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Recombination VTT and method for detecting vaccinia virus neutralizing antibody by using same

A vaccinia virus and antibody technology, applied in the field of immunology, can solve the problems of strong subjectivity in result analysis, long detection cycle, and high detection cost, and achieve the effects of easy promotion, short detection cycle, and low detection cost

Inactive Publication Date: 2012-03-28
NAT INST FOR FOOD & DRUG CONTROL
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

The main disadvantages of this method are long detection period (4-7 days); low detection throughput; highly subjective analysis of results; need to analyze in 6-well cell culture plate, so the sample volume is large
In 2008, Prof. Maria used a 96-well plate to analyze the titer of neutralizing antibodies to reduce the sample requirement, but manual interpretation of the data was still required after magnification of 12.5 times (Borges MB, Kato SE, Damaso CR, Moussatche N, da Silva Freire M, Lambert Passos SR, et al. Accuracy and repeatability of a micro plaque reduction neutralization test for vaccinia antibodies. Biologicals 2008Mar; 36(2): 105-10)
However, the main disadvantage of this method is that the infection inhibition rate of MVA-gfp cells is detected by flow cytometry, and the detection cost is high (Cosma A, Buhler S, Nagaraj R, Staib C, Hammarin AL, Wahren B, et al.Neutralization assay Using a modified vaccine virus Ankara vector expressing the green fluorescent protein is a high-throughput method to monitor the humoral immune response against vaccine virus. Clinical and diagnostic laboratory immunology 2004Mar;11(2):406-10)

Method used

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  • Recombination VTT and method for detecting vaccinia virus neutralizing antibody by using same
  • Recombination VTT and method for detecting vaccinia virus neutralizing antibody by using same
  • Recombination VTT and method for detecting vaccinia virus neutralizing antibody by using same

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0050] Example 1: Construction of recombinant vaccinia virus Tiantan strain comprising firefly luciferase gene

[0051] The construction process of the vaccinia virus Tiantan strain containing the firefly luciferase gene is as follows: First, construct the recombinant plasmid pSCluc containing the firefly luciferase gene using the pSC59 plasmid as a framework; use lipofect2000 liposome technology to transfect the vaccinia virus Tiantan strain (vaccine strain) Infected chicken embryo fibroblasts (CEF), through the X-Gal gene blue-white selection process to obtain recombinant replicative vaccinia virus Tiantan strain rTV-Fluc.

[0052] 1. Construction of recombinant vaccinia virus shuttle plasmid (pSCluc) comprising luciferase gene

[0053] 1.1 Luciferase gene PCR amplification

[0054] Use PrimeSTAR high-fidelity enzyme (TAKARA, Cat: DR010A) to contain the plasmid pLUCF (John T. Schiller, National Cancer Institute) containing the luciferase gene, and the plasmid containing the...

Embodiment 2

[0144] Example 2: Detection of vaccinia virus neutralizing antibody

[0145] The vaccinia virus neutralizing antibody detection method of the present invention is established based on the vaccinia virus neutralizing antibody in the sample inhibiting the expression of the recombinant virus firefly luciferase reporter gene.

[0146] This example illustrates the principle of the detection method of the present invention as follows: Vero cells are infected with the rTV-Fluc virus constructed in Example 1 to express firefly luciferase, which catalyzes the luminescent reaction of the substrate luciferin. Since the luminescence value is proportional to the amount of rTV-Fluc virus that infects Vero cells, the luminescence value that can be read by a microplate luminometer reflects the viral infection dose (see figure 1 A). The vaccinia virus-specific neutralizing antibody contained in the serum to be tested can block the cell receptor binding site on the surface of the rTV-Fluc viru...

Embodiment 3

[0168] Embodiment 3 Comparison of the inventive method and the plaque inhibition method

[0169] Plaque inhibition is the most traditional neutralizing antibody detection method and is considered the "gold standard". Therefore, the method of the present invention is compared with the plaque inhibition method to determine the specificity and sensitivity of the method.

[0170] In this example, the positive control sample used was the high-titer rabbit serum immunized with the Tiantan strain of vaccinia virus provided by Beijing Tiantan Biological Products Co., Ltd., labeled as HCS.

[0171] 1. Standardization of Plaque Inhibition Assay

[0172] 1) Serum was inactivated at 56°C for 60 minutes;

[0173] 2) using 3% DMEM medium to dilute the serum in a 5-fold gradient at 1:20, 1:100, 1:500, 1:2500, 1:12500;

[0174] 3) Mix 400 μl of vaccinia virus (rTV-Fluc) at three dilutions of 17, 33 and 66 pfu / ml with 400 μl of serum at each dilution, and incubate at 37°C for 1 hour;

[01...

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Abstract

The invention relates to recombination VTT (Vaccinia Tian Tan), and also relates to a vaccinia virus neutralizing antibody detection method and the purpose of the recombination VTT, wherein, the recombination VTT comprises luciferase genes and is used for detecting a vaccinia virus neutralizing antibody.

Description

Technical field: [0001] The invention relates to the field of immunology, more specifically to the detection of vaccinia virus neutralizing antibodies. Background technique [0002] Vaccinia virus is the most familiar member of the Orthopoxvirus genus in the Poxviridae family, and it is also the most structurally complex virus known so far. The vaccinia virus Tiantan strain (Vaccinia Tian Tan, VTT) has been used as a vaccine strain for smallpox virus disease for a long time in a large number of people in my country. It has the characteristics of relatively weak virulence, safe use and clear biological properties. Ideal strains for viral live vaccines and eukaryotic expression vectors (McCurdy LH, Rutigliano JA, Johnson TR, Chen M, Graham BS. Modified vaccine virus Ankara immunization protects against lethal challenge with recombinant vaccine virus expressing murine interleukin-4. Journal of virology 2004Nov; 78(22): 12471-9; Dai K, Liu Y, Liu M, Xu J, Huang W, Huang X, et al....

Claims

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Application Information

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IPC IPC(8): C12N7/01G01N33/569C12R1/93
Inventor 王佑春刘强
Owner NAT INST FOR FOOD & DRUG CONTROL
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