Method for detecting content of D antigen in poliovirus type III

A poliovirus and poliovirus technology, applied in the field of antigen detection, can solve the problem that the poliovirus detection system cannot effectively distinguish

Inactive Publication Date: 2017-01-04
SINOVAC BIOTECH
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  • Abstract
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  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0004] The existing poliovirus detection systems on the market are generally aimed at the linear epitope of the poliovirus antigen. Since this linear epitope does not involve the spatial conformation of the virus, it is widely present in the D antigen and the C antigen. Poliovirus detection systems cannot effectively differentiate between the two

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  • Method for detecting content of D antigen in poliovirus type III
  • Method for detecting content of D antigen in poliovirus type III
  • Method for detecting content of D antigen in poliovirus type III

Examples

Experimental program
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Effect test

Embodiment 1

[0022] Example 1 Preparation of Type III Poliovirus Polyclonal Antibody

[0023] The inactivated poliovirus type Ⅲ was emulsified with Freund's adjuvant, and immunized cattle and New Zealand white rabbits. Bovine immunization dose: 10ml of inactivated type III poliovirus mixed with 10ml of complete Freund's adjuvant for the first immunization by emulsification, followed by emulsification of 5ml with incomplete Freund's adjuvant of equal volume. Rabbit immunization dosage: 2ml of inactivated poliovirus type Ⅲ and 2ml of Freund's complete adjuvant mixed in equal volume for the first immunization, followed by emulsification immunization with 1ml and equal volume of Freund's incomplete adjuvant. Immune site: multiple points under the skin on the back of the neck. Immunization schedule: 0, 2, 4, 6 weeks. Blood was collected at 7 weeks, and serum was separated.

[0024] Indirect ELISA was used to detect the antibody titers of polyantiserum against polio Ⅰ, Ⅱ, Ⅲ, and to examine th...

Embodiment 2

[0030] Example 2 Establishment and linearity verification of a detection method for type III poliovirus D antigen

[0031] 1. Coating: Dilute the poliovirus type III bovine polyclonal antibody prepared in Example 1 at 1:4000 and mix evenly, add 100 μl / well to the microtiter plate, incubate overnight at 4°C; wash the plate 3 times, and pat dry .

[0032] 2. Blocking: prepare PBST-20 blocking solution containing 1% skimmed milk powder, 200 μl per well, and incubate at 37° C. for 120 minutes.

[0033] 3. D-antigen internal reference substance dilution: Use the national polio D-antigen standard substance to calibrate the D-antigen reference substance D-antigen content, and dilute the reference substance to make the concentrations 3, 1.5, 0.75, 0.375, and 0.1875 DU / ml.

[0034] 4. Adding samples: add the diluted standard substance to the microplate, 100 μl per well, add multiple wells for each dilution, and make the test sample dilution control well at the same time; incubate at...

Embodiment 3

[0043] Example 3 Precision verification of poliovirus type III D antigen detection system

[0044] In this example, the test sample type III poliovirus stock solution (Beijing Kexing Biological Products Co., Ltd.) was made in duplicate, one of which was not subjected to any treatment as a control (D antigen), named sample A; the other was prepared in a 56°C water bath 30min (H antigen), named sample B; mix sample A and sample B according to the volume ratio of 1:1, that is, the mixed sample of D antigen and H antigen in equal proportion, named sample C. This embodiment is operated by a single person, and for the test sample A, three independent operations are respectively carried out for detection. Operation steps are with embodiment 2. According to the OD value of statistical detection, the corresponding D antigen content is calculated according to the linear equation of the standard, and the average value, standard deviation and coefficient of variation are calculated.

[...

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Abstract

The invention provides a method for detecting the content of D antigen in poliovirus type III and belongs to the technical field of antigen detection. Through matching of a poliovirus type III specific polyclonal antibody and a monoclonal antibody, the content of D antigen in poliovirus type III can be detected in a highly targeted mode without detecting C antigen. The method is easy to operate, the detection result can truly reflect the content of effective antigen in poliovirus, a basis can be well provided for follow-up poliomyelitis vaccine immunogenicity testing usage and the final concentration of a vaccine product, and the quality of poliomyelitis vaccine can be improved finally. The method has a high economic value and a high market application value.

Description

technical field [0001] The invention relates to the technical field of antigen detection, in particular to a method for detecting the content of type III poliovirus D antigen. Background technique [0002] Poliovirus (hereinafter referred to as poliovirus) is an acute infectious RNA virus that seriously endangers children's health. Most of the patients are children aged 1 to 6. The main symptoms are fever, general discomfort, severe limb pain, and flaccid paralysis with irregular distribution and varying severity, commonly known as polio. At present, there is no effective treatment for children with symptoms, and vaccination is usually used to protect children from the disease. In the polio vaccine, the D antigen is the main component of the vaccine. It has strong immunogenicity and can effectively stimulate the immune response of the human body. Therefore, the preparation and detection level of the D antigen directly affect the protective effect of the vaccine. Currently,...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): G01N33/577
CPCG01N33/56983G01N2333/105
Inventor 戈小琴张颖程会欣陈慧芬蔡芳高强尹卫东
Owner SINOVAC BIOTECH
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