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Composite immunoaffinity column for bisphenol A and its analogues and preparation and application thereof

An analog, immunoaffinity technology, applied in the field of composite immunoaffinity column and its preparation, can solve the problems of large amount of organic solvent, complicated operation, poor selectivity, etc., and achieve improved purification efficiency, high purity and high yield. Effect

Inactive Publication Date: 2019-03-05
北京维德维康生物技术有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

These methods have disadvantages such as poor selectivity, large amount of organic solvent used, complex operation, and poor reproducibility among different matrices. Therefore, it is necessary to establish a sample pretreatment method with good selectivity, high sensitivity, simple operation, and rapid

Method used

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  • Composite immunoaffinity column for bisphenol A and its analogues and preparation and application thereof
  • Composite immunoaffinity column for bisphenol A and its analogues and preparation and application thereof
  • Composite immunoaffinity column for bisphenol A and its analogues and preparation and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0041] Example 1. Preparation of hapten of bisphenol A and its analogues

[0042] Add dimethylformamide (DMF) and NaH to any one of bisphenol A, bisphenol AF, bisphenol S, bisphenol F, and bisphenol B. The ratio of the raw materials, DMF and NaH is 1000mg: 7ml: 1400mg, the required reaction temperature is an ice bath reaction, remove the ice bath when there are no more bubbles, and then warm the reaction for 40 min to obtain a reaction solution; add 200mg of ethyl 4-bromobutyrate to the reaction solution, 40 The reaction was carried out in an oil bath at ℃, 25ml ethyl acetate was extracted twice, the ethyl acetate phases were combined, evaporated to dryness, 12ml ethyl acetate was added to dissolve the residue, 2 times the weight of silica gel was added to it, and the silica gel was evaporated to dryness. Load the column.

[0043] The silica gel sample mixed with the reaction solution and silica gel is added using the dry sample addition method, and rinsed with a petroleum ether: ...

Embodiment 2

[0055] Example 2: Preparation and structural identification of artificial antigens of bisphenol A and its analogs

[0056] 1. Synthesis of immunogen

[0057] (1) Dissolve 9.55mg of bisphenol A and its analogue hapten (any one of formula I (1-5)) with 1.5mL DMF, stir at 200rpm for 10min, add 21.5mg of EDC to dissolve, then add 13mg of NHS, room temperature Stirring (500rpm) for 2-3h activation.

[0058] (2) Weigh 50mg of BSA and dissolve it in 3.5mL 0.1M sodium bicarbonate solution, stir at 200rpm for 10min to fully dissolve it, cool to 0-4℃ in an ice bath, and under stirring at 1000rpm, add the reaction solution of step 1 dropwise (1mL / min), 500rpm stirring reaction 24h.

[0059] (3) Put the reaction product into distilled water to rinse the dialysis bag (10cm), 1L0.01M PBS (1×, pH7.2) 4℃ stirring (100rpm) and dialyze for 3d, change the liquid 3 times a day (once in the morning, once in the evening) , Change the medium 9 times in total, centrifuge the dialysis product at 5000 rpm f...

Embodiment 3

[0064] Example 3. Preparation of monoclonal antibodies by immunizing animals with artificial antigens of bisphenol A and its analogs

[0065] 1. Animal immunity

[0066] Using the immunogen prepared in Example 2 (bisphenol A and its analogues (any one of formula I (1-5))-BSA) at 100 μg / head, dissolve the immunogen and Freund's complete adjuvant in saline Mix in equal volume, immunize 6-8 week-old Balb / c female mice by subcutaneous injection on the back of the neck, and mix with equal volume of immunogen and incomplete Freund’s adjuvant on the 7, 14, and 28 days after the initial immunization, and each additional immunization , 3 days before fusion, 100 μg of immune complexes per mouse, without Freund’s adjuvant, was immunized again.

[0067] 2. Cell Fusion and Cloning

[0068] According to the conventional method, take the spleen cells of the immunized mouse and mix with the mouse myeloma cells (SP2 / 0) in the logarithmic growth phase, and then slowly add the preheated fusion agent (P...

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Abstract

The invention discloses an immunoaffinity column for simultaneous purification of bisphenol A and its analogues and a preparation method and application thereof. An artificial antigen of bisphenol A and its analogues provided herein is an antigen prepared by coupling bisphenol A shown as any one of formulas I (1-5) and its analogue hapten to a carrier protein; an antibody prepared by immunizationis used to prepare the immunoaffinity column. The immunoaffinity column for simultaneous purification of bisphenol A and its analogues allows various components under test to be highly purified through single extraction; matrix effect of a sample can be substantially completely eliminated; purifying efficiency is greatly improved. The immunoaffinity column can be combined with LC-MS / MS detection technique to estimate exposure of food endocrine disruptors.

Description

Technical field [0001] The invention belongs to the field of purification and detection of endocrine interferon in food, and relates to a composite immunoaffinity column for purifying bisphenol A and its analogs at the same time, and a preparation method and application thereof. Background technique [0002] In recent years, cases of congenital malformations, underdevelopment and dementia in fetuses and infants are not uncommon. Many studies have shown that the main reason for these phenomena is that the human endocrine system is interfered by some chemical substances in the environment, causing the imbalance of natural hormone levels in the body. These chemicals that can interfere with hormone balance in the body are generally wastes produced and discharged by humans in production and life, so they are called environmental endocrine disrupting chemicals (EDCs). Bisphenol A (BPA) is a typical environmental endocrine disruptor. Because of its stable chemical properties, high temp...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): G01N33/558C07K14/765C07K14/77C07K14/795C07K14/47C07K16/44
CPCC07K14/47C07K14/765C07K14/77C07K14/795C07K16/44C07K19/00G01N33/558
Inventor 聂靖东邵兵陶晓奇吴雨洋杨柳刘薇
Owner 北京维德维康生物技术有限公司
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