Estriol hapten and antibody and application thereof in fast sensor method for uncojugated estriol detection

An estriol and hapten technology, applied in the field of biosensors, can solve the problems of difficulty in distinguishing structural analogs, insufficient antibody specificity, insufficient sensitivity, etc., so as to improve the ability to catalyze hydrogen peroxide, improve specific performance, The effect of increasing sensitivity

Active Publication Date: 2016-06-01
广州捷达生物科技有限公司
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  • Abstract
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  • Claims
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Problems solved by technology

[0005] Although the immunological rapid detection method of estriol has been reported above, there are still several problems: (1) the specificity of the antibody is not enough, and hormones such as estradiol and progesterone are structurally similar to estriol. In general, it is difficult to distinguish these structural analogs, especially estradiol, which is likely to cause interference; (2) The sensitivity is not enough. The highest sensitivity of the existing immunoassay methods is generally 0.1ng / mL, due to the presence of matrix interference in the immunoassay method However, it is often necessary to dilute the sample to eliminate matrix interference, so the actual detection sensitivity is usually reduced by hundreds of times, making the existing method unable to meet the demand

Method used

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  • Estriol hapten and antibody and application thereof in fast sensor method for uncojugated estriol detection
  • Estriol hapten and antibody and application thereof in fast sensor method for uncojugated estriol detection
  • Estriol hapten and antibody and application thereof in fast sensor method for uncojugated estriol detection

Examples

Experimental program
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Embodiment 1

[0053] This example provides a preparation method of estriol immune hapten and competition hapten.

[0054] The synthetic route map of estriol immune hapten is shown in figure 1 shown. Take 0.5g (1.7mM) estriol in a 25mL round bottom flask, add 3mL of anhydrous DMF, stir and dissolve under nitrogen protection at 0°C. Then add 45.0 mg (1.9 mM) NaH, stir for 20 min, and finally add 0.17 mg (1.7 mM) succinic anhydride, and continue the reaction at 0° C. for 30 min. (Refer to ZhaoDangetal, 2015) Adjust the pH of the reaction solution to about 3 with 1M HCl. Spot the silica gel plate to monitor the reaction, developer methanol: chloroform = 1:5 (volume ratio, the same below), raw material Rf = 0.7, product Rf = 0.1. 200-300 mesh silica gel passes through the column, developer methanol: chloroform = 1:10 After evaporating the solvent to dryness, the quality of the product (estriol immune hapten) obtained is 0.24g, and the mass spectrometry identification figure is shown in figu...

Embodiment 2

[0056] Example 2 Polymer horseradish peroxidase (MHRP) was labeled on the estriol monoclonal antibody.

[0057] Screening of estriol monoclonal antibody: the mice after 5 times of immunization were killed, and spleen cells were taken to fuse with mouse SP2 / 0 cells (myeloma cells and spleen cells at a ratio of 5-10:1). Positive hybridoma cells were screened by indirect ELISA and indirect competition ELISA, and the wells showing positive and competitive inhibition reactions were positive wells, and were used for further subcloning. Under sterile conditions, the cells in the positive wells were eluted, and the cells were transferred to a 96-well culture plate pre-plated with feeder cells using an elbow pipette. Each original positive well clone was counted by a cell counting plate and limitedly diluted to each well. 1 cell. One 96-well cell culture plate was limitedly diluted for each positive original clone. After the cells adhere to the wall and cover 1 / 2 to 1 / 3 of the bottom...

Embodiment 3

[0060] This embodiment provides a disposable one-step electrodeposition immunobiological sensor for detecting histamine, which uses an electrode with a Prussian blue-chitosan-nano-gold composite film on the surface.

[0061] Nano gold and Prussian blue (Fe4[Fe(CN) 6 ] 3 ) molar concentration ratio is 1:1000, and the chitosan content is 0.02%, and the preparation method is as follows:

[0062] Dissolve 0.05g chitosan (deacetylation degree ≥ 95%, viscosity 100-200mpa.s) in 100mL 1.0% acetic acid solution, stir at room temperature for 3 hours to completely dissolve chitosan, and prepare 0.05% chitosan sugar solution. 0.0406gFeCl 3 , 0.0822gK 3 [Fe(CN) 6 ], 0.7455g KCl and 1mL concentrated hydrochloric acid were added to 100mL of the above-mentioned chitosan solution, and ultrasonically dispersed at room temperature until a stable dark green dispersion was obtained; then the prepared 1mL nano-gold colloid was added to the above-mentioned solution that had been mixed , and th...

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Abstract

The invention relates to the technical field of biosensors and discloses an estriol hapten and antibody and the application thereof in a fast sensor method for uncojugated estriol detection. The structure of the hapten is scientifically designed, so that sensitivity and specificity of the antigen-antibody are improved; electro-deposition of a prussian blue-chitosan-nanogold composite membrane on the surface of an electrode is conducted, the estriol antigen is fixed to the surface of the membrane, a novel horse radish peroxidase labeled estriol antibody and a sample to be tested are added after an unbound site is sealed, the sample and a fixed antigen compete for a free enzyme labeled antibody, hydrogen peroxide is added after washing, a current signal is measured, a cyclic voltammetry estriol standard curve is obtained, the fast sensor method for uncojugated estriol detection is provided successfully, detection range is 0.001-100 ng / mL, and detection limit is 0.001 ng / mL. The method has the advantages of being easy to operate, high in sensitivity, quick, convenient and the like and can be well applied to ultra-sensitive and fast detection of estriol in the sample.

Description

technical field [0001] The invention relates to the technical field of biosensors, and more specifically, provides an estriol hapten and an antibody and their application in a rapid sensor method for detecting free estriol. Background technique [0002] Estriol is a sex hormone, which is mainly converted from estrone and estradiol in the liver, and only a small amount is secreted by the ovary. Dehydroepiandrosterone produced in the adrenal glands of the fetus is produced by aromatization in the placenta after 16α hydroxylation in the liver. As the pregnancy progresses, the excretion in urine increases gradually and decreases sharply after delivery. As a mild sex hormone agent, it can be taken orally to treat diseases such as menopausal disorders. Estriol has a significant effect on maintaining female side characteristics and protein assimilation. Clinical veterinarians are used for estrus synchronization, livestock growth promotion and feed conversion rate improvement. Es...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07J1/00C07J41/00C07K14/765C07K14/77C07K14/795C07K16/44G01N33/577G01N27/327
CPCC07J1/0051C07J41/0072C07K14/765C07K14/77C07K14/795C07K16/44C07K19/00G01N27/3278G01N33/54366
Inventor 袁利鹏刘波王宇戚平梁智安邱腊凤薛奕菲瑶君梁惠如
Owner 广州捷达生物科技有限公司
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