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Amantadine monoclonal antibody hybridoma cell strain and application thereof

A hybridoma cell line and monoclonal antibody technology, applied in biochemical equipment and methods, instruments, microorganisms, etc., can solve the problems of cumbersome processing, expensive instruments, and complicated operations

Inactive Publication Date: 2015-12-02
JIANGNAN UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Although the instrument detection method is accurate, the instrument is expensive, the operation is complicated, and the sample pretreatment is cumbersome

Method used

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  • Amantadine monoclonal antibody hybridoma cell strain and application thereof

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Experimental program
Comparison scheme
Effect test

Embodiment 1

[0023] Embodiment 1: the preparation of hybridoma cell line 1E5

[0024] (1) Synthesis of complete antigen

[0025] Add amantadine 6-bromohexanoic acid to toluene, heat up to 130°C and reflux for 48 hours under nitrogen protection; concentrate under reduced pressure, add water, adjust the pH of the aqueous phase to about 2 with hydrochloric acid, extract with ethyl acetate, wash with saturated NaCl, Dry and concentrate to obtain the product amantadine hapten. Take 10mg of amantadine hapten, add 2mL of ultrapure water to dissolve, then add NHS (N-hydroxysuccinimide) and EDC (carbodiimide) respectively; the molar ratio of hapten:NHS:EDC is 1:2 ︰2, at room temperature, mix well, stir for 6h, then react for 12h at room temperature 25°C for activation. Take 25mg bovine serum albumin, the molar ratio of hapten and bovine serum albumin is 80:1, add 5mL0.1MpH9.6 carbonate buffer. The activated amantadine hapten solution was slowly added dropwise to the protein solution, and reacted...

Embodiment 2

[0033] Example 2 Preparation and Identification of Monoclonal Antibody

[0034] Take 8-10 week-old BALB / c mice, and inject 1 mL of paraffin oil into each mouse; 7 days later, each mouse is injected with 1×10 6 Hybridoma cell line 1E5, ascitic fluid was collected from the seventh day, the ascites fluid was purified by octanoic acid-ammonium sulfate method, and the monoclonal antibody obtained was stored at -20°C.

[0035] Determination of the IC of monoclonal antibodies against amantadine using indirect competition ELISA and indirect ELISA 50 1ng / mL, the affinity constant is 8.04×10 9 L / mol, indicating that it has good sensitivity and affinity for amantadine, and can be used for amantadine total immunoassay detection and the preparation of an affinity column.

Embodiment 3

[0036] Example 3 Antibody Application

[0037] The amantadine monoclonal antibody hybridoma cell line 1E5 is applied to the ELISA addition recovery test of amantadine by the monoclonal antibody prepared by in vivo ascites, and the specific steps are as follows:

[0038] (1) Coat 96-well ELISA plate with 1.5 μg / mL Am-OVA diluted with carbonate buffer solution (CBS) as the original coating, 100 μL per well, after coating at 37°C for 2 hours, wash the plate with PBST washing solution Three times, 250 μL per well each time, 3 min each time, pat dry;

[0039] (2) Block with CBS containing 0.01% gelatin, 200 μL per well, block for 2 hours at 37°C, wash the plate three times with PBST washing solution, 250 μL per well for 3 minutes each time, and pat dry;

[0040] (3) Prepare amantadine standard solutions of 0, 0.03, 0.1, 0.3, 1, 3, 9, and 27 ng / mL in phosphate buffered saline (PBS). Add the amantadine standard solution and the sample extract to be tested to the sealed ELISA plate,...

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Abstract

The invention discloses an amantadine monoclonal antibody hybridoma cell strain and application thereof, and belongs to the field of immunological detection in food safety. After amantadine complete antigens and equivalent Freund's immunologic adjuvants TM are mixed evenly, BALB / c mice are immunized through back multipoint injection. Splenocytes of the immune mice are fused with myeloma cells of the mice according to a PEG (polyethylene glycol) method, and the hybridoma cell strain named as 1E5 is obtained through indirect ELISA (enzyme-linked immune sorbent assay), indirect competition ELISA screening and three-time subcloning. The hybridoma cell strain is collected in China general microbiological culture collection center, and the collection number is CGMCC No.10865. Monoclonal antibodies secreted by the cell strain 1E5 have high affinity and detection sensitiveness on amantadine, the affinity constant is 8.04*109L / mol, the IC50 value of detection sensitiveness is 1ng / mL, and the monoclonal antibodies can be used for amantadine content detection in food safety.

Description

technical field [0001] The invention relates to an amantadine monoclonal antibody hybridoma cell line and the anti-amantadine monoclonal antibody produced therefrom and application thereof, belonging to the field of food safety immunology detection. Background technique [0002] Amantadine is an antiviral drug and anti-shock paralysis drug. Due to its strong toxic and side effects, the Ministry of Agriculture clearly prohibited the sale and use of amantadine and other antiviral veterinary drugs as early as 2005. [0003] The current detection methods for amantadine mainly include instrumental detection methods and immunological detection methods. Although the instrumental detection method is accurate, the instrument is expensive, the operation is complicated, and the sample pretreatment is cumbersome. Compared with instrumental detection methods, immunoassay methods have the characteristics of low cost, high throughput, high sensitivity, and low requirements for technicians...

Claims

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Application Information

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IPC IPC(8): C12N5/20C07K16/44G01N33/577C12R1/91
Inventor 匡华彭双胥传来徐丽广马伟刘丽强宋珊珊胡拥明
Owner JIANGNAN UNIV
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