Hybridoma cell strain, anti-salbutamol monoclonal antibody generated by hybridoma cell strain and application

A hybridoma cell line and monoclonal antibody technology, applied in the field of immunochemistry, can solve the problems of low reliability, low specificity and high technical requirements of detection results, and achieve simple method, good specificity and low cross-reaction rate. Effect

Active Publication Date: 2013-10-09
江苏奥的特生物技术有限公司
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AI Technical Summary

Problems solved by technology

Instrumental analysis relies on instruments with high technical requirements, long detection time, and low recovery rate. The cost is high and the reliability of the detection results is not high.
The anti-salbutamol monoclonal antibody used in the immunoassay technique has low specificity, and the cross-reactivity rate with the salbutamol analog clenbuterol reaches 50.04%, and the cross-reaction rate with ractopamine reaches 50%. The use of the kit is limited, that is, once the test is positive, it is still necessary to further determine which substance exceeds the standard

Method used

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  • Hybridoma cell strain, anti-salbutamol monoclonal antibody generated by hybridoma cell strain and application
  • Hybridoma cell strain, anti-salbutamol monoclonal antibody generated by hybridoma cell strain and application
  • Hybridoma cell strain, anti-salbutamol monoclonal antibody generated by hybridoma cell strain and application

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Experimental program
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Effect test

Embodiment 1

[0033] The preparation of embodiment 1 immunogen and coating former

[0034] Preparation of immunogen (Sal-BSA, Sal-HSA) and coating agent (Sal-OVA) by EDC method:

[0035] 1. Preparation of immunogens Sal-BSA and Sal-HSA

[0036] (1) Weigh 20 mg of BSA / HSA and dissolve it in 4 mL of PBS buffer solution with a concentration of 0.01M and pH 7.4 to obtain liquid A;

[0037] (2) Weigh 17mg EDC and dissolve it in 200uL distilled water to obtain liquid B;

[0038] (3) Weigh 10mg of albuterol sulfate and dissolve it in 200uL of PBS buffer solution with a concentration of 0.01M and pH7.4 to obtain liquid C;

[0039] (4) Slowly add liquid B to liquid C dropwise for 10 minutes. After the dropwise addition, shake at 15°C for 1 hour to obtain a mixed reaction solution.

[0040] (5) Slowly add the mixed reaction liquid in step (4) into liquid A for 20 minutes, shake at 15°C after the addition, and react for 2 hours to obtain the reaction product.

[0041] (6) Dialyze the reaction prod...

Embodiment 2

[0053] Embodiment 2 Preparation of anti-salbutamol monoclonal antibody

[0054] an animal immune

[0055] Balb / c mice were used as immunized animals, the immunogen was Sal-HSA or Sal-BSA, each immunization dose was 50 μg / mouse, and immunized three times.

[0056] 2 Screening of immunized mouse serum

[0057] 1. The above-mentioned immunized mice were 7-10 days after the last immunization, and the serum antibody titer was measured by indirect ELISA method, and the sensitivity was measured by indirect competitive ELISA method (Yang Liguo, "Enzyme Immunoassay Technology", Nanjing University Press, 1998). Mice with high serum titers and high sensitivity were selected for booster immunization.

[0058] 2. Indirect ELISA and indirect competitive ELISA

[0059] Coating buffer: 0.05mol / L, carbonate (Na 2 CO 3 -NaHCO 3 ).

[0060] Buffer A: 0.01 mol / L, pH7.4 PBS buffer.

[0061] Washing solution: Add Tween at a final concentration of 1‰ (volume concentration) to buffer A.

[0...

Embodiment 3

[0137] Embodiment 3 drug cross reaction test

[0138] According to the indirect competition ELISA method in Example 2, the anti-albuterol monoclonal antibody 8D7' was subjected to competition tests with albuterol hemisuccinic acid structural analogues such as clenbuterol and ractopamine. Dilute these standards into different concentrations for indirect competition ELISA experiments, draw inhibition curves, and calculate ICs of competitors 50 Value and cross-reaction rate (cross-reaction rate = salbutamol IC50 value ÷ IC50 value of the substance to be tested × 100%), the highest cross-reaction rate is 2.7%. The monoclonal antibody of salbutamol reported in the literature and the cross-reactivity rate of clenbuterol reach 50.04%, and the monoclonal antibody of anti-salbutamol prepared by the present invention only reacts with salbutamol, therefore, prepared anti-salbutamol monoclonal antibody of the present invention The cloned antibody 8D7' has higher specificity, and the spec...

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Abstract

The invention provides a hybridoma cell strain, an anti-salbutamol monoclonal antibody generated by the hybridoma cell strain and application, and belongs to the technical field of immunochemistry. The invention discloses a hybridoma cell strain 8D7 which generates the anti-salbutamol monoclonal antibody and is stored with a number of CGMCC (China General Microbiological Culture Collection Center) No., 7953, an anti-salbutamol monoclonal antibody 8D7' secreted by the hybridoma cell strain 8D7, and application of the anti-salbutamol monoclonal antibody in preparation of an enzyme linked immunosorbent assay kit and a colloidal gold test strip used for detecting salbutamol. The hybridoma cell strain 8D7 can efficiently and stably secrete the anti-salbutamol monoclonal antibody; the anti-salbutamol monoclonal antibody has high titer, specificity and sensitivity, can be produced as mass, and realizes the effect of quickly and sensitively detecting a salbutamol medicine residual in urine, muscle and viscera. By adopting the anti-salbutamol monoclonal antibody for detecting the salbutamol residual in a sample, toxic agents are not needed during treating the sample, and the operation is simple.

Description

technical field [0001] The invention relates to the technical field of immunochemistry, in particular to a hybridoma cell line, an anti-salbutamol monoclonal antibody produced by it and an application thereof. Background technique [0002] Salbutamol (salbutamol, Sal) is also known as albuterol and surbutamol. Its chemical name is 4-hydroxy-5-hydroxymethyl-α-(tert-butylamine) methylphenylethanolamine, and its molecular formula is C 13 h 21 NO 3 , molecular weight 239, is an oral potent β2 adrenergic stimulant. In human and veterinary clinics, β2 adrenergic stimulants such as clenbuterol and salbutamol are mainly used as drugs for the prevention and treatment of asthma and bronchospasm. In addition, this kind of substance also has a certain nutrient redistribution effect, and is often used as a growth promoter for the production of pigs, cattle, chickens and other livestock and poultry. But this stimulant caused a series of food poisoning incidents in Spain, France, Italy...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N5/20C07K16/18G01N33/577C12R1/91
Inventor 唐宏王晓艳杨利张浩明崔迎利
Owner 江苏奥的特生物技术有限公司
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