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Monoclonal antibody, enzyme-linked immunosorbent assay method and kit for detecting T-2 and HT-2 toxin

An enzyme-linked immunosorbent reagent and monoclonal antibody technology, which is applied in the field of detection analysis and immunology, can solve the problems of poor sensitivity and specificity, low sensitivity, and many steps of antibodies, and achieve simple sample processing methods, high recognition sensitivity, and antibody specificity. good sex effect

Inactive Publication Date: 2012-11-07
HUAZHONG AGRI UNIV
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AI Technical Summary

Problems solved by technology

Another type of antigen is used to detect metabolites of T-2 toxin. Chu et al. (1987) hydrolyzed the R8 side chain of T-2 to generate T-2tetraol and T-2tetraol tetraacetate, and then enzymatically hydrolyzed T-2tetraol Tetraacetate generates the intermediate metabolite 3-AcNEOS of T-2 toxin, and finally synthesizes the complete antigen 3-AcNEOS-HS-BSA to obtain cell line H159B1D5, and the obtained antibody can recognize T-2 toxin and its metabolite (T-2 , Ac-T-2, 3-OH-T-2, DAS), this method is more complicated, and the steps of reaction are more, especially the productive rate of T-2tetraol is lower than 10%, and by-product is many, needs to expend a large amount of T-2 toxin, these defects severely limit the application of this method
1D7 Antibody IC by Yang Chuanhe (1990) 50 It is 180ng / mL, and its sensitivity is relatively low compared with the limit standard of T-2. The sample detection requires cumbersome extraction and purification methods to meet the detection requirements, and requires a long sample processing time, which is not conducive to rapid screening
[0004] It can be seen that the sensitivity and specificity of existing antibodies are poor, and cannot fully meet the detection requirements of T-2 and HT-2 toxins

Method used

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  • Monoclonal antibody, enzyme-linked immunosorbent assay method and kit for detecting T-2 and HT-2 toxin
  • Monoclonal antibody, enzyme-linked immunosorbent assay method and kit for detecting T-2 and HT-2 toxin
  • Monoclonal antibody, enzyme-linked immunosorbent assay method and kit for detecting T-2 and HT-2 toxin

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Experimental program
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Embodiment 1

[0031] The preparation of embodiment 1 immunogen and coating former

[0032] Hapten synthesis: 10 mg of T-2 toxin was dissolved in 0.4 ml of pyridine, 210 mg of HG (or HS) was added, and the reaction was stirred for 4 hours in a steam bath. After the reaction, the solvent was blown dry with nitrogen, and the reactant was redissolved in 5ml of chloroform, washed 4 times with 5ml of water, and then dried with nitrogen, and the product was T-2HG (or T-2HS). The product was transferred to 0.2ml DMF, 10-fold molar concentration of carbodiimide (DCC) and 5-fold molar concentration of N-hydroxysuccinimide (NHS) were added, stirred for 1 h, and dried with nitrogen gas.

[0033] Synthesis of immunogen: transfer the hapten T-2HG into 0.2ml of DMF, add the reaction solution dropwise to 25mg BSA (10ml 0.1mol / L phosphate) solution, stir magnetically for 24h, and put the reaction solution into In a dialysis bag, dialyze in phosphate buffered saline (PBS) for 3 days, and change the dialysat...

Embodiment 2

[0035] The preparation of embodiment 2 monoclonal antibody

[0036] 2.1 Immunization of mice

[0037]Referring to the method in Xue Qingshan's "Principles and Techniques of In Vitro Culture" Science Press, 2001 edition: the T-2HG-BSA conjugate prepared in Example 1 was used as the immunogen to immunize Balb / c female mice. In the first immunization, the antigen emulsified with Freund's complete adjuvant was injected subcutaneously on the back of the neck of the mice, and then the antigen emulsified with Freund's incomplete adjuvant was used for booster immunization every 15 days (the interval between the first immunization and the second immunization was 21 days). After three times of immunization, blood was collected from the tail vein on the 8th day, coagulated at 37°C for 0.5h, left standing at 4°C overnight, and centrifuged at 4000r / min for 5min to separate the serum. The antibody titer was detected by indirect ELISA method, and the specificity of serum to the drug was det...

Embodiment 3

[0042] The establishment of embodiment 3 indirect competition ELISA detection method

[0043] 3.1 Reagent preparation

[0044] Carbonate buffer (pH9.6): Accurately weigh Na 2 CO 3 1.59g, NaHCO 3 2.93g, dissolved in a small amount of ultrapure water, and adjusted to 1000mL.

[0045] Washing solution (pH7.4): Accurately weigh 8.00g of NaCl, KH 2 PO 4 0.20g, Na 2 HPO 4 12H 2 O 2.90g, KCl 0.20g, dissolved in a small amount of ultrapure water, Tween 200.50mL was added, and the volume was adjusted to 1000mL.

[0046] Phosphate buffer (pH7.4): Accurately weigh 8.00g of NaCl, KH 2 PO 4 0.20g, Na 2 HPO 4 12H 2 O 2.90g, KCl 0.20g, dissolved in a small amount of ultrapure water, and the volume was adjusted to 1000mL.

[0047] Blocking solution: Accurately weigh 10.00 g of ovalbumin, add 1000 mL of phosphate buffer, stir and mix until the protein is completely dissolved.

[0048] Substrate mixture: Accurately absorb 10 mL of Substrate B solution (purchased from Wuhan Feiyua...

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Abstract

The invention discloses a monoclonal antibody capable of identifying T-2 and HT-2 toxin. The monoclonal antibody is secreted by a hybridoma cell T-2, which is preserved in China Center for Type Culture Collection, and has a preservation number of CCTCC NO:C201189. The invention also discloses an enzyme-linked immunosorbent assay method and a kit for detecting T-2 and HT-2 toxin, and application thereof to detection of HT-2 and T-2 toxin. Compared with prior art, the monoclonal antibody prepared by the invention has high identification sensitivity, good antibody specificity, IC50 values of 1.46 mug / L and 26.26 mug / L; the enzyme-linked immunosorbent assay method and the kit established by the invention have high detection precision and good accuracy; and a sample treatment method is simple and beneficial for rapid screening.

Description

technical field [0001] The invention belongs to the technical field of detection analysis and immunology, and in particular relates to a monoclonal antibody for detecting T-2 and HT-2 toxins, an ELISA method and a kit. Background technique [0002] T-2 toxin and HT-2 toxin are class A trichothecenes, produced by Fusarium Poaea (F.poae) and Fusarium sporotrichioides (F.sporotrichioides) It is produced under storage conditions and widely exists in nature. It contaminates corn, wheat, oats, rye and other grains, and corn pollution is the most common. T-2 toxin and HT-2 toxin can inhibit the synthesis of DNA, RNA and protein, interfere with the energy metabolism of organisms, have carcinogenicity and teratogenicity, and are extremely harmful to humans and animals. -2 and HT-2 toxin detection methods are very important. The maximum tolerated daily intake (PMTDI) of T-2 and HT-2 toxins stipulated by the FAO / WHO Joint Expert Committee on Food Additives (JACFA) is 60ngKg (JECFA, 2...

Claims

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Application Information

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IPC IPC(8): C07K16/14G01N33/577
Inventor 袁宗辉常芳芳彭大鹏王玉莲陈冬梅刘振利
Owner HUAZHONG AGRI UNIV
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