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IFA neutralizing antibody detection method of PRRSV

An antibody detection and cell technology, applied in the biological field, can solve the problems of inability to evaluate PRRSV-specific antibody response and development

Pending Publication Date: 2021-02-12
XINXIANG UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Therefore, such systems cannot assess PRRSV-specific antibody responses against other PRRSV envelope proteins or nonstructural proteins (nssps)
It should be noted here that a system using multiple PRRSV antigens may not be developed due to the enormous effort required to express and purify multiple ELISA plate-coating antigens

Method used

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  • IFA neutralizing antibody detection method of PRRSV
  • IFA neutralizing antibody detection method of PRRSV
  • IFA neutralizing antibody detection method of PRRSV

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0027] Embodiment 1PRRSV separation and identification

[0028] Rinse the suspected PRRSV disease tissue (lymph, liver, spleen, lung) with PBS, cut it into pieces, add 1mL PBS to 1g of the tissue sample, freeze and thaw three times, and make the disease material grinding liquid (used as PRRSV virus liquid), Use the RNA extraction kit to extract the RNA of PRRSV from the above disease materials, and then reverse transcribe the RNA into cDNA.

[0029] Use DNAMAN software to design PRRSV target gene primers, and the specific primer sequences are as follows:

[0030] PRRSV-F: 5'-GGCCAGCCAGTCAATCAG-3'; SEQ ID NO.1;

[0031] PRRSV-R: 5'-GGCAAACTAAACTCCACAGTG-3'; SEQ ID NO.2.

[0032] Using the designed primers, PCR amplification was carried out using the above-mentioned extracted PRRSV lymph, liver, spleen, and lung cDNA as templates. The system was 25 μL, and the reaction conditions were: pre-denaturation at 98°C for 5 minutes; denaturation at 95°C for 30 seconds, annealing at 55...

Embodiment 2

[0033] Example 2 establishes a stable IFA neutralizing antibody detection method

[0034] Marc145 cells were digested and centrifuged to prepare 1.0×10 5 Cells / ml cell suspension, 100 μl per well spread on 96-well plate, placed at 37°C 5% CO 2 Cultivate in the incubator for 12 hours. After the cells are completely adhered to the wall, incubate an equal volume of PRRSV virus solution (50 μl) and clinical serum at 37°C for 1 hour, and then inoculate them on Marc145 cells in a 96-well plate. After culturing for 48 hours, take out after the cells are full. 96-well plate, fixed with pre-cooled absolute ethanol. Add PRRSV standard positive serum, incubate at 37°C for 1h, then add goat anti-pig IgG-FITC, incubate at 37°C for 1h, observe under a fluorescent microscope, negative for specific green fluorescence, otherwise positive. The serum identified as positive was serially diluted, and the above operation was repeated to obtain the antibody titer.

[0035] (1) Plating: Marc145 ce...

Embodiment 3I

[0040] Embodiment 3IFA reaction conditions

[0041] 1) TCID of PRRSV 50 determination

[0042] (1) Spread the Marc145 cell suspension on a 96-well plate, 100 μL per well, so that the cell volume reaches 2-3×10 5 cells / mL, cultivated for 12 hours until the cells were completely attached to the wall;

[0043] (2) In the penicillin bottle or the centrifuge tube, the PRRSV virus liquid is diluted 10 times continuously, starting from 10 -1 -10 -10 ;

[0044] (3) Inoculate the diluted virus onto a 96-well plate in which the cells grow into a single layer, inoculate a vertical row of 8 wells for each dilution, and inoculate 100 μL in each well;

[0045] (4) The remaining two vertical rows are not exposed to poison, and a normal cell control is established (100 μL of maintenance solution per well, and the maintenance solution is a DMEM medium with a fetal bovine serum content of 2%);

[0046] (5) After culturing for 48 hours, the cells were taken out and fixed, and placed at -20...

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Abstract

The invention discloses an IFA neutralizing antibody detection method for PRRSV, and belongs to the technical field of molecular biological detection. The invention discloses an IFA neutralizing antibody detection method of PRRSV. The method comprises the following steps: inoculating an isopyknic PRRSV virus solution and clinical serum onto Marc145 cells, immobilizing, adding a primary antibody, adding a secondary antibody, and observing a result. According to the method, target cells are infected after interaction of whole viruses and antibodies, and the situation of interaction of the viruses and the neutralizing antibodies is truly reflected.

Description

technical field [0001] The invention relates to the field of biotechnology, and more specifically relates to a method for detecting a PRRSV IFA neutralizing antibody. Background technique [0002] Porcine reproductive and respiratory syndrome virus (PRRSV) is a positive-sense enveloped RNA virus. Virions consist of a nucleocapsid core constructed of a nucleocapsid protein (encoded by open reading frame 7 or ORF7) combined with viral RNA. The nucleocapsid is surrounded by a lipid envelope, which contains six structural proteins: glycoproteins GP2 (ORF2a), GP3 (ORF3), GP4 (ORF4) and GP5 (ORF5), and non-glycosylated proteins M (ORF6) and E(ORF2b). GP5 and M were found to be the most abundant proteins in the envelope, while other envelope proteins were present in lower amounts. Therefore, the antibody response after PRRSV infection is very complex, which can be generally divided into neutralizing antibody (NA) and non-neutralizing antibody (NNA). Some studies have shown that ...

Claims

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Application Information

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IPC IPC(8): G01N33/68G01N33/58G01N33/569G01N33/543
CPCG01N33/6854G01N33/56983G01N33/582G01N33/543G01N2333/08G01N2469/20
Inventor 王选年李鹏冯春花王利平高小静李红孙国鹏岳锋朱艳平张艳芳郭东光齐永华潘鹏涛
Owner XINXIANG UNIV
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