Double-antibody sandwich ELISA antigen detection kit for Porcine deltacoronavirus (PDCoV) N protein

A double antibody sandwich, coronavirus technology, applied in the field of biotechnology detection, can solve the problems of lack of PDCoV antigen immunology related methods, etc., and achieve the effect of high linear coefficient and wide detection range

Inactive Publication Date: 2019-10-22
YANGZHOU UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

At present, the etiological detection methods for clinical diagnosis of PDCoV infection are mainly RT-PCR, qRT-PCR and other detection technologies for viral nucleic acids, and there is a lack of immunological methods that directly target PDCoV antigens.

Method used

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  • Double-antibody sandwich ELISA antigen detection kit for Porcine deltacoronavirus (PDCoV) N protein
  • Double-antibody sandwich ELISA antigen detection kit for Porcine deltacoronavirus (PDCoV) N protein
  • Double-antibody sandwich ELISA antigen detection kit for Porcine deltacoronavirus (PDCoV) N protein

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Experimental program
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Effect test

Embodiment 1

[0019] Embodiment 1 Preparation of anti-porcine delta coronavirus N protein mouse monoclonal antibody

[0020] Using the recombinant N protein prepared by a disclosed recombinant porcine delta coronavirus N protein preparation method as the immunogen and detection source (CN 109880843 A), using lymphocyte hybridoma technology, successfully prepared 6 strains of anti-PDCoV N protein Specific monoclonal antibodies 1A2, 4C5, 5C5, 8C12, 8E8 and 10A7. Using PK1 cells infected by PDCoV CHN / Tianjin / 2016 strain as antigen, Western Blot identification was carried out on 6 monoclonal antibodies, the results are as follows Figure 1 to Figure 6 .

[0021] The specific steps for preparing PDCoV N protein-specific monoclonal antibodies are as follows:

[0022] (1) Recombinant N protein was used as the coating antigen, the optimal coating concentration of antigen and the optimal dilution of serum were determined by checkerboard titration, and an indirect ELISA method was established to de...

Embodiment 2

[0032] Example 2 Establishment of Porcine Delta Coronavirus N Protein Double Antibody Sandwich ELISA Antigen Detection Kit

[0033] The porcine delta coronavirus N protein double antibody sandwich ELISA antigen detection kit of the present invention can be obtained by the following steps:

[0034] (1) Monoclonal Antibody Antigen Binding Epitope Analysis

[0035] The antigen-binding sites of different strains of monoclonal antibodies were analyzed by ELISA overlay test. The recombinant N protein antigen was coated with a microtiter plate at a concentration of 1 μg / mL, and the monoclonal antibody was diluted according to the monoclonal antibody dilution of 100% saturated antigen ascites , add single or two monoclonal antibodies of different combinations to each well, keep the same amount of monoclonal antibodies of different strains, perform indirect ELISA detection, and read the OD 450nm value. Calculate the additivity index (A.I) of each monoclonal antibody combination accord...

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Abstract

The invention belongs to the field of biotechnology detection and particularly relates to a double-antibody sandwich ELISA antigen detection kit for Porcine deltacoronavirus (PDCoV) N protein. The kitcomprises a monoclonal antibody PDCoV-N-1A2 ELISA plate, positive and negative controls, an HRP labeled detection antibody PDCoV-N-5C5, a sample diluent, a color developing solution and a washing solution, wherein the antibodies PDCoV-N-1A2 and PDCoV-N-5C5 are secreted by hybridoma cell strains PDCoV-N-1A2 and PDCoV-N-1A2, respectively. The kit of the invention can specifically detect PDCoV N protein by the double-antibody sandwich ELSIA method established by using 1A2 as a capture antibody and using hybridization 5C5 (HRP coupled) as a detection antibody, and provides a new detection means for the clinical diagnosis and epidemiological investigation of PDCoV infection.

Description

technical field [0001] The invention belongs to the field of biotechnology detection, in particular to a double-antibody sandwich ELISA antigen detection kit for porcine delta coronavirus N protein. Background technique [0002] Porcine deltacoronavirus (Porcine deltacoronavirus, PDCoV) is a newly discovered coronavirus that can cause diarrhea in pigs, especially newborn piglets. Very similar, easy to confuse, infection can lead to higher morbidity and mortality, and it is one of the important reasons for the death of newborn piglets in many countries and regions in recent years. PDCoV was first discovered by Hong Kong scholars in 2012 (WooPC, et al. virology, 2012,86(7):3995-4008.), the first large-scale epidemic occurred in the United States in 2014 (Li G, et al.Full-Length GenomeSequence of Porcine Deltacoronavirus Strain USA / IA / 2014 / 8734.Genomeannouncements, 2014 ,2(2):e00278-14.), and subsequently detected in South Korea, China and other places (Lee S, etal. Complete ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): G01N33/68G01N33/577G01N33/558
CPCG01N33/6854G01N33/577G01N33/558G01N2333/17
Inventor 张小荣钱炳旭吴艳涛郭梦娇张成成曹永忠陈杨
Owner YANGZHOU UNIV
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