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Preparation method for monoclonal antibody specifically binding vardenafil and analog thereof

A monoclonal antibody and vardenafil technology, applied in the field of immunochemistry, can solve the problems of weak vardenafil research and development, PDE-5 drug dosage exceeding the safe range, etc., and achieve the effect of convenient construction

Inactive Publication Date: 2009-10-14
NANCHANG UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0004] ①The interaction between PDE-5 inhibitors and nitrate drugs leads to severe hypotension; ②Many PDE-5 inhibitor analogues have unknown risk factors; ③Illegally added doses of PDE-5 drugs exceed the safe range
[0007] Compared with several other PDE-5 drugs, the research and development of detection methods for vardenafil and its related analogues is relatively weak, and there is no statutory detection method for vardenafil in China

Method used

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  • Preparation method for monoclonal antibody specifically binding vardenafil and analog thereof
  • Preparation method for monoclonal antibody specifically binding vardenafil and analog thereof

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specific Embodiment approach 1

[0033] Specific implementation mode one. The synthetic method of vardenafil artificial immunization antigen and artificial detection antigen:

[0034]①Preparation of cationized bovine serum albumin (M-BSA): Dissolve 300mg bovine serum albumin (BSA) and 400mg carbodiimide (EDC) in 15mL distilled water, add 1mL of 10% ethylenediamine aqueous solution, and use 1N The pH value was adjusted to 5.5 with hydrochloric acid, and the reaction was stirred slowly at room temperature for 2 hours. 200 mg of EDC was added, and the pH value was adjusted to 5.5 with 1N hydrochloric acid, and the reaction was continued to stir slowly overnight at room temperature. Dialyze with distilled water at 4°C for 3 days, freeze-dry in vacuum, and store at 4°C for later use;

[0035] ②Preparation of cationized ovalbumin (M-OVA): Dissolve 100mg ovalbumin (OVA) and 300mg carbodiimide (EDC) in 10mL distilled water, add 1mL 10% ethylenediamine aqueous solution, adjust with 1N hydrochloric acid The pH value...

specific Embodiment approach 2

[0038] Specific embodiment two: Using vardenafil artificial immune antigen to immunize mice:

[0039] ①After emulsifying vardenafil artificial immune antigen with an equal amount of complete Freund's adjuvant, subcutaneously immunize four 4-week-old female BALB / c mice by subcutaneous multi-point injection (100 μg / 0.2 mL per mouse) ;

[0040] ② Four weeks after the initial immunization, the artificial immune antigen of vardenafil was emulsified with an equal amount of Freund's incomplete adjuvant, and the mice were boosted by subcutaneous injection (100 μg / mouse); after that, boosted immunization once every three weeks;

[0041] ③Three days before the fusion, vardenafil artificial immune antigen aqueous solution (50 μg / rat) was injected into the tail vein. Prepare for cell fusion experiments;

specific Embodiment approach 3

[0042] Specific implementation mode three. Screening of anti-vardenafil monoclonal antibodies using hybridoma cell technology:

[0043] ① Put 1×10 8 splenocytes with 2 x 10 7 Myeloma cells SP2 / 0 were mixed in a 50mL fusion tube, and DMEM medium was added to 30mL. Centrifuge at 1000rpm for 7min, and suck up the supernatant as much as possible;

[0044] ② Tap the bottom of the fusion tube lightly on the palm of your hand to loosen and evenly precipitate the cells, and preheat in a 40°C water bath. Add 1 mL of 50% PEG (pH 8.0) preheated to 40°C within 1 minute with a 1 mL pipette, and shake gently while adding. Use a 5mL pipette to add 20-30mL of incomplete DMEM medium preheated to 37°C within 90 seconds, and let stand at room temperature for 10min. Centrifuge at 800rpm for 7 minutes, discard the supernatant;

[0045] ③Add 5mL of complete HAT medium, blow and aspirate the pelleted cells gently, suspend and mix well, then add HAT medium to 80-100mL. Dispense to a 96-well ce...

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Abstract

The invention relates to a preparation method for a monoclonal antibody specifically binding vardenafil and an analog thereof. The preparation method comprises the steps of: synthesizing artificial immunizing antigens and artificial testing antigens, screening mice under immune state and the monoclonal antibody, and conducting structural specificity identification of the monoclonal antibody, specifically, mixing M-BSA with the vardenafil, regulating pH, adding a glutaraldehyde coupling agent, slowly stirring with a magnetic stirrer for reaction, dialyzing with distilled water, freezing and drying under vacuum condition, and synthesizing the artificial immunizing antigens; mixing M-OVA with the vardenafil, regulating pH, adding a glutaraldehyde coupling agent, slowly stirring with a magnetic stirrer for reaction, dialyzing with distilled water, freezing and drying under vacuum condition, and synthesizing the artificial testing antigens; and using a competitive inhibition ELISA method to detect the coasensual reaction between the monoclonal antibody and the vardenafil and piperidenafil and identifying the monoclonal antibody which has high coasensual reaction rate and can specifically bind the vardenafil and the analog thereof. The affinity equilibrium constant of the monoclonal antibody prepared by the method on the vardenafil and the analog thereof is higher than 10M.

Description

technical field [0001] The invention belongs to the technical field of immunochemistry, and in particular relates to a preparation method of vardenafil artificial antigen. In the artificial antigen, the group necessary for the efficacy of vardenafil is completely retained and exposed outside the carrier protein; based on this artificial immune antigen and detection antigen, hybridoma technology is used to screen and identify vardenafil to specifically recognize vardenafil A monoclonal antibody to a group essential for pharmacodynamics. It lays the foundation for the construction of convenient, fast, cheap and efficient vardenafil and its structural analogue immunoassay reagents. Background technique [0002] At the end of the last century, Pfizer of the United States planned to develop a phosphodiesterase-5 (PDE-5) inhibitor drug for the treatment of angina pectoris. During phase I and II clinical trials, it was found that some subjects' impotence symptoms were cured. This...

Claims

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Application Information

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IPC IPC(8): C12P21/08C07K16/44
Inventor 许杨郭杰标黄志兵
Owner NANCHANG UNIV
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