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Egg-yolk antibody for resisting soluble egg antigen of Japanese blood flukes, preparation method thereof and application thereof

A chicken egg yolk antibody and schistosome technology, applied in the field of schistosomiasis, can solve the problems of inability to distinguish current symptoms from past infections, insufficient objective diagnostic criteria, and low circulating antigen content in circulating antigen detection methods

Inactive Publication Date: 2010-02-10
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. 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; the diagnostic standards are not objective enough, which can easily lead to human errors, etc.
Many effector functions of Ig are mediated through the Fc region, so IgY (ΔFc) cannot activate the mammalian complement system, and does not bind to RF factors, rheumatoid factors, SPA, SPG, etc.

Method used

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  • Egg-yolk antibody for resisting soluble egg antigen of Japanese blood flukes, preparation method thereof and application thereof
  • Egg-yolk antibody for resisting soluble egg antigen of Japanese blood flukes, preparation method thereof and application thereof
  • Egg-yolk antibody for resisting soluble egg antigen of Japanese blood flukes, preparation method thereof and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0071] Preparation of Schistosoma japonicum SEA

[0072] Oncomelania snails (mainland strain) infected with Schistosoma japonicum passed down in our laboratory escaped cercariae at 25°C under sufficient light, mixed the cercariae escaped from at least 20 snails, and infected New Zealand white rabbits through the skin (Tongji Medical College experiment Provided by the Animal Center, clean grade), the infection rate was 800 cercariae / rabbit, the infected rabbit was anesthetized 45 days after infection, the heart was perfused with normal saline, and then the liver was dissected and the liver was separated and collected. The collected eggs were repeatedly homogenized in an ice bath, precipitated at 4°C for 48 hours, and then centrifuged at 10,000×g for 1 hour. Aspirate the supernatant, collect and aliquot it, which is the soluble egg antigens (SEA).

[0073] The determination of SEA protein content adopts BCA method (BCA Protein Assay Kit, American BIPEC). Add 20 μl of standard ...

Embodiment 2

[0075] Immunization and egg collection

[0076] Mix 0.5ml of 60μg / ml SEA with an equal volume of complete Freund's adjuvant, fully emulsify and immunize via subwing vein. 10 days after the first immunization, use the antigen plus incomplete Freund's adjuvant to subcutaneously boost the immunization on the back of the chicken for 3 times, and the immunization dose is 30 μg SEA / bird, with an interval of 10 days between each time. The eggs are collected from 7 days after the first immunization and marked. Store in a refrigerator at 4°C.

Embodiment 3

[0078] Extraction and Purification of IgY by Polyethylene Glycol Method

[0079] Get the eggs 60 days after the initial immunization, remove the eggshell, wash the egg white outside the yolk with deionized water, remove the yolk coat, and mix the yolk with twice the volume of 0.01mol / L pH 7.6 Phosphate Buffer Solution (Phosphate Buffer Solution , PBS) and stir well. Add 5% PEG and stir until PEG is completely dissolved. Centrifuge at room temperature (4420×g, 20 min). Filter the supernatant, continue to add PEG to 20%, stir well to dissolve and then centrifuge at room temperature (12,000×g, 10 min). Remove the supernatant and redissolve the pellet with 1 / 6 of the original yolk volume in PBS. Place in a conventionally treated dialysis bag, dialyze in distilled water at 4°C for 2 hours, and change the water 3 to 4 times during the period. Finally, concentrate in polyethylene glycol 6000 (PEG 6000) to 1 / 10 of the original volume, collect and aliquot and store at -20°C for fut...

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Abstract

In the invention, a method of hypodermic multi-point injection of specific antigens of blood flukes is used to immunize a hen, the specific IgY is extracted, purified and identified from egg yolks, and an ELISA diagnosis system IgY-ELISA is established by combining the monoclonal antibody technique. The invention establishes a method for detecting circulating antigens of blood flukes, which is novel, highly efficient, sensitive and specific and also has a curative effect evaluation action. Moreover, the invention also initially provides a novel technique and means for detecting other pathogensand diagnosing infectious diseases.

Description

technical field [0001] The present invention relates to the diagnosis technology of parasite, particularly relates to schistosoma japonicum 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 still reaches 200 million every year. Schistosomiasis japonicum is prevalent in my country. . Schistosomiasis is prevalent in 370 counties and cities in 13 provinces, municipalities, and autonomous regions including Hunan, Hubei, Jiangxi, Anhui, Jiangsu, Yunnan, Sichuan, Zhejian...

Claims

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Application Information

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IPC IPC(8): C07K16/18C07K16/02C07K1/14G01N33/53
Inventor 刘文琪
Owner HUAZHONG UNIV OF SCI & TECH
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