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A double-antibody sandwich ELISA diagnostic kit for detecting bovine rotavirus and its application

A bovine rotavirus and kit technology, applied in the field of medicine, can solve the problems of false positives, easy contamination, etc., and achieve the effect of good specificity

Active Publication Date: 2021-01-15
NORTHEAST AGRICULTURAL UNIVERSITY
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Although the PCR method has strong specificity and high sensitivity, it is prone to contamination, leading to false positive results

Method used

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  • A double-antibody sandwich ELISA diagnostic kit for detecting bovine rotavirus and its application
  • A double-antibody sandwich ELISA diagnostic kit for detecting bovine rotavirus and its application
  • A double-antibody sandwich ELISA diagnostic kit for detecting bovine rotavirus and its application

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0048]Example 1 Screening and establishment of hybridoma cells secreting monoclonal antibodies against bovine rotavirus VP6 protein

[0049]1 Test materials

[0050]1.1 Strains, cells and viruses

[0051]The recombinant strain pProHTa-NCDV-VP6 / Rosetta expressing bovine rotavirus VP6 protein was constructed and stored by our laboratory (this recombinant strain has been recorded in the following literature: "Establishment of an indirect ELISA method for the detection of bovine rotavirus VP6 protein", Zhang Yiting Et al., Chinese Veterinary Science, 2010, 40(10), 1039-1043); BRV NCDV cell-adapted strain was purchased from the China Veterinary Drug Administration, and MA104 cells are stored in our laboratory.

[0052]1.2 Experimental animals

[0053]SPF BALB / c mice and healthy female rabbits weighing 2 to 3 kg were purchased from Liaoning Changsheng Biotechnology Co., Ltd.

[0054]1.3 Reagent

[0055](1) Solution I: 12g urea, 1.56g NaH2PO4·2H2O and 0.12g Tris-Base were dissolved in 100mL three-distilled wat...

Embodiment 2

[0173]Example 2 Establishment of double antibody sandwich ELISA method

[0174]1 Test materials

[0175]1.1 Strains, cells and viruses

[0176]The BRV NCDV cell-adapted strain was purchased from China Veterinary Drug Administration, and MA104 cells are kept in our laboratory.

[0177]1.2 Main reagents

[0178](1) PBST washing solution: KH2PO4 0.2g, Na2HPO4·12H2O 2.9g, sodium chloride 8.0g, KCl 0.2g, 1L deionized water, Tween-20 0.5mL, pH adjusted to 7.4.

[0179](2)Na2CO3Coating diluent: NaHCO3 2.93g, Na2CO3 1.5g, 1000mL deionized water dissolved, concentrated HCl and NaOH to adjust the pH to 9.6.

[0180](3) Blocking solution: 1g skimmed milk dissolved in 20mL PBS buffer (KH2PO4 0.2g, Na2HPO4·12H2O 2.9g, sodium chloride 8.0g, KCl 0.2g, 1L deionized water) dissolved, and stored at 4°C.

[0181](4) PBS buffer: KH2PO4 0.2g, Na2HPO4·12H2O 2.9g, sodium chloride 8.0g, KCl 0.2g, 1L deionized water.

[0182](4) TMB substrate color-developing solution: mix A and B at a ratio of 1:1, prepare them for use now, avoid li...

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Abstract

The invention discloses a double-antibody sandwich type ELISA (enzyme-linked immuno sorbent assay) diagnosis kit for detecting BRV (bovine rotavirus) and application thereof. The double-antibody sandwich type ELISA diagnosis kit contains a monoclonal antibody 4D10 excreted by a hybridoma cell strain with collection number of CGMCC NO.14726; futhermore, a purified rabbit-resistant anti-BRV polyclonal antibody is used as a capturing antibody, the monoclonal antibody 4D10 is used as a detection antibody, and the BRV double-antibody sandwich type ELISA method is established. Proofed by sensitivity experiment resultss, the minimum detection amount of BRV is 9.884*10<4>TCID50 / mlL; proofed by specificity experiment results, the specificity is good, and the cross reaction with PoRV (porcine rotavirus), PEDV (porcine epidemic diarrhea virus), BPV (bovine papilloma virus) and TGEV (eransmissible gastroenteritis virus) is avoided; proofed by between-run and within-run repeatability experiment results, the repeatability is good. Compared with the RT-PCR (reverse transcription-polymerase chain reaction) method, when the established double-antibody sandwich type ELISA method has a satisfactionrate of 100% iswhen being used for detecting 95 clinical samples, the satisfaction rate is 100%. The established double-antibody sandwich type ELISA method has the advantages that the specificity, sensitivity and repeatability are good; the method can be used for quickly detecting the BRV.

Description

Technical field[0001]The invention relates to a virus detection kit, in particular to a double antibody sandwich ELISA diagnostic kit for bovine rotavirus detection prepared based on the double antibody sandwich ELISA detection principle. The invention belongs to the field of medical technology.Background technique[0002]Rotavirus (Rotavirus, RV) is a member of the family Reoviridae (Reoviridae) and Rotavirus. It has seven different groups A, B, C, D, E, F, and G. Among them, group A is one of the important pathogens that cause diarrhea in livestock, and the severity of rotavirus infection is age-dependent, and it is most common in young animals clinically. After a calf infected with rotavirus undergoes an incubation period of 2-4 days, symptoms usually start from sudden fever and vomiting, followed by watery diarrhea that lasts for 3-8 days, leading to dehydration, electrolyte imbalance, and even severely infected calves. Symptoms of convulsions. BRV infection causes decline in catt...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N5/20C07K16/10G01N33/569G01N33/543C12R1/91
CPCC07K16/10C07K2317/33C07K2317/35G01N33/543G01N33/56983G01N2469/10
Inventor 乔薪瑗姜艳平李一经王丽唐丽杰徐义刚崔文
Owner NORTHEAST AGRICULTURAL UNIVERSITY
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