Bromhexine hapten, bromhexine artificial antigen, bromhexine antibody and preparation method and application of bromhexine artificial antigen

An artificial antigen, bromhexine technology, applied in the preparation methods of peptides, the preparation of carboxylic acid amides, chemical instruments and methods, etc., can solve the problems of lack of bromhexine hapten antibodies, etc. Accurate effect

Active Publication Date: 2022-06-21
SOUTH CHINA AGRI UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0004] The object of the present invention is to overcome the defects and deficiencies that lack bromhexine hapten, artificial antige...

Method used

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  • Bromhexine hapten, bromhexine artificial antigen, bromhexine antibody and preparation method and application of bromhexine artificial antigen
  • Bromhexine hapten, bromhexine artificial antigen, bromhexine antibody and preparation method and application of bromhexine artificial antigen
  • Bromhexine hapten, bromhexine artificial antigen, bromhexine antibody and preparation method and application of bromhexine artificial antigen

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0071] Preparation and identification of embodiment 1 bromhexine hapten

[0072] 1. Preparation of bromhexine hapten (BOX-SA)

[0073] Add 80 mg of bromhexine to a round bottom flask, add 3 mL of dimethyl sulfoxide to dissolve, then add 60 mg of succinic anhydride and 8 mg of pyridine, and react at room temperature for 6 h. The reaction was monitored by TLC until the reaction was completed, and the developing solvent was petroleum ether-ethyl acetate (3:1, v / v). Purify by silica gel column chromatography to obtain the reaction product. After the collected product is purified by a rotary evaporator and spin-dried, it is extracted twice with ethyl acetate and saturated sodium chloride, and anhydrous sodium sulfate is added to the ethyl acetate phase to dry. After concentration, the bromhexine hapten (BOX-SA) can be obtained. The synthetic route of BOX-SA is as follows figure 1 shown.

[0074] 2. Identification of bromhexine hapten (BOX-SA)

[0075] BOX-SA proton nuclear mag...

Embodiment 2

[0080] Synthesis and identification of embodiment 2 bromhexine artificial antigen

[0081] 1. Synthesis of bromhexine artificial antigen

[0082] The bromhexine hapten (BOX-SA) prepared in Example 1 was coupled to keyhole limpet hemocyanin (KLH), chicken ovalbumin (OVA) and lactoferrin (LF) by the active ester method.

[0083] (1) Dissolve 10 mg of BOX-SA prepared in Example 1, 5.15 mg of NHS and 7.67 mg of EDC in 200 uL DMF, and stir at room temperature for 4 to 6 hours in the dark to obtain bromhexine hapten activation solution;

[0084] (2) Weigh 10 mg of keyhole limpet hemocyanin (KLH), lactoferrin (LF) and chicken ovalbumin (OVA) respectively and add to 2 mL of PBS buffer (0.01moL / L, pH=7.4);

[0085] (3) The bromhexine hapten activation solution obtained in step (1) was slowly added dropwise to the carrier protein buffer solution obtained in step (2), and stirred at 4° C. for 12 hours;

[0086] (4) Dialyze with PBS buffer solution for three days, 3 times a day. After t...

Embodiment 3

[0093] The preparation of embodiment 3 antibody

[0094] 1. Preparation of polyclonal antibodies

[0095] The bromhexine artificial antigen BOX-SA-KLH, BOX-SA-LF of the BOX-SA coupling keyhole limpet hemocyanin (KLH) and lactoferrin (LF) that embodiment 2 prepares is used as immunogen, and equal amount The immune adjuvant (complete Freund's adjuvant for the first immunization, and incomplete Freund's adjuvant for subsequent booster immunizations) was emulsified evenly and the animals were immunized. New Zealand white rabbits weighing 2.5 to 3 kg were immunized by subcutaneous injections on the back, subcutaneous injections in various parts, leg muscles and ear veins respectively. The second immunization was done after 4 weeks, and the booster immunization was performed every 3 weeks thereafter. One week after the third booster immunization, blood was collected from the ear vein, and the serum titer was determined by indirect competitive ELISA. When the titer no longer rises,...

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Abstract

The invention discloses a bromhexine hapten, an artificial antigen, an antibody as well as a preparation method and application of the bromhexine hapten and the artificial antigen. The structural formula of the bromhexine hapten is shown as a formula (I), the hapten is used for preparing an artificial antigen and an antibody for detecting bromhexine, the antibody has high-sensitivity and high-specificity recognition capability on bromhexine, the semi-inhibitory concentration is 4.57 ng/mL, the lowest detection limit is 0.41 ng/mL, the quantitative detection range is 0.85-35.11 ng/mL, and the hapten can be used for detecting bromhexine. A core raw material is provided for establishing an immunoassay method for specifically detecting bromhexine. Meanwhile, the invention also establishes a bromhexine immunoassay method with higher specificity and sensitivity, in addition, a kit for detecting bromhexine residues is developed by utilizing the artificial antigen and the antibody, and the kit has the characteristics of high specificity, high sensitivity, high precision, high accuracy and the like.

Description

technical field [0001] The invention relates to the technical field of food detection, and more specifically relates to a bromhexine hapten, an artificial antigen, an antibody and a preparation method and application thereof. Background technique [0002] Bromhexine is an expectorant, which can directly act on bronchial glands, promote the release of lysosomes in mucus-secreting cells, and cause the mucopolysaccharide fibers in sputum to differentiate and crack. It is mainly used for acute and chronic bronchitis, asthma, Symptoms such as bronchiectasis and emphysema. Long-term overdose may cause greater irritation to the gastrointestinal mucosa, resulting in nausea, stomach discomfort and other symptoms. Because of its strong expectorant effect, it is often added to homemade herbal tea by many unscrupulous merchants to promote its effect, which has caused great harm to consumers' health and market order. [0003] At present, there is no standard detection method for bromhe...

Claims

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Application Information

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IPC IPC(8): C07C233/43C07C231/02C07K14/795C07K14/77C07K14/79C07K1/107C07K16/44G01N33/53G01N33/577
CPCC07C233/43C07C231/02C07K14/795C07K14/77C07K14/79C07K16/44G01N33/94G01N33/577C07C2601/14C07K2317/76C07K2317/33
Inventor 雷红涛王子安潘康亮王锦关甜沈兴李向梅徐小艳韦晓群徐振林杨金易沈玉栋
Owner SOUTH CHINA AGRI UNIV
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