Immunoassay and diagnostic reagent for malaria

A technology for diagnostic reagents and immunodetection methods, which is applied in the field of preparation of Plasmodium surface proteins, can solve the problems of increased false positive signals, ineffective false positive signals, and increased preparation costs.

Inactive Publication Date: 2003-03-26
LG CORP
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  • Abstract
  • Description
  • Claims
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Problems solved by technology

[0017] However, when they are used as diagnostic reagents, false positive signals are also increased due to the protein being a fusion partner
Although it was solved by cutting the fusion protein (Chang et al., Biotechnology 3, 985-990, 1985; Ellinger et al., Virology, 180, 811-813, 1991), there was no further reduction of false positive signals. What effect, and the yield is reduced, and the cost of preparation is increased, because the cleavage and purification methods to remove the fusion protein from the antigen are very complicated

Method used

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  • Immunoassay and diagnostic reagent for malaria
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  • Immunoassay and diagnostic reagent for malaria

Examples

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Embodiment 1-1

[0128] The invention will be further illustrated by the following examples. It is clear to those skilled in the art that these embodiments are only a more detailed description of the present invention, but the present invention is not limited to these embodiments. Example 1-1 prepares cDNA with malaria positive blood

[0129] RNA from malaria-positive blood was purified using TRI Reagent™ (Sigma Co.). The following purification methods were provided by Sigma Co.

[0130] At room temperature, let 0.1ml of TRI reagent TM and 0.1ml of malaria-positive blood stand for 5 minutes, add 20 microliters of BCP to the mixture, let the final mixture stand for 5 minutes at room temperature, and centrifuge at 12,000 rpm and 4°C for 15 minutes , the centrifuged product was divided into 3 layers. Transfer the top layer containing RNAs to a new test tube, then add 50 μl of isopropanol to the test tube, let it stand at room temperature for 5 minutes, then centrifuge the resulting mixture at ...

Embodiment 1-2

[0132] 4 microliters of RT buffer, 2 microliters of 0.1M DTT (DL-dithiothreitol) (Sigma, Cal No. D5545), 1 microliter of 10mM dNTP, 1 microliter of reverse transcriptase (Gibco Co), 1 Microliter RNase inhibitor (Promega) and microliter deionized distilled water were added to the above mixture, reacted at 42° C. for 1 h, and then reacted at 70° C. for 10 minutes to prepare cDNA. Embodiment 1-2 prepares the recombinant plasmid containing gene encoding PV200C

[0133] Using the cDNAs prepared in Example 1-1 as a template and a pair of the following primers SEQ.ID.NO: 2 and SEQ.ID.NO: 4, polymerase chain reaction was carried out as follows.

[0134] The reaction mixture contains 5 μl of PCR buffer, 5 μl of 2.5mM dNTP, 1 μl of sensitive primer, 1 μl of anti-sensitive primer, 2 μl of cDNA template and 35 μl of deionized distilled water, and react at 94°C for 30 seconds . Then 1 microliter of Vent polymerase (BioLab) was added to the mixture, and the reaction was repeated 36 times ...

Embodiment 1-3

[0135] Vent polymerase (BioLab) PCR was performed under the same conditions as above. The enhanced DNAs can be confirmed by electrophoresis in 1% agarose gel, and the enhanced DNAs (referring to Pv200-ct657) and pBluscript KS(+) (Stratagene) vector are cut with restriction enzymes, using T4 DNA ligase (Promega ). EcoR V and Pv200-ct657 were embedded in pBluscript KS(+) vector. As a result, a recombinant plasmid (ie, pBC-Pv200ct657) was obtained. E. coli strain JM109 was then transformed with pBC-Pv200ct657. The preparation of embodiment 1-3 expression vector pYLJ-MSP

[0136] Using pBC-Pv200-ct657 prepared in Example 1-2 as a template, and a pair of primers SEQ.ID.NO: 5 and SEQ.ID NO: 6 for polymerase chain reaction. PCR was carried out under the same conditions as above. The enhanced DNAs (ie PV200-19) were confirmed by electrophoresis in a 1% agarose gel.

[0137] In order to obtain the leader peptide sequence of yeast α factor linked to the N-terminus of PV200C polype...

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Abstract

The present invention relates to an immunoassay and diagnostic reagent for malaria by using antigens of malarial Protozoa. More preferably, the present invention relates to an immunoassay and diagnostic reagent for malaria which detect malaria-specific antibodies in blood by using Merozoite Surface Protein of Plasmodium vivax. The immunoassay and diagnostic reagent detecting malaria-specific antibodies in blood according to the present invention have high specificity and sensitivity and are useful in diagnosing a type of malaria where latent period is long and number of Protozoa in blood if few. Also, the present invention relates to a preparation method of the surface protein of malarial Protozoa using yeast or E.Coli. Preferably, the present invention provides an expression vector comprising genes of Merozoite Surface Protein of Plasmodium Vivax and histidine residues, as well as transformants transformed with the expression vector. Also, the present invention provides a method for preparing Merozoite Surface Protein of malarial Protozoa by using the transformant. The surface protein Merozoite Surface Protein of malarial Protozoa prepared from yeast or E.Coli transformant according to the present invention has high sensitivity and specificity to antibody as well as high purity. Also, the surface protein prepared by the preparation method of the present invention has markedly low pseudo-positive signals, and is useful in diagnosing malaria.

Description

technical field [0001] The invention relates to a malaria immune detection and diagnosis reagent. In particular, the present invention relates to a malaria immunoassay and diagnostic reagent for detecting malaria-specific antibodies in blood using plasmodium antigens. The invention also relates to a malaria immunodetection and diagnosis reagent for detecting malaria specific IgM and / or IgG in blood by using plasmodium antigen. In addition, the invention also relates to a method for preparing Plasmodium surface protein by using yeast or Escherichia coli. technical background [0002] Malaria is a disease caused by Plasmodium parasites that infect the red blood cells of the body and are carried by mosquitoes. One billion people in the world are at risk of malaria, and half a billion people are infected with malaria every year. [0003] Two million people die from malaria every year. Malaria is widespread throughout the world. However, in some areas malaria has been eradic...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07K14/445G01N33/569
CPCG01N2333/445G01N33/56905C07K14/445A61P33/06Y02A50/30
Inventor 林国镇孙美进柳承范李相翊吴宰勳李承远金炯澈
Owner LG CORP
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