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Enzyme-linked immunosorbent assay method for enterovirus 71-specific antibody

An enzyme-linked immunosorbent assay and enterovirus technology, which is used in the detection of enterovirus type 71 antibodies by enzyme-linked immunosorbent assay, and the qualitative or quantitative detection of enterovirus type 71-specific antibodies, which can solve the problem of poor sensitivity and specificity. , the test cycle becomes longer, etc., to reduce the effect of false positives

Inactive Publication Date: 2012-07-04
ZHEJIANG PUKANG BIOTECH
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

The most commonly used method is still the neutralization experiment, but the sensitivity and specificity of this method are poor
Moreover, this method requires cell culture during the detection process, which leads to a longer test cycle, and it often takes about one month to get the result, which has certain limitations in the actual application process.

Method used

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  • Enzyme-linked immunosorbent assay method for enterovirus 71-specific antibody
  • Enzyme-linked immunosorbent assay method for enterovirus 71-specific antibody
  • Enzyme-linked immunosorbent assay method for enterovirus 71-specific antibody

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0034] Detection of EV71 Antibody Titer in Mouse Serum by Immobilized and Purified EV71 Virus Antigen

[0035] 1) Dilute the purified EV71 virus antigen to 2 μg / ml with carbonate buffer (pH9.6), add 100 μl / well to the wells of the microplate, incubate overnight at 4°C, wash with PBS Wash the plate 3 times;

[0036] 2) Add 200 microliters / well of blocking solution to the wells of the microtiter plate, incubate at 37°C for 1 hour, and wash the plate 3 times with PBS washing solution;

[0037] 3) Add 100 microliters / well of 5% sucrose to the wells of the microplate plate, incubate at 37°C for 0.5-1 hour, pat dry, and store at 4°C until use;

[0038] 4) Dilute the serum to be tested 10 times with 0.9% normal saline, and then use PBS containing 1% BSA to make a series of dilutions of the serum: 1:20, 1:40, 1:80, 1:160, 1: 320, 1:640, 1:1280, 1:2560, according to the amount of 100 μl / well, add to the well of the pre-prepared microplate plate, incubate at 37°C for 1 hour, wash the ...

Embodiment 2

[0056] Immobilized and purified EV71 virus antigen to detect the positive rate of EV71 antibody in mouse serum

[0057] 1) Dilute the purified EV71 virus antigen to 3 micrograms / ml with carbonate buffer (pH9.6), add 100 microliters / well to the wells of the microplate, incubate overnight at 4°C, wash with PBS Wash the plate 3 times;

[0058] 2) Add 200 microliters / well of blocking solution to the wells of the microtiter plate, incubate at 37°C for 1 hour, and wash the plate 3 times with PBS washing solution;

[0059] 3) Add 100 microliters / well of 5% sucrose to the wells of the microplate, incubate at 37°C for 0.5-1 hour, pat dry, and store at 4°C for later use

[0060] 4) Dilute 75 mouse positive sera respectively by 100 times, add 100 microliters / well to the wells of the pre-prepared microplate plate, incubate at 37°C for 1 hour, wash the plate with PBS washing solution 4 times, and set up at the same time 2 negative control wells, and 1 blank well;

[0061] 5) Add an enzy...

Embodiment 3

[0071] Detection of EV71 Antibody Positive Rate in Mouse Serum by Immobilized Virus-Specific Antigen Epitope Peptide

[0072] 1) Dilute the purified EV71 virus antigen to 3 micrograms / ml with carbonate buffer (pH9.6), add 100 microliters / well to the wells of the microplate, incubate overnight at 4°C, wash with PBS Wash the plate 3 times;

[0073] 2) Add 200 microliters / well of blocking solution to the wells of the microtiter plate, incubate at 37°C for 1 hour, and wash the plate 3 times with PBS washing solution;

[0074] 3) Add 100 microliters / well of 5% sucrose to the wells of the microplate, incubate at 37°C for 0.5-1 hour, pat dry, and store at 4°C for later use

[0075] 4) Dilute 75 sera of mice inoculated with EV71 virus 50 times respectively, add 100 microliters / well to the wells of the pre-prepared microplate plate, incubate at 37°C for 1 hour, wash the plate 4 times with PBS washing solution , and set up 2 negative control wells and 1 blank well at the same time;

...

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PUM

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Abstract

The invention relates to the field of biotechnology, in particular to an enzyme-linked immunosorbent assay method for an enterovirus 71 (EV71 for short)-specific antibody in serum. Through a method of indirect enzyme-linked immunosorbent assay (ELISA), a prepared EV71 viral antigen is immobilized on an ELISA plate, and the antibody is detected through specific adsorption of antigen and antibody. Since the coated antigen comprises a specific antigen epitope for the EV71 virus, the method can be used for detecting specific antibody for the EV71 virus in the serum, and false positive of serum detection is reduced. The enzyme-linked immunosorbent assay method for the enterovirus 71-specific antibody is suitable for detecting a large batch of animal or human serum samples, and is suitable for large-scale serological and epidemiological investigation.

Description

technical field [0001] The invention belongs to the field of biotechnology, in particular to the qualitative or quantitative detection of enterovirus 71 (Enterovirus 71, referred to as EV71) specific antibody, and the detection method of enterovirus 71 antibody by enzyme-linked immunosorbent assay. Background technique [0002] Infectious diseases caused by enteroviruses mostly occur in children under 5 years old, and can cause herpes on hands, feet, mouth and other parts. The main pathogens causing HFMD are Enterovirus 71 and Coxsackievirus A16. A large number of epidemiological studies have shown that enterovirus 71 infection mostly occurs in infants under the age of 5, and its infection may cause a variety of serious diseases related to the nervous system, including fatal encephalitis, aseptic meningitis and acute paralysis. Therefore, the specific diagnosis of EV71 virus has very important clinical significance. [0003] The EV71 virus is a member of the Enterovirus g...

Claims

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Application Information

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IPC IPC(8): G01N33/68G01N33/543G01N33/535
Inventor 高孟罗永能周康凤姜云水毛子旭王一虎唐彩华朱莲高丽美庄昉成毛子安毛江森
Owner ZHEJIANG PUKANG BIOTECH
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