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Identification detecting method for Cox A16 and EV71 viral antigens

A virus antigen, differential detection technology, applied in the field of biology, can solve the problems of difficult diagnosis and delay of optimal treatment time in severe cases

Inactive Publication Date: 2016-07-13
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 detection and diagnosis of CoxA16 and EV71, the main pathogens of hand, foot and mouth disease, currently mainly rely on viral nucleic acid detection and virus isolation and culture, which are not yet available in primary hospitals and CDCs, and some severe cases do not have typical siblings clinically. Therefore, it is difficult to diagnose severe cases in the early stage of the outbreak, and they are often diagnosed as pneumonia, which delays the best treatment time

Method used

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  • Identification detecting method for Cox A16 and EV71 viral antigens
  • Identification detecting method for Cox A16 and EV71 viral antigens
  • Identification detecting method for Cox A16 and EV71 viral antigens

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0054] The phosphate buffer used below is 0.01mol / L phosphate buffer, which is prepared by adding 2.9 g of disodium hydrogen phosphate, 0.2 g of potassium dihydrogen phosphate and 8 g of sodium chloride to 1000 mL of water for injection.

[0055] The identification and detection of CoxA16 virus antigen, through the following steps:

[0056] Step 1. Prepare horseradish peroxidase-labeled CoxA16 antibody or EV71 antibody:

[0057] ①Take a commercially available protein A / G affinity chromatography column, and after equilibrating at room temperature for 30 minutes, equilibrate the column with an equilibration solution (phosphate buffer) 5 times the volume of the column;

[0058] ② Mix the anti-CoxA16 serum or anti-EV71 serum with the equilibrium solution (phosphate buffer) at a volume ratio of 1:1, and then load the sample, and use 10 times the column volume of the equilibrium solution (phosphate buffer) to elute the impurity protein, leaving target protein;

[0059] ③Use 10 tim...

Embodiment 2

[0082] The identification and detection of EV71 virus antigen, through the following steps:

[0083] Same as Example 1, only the CoxA16 antigen positive control sample in step 4 is replaced with EV71 antigen positive control sample, and the horseradish peroxidase-labeled CoxA16 antibody (CoxA16-IgG-HRP) in step 5 is replaced with horseradish peroxide Enzyme-conjugated EV71 antibody (EV71-IgG-HRP).

[0084] The test results are as follows:

[0085]

[0086] And make the following judgments:

[0087] Positive control average A value 0.983-negative control average A value 0.05=0.933, greater than 0.4, the test is established;

[0088] Cutoff value = average A value of negative control 0.05×2.1=0.105;

[0089] If the A value of sample 1 suspected to contain CoxA16 and / or EV71 virus antigen is 0.553≥Cutoff value, the sample 1 suspected to contain CoxA16 and / or EV71 virus antigen is determined to be positive; it is determined to be EV71 infection or identified as EV71 virus; ...

Embodiment 1 and 2

[0092] Advantages of Embodiments 1 and 2:

[0093] (1) From the analysis of the detection time, compared with the current detection method, the detection time of the detection method of the present invention is shortened from 3 to 5 days of the cell culture separation method to 3 to 5 hours, and the detection range is expanded from the live virus of the cell culture method To inactivate the virus, the detection safety is greatly increased, and it has greater practicability regardless of the requirements for operators and the environment. Compared with nucleic acid detection methods, it is easy to operate, and the requirements for equipment, venues, and samples are significantly lower than nucleic acid detection methods, and it can also be popularized and used in grassroots units.

[0094] (2) One test panel can detect or identify at least 44 samples and two viruses of CoxA16 and EV71 at the same time, which is convenient, fast, sensitive and specific.

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Abstract

The invention provides an identification detecting method for Cox A16 and EV71 viral antigens.The method comprises the steps that the to-be-detected COX A16 viral antigen or / and the to-be-detected EV71 viral antigen, positive antigen controls and negative antigen controls are added into detection holes of an ELISA plate respectively; a horse radish peroxidase-labelled Cox A16 antibody and / or EV71 antibody, a TMB color developing solution A and a TMB color developing solution B are added into other holes of the ELISA plate; after a stop solution is added into all the holes of the ELISA plate, an absorbance (A) value is detected on an ELISA instrument, and obtained detection results are judged according to the conditions.The method is good in specificity, high in sensitivity, convenient and rapid to operate and suitable for the two viral antigens Cox A16 and EV71.According to the method, the detection time can be shortened to several hours from 3-5 days of a general lesion cell culture method, and the infected antigen of a patient is determined in a short time; in addition, detection can be achieved in primary hospitals and the centers for disease control and prevention, symptomatic treatment and prevention and control over the epidemic situation can be performed in time, and the detection method for the Cox A16 and EV71 viral antigens is good in specificity, high in sensitivity and convenient and rapid to operate.

Description

technical field [0001] The invention relates to a method for identifying and detecting CoxA16 and EV71 virus antigens, belonging to the technical field of biology. Background technique [0002] Hand-foot-mouth disease (HFMD) is a long-term viral infectious disease that is generally prevalent in infants and preschool children. Usually, the clinical symptoms of most patients are not very serious. It is a mild cold symptom, accompanied by herpes on the palms, soles of the feet, and oral mucosa. The course of the disease is usually limited to 7 to 10 days. However, reports in recent years have shown that the infection of this type of virus can be accompanied by many severe manifestations, mainly severe lesions of the nervous system and respiratory system, and may cause the death of the patient due to the failure of the respiratory system. What's more serious is that such severe lesions are more common in infants and young children. The male to female sex ratio of the reported ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): G01N33/569
CPCG01N33/56983G01N2333/085
Inventor 谢忠平李华龙润乡刘正玲杨婷罗芳宇谢天宏宋霞杨蓉岳磊
Owner INST OF MEDICAL BIOLOGY CHINESE ACAD OF MEDICAL SCI
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