阅读说明：本技术 一种高通量检测eb病毒感染效率/抗体阻断eb病毒感染效率的方法 (Method for high-throughput detection of EB virus infection efficiency/EB virus infection blocking efficiency by antibody ) 是由 张晓� 曾益新 曾木圣 徐淼 冯启胜 于 2020-06-02 设计创作，主要内容包括：本发明公开了一种可用于高通量检测EB病毒中和实验结果的方法。所述方法主要以高内涵细胞成像仪作为检测仪器,对病毒感染细胞的感染效率或中和抗体及多抗血清的阻断病毒感染细胞的效率进行检测评估。为检测EB病毒的感染效率或评估中和抗体或多抗血清的中和能力提供了简便的高通量的检测方法,为临床样本的检测、EB病毒预防性疫苗的开发以及中和抗体的筛选提供基本的技术体系支持。(The invention discloses a method for detecting EB virus neutralization experimental results in high throughput. The method mainly uses a high content cell imaging instrument as a detection instrument to detect and evaluate the infection efficiency of virus infected cells or the efficiency of blocking virus infected cells by neutralizing antibodies and multiple antiserums. Provides a simple and convenient high-flux detection method for detecting the infection efficiency of the EB virus or evaluating the neutralizing capacity of a neutralizing antibody or multiple antiserums, and provides basic technical system support for the detection of clinical samples, the development of EB virus preventive vaccines and the screening of neutralizing antibodies.)

1. A method for detecting EB virus infection efficiency in high throughput comprises the following steps:

s1: preparing a cell sample to be detected infected by the EB virus;

s2: and (3) detecting the cell sample to be detected by using a high content detection instrument, and calculating the half inhibitory concentration.

2. The method according to claim 1, wherein the test cell sample is prepared in step S1 by: the cell sample to be tested is infected with the virus liquid in a 96-well plate and tested after culture.

3. The method according to claim 2, wherein the test cell sample is prepared in step S1 by: infection of 10 with 20. mu.l of virus fluid in 96-well plates⁴And (3) infecting the cell sample to be detected in a carbon dioxide incubator at 37 ℃ for 120min, supplementing a culture medium until the final volume is 200 mu l, and culturing for 48 hours to be detected.

4. The method of any one of claims 1 to 3, wherein the step S2 is performed by using a high content detection instrument to detect the cell sample to be detected by using a GFP fluorescence detection module.

5. A method for detecting the efficiency of blocking EB virus infection by multiple antiserum/antibody in a high-throughput manner comprises the following steps:

s1: respectively mixing virus liquid and the antibody diluted according to multiple gradient, and incubating;

s2: adding an equal amount of cell samples to be detected into the mixed solution of the virus solution and the antibody, and culturing;

s3: and (3) detecting the cell sample to be detected by using a high content detection instrument, and calculating the half inhibitory concentration according to the sample dilution and the EB virus infection efficiency.

6. The method of claim 5, wherein the blocking of EB virus infection is measured by detecting the neutralizing titer of multiple antisera/antibodies against EB virus.

7. The method of claim 5, wherein step S1 is performed by mixing 20 μ l of virus solution with the antibody diluted by a multiple gradient in a 96-well plate and incubating.

8. The method according to claim 5, wherein 10 is added to the mixture of the virus solution and the antibody in step S2⁴And infecting the cells to be detected in a carbon dioxide incubator at 37 ℃ for 100-150 min, supplementing a culture medium until the final volume is 200 mu l, and culturing for 48 hours to be detected.

9. The method according to any one of claims 5 to 8, wherein the step S3 is performed by using a high content detection instrument to detect the cell sample to be detected, wherein a GFP fluorescence detection module is used.

10. The method of claim 9, wherein the specific parameters of the GFP fluorescence detection module are: the exposure time was 80ms, the exposure intensity was 50%, and the focal height was 11 μm.

Technical Field

The invention relates to the technical field of biomedicine, in particular to a method for detecting EB virus infection efficiency/blocking EB virus infection by an antibody in a high-throughput manner.

Background

An important research index in the development process of viral prophylactic vaccines is to detect the response of neutralizing antibodies induced by candidate vaccines, and the titer evaluation of the neutralizing antibodies and the detection of the neutralizing titer of clinical serum mainly depend on a high-throughput detection method, so that a large number of samples can be detected at one time in a short time, and the high throughput of the neutralizing detection method is required.

