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Fluorescence polarization immunoassay method for detecting SM2 (sulfamethazine) on basis of egg yolk antibodies

A sulfamethazine, egg yolk antibody technology, applied in analytical materials, measuring devices, instruments, etc., can solve the problems of low production of monoclonal antibodies, large animal damage, complicated operation, etc., to overcome the high production cost and animal damage. Small, stable effect

Inactive Publication Date: 2015-12-16
CHINA AGRI UNIV
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  • Description
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  • Application Information

AI Technical Summary

Problems solved by technology

[0004] The purpose of the present invention is to establish a sulfamethazine based on egg yolk antibody in view of the shortcomings of the existing sulfamethazine detection technology, such as complicated operation, long time consumption, low yield of monoclonal antibody, and great damage to animals during collection. Fluorescence polarization immunoassay for pyrimidines

Method used

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  • Fluorescence polarization immunoassay method for detecting SM2 (sulfamethazine) on basis of egg yolk antibodies
  • Fluorescence polarization immunoassay method for detecting SM2 (sulfamethazine) on basis of egg yolk antibodies
  • Fluorescence polarization immunoassay method for detecting SM2 (sulfamethazine) on basis of egg yolk antibodies

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Experimental program
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Effect test

preparation example Construction

[0038] The preparation method of the yolk antibody used in the following examples is as follows: use sulfamethazine as the immunogen to carry out multi-point immunization on the subcutaneous back of laying hens, the immunization dose is 0.3mL (the concentration of the immunogen is 1mg / mL), Afterwards, booster immunizations are carried out at intervals of 2-3 weeks each time, the immunization dose is halved, and the number of immunizations is seven times.

[0039] The change of the titer of the prepared yolk antibody to sulfamethazine is as follows: after the first three booster immunizations, the titer increased significantly until the titer of the antibody was 1:2 16 , decreased slightly after a period of time, but reached the highest titer of 1:2 after the fourth booster immunization 18 , and remained at this level thereafter. After the last booster immunization, the titer of egg yolk antibody can maintain a high level for 30 weeks.

example 1

[0040] Example 1, sulfamethazine fluorescent marker SM 2 - Preparation of GA-EDF

[0041] Step 1: Using fluorescein isothiocyanate and ethylenediamine as raw materials, fluoresceinthiocarbamylethylenediamine (EDF) was synthesized. The specific synthesis method is: take 60 mg of FITC and dissolve it in 10 mL of methanol solution, and then add Mix 100 μL triethylamine; add dropwise into 50 mL methanol solution containing 100 mg ethylenediamine, shake while adding, and continue shaking for 1 hour after dropping; filter the above mixed solution, and wash the orange color attached to the filter paper with 10 mL methanol Precipitate; store the obtained precipitate in a dry place protected from light.

[0042] Step 2: Hapten SM 2 - Preparation of GA

[0043] 2.8-3.2mg SM 2 Dissolve in 250uL chloroform, add 1.3-1.7mg glutaric anhydride, stir at room temperature for 8-10 hours to obtain hapten SM 2 -GA, SM can be obtained after rotary evaporation of the obtained reaction solution ...

example 2

[0046] Example 2, sulfamethazine fluorescent marker (SM 2 -FITC, SMR-FITC, SMX-FITC, SM 2 - Preparation of DTAF, SMR-DTAF, SMX-DTAF)

[0047] to SM 2 Synthesis of -FITC as an example:

[0048] Weigh 5mgSM 2 Dissolve in 250 μL of methanol, add 25 μL of triethylamine; after dissolving, add 4 mg of FITC until the red solution is completely dissolved; react overnight in the dark at room temperature; take 50 μL of the reaction solution and separate it with a thin-layer chromatography plate, and the selected developing agent is dichloromethane : Methanol (4:1, V / V); the yellow band with Rf=0.05 was scraped off, extracted with 1 mL of methanol, and the resulting liquid was stored at 4°C. SMR-FITC, SMX-FITC, SM 2 -DTAF, SMR-DTAF, and SMX-DTAF have similar synthesis methods.

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Abstract

The invention provides a fluorescence polarization immunoassay method for detecting SM2 (sulfamethazine) on the basis of egg yolk antibodies. A proper SM2 hapten is selected to be coupled with specific fluorescein, and various fluorescence indicators are synthesized; the egg yolk antibodies are adopted to replace traditional monoclonal and polyclonal antibodies; a competitive immune reaction is established through the fluorescence indicators, the egg yolk antibodies of SM2 and a standard substance, and a standard curve is drawn; a to-be-tested sample has the competitive immune reaction with the fluorescence indicators and the egg yolk antibodies of SM2, a fluorescence polarization value of the system is obtained through measurement, and the concentration of SM2 in the to-be-tested sample is acquired. According to the method, all that is required is to add a sample, separation and washing operations are not required, and a detection result can be acquired in ten minutes. The stability of the egg yolk antibodies of SM2 is high, the yield is high, and little harm is caused to animals; the defects that manufacturing costs of monoclonal antibodies and polyclonal antibodies required in a traditional method are high, animal welfare requirements cannot be met and the like are overcome.

Description

technical field [0001] The invention belongs to the field of immune analysis technology and veterinary drug residue detection, and in particular relates to a fluorescent polarization immunoanalysis method for detecting sulfamethazine based on egg yolk antibody. Background technique [0002] Sulfamethazine (Sulfamethazine, SM 2 ) is a synthetic broad-spectrum antibacterial drug that has inhibitory effects on most Gram-positive and Gram-negative bacteria. The treatment of bacterial diseases such as respiratory tract infection and intestinal infection caused by it. Therefore, it is widely used in animal husbandry, and the residue phenomenon in animal food is relatively serious. And it has a strong toxic effect, long-term or excessive use can cause damage to the urinary system, hematopoietic system, nervous system, etc., and even has the possibility of carcinogenesis. Both my country and the European Union have stipulated that the maximum residue limit of the total amount of ...

Claims

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Application Information

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IPC IPC(8): G01N33/533
CPCG01N33/533
Inventor 王战辉沈建忠盛雅洁张会艳张素霞温凯史为民江海洋
Owner CHINA AGRI UNIV
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