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Hybridoma cell, monoclonal antibody capable of secreting anti-Plasmodium lactate dehydrogenase monoclonal antibody and its application

A technology of hybridoma cells and protozoan lactic acid, applied in the field of monoclonal antibodies and hybridoma cells, can solve the problem of inability to differentially diagnose vivax malaria and mixed infections, inability to accurately confirm the infection period, and inability to detect vivax malaria, etc. question

Active Publication Date: 2017-06-27
GUANGDONG FAPON BIOTECH CO LTD +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Using histidine-rich protein II (HRP-II) as the target antigen diagnostic reagent has several disadvantages: 1, it can only be used for the detection of falciparum malaria, and cannot detect the widespread vivax malaria; 2, when the patient's blood After the live worms disappear, HRP-II still exists in the body for a long time, and the infection period cannot be accurately confirmed; 3 The cross-reaction of anti-HRP-II monoclonal antibody and rheumatoid factor will cause false positive reaction
The falciparum malaria detection kit with Plasmodium histidine-rich protein II (HRP-II) as the target antigen can only be used for the detection of falciparum malaria, and cannot be detected for vivax malaria, and it is easy to cross-react with rheumatoid factor and cause false positive reaction
The combined detection product of Plasmodium falciparum histidine-rich protein II (HRP-II) and non-falciparum lactate dehydrogenase (LDH) target antigen can only diagnose Plasmodium falciparum and non-Plasmodium falciparum infections, but cannot diagnose Plasmodium vivax and mixed infections differential diagnosis

Method used

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  • Hybridoma cell, monoclonal antibody capable of secreting anti-Plasmodium lactate dehydrogenase monoclonal antibody and its application
  • Hybridoma cell, monoclonal antibody capable of secreting anti-Plasmodium lactate dehydrogenase monoclonal antibody and its application
  • Hybridoma cell, monoclonal antibody capable of secreting anti-Plasmodium lactate dehydrogenase monoclonal antibody and its application

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Experimental program
Comparison scheme
Effect test

Embodiment 1

[0038] Establishment of hybridoma cell line and preparation of anti-Plasmodium lactate dehydrogenase protein monoclonal antibody.

[0039] 1. Antigen immunity.

[0040] Mix the recombinant Plasmodium lactate dehydrogenase protein (LDH) antigen (1.2mg / mL, produced by Shenzhen Feipeng Biological Co., Ltd., product name: 28D-MA1) with Freund's complete adjuvant (SIGMA, F5881) in equal volumes , to obtain an oily emulsion. This emulsion is subcutaneously administered to the back site of BALB / c mice (Experimental Animal Center of Guangzhou Province, 6 weeks old female, 5) with the dose of every 0.2 milliliters, after the first immunization 14 days, the abdominal cavity boosts immunity (antigen and Fu Incomplete adjuvant (SIGMA, F5506) was mixed in equal volumes), after boosting immunization to four injections, the tail blood was collected for titer detection, and the titer reached the fusion requirement.

[0041] Three days before the fusion, the same dose of antigen mixed with a...

Embodiment 2

[0062] Preparation of colloidal gold rapid detection test paper for malaria.

[0063] 1. Preparation of nitrocellulose membrane.

[0064] Coating buffer preparation: 0.01M PBS buffer at pH 7.2 containing 6% methanol was used as the coating buffer, filtered through a 0.22μ membrane, placed at 4°C for use, and valid for one week. 1000mL 0.01M PH 7.2 PBS buffer solution with 6% methanol Recipe: NaCL 8g, KCL 0.2g, NaCl 2 HPO 4 12H 2 O 2.9g, KH 2 PO 4 0.2g, methanol 60mL, distilled deionized water to 1000mL.

[0065] Preparation of nitrocellulose membrane: Dilute anti-Plasmodium falciparum lactate dehydrogenase protein (LDH) monoclonal antibody 1G2 to 1-5 mg / mL with coating buffer, adjust the machine, draw the line as T1 line, and T1 line is The first detection line, T1 line is close to the end of the gold standard pad, about 5mm away from the end of the gold standard pad. Dilute the anti-Plasmodium vivax lactate dehydrogenase protein (LDH) monoclonal antibody 3G6 with coati...

Embodiment 3

[0090] Malaria colloidal gold rapid test kit.

[0091] 1. Malaria colloidal gold rapid test kit includes:

[0092] ①A pack of test strips (10 strips / pack)

[0093] ②One bottle of sample diluent (10mL / bottle)

[0094] Preparation of relevant solutions.

[0095] Sample diluent: The sample diluent is 8% NaCl solution. Preparation method: 80gNaCL, add distilled water to make up to 1000mL.

[0096] 2. Detection of Plasmodium lactate dehydrogenase protein (LDH) by colloidal gold method.

[0097] (1) Directly put 20 μL of collected venous whole blood into a plastic test tube containing 180 μL of diluent, mix well, take 120 μL of dissolved sample and add it to the sample hole of the test paper card, and wait for 15 minutes to observe the result.

[0098] (2) Judgment of results: When the test strip shows a purple-red quality control line visible to the naked eye, but no visible purple-red detection line appears, the result is judged as negative; when the test strip shows a purple...

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Abstract

The invention relates to hybridoma cells capable of secreting an anti-plasmodial lactate dehydrogenase monoclonal antibody, a monoclonal antibody and an application of the monoclonal antibody. The number of the hybridoma cells are three strains, are collected in Collection Center of Wuhan University in Luojiashan, Wuchang, Wuhan City of China Center for Type Culture Collection (CCTCC) and have collection numbers of CCTCC No: C2014185, CCTCC No: C2014184 and CCTCC No: C2014181. In addition, the invention also relates to a malaria test strip having high sensitivity and strong specificity and a malaria detection kit containing the malaria test strip. By the malaria detection kit, plasmodium falciparum can be accurately and quickly detected and the malaria detection kit has significant advantages of specificity and sensitivity compared to the traditional detection method and also can be applied in the detection of tertian malaria and mixed infection.

Description

technical field [0001] The invention relates to the field of immune detection, in particular to a hybridoma cell capable of secreting anti-plasmodium lactate dehydrogenase monoclonal antibody, monoclonal antibody and application. Background technique [0002] Malaria is a serious parasitic disease that endangers human health. It is an infectious disease transmitted by Anopheles mosquitoes. . Malaria is widely prevalent in tropical, subtropical and temperate marginal areas. Among all vector-borne diseases, malaria is one of the diseases with the highest morbidity and mortality. Worldwide, the number of patients with clinical symptoms alone is between 300 million and 500 million per year, and the number of deaths due to malaria is between one and three million per year. Among the cases of morbidity and death, 90 % are poor countries in Africa and Asia. According to incomplete statistics, it is estimated that 250,000 to 300,000 people suffer from the disease every year in Ch...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N5/20C07K16/40G01N33/577G01N33/558C12R1/91
CPCY02A50/30
Inventor 龚春喜王益琼杨耿周梁巍腾汪春霞范凌云彭亮刘莉莉
Owner GUANGDONG FAPON BIOTECH CO LTD
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