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Detection method for measuring titer of antigens of enterovirus71 inactivated vaccine

A technology of inactivated vaccines and enteroviruses, applied in the biological field, can solve the problems of inconvenient research and development of EV71 inactivated vaccine production process, inability to correctly detect the antigen content of inactivated vaccines, and inability to monitor effectively, instantly and correctly, and achieve Shortened detection time, good specificity, and high sensitivity

Inactive Publication Date: 2010-11-10
ZHEJIANG PUKANG BIOTECH
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, the cycle of this method is long, and it often takes several days to get the results, and because CPE can only detect infectious viruses, it cannot correctly detect the antigen content in inactivated vaccines, such as vacuolar viruses, inactivated viruses, etc. Therefore, effective, real-time and correct monitoring cannot be carried out in the production process of the vaccine, which brings some inconvenience and bottlenecks to the research and development of the production process of the EV71 inactivated vaccine

Method used

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Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0027] Example 1: Preparation and purification of rabbit anti-enterovirus type 71 virus serum antibody

[0028] 1) Dilute the concentration of the virus to a LogTCID50 / ml (infection titer) of 8.0, draw 2 ml of the virus and inoculate it in New Zealand big-eared white rabbits, and inject it subcutaneously in 4 parts;

[0029] 2) After 14 days, booster immunization with the same dose;

[0030] 3) Blood collection at the 4th week after the initial injection of immunization;

[0031] 4) Separate the serum and store it below -20°C until use;

[0032] 5) Take a commercially available Protein A affinity antibody purification column and place it at room temperature for equilibrium for 30 minutes, equilibrate the column with an equilibrium solution (20 millimolar phosphate buffer) of five times the volume of the column, and mix the column in 4) After the serum is thawed, the sample is passed through the column, and the impurity protein is eluted with 10 times the column volume of the...

Embodiment 2

[0034] The pre-coating of embodiment 2 enzyme plate

[0035] 1) Coating of ELISA plate: Dilute the rabbit anti-EV71 serum antibody to 10 micrograms / ml with carbonate buffer (pH9.0), add 100 microliters / well to the surface of the ELISA plate, and put Incubate at 4°C for more than 20 hours, wash the plate 3 times with PBS washing solution;

[0036] 2) Blocking of ELISA plate: Add blocking solution at 200 microliters / well, incubate at 37°C for 1 hour, wash the plate with PBS washing solution 3 times, dry in the air, and store in refrigerator at 4°C for later use.

Embodiment 3

[0037] The mensuration of embodiment 3 enterovirus 71 type virus antigens

[0038] 1) First, serially dilute the antigen sample to be tested with PBS diluent at different dilutions: 1:90, 1:270, 1:810, 1:2430, 1:7290, 1:14580, 1:29160, 1: The specific dilution method of 58320 is as follows: Take 100 microliters of the EV71 virus antigen sample to be tested and add 900 microliters of PBS diluent, mix well to form a 1:10 sample; take 100 microliters of 1:10 sample and add 800 microliters of PBS diluent, mix Evenly make a 1:90 sample, and so on for dilution; use a micropipette to add the diluted EV71 virus antigen sample to the well of the pre-coated enzyme plate sequentially starting from the low dilution (1:90), 100 microliters / Add two wells for each diluted sample, and at the same time set up two negative wells in the other wells of the ELISA plate, and leave two wells as blank zero wells, put them in a shaker at 37°C, shake at 180 rpm for 1 hour , wash the plate 4 times wit...

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Abstract

The invention relates to the technical field of biology, in particular to an enzyme-linked immunological detection method for measuring the titer of antigens of an enterovirus71 inactivated vaccine. The method adopts goat-anti-mouse immunoglobulin G antibody labeled with horse radish peroxidase as an antibody so as to be beneficial to amplifying developing signals and improving sensitivity of detection; generally, the detected titer of the virus is 3.7-4.3 LogTCID50 / ml virus antigen, and when detection is carried out on the EV71 inactivated vaccine, a series of complex and time-consuming work, such as cell culture and the like, do not need to be carried out, thus shortening the detection time to 4 hours from the general days; and when being adopted for enzyme-linked immunological reaction, the method has the advantages of low background, and the A value of a negative control hole is generally less than 0.08. The detection method established by the invention is an enzyme-linked immunological detection method for measuring the titer of antigens of an enterovirus71 inactivated vaccine, which has good specificity, high sensitivity, good repeatability, simpleness and convenience.

Description

technical field [0001] The invention belongs to the field of biotechnology, in particular to an enzyme-linked immunoassay method for sensitive and specific determination of enterovirus 71 inactivated vaccine antigen titer for enterovirus 71 (EV71) Background technique [0002] In recent years, hand, foot and mouth disease has ravaged most parts of our country and brought great harm to people. A large number of epidemiological studies have shown that enterovirus 71 is one of the main pathogens causing HFMD. EV71 belongs to the Picornaviridae family, a member of the Enterovirus genus. The infection of this virus mostly occurs in infants under 5 years old, and can cause herpes on the hands, feet, mouth and other parts. Individual patients can cause complications such as myocarditis, pulmonary edema, and aseptic meningoencephalitis. morbidity and mortality rates. Therefore, it is of great significance to develop a safe and effective EV71 vaccine to prevent and control the spr...

Claims

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Application Information

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IPC IPC(8): G01N33/577G01N33/569G01N33/543G01N21/31G01N33/531
Inventor 高孟周康凤姜云水罗永能唐彩华高丽美朱莲王平毛子安毛江森
Owner ZHEJIANG PUKANG BIOTECH
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