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Chicken yolk antibody-magnetic bead ELISA (Enzyme-Linked Immuno Sorbent Assay) method for detecting schistosoma japonica circulating antigen

A technique for schistosomiasis and chicken egg yolk, which is applied in the field of detection of circulating antigens of Schistosoma japonicum, can solve the problems of low sensitivity, instability and uneven levels of the detection system, and achieves improved sensitivity, improved specificity, high integration and coordination. Effect

Inactive Publication Date: 2011-05-04
HUAZHONG UNIV OF SCI & TECH
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0006] (1) The curative effect evaluation value of the current schistosomiasis detection system is not ideal, the main reason is that a large number of existing detection reagents are used to detect antibodies, and antibodies can still be detected 1-3 years or even more than ten years after the treatment of patients. Detected in the serum, it is impossible to distinguish current symptoms from past infections, so it is necessary to provide an effective diagnostic system based on the detection of antigens for efficacy evaluation
[0007] (2) The main difficulty in the detection of circulating antigens is that the content of circulating antigens in patients, especially those in the chronic phase, is very small, and the sensitivity of the current detection system is low, especially in the evaluation of a large number of mildly endemic areas in my country. There is no very effective method
[0008] (3) There are many types of diagnostic reagents for schistosomiasis, and the levels are uneven, and the quality of reagents has problems of production differences and instability between batches; diagnostic standards are not objective enough, and there are human errors, etc.
There are no reports and mature kits on the application of immunomagnetic bead technology in the immunological diagnosis of schistosomiasis

Method used

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  • Chicken yolk antibody-magnetic bead ELISA (Enzyme-Linked Immuno Sorbent Assay) method for detecting schistosoma japonica circulating antigen
  • Chicken yolk antibody-magnetic bead ELISA (Enzyme-Linked Immuno Sorbent Assay) method for detecting schistosoma japonica circulating antigen
  • Chicken yolk antibody-magnetic bead ELISA (Enzyme-Linked Immuno Sorbent Assay) method for detecting schistosoma japonica circulating antigen

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0087] Embodiment 1 The method that specific IgY is labeled on the magnetic nano-bead

[0088]The method for labeling specific IgY on nano-magnetic beads in the present invention is: after washing the magnetic beads with a diameter of 800 nm with distilled water, resuspend in 0.1M PBS (pH7.4, containing 1% BSA) and adjust the concentration of magnetic beads to 10 mg / ml; take 5ml magnetic bead suspension, add 50mg carbodiimide, shake at 37°C for 15min, wash twice with PBS, make 2.5ml suspension, add chicken yolk against soluble egg antigens (SEA) of Schistosoma japonicum eggs Immunoglobulin IgY0.5ml, placed on a shaker at 37°C and incubated for 90min; added 0.01M glycine (pH8.2, containing 1% BSA) buffer to block overnight; resuspended in 5ml of PBS solution, stored at 4°C for later use.

Embodiment 2

[0090] Clinical small sample detection application and result of IgY-IMB-ELISA method of the present invention

[0091]

[0092]

[0093] Note: The serum of patients with chronic schistosomiasis was obtained from Wuhan CDC Schistosomiasis Research Institute; the serum of patients with acute schistosomiasis was obtained from the Parasitic Disease Detection Center of Tongji Medical College, Huazhong University of Science and Technology; both were positive for schistosomiasis eggs in feces. The sera of lung fluke patients and liver fluke patients were obtained from the endemic areas of Guangxi and Guangzhou, respectively; the sera of normal people were obtained from healthy people in Shandong, a non-endemic area of ​​schistosomiasis.

Embodiment 3

[0095] The kit for detecting schistosomiasis antigen provided by the invention comprises:

[0096] (1) 25 ml of chicken yolk immunoglobulin IgY-labeled nano-magnetic beads against Schistosoma japonicum egg soluble antigen;

[0097] (2) 1 bottle of standard product (freeze-dried product); dilute to 1ml with double distilled water before use, and prepare within 15 minutes before use;

[0098] (3) 1 bottle of sample diluent: 25ml;

[0099] (4) 1 bottle of detection antibody: 50ml;

[0100] (5) 1 bottle of washing liquid: 500ml;

[0101] (6) 1 bottle of substrate solution: 20ml;

[0102] (7) 1 bottle of stop solution: 20ml;

[0103] (8) A copy of instruction manual.

[0104] Instructions for use of the kit for detecting schistosomiasis antigens provided by the present invention: each reagent is equilibrated to room temperature before use. Each reaction should be provided with 2 standard tubes and sample tubes to be tested. Add 200 μl / tube of IgY-coated magnetic beads to the...

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Abstract

The invention provides a nano magnetic bead labeled by chicken yolk immune globulin IgY and used for resisting the soluble antigen of schistosoma japonica ovums and a chicken yolk antibody-magnetic bead ELISA (Enzyme-Linked Immuno Sorbent Assay) method for detecting schistosoma japonica circulating antigen. The method comprises the steps of: immunizing a hen with specific antigens of schistosoma japonica with a method of subcutaneous multi-point injection, and extracting, purifying and identifying specific IgY from an egg yolk; and coupling the polyclone IgY on a magnetic bead, and detecting the circulating antigen of the schistosoma by using the magnetic bead-IgY polyclone antibody as a capture antibody and using the specific monoclone IgY antibody as a detecting antibody. The method not only can be used for capturing more antigens by using the magnetic bead and the IgY to increase the sensibility, but also benefits the increase of detecting specificity by using the monoclonal antibody. Thus, the high combination and coordination of sensibility and specificity, required in immunology diagnosis, are realized.

Description

technical field [0001] The invention relates to a detection technology of Schistosoma japonicum circulating antigen, in particular to a chicken egg yolk antibody-magnetic bead ELISA diagnostic technology for detecting Schistosoma japonicum circulating antigen. Background technique [0002] Schistosoma is a helminth parasitic in the veins, which can infect a variety of mammals including humans and livestock, causing schistosomiasis. If patients with schistosomiasis are not diagnosed and treated in time, it may cause advanced schistosomiasis mainly caused by liver cirrhosis and ascites. And die. At present, schistosomiasis is still a parasitic disease that seriously endangers human health and economic development. The disease is prevalent in 74 countries and regions all over the world, and the number of infected people reaches 200 million every year. Schistosomiasis japonicum is prevalent in my country. Schistosomiasis is prevalent in 370 counties and cities in 13 provinces,...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): G01N33/577G01N33/543G01N33/531
Inventor 刘文琪管晓虹沈继龙李雍龙冯振卿雷家慧吴玉龙
Owner HUAZHONG UNIV OF SCI & TECH
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