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Anti-morphine monoclonal antibody, cell line producing the antibody, morphine detection kit and production method thereof

A monoclonal antibody and kit technology, applied in the biological field, can solve the problems of late window period, inconvenient sampling, dirty samples, etc., and achieve the effect of detection limit advantage, strong affinity and high specificity

Active Publication Date: 2016-09-28
HANGZHOU CLONGENE BIOTECH
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

The traditional morphine detection kit uses urine as the detection sample, which has the disadvantages of dirty sample, inconvenient sampling, late window period, and easy adulteration. The new saliva detection kit overcomes the above shortcomings and can be detected within 24 hours after drug abuse , can replace traditional urine testing kits, and meet the needs of drug testing in public security, conscription, entry and exit, drug rehabilitation centers, etc.

Method used

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  • Anti-morphine monoclonal antibody, cell line producing the antibody, morphine detection kit and production method thereof
  • Anti-morphine monoclonal antibody, cell line producing the antibody, morphine detection kit and production method thereof
  • Anti-morphine monoclonal antibody, cell line producing the antibody, morphine detection kit and production method thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0041] Embodiment 1: Preparation of morphine artificial antigen

[0042]

[0043] The above formula is the synthetic route of morphine artificial antigen.

[0044] 1-1. Preparation of morphine hapten: use morphine hydrochloride as raw material, carry out nucleophilic substitution reaction with ethyl bromoacetate after dissociation, and obtain carboxyl-containing morphine hapten.

[0045] Dissolve 350mg of morphine hydrochloride in 10ml of water, adjust the pH to 10 with concentrated ammonia water, a large amount of solids precipitate, place it at 4°C for 30 minutes, and centrifuge at 10,000 rpm to remove the clear liquid The water and ethanol were transferred to a round bottom flask, and concentrated under reduced pressure at 50°C to obtain 288 mg of white solid morphine.

[0046] In a 100ml three-necked flask, 285mg (1mmol) of morphine and 10ml of DMF were added, and under nitrogen protection, 330mg (2.4mmol) of potassium carbonate and 184mg (1.1mmol) of ethyl 4-bromoacet...

Embodiment 2

[0050] Example 2: Screening of anti-morphine monoclonal antibody hybridoma cell lines

[0051] Step (1) mouse immunization

[0052] Morphine artificial antigen, Freund's adjuvant, and 3% Tween-80 saline were mixed according to the volume of 1:1:1 to obtain emulsion by stirring method. Select 8-week-old BALB / c female mice for subcutaneous immunization combined with intrasplenic and intravenous immunization for a total of 5 immunizations. The immunization time is the 1st day, the 14th day, the 21st day, the 22nd day, and the 23rd day. The specific steps Eight-week-old BALB / c female mice were immunized for the first time, and 50 μg of morphine artificial antigen emulsion (complete Freund's adjuvant was used as the adjuvant) was injected into the subcutaneous part near the lymph nodes; for the second intrasplenic immunization, Morphine artificial antigen emulsion with an immune dose of 20 μg (incomplete Freund’s adjuvant is used as an adjuvant); the third, fourth, and fifth times...

Embodiment 3

[0078] Example 3: Performance testing of anti-morphine monoclonal antibody

[0079] 3.1 Anti-morphine monoclonal antibody subtype identification

[0080]The subtype of the anti-morphine monoclonal antibody prepared in Example 1 was identified with reference to the monoclonal antibody typing kit. The anti-morphine monoclonal antibody belonged to IgG1 subtype, and the light chain was kappa chain.

[0081] 3.2 Determination of affinity constant of anti-morphine monoclonal antibody

[0082] Example of Affinity Determination Using Antibody Competitive Binding to Antigen

[0083] ① Dilute the morphine monoclonal antibody (MOP-BSA) to 1 μg / ml with carbonate buffer solution with a pH of 9.6 and a concentration of 0.05M, and then add 100 μl of diluted morphine artificial antigen to each well of a 96-well microtiter plate, Coat overnight at 4°C (2 plates); then wash the plate 3 times with PBS buffer containing 0.05% Tween-20 and pat dry; ② Add 200 μl 0.01mol / L pH7.2 phosphate containi...

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Abstract

The invention provides a hybridoma cell strain MOP1.0 capable of generating an anti-morphine monoclonal antibody, wherein the cell strain is preserved in China Center for Type Culture Collection with a preservation number of CCTCCNO. C2013159. The invention also provides the anti-morphine monoclonal antibody secreted by the hybridoma cell strain MOP1.0. The invention also provides a kit for detecting morphine in saliva. The invention further provides a detection method and a manufacturing method of the morphine detection kit. The hybridoma cell strain MOP1.0 has the benefit that the artificial antigen of obtained morphine keeps the structural specificity of morphine by utilizing ethyl bromoacetate as a cross-linking agent to activate the morphine. Through the adoption of a sandwich immune method, the using amount of antigens is effectively reduced, and the immunization time is shortened. The hybridoma cell strain MOP1.0 is high in secretion yield, and the anti-morphine monoclonal antibody obtained through secretion of the hybridoma cell strain MOP1.0 has the characteristics of high affinity, high specificity and high sensitivity. A detection product provided by the invention is more advantageous in the aspects of sensibility, specificity, detection limit and the like.

Description

technical field [0001] The invention belongs to the field of biotechnology, and in particular relates to an anti-morphine monoclonal antibody and a hybridoma cell line secreting and producing the antibody. [0002] The invention also relates to a morphine detection kit and a production method thereof. Background technique [0003] Morphine is the main component of opium, heroin, etc. It has analgesic and hypnotic effects. In medicine, it has always been a narcotic analgesic. It is regarded as the most effective traditional analgesic for relieving severe pain. Its hydrochloride, Sulfate, acetate and tartrate forms. The biggest disadvantage of morphine is that it is easy to become addicted, so it is abused. Morphine has the function of inhibiting the central nervous system, and eaters are prone to tolerance and physical dependence. After taking it, the human body will produce a sense of euphoria and hallucinations. Once you stop using it, the functions of the top ten syste...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N5/20C07K16/44G01N33/94G01N33/577G01N33/558
Inventor 殷秀飞陈国琴郑曙剑
Owner HANGZHOU CLONGENE BIOTECH
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