CN1861631A - Coupling compound of tetracycline, preparation process and application thereof - Google Patents

Abstract

The invention discloses a tetracycline conjugate of the general formula (I), which is composed of a tetracycline hapten coupled with an immunogenic carrier substance bovine serum albumin or ovalbumin. Wherein n is the molecular number of tetracycline combined with one bovine serum albumin molecule, and said n is an integer of 1-15; BSA is bovine serum albumin, and its molecular weight ranges from 6.6KDa to 6.9KDa. The invention also discloses the preparation method of the conjugate, that is, the tetracycline is connected with the immunogenic carrier substance to form a conjugate capable of inducing the animal immune system to produce antibodies. The tetracycline conjugate of the present invention prepares antiserum with a titer of more than 1:50,000 by immunizing New Zealand white rabbits, and the minimum detection limit is 1ppb. The invention has the characteristics of simple, fast, specific and accurate method, and provides a basis for preparing the tetracycline enzyme-linked immunoassay kit.

Description

Conjugate of tetracycline and its preparation method and application

technical field

The invention relates to a conjugate of antibiotics and a preparation method and application thereof, in particular to a tetracycline conjugate and a preparation method and application thereof. The invention belongs to the field of antibiotic immunoassay.

Background technique

The following titles involved in the present invention apply to the entire specification and claims:

Bovine Serum Albumin (BSA for short): a product of Sigma

Ovalbumin (OVA for short): a product of Sigma

Phosphate Buffered Saline (PBS for short) (0.01M, pH=7.40)

Dialysis membrane: Bioshorp products

1,1'-carbonyldiimidazole (1,1'-carbonyldiimidazole, referred to as CDI): Fluka company product

Tetracycline: product of Sigma

Hydroxysuccinimide (NHS for short): a product of Sigma

Ethyl[3-(dimethylamino)propyl]carbodiimide (EDC for short): product of Sigma

p-aminobenzoic acid (4-aminobenzoic acid): product of Fluka

Tetracycline antibiotics include aureomycin, doxycycline, metacycline, oxytetracycline, and tetracycline. Tetracycline antibiotics are broad-spectrum antibiotics, which have antibacterial effects on Gram-positive and negative bacteria, Rickettsia, etc. Its mechanism of action is mainly to bind to the end of the 30S ribosomal subunit, thereby interfering with bacterial protein synthesis. Due to its good oral absorption, excellent antibacterial properties, stable drug resistance and low price, tetracycline antibiotics are widely used as drug additives in livestock and poultry production to prevent intestinal infection and promote growth, but excessive use inevitably makes Related antibiotics such as maternal metabolites remain in animal muscles, eggs, milk, organs, and tissues, and then affect human health through the food chain. The toxic reactions of tetracyclines are mainly manifested in the damage to the stomach, intestines, and liver, as well as the staining of teeth, and can also cause allergic reactions, superinfection, and teratogenic effects. Yan Jun et al reported that oxytetracycline can lead to hepatomegaly, jaundice, fatty liver and so on. Tetracyclines can cause severe liver damage and even death in pregnant women.

Tetracycline antibiotics, especially tetracycline, are the most widely used pharmaceutical additives in my country, and their residues in animal foods often exceed the prescribed limit, causing great harm to human health. With the deepening of research on tetracyclines, especially the development and utilization of tetracyclines as preservatives, preservatives and other new uses, the analysis of tetracycline residues in animal foods is becoming more and more important; moreover, since China joined the WTO, my country The export of livestock and poultry products has repeatedly encountered green barriers, hindering trade and causing great economic losses to the entire industry. In order to cope with the challenges brought by drug residues to the development of the entire industry and food safety, the Chinese government and relevant departments have stepped up the detection of drug additives, revised and promulgated more stringent laws and regulations. To monitor and enforce the law on the production process of livestock products, one is to formulate standards for maximum residue limits, and the other is to improve residue analysis methods and technologies, and develop rapid, accurate and highly sensitive detection methods.

