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Hybridoma cell strain YNEMC3, monoclonal antibody generated thereby, and application of monoclonal antibody

A hybridoma cell line and monoclonal antibody technology, applied in the biological field, can solve the problems of inability to distinguish microcystins, false positives in screening, poor selectivity of monoclonal antibodies, etc., achieve high sensitivity, eliminate false positives in screening, and quantitatively accurate Effect

Inactive Publication Date: 2013-10-16
云南省环境监测中心站
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  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

The existing domestic kits for MC-LR detection are mainly provided by the Institute of Hydrobiology, Chinese Academy of Sciences and Jiangsu Suwei Microbial Research Co., Ltd. Formed by coupling protein on the carboxyl group, such monoclonal antibody has poor selectivity, cannot distinguish various isomers of microcystin, and can only obtain the total amount of microcystin
Second, the monoclonal antibodies prepared in the past were all immunized with carrier protein-coupled microcystin to mice, and then used another carrier protein as a detection antigen to screen positive hybridoma cell lines resistant to microcystin. Possibility of false-positive screening caused by the same antigenic epitope of two carrier proteins

Method used

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  • Hybridoma cell strain YNEMC3, monoclonal antibody generated thereby, and application of monoclonal antibody
  • Hybridoma cell strain YNEMC3, monoclonal antibody generated thereby, and application of monoclonal antibody
  • Hybridoma cell strain YNEMC3, monoclonal antibody generated thereby, and application of monoclonal antibody

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0021] Example 1: Preparation of hybridoma cell line YNEMC3 (CCTCC NO: C201298)

[0022] 1. Preparation of Microcystin LR

[0023] Fresh algae samples were taken from Dianchi Lake, screened and freeze-dried to obtain cyanobacterial algae powder; the cyanobacterial cell wall was destroyed with acetic acid solution, so that algal toxins overflowed the cells; using SPEC 18 The column is concentrated and enriched to obtain the algal toxin extract; the algal toxin extract is separated by liquid chromatography to collect the MC-LR separation solution; the separation solution is concentrated and then used with C 18 Column purification, collecting the crude MC-LR extract; further separation by fluorescence-silica gel-thin layer plate to obtain refined microcystins; detected by liquid chromatography-mass spectrometer (LC-MS) with PDA detector , under the reference of MC-LR standard sample, the external standard method is used for quantification, and the purity and concentration of MC-...

Embodiment 2

[0061] Example 2: Preparation of Microcystin LR-Specific Monoclonal Antibody

[0062] 1. Preparation of ascites: Female Balb / c mice were selected, and 0.5 mL of liquid paraffin was intraperitoneally injected into the mice one week before the inoculation of hybridoma cells. After the hybridoma cells screened in step 5 of Example 1 were expanded and cultured, the supernatant was discarded by centrifugation, and the cell concentration was adjusted to 2 × 10 6 per mouse / mL, each mouse was intraperitoneally injected with 0.5 mL;

[0063] On the 13th day after the inoculation of hybridoma cells, the abdomen of the mice was obviously distended. After disinfecting the skin of the lower abdomen with alcohol cotton balls, the ascites was extracted with a 5mL syringe with a needle. Place the collected ascites in a centrifuge tube at 5000rpm for 10min, and collect the supernatant, which is the ascites containing monoclonal antibodies. Add 50% glycerol to the obtained ascites and place th...

Embodiment 3

[0072] Example 3: Competitive ELISA detection of microcystin LR

[0073] Using the extracted and purified MC-LR as the antigen-coated ELISA plate, the detection results of the competition ELISA between the monoclonal antibody secreted by the hybridoma cell line YNEMC3 and the MC-LR standard of different concentrations showed good experimental results, and the detection limit was 0.1 μg / L. Four wells were set for each concentration, and the corrected absorbance value after deducting the average absorbance value of the blank control from the average absorbance value of the four wells was the ordinate, and the logarithm of the concentration was the abscissa to draw a curve. The results are shown in Figure 5 . The results in the figure show that the monoclonal antibody MC-LR prepared with the hybridoma cell line YNEMC3 has a linear relationship with the competitive ELISA detection results of MC-LR standard samples of different concentrations, indicating that the prepared monoclo...

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Abstract

The invention discloses a hybridoma cell strain YNEMC3 and a monoclonal antibody generated thereby. The preservation number of the murine microcystin LR monoclonal antibody hybridoma cell strain YNEMC3 in China Center for Type Culture Collection is CCTCC NO:C201298. The results of experiments about the application of the monoclonal antibody in the murine microcystin LR detection show that the monoclonal antibody is specific to the murine microcystin LR and greatly eliminates the interferences of other microcystins in order to obtain an accurate determination result.

Description

technical field [0001] The invention provides a hybridoma cell line YNEMC3, a monoclonal antibody secreted and produced by the hybridoma cell line and applications thereof, belonging to the field of biotechnology. Background technique [0002] The eutrophication of lakes leads to the abnormal proliferation of algae and the release of biological toxins (Algae Toxin), these secondary metabolites seriously endanger the safety of human water and other aquatic organisms. Among them, Microcystins (MCs) produced by Microcystis (Microcystis) have attracted widespread attention. Microcystin-LR (Microcystin-LR, MC-LR) is currently known to be the most acutely toxic and most harmful freshwater cyanobacterial toxin. Its chemical structure is shown in formula 1: [0003] [0004] At present, most of the detection methods of algal toxins in water are HPLC or LC-MS. This type of detection technology has cumbersome sample pretreatment, long detection time, high cost, high technical req...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N5/20C07K16/14G01N33/577C12R1/91
Inventor 杨良李作生金玉张爱张婉静张榆霞李颖黄微王志慧
Owner 云南省环境监测中心站
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