Immunofluorescence quantitative detection kit for swine reproductive and respiratory syndrome antibodies and use method thereof

A technology for quantitative detection of respiratory syndrome, applied in the field of quantitative detection of porcine reproductive and respiratory syndrome antibodies, can solve the problems of time-consuming, labor-intensive, poor sensitivity, and long time-consuming, so as to improve detection sensitivity, sensitivity, and immunogenicity Good results

Inactive Publication Date: 2018-11-23
ANHUI JIUCHUAN BIOTECH
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0003] At present, the traditional ELISA detection method is mostly used clinically for the detection of PRRSV antibodies, which is time-consuming and has low specificity and sensitivity. Although molecular diagnostic methods have high sensitivity and specificity, they are not suitable for rapid clinical diagnosis and are time-consuming. Laborious; the emerging immunoluminescence method requires expensive equipment and experienced operators, and is not suitable for farm use
[0004] The above methods have disadvantages such as time-consuming, labor-intensive, and poor sensitivity.

Method used

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  • Immunofluorescence quantitative detection kit for swine reproductive and respiratory syndrome antibodies and use method thereof
  • Immunofluorescence quantitative detection kit for swine reproductive and respiratory syndrome antibodies and use method thereof
  • Immunofluorescence quantitative detection kit for swine reproductive and respiratory syndrome antibodies and use method thereof

Examples

Experimental program
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Effect test

Embodiment 1

[0041] An immunofluorescence quantitative detection kit for porcine reproductive and respiratory syndrome antibodies, which is composed of a test paper card, a diluent and a standard substance. Membrane and absorbent pad, the nitrocellulose membrane is coated with recombinant PRRSV capsid protein (N protein) and envelope glycoprotein (GP) as detection lines 5 protein) and goat anti-mouse IgG antibody as a control line.

[0042] The diluent is a 0.05M TB buffer containing 1% BSA and 2% sucrose at pH 7.5; the standard preparation method is as follows: after taking multiple positive sera from pigs infected with PRRSV, mixing them, and putting them in a Protein A tube. After purification by column affinity chromatography, it was concentrated to a final concentration of 1.0 mg / mL, and its titer was 20 U.

[0043] The preparation method of the test paper card comprises the following steps:

[0044] A. Soak the glass fiber membrane in 0.05M TB buffer containing 1.0% Tween-20 and 1%...

Embodiment 2

[0067] The use method of the immunofluorescence quantitative detection kit for porcine reproductive and respiratory syndrome antibody in Example 1, comprising the following steps:

[0068](1) Constructing the standard curve: Dilute the standard product to a series of concentrations with diluent, followed by 0.01mg / mL, 0.05mg / mL, 0.1mg / mL, 0.25mg / mL, 0.5mg / mL, 1.0mg / mL, The corresponding titers are 0.1U, 0.5U, 2U, 5U, 10U, 20U; accurately draw 100 μL of standard sample, add it to the sample pad hole of the test paper card, and use a fluorescence immunochromatography analyzer for detection after 15 minutes . Each concentration standard was tested twice, and the average value of the fluorescence intensity ratio (T / C) of the test line and the quality control line was taken. Taking the titer of the standard product as the abscissa and the average value of T / C as the ordinate, the standard curve obtained is as follows Figure 5 As shown, the linear equation is y=0.3427x+0.1362, wh...

Embodiment 3

[0071] Kit performance indicators and clinical testing

[0072] 1. Specificity and Sensitivity Assays

[0073] Use the kit in Example 1 to measure 100 cases of negative sera, take M+3×SD as the critical value of yin and yang, where M is the average of the titers of 100 cases of negative sera, and SD is the titer of 100 cases of negative sera. The standard deviation of the valence was obtained, and the Cut-off value of the kit was obtained as 0.24U.

[0074] (1) Specificity

[0075] Specificity: The ratio of the number of samples with the detection result less than the Cut-off value in the clinical negative serum to the total number of clinical negative samples.

[0076] Using the detection kit prepared in Example 1 and the kit prepared with a single antigen as the detection line, 30 clinical negative sera were simultaneously measured, and the specific detection results of several kits were compared. The preparation process of the kit prepared by the single antigen as the de...

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Abstract

The invention discloses an immunofluorescence quantitative detection kit for swine reproductive and respiratory syndrome antibodies and a use method thereof. The kit consists of a test paper card, a diluent and a standard product, wherein the test paper card consists of a sample pad, a combination pad, a nitrocellulose membrane and a water suction pad which are sequentially and mutually in lap joint on a PVC plate; a mixture of glycoprotein (GP5 protein) and a recombinant PRRSV capsid protein (N protein) used as a detection line and a goat anti mouse IgG antibody used as a quality control lineare coated on the nitrocellulose membrane; an anti-swine IgG monoclonal antibody marked by rare earth fluorescent microspheres is coated on the combination pad. The detection kit disclosed by the invention uses an indirect method for realizing the quantitative detection of the antibody level in blood serum; detection antigen uses self-made mixed solution of recombinant N protein and recombinant GP5 protein as an antigen pool. Compared with a kit using a single antigen as the detection antigen, the kit has the advantage that the sensitivity is obviously improved. Compared with conventional ELISA, the kit has the advantages of high specificity, high sensitivity, high repeatability and high stability.

Description

technical field [0001] The invention relates to quantitative detection of porcine reproductive and respiratory syndrome antibodies, in particular to an immunofluorescence quantitative detection kit for porcine reproductive and respiratory syndrome antibodies and a method for using the same. Background technique [0002] Porcine reproductive and respiratory syndrome (PRRS) is a severe infectious disease caused by porcine reproductive and respiratory syndrome virus (PRRSV) of the family Arteriviridae. The disease is mainly characterized by reproductive disorders such as premature birth, abortion, and stillbirth of pregnant sows and respiratory diseases of piglets. Due to the cyanosis and purple ear of some affected pigs, it is also called "blue ear disease". The disease spreads very fast and has a high fatality rate, which brings huge economic losses to animal husbandry production. Early detection and early diagnosis are of great significance to the prevention and treatment o...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): G01N33/68G01N33/577G01N33/558G01N33/533G01N21/64
CPCG01N21/6408G01N21/6428G01N33/533G01N33/558G01N33/577G01N33/6854G01N33/6893G01N2800/26
Inventor 许高涛凡玉芳单雪芹蒋敏之王亚男刘家炉徐文俊赵雨高耀辉夏兵兵
Owner ANHUI JIUCHUAN BIOTECH
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