In the detection of tetracycline antibiotic drug residues, commonly used methods include instrumental methods such as high performance liquid chromatography, gas chromatography, and liquid chromatography-mass spectrometry. These methods are accurate, stable and reliable, and can be used as standard methods. However, the instrument method is expensive, time-consuming, and causes organic solvent pollution. It requires large-scale equipment and specialized technical personnel, so it is difficult to be used in field operations. Enzyme-linked immunoassay (ELISA) has the advantages of sensitivity, rapidity, good specificity, small sample volume, and does not require specialized personnel to operate, which makes ELISA an ideal detection method for routine scanning. However, ELISA requires high-quality antibodies. Tetracycline antibiotics are small molecular organic compounds that are not immunogenic and are called haptens. Therefore, these compounds must be converted into immunogens (also known as complete antigens) that can trigger the animal's immune system to produce antibodies. After searching, there are no reports on the synthesis of tetracycline immunogens and antibody preparation in the world, and most of the domestic ELISA kits for detecting tetracycline drug residues are purchased abroad, with short shelf life and high prices, which are far from meeting the detection needs. Therefore, it is very necessary to study the synthesis of tetracycline immunogen and antibody preparation.

Contents of the invention

Aiming at the deficiencies of the above-mentioned prior art, the problem to be solved by the present invention is to provide an immunogen capable of triggering the animal immune system to produce antibodies with specific responses to tetracycline, ie a conjugate of tetracycline and a preparation method thereof. At the same time, the present invention also provides the application of the tetracycline conjugate as an immunogen in the preparation of tetracycline specific reaction antibody.

The tetracycline conjugate of the present invention is composed of a tetracycline hapten coupled with an immunogenic carrier substance bovine serum albumin or ovalbumin.

Wherein: the above-mentioned immunogenic carrier substance is preferably bovine serum albumin.

The tetracycline conjugate of the present invention has a general structural formula such as (I)

Wherein: n: is the molecular number of tetracycline combined with one bovine serum albumin molecule, said n is an integer of 1 to 15, BSA is bovine serum albumin (Bovine Serum Albumin), and the molecular weight range is 6.6KDa to 6.9KDa;

The above-mentioned conjugates show the following physical and chemical characteristics:

(1) Appearance: light yellow powder solid;

(2) Ultraviolet absorption spectrum: 276nm, 320nm.

In the tetracycline conjugate mentioned above, said n is preferably an integer of 1-10, and the molecular weight range of BSA is preferably 6.7-6.8 KDa.

The preparation method of the above-mentioned tetracycline conjugate is as follows: the tetracycline is connected with the carrier substance that produces the immunogen, combined into a conjugate capable of inducing the animal immune system to produce antibodies, and the biological activity of the conjugate remains unchanged.

The preparation method of the above-mentioned tetracycline conjugate is specifically completed by the following steps:

(1) Preparation of solution A: Dissolve tetracycline and 1,1'-carbonyldiimidazole (CDI) in anhydrous acetone at a molar ratio of 1:1 to 1:3, feed nitrogen, and under nitrogen protection , 37°C±1°C and react in the dark for 3±1 hours to generate active intermediates of tetracycline and 1,1’-carbonyldiimidazole for later use; at the same time, the molar ratio of tetracycline to bovine serum albumin is 20:1- The amount of 35:1, weigh bovine serum albumin for later use;

(2) Preparation of solution B: Rotate the acetone in solution A after the above reaction to dryness, add pH = 8.5 ± 0.5, and dissolve the tetracycline and 1,1'-carbonyldiimidazole formed by borax buffer solution with a concentration of 0.5M Then add the above-mentioned bovine serum albumin to make the final concentration of bovine serum albumin 10.0±5mg/ml, under nitrogen protection, 25°C±5°C and avoid light for 30-48 hours to obtain solution B;

(3) Use a phosphate buffer solution with a pH of 7.30-7.50 and a concentration of 0.01M-0.02M at 0-4°C to stir the dialysis solution B for 70-80 hours, then use distilled water for dialysis for 20-30 hours, every 6 hours Change the dialysate once;

(4) Freeze-dry the dialyzed solution to obtain a light yellow tetracycline conjugate.

