Method for detecting trichinella circulating antigen by utilizing IgY-McAb sandwich ELISA (enzyme-linked immuno sorbent assay)

Abstract

The invention discloses a method for detecting a trichinella circulating antigen by utilizing IgY-McAb sandwich ELISA (enzyme-linked immnuo sorbent assay). With an anti-trichinella muscle larval ES antigen egg yolk antibody IgY as a capture antibody and an anti-trichinella ES antigen McAb as a detection antibody, the preparation method of the IgY comprises the following steps of: adding the trichinella muscle larval into a culture medium to carry out sterile culture, purifying and dialyzing supernate and then concentrating and drying to obtain the ES antiagen, determining protein concentration and then immunizing roman legehenne by utilizing the ES antigen, collecting the IgY from the produced egg yolk by adopting a saturate ammonium sulphate precipitation method, purifying and determining concentration, and sub-packaging for later use; and the preparation method of the McAb comprises the following steps of: establishing a hybridoma cell strain secreting the anti-trichinella muscle larval ES antigen, cloning by adopting a limiting dilution method, purifying McAb by applying an octanoic acid-ammonia sulphate method, determining the concentration and sub-packaging for later use. The invention has the advantage that a new method which has high sensitivity and specificity and has wide application prospect is provided for early detection of trichinella CAg in blood serum of an infected animal.

Description

technical field [0001] The present invention relates to biology, parasitology, serology and immunology technology, especially relates to egg yolk immunoglobulin (Egg Yolk Immunoglobulin, IgY)-monoclonal antibody (Monoclonal Antibody, McAb) sandwich ELISA (Enzyme Linked Immunosorbent Assay, ELISA) is a method for detecting circulating antigens of Trichinella spiralis in serum. Background technique [0002] Trichinellosis (trichinellosis) is a worldwide distribution of zoonotic parasitic diseases that seriously endanger human health, mainly caused by eating raw or semi-raw meat and its products containing infective larvae of Trichinella spiralis. More than 150 species of animals are known to be naturally infected with Trichinella spiralis ( Trichinella spiralis ), popular in 66 countries (or regions). In my country, except Hainan Province, other provinces, municipalities and autonomous regions have reported that animals are infected with Trichinella spiralis. In recent years...

AI Technical Summary

Problems solved by technology

Because the traditional detection method of Trichinella spiralis infection mainly relies on muscle biopsy, the detection rate of this method is low and it is difficult to implement. Trichinella antibodies can only be detected 3 to 4 weeks after infection, and at this time some severely infected patients may have died due to acute complications, so the detection of trichinella antibodies cannot be used for early diagnosis of trichinellosis

The results of the study showed that the Excretory-Secretory (ES) and the sheds on the surface of Trichinella spiralis larvae during the migration and development of the host can enter the blood and become Circulating Antigens (CAg). Antigen-like content is proportional to the intensity of infection, and its appearance time is earlier than that of specific antibodies, and its half-life is also significantly shorter than that of specific antibodies. Therefore, the detection of CAg in serum is expected to be used for early detection, past and current However, the CAg content in serum is usually low, and the detection rate is only 19%-54% when using conventional methods, which greatly limits the popularization and application of CAg detection. Therefore, it is urgent to establish A New Method for Detecting CAg of Trichinella spiralis with High Sensitivity and Specificity

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