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BRV serum neutralizing antibody titer level detection method based on IFA

A detection method and antibody titer technology, applied in the biological field, can solve problems such as difficulty in accurately detecting antibody titer levels, and achieve representative, specific, and short detection cycles

Pending Publication Date: 2021-10-26
JINYUBAOLING BIO PHARMA CO LTD
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  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

However, since the inoculation and passage of BRV needs to rely on trypsin, clinical serum samples themselves have a neutralizing effect on trypsin, thereby inhibiting the growth of BRV in cells and affecting the generation of cytopathy induced by BRV (or even unable to produce cytopathy ), so it is difficult to accurately detect the BRV neutralizing antibody titer level in clinical serum samples in the detection of BRV serum neutralizing antibody titer level (such as serum neutralization test, micro serum neutralization test or ELISA)

Method used

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  • BRV serum neutralizing antibody titer level detection method based on IFA
  • BRV serum neutralizing antibody titer level detection method based on IFA
  • BRV serum neutralizing antibody titer level detection method based on IFA

Examples

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Effect test

Embodiment 1

[0031] Embodiment 1: BRV virus collection, identification and cultivation

[0032] 1.1. Collect the excreta of calves suspected of being infected with BRV from Hohhot, Inner Mongolia. After repeated dissolving with 1ml PBS, freeze-thaw three times, 3000r / min, centrifuge for 10min and take the supernatant as a virus sample. Use a nucleic acid extraction kit ( Tiangen), extract the double-stranded RNA in the virus sample, and then reverse-transcribe the RNA into cDNA, and use the BRV target gene primer designed by DNAman (the length of the amplified target band should be 1062bp) to identify the cDNA. Wherein the BRV target gene primer sequence is:

[0033] BRV-F: 5'GGCTTTAATTGCGAGAATTTCC 3' (SEQ ID NO: 1);

[0034] BRV-R: 5'GGTCTCATCATTCAACTCTAAT 3' (SEQ ID NO:2);

[0035] 1.2. Use the above primers to perform PCR amplification using the reverse-transcribed cDNA as a template. The specific amplification system is: 2 μl template, 0.5 μl BRV upstream primer (BRV-F), 0.5 μl BRV d...

Embodiment 2

[0038] Embodiment 2: Preparation of BRV neutralizing poison

[0039] 2.1. MA104 monolayer cells (preserved in the laboratory of Jinyu Baoling Biopharmaceutical Co., Ltd., commercially available) were prepared into 2×10 cells by digestion and passage. 5 cells / ml cell suspension, according to 150 μL of cell suspension spread in the wells of 96-well plate, at 37 ℃, 5% CO 2 After culturing in the incubator for 24 h, after the cells grew into a single layer, the surface layer of the cells was washed with DMEM maintenance solution containing 5 μg / ml trypsin (gibco company), and 100 μL of DMEM maintenance solution containing 5 μg / ml trypsin was added to each well. Obtain cell plates plated with MA104 monolayer cells.

[0040] 2.2. The BRV virus liquid obtained in Example 1 was serially diluted 10 times with DMEM maintenance solution containing 5 μg / ml trypsin, from 10 -1 ~10 -8 Inoculate the virus solution of each diluted titer into the wells of the monolayer cell plate treated in...

Embodiment 3

[0048] Embodiment 3: Determination of neutralization reaction conditions in the IFA neutralizing antibody detection method of BRV

[0049] 3.1. Determination of trypsin concentration in maintenance solution when clinical serum samples are diluted

[0050] Negative for BRV neutralizing antibody using DMEM maintenance solution containing 20 μg / ml, 40 μg / ml, 60 μg / ml, 80 μg / ml, 100 μg / ml, 120 μg / ml, 140 μg / ml, 160 μg / ml, 180 μg / ml trypsin Serum samples (gibco fetal bovine serum) were serially diluted 2-fold to a dilution of 1:128 to obtain serial dilutions of serum samples obtained by dilution with DMEM maintenance solution containing different concentrations of trypsin. Serum samples of each dilution were mixed with an equal amount of 200TCID 50 After neutralizing BRV neutralizing virus (prepared in Example 2) for 1 h, inoculate 100 μl of the neutralized mixed solution into the wells of the cell plate (prepared in step 2.1 in Example 2) covered with MA104 monolayer cells, and ...

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Abstract

The invention discloses a BRV serum neutralizing antibody titer level detection method based on IFA, and belongs to the technical field of biology. According to the method provided by the invention, two DMEM maintenance solutions containing different concentrations of pancreatin are adopted to carry out two-stage two-fold dilution on a clinical serum sample on the basis of a method for detecting a neutralizing antibody by IFA to prepare a to-be-detected serum sample for co-incubation with a BRV neutralizing virus; and the influence on the growth of the BRV in the cell due to the neutralization of the pancreatin by the clinical serum sample can be effectively avoided so as to accurately detect the BRV serum neutralizing antibody titer level, and the method further has characteristics of rapidness, and can be effectively used for guiding the BRV vaccine production and the quality monitoring.

Description

technical field [0001] The invention belongs to the field of biotechnology, in particular to an IFA-based method for detecting the titer level of BRV serum neutralizing antibodies, in particular to a method for quickly and accurately detecting the titer level of BRV serum neutralizing antibodies based on IFA. Background technique [0002] Bovine rotavirus (Bovine rotavirus, BRV) is a non-enveloped double-stranded RNA virus of the genus Rotavirus in the Reoviridae family. It is a symmetrical icosahedron with a diameter of about 60-80 nanometers. It can be seen under a microscope Typical "wheel shape". The diarrhea caused by the virus has brought huge economic losses to the dairy farming industry. It is manifested in the fact that there is no specific medicine for infected calves and often causes high mortality. [0003] At present, the prevention of BRV infection is mainly based on virus vaccines (bovine rotavirus, bovine coronavirus dual inactivated vaccine disclosed in pat...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): G01N33/533G01N33/537G01N33/569G01N21/64
CPCG01N33/533G01N33/537G01N33/56983G01N21/6486G01N21/6428G01N2021/6439
Inventor 杨青春陈坚屠洁刘玉梅黄海碧刘建奇王秉昆吉格木德宋庆庆赵丽霞王凯李晓燕李超俎红丽宋志刚田志辉杜宇荣刘东霞
Owner JINYUBAOLING BIO PHARMA CO LTD
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