adjusted so that the confluence of the cells is around 30%-50%.

[0047] The virus was serially diluted 3.2 times to 3.2 -14 Dilution: Infect PG4 cells with prepared X-MuLV virus of different dilutions: suck off the cell culture medium, add 50 μL of virus solution, add 200 μL of ...

Embodiment 2

[0049] Example 2: Linearity and range of X-MuLV tissue half infection method

[0050] According to the operation process of Example 1, the experimental operator carried out three independent serial dilutions, each serial dilution contained 5 different dilution factors, and calculated the virus titer of each dilution series: draw a standard curve, calculate the linear correlation coefficient , determine the virus detection range, standard curve and linear correlation coefficient results see figure 2 .

[0051] Depend on figure 2 It can be seen that within the range of the measured virus titer, the standard curves of the three independent serial dilutions have a good linear relationship, and the results are stable and reproducible.

Embodiment 3

[0052] Embodiment 3: The same operator carries out the comparison of X-MuLV virus infection PG4 cell experiment at different times

[0053] According to the operation process of Example 1, the same experimenter carried out the titer determination experiment of X-MuLV at different times of the same day and the next day, and each experiment was repeated in three groups, and each group was made with eight duplicate holes. The experimental results Such as image 3 shown.

[0054] Experimental results: X-MuLV virus infection of PG4 cells at three time points showed a good linear relationship under different dilution factors, and the results were stable and reproducible.

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Abstract

The invention discloses a method for staining and determining titer of an allophilic mouse leukemia virus by using a tissue half-infection method, which takes a PG4 cell as an indicator cell for detecting X-MuLV, accurately and quantitatively determines the titer of the X-MuLV by using TCID50 assay combined with a crystal violet staining method, and comprises the following steps: continuously diluting the virus; adding viruses with different dilution degrees into the cell holes; adding a cell growth solution into each pore in the pore plate, transferring into a cell incubator, and culturing for 5-7 days; terminating the culture, dyeing, and reading the number of CPE pores; and calculating virus titer. The method can overcome the defect of multiple operation procedures of a plaque determination method, and is more convenient to operate; meanwhile, in combination with cell staining, subjective errors of different persons can be effectively avoided when the cytopathic effect is observed through a microscope, and the product has the advantages of being high in sensitivity, clear in background, good in stability, high in repeatability and the like.

Description

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Claims

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Application Information

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Owner SUZHOU YAOMING KANGDE INSPECTION TESTING
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