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Use of Plasmodium Randomized Recombinant Antigen-1 in the Preparation of a Kit for Screening and/or Evaluating the Prevalence of Falciparum Malaria

A technology of recombinant antigens and kits, which can be used in the fields of resistance to vector-borne diseases, biological testing, and material inspection products, which can solve the problems of high testing environment and difficult implementation.

Active Publication Date: 2017-10-10
THE INST OF BASIC MEDICAL SCI OF CHINESE ACAD OF MEDICAL SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0006] A simple and feasible screening method is needed to achieve the above goals, but the current methods for malaria screening are still limited. For the high-throughput nucleic acid detection technology developed in recent years, see Cheng, Z., et al., A novel, sensitive assay for high- Throughput molecular detection of plasmodia for active screening of malaria for elimination.J Clin Microbiol,2013.51(1):p.125-30, such as detection of Plasmodium 18S ribosomal RNA in blood, has high sensitivity and specificity, but The requirements for testing environment and testing instruments are still high, and it is still difficult to implement in epidemic areas with relatively backward economy and limited sanitation conditions.

Method used

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  • Use of Plasmodium Randomized Recombinant Antigen-1 in the Preparation of a Kit for Screening and/or Evaluating the Prevalence of Falciparum Malaria
  • Use of Plasmodium Randomized Recombinant Antigen-1 in the Preparation of a Kit for Screening and/or Evaluating the Prevalence of Falciparum Malaria
  • Use of Plasmodium Randomized Recombinant Antigen-1 in the Preparation of a Kit for Screening and/or Evaluating the Prevalence of Falciparum Malaria

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0034] Example 1: Synthesis and detection of antigenic protein

[0035] M.RCAg-1 protein was prepared and purified by Webster Bohui Chromatography Technology Co., Ltd. and the Institute of Process Engineering, Chinese Academy of Sciences. The protein prepared by the company was poor in stability and easy to degrade; the protein solution of the Institute of Process Engineering added stabilizers to stabilize the protein Good sex. Experimental detection, protein degradation and the effect of stabilizers on antibody recognition:

[0036] The amount of protein was detected by ordinary SDS-polyacrylamide gel electrophoresis.

[0037] Solution preparation:

[0038] 【1.5M Tris·HCl(pH8.8)】

[0039] Tris 18.6g

[0040] Deionized water 50ml

[0041] Adjust the pH to 8.8 with 1N HCl, and make up to 100ml with deionized water.

[0042] 【1.0M Tris·HCl(pH6.8)】

[0043] Tris base 12.1g

[0044] Deionized water 50ml

[0045] Adjust the pH to 6.8 with 1N HCl, and make up to 100ml with ...

Embodiment 2

[0069] Example 2, Indirect ELISA Detection of M.RCAg-1 Antibody Recognition Situation to Normal People, Plasmodium vivax and Plasmodium falciparum

[0070] Solution preparation:

[0071] [Coating solution] 0.1M carbonic acid coating buffer, pH9.2

[0072] Na 2 CO 3 0.68g

[0073] NaHCO 3 3.67g

[0074] Add distilled water to 500ml

[0075] [Blocking solution] 3% (v / v) goat serum, dissolved in PBS

[0076] 【Color developing solution】

[0077] Chromogenic solution A (stock solution): citric acid 6.2g

[0078] Na 2 HPO 4 12H 2 0 25g

[0079] Add distilled water to 500ml

[0080] Chromogenic solution B (stock solution): citric acid 1.05g

[0081] EDTA0.093g

[0082] Add distilled water to 500ml

[0083] TMB stock solution:

[0084] 3,3’,5,5’Tetramethylbenzidine (TMB) 140mg dissolved in 20ml DMSO

[0085] 10ml chromogenic solution=(5ml A stock solution+5ul H 2 o 2 )+(4.8ml B stock solution+0.2ml TMB stock solution)

[0086] Experimental steps:

[0087] a) Coat...

Embodiment 3

[0101] Embodiment 3, the experimental result that indirect ELISA is carried out screening to the resident of malaria endemic area (endemic area)

[0102] There are two forms of collection: ①venous blood collection, anticoagulation, and centrifugation to obtain serum; ②fingertip blood collection, dripping on blood collection filter paper, and drying naturally to form a dried blood sheet.

[0103] data processing:

[0104] a) Each test plate has the same negative control and positive control, and the test results of all samples are expressed in the form of ratio, recorded as OD%;

[0105] OD%=sample OD value / (X+3SD) 阴性对照OD值

[0106] b) Serum test results of healthy people in non-epidemic areas serve as normal controls, (X+3SD) 正常对照OD％ As the cutoff value for judging positive and negative, it is judged as positive if it is higher than this value, otherwise it is judged as negative;

[0107] c) After comparing all samples with the cutoff value, record the number of false posit...

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Abstract

The invention relates to application of plasmodium random recombinant antigen-1 to preparation of a kit for screening and / or evaluating the epidemic intensity of severe malaria. In addition, the invention relates to an indirect ELISA (Enzyme-linked Immuno Sorbent Assay) kit for detecting plasmodium antibodies in blood serum to be detected, wherein the kit comprises the 0.1 microgram / 100 microliters of plasmodium random recombinant antigen-1 covering a 96 porous plate, and the blood serum to be detected is diluted with PBS to be 1 to 200.

Description

technical field [0001] The invention relates to the detection field of Plasmodium. Specifically, the use of random recombinant antigen-1 (M.RCAg-1) of malaria falciparum in the preparation of a kit for screening and / or evaluating the epidemic intensity of malaria falciparum. Background technique [0002] Malaria is an infectious disease that seriously endangers human health. The Plasmodium that infects humans mainly includes Plasmodium falciparum, P.vivax, P.malariae and ovale Plasmodium (P.ovale) 4 species, of which the most serious damage is Plasmodium falciparum. The life cycle of Plasmodium is complex. Humans and Anopheles mosquitoes have two hosts. Plasmodium is divided into liver stage and red inner stage in the human body. The red inner stage has four different forms: ring stage, trophozoite stage, and schizont stage During the merozoite and merozoite stages, the antigens in different stages are variable, which also increases the difficulty of malaria prevention and...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): G01N33/569G01N33/68
CPCG01N33/56905G01N33/68G01N2333/445Y02A50/30
Inventor 王恒姚梅雪蔡启良
Owner THE INST OF BASIC MEDICAL SCI OF CHINESE ACAD OF MEDICAL SCI