In the above-mentioned preparation method of tetracycline conjugates: the molar ratio of tetracycline to 1,1'-carbonyldiimidazole (CDI) in step (1) is preferably 1:2.

In the above-mentioned preparation method of tetracycline conjugates: the molar ratio of tetracycline to bovine serum albumin in step (1) is preferably 30:1.

In the above method for preparing tetracycline conjugates: the pH of the phosphate buffer in step (3) is preferably 7.40, and the concentration is preferably 0.01M.

The tetracycline conjugate of the present invention is used as an immunogen in the preparation of a tetracycline specific reaction antibody.

The technical scheme of the present invention can successfully couple the hapten tetracycline to the carrier protein, especially the bovine serum albumin, thereby synthesizing the complete immunogen-tetracycline conjugate capable of triggering an immune response and producing antibodies in animals.

The tetracycline conjugate of the present invention is used as the immunogen to immunize the New Zealand white rabbit, and the antibody with specific reaction to the hapten tetracycline is successfully obtained. As identified by enzyme-linked immunoassay, the tetracycline-specific reaction antibody prepared by using the tetracycline conjugate of the present invention as an immunogen has an antiserum titer of 1:50,000 or more and a minimum detection limit of 1 ppb.

The successful preparation of the above-mentioned tetracycline conjugates and high-titer tetracycline specific reaction antibodies provides a basis for the preparation of tetracycline enzyme-linked immunoassay kits. In practical application, the prepared tetracycline-specific reaction antibody can be plated in a microwell plate to detect tetracycline residues in animal-derived foods. Because the method of the invention has the characteristics of simplicity, rapidity, specificity and accuracy, it can be used for preliminary scanning and detection in food inspection and quarantine. This not only saves a lot of inspection time, but also can be used for on-site operations, thus making up for the shortcomings of the instrument method, which takes a long time, requires the support of large-scale instruments and equipment, requires specialized technical personnel to operate, and is difficult to use on-site. Therefore, the synthesis of the conjugated hapten tetracycline and carrier protein, especially bovine serum albumin, and the successful preparation of antiserum laid the foundation for this rapid test method.

Description of drawings

Figure 1 UV absorption spectrum:

Among them: 1: Tetracycline conjugate 2: Tetracycline 3: Mechanical mixing of tetracycline and BSA 4: BSA

Detailed ways

Example 1

(1) Preparation of solution A: Dissolve 40 mg of tetracycline in 8 ml of anhydrous acetone, pass through nitrogen, then quickly add 29 mg of 1,1'-carbonyldiimidazole, and react in the dark at 37°C for 3 hours under nitrogen protection. Reaction generates the active intermediate of tetracycline and 1,1'-carbonyldiimidazole (CDI), for subsequent use; meanwhile, take 200mg bovine serum albumin (molecular weight is 6.8Kda) for subsequent use;

(2) Preparation of solution B: the acetone in the above-mentioned reacted solution A was spun to dryness, and 15ml of pH=8.5 was added, and the concentration was 0.5M borax buffer solution to dissolve the generated tetracycline and the activity of 1,1'-carbonyldiimidazole intermediate, and then add the above-mentioned bovine serum albumin, so that the final concentration of bovine serum albumin is 13mg/ml, under the protection of nitrogen, 30 ℃ dark reaction for 48 hours; Stir dialysis solution B at -4°C for 72 hours, then dialyze with distilled water for 24 hours, and change the dialysate every six hours;

(3) Freeze-dry the dialyzed solution to obtain a pale yellow tetracycline conjugate, and the absorption peak of the product is determined to be located at 276nm and 320nm by ultraviolet absorption spectrum.