Disclosure of Invention

The first aspect of the invention aims to provide a method for detecting EB virus infection efficiency in a high-throughput manner.

The second aspect of the invention aims to provide a method for detecting the efficiency of blocking EB virus infection by multiple antiserum/antibody in high throughput.

The technical scheme adopted by the invention is as follows:

in a first aspect of the present invention, there is provided a method for high-throughput detection of EB virus infection efficiency, comprising the following steps:

s1: preparing a cell sample to be detected infected by the EB virus;

s2: and (3) detecting the cell sample to be detected by using a high content detection instrument, and calculating the half inhibitory concentration.

Preferably, according to the method of the first aspect of the present invention, the preparation method of the test cell sample in step S1 is: the cell sample to be tested is infected with the virus liquid in a 96-well plate and tested after culture.

More preferably, according to the method of the first aspect of the present invention, the method for preparing the test cell sample in step S1 is: infection of 10 with 20. mu.l of virus fluid in 96-well plates⁴And (3) infecting the cell sample to be detected in a carbon dioxide incubator at 37 ℃ for 120min, supplementing a culture medium until the final volume is 200 mu l, and culturing for 48 hours to be detected.

According to the method of the first aspect of the present invention, the GFP fluorescence detection module is selected for detecting the cell sample to be detected by the high content detection instrument in step S2.

More specifically, according to the method of the first aspect of the present invention, the specific parameters of the GFP fluorescence detection module are: the exposure time was 80ms, the exposure intensity was 50%, and the focal height was 11 μm.

More specifically, according to the method of the first aspect of the present invention, in the step S2, when the high content detection instrument is used to detect the cell sample to be detected, the objective lens is a 20 × air lens.

According to the method of the first aspect of the present invention, the cell test sample is placed in a Nunc 96 cell plate.

In a second aspect of the present invention, there is provided a method for high throughput detection of the efficiency of blocking epstein barr virus infection by multiple antisera/antibodies, comprising the steps of:

s1: respectively mixing virus liquid and the antibody diluted according to multiple gradient, and incubating;

s2: adding an equal amount of cell samples to be detected into the mixed solution of the virus solution and the antibody, and culturing;

s3: and (3) detecting the cell sample to be detected by using a high content detection instrument, and calculating the half inhibitory concentration according to the sample dilution and the EB virus infection efficiency.

According to the method of the second aspect of the present invention, the efficiency of blocking EB virus infection is measured by detecting the neutralizing titer of multiple antisera/antibodies against EB virus.

According to the method of the second aspect of the present invention, step S1 is performed by mixing 20 μ l of virus solution with the antibody diluted in the fold gradient, respectively, in a 96-well plate and incubating.

According to the method of the second aspect of the present invention, step S2 is to add 10 to the mixture of virus solution and antibody⁴And infecting the cells to be detected in a carbon dioxide incubator at 37 ℃ for 100-150 min, supplementing a culture medium until the final volume is 200 mu l, and culturing for 48 hours to be detected.

According to the method of the second aspect of the present invention, in the step S3, a GFP fluorescence detection module is selected for detecting the cell sample to be detected by the high content detection instrument.

According to the method of the second aspect of the present invention, the specific parameters of the GFP fluorescence detection module are: the exposure time was 80ms, the exposure intensity was 50%, and the focal height was 11 μm.

According to the method of the second aspect of the invention, in the detection of the neutralizing titer of the multiple antisera, the serum is diluted 8 gradients by 10-fold dilution from the original concentration as an initial gradient, and then incubated with 20. mu.l of virus solution in a 96-well plate, incubated in a 37-degree carbon dioxide incubator for 2 hours, added with 104 infected cells, and then infected in a 37-degree carbon dioxide incubator for 2 hours, supplemented with culture medium to a final volume of 200. mu.l, and cultured for 48 hours, and then the serum is tested.

According to the method of the second aspect of the present invention, in detecting the neutralizing titer of the monoclonal antibody, the antibody is first diluted to 8 gradients by 5-fold dilution with 5mg/ml as an initial gradient, then incubated with 20. mu.l of virus solution in a 96-well plate, incubated in a 37 ℃ carbon dioxide incubator for 2 hours, then added with 104 infected cells, and then supplemented with culture medium to a final volume of 200. mu.l after 2 hours of infection in a 37 ℃ carbon dioxide incubator, and cultured for 48 hours, and then assayed.

