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A hybridoma cell line secreting acyclovir monoclonal antibody and its preparation method

A hybridoma cell line, monoclonal antibody technology, applied in the fields of botanical equipment and methods, biochemical equipment and methods, cells modified by introducing foreign genetic material, etc. Large, toxic and side effects, etc., to achieve the effect of good detection sensitivity and specificity

Active Publication Date: 2021-03-02
JIANGNAN UNIV +1
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  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0002] Acyclovir is a synthetic purine nucleoside analog, mainly used to treat various infections caused by herpes virus (especially herpes simplex virus, varicella zoster virus), and has become the drug of choice for severe herpes virus infection However, its residue and accumulation have certain toxic and side effects on the nervous system, blood system, digestive system, etc.
[0003] In order to prevent the abuse of acyclovir in animal feed and the abuse of acyclovir in other food products, acyclovir will form drug residues in the human body and seriously endanger human health. High method for detection of acyclovir residues in food
[0004] At present, the methods for detecting acyclovir are mainly liquid chromatography (HPLC), liquid chromatography tandem mass spectrometry (LC-MS / MS), ultraviolet-visible spectrophotometry, etc., but these methods are not effective in the actual sample analysis process. Often due to the complex components of acyclovir and the low content of target substances, the interference of impurities is large, which affects the accuracy and accuracy of the entire process

Method used

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  • A hybridoma cell line secreting acyclovir monoclonal antibody and its preparation method
  • A hybridoma cell line secreting acyclovir monoclonal antibody and its preparation method

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0038] Embodiment 1: the synthesis of complete antigen of acyclovir

[0039] Take 4 mg of acyclovir, add 30 μL of 5% glutaraldehyde aqueous solution, stir at room temperature, and activate for 10 min; another 10 mg of BSA (bovine serum albumin) is dissolved in 2 mL of 0.05 M, pH 9.6 CB (carbonate buffer solution) ) solution, the above activation solution was added dropwise to the KLH solution, stirred at room temperature for 1.5 hours, dialyzed at 4°C for three days, and stored at -20°C. (The ultraviolet characterization effect of the complete antigen of acyclovir is as follows figure 1 )

Embodiment 2

[0040] Embodiment 2: the preparation of the hybridoma cell line that secretes acyclovir monoclonal antibody

[0041] 1. Acquisition of animal immunity

[0042]Select healthy 6-8 week-old Balb / C mice for immunization, mix the obtained acyclovir complete antigen with an equal amount of oil, and then add emulsifier to obtain incomplete Freund's adjuvant after emulsification. Complete Freund's adjuvant was obtained by adding mycobacteria into complete Freund's adjuvant; the obtained Freund's adjuvant was subcutaneously injected in the back to immunize BALB / c mice for several times, and the complete Freund's adjuvant was used for the first immunization, and then Two times of booster immunization with incomplete Freund's adjuvant; on the 7th day after the end of the three times of immunization, blood was collected from the mice, and the immune titer and immunosuppressive ability of the mouse serum were detected by indirect ELISA, and the abcicilin in the serum was deleted. The immu...

Embodiment 3

[0053] Example 3: Preparation and Identification of Acyclovir Monoclonal Antibody

[0054] 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 For the hybridoma cells secreting acyclovir monoclonal antibody, the ascites was collected from the seventh day, and the ascites was purified by octanoic acid-ammonium sulfate method, and the obtained monoclonal antibody was stored at -20°C.

[0055] The mouse monoclonal antibody subtype identification kit was used to identify the immunoglobulin subtype of the monoclonal antibody purified from ascitic fluid, and the subtype was IgG2b. (The subtype identification results of monoclonal antibodies are shown in Table 1)

[0056] Using indirect competition ELISA, the IC50 of the monoclonal antibody acyclovir was determined to be 35 μg / L, indicating that it has good sensitivity to acyclovir and can be used for acyclovir immunoassay detection.

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Abstract

The invention relates to a hybridoma cell line secreting acyclovir monoclonal antibody and a preparation method thereof, belonging to the field of food safety immunoassay. The preservation number of the hybridoma cell line is: CGMCC No.14695. BALB / c mice were first immunized with complete Freund's adjuvant, boosted with incomplete Freund's adjuvant, and adjuvant-free adjuvant BALB / c mice were immunized by shock immunization with Lowe's complete antigen; the immunized mouse splenocytes with high titer and low IC50 were fused with mouse myeloma cells by PEG method, and were screened by indirect competition ELISA and subcloned three times , the resulting cell lines. The monoclonal antibody secreted by this cell line has good specificity and detection sensitivity to acyclovir, and can be used for the detection of acyclovir residues in food.

Description

technical field [0001] The invention relates to a hybridoma cell line secreting acyclovir monoclonal antibody and a preparation method thereof, belonging to the technical field of food safety immunoassay. Background technique [0002] Acyclovir is a synthetic purine nucleoside analog, mainly used to treat various infections caused by herpes virus (especially herpes simplex virus, varicella zoster virus), and has become the drug of choice for severe herpes virus infection However, its residue and accumulation have certain toxic and side effects on the nervous system, blood system, and digestive system. [0003] In order to prevent the abuse of acyclovir in animal feed and the abuse of acyclovir in other food products, acyclovir will form drug residues in the human body and seriously endanger human health. High method for the detection of acyclovir residues in food. [0004] At present, the methods for detecting acyclovir are mainly liquid chromatography (HPLC), liquid chrom...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N5/20C12N15/06C07K16/44G01N33/577
CPCC07K16/44C12N5/163G01N33/577G01N33/9446
Inventor 匡华王忠兴胥传来徐丽广马伟刘丽强吴晓玲宋珊珊胡拥明
Owner JIANGNAN UNIV
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