Example 2

(1) Preparation of solution A: Dissolve 40 mg of tetracycline in 8 ml of anhydrous acetone, pass through nitrogen, then quickly add 16 mg of 1,1'-carbonyldiimidazole, and react in the dark at 37°C for 2 hours under nitrogen protection. Reaction generates the active intermediate of tetracycline and 1,1'-carbonyldiimidazole (CDI), for subsequent use; meanwhile, take 240mg bovine serum albumin (molecular weight is 6.7Kda) for subsequent use;

(2) Preparation of solution B: the acetone in the above-mentioned reacted solution A was spun to dryness, and 20ml of pH=9.0 was added, and the concentration was 0.5M borax buffer solution to dissolve the generated tetracycline and 1,1'-carbonyldiimidazole activity Intermediate, and then add the above-mentioned bovine serum albumin, so that the final concentration of bovine serum albumin is 12 mg/ml, under the protection of nitrogen, 25 ℃ dark reaction for 36 hours; Stir dialysis solution B at -4°C for 70 hours, then dialyze with distilled water for 20 hours, and change the dialysate every six hours;

(3) Freeze-dry the dialyzed solution to obtain a pale yellow tetracycline conjugate, and the absorption peak of the product is determined to be located at 276nm and 320nm by ultraviolet absorption spectrum.

Example 3

(1) Preparation of solution A: Weigh 20 mg of tetracycline and dissolve it in 5 ml of anhydrous acetone, pass through nitrogen, then quickly add 10 mg of 1,1'-carbonyldiimidazole, and react in the dark at 37°C for 4 hours under the protection of nitrogen, Reaction generates the active intermediate of tetracycline and 1,1'-carbonyldiimidazole (CDI), for subsequent use; meanwhile, take 155mg bovine serum albumin (molecular weight is 6.9Kda) for subsequent use;

(2) Preparation of Solution B: Rotate the acetone in the A solution after the above reaction to dryness, add 15ml pH=8.0, and the concentration is 0.5M borax buffer solution to dissolve the generated tetracycline and 1,1'-carbonyldiimidazole activity intermediate, and then add the above-mentioned bovine serum albumin, so that the final concentration of bovine serum albumin is 10mg/ml, under the protection of nitrogen, 20 ℃ dark reaction for 40 hours; Stir dialysate solution B at -4°C for 75 hours, then dialyze with distilled water for 30 hours, and change the dialysate every six hours;

(3) Freeze-dry the dialyzed solution to obtain a pale yellow tetracycline conjugate, and the absorption peak of the product is determined to be located at 276nm and 320nm by ultraviolet absorption spectrum.

Example 4

Preparation, purification and detection of antibodies

1. Antibody Preparation

The tetracycline conjugate prepared in Example 1 above was selected as the immunogen to carry out animal immunization experiments to prepare antibodies.

Take 1ml of the tetracycline conjugate solution of 1mg/ml, add an equal volume of Freund's complete adjuvant, fully emulsify, and inject into four male healthy New Zealand white rabbits weighing 2kg at multiple points subcutaneously, 1ml/only, 15 Two days later, the same amount of antigen was fully emulsified with Freund's incomplete adjuvant for the second immunization. After the second immunization, a booster immunization was given every 15 days, and the amount of antigen was halved, for a total of 5 immunizations. Seven days after the last immunization, blood was collected from the heart, left at room temperature for 1 hour, overnight at 0-4°C, centrifuged at 13,000 rpm for 15 minutes, and serum was collected and stored at -20°C for later use.

2. Antibody Purification

Add saturated ammonium sulfate to the antiserum prepared above under stirring until the final concentration of the ammonium sulfate solution is 50% by volume, and place it at 0-4°C overnight, and a precipitate precipitates; centrifuge at 13000 rpm for 15 minutes, discard For the supernatant, add 0.01M, pH7.4 PBS to the precipitate until the precipitate is dissolved, then add saturated ammonium sulfate solution until the final concentration of the ammonium sulfate solution is 33% by volume, place it overnight at 0-4°C, and there will be precipitation Centrifuge at 13,000 rpm for 15 minutes, discard the supernatant, and add 0.01M PBS with pH 7.4 to the precipitate until the precipitate dissolves. The above-mentioned purified product was dialyzed with 0.01M, pH7.4 PBS at 0-4°C, and the dialysate was changed 3 times, then sodium azide with a mass volume percentage of 0.02% was added, and stored at -20°C for future use.

