Preparation method of diethylstilbestrol antibody

A technology of diethylstilbestrol and antibody, which is applied in the field of antibody preparation, can solve the problems of high detection cost and difficult detection, and achieve the effect of simple preparation method, low cost and high immunogenicity

Inactive Publication Date: 2010-12-15
SOUTH CHINA AGRI UNIV
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  • Abstract
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Problems solved by technology

[0006] The object of the present invention is to provide a method for preparing diethylstilbestrol (DES) antibody according to the problems of high detection difficulty and high detection cost in existing diethylstilbestrol detection, and the diethylstilbestrol (DES) antibody prepared by this method can be used to prepare diethylstilbestrol rapid detection ELISA reagent box

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  • Preparation method of diethylstilbestrol antibody
  • Preparation method of diethylstilbestrol antibody
  • Preparation method of diethylstilbestrol antibody

Examples

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Embodiment 1

[0026] Preparation of hapten loading reagent: add anhydrous potassium carbonate and neutral alumina to distilled water, so that potassium carbonate, neutral alumina and water are 1:3 (w / v) and 1:2 (w / v) respectively , Stir magnetically at 60-70°C for 1 hour, then dry in an oven at 60°C, activate at 130-140°C for 3 hours before use.

[0027] Preparation of diethylstilbestrol-mono-ethylbutyrate-ether (DES-mono-ethylbutyrate-ether, DES-MEBE): 25mL chromatographically pure acetone, 0.55g DES, 1.86g loading reagent, 0.15mL 4-bromobutyric acid Ethyl ester was added into a three-necked flask, and refluxed for 8 hours under the conditions of protecting from light, water bath, and N2. The progress of the reaction was monitored by high performance liquid chromatography (HPLC). HPLC conditions: BDS-C18 chromatographic column, 5 μm, 4.6×250 mm; mobile phase: methanol-water (75:25, v / v); flow rate: 1.0 mL / min; column temperature: 25° C.; injection volume: 10 μL. For HPLC spectra related ...

Embodiment 2

[0031] DES-MCPE was cross-linked with carrier protein BSA (immunogen) or OVA (coating agent) by mixed anhydride method. First activate DES-MCPE: Accurately weigh 35.0mg DES-MCPE and dissolve it in 1mL of anhydrous dioxane, add 2 to 18μL tri-n-butylamine, stir and react at 4°C for 20min, then add 13μL isobutyl chloroformate 4 The reaction was stirred at ℃ for 30 min. Preparation of protein solution: Dissolve protein (BSA 110 mg or OVA 70 mg) in 3 mL of dioxane, add 3 mL of distilled water and 66 μL of 1 mol / L NaOH. Cross-linking of DES-MCPE and carrier protein: Slowly add DES-MCPE activation solution into the protein solution, and use 1mol / L NaOH to control the pH value at about 8.0. After the dropwise addition was completed, the mixture was stirred and reacted overnight at 4°C in the dark. After the reaction was completed, the reaction solution was put into a treated dialysis bag, dialyzed with 1000 mL of dialysate at 4°C, and the dialysate was replaced every 12 hours. The ...

Embodiment 3

[0033] Choose healthy male New Zealand white rabbits with a body weight of about 3.5kg and observe them for a week. Dilute the immunogen to 0.5 mg·mL with sterile saline -1 Concentration, with a dose of 0.10 mL (50 μg of immunogen), the immunogen and an equal volume of Freund's complete adjuvant were fully mixed and emulsified before immunization, and then injected subcutaneously at multiple points on both sides of the back and spine of rabbits. A booster immunization was carried out every 4 weeks. During the booster immunization, the immunogen was emulsified with Freund's incomplete adjuvant. On the 10th day after the two immunizations, 1-2ml blood was collected from the ear vein, the serum was separated by centrifugation, and the antibody titer was detected by indirect ELISA. After the serum titer stabilized, a large number of antiserum was collected. Calculation method of serum titer: negative control OD450 value is recorded as N, positive control OD 450 The value is rec...

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Abstract

The invention discloses a preparation method of a diethylstilbestrol antibody. The preparation method of the diethylstilbestrol antibody comprises the following steps of: (1) performing a reaction of diethylstilbestrol and 4-bromine butyric acid hexylester, exposing a carboxyl group by saponifying and hydrolyzing, and synthesizing diethylstilbestrol artificial haptene; (2) performing a reaction of the diethylstilbestrol artificial haptene and bovine serum albumin by a mixed anhydride method to obtain diethylstilbestrol artificial immunogen; and (3) emulsifying the diethylstilbestrol artificial immunogen and Freund's complete adjuvant (initial immunization) or Freund's incomplete adjuvant, subcutaneously injecting a rabbit, and separating blood serum when the antibody titer is stable to obtain the diethylstilbestrol antibody. The preparation method of the diethylstilbestrol antibody has the advantages of simpleness, convenience and low cost; and the prepared diethylstilbestrol antibody has high specificity, high antibody titer and indirect ELISA experimental antibody titer of over 1 million, can reduce detection cost when used for immunologically detecting diethylstilbestrol, and is easy to promote and use.

Description

technical field [0001] The invention relates to the field of antibody preparation, in particular to a method for preparing diethylstilbestrol antibody. Background technique [0002] Diethylstilbestrol (DES) is a synthetic estrogen. The chemical name is 1,2-bis(4-hydroxyphenyl)-1,2-diethylethylene. Its physical properties are colorless crystals or white crystalline powder, soluble in ethanol, ether, chloroform, fatty oil or strong alkali solution, almost insoluble in water. The chemical structure is shown in formula (I). [0003] [0004] Formula (I) [0005] DES can increase animal body weight and protein deposition, while reducing fat deposition. It has been used as a growth promoter in agriculture. Due to the carcinogenicity and estrogen-like effects of DES, the "People's Republic of China Animal and Animal Source Food Residue Monitoring Plan" formulated in 1999 stipulated that diethylstilbestrol should not be detected in pork, livestock and poultry. The detection...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07K16/26C07K16/06
Inventor 刘雅红岳磊单国强
Owner SOUTH CHINA AGRI UNIV
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