CN103439490A - Method of using fluorescent microsphere to mark ractopamine monoclonal antibody - Google Patents

Abstract

The invention belongs to the field of biotechnology, and discloses a method for labeling ractopamine monoclonal antibody with fluorescent microspheres, specifically: coupling amino-modified fluorescent microspheres with active esterified biotin, and combining streptavidin with the above-mentioned coupling After the conjugate was mixed and reacted at a ratio of 1000:1, the conjugate of fluorescent microspheres and biotin-streptavidin was obtained, and finally biotinylated ractopamine monoclonal antibody was added to the fluorescent microspheres-biotin- The target product can be obtained by reacting in the conjugate solution of streptavidin. The novel conjugates of the fluorescent microsphere-labeled ractopamine monoclonal antibody obtained in the present invention can be effectively applied to the ractopamine fluorescent microsphere test strips, correspondingly improving the specificity and sensitivity of the test strips, thereby rapidly and accurately Qualitative and quantitative detection of ractopamine residues in animal foods.

Description

Method for labeling ractopamine monoclonal antibody with fluorescent microspheres

technical field

The invention belongs to the field of detection of beta ₂ -receptor agonist residues in food safety, in particular to a conjugate of ractopamine monoclonal antibody labeled with fluorescent microspheres and a preparation method thereof.

Background technique

In recent years, many unscrupulous businesses have added ractopamine to animal feed as a substitute for clenbuterol hydrochloride. When fed to animals for a long time, ractopamine, like clenbuterol hydrochloride, has the same nutritional redistribution effect on animