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Mouse hybridoma cells and rapid diagnosis test strip for distinguishing and detecting plasmodium falciparum, plasmodium vivax, plasmodium malaria and plasmodium ovale

A technology for hybridoma cells and malaria ovale, applied in the field of bioengineering, can solve the problem of inability to effectively distinguish various pathogenic species of malaria infection, and achieve the effects of clear and intuitive result judgment, easy industrial production, and low cost.

Pending Publication Date: 2022-01-07
中国疾病预防控制中心寄生虫病预防控制所国家热带病研究中心
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  • Abstract
  • Description
  • Claims
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AI Technical Summary

Problems solved by technology

[0004] The object of the present invention is to provide a mouse hybridoma cell and a rapid diagnostic test strip for distinguishing and detecting falciparum malaria, vivax malaria, malaria ovale, and ovale malaria. The rapid diagnostic test strips for differentiating and detecting Plasmodium falciparum, Plasmodium vivax, Plasmodium ovale, and Plasmodium ovale need to solve the technical problem that the existing rapid diagnosis technology for malaria cannot effectively distinguish the species of pathogens infected by malaria

Method used

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  • Mouse hybridoma cells and rapid diagnosis test strip for distinguishing and detecting plasmodium falciparum, plasmodium vivax, plasmodium malaria and plasmodium ovale

Examples

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Embodiment 1

[0050] Example 1 Preparation of Plasmodium falciparum rich histone II antigen

[0051] Histidine-rich protein-II (HRP-II) is a water-soluble glycoprotein synthesized by Plasmodium falciparum in the erythroid stage. It is an ideal diagnosis because it contains large segments of amino acid repeats arranged in tandem. target molecule.

[0052] A pair of primers were designed and synthesized according to the published conserved sequence of the HRP-II gene of Plasmodium falciparum (GenBank accession number: FJ871325.1) and primer design principles:

[0053] P1: 5'-actcaagcacatgtagatgatg-3'; (shown in SEQ ID NO.1)

[0054] P2: 5'-taatggcgtaggcaatgtgtg-3'. (shown in SEQ ID NO.2)

[0055] Using anticoagulated whole blood from Plasmodium falciparum-infected patients as the template for PCR amplification: take 20 μL of whole blood and add 200 μL of red blood cell lysate (50 mmol / L NaCl, 0.015% saponin, 1 mmol / L EDTA), shake and mix well at room temperature. Set aside for 10 min to f...

Embodiment 2

[0057] Example 2 Preparation of Plasmodium falciparum-rich histone II monoclonal antibody

[0058] Immunization: Each BALB / c mouse was injected intraperitoneally for the first time with a suspension of 100 μg of the purified Plasmodium falciparum-rich histone II recombinant fusion protein + Freund's complete adjuvant purified above (Example 1), and then injected with 100 μg of purified P. falciparum adjuvant every 1 month. The suspension of recombinant fusion protein + Freund's incomplete adjuvant was used once for a total of 2 times, and the antigen was injected directly through the tail vein 3 days before the mouse was killed and the spleen was taken for cell fusion.

[0059] Generation and Screening of Hybridoma Cells The fusion of SP2 / 0 tumor cells and spleen cells of immunized mice and the cloning of hybridoma cells were performed according to conventional methods in the art. Using the purified Plasmodium falciparum histone-rich protein II recombinant fusion protein and g...

Embodiment 3

[0069] Example 3 Preparation of Plasmodium Lactate Dehydrogenase Antigen

[0070] There are 4 species of Plasmodium parasites in human body, namely Plasmodium vivax, Plasmodium malaria, Plasmodium falciparum and Plasmodium ovale. Early studies found that Plasmodium lactate dehydrogenase (LDH) and host lactate dehydrogenase are quite different in electrophoretic properties, enzymatic kinetic properties, and immunological properties. Anti-LDH antibodies are genus-specific to Plasmodium, and some anti-LDH antibodies have certain cross-reactions with lactate dehydrogenase antigens of Plasmodium from different species, and have a certain degree of cross-reaction with the three LDH isoenzymes (A4, B4) of mammals. No cross-reaction with C4). Therefore, Plasmodium lactate dehydrogenase is an ideal diagnostic target molecule.

[0071] Design and synthesize a pair of primers according to the published conserved sequence of Plasmodium LDH gene (GenBank: KX885921.1) and primer design pr...

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Abstract

The invention provides eight mouse hybridoma cells. Meanwhile, the invention provides a rapid diagnosis test strip which is composed of a sample pad, a colloidal gold probe pad, a cellulose membrane and a water absorption pad, colloidal gold contains colloidal gold labeled antibodies, and four detection lines and a quality control line are arranged on the cellulose membrane, the colloidal gold probe pad comprises a monoclonal antibody which is labeled by colloidal gold and is specifically bound with histone-rich protein II of plasmodium falciparum and monoclonal antibodies which are labeled by colloidal gold and are specifically bound with plasmodium lactate dehydrogenase, plasmodium malaria lactate dehydrogenase and plasmodium ovale lactate dehydrogenase, the four detection lines comprise the monoclonal antibody which is specifically bound with the histone-rich protein II of the plasmodium falciparum, the monoclonal antibody which is specifically bound with the plasmodium malaria lactic dehydrogenase, the monoclonal antibody which is specifically combined with plasmodium ovale lactic dehydrogenase and the monoclonal antibody which is specifically combined with the lactic dehydrogenase of the four plasmodium. The rapid diagnosis test strip has the advantages of simplicity, sensitivity, specificity and rapidness.

Description

technical field [0001] The invention belongs to the field of bioengineering, and relates to a colloidal gold immunochromatographic test strip, in particular to a mouse hybridoma cell and a method based on the detection of circulating antigens that can rapidly detect four human malaria parasites (P. Plasmodium, Plasmodium ovale, and Plasmodium ovale). Background technique [0002] Malaria is one of the parasitic diseases that seriously endanger human health and is one of the three major global public health problems listed by the World Health Organization. In 2019, malaria was still endemic in 87 countries around the world, with a total of 229 million reported cases, an increase of about 1 million cases compared with 2018, and 409,000 deaths from malaria. China has been an endemic area of ​​malaria since ancient times, seriously endangering people's lives and health and restricting social and economic development. From 2011 to 2016, the cumulative number of imported malaria...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N5/20C07K16/20C07K16/40G01N33/577G01N33/573G01N33/569G01N33/558G01N33/58G01N33/543C12R1/91
CPCC07K16/205C07K16/40G01N33/577G01N33/573G01N33/587G01N33/56905G01N33/558G01N33/54346G01N2333/445G01N2469/10G01N2333/904Y02A50/30
Inventor 石锋汪俊云高春花杨玥涛贾孝凯危芙蓉王颖张璟
Owner 中国疾病预防控制中心寄生虫病预防控制所国家热带病研究中心
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