According to the method of the second aspect of the present invention, in the step S3, when the high content detection instrument is used to detect the cell sample to be detected, the objective lens is a 20 × air lens.

According to the method of the second aspect of the invention, the cell test sample is placed in a Nunc 96 cell plate.

The invention has the beneficial effects that:

the invention realizes high-throughput detection of EB virus infection efficiency determination, neutralizing antibody neutralization titer determination and clinical serum neutralization titer determination by using a specific detection method and an experimental system. The time required by detection is greatly shortened, the using amount of virus and serum is reduced, the detection efficiency is improved, and a high-flux simple detection method is provided for screening EB virus specific neutralizing antibodies and evaluating the neutralizing titer of vaccine immunity clinical serum.

The invention also verifies the accuracy of the method by the existing flow cytometry detection method of EB virus infection efficiency, and in the same sample and the same batch detection result, the correlation analysis is carried out on the half inhibitory concentration detected by the two methods, and the R of the half inhibitory concentration is²The value is 0.8942, and the analysis results show that the correlation between the two methods is better. But 96 samples can be detected at one time by adopting a high content detection instrument: the flow cytometry requires sample loading of one flow tube and one flow tube, and the sample is transferred to the flow tube, so that time consumption and consumable material waste are caused, and large system errors are easily caused for large-scale detection due to the time consumption of the detection method.

The inventor of the invention finds out after a long time of practice, and also provides specific parameters of the number of cells used in the experimental stage and the detection stage of the method and the volume of the virus used in infection.

Drawings

FIG. 1 shows the infection efficiency of ten batches of EB virus detected by a high content instrument.

FIG. 2 shows the infection efficiency of ten batches of EB virus detected by flow cytometry.

FIG. 3 is a correlation analysis of the infection efficiency of ten batches of EB virus detected by two methods. A is a scatter diagram for detecting infection efficiency of ten batches of EB viruses by two methods; the B diagram is a linear correlation diagram of the two methods.

FIG. 4 shows that the neutralizing antibody 72A1 can be detected by high content instrument to neutralize and block EB virus with different infection efficiency.

FIG. 5 shows the flow cytometry detection of the neutralization blocking efficiency of neutralizing antibody 72A1 on EB virus with different infection efficiency.

FIG. 6 is a correlation analysis of neutralization blocking efficiency of neutralizing antibody 72A1 on EB virus with different infection efficiency detected by two methods.

FIG. 7 shows that 32 clinical sera were tested for neutralization titer against EB virus infection by a high content instrument. Wherein, the A picture is the neutralization titer of 1-8 clinical sera for blocking EB virus infection; wherein the B picture is the neutralization titer of blocking EB virus infection by 9-16 parts of clinical serum; wherein the chart C is the neutralization titer of the 17 th to 24 th clinical serum for blocking EB virus infection; wherein the D picture is the neutralization titer of the 25 th to 32 th clinical serum for blocking EB virus infection.

FIG. 8 shows the flow cytometry detection of 32 clinical sera for blocking the neutralization titer of EB virus infection. Wherein, the A picture is the neutralization titer of 1-8 clinical sera for blocking EB virus infection; wherein the B picture is the neutralization titer of blocking EB virus infection by 9-16 parts of clinical serum; wherein the chart C is the neutralization titer of the 17 th to 24 th clinical serum for blocking EB virus infection; wherein the D picture is the neutralization titer of the 25 th to 32 th clinical serum for blocking EB virus infection.

Figure 9 correlation analysis of two methods to detect neutralization titers of 32 clinical sera blocking EB virus infection. A picture is a scatter diagram for detecting 32 clinical serum blocking EB virus infection titer by two methods; the B picture is a linear correlation graph for detecting 32 clinical sera to block the EB virus infection neutralizing titer.

Detailed Description

The invention will be further illustrated with reference to the following specific examples. These examples are given for illustrative purposes only and do not limit the scope of the invention. The materials and reagents used in the following examples are commercially available without specific reference. The virus production cell line CNE2-EBV and the virus infection cell line AKATA-Neg are preserved in the room, the antibody 72A1 is secreted by HB168 hybridoma cell line preserved in ATCC, and the ATCC number is 72A1(HB168TM), and the serum of nasopharyngeal carcinoma patient is preserved in specimen bank of center for tumor prevention and treatment of Zhongshan university.