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Anti-methadone metabolite eddp hybridoma cell line and its preparation method and application

A hybridoma cell line and a methadone-resistant technology, applied in biochemical equipment and methods, microorganism-based methods, chemical instruments and methods, etc., can solve the problem of low reliability of test results, high technical requirements for instrument analysis, and long detection time. and other problems, to achieve the effect of high sensitivity, good specificity and high sensitivity

Active Publication Date: 2018-04-13
HANGZHOU ALLTEST BIOTECH
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Instrumental analysis relies on high technical requirements, long detection time, high cost, and low reliability of test results

Method used

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  • Anti-methadone metabolite eddp hybridoma cell line and its preparation method and application
  • Anti-methadone metabolite eddp hybridoma cell line and its preparation method and application

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 2

[0057] Example 2 Performance Detection of Anti-Methadone Metabolite EDDP Monoclonal Antibody

[0058] 2.1 Reactivity test of anti-methadone metabolite EDDP monoclonal antibody

[0059] Indirect ELISA method detects the anti-methadone metabolite EDDP monoclonal antibody prepared in Example 1: Dilute the methadone metabolite EDDP artificial antigen (KET-BSA) to 1ug / mL with pH9.6 concentration of 0.05M carbonate buffer, then Add 100ul diluted methadone metabolite EDDP artificial antigen to each well of a 96-well ELISA plate, and coat overnight at 4°C; then wash the plate 3 times with PBS buffer containing 0.05%tween20; then add 100ul concentration of 1ug / 1 ml of anti-methadone metabolite EDDP monoclonal antibody solution, make doubling dilution, and set up a negative control at the same time, after reacting at 37°C for 60 minutes, wash the plate once with PBS buffer containing 0.05%tween20; add PBS buffer to sheep Dilute anti-mouse IgG-HRP to 5000 times volume, then add 100 μl ...

Embodiment 3

[0062] Embodiment 3 Establishment and evaluation of methadone metabolite EDDP colloidal gold test strip method

[0063] 1. Colloidal gold preparation

[0064] First prepare 20ml of 0.1g / L chloroauric acid solution, heat to boiling with a constant temperature electromagnetic stirrer, add 0.4ml of 1% trisodium citrate solution, continue heating and stirring for 10min, and replenish to the original volume with distilled water after cooling to normal temperature, to obtain Colloidal gold solution;

[0065] 2. Colloidal gold-labeled antibody protein

[0066] With 0.1mol / L K 2 CO 3 Adjust the pH value of the colloidal gold solution to 9.0, add dropwise 0.5 mg of anti-methadone metabolite EDDP monoclonal antibody protein; continue stirring for 3 minutes, add dropwise the BSA solution with a mass volume ratio of 5% until the mass volume ratio of the BSA solution is 1%, continue Stir for 10 minutes to obtain an antibody solution; wherein the anti-methadone metabolite EDDP monoclona...

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Abstract

The invention discloses a hybridoma cell strain resistant to methadone metabolite EDDP, which is characterized in that the hybridoma cell strain resistant to methadone metabolite EDDP is named hybridoma cell strain 9G2.5, and was preserved in China Typical Culture Collection Center, the preservation number is CCTCC NO: C201507. The hybridoma cell strain can produce highly specific anti-methadone metabolite EDDP monoclonal antibody, and has high production efficiency. The secreted anti-methadone metabolite EDDP monoclonal antibody has high specificity, sensitivity, and low cross-reaction rate, and can be used to prepare colloidal gold test strips for the detection of methadone metabolite EDDP.

Description

technical field [0001] The invention belongs to the field of biotechnology, and in particular relates to an anti-methadone metabolite EDDP hybridoma cell line and the application of the monoclonal antibody produced therefrom. Background technique [0002] The methadone metabolite EDDP is the biotransformation of methadone in the liver, which forms a pharmacologically inactive pyrrolidine derivative through demethylation, and its chemical name is 2-ethylidene-1,5-dimethyl-3 ,3-Diphenylpyrrolidine (EDDP). [0003] Methadone is a synthetic narcotic drug, which is one of the narcotic drugs strictly controlled by the state. Methadone can be used to treat heroin and opiate addiction. Methadone is a long-acting opioid drug. Unlike heroin and other drugs, drug addicts will not develop drug dependence after taking methadone, that is to say, they will not produce painful symptoms after drug withdrawal. Since methadone is a special drug, Therefore, the custody of it is particularly ...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N5/20C07K16/44G01N33/577C12R1/91
Inventor 苏娟高飞陈东
Owner HANGZHOU ALLTEST BIOTECH
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