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Enterovirus 71 type latex agglutination detection kit, preparation and application

A detection kit and enterovirus technology, applied in the field of pathogen biology detection, can solve the problems of high price, long detection time and high cost, and achieve the effects of low price, simple operation and high biological safety.

Inactive Publication Date: 2015-03-11
绍兴市疾病预防控制中心
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

All of the above methods use ELISA to detect EV71 antigen, which is suitable for clinical diagnosis. Due to the long detection time and the need for professional equipment, the price is expensive, the operation steps are cumbersome and time-consuming, and need a plate washer, row gun, enzyme labeling, etc. Expensive equipment, not suitable for promotion and use in ordinary township medical institutions
Hunan Kangrun Pharmaceutical Co., Ltd. has disclosed the patent of "Enterovirus 71 colloidal gold detection test strip and its preparation method and application" (application number 201210151666.8), which uses colloidal gold labeling method to detect EV71 antibody. Simple and fast, but high cost, not conducive to large-scale seroepidemiological survey results

Method used

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  • Enterovirus 71 type latex agglutination detection kit, preparation and application
  • Enterovirus 71 type latex agglutination detection kit, preparation and application
  • Enterovirus 71 type latex agglutination detection kit, preparation and application

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0042] Preparation of EV71 monoclonal antibody

[0043] The key raw materials of this product are EV71 monoclonal antibody and VP1 purified protein, both of which are developed and prepared at the Wuhan Institute of Virology, Chinese Academy of Sciences.

[0044] Preparation of EV71 monoclonal antibody: Express and purify EV71 VP1 protein in prokaryotic, immunize BALB / c mice respectively, carry out cell fusion with conventional hybridoma cell technology, screen positive hybridoma cells by ELISA method and measure titer, screen out the anti-EV71 Hybridoma cells with high antibody secretion and high affinity are ready to be put into production as original cell lines, and stored in liquid nitrogen. Take out the cell seeds screened by the above method from the -173°C liquid nitrogen tank, melt them as soon as possible, cultivate at 37°C to obtain the first-grade seed liquid, and cultivate the second-grade seed liquid under the same conditions, and then transfer the seed liquid to ...

Embodiment 2

[0046] Production of EV71 VP1 purified protein, that is, cloning the VP1 gene of the EV71-C4 standard strain into the prokaryotic expression vector pET-28a, prokaryotic expression of the EV71 virus VP1 protein in BL21 Escherichia coli, and Ni column for affinity purification: as Figure 1-Figure 3 As shown, select the positive colony on the plate and inoculate it on the Kana plate, shake culture at 37°C until OD600 is approximately equal to 0.6, add IPTG to a final concentration of 0.4mM, continue to cultivate for 3h, take 1ml of bacterial liquid sample under sterile conditions, 10000r / min centrifuged for 1 min, and the precipitate was subjected to SDS-PAGE electrophoresis according to the conventional method to observe the protein expression. Take the bacterial culture induced for 3 hours and centrifuge at 8000r / min for 10min, collect the bacteria, add 1 / 10 of the original culture volume of buffer A to resuspend, and apply strong ultrasonic treatment for about 8 times, 10s / ti...

Embodiment 3

[0048] The establishment of a rapid two-way latex agglutination detection kit for enterovirus 71 antigen antibody, such as Figure 4-Figure 7 Shown:

[0049] 1. Coupling the purified protein or monoclonal antibody to latex particles by carbodiimide. The specific steps are: a. Take 125 μL of 10% carboxylated latex and put it into a centrifuge tube; b. Wash 3 times with carbonate buffer (pH9.6); c. Wash 3 times with phosphate buffer (pH4.5); d. Add Water-soluble carbodiimide (EDC) reacted in phosphate buffer (pH4.5) for 3 hours at room temperature; e. Washed 3 times with boric acid buffer (pH8.0); f. Added VP1 protein antigen to the latex suspension or mixed Monoclonal antibody, felt on a shaker for 6 hours; g. The latex antibody detection reagent was suspended in the storage solution.

[0050] 2. Following the principle of fixing other conditions and changing one of the conditions, explore the optimal coupling protein amount, optimal coupling time, optimal latex concentration...

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Abstract

The invention relates to an enterovirus 71 type latex agglutination detection kit, preparation and application. The kit comprises the following substances: (1) a carboxylated latex reagent sensitizing a prokaryotic expression enterovirus 71 type VP1 specific antigen; (2) a carboxylated latex reagent sensitizing three strains of monoclonal mixed antibodies aiming at enterovirus 71 type VP1; (3) enterovirus 71 type positive-negative standard serum, an inactivated virus solution and PBS; and (4) a toothpick or a plastic cement rod which contains a glass slide platform for using the sentization latex to perform aggregation reaction and is used to uniformly mix latex and to-be reacted serum. The kit can detect EV71 antigen in samples of different sources, and overcomes the disadvantages that the sensitized antigen is less in amount and unstable, sensitized protein is easy to fall off, and the like when protein sensitizes common latex. The detection method is applicable to enterovirus 71 type serum epidemiological investigation, clinic assistant diagnosis, regulation and control on EV71 virus replication, anti-virus therapy medicine screening and other scientific research fields.

Description

technical field [0001] The invention belongs to the technical field of pathogen biological detection, and in particular relates to a rapid two-way latex agglutination detection kit for enterovirus 71 antigen antibody and its preparation method and application. Background technique [0002] Hand, foot and mouth disease is a global infectious disease and has become one of the infectious diseases that seriously threaten children's health in recent years. It is characterized by fever and rashes or herpes on the hands, feet, and throat. A small number of patients may be complicated by aseptic meningitis, encephalitis, acute flaccid paralysis, respiratory tract infection, and myocarditis. Individual children with severe disease progress rapidly and are prone to death. The fatality rate of children with severe disease is 10%-25%. [0003] HFMD is mainly caused by Coxsackievirus (Cox, A group 16, 4, 5, 7, 9, 10, B group 2, 5, 13), Echo virus (Echo) and EV71 Caused, of which EV71 a...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): G01N33/577G01N33/569
CPCG01N33/56983G01N33/577
Inventor 钦博屠春雨傅利军何婷婷
Owner 绍兴市疾病预防控制中心
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