Newcastle disease virus antibody magnetic immunochemiluminescence detection kit and application thereof

A chemiluminescence detection and Newcastle disease virus technology, applied in the field of immunological detection, can solve the problems of low sensitivity and accuracy, low specificity of Newcastle disease virus antibody detection, etc., so as to improve the detection throughput and reduce human operation errors. , the effect of high sensitivity

Pending Publication Date: 2020-01-10
HUAZHONG UNIV OF SCI & TECH +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0005] The invention solves the technical problems of low detection specificity, low sensitivity and low accuracy of Newcastle disease virus antibody in the prior art, and provides a Newcastle disease virus antibody magnetic immunochemiluminescent detection kit and its application

Method used

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  • Newcastle disease virus antibody magnetic immunochemiluminescence detection kit and application thereof
  • Newcastle disease virus antibody magnetic immunochemiluminescence detection kit and application thereof
  • Newcastle disease virus antibody magnetic immunochemiluminescence detection kit and application thereof

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preparation example Construction

[0034] The invention provides a method for preparing a magnetically labeled Newcastle disease antigen, comprising the following steps:

[0035] (1) Mix the superparamagnetic microspheres in the acid buffer 4-morpholineethanesulfonic acid (MES, pH5.0~5.5), and add EDC and NHS to activate the carboxyl groups on the magnetic microspheres;

[0036] (2) remove the activator and by-products after the activation reaction in step (1), change the buffer system to the PBS buffer system, and drop into the Newcastle disease antigen in the activated magnetic microspheres described in step (1);

[0037] (3) Perform site blocking on the magnetic-labeled antigen reaction product described in step (2), and store the magnetic-labeled antigen product in the magnetic particle storage buffer after the sealing is completed to obtain the final product.

[0038] Preferably, the superparamagnetic nanoparticles are superparamagnetic ferric oxide nanoparticles or superparamagnetic ferric oxide nanoparticl...

Embodiment 1

[0070] Embodiment 1: Preparation of specific components of the kit

[0071] 1. Preparation method of enzyme-labeled antibody (1)

[0072] A. Alkaline phosphatase activation

[0073] Use DMSO as solvent to prepare 10mM SMCC solution; add 240μL 5mg / mL alkaline phosphatase to 29.5μL 10mM SMCC solution, mix well and let stand at room temperature for 15-30min; The desalting column removes impurities and is concentrated by ultrafiltration. The absorbance of the product is measured at 280nm. The enzyme concentration is: ABS280*dilution factor / 1.0, the enzyme quality: enzyme concentration*product volume (mg / mL), and stored at 2-8°C.

[0074] B. Reduction of antibodies

[0075] Using 0.01M PBS as solvent, prepare 0.1M reducing agent dithiothreitol (DTT); take 1.2mg antibody, add 16μL 0.1M DTT solution to it, mix well and let stand at room temperature for 15min~30min; Mix and concentrate by ultrafiltration, measure the absorbance at 280nm, the protein concentration is: ABS280*dilutio...

Embodiment 2

[0102] Embodiment 2: the formation of kit

[0103] A magnetic immunochemiluminescence detection kit for detecting Newcastle disease virus antibody was set up so that it contained the following components:

[0104] (1) Alkaline phosphatase-labeled secondary antibody

[0105] (2) Magnetically labeled Newcastle disease antigen

[0106] (3) Newcastle disease virus antibody standard solution

[0107] (4) Sample diluent

[0108] (5) Chemiluminescence substrate solution

[0109] (6) Concentrated washing solution

[0110] The enzyme-labeled antibody diluent used in the present invention is pH 7.2-pH 7.4, containing 0.1% BSA (M:V) and 15% bovine serum (V:V), 0.05M Tris buffered saline solution.

[0111] The magnetic label Newcastle disease antigen diluent is pH 7.2-7.4, containing 1% TritionX-100 (M:V) and 0.5% BSA (M:V), 0.05M Tris buffered saline solution.

[0112] Newcastle disease virus antibody standard solution, the concentrations are: 0.3AU / mL, 0.6AU / mL, 1.2AU / mL, 2.4AU / mL...

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Abstract

The invention discloses a Newcastle disease virus antibody magnetic immunochemiluminescence detection kit and application thereof, and belongs to the field of immunological detection. The kit containsan enzyme-labeled anti-IgY antibody, a magnetic-labeled Newcastle disease virus antigen and a luminous substrate solution. The method comprises the following steps of: uniformly mixing a magnetic-labeled Newcastle disease virus antigen with a diluted serum sample and a standard solution; forming a magnetic-labeled Newcastle disease virus antigen and Newcastle disease virus antibody compound; performing magnetic separation, cleaning precipitates, adding an enzyme-labeled antibody into the precipitates; combining the precipitates with an antibody on the compound; carrying out magnetic separation and precipitate cleaning, then adding a luminous substrate solution, enabling the luminous substrate to react with an enzyme on the enzyme-labeled antibody bound to the compound, detecting a luminous value, drawing a standard curve according to the luminous value of the added standard substance solution, and calculating the content of the antibody in the sample to be detected according to the luminous value of the added sample to be detected. The Newcastle disease virus antibody magnetic immunochemiluminescence detection kit has the effects of high sensitivity, high accuracy and high specificity.

Description

technical field [0001] The invention belongs to the field of immunological detection, and in particular relates to a Newcastle disease virus antibody magnetic immunochemiluminescence detection kit and application thereof. Background technique [0002] Newcastle disease virus (NDV), sometimes referred to as avian paramyxovirus type 1 (APMV-1), is a mumps virus member of the Paramyxoviridae family. The genome of NDV is a single-stranded negative-sense RNA with a total length of 15156kb, and the molecular weight is 5.2×10 6 ~5.6×10 6 Among them, six specific structural proteins can be encoded. Virus particles have a double-layer envelope structure, which is derived from the lipids of the outer membrane of the infected body's cells and the glycoprotein of the virus. Virus particles contain a multi-coiled bamboo-like capsid, and the glycoprotein on the surface of the envelope has hemagglutinin and ceramidase activities. [0003] Newcastle disease is commonly known as "chicken...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): G01N33/569G01N33/68G01N33/535G01N21/76
CPCG01N33/56983G01N33/6854G01N33/535G01N21/76G01N2333/125
Inventor 杨海计梦琴杨宝凤张德军成诗琦
Owner HUAZHONG UNIV OF SCI & TECH
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