Competitive ELISA qualitative and quantitative detection method of oil adjuvant vaccine

A quantitative detection method and quantitative detection technology are applied in the field of foot-and-mouth disease synthetic peptide vaccine detection, which can solve the problems of no universal and accurate method for quantitative detection and can not respond well to effective antigen content, etc. The effect of detection sensitivity

Inactive Publication Date: 2017-05-24
SHANGHAI SHEN LIAN BIOMEDICAL CORP
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  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

Can not reflect the effective antigen content in synthetic peptide vaccine against foot-and-mouth disease
[0004] At present, there is no univer...

Method used

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  • Competitive ELISA qualitative and quantitative detection method of oil adjuvant vaccine
  • Competitive ELISA qualitative and quantitative detection method of oil adjuvant vaccine
  • Competitive ELISA qualitative and quantitative detection method of oil adjuvant vaccine

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Experimental program
Comparison scheme
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Embodiment 1

[0034] This embodiment provides a competitive ELISA qualitative and quantitative detection method for oil adjuvant vaccines, specifically using the following steps:

[0035] 1. Antigen coating: 3 μg / mL artificially synthesized FMD antigen, 100 μL per well, was immobilized onto the ELISA plate;

[0036] 2. Dilute the antibody: Dilute the antibody with known titer to a certain ratio, and the antibody titer after dilution is 1:250000;

[0037] 3. Dilution of the antigen to be tested (the antigen to be tested is a finished product of a synthetic peptide antigen, and the antigen concentration of the antigen to be tested is 39.25 μg / mL as measured by HPLC method) and the standard antigen: on the serum dilution plate, dilute the antigen to be tested with the diluent The test antigen was diluted 1:100, and the standard antigen was diluted according to the diluted concentrations of 1000ng / mL, 500ng / mL, 200ng / mL, 100ng / mL, 50ng / mL, 10ng / mL, and 5ng / mL;

[0038] 4. Antigen-antibody reac...

Embodiment 2

[0045] This embodiment provides a competitive ELISA qualitative and quantitative detection method for oil adjuvant vaccines, specifically using the following steps:

[0046] 1. Antigen coating: 3 μg / mL artificially synthesized FMD antigen, 100 μL per well, was immobilized onto the ELISA plate;

[0047] 2. Dilute the antibody: Dilute the antibody with known titer to a certain ratio, and the antibody titer after dilution is 1:1100000;

[0048] 3. Antigen to be tested (the antigen to be tested is the aqueous phase sample after vaccine demulsification, and the demulsification efficiency is 89.2%, after purification, adopting HPLC method to record its antigen concentration is 148.83ng / mL) and standard antigen dilution : On the serum dilution plate, dilute the antigen to be tested with the diluent at 1:100, and dilute the known standard antigen. The dilution concentrations are 1000ng / mL, 500ng / mL, 200ng / mL, 100ng / mL, 50ng / mL , 10ng / mL, 5ng / mL, 1ng / mL;

[0049] 4. Antigen-antibody ...

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Abstract

The invention provides a competitive ELISA qualitative and quantitative detection method of an oil adjuvant vaccine. The method comprises the following steps of coating an antigen, diluting an antibody, diluting the antigen, drawing an antigen standard curve and quantitatively detecting the to-be-detected antigen. The method comprises the specific steps: firstly enabling the to-be-detected antigen to react with the antibody with known concentration to completely neutralizing the antibody and the antigen; and then enabling the neutralized solution to react with the antigen adsorbed in an ELISA plate in a solid-phase way to determine the concentration of the to-be-detected antigen. The standard curve slope obtained by the invention is able to be greater than that obtained through an indirect competition method, thereby greatly improving the detection sensitivity; the detection method disclosed by the invention is extensive in detection, and capable of detecting the synthetic peptide antigen finished product, semi-finished product and vaccine antigen; and an aqueous-phase sample obtained after vaccine demulsification can be determined without performing the purification treatment; the spent time is short in comparison with other detection method; and the lowest detection limit of the antigen sample according to the invention can achieve 1ng/mL-1.5ng/mL.

Description

technical field [0001] The invention relates to the technical field of detection of foot-and-mouth disease synthetic peptide vaccines, in particular to a competitive ELISA qualitative and quantitative detection method for oil adjuvant vaccines. Background technique [0002] Foot-and-mouth disease synthetic peptide vaccine is a protective short peptide synthesized artificially according to the amino acid sequence of natural protein. Existing quantitative determination methods usually include Lowry method, BCA method, HPLC and other methods. The existing quantitative methods are mainly aimed at the purified antigen. If it is used for antigen detection after vaccine demulsification, due to the complex composition of the aqueous phase of the vaccine, it usually takes a lot of time to purify the sample to achieve the detection purpose, and many conventional The detection sensitivity of the method is limited and cannot meet the detection requirements. [0003] Enzyme-linked immu...

Claims

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Application Information

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IPC IPC(8): G01N33/68
CPCG01N33/68
Inventor 刘自立马贵军姬明放
Owner SHANGHAI SHEN LIAN BIOMEDICAL CORP
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