An immune test strip for rapid detection of Plasmodium falciparum, Plasmodium vivax, Plasmodium malariae and Plasmodium ovale
A kind of technology of falciparum malaria and test strips, which is applied to the epidemic test strips of malaria ovale and malaria ovale, colloidal gold immunochromatographic test strips, rapid detection of falciparum malaria and vivax malaria, which can solve the problem of ineffective distinction Malaria infects various pathogen species and other issues, achieving the effect of intuitive and clear judgment of results, high sensitivity and specificity, and easy preservation
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Embodiment 1
[0042] Example 1 Preparation of Plasmodium falciparum histone-rich II antigen
[0043] Histidine-rich protein-II (HRP-II) is a water-soluble glycoprotein synthesized by Plasmodium falciparum in the red inner stage. Because it contains a large segment of amino acid repeating sequence arranged in series, it is an ideal diagnosis target molecule.
[0044] A pair of primers were designed and synthesized according to the published conserved sequence of Plasmodium falciparum HRP-II gene (GenBank accession number: FJ871325.1) and primer design principles:
[0045] P1: 5'-actcaagcacatgtagatgatg-3';
[0046] P2: 5'-taatggcgtaggcaatgtgtg-3'.
[0047] Use anticoagulated whole blood of patients infected with Plasmodium falciparum as a template for PCR amplification: take 20 μL whole blood plus 200 μL red blood cell lysate (50 mmol / L NaCl, 0.015% saponin, 1 mmol / L EDTA) and shake and mix well at room temperature Set aside for 10 minutes to fully dissolve red blood cells, centrifuge at 1...
Embodiment 2
[0049] Example 2 Preparation of Plasmodium falciparum histone-rich II monoclonal antibody
[0050] Immunization: Each BALB / c mouse was injected intraperitoneally with 100 μg of the above-mentioned (Example 1) purified Plasmodium falciparum histone II recombinant fusion protein + Freund’s complete adjuvant suspension, and then injected 100 μg every other month Suspension of purified recombinant fusion protein + Freund's incomplete adjuvant once, twice in total, and immunization was boosted once by direct injection of antigen through tail vein 3 days before the mice were killed and spleens were taken for cell fusion.
[0051] Production and screening of hybridoma cells The fusion of SP2 / 0 tumor cells and splenocytes of immunized mice and the cloning of hybridoma cells were carried out according to conventional methods in the art. The above-mentioned purified Plasmodium falciparum Plasmodium Plasmodium Ⅱ recombinant fusion protein and glutathione-S-transferase (GST) protein were ...
Embodiment 3
[0061] Example 3 Preparation of Plasmodium Lactate Dehydrogenase Antigen
[0062]There are 4 species of Plasmodium parasites in the human body, namely Plasmodium vivax, Plasmodium malariae, Plasmodium falciparum and Plasmodium ovale. Early studies have found that malaria parasite lactate dehydrogenase (LDH) is quite different from host lactate dehydrogenase in terms of electrophoretic properties, enzyme kinetics, and immunological properties. Anti-LDH antibodies have genus-specificity to Plasmodium, and some anti-LDH antibodies have certain cross-reactivity with Lactate dehydrogenase antigens from different species of Plasmodium, while they have a certain cross-reactivity with the three LDH isoenzymes (A4, B4) of mammals. and C4) no cross-reactivity. Therefore, Plasmodium lactate dehydrogenase is an ideal diagnostic target molecule.
[0063] A pair of primers were designed and synthesized according to the published conserved sequence of Plasmodium LDH gene (GenBank: KX885921...
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