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EV71 early diagnosis method and reagent kit

A kit and antibody technology, applied in the field of molecular immunology, can solve problems such as the lack of pathogens

Inactive Publication Date: 2012-02-15
湖北新纵科病毒疾病工程技术有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, there is currently a lack of specific antibodies against pathogens

Method used

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  • EV71 early diagnosis method and reagent kit
  • EV71 early diagnosis method and reagent kit
  • EV71 early diagnosis method and reagent kit

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0044] Expression and purification of recombinant protein antigens

[0045] Express the cloned gene sequence with the pET28a vector, and follow GE's HisTrap TM The FF purification procedure was used for purification, and the molecular weight of the target protein was about 35kDa. The present invention expresses, purifies and isolates the target protein ( figure 1 )As shown in Table 1.

[0046] Table 1: Amino acid sequence of the recombinant protein of the present invention

[0047] ID (locus)

Embodiment 2

[0049] Antibody production by immunizing animals with recombinant protein antigens

[0050] Use the recombinant protein prepared in Example 1 to immunize rabbits, the dosage is determined according to the body weight of the rabbit, the first antigen amount is 50ug / 20g, mixed with complete adjuvant in equal volume; the antigen amount for subsequent immunization is 25ug / 20g, equal volume with incomplete adjuvant Mixed; ELISA to evaluate the immune effect, after reaching the expected antibody titer, intravenously strengthened with a dose of 50ug / 20g.

[0051] The spleen was taken to separate splenocytes, fused with myeloma cells in a certain ratio, and then screened to obtain multiple strains of antibodies.

Embodiment 3

[0053] Detection application

[0054] (1) Detection of recombinant antigens in patient serum samples

[0055] Use the treated recombinant antigen prepared in Example 1 to coat and react with the patient's serum. As a result, the recombinant antigen of the present invention can recognize the early specific antibody against EV71 in the patient's serum and bind to it. After washing, add HRP enzyme-linked Secondary antibody reaction: Finally, add a chromogenic substrate for detection.

[0056] The results show that the recombinant antigen of the present invention can specifically combine with the antibody against EV71 virus.

[0057] (2) Antibody (monoclonal antibody and polyclonal antibody) detection of pathogens

[0058] For the antibodies (polyclonal antibody and monoclonal antibody) obtained in Example 2, the ELISA method was used to react with the patient's serum to detect the combination of the virus in the patient's blood and detect the pathogen.

[0059] The results sho...

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Abstract

The invention relates to an EV71 early diagnosis method and a reagent kit. Particularly, recombinant protein from EV71 viruses is used as antigen, antibodies (monoclonal antibodies and polyclonal antibodies) generated after the animal immunization by the recombinant protein is combined with enzyme linked immunization and other methods for detecting early anti-EV71 antibodies IgM or IgG in human serum samples or directly detecting EV71 pathogens in the serum samples.

Description

technical field [0001] The invention belongs to the field of molecular immunology, and in particular relates to a diagnostic method and kit for enterovirus. Background technique [0002] Hand-foot-mouth disease (HFMD), also known as exanthematous vesicular stomatitis, is a common global infectious disease caused by enteroviruses. Virological and epidemiological studies have confirmed that human enterovirus 71 (Entrovirus 71, EV71) is the main pathogen of human hand, foot and mouth disease that broke out in recent years. The EV71 virus belongs to the Picornaviridae family. The virus particle has a typical icosahedral structure, and its genome is a single-stranded positive-strand RNA with a length of about 7,408 nucleotides. The virus is highly contagious and contagious. In addition to hand, foot and mouth disease, it can also cause aseptic meningitis (aseptic meningitis), brain stem encephalitis (brain stem encephalitis) and other neurological diseases. [0003] Diagnostic ...

Claims

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Application Information

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IPC IPC(8): C07K7/06C07K7/08C07K16/10G01N33/577G01N33/569
Inventor 王华林张涛王志英周敏苏正元胡志红
Owner 湖北新纵科病毒疾病工程技术有限公司
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