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Anti-thiram monoclonal antibody, test paper for fast testing thiram and application thereof

A monoclonal antibody and cloning antibody technology, which is applied in the field of anti-thiram monoclonal antibody and detection of thiram composition, can solve the problems affecting the accuracy of analysis results, high technical requirements for instruments, expensive equipment and other problems, and achieve easy operation , low detection cost, and convenient storage

Inactive Publication Date: 2010-09-08
PEOPLES REPUBLIC OF CHINA BEIJING ENTRY EXIT INSPECTION & QUARANTINE BUREAU +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0004] Chromatographic technology is very effective, accurate and sensitive for thiram detection, but it also has the following disadvantages: (1) sample pretreatment needs to be derivatized, and the procedure is cumbersome and time-consuming; (2) professional technicians are required to operate, and operators must be rich In order to obtain reliable analysis results, it is necessary to understand the various interference factors that affect chromatographic analysis and the advantages and disadvantages of the pretreatment method used; (3) It requires expensive instruments and equipment, and it is difficult for small and medium-sized cities and small and medium-sized enterprises to obtain reliable analysis results; (4) High technical requirements for instrument maintenance and maintenance
Whether the maintenance is proper will directly affect the accuracy of the analysis results; (5) The detection cost is high

Method used

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  • Anti-thiram monoclonal antibody, test paper for fast testing thiram and application thereof
  • Anti-thiram monoclonal antibody, test paper for fast testing thiram and application thereof
  • Anti-thiram monoclonal antibody, test paper for fast testing thiram and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0049] Embodiment 1: the preparation of the immune colloidal gold test paper that detects thiram

[0050] 1. Preparation of thiram double-carrier protein conjugates

[0051] 1) Weigh 40 mg of thiram and dissolve it in 20 mM PBS solution containing 0.02% Tween-20 to prepare a thiram solution.

[0052] 2) Under ice-water bath and magnetic stirring, add thiram solution dropwise to 2.4ml 1mol / l HCl for diazotization (Itaru Y, Kohji I. Enzyme immunoassay forclenbuteol, an β 2 -adrnergicstimulant [J]. Journal ofimmunoassay, 1982, 3 (2): 155-171) reaction, continue to react for 5 minutes after addition.

[0053] 3) Under magnetic stirring, add the diazotization product dropwise to human serum albumin (HSA), bovine serum albumin (BSA) and ovalbumin (OVA) solutions, and adjust the pH value to 9.5 with 11mol / 1NaOH , stirred slowly overnight at 4°C to obtain three linking products, namely thiram-HSA, thiram-BSA, and thiram-OVA.

[0054] 4) The connection product thiram-HSA, thiram-BSA...

Embodiment 2

[0081] Example 2: Sensitivity and specificity detection of anti-thiram double monoclonal antibody

[0082] Coat the thiram double-bovine serum albumin conjugate on the microwell plate, the coating amount is 10ng / well; use the anti-thiram double monoclonal antibody and HRP enzyme to label to obtain the anti-thiram double monoclonal antibody enzyme-labeled reagent , used at 1:1000; PBST (0.5% Tween-20, 0.02M phosphate buffered saline at pH 7.4) was used as washing solution; TMB color development system was used for color development; 0.2M concentrated sulfuric acid was used for color development termination. Use this as a thiram double detection system for detection, and use a microplate reader to read the results.

[0083] Selection buffer (phosphate buffer of 0.01MpH7.4, formula: NaCl 8g, KCl0.2g, NaCl 2 HPO 4 12H 2 O 2.9g, KH 2 PO 4 0.2g, dilute to 1000ml with distilled water), add different contents of thiram and concentration of 3000ppb thiram, ferbam, thiram, propine...

Embodiment 3

[0088] Embodiment 3: the specificity and sensitivity detection of immune colloidal gold test paper

[0089] 1. Specific detection

[0090] The samples with concentration of 3000ppb were prepared by ziram, ferbam, arsine, propineb, mancozeb, dithiocyanomethane, chlorothalonil, promethadine and acetoallin. Directly draw 80 μl and add the immunocolloidal gold test paper for detecting thiram of the present invention. The test results (see table 2) show that detection of ziram, ferbam, methazine, propineb, mancozeb, dithiocyanomethane, chlorothalonil, chlorhydrin, ethyl allicin When taking samples, a red band appears on the test paper detection line, and a red band appears on the quality control line at the same time. There is no cross-reaction between methane, chlorothalonil, promethadine and allicin and the test paper of the present invention, which shows that the test paper of the present invention has good specificity.

[0091] Table 2: the result of specific detection of te...

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Abstract

The invention belongs to the field of pesticide detection and can fast detect the residual pesticide with an immunochemical technology. More particularly, the invention relates to a hybridoma cell line with the preservation number of CGMCC No.3580, and an anti-thiram monoclonal antibody generated by the hybridoma cell line. The invention further relates to a composite for detecting the thiram, a test paper for detecting the thiram, a preparation method of the test paper, an enzyme-coupled immunization agent for detecting the thiram, and application of the anti-thiram monoclonal antibody in detecting the thiram. The monoclonal antibody has high sensitivity and specificity. The test paper has the obvious advantages of high sensitivity, high specificity, convenient operation, fast and exact detection, etc.

Description

technical field [0001] The invention belongs to the field of pesticide detection and uses immunochemical technology to quickly detect residual pesticides. Specifically, the invention relates to a hybridoma cell line and an anti-thiram monoclonal antibody produced by the hybridoma cell line. The present invention also relates to a composition for detecting thiram, a test paper for detecting thiram, a preparation method of the test paper, an enzyme-linked immunosorbent reagent for detecting thiram, and the anti-thiram monoclonal antibody in the detection of thiram Familia in the application. Background technique [0002] Thiram, CAS registration number is 137-26-8 chemical name is tetramethyl thiuram disulfide, belongs to thiocarbamate pesticide, is a kind of fungicide with protective effect, its antibacterial spectrum Wide range, can treat seeds and soil, control cereal smut and seedling blight of various crops, and can control some fruit trees and vegetable diseases after s...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): G01N33/577G01N33/558G01N33/544
Inventor 王金花罗海峰
Owner PEOPLES REPUBLIC OF CHINA BEIJING ENTRY EXIT INSPECTION & QUARANTINE BUREAU
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