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Malaria recombinant antigen, IgY immune body and malaria detection kit

A technology of recombinant antigens and antibodies, applied in measurement devices, resistance to vector-borne diseases, biochemical equipment and methods, etc., can solve the problems of high cost, short half-life, low sensitivity, etc., and achieve significant social and economic benefits. The effect of reducing application cost and avoiding cross-reaction

Inactive Publication Date: 2010-09-22
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

So far, the main problems of existing methods of this type are: 1) low sensitivity: since monoclonal antibodies can only recognize a single epitope and antigen, the sensitivity is lower than that of blood smear microscopy: at 82%- Between 95.2%, the lowest is only 57%, especially when the worm blood rate is lower than 0.01%, the sensitivity obviously drops to 50%; 2) low specificity: due to the influence of differences and variations between strains in endemic areas Still not ideal: generally around 93%, the lowest is only 76.9%; 3) cross-reactivity: the use of monoclonal antibodies tends to cross-react with rheumatoid factor, heterophilic antibody, and antinuclear antibody; the false positive rate of detection Higher; 4) Short half-life: Most commercial rapid detection kits require storage at 2-30°C, but this condition is difficult to guarantee when used in epidemic areas; ideal kits should be able to store at 40-50°C 5) High cost: especially the research and development and production costs of monoclonal antibodies are difficult to significantly reduce, which is still unacceptable to people in developing countries where malaria is rampant
[0007] There is no report on the use of IgY in the immunodiagnosis of malaria in the prior art, especially the application of the specific IgY antibody obtained by using the antigen of the present invention in the immunodiagnosis of malaria has not been reported

Method used

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  • Malaria recombinant antigen, IgY immune body and malaria detection kit
  • Malaria recombinant antigen, IgY immune body and malaria detection kit
  • Malaria recombinant antigen, IgY immune body and malaria detection kit

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0032] Example 1: Preparation of Plasmodium falciparum recombinant antigen

[0033]From the analysis of the confirmed specific epitopes of falciparum malaria combined with the malaria genome database, 9 epitopes including five major antigens from RESA, MSA1, CSP, MSA1, and MAg-1 were finally selected, and their sequences are respectively SEQ ID Nos: 1-9, the epitope connection sequence of the antigen designed in the present invention is 9-6-9-8-6-7-7-3-9-6-4-9-1-5-6-2 (numbers are corresponding serial number). Design the polynucleotide sequence encoding the corresponding epitope according to the codon preference of Escherichia coli, according to the primer overlap method known to those skilled in the art, introduce BamH I and Bcl II endonuclease sites respectively at the front and rear ends of the epitope coding sequence, After PCR amplification, the amplified sequence is concatenated in order to form the coding sequence of the recombinant antigen (plus the start codon ATG, i...

Embodiment 2

[0034] Embodiment 2: Preparation of anti-falciparum malaria (Plasmodium falciparum) recombinant antigen IgY

[0035] (1) Antigen immunization Laihang chicken

[0036] 200 μl of the antigen (0.5 mg / ml) obtained in Example 1 was mixed well with an equal volume of Freund's adjuvant to immunize Leghorn chickens (purchased from the School of Veterinary Medicine, China Agricultural University). At week 0, chickens were immunized with complete Freund's adjuvant for primary immunization, and the chicken neck was injected subcutaneously at multiple points; at weeks 3, 5, and 11, chickens were immunized with incomplete adjuvant at multiple points in the leg and wing muscles. Eggs were collected daily and stored at 4°C after labeling for later use.

[0037] (2) Rough purification of IgY

[0038] Wash the fresh Laihang eggs obtained in step (1) and immerse them in 0.1% bromogeramine solution for disinfection, then wipe the shells of the eggs with 75% alcohol cotton. After removing the ...

Embodiment 3

[0044] Example 3: Activity Identification of IgY Antibody

[0045] (1) Determination of the titer of IgY antibody in immunized Laihang chicken eggs:

[0046] Indirect enzyme-linked immunoassay (ELISA) was used to measure the antibody level of immunized laying hens, the recombinant antigen was diluted with coating solution, added to the ELISA plate at 0.1ug / well, and coated overnight at 4°C. Pour off the coating solution and wash 5 times with PBST, then blot dry with absorbent paper; block the plate with PBS containing 3% BSA, 200ul per well, incubate at 37°C for 1 hour and wash as before; dilute the IgY antibody to be tested in a 2-fold gradient Add 100ul of antibody diluent to each well, incubate at 37°C for 2 hours and wash as before; add 100ul II anti-HRP-labeled goat anti-chicken IgG to each well at a dilution of 1:10000 (purchased from Promega, G1351), and incubate at 37°C for 2 hours After 1 hour, wash as before; then add 100ul of chromogenic solution to each well, and ...

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Abstract

The invention relates to a malaria recombinant antigen, an IgY immune body and a malaria detection kit. In the invention, the malaria antigen with the sequence as shown in SEQ ID No: 11 is designed; the IgY immune body is obtained from an antigen-immunized chicken; and a malaria infection detection kit containing the IgY immune body is prepared. The sensitivity of the detection kit of the invention is 1000 times higher than that of the similar rapid detection card in the prior art.

Description

technical field [0001] The present invention relates to a novel malaria recombinant antigen. Specifically, the present invention relates to a new malaria antigen, the IgY antibody obtained by immunizing chickens with the antigen, and a kit for detecting malaria infection containing the IgY antibody. Background technique [0002] Malaria is one of the three most serious infectious diseases in the world, threatening the health of nearly 4 billion people living in tropical and subtropical regions. Among them, falciparum malaria infects 300-500 million people every year and kills millions of people. Malaria thus severely hampers economic growth in developing countries. Due to the particularity of malaria treatment drugs, accurate and rapid diagnosis can not only detect and treat malaria patients in time, greatly reduce the fatality rate, but also save the lives of patients infected with other infectious diseases in time through early elimination of malaria. At the same time, ...

Claims

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Application Information

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IPC IPC(8): C07K14/445C12N15/30C07K16/20C07K16/02G01N33/569C12R1/90
CPCY02A50/30
Inventor 王恒蔺亚晖
Owner THE INST OF BASIC MEDICAL SCI OF CHINESE ACAD OF MEDICAL SCI
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