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Method for rapidly detecting neutralizing antibody of virus and kit therefor

A virus and antibody technology, applied in biological testing, measuring devices, material inspection products, etc., can solve the problems of difficult screening requirements, heavy workload, and inability to accurately detect neutralizing antibodies

Inactive Publication Date: 2010-02-10
XIAMEN UNIV +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, in practical application, the former method has a long experimental cycle and a large workload, and there are also subjective errors in manual judgment, which is difficult to meet the needs of fast and high-throughput screening; the latter method is more sensitive and can be used for automated analysis. The judgment is more objective, but its specificity depends on the preparation of the antigen and cannot be used to accurately detect neutralizing antibodies

Method used

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  • Method for rapidly detecting neutralizing antibody of virus and kit therefor
  • Method for rapidly detecting neutralizing antibody of virus and kit therefor
  • Method for rapidly detecting neutralizing antibody of virus and kit therefor

Examples

Experimental program
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Effect test

Embodiment 1

[0138] Example 1. Cultivation of enterovirus

[0139] The enterovirus strain used as an example in this embodiment is JS06-52-3 (preserved by the National Research Center for Infectious Disease Diagnostic Reagents and Vaccine Engineering Technology), and a nearly full-length 7312bp sequence (Genbank No: FJ 600325) was obtained by RT-PCR. ), belongs to the EV71C4 subtype, has a nucleotide sequence homology of 97.7% and an amino acid sequence homology of 97.6% with the EU703814 virus strain. Other enteroviruses can also be used.

[0140] The present invention infects cells in RD cells (ATCC, CCL-136 TM ) (ATCC is the supplier) produces EV71 virus.

[0141] experimental method:

[0142] The RD cells were cultured in a 10cm cell culture dish with a MEM medium (10% FBS, 2mM L-glutamine, 0.1mM MEM Non-Essential Amino Acids and 1% penicillin-streptomycin), and the confluence rate was about 80%.

[0143] After 10 hours, the cells in each dish were replaced with serum-free MEM medium (additi...

Embodiment 2

[0145] Example 2. Enterovirus infection of cells

[0146] The harvested enterovirus was infected with RD cells, and the infection of RD cells was detected. In this embodiment, the EV71 virus infection is taken as an example, and the detection of other enterovirus infections may also be included.

[0147] experimental method:

[0148] RD cells are cultured in 96-well cell culture plates, about 2×10 4 / Well, the medium is MEM medium (add 10% FBS, 2mM L-glutamine, 0.1mM MEM Non-EssentialAmino Acids and 1% penicillin-streptomycin), the confluence rate is about 80%. After culturing for 10 hours, 50 μL of EV71 virus (diluted by 0, 10, 100, 1000, 10000 times) was added to each well. After culturing for 14 hours, aspirate the medium in each well, add 100μL of fixative (PBS solution containing 0.2% glutaraldehyde) to each well, and let stand for 1h at room temperature in the dark; after 1h, aspirate the fixative. Add 100μL of 1% TritonX-100 solution to permeabilize the cells and let stand ...

Embodiment 3

[0151] Example 3. Using Elispot, a spot detection instrument, to detect cell infection by enterovirus

[0152] In the present invention, the EV71 virus is taken as an example. The EV71 virus is infected with RD cells. After infection, the detection by the enzyme-linked immunospot method can make the virus-infected cells produce a signal that is different from the uninfected cells. This signal can be observed under visible light, and it does not require excitation by a special light source (such as a laser, a mercury lamp with a limited wavelength range, etc.), and is suitable for detection with a spot detection instrument Elispot. Currently, the traditional TCID 50 The method is to observe the diseased cells with naked eyes, which is laborious and time-consuming, and is not suitable for high-throughput detection experiments; and lack of objectivity, there are certain subjective errors, which easily affect the reproducibility of experimental results. Elispot, a spot detection inst...

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Abstract

The invention relates to a method for rapidly detecting neutralizing antibody of enterovirus with high efficiency and a kit therefor. The method is suitable for high-throughput detection of the neutralizing antibody of the enterovirus, and is characterized by combining enzyme linked immune spot assay with a spot detecting instrument to detect cells infected by the enterovirus. The invention also discloses the usage of the method in the aspects such as neutralizing enterovirus monoclonal antibody, neutralizing titer for detecting the enterovirus monoclonal antibody, etc.

Description

Technical field [0001] The invention relates to the field of virus antibody detection. More specifically, the present invention relates to methods and kits for detecting neutralizing antibodies to human enteroviruses through enzyme-linked immunospot color development and automatic scanning equipment. Background technique [0002] Human Enterovirus (EV) includes poliovirus (Poliovirus), Coxsackievirus (Coxsackievirus), Echo virus (entericcytopathogenic human orphan virus, ECHO) and new enterovirus (Enterovirus 68-71 and Type 73-102), which caused a variety of diseases from the respiratory system to the central nervous system to bring great threats and serious harm to human health, especially the hand, foot and mouth disease, morbidity and death caused by the new enterovirus EV71 The rate has been increasing year by year in recent years. Therefore, the development of rapid diagnostic reagents and safe and effective preventive or therapeutic vaccines is of great significance. The...

Claims

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Application Information

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IPC IPC(8): G01N33/569G01N33/577G01N33/543G01N33/52
Inventor 程通蔡毅君何德磊陈毅歆张军夏宁邵
Owner XIAMEN UNIV
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