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TCMTB monoclonal antibody hybridoma cell strain and application thereof

A hybridoma cell line and antibody cloning technology, which is applied to biochemical equipment and methods, instruments, microorganisms, etc., can solve the problems of high cost, cumbersome operation, and time-consuming, and achieve good detection sensitivity and specificity

Active Publication Date: 2015-11-25
JIANGNAN UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, these methods have the disadvantages of cumbersome operation, time-consuming, and high cost, and cannot realize the rapid detection of a large number of samples. Therefore, it is of great significance to establish a fast and simple detection method for thiocyanate

Method used

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  • TCMTB monoclonal antibody hybridoma cell strain and application thereof
  • TCMTB monoclonal antibody hybridoma cell strain and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0036] The preparation of embodiment 1 hybridoma cell line 1B2

[0037] (1) Synthesis of complete antigen

[0038] Weigh 28.6 mg of TCMTB analogue 2-benzothiazolethioacetic acid (BTAC), 38.3 mg of EDC (carbodiimide), 23.0 mg of NHS (N-hydroxysuccinimide), and dissolve them in 1 mL of anhydrous DMF (called Solution A), stirred at room temperature for 8 h. Weigh 112 mg of BSA (bovine serum albumin) and dissolve it in 20 mL of 0.01 mol / LPBS with pH 7.4 (referred to as solution B). Add solution A to solution B drop by drop at room temperature, and react overnight at room temperature to obtain a conjugate The BTAC-BSA mixture is the complete antigen of thiocyanate, and the complete antigen and uncoupled small molecule hapten are separated by dialysis, and identified by ultraviolet absorption scanning method;

[0039] (2) Animal immunity

[0040] Healthy BALB / c mice aged 6-8 weeks were selected for immunization. After mixing and emulsifying the complete antigen of thiothiocyanat...

Embodiment 2

[0045] The preparation of embodiment 2 thiophene monoclonal antibodies

[0046] Take 8-10 week-old BALB / c mice, and inject 1 mL of paraffin oil into each mouse; 7 days later, each mouse is injected with 1×10 6 Hybridoma cell line 1B2, ascitic fluid was collected from the seventh day, the ascites fluid was purified by octanoic acid-ammonium sulfate method, and the monoclonal antibody obtained was stored at -20°C.

[0047] Determination of the IC of Thiophanidine Monoclonal Antibody Using an Indirect Competitive ELISA 50 It is: 7μg / L, indicating that it has good sensitivity to thiocyanate and can be used for immunoassay detection of thiocyanogen.

Embodiment 3

[0048] The application of embodiment 3 thiophene monoclonal antibody

[0049] The monoclonal antibody prepared by the hybridoma cell line 1B2 through the in vivo ascites was applied to the rapid detection of thiocyanate, and the specific steps were as follows:

[0050] (1) Coating: Dilute the coated original TCMTB-OVA with 0.05M pH9.6 carbonate buffer starting from 2μg / mL, 100μL / well, and react at 37°C for 2h;

[0051] (2) Washing: Pour off the solution in the ELISA plate, spin dry, and wash 3 times with washing solution, 3 minutes each time;

[0052] (3) Blocking: After patting dry, add carbonate buffer containing 0.01% gelatin as blocking solution, 200 μL / well, react at 37°C for 2 hours; wash and dry for later use;

[0053] (4) Adding samples: Dilute the antiserum starting from 1:1000, and add it to the coated wells of each dilution, 100 μL / well, and react at 37°C for 1 hour; after fully washing, wash with PBS containing 0.01% gelatin Add 1:3000 diluted HRP-goat anti-mouse...

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Abstract

The invention relates to a TCMTB monoclonal antibody hybridoma cell strain and application thereof, and belongs to the field of food safety immunology detection. The TCMTB monoclonal antibody hybridoma cell strain 1B2 is preserved in the general microorganism center of the China committee for culture collection of microorganisms, and the collection number is CGMCC No.10862. TCMTB complete antigens and an equal amount of a Freund's immunologic adjuvant are uniformly mixed, and the mixture is subcutaneously injected to the backs of BALB / c mice for immunization. Splenocytes of the immunized mice are fused with myeloma cells of the mice according to a PEG method, and the hybridoma cell strain 1B2 is obtained through indirect ELISA, indirect competition ELISA screening and three times of subcloning. Monoclonal antibodies excreted from the cell strain 1B2 have very good specificity and detection sensitivity (the IC50 value is 7 [mu]g / L) on TCMTB, and can be used for transfer volume detection of TCMTB in food contacted related materials.

Description

technical field [0001] The invention relates to a thiocyanogen monoclonal antibody hybridoma cell line and an application thereof, belonging to the field of food safety immunology detection. Background technique [0002] Thiothiocyanate (TCMTB) is a very economical and effective green fungicide. It belongs to the same type of product as BUSAN30L of Buckmam Company of the United States and BIOCIDEMT30 of Progiven Company of France. Bacteria, fungi and algae all have extremely strong killing and control effects, and have special effects on seed dressing, soaking and spraying to treat diseases such as anthrax, rice blast, damping-off, damping off, and citrus canker. Therefore, thiophene and its Metabolites have large residues in plants and soil. [0003] At present, the methods for detecting thiophene mainly include gas chromatography (GC), liquid chromatography (HPLC), spectrophotometry, electrochemical determination, gas chromatography and other methods. However, these meth...

Claims

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Application Information

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IPC IPC(8): C12N5/20C07K16/44G01N33/577
Inventor 匡华胥传来余丽徐丽广马伟刘丽强宋珊珊吴晓玲
Owner JIANGNAN UNIV
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