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Monoclonal antibody and kit for detecting tick-borne encephalitis virus

A monoclonal antibody and encephalitis virus technology, applied in the direction of resistance to vector-borne diseases, measuring devices, instruments, etc., can solve the problems of high false positives and up to 10 months

Active Publication Date: 2016-03-30
MICROBE EPIDEMIC DISEASE INST OF PLA MILITARY MEDICAL ACAD OF SCI
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  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0003] Tick-borne encephalitis virus-specific IgM appears within one week after the illness, reaches a peak in about two weeks, and then declines rapidly, which is suitable for early diagnosis of tick-borne encephalitis virus. The IgM produced by encephalitis virus can exist for up to 10 months. For IgM-positive samples, specific IgG detection for tick-borne encephalitis is required to confirm the pathogen
The specific IgG of dengue virus, yellow fever virus and Japanese encephalitis virus of the genus Flavivirus cross-reacts with tick-borne encephalitis virus, and the method for detecting IgG antibodies has a high false positive rate

Method used

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Examples

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preparation example Construction

[0042] The preparation method of the virus antigen sheet used in following embodiment is as follows:

[0043] 1) Stay 25cm 2 When the BHK21 cells (baby hamster kidney passage cells) in the square bottle grow to about 80%, infect the cells with 1ml of the virus strain, after 1 hour, suck out the adsorption solution, and add the cell maintenance solution (DMEM culture solution containing 2% FBS) , placed in a 37°C incubator for cultivation;

[0044] 2) When 25%-50% of the cells just appear cytopathic, digest the cells according to the method of cell passage, suspend the cells with an appropriate volume of cell culture medium (DMEM medium containing 10% FBS), add 40ul to each antigen well Cell suspension, put into the incubator and continue to cultivate for 8 hours;

[0045]3) Take a container containing PBS solution, slowly put the antigen sheet cultured for 8 hours in the previous step, soak for 1 min, take out the antigen sheet and put it on the workbench to dry;

[0046] 4...

Embodiment 1

[0048] Example 1. Screening and preparation of monoclonal antibodies for detection of tick-borne encephalitis virus

[0049] 1. Preparation and purification of immunogen-

[0050] Tick-borne encephalitis virus strain Senzhang was used to inoculate BHK-21 cells at 37°C, 5% CO 2 Cultivate in an incubator, and when more than 75% of the cells have lesions, centrifuge the culture solution at 4°C and 10,000 rpm for 30 minutes, and take the supernatant; use β-propiolactone (purchased from Seebio, Cat. No. 168-21011) to inactivate the virus, Obtain the immunogen and store it in a -70°C refrigerator for later use.

[0051] 2. Animal immunity

[0052] 1. Measure the total protein content of the immunogen obtained in step 1, and adjust the protein concentration to 0.5 mg / mL with antibody diluent.

[0053] 2. Basic immunization: emulsify the 0.5 mg / mL immunogen solution obtained in step 1 with an equal volume of Freund's complete adjuvant, and then subcutaneously inject 12-week-old fem...

Embodiment 2

[0072] Example 2, Screening and Preparation of Monoclonal Antibodies for Detection of Flaviviruses

[0073] 1. Preparation and purification of immunogen

[0074] Same as Step 1 in Example 1.

[0075] 2. Animal immunity

[0076] Same as Step 2 in Example 1.

[0077] 3. Cell fusion and cloning

[0078] From the second booster immunization, on the 3rd day after each immunization, blood was collected from the mouse orbit to determine the antibody titer, and the mouse with the best serum titer was selected, and splenocytes were taken to fuse with SP2 / 0 myeloma cells; Screen hybridoma cell lines that secrete monoclonal antibodies by limiting dilution method; use indirect non-competitive ELISA to screen out the monoclonal that stably secretes the monoclonal antibody with the highest titer (titer 1:10240) against the immunogen obtained in step 1 The hybridoma cell line, named 2A10, was preserved on August 19, 2013 in the General Microbiology Center of China Committee for Culture C...

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Abstract

The invention discloses monoclonal antibodies and a monoclonal antibody kit for detecting a tick-borne encephalitis virus. The monoclonal antibody 2B5 for detecting the tick-borne encephalitis virus is generated by a hybridoma cell strain 2B5 with a preservation number of CGMCC No. 8059, and the monoclonal antibody 2A10 is generated by a hybridoma cell strain 2A10 with a preservation number of CGMCC No. 8060. Experiments show that the lowest detection limit of a dual-antibody sandwich enzyme-linked immunosorbent assay for detecting the tick-borne encephalitis virus is 4*103PFU / mL, and the dual-antibody sandwich enzyme-linked immunosorbent assay is established by adopting the monoclonal antibody 2B5 as a capturing antibody and the monoclonal antibody 2A10 marked by biotin as a detection antibody; moreover, the monoclonal antibody 2B5 has no crossing reaction with a Japanese encephalitis virus, a dengue virus type-4 and a yellow fever virus, has advantages of specificity, sensitivity and accuracy and can be widely used for detecting the tick-borne encephalitis virus in laboratory research.

Description

technical field [0001] The invention relates to a monoclonal antibody for detecting tick-borne encephalitis virus and a kit thereof. Background technique [0002] Tick-borne encephalitis virus (TBEV), also known as Russian spring-summer encephalitis virus (Russian Spring-Summer encephalitis virus), domestically known as forest encephalitis virus. The virus belongs to the Flaviviridae family and is a single-stranded positive-sense RNA virus. The virus is highly pathogenic and has a high fatality rate. It can be transmitted through aerosols, can be cultured in large quantities, and can be stored for a long time at low temperature. At present, there is no specific treatment drug, and it is listed as a classic biological warfare agent. The northeast, northwest, and southwest of my country are all high-risk areas for tick-borne encephalitis, and Heilongjiang Province in the northeast is the most serious. Due to the non-specific clinical symptoms of tick-borne encephalitis, the ...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): G01N33/577
CPCC07K16/1081G01N33/56983G01N33/577G01N2333/185Y02A50/30
Inventor 李裕昌康晓平杨银辉吴晓燕张雨李靖户义祝庆余张晓松
Owner MICROBE EPIDEMIC DISEASE INST OF PLA MILITARY MEDICAL ACAD OF SCI
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