3. Antibody ELISA

(1) Potency determination: the method adopts conventional indirect enzyme-linked immunosorbent assay method:

On a 96-well microtiter plate, coat it with 100 μl/well tetracycline-ovalbumin conjugate (10 μg/ml), place it overnight at 0-4°C, and then wash it with PBST (1000ml pH7.4, concentration 0.01M PBS + volume percentage is 0.05% Tween20) wash plate four times; use 250 μl/well blocking solution (1000ml PBST + mass volume percentage is 1% ovalbumin) to block, place at room temperature for 3 hours, wash plate; after washing away blocking solution, add 100ul antiserum per well, place at room temperature for 2 hours, and wash the plate; after washing away the antiserum, add 1:1000 horseradish peroxidase-labeled goat anti-rabbit IgG 100ul to each well, place at room temperature for 1 hour, and wash the plate; The substrate o-phenylenediamine was added for color development, left at room temperature for 10 min, and then 2M HCl was added to stop. Microplate reader A492nm detection.

It is determined that the tetracycline conjugate antibody titer of the present invention reaches 1:51200.

The determination of the titer is based on the highest serum dilution ratio with a P/N greater than 2:1 as the ELISA titer of the antibody.

Wherein: the above-mentioned P is the absorbance value measured at a certain dilution factor of the substitute test serum, and the above-mentioned N is the absorbance value measured at the corresponding dilution factor of the negative control.

(2) Specificity determination:

The determination procedure is similar to the titer determination. Under the above-mentioned optimum coating antigen and antibody concentration conditions, tetracycline solution (from 200ppm-0.1ppb) is added at the same time as the antibody is added to compete with the coating antigen for limited antibody binding. Tetracycline drug The higher the concentration, the less the antibody binds to the coated antigen, resulting in lighter color development and lower absorbance values. Then compare it with the blank control (the absorbance value of only adding the antibody, without adding tetracycline drug) to determine the specificity of the antibody.

By determining the specificity of the antibody is better, the minimum detection limit can reach 1ppb.

Example 5

Prepare the conjugate of tetracycline and ovalbumin used in the above enzyme-linked immunoassay:

(1) Preparation of solution A: Weigh 12 mg of sodium nitrite and dissolve it in 0.35 ml of distilled water, then weigh 20 mg of p-aminobenzoic acid and dissolve it in 2.2 ml of 0.2M hydrochloric acid, stir in an ice bath, and dissolve the sodium nitrite solution Add it dropwise to the p-aminobenzoic acid solution, and react in the dark for 1 hour to obtain a colorless active p-aminobenzoic acid intermediate, which is set aside;

(2) Preparation of solution B: Weigh 27 mg of tetracycline and dissolve it in 10 ml of ice-cold borax buffer solution (pH=9.0, containing 0.15 M sodium chloride), stir in an ice bath, and add 1.5 ml of the above-mentioned A solution dropwise to In this solution, react in the dark for 2 hours to obtain a dark red tetracycline active intermediate, which is set aside.

(3) Preparation of the conjugate of tetracycline and ovalbumin: add H ₃ BO ₃ crystals to solution B to adjust its pH to 7.4, then add 86 mg of ovalbumin (molecular weight 4.3 Kda), and then add 120 mg of ethyl [3-(Dimethylamino)propyl]carbodiimide, 36 mg of hydroxysuccinimide, stirred at room temperature for 3 hours to obtain a dark red solution.

Using PBS buffer solution with pH 7.4 and concentration 0.01M at 0-4°C, stir the solution prepared in the dialysis step (3) for 72 hours, then dialyze with distilled water for 30 hours, and change the dialysate every 6 hours.

The dialysate was centrifuged at 3000 rpm for 30 minutes. Freeze-dry the supernatant to obtain a deep red solid, which is the conjugate of tetracycline and ovalbumin; its UV absorption is 277nm, 410nm, 524nm, and its structure is as follows:

Wherein: the above n: is the number of tetracycline molecules bound to one ovalbumin molecule, and the n is an integer of 1-15.

Claims (10)

1. the conjugate of a tsiklomitsin is made of tsiklomitsin haptens and immunogenic carrier substance bovine serum albumin of generation or ovalbumin coupling.

2. the conjugate of tsiklomitsin as claimed in claim 1, the immunogenic carrier substance of wherein said generation is a bovine serum albumin.

3. the conjugate of tsiklomitsin as claimed in claim 2, its general structure is as (I)

Wherein: n: for the molecule number of a bovine serum albumin molecule bonded tsiklomitsin, described n is an integer 1～15, BSA is bovine serum albumin (Bovine Serum Albumin), molecular weight ranges is 6.6KDa～6.9KDa;

Above-mentioned conjugate demonstrates following physical chemical characteristics:

(1) outward appearance: pale yellow powder solid;

(2) ultra-violet absorption spectrum: 276nm, 320nm.

4. the conjugate of tsiklomitsin as claimed in claim 3, it is characterized in that: described n is an integer 1～10, and the bovine serum albumin molecular weight ranges is 6.7～6.8KDa.

5. the preparation method of the conjugate of each described tsiklomitsin in the claim 1～4, it is characterized in that: tsiklomitsin and the immunogenic carrier substance of generation are coupled together, be combined into and have the conjugate that brings out animal immuning system generated antibody, and keep the biological activity of this conjugate constant.

6. as the preparation method of the conjugate of tsiklomitsin as described in the claim 5, finish by following steps:

(1) preparation of solution A: with tsiklomitsin and 1,1 '-carbonyl dimidazoles (CDI) is that 1: 1～1: 3 ratio is dissolved in the anhydrous propanone in molar ratio, feed nitrogen, under nitrogen protection, 37 ℃ ± 1 ℃ lucifuge was reacted 3 ± 1 hours, reaction generates tsiklomitsin and 1, and the active intermediate of 1 '-carbonyl dimidazoles is standby; Simultaneously, be 20 with the mole ratio of tsiklomitsin and bovine serum albumin: 1-35: 1 amount, it is standby to take by weighing bovine serum albumin;

(2) preparation of solution B: with the rotation of the acetone among above-mentioned reacted solution A evaporate to dryness, add pH=8.5 ± 0.5, concentration is the tsiklomitsin and 1 that the borax buffer solution dissolving of 0.5M generates, the active intermediate of 1 '-carbonyl dimidazoles, and then adding above-mentioned bovine serum albumin, the final concentration that makes bovine serum albumin is 10.0 ± 5mg/ml, under nitrogen protection, 25 ℃ ± 5 ℃ lucifuges were reacted 30～48 hours, got solution B;

(3) be 7.30-7.50 with pH, concentration be the phosphoric acid buffer of 0.01M-0.02M under 0-4 ℃, stirred dialysis solution B 70～80 hours, used distill water dialysis then instead 20～30 hours, changed a dialyzate in per 6 hours;

(4) solution after the freeze-drying dialysis obtains the conjugate of flaxen tsiklomitsin.

7. the preparation method of the conjugate of tsiklomitsin as claimed in claim 6, it is characterized in that: the described tsiklomitsin of step (1) and 1, the mol ratio of 1 '-carbonyl dimidazoles (CDI) is 1: 2.

8. the preparation method of the conjugate of tsiklomitsin as claimed in claim 6, it is characterized in that: the mole ratio of described tsiklomitsin of step (1) and bovine serum albumin is 30: 1.

9. the preparation method of the conjugate of tsiklomitsin as claimed in claim 6, it is characterized in that: the pH of the described phosphoric acid buffer of step (3) is 7.40, and concentration is 0.01M.

In the claim 1～4 conjugate of each described tsiklomitsin as the application of immunogen in preparation tsiklomitsin specific reaction antibody.