EV71 virus neutralization epitope detection kit or reagent and preparation method thereof

A technology of EV71 and reagent kit, which is applied in the direction of measuring devices, instruments, scientific instruments, etc., can solve problems such as laborious, time-consuming, and unsatisfactory, and achieve the effects of wide applicability, guaranteed specificity, and reduced use

Active Publication Date: 2009-12-09
SINOVAC BIOTECH
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

However, these methods are time-consuming and labor-intensive, and cannot meet t...

Method used

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  • EV71 virus neutralization epitope detection kit or reagent and preparation method thereof
  • EV71 virus neutralization epitope detection kit or reagent and preparation method thereof
  • EV71 virus neutralization epitope detection kit or reagent and preparation method thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0027] Example 1: Preparation, purity and activity identification of EV71 purified solution

[0028] Inoculate Vero cells with C4 genotype EV71, harvest the culture fluid after culturing, purify EV71 virus after inactivation, and prepare EV71 purified fluid after sterilizing and filtering to obtain immune antigen. (See: Bishop NE, Hago DL, Borovec SV et al.Rapid and efficient purification of hepatitis Avirus from cell culture (rapid and efficient purification of hepatitis Avirus from cell culture). J VirolMethods, 1994, 47: 203-216)

[0029] For the EV71 purified solution prepared above, the purity of its antigen was identified by HPLC method, and the result was greater than 95%, and the purified solution was placed at 2-8°C. Among them, the HPLC detection method includes the following process: use PBS to equilibrate the column for 40 minutes to the baseline level and then load the sample; use a sample needle to draw 100ul EV71 purified liquid, and inject it into the sample in...

Embodiment 2

[0033] Example 2: Preparation, titer detection and purification of anti-EV71 polyclonal antibody

[0034] The EV71 purified solution obtained in Example 1 was mixed and emulsified in equal volumes with complete Freund's adjuvant for immunization of New Zealand white rabbits, thereby preparing anti-EV71 serum (containing anti-EV71 polyclonal antibody).

[0035] Use the indirect ELISA method to detect the antibody titer against the three genotypes A, B, and C of the anti-EV71 serum, and use the micro-neutralization assay to detect the neutralization test of the anti-EV71 polyclonal antibody against the three genotypes A, B, and C of the EV71 virus .

[0036] Taking the C4 genotype as an example, the indirect method titer detection of the anti-EV71 polyclonal antibody is carried out as follows: the EV71 purified solution obtained in Example 1 is diluted 100 times and coated on a microtiter plate (100ul / well), and coated with Overnight; wash the plate 3 times, block the microtite...

Embodiment 3

[0049] Embodiment 3: Preparation of anti-EV71 monoclonal antibody

[0050] BALB / c mice were immunized three times with EV71 purified solution, spleen cells from BALB / c mice with the highest titer by indirect ELISA were fused with SP2 / 0 myeloma cells in proportion, and fusion hybrid tumor cells were cultured on HAT medium . Subcloning was performed three times, and the indirect ELISA titer of each cell well was detected for each subcloning, and the wells with an OD value greater than 0.4 were cloned. Indirect detection method: Enzyme-labeled plates were coated with purified EV71 genotypes A, B, and C at a ratio of 1:100, cell culture supernatant was added, and goat anti-mouse IgG-HRP (Jackson Immunoresearch Company) was used as an enzyme-labeled reagent.

[0051] Finally, the inventor of the present application obtained an ideal hybridoma cell line through the above screening, and the applicant obtained the ideal hybridoma cell line on March 20, 2009 at the General Microorgani...

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Abstract

The invention relates to an EV71 virus neutralization epitope detection kit or a reagent and a preparation method thereof. Particularly, the invention performs quantitative detection on EV71 virus antigen by adopting an HD6 monoclonal antibody with spectral activity. The kit or the reagent has the advantages of easy operation, high sensitivity and the like.

Description

technical field [0001] The invention relates to a virus antigen content detection kit and a preparation method thereof, in particular to an ELISA kit for quantifying the EV71 virus antigen content and a preparation method thereof. Background technique [0002] Hand, foot and mouth disease is a global infectious disease, and there are reports of the prevalence of this disease in most parts of the world. The disease was first reported in New Zealand in 1957, Coxsackie virus was isolated in 1958, and the name of hand, foot and mouth disease was proposed in 1959. The early pathogen of HFMD was mainly Cox A16 type, but in 1969, the EV71 virus was isolated and confirmed for the first time in fecal samples of infants with central nervous system diseases in the United States. Since then, EV71 infection and Cox A16 infection have appeared alternately, becoming the main pathogen of HFMD. In the mid-1970s, Bulgaria and Hungary successively broke out EV71 epidemics with the central ne...

Claims

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Application Information

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IPC IPC(8): G01N33/577G01N33/569
Inventor 蔡芳高强张小梅尹卫东
Owner SINOVAC BIOTECH
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