A hybridoma cell line c1 secreting anti-paromomycin monoclonal antibody and its application

A hybridoma cell line and paromomycin-resistant technology, which is applied in the field of immunochemistry and can solve problems such as no paromomycin ELISA method

Active Publication Date: 2019-04-30
DELISI GROUP +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Most aminoglycoside antibiotics have corresponding ELISA methods, but there is no ELISA method for paromomycin

Method used

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  • A hybridoma cell line c1 secreting anti-paromomycin monoclonal antibody and its application
  • A hybridoma cell line c1 secreting anti-paromomycin monoclonal antibody and its application
  • A hybridoma cell line c1 secreting anti-paromomycin monoclonal antibody and its application

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0013] Example 1: Synthesis of Immunogens and Coating Gens

[0014] The immunogen was coupled by the glutaraldehyde method: 31.96 mg of paromomycin was dissolved in 4 mL of 0.01 M phosphate buffer solution, 200 μL of 1% glutaraldehyde aqueous solution was added and stirred for 15 min. 20 mg of bovine serum albumin was dissolved in 2 mL of 0.01M phosphate buffer solution, reacted at 4°C for 1 h, added an appropriate amount of sodium borohydride to terminate the reaction, and incubated at 4°C for 2 h. The reaction mixture was dialyzed to remove free paromomycin to obtain the immunogen. The coating was synthesized by the carbodiimide method: 10 mg ovalbumin was dissolved in 1 mL pH 4.7 0.1 M MES (morpholine ethanesulfonic acid) buffer solution, 2 mg EDC and 1.2 mg NHS were added, and reacted at room temperature for 2 h. 19.03 mg paromomycin dissolved in 1 mL 0.05M KH 2 PO 4 , the activated protein solution was slowly added dropwise into the paromomycin solution, and reacted fo...

Embodiment 2

[0015] Example 2: Immunization of mice

[0016] The immunogen was diluted to an appropriate concentration with physiological saline, emulsified with an equal volume of Freund's adjuvant, and injected at multiple points on the back of 6-8 week-old BALB / c mice. For the first immunization, complete Freund's adjuvant was used at a dose of 100 μg. The following four booster immunizations used incomplete Freund's adjuvant, the immunization dose was 50 μg, and the immunization interval was 21 days. After the third immunization, blood was collected from the tail vein of the mice, and the sera were tested by indirect ELISA. After the fifth immunization, the mice with high titer and inhibition were screened for intraperitoneal immunization, and the spleens of the mice were taken out three days later for cell fusion.

Embodiment 3

[0017] Example 3: Cell Fusion and Screening

[0018] Mouse splenocytes and tumor cells were fused under the action of PEG 1500 to form hybridoma cells. Detection was carried out on the seventh day after fusion, and the cell lines with high titer and good paromomycin recognition were screened for subcloning, and so on, 5 times of subcloning were performed to obtain pure cell lines.

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Abstract

Disclosed are a hybridoma cell strain C1 for secreting an anti-paromomycin monoclonal antibody and the use thereof, belonging to the technical field of immunochemistry. A murine monoclonal cell strain C1 has been prepared through the conventional cell fusion technologies and deposited in the China General Microbiological Culture Collection Centre, and the deposit number is CGMCC No. 12025. The monoclonal antibody secreted by the cell strain is determined by the indirect competitive enzyme-linked immunosorbent assay, and the IC50 to paromomycin and neomycin is 0.61 and 2.43 μg / L respectively. The range of the recovery rate of paromomycin and neomycin in animal foodstuff is 64.56-105.85% and 54.08-100.55%. The hybridoma cell strain C1 provides a raw material for the immunological detection of the paromomycin residue in animal foodstuff, and has a practical application value.

Description

technical field [0001] The invention relates to a mouse-derived hybridoma cell strain C1 and the secreted monoclonal antibody capable of recognizing paromomycin, which can be used for the detection of paromomycin residues in food and belongs to the technical field of immunochemistry. Background technique [0002] Paromomycin is a broad-spectrum aminoglycoside antibiotic secreted by Streptomyces. The structure of paromomycin is very similar to neomycin, the only difference is that the amino group at position 6 of neomycin is replaced by a hydroxyl group. The antibacterial spectrum of paromomycin is similar to that of kanamycin, and it has a strong killing effect on amoeba, and it also has antibacterial activity against some protozoa, including: Leishmania, amoeba dysenteriae, cryptosporidium insect. Clinically, paromomycin is used to treat amoebiasis, giardiasis and leishmaniasis. In animal husbandry, it is used to treat bacterial infections, including histomoniasis of tur...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N5/20C07K16/44G01N33/577G01N33/543
CPCC07K16/44
Inventor 徐丽广陈燕妮胥传来匡华刘丽强宋珊珊吴晓玲郑乾坤
Owner DELISI GROUP
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