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Quantitative determination kit for neutralizing antibodies of virus and application thereof

A quantitative detection and kit technology, applied in the biological field, can solve the problems of increasing the difficulty of neutralizing antibodies, inconspicuous cell lesions, laborious and other problems, and achieve the effect of shortening the detection time

Inactive Publication Date: 2014-09-24
JIANGSU PROVINCIAL CENT FOR DISEASE PREVENTION & CONTROL
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

The current method for detecting SFTS virus neutralizing antibodies is mainly by observing cytopathic changes or immunofluorescence, which is time-consuming, laborious, and has poor sensitivity, and it cannot detect a large number of samples at the same time
In addition, the cytopathic changes are not obvious after SFTS virus infection of cells (such as Vero), which increases the difficulty of detecting neutralizing antibodies

Method used

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  • Quantitative determination kit for neutralizing antibodies of virus and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0024] Example 1: Preparation of NP protein monoclonal antibody and polyclonal antibody

[0025] Amplify the SFTS virus NP gene by RT-PCR, insert the reading frame into the multiple cloning site of the expression plasmid pQE30 (Qiagen, Germany), obtain the prokaryotic expression plasmid pQE30-SFTS-NP of the NP gene, and transform E.Coli M15 bacterial strain (Qiagen , Germany), induced expression of 6His-NP recombinant protein. After the NP recombinant protein was purified and quantified, BALB / c mice were immunized according to conventional methods to prepare monoclonal and polyclonal antibodies against SFTS virus NP protein.

Embodiment 2

[0026] Embodiment 2:. Determination of virus titer

[0027] Tissue cell half infectious dose (TCID50) assay was used. The virus solution frozen at -70°C was diluted 100 times. Take the 1:100-fold diluted virus liquid, and perform 1 / 2 log dilution on a 96-well cell culture plate, that is, 10 －2 、10 －2.5 、10 －3 、10 －3.5 ...10 －7 . Each well contains 100 μl of virus solution, and each dilution is repeated in 4 wells.

[0028] Take the cells in the logarithmic growth phase, digest with LEDTA-trypsin, disperse the cells with cell culture medium, add virus dilution to 10mL, count the cells with a cell counting plate, and dilute the cells to 1.5×10 5 cells / mL for later use. Add 100 μl cells (1.5x10 4 cells / well). It is best to use one column of the cell culture plate as the normal cell (CC) control. Cultivate at 37°C, 5% CO2 incubator for 20-24h.

[0029] The cells were washed twice with PBS (pH 7.2), 100 μl of 80% ice acetone was added to each well, and the cells were fi...

Embodiment 3

[0031] Example 3: Detection of neutralizing antibodies

[0032] Serum neutralizing antibody titers were tested on 96-well cell culture plates. Add 50 μl of MEM cell culture medium (without bovine serum) to each well of a 96-well cell plate. In the first 11 wells (A1~A11) in the first row, add 40 μl of virus diluent to make it 90 μl / well, add 10 μl of the serum to be tested in the first row A1~A11, and the serial dilution (A~H) is 1:10, 1:20, 1:40...1:1280. Dilute virus to 100TCID 50 / 50mL. Add 50 μl virus working solution to each well except CC wells (E12, F12, G12, H12). Wells A12, B12, C12 and D12 were used as virus inoculated control (VC) wells. Add 50 μl of MEM cell culture medium (without bovine serum) to CC wells. In addition, select 1 column of wells to verify the titer of the virus working solution, add 200TCID50 / 100mL to each well, and make serial doubling dilutions to make it 100TCID50, 50, 25, 12, 6,...0.7, and then add 50μl to each well The volume of the MEM...

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Abstract

The invention belongs to the field of a biotechnology and particularly relates to a quantitative determination kit for neutralizing antibodies of virus and application of the quantitative determination kit to detection of the neutralizing antibodies of virus of severe fever with thrombocytopenia syndrome. Virus NP proteins of the severe fever with thrombocytopenia syndrome are subjected to prokaryotic expression to prepare a rat source monoclonal antibody or polyclonal antibody aiming at NP proteins. The tissue cell half infection amount (TCIF50) of determined viruses is measured by detecting the NP proteins through an enzyme linked immunosorbent assay. A specimen to be detected and the equal amount of virus liquid are mixed and inoculated with cells. The monoclonal antibody or the polyclonal antibody of the NP proteins is monoclonal antibody; a specimen neutralizing antibody valence is detected by a double-antibody enzyme linked immunosorbent assay. The quantitative determination kit can be applied to the aspects of clinical immune effect evaluation of severe fever with thrombocytopenia syndrome vaccines, blood serum epidemiologic studies of the severe fever with thrombocytopenia syndrome of crowds or faunas, in-vitro valence determination and estimation of the manually-prepared virus-neutralizing antibodies of the severe fever with thrombocytopenia syndrome. The method provided by the invention has the characteristics of sensitivity, rapidness, specificity, high flux and the like.

Description

technical field [0001] The invention belongs to the field of biotechnology, and in particular relates to a virus neutralizing antibody quantitative detection kit and its application in the quantitative detection of fever with thrombocytopenia syndrome virus neutralizing antibody. Background technique [0002] Fever with thrombocytopenia syndrome (Severe fever with thrombocytopenia syndrome, SFTS) is a newly discovered hemorrhagic disease caused by bunya virus in my country in recent years, and the pathogen is called SFTS virus. The disease is mainly distributed in rural areas in mountainous and hilly areas, and is sporadic, and the crowd is generally susceptible. The clinical manifestations are fever, with body temperature above 38°C, accompanied by fatigue, nausea, and vomiting. Some cases have headache, muscle aches, and diarrhea. The vast majority of patients have a good prognosis, but some cases are critically ill, with disturbance of consciousness, skin ecchymosis, gas...

Claims

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Application Information

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IPC IPC(8): G01N33/96G01N33/569
CPCG01N33/56983G01N33/94
Inventor 祁贤汤奋扬焦永军鲍倡俊崔仑标李志锋周明浩
Owner JIANGSU PROVINCIAL CENT FOR DISEASE PREVENTION & CONTROL
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