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Hybridoma cell strain for secreting spirolactone and metabolite monoclonal antibody thereof and application of hybridoma cell strain

A hybridoma cell line, monoclonal antibody technology, applied in microorganisms, analytical materials, biological tests, etc., can solve the problems of time-consuming, high cost, and complicated steps.

Active Publication Date: 2022-03-15
JIANGNAN UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0003] The content analysis methods of spironolactone and its metabolites include instrumental methods such as liquid chromatography-mass spectrometry (LC-MS). Metabolite detection method is of great significance

Method used

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  • Hybridoma cell strain for secreting spirolactone and metabolite monoclonal antibody thereof and application of hybridoma cell strain
  • Hybridoma cell strain for secreting spirolactone and metabolite monoclonal antibody thereof and application of hybridoma cell strain
  • Hybridoma cell strain for secreting spirolactone and metabolite monoclonal antibody thereof and application of hybridoma cell strain

Examples

Experimental program
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Embodiment 1

[0049] Synthesis of embodiment 1 spironolactone and its metabolite hapten

[0050] Dissolve 1 g of spironolactone (or canrenone) and 3 g of CMO in 10 mL of DMF, and heat in a water bath at 60° C. for 8 h. The organic solvent was removed by rotary evaporation under reduced pressure to obtain a yellow precipitate. Wash three times with deionized water and 1M HCl, add ethanol for recrystallization, and obtain light yellow or white needle-like crystals, namely the hapten Spi-COOH (or Can-COOH) of spironolactone and its metabolites.

[0051] The reaction equation is as follows:

[0052]

Embodiment 2

[0053] The synthesis of embodiment 2 spironolactone and its metabolite complete antigen

[0054] (1) Preparation of the immunogen spironolactone and its metabolite-BSA: Weigh 3.4mg of prepared Can-COOH, dissolve it in 200μL DMF, add 4.6mg N-hydroxysuccinimide and 7.7mg 1- Ethylcarbodiimide hydrochloride, react at room temperature for 4 hours, and the resulting mixture is called solution A; then weigh 10 mg of bovine serum albumin BSA and dissolve it in 2 mL of carbonate buffer, called solution B; slowly drop solution A Pour into solution B, react at room temperature for 12 hours under stirring, and dialyze with 0.01mol / L phosphate buffer solution PBS for 3 days to obtain the conjugated spironolactone and its metabolite -BSA, which should be frozen at -20°C for later use;

[0055] (2) Preparation of coated prospironolactone and its metabolite-OVA: Weigh 1.7mg of the prepared Spi-COOH, dissolve it in 200μL of DMF, add 2.3mg of N-hydroxysuccinimide and 3.4mg of 1 - Ethylcarbodii...

Embodiment 3

[0056] Example 3 Preparation of hybridoma cell lines secreting spironolactone and its metabolite monoclonal antibody

[0057] 1. Immunization of mice: healthy BALB / c mice aged 6-8 weeks were selected for immunization. The complete antigen of spironolactone and its metabolite was mixed and emulsified with an equal amount of Freund's adjuvant, and BALB / c mice were immunized by subcutaneous injection in the back. Complete Freund's adjuvant was used for the first immunization, followed by incomplete Freund's adjuvant. The interval between the first immunization and the second booster immunization was 28 days, and the interval between multiple booster immunizations was 21 days. Blood was collected 7 days after the third immunization (5 μL of mouse tail blood collection + 995 μL antibody diluent = antiserum), and ic-ELISA was used to measure the titer and inhibition of the mouse serum, and the mice with high titer and inhibition were selected, and the mice with high titer and inhib...

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Abstract

The invention relates to a hybridoma cell strain secreting spirolactone and metabolite monoclonal antibodies thereof, the hybridoma cell strain is named as a monoclonal cell strain PRM, and the hybridoma cell strain is preserved in China General Microbiological Culture Collection Center on May 13, 2021, the preservation address is No.3, Yard 1, Beichen West Road, Chaoyang District, Beijing, and the preservation number is CGMCC No.22341. The hybridoma cell strain disclosed by the invention can efficiently and stably secrete the monoclonal antibody of the spirolactone and the metabolite of the spirolactone, provides a powerful detection method and means for detecting the residual quantity of the spirolactone and the metabolite of the spirolactone in animal-derived food and cosmetics, and has relatively good sensitivity and specificity.

Description

technical field [0001] The invention relates to the technical field of immunoassay, in particular to a hybridoma cell line secreting spironolactone and its metabolite monoclonal antibody and application thereof. Background technique [0002] Spironolactone, also known as spironolactone, is a mineralocorticoid antagonist developed by Pfizer. The chemical structure of spironolactone is similar to that of aldosterone. It is the earliest clinically used aldosterone receptor antagonist. It can competitively bind to the aldosterone receptor in the cytoplasm, antagonize the potassium-expelling and sodium-retaining effect of aldosterone, promote water excretion, and reduce K + secretion. Since the diuretic effect of this drug is related to the level of aldosterone in the body, it is a potassium-sparing diuretic. Clinically, in the treatment of hypertension, it is often used in combination with diuretics such as diuretics to prevent hypokalemia. Spironolactone has a low blood conc...

Claims

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Application Information

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IPC IPC(8): C12N5/20C07K16/44G01N33/577
CPCC07K16/44G01N33/577G01N33/94C07K2317/33C07K2317/76Y02A50/30
Inventor 胥传来王鹏匡华徐丽广孙茂忠吴晓玲刘丽强马伟朱建平郝昌龙宋珊珊胡拥明吴爱红郭玲玲胥欣欣
Owner JIANGNAN UNIV
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