Ketamine hybridoma cell strain and preparation method and application thereof

A hybridoma cell line, anti-ketamine technology, applied in the direction of biochemical equipment and methods, chemical instruments and methods, material inspection products, etc., can solve problems such as unsatisfactory, sensitivity limit of ketamine monoclonal antibody, etc., and achieve high sensitivity effect

Active Publication Date: 2014-07-09
HANGZHOU AORUI BIOMEDICINE TECH
View PDF1 Cites 0 Cited by
  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0005] Ketamine colloidal gold test strips have the advantages of rapidity and simple operation, and provide an effective on-site detection method. Due to the limited sensitivity of its core raw material, ketamine monoclonal antibody, the detection threshold of existing commercial products is around 1000ng / ml , can no longer meet the detection threshold of 100ng / ml, so it is urgent to develop a highly sensitive ketamine antibody to meet the detection needs

Method used

the structure of the environmentally friendly knitted fabric provided by the present invention; figure 2 Flow chart of the yarn wrapping machine for environmentally friendly knitted fabrics and storage devices; image 3 Is the parameter map of the yarn covering machine
View more

Image

Smart Image Click on the blue labels to locate them in the text.
Viewing Examples
Smart Image
  • Ketamine hybridoma cell strain and preparation method and application thereof
  • Ketamine hybridoma cell strain and preparation method and application thereof
  • Ketamine hybridoma cell strain and preparation method and application thereof

Examples

Experimental program
Comparison scheme
Effect test

preparation example Construction

[0049] Step (1). Preparation of ketamine artificial antigen:

[0050] 1-1. Preparation and detection of ketamine hapten: use ketamine hydrochloride as raw material, carry out substitution reaction with ethyl bromoacetate after dissociation to introduce ester group, and obtain ketamine ester; hydrolyze under alkaline conditions to obtain carboxyl-containing ketamine hapten :

[0051] Dissolve 200mg of ketamine hydrochloride in 10ml of deionized water, add ammonia water to adjust the pH value to 10, add 90ml of dichloromethane to extract 3 times, collect the organic phase, dry with anhydrous magnesium sulfate, filter, and evaporate the solvent under reduced pressure to obtain free ketamine ; Free ketamine is detected by thin-layer chromatography: the chromatographic liquid is petroleum ether: ethyl acetate=1:1, product R f =0.3~0.4.

[0052] Dissolve free ketamine in 10ml of acetone, add 121μl of ethyl bromoacetate and 300mg of potassium carbonate, reflux at 70°C for 16h; coo...

Embodiment 2

[0077] Example 2 Performance testing of anti-ketamine monoclonal antibody

[0078] 2.1 Anti-ketamine monoclonal antibody subtype identification

[0079] The subtype of the anti-ketamine monoclonal antibody prepared in Example 1 was identified with reference to Monoclonal antibody Isotyping Kits (pierce #37501). The anti-ketamine monoclonal antibody belonged to the IgG1 subtype, and the light chain was a kappa chain.

[0080] 2.2 Reactivity test of anti-ketamine monoclonal antibody

[0081] The indirect ELISA method was used to detect the anti-ketamine monoclonal antibody prepared in Example 1: Dilute the ketamine artificial antigen (KET-BSA) to 1 ug / mL with a carbonate buffer solution with a pH of 9.6 and a concentration of 0.05M, and then in a 96-well microtiter plate Add 100ul diluted ketamine artificial antigen to each well, and coat overnight at 4°C; then wash the plate 3 times with PBS buffer containing 0.05%tween20; then add 100ul anti-ketamine monoclonal antibody solut...

Embodiment 3

[0084] Example 3 Establishment and Evaluation of Ketamine Colloidal Gold Test Strip Method

[0085] 1. Colloidal Gold Preparation

[0086] 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;

[0087] 2. Colloidal gold-labeled antibody protein

[0088] With 0.1mol / L K 2 CO 3 Adjust the pH value of the colloidal gold solution to 9.0, add 0.5 mg anti-ketamine monoclonal antibody protein dropwise; after continuing to stir 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%, and continue stirring for 10 min. The antibody solution is obtained; wherein the anti-ketamine monoclonal antibody protein is the anti-ketamine mo...

the structure of the environmentally friendly knitted fabric provided by the present invention; figure 2 Flow chart of the yarn wrapping machine for environmentally friendly knitted fabrics and storage devices; image 3 Is the parameter map of the yarn covering machine
Login to view more

PUM

No PUM Login to view more

Abstract

The invention discloses a Ketamine hybridoma cell strain and a preparation method and an application thereof. The detection threshold value of the existing Ketamine colloidal gold test paper strip is 1,000 ng / ml. The hybridoma cell strain is named as hybridoma cell strain 3E7.1 and has the preservation number of CCTCC NO: C2012157. The hybridoma cell strain 3E7.1 is applied to the secretion of an anti-ketamine monoclonal antibody. The preparation method of the anti-ketamine monoclonal antibody secreted by the hybridoma cell strain 3E7.1 comprises the following steps: preparing a ketamine artificial antigen; mice immunization; cell fusion; and screening a hybridoma cell strain 3E7, carrying out clonal culture to obtain the hybridoma cell strain 3E7.1 and purifying to obtain the anti-ketamine monoclonal antibody. The ketamine artificial antigen prepared by the invention facilitates the generation of a ketamine antibody, and the antibody preparation period is short; the anti-ketamine monoclonal antibody secreted by the hybridoma cell strain 3E7.1 has the characteristics of high sensitivity; and the Ketamine hybridoma cell strain is suitable for 1,000 ng / ml-threshed value detection.

Description

technical field [0001] The invention belongs to the field of biotechnology, and specifically relates to an anti-ketamine hybridoma cell line, the application of the anti-ketamine hybridoma cell line as secreting anti-ketamine monoclonal antibody, and preparation of the anti-ketamine hybridoma cell line and secretion of anti-ketamine monoclonal antibody Antibody method. Background technique [0002] Ketamine (KET), the chemical name is 2-o-chlorophenyl-2-methylamino-cyclohexanone, and its structural formula is: [0003] [0004] Ketamine (Ketamine) is a non-barbiturate intravenous anesthetic, mainly used for surgical anesthesia or induction of anesthesia. Ketamine has a certain degree of excitement, hallucinations and dependence. After taking it, it will produce a state of separation of consciousness and sensation, leading to neurotoxic reactions, hallucinations and schizophrenia. ﹑Motor dysfunction, depression, and strange and dangerous behaviors, causing serious damage...

Claims

the structure of the environmentally friendly knitted fabric provided by the present invention; figure 2 Flow chart of the yarn wrapping machine for environmentally friendly knitted fabrics and storage devices; image 3 Is the parameter map of the yarn covering machine
Login to view more

Application Information

Patent Timeline
no application Login to view more
Patent Type & Authority Patents(China)
IPC IPC(8): C12N5/20C07K16/44G01N33/94G01N33/577
Inventor 陈东邵越水郑孝君
Owner HANGZHOU AORUI BIOMEDICINE TECH
Who we serve
  • R&D Engineer
  • R&D Manager
  • IP Professional
Why Eureka
  • Industry Leading Data Capabilities
  • Powerful AI technology
  • Patent DNA Extraction
Social media
Try Eureka
PatSnap group products

Browse by: Latest US Patents, China's latest patents, Technical Efficacy Thesaurus, Application Domain, Technology Topic.

© 2024 PatSnap. All rights reserved.Legal|Privacy policy|Modern Slavery Act Transparency Statement|Sitemap