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Isokwas tenuazonowy artificial antigen and antibody as well as preparation method and application thereof

A technology of streptosporin and artificial antigen, which is applied in the field of artificial antigen and antibody of streptomycin and its preparation, and achieves the effects of simple operation, high specificity and high accuracy

Active Publication Date: 2014-05-14
SOUTH CHINA AGRI UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0004] The technical problem to be solved in the present invention is to overcome the existing technical deficiencies in the detection of alternarinic acid, to provide an artificial antigen and antibody to alternarinic acid and its preparation method and application

Method used

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  • Isokwas tenuazonowy artificial antigen and antibody as well as preparation method and application thereof
  • Isokwas tenuazonowy artificial antigen and antibody as well as preparation method and application thereof
  • Isokwas tenuazonowy artificial antigen and antibody as well as preparation method and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0047] Example 1 Synthesis and Identification of Immunogen ITeAHGA-BSA

[0048] 1. Synthesis of enzyme-linked protein by active ester method

[0049] S1. Dissolve 1 mmol of the hapten ITeAHGA in 500 μL of DMF, stir and add 3 mmol of DCC and 4 mmol of N-hydroxysuccinimide (NHS). Magnetic stirring was carried out at 4°C overnight, and the supernatant was taken after centrifugation as solution A;

[0050] S2. Weigh 1 / 80 mmol of BSA and dissolve it in PBS with a pH value of 8.0, stir and dissolve to obtain liquid B;

[0051] S3. Under magnetic stirring at 4°C, gradually drop all the obtained liquid A into the obtained liquid B, react at 4°C for 12 hours, and dialyze with physiological saline at 4°C for 3 days to obtain alternaria isoic acid ITeAHGA-BSA Artificial antigen (immunogen), the structural formula is attached figure 1 shown.

[0052] The resulting immunogen was divided into small tubes at 1 mg / mL and stored at -20°C.

[0053] 2. Identification of ITeAHGA-BSA artifi...

Embodiment 2

[0055] Example 2 Synthesis and identification of coated original ITeAH-OVA

[0056]1. Synthesis of enzyme-linked protein by glutaraldehyde method

[0057] S1. Weigh 1mmol of the hapten ITeAH and 1 / 80mmol of OVA and dissolve them together in a PBS solution with a pH value of 8.0, stir evenly at 25°C, add 50-200μL of 25% (volume ratio) glutaraldehyde drop by drop into the above mixed solution, and stirred at 25°C for 3h after the dropwise addition.

[0058] S2. After the reaction, take the supernatant and put it into a dialysis bag (molecular weight cut-off 10000Da), and dialyze it with 0.1mol / L, pH8.0 PBS solution for three days at 4°C, and change the dialysate every 12 hours to obtain isofine Alternaris acid ITeAH-OVA artificial antigen (coating source), the structural formula is attached figure 2 shown.

[0059] The obtained coated original 1mg / mL was divided into small tubes and stored at -20°C.

[0060] 2. Identification of ITeAH-OVA artificial antigen (coating sourc...

Embodiment 3

[0062] Example 3 Preparation and Identification of Monoclonal Antibody to Alternaris Acid

[0063] 1. Animal immunity

[0064] In this example, four 7-week-old, healthy female Balb / c mice (purchased from Guangdong Medical Experimental Animal Center) were selected and numbered respectively. Immunized with the immunogen ITeAHGA-BSA prepared in Example 1.

[0065] First immunization: Take 0.5 μg of artificial antigen ITeAHGA-BSA and emulsify with an equal volume of complete Freund's adjuvant, and adopt multi-point immunization in the abdomen.

[0066] Booster immunization: After the first immunization, take 0.5 μg of artificial antigen and emulsify with the same amount of Freund's incomplete adjuvant, and boost the immunization at intervals of two weeks. One week after each booster immunization, blood was collected from the tail, and the antiserum titer was determined by ELISA method.

[0067] After the titer stabilized, select a mouse with the best immune effect to boost th...

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Abstract

The invention provides an isokwas tenuazonowy artificial antigen and an antibody as well as a preparation method and an application thereof, which belongs to the field of food security. The molecular structure of the isokwas tenuazonowy artificial antigen is shown in the specification. The isokwas tenuazonowy artificial antigen is prepared by coupling carrier protein by using a glutaraldehyde method or by using an active ester method. The titer of the antibody prepared from the artificial antigen provided by the invention can be 1:32,000, the lowest detection limit is 0.01ng / mL, the half inhibiting concentration is 0.77ng / mL, the generated antibody has the advantages of high specificity, high sensitivity and high accuracy, thereby laying a foundation for building up a rapid, accurate and sensitive isokwas tenuazonowy immunodetection method.

Description

Technical field [0001] The invention involves food safety.More specifically, it involves an artificial antigen and antibodies and preparation methods and applications of alien sported spores. Background technique [0002] ISO-Tenuazonic Acid (ITEA, Chemical Name, 3-acetyl-5-lycopillin-2-ketone), which is a secondary metabolism generated by chain cellsOne of the toxins in the object is also a tetra valence compound with Tenuzonicacid (TEA).As a similar division of the sported spore ketone acid, it has a toxicity similar to TEA and can be secreted by chains at the same time. It has reported that the toxin has antitumoric, weak antiviral activity, and broadSpectrum, fast, efficient weeding and other biological activity.In recent years, the further biology and toxicology research of various countries has found that the situation of the toxins contaminated crops (such as tomatoes, apples, grains, etc.) is quite common, but because people's average daily intake is very low, it fails to...

Claims

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Application Information

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IPC IPC(8): C07D207/38C07K14/765C07K14/77C07K14/795C07K16/14G01N33/569
CPCC07D207/38C07K1/1077C07K14/765C07K14/77C07K14/795C07K16/44C07K19/00G01N33/56911
Inventor 王弘孙远明王雅丽沈玉栋肖治理杨星星杨金易徐振林雷红涛
Owner SOUTH CHINA AGRI UNIV
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