Antibody against lactate dehydrogenase of plasmodium vivax, related preparation method, hybridoma cell strain and application

A hybridoma cell line, protozoan lactic acid technology, applied in biochemical equipment and methods, oxidoreductase, microbial-based methods, etc., to achieve low cost, high specificity, and responsive effects

Active Publication Date: 2010-12-22
SHANGHAI MUNICIPAL CENT FOR DISEASE CONTROL & PREVENTION
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  • Abstract
  • Description
  • Claims
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AI Technical Summary

Problems solved by technology

[0005] However, the commercialized detection kits currently developed at home and abroad can only detect Plasmodium falciparum and non-Plasmodium falciparum infections, and lack species specificity for most endemic areas in my country where Plasmodium vivax infection dominates.
Therefore, there is an urgent need to provide species-specific detection of P. vivax infection to make up for the lack of specific detection methods for P. vivax

Method used

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  • Antibody against lactate dehydrogenase of plasmodium vivax, related preparation method, hybridoma cell strain and application
  • Antibody against lactate dehydrogenase of plasmodium vivax, related preparation method, hybridoma cell strain and application
  • Antibody against lactate dehydrogenase of plasmodium vivax, related preparation method, hybridoma cell strain and application

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0050] Preparation of Example 1 Antigen (amino acid sequence shown in SEQ ID NO: 2)

[0051] 1.1 Extraction of Plasmodium Genomic DNA

[0052] The DNA extraction kit (Omega) was used to extract the genomic DNA of Plasmodium from the blood samples of vivax malaria patients in Shanghai, according to the instruction manual, and the extracted DNA was stored at -20°C.

[0053] 1.2 Primer design and PCR amplification

[0054] Specific primers were designed according to the target gene sequence in GenBank (Accession No. DQ060151). The sequence of the upstream primer is the nucleotide sequence shown in SEQ ID NO:3, and the sequence of the downstream primer is the nucleotide sequence shown in SEQ ID NO:4. EcoR Ⅰ and Xho Ⅰ restriction sites were introduced at the 5′ ends of the two primers respectively. Primers were synthesized by Shanghai Yingjun Biotechnology Co., Ltd. Using the extracted genomic DNA as a template, the reaction conditions were 95°C for 5 min; 94°C for 1 min, 54°C ...

Embodiment 2

[0063] Embodiment 2, the preparation of monoclonal antibody

[0064] 2.1 Animal immunity

[0065] 2.1.1 Animals: female, 5 Balb / c mice aged 6-8W (purchased from Shanghai SLIKE Experimental Animal Co., Ltd. (Shanghai) 2007-0005).

[0066]2.1.2 Antigen: the purified rePv-LDH recombinant antigen prepared in Example 1, 50 μg / animal / time.

[0067] 2.1.3 Immunization route and procedure: initial immunization: rePv-LDH antigen was fully mixed with an equal volume of complete Freund's adjuvant to form water-in-oil, and injected intradermally at multiple points on the back of the mouse. After 3 weeks for the second immunization, equal volumes of rePv-LDH antigen and incomplete Freund's adjuvant were mixed, and multi-point intradermal injection was performed on the back. Then immunize once every 3 weeks (intraperitoneal without adjuvant), and 20 μl of tail blood was collected on the 5th to 7th day after immunization, and the antibody titer was measured by ELISA method. When the antib...

Embodiment 3

[0081] Embodiment 3, identification of monoclonal antibody

[0082] 3.1 Antibody Subclasses

[0083] Assays were performed using a mouse monoclonal antibody subclass detection kit (Sigma). Dilute goat anti-mouse IgG1, IgG2a, IgG2b, IgG3, IgA and IgM with coating solution 1:2000 respectively, coat with 100 μl / well, incubate overnight at 4°C or 2h at 37°C; wash 3 times with washing solution PBST for 3 minutes Each time, add 100 μl of monoclonal antibody cell culture supernatant to be detected in each well, incubate at 37°C for 1 hour; wash 3 times as above, add horseradish peroxidase-labeled goat anti-mouse multivalent immunoglobulin diluted 1:5000 (G, A, M) antibody, 100 μl per well, incubate at 37°C for 1 h; wash three times as above, add 100 μl of freshly prepared TMB substrate solution to each well, and incubate at 37°C in the dark for 20 minutes; add 50 μl 2M H to each well 2 SO 4 Stop the reaction. Read OD on microplate reader 450 For the absorbance value, the subtype...

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Abstract

The invention provides an antibody against lactate dehydrogenase of plasmodium vivax, which is a monoclonal antibody or polyclonal antibody specific to proteins shown in SEQ ID NO: 2. More preferably, the monoclonal antibody or polyclonal antibody is obtained by using a fusion protein formed by the protein shown in SEQ ID NO: 2 and the known label as an antigen immune animal; the monoclonal antibody is produced by a hybridoma cell strain of which the collection number is CGMCC No.3897 or No.3898. The invention also provides a hybridoma cell strain producing the antibody against the lactate dehydrogenase of plasmodium vivax, a preparation method and use of the antibody against the lactate dehydrogenase of plasmodium vivax, a related immunoassay method, a kit prepared from the antibody, etc. The antibody against the lactate dehydrogenase of plasmodium vivax can specifically detect the lactate dehydrogenase of plasmodium vivax, can be applied to specificity detection of vivax infection, has the advantages of high specificity, sensitive response and low cost, and is applicable to high-throughput detection.

Description

technical field [0001] The present invention relates to the technical fields of genetic engineering and immunology, more specifically, to the technical field of monoclonal antibodies and polyclonal antibodies, in particular to a Plasmodium vivax lactate dehydrogenase (Pv-LDH) antibody, related preparation methods, Hybridoma cell lines and applications. Background technique [0002] Malaria is a serious parasitic disease caused by Plasmodium infection. There are four types of Plasmodium that infect humans: Plasmodium falciparum (P.f), Plasmodium vivax (P.vax, P.v), Plasmodium malariae (P.m) and Plasmodium ovale (P. ovale, P.o). According to WHO statistics, malaria is prevalent and spreading in 107 countries and regions around the world, and 3.2 billion people are threatened by malaria. Every year, 300 to 500 million people get sick, and the death toll reaches more than 1 million. [0003] Rapid and accurate diagnosis is key to effective malaria control and is one of the m...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07K16/40C12N5/20C12N9/04G01N33/577G01N33/569C12R1/91
CPCY02A50/30
Inventor 江莉蔡黎马晓疆王真渝蒋守富江西均张小萍
Owner SHANGHAI MUNICIPAL CENT FOR DISEASE CONTROL & PREVENTION
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