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Colloidal gold test paper for detecting IBV, and preparation method thereof

A technology of colloidal gold test paper and detection line, which is applied in measuring devices, immunoassays, instruments, etc., to achieve the effects of good specificity, clear and easy to distinguish results, and simple and fast operation

Pending Publication Date: 2020-09-25
ZHENGZHOU UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

At present, many ELISA methods for IBV infection detection have been established at home and abroad; however, studies have found that most of these detection methods are based on antibody detection, which has certain limitations; therefore, it is urgent to develop a method for detecting IBV antigens. method, in order to achieve a more intuitive, fast and accurate detection of virus infection

Method used

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  • Colloidal gold test paper for detecting IBV, and preparation method thereof
  • Colloidal gold test paper for detecting IBV, and preparation method thereof
  • Colloidal gold test paper for detecting IBV, and preparation method thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0030] Example 1 Immunogen selection and preparation

[0031] 1. Selection of immunogen

[0032] Based on a large number of studies, it was found that, firstly, among the proteins encoded by the avian infectious bronchitis genome, the nucleocapsid protein, namely N protein, is highly conserved; secondly, N protein is an important immunogenic protein that induces the body's immune response.

[0033] Based on the above two points, research and screening of the N protein has become the preferred target protein for the detection of IBV.

[0034] 2. N protein preparation of immunogen

[0035] (1) Clone the N protein gene sequence of avian infectious bronchitis virus and connect it into the expression vector pGEX-6p-1 to obtain the recombinant expression vector pGEX-6p-1-N;

[0036] (2) The recombinant expression vector pGEX-6p-1-N was treated with CaCl 2 competent cells E. coli In BL21(DE3), positive recombinant Escherichia coli BL21(DE3) was obtained;

[0037] (3) Inoculate ...

Embodiment 2

[0040] Example 2 Preparation and Identification of Monoclonal Antibody

[0041] 1. Animal immunity

[0042] (1) Add the immunogen N protein prepared in Example 1 into Freund's complete adjuvant (used for the first immunization) or Freund's incomplete adjuvant, and emulsify to make the immune antigen;

[0043] (2) Immunize 4-8 week-old female BALB / c mice (3 mice / group) by multi-point subcutaneous injection on the back, with an immune dose of 50 μg / mouse;

[0044] (3) After the first immunization, BALB / c mice were boosted with the same method and dose after emulsification with Freund's incomplete adjuvant and immune antigen every two weeks;

[0045] (4) After four immunizations, 3 to 4 days before cell fusion, BALB / c mice were boosted with an immunogen without adjuvant by tail vein injection, and the immunization dose was 100 μg / mouse;

[0046] (5) One week after the last booster immunization, the tail-cut blood was collected from each mouse, and then the potency and sensitivi...

Embodiment 3

[0063] Example 3 Purification of Antibodies

[0064] Antibody purification was carried out by caprylic acid-ammonium sulfate method, and the operation method was as follows:

[0065] (1) Take out the frozen monoclonal antibody ascites or antiserum, and centrifuge at 6000 r / min for 30 minutes to obtain the supernatant, which is diluted 5 times with sodium acetate buffer (0.06mol / L pH4.8).

[0066] (2) Adjust the pH to 4.5 with NaOH (5mol / L), stir slowly at room temperature for 0.5 h, and add n-octanoic acid to a final concentration of 25 μL / mL.

[0067] (3) Centrifuge at 6000r / min for 30min at 4°C and keep the supernatant.

[0068] (4) Filter the obtained supernatant with medium-speed filter paper, add 10×PBS buffer solution (pH7.4), the volume is 1 / 10, mix well and adjust the pH to 7.4.

[0069] (5) Add solid ammonium sulfate to the mixture at 4°C, the addition ratio is 0.2778 g / mL, and can be added several times, and the addition is completed within 30 minutes.

[0070] (6...

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Abstract

The invention discloses colloidal gold test paper for detecting IBV. The colloidal gold test paper comprises a support plate, a coating film, a sample pad, a gold-labeled antibody pad, water absorbingpaper, a detection line and a quality control line, and is characterized in that the gold-labeled antibody pad is labeled with an anti-IBV IgG antibody, the detection line is coated with an anti-IBVN protein monoclonal antibody, and the quality control line is coated with a goat anti-mouse IgG antibody. The invention also discloses an index method of the colloidal gold test paper for detecting IBV. The test paper prepared by the invention is based on the principle of a double-antibody sandwich ELISA antigen detection method, is used for detecting IBV antigens so as to intuitively, quickly, simply and conveniently detect virus infection and achieve good specificity and the purpose of accurate and quick detection, can provide technical support for immune monitoring and epidemiological monitoring research of avian infectious bronchitis, and has important significance in prevention and control of avian diseases.

Description

technical field [0001] The invention relates to animal virus detection technology, in particular to a colloidal gold test paper for detecting IBV, and also relates to a preparation method of the colloidal gold test paper. Background technique [0002] Infectious bronchitis (IB) is an acute and highly contagious infectious disease of chickens caused by infectious bronchitis virus (IBV), and it is one of the major infectious diseases that seriously endanger the chicken industry. . IBV is a highly variable coronavirus with numerous serotypes and lacks cross-protection among different serotypes. The spread of IBV has caused severe economic losses to the global poultry industry. The evolution of the N gene of IBV is the most conservative, and the N protein is an important immunogenic protein that induces the body's immune response. The preparation of monoclonal antibodies against N protein and the establishment of a rapid diagnostic method for IBV colloidal gold based on monoc...

Claims

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Application Information

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IPC IPC(8): G01N33/569G01N33/558G01N33/543
CPCG01N33/56983G01N33/558G01N33/54306G01N2333/165G01N2469/10
Inventor 王爱萍周景明李佳楠祁艳华刘红亮陈玉梅梁超
Owner ZHENGZHOU UNIV
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