Detection method of EV71 virus antigen

A technology of EV71 and virus antigen, which is applied in the field of biology, can solve the problems of delaying the best treatment time and difficulty in making a clear diagnosis of severe cases, so as to facilitate early diagnosis and control of the epidemic, shorten the detection time, and reduce the number of steps.

Active Publication Date: 2010-02-24
INST OF MEDICAL BIOLOGY CHINESE ACAD OF MEDICAL SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0005] The diagnosis of EV71 mainly relies on virus nucleic acid detection and virus isolation and culture, which are currently not available in primary hospitals and CDCs. In addition, some severe cases do not have typical clin

Method used

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  • Detection method of EV71 virus antigen
  • Detection method of EV71 virus antigen
  • Detection method of EV71 virus antigen

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0050] One-step, separation and purification of EV71 virus antibody

[0051] 1) Take a commercially available protein A / G affinity chromatography column, place it at room temperature to equilibrate for 30 minutes, and equilibrate the column with an equilibration solution 5 times the volume of the column;

[0052] 2) Mix the commercially available anti-EV71 serum with the conventional balance solution at a volume ratio of 1:1 and load the sample, and use 10 times the column volume of the balance solution to elute the impurity protein, leaving the target protein;

[0053] 3) The target protein was eluted from the affinity column with a conventional eluent of 10 times the column volume, the elution peak was collected, and dialyzed overnight with conventional phosphate buffered saline (PBS) to obtain the EV71 virus antibody;

[0054] 4) Use the lowry method to measure the protein content, and store it below -20°C after labeling;

[0055] Two steps, preparation of biotin-labeled E...

Embodiment 2

[0101] One-step, separation and purification of EV71 virus antibody

[0102] With embodiment 1;

[0103] Two steps, preparation of biotin-labeled EV71 antibody

[0104] 1) According to the ratio of biotin: EV71 virus antibody = 1: 20 molar ratio, commercially available biotin was mixed with the one-step EV71 virus antibody, and placed at 5° C. for 2 hours;

[0105] 2) Dialyze the mixture of the above step 1) with phosphate buffered solution (PBS) at 2°C overnight, add an equal volume of glycerol and mix well to obtain biotin-labeled EV71 antibody, and store it below -20°C for future use;

[0106] 3) Dilute the biotin-labeled EV71 antibody in step 2) according to the dilution ratio of 1:100, 1:200, 1:400...1:5000, and determine their optimum by conventional checkerboard test. Using the dilution, the dilution of the biotin-labeled EV71 antibody is 1:500-3000;

[0107] Three steps, the EV71 virus antigen to be tested is obtained through the following methods:

[0108] 1) Coll...

Embodiment 3

[0127] One-step, separation and purification of EV71 virus antibody

[0128] With embodiment 1;

[0129] Two steps, preparation of biotin-labeled EV71 antibody

[0130] 1) According to the ratio of biotin: EV71 virus antibody = 1:5 molar ratio, commercially available biotin was mixed with the one-step EV71 virus antibody, and placed at 8° C. for 1 hour;

[0131] 2) Dialyze the mixture of the above step 1) with phosphate buffered solution PBS at 8°C overnight, add an equal volume of glycerol and mix well to obtain biotin-labeled EV71 antibody, and store it below -20°C for future use;

[0132] 3) Dilute the biotin-labeled EV71 antibody in step 2) according to the dilution ratio of 1:100, 1:200, 1:400...1:5000, and determine their optimum by conventional checkerboard test. Using the dilution, the dilution of the biotin-labeled EV71 antibody is 1:500-3000;

[0133] Three steps, the EV71 virus antigen to be tested is obtained through the following methods:

[0134] 1) Collect t...

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Abstract

The invention provides a detection method of EV71 virus antigen, which comprises that: respectively adding EV 71 virus antigen, antigen positive control and antigen negative control into holes of an elisa plate, successively adding biotin-labeled EV71 antigen of which the dilution is 1:500 to 3000, horse radish peroxidase-labeled streptavidin of which the dilution is 1:2000 to 10000, and TMB color development liquid A and B into holes of the elisa plate, placing the elisa plate on an enzyme immunoassay instrument after adding a stop solution to each hole of the elisa plate, and detecting the absorbance A value after zeroing through a blank hole under the wave length of 450 nm to obtain the detection result. The detection can shorten the 3 to 5 days of the common cell cultivation pathologic-change method into a plurality of hours, can confirm the antigen infected by the patient in a short time, complete the detection in a basal-level hospital and a control center, and timely carry out symptomatic treatment and prevent and control the epidemic situation, and is the detection method of EV71 virus antigen of good particularity, high sensitivity, convenience and high speed.

Description

technical field [0001] The invention relates to a detection method of enterovirus 71 virus antigen, which is the main pathogenic virus of hand, foot and mouth disease, and belongs to the technical field of biology. Background technique [0002] Hand-foot-mouth disease (HFMD) is mainly an epidemic disease caused by enterovirus 71 (hereinafter referred to as EV71) or coxsackie virus A16 (COX A16) infection, and the clinical manifestations of infection are diverse. , from asymptomatic infection, common hand, foot and mouth disease, and angina, to severe cases such as neurogenic pulmonary edema, respiratory failure, and central nervous system infection, and some severe cases progress rapidly and even die. Hand, foot and mouth disease is widely prevalent in my country and the world, and the patients are mainly preschool children, especially children under 3 years old with a high incidence rate. Compared with hand, foot and mouth disease caused by other enteroviruses, the proport...

Claims

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Application Information

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IPC IPC(8): G01N33/569G01N33/543G01N33/52G01N1/34C07K14/085C07K1/14
Inventor 李琦涵谢忠平龙润乡董承红杨蓉白惠珠李华崔萍芳刘龙丁
Owner INST OF MEDICAL BIOLOGY CHINESE ACAD OF MEDICAL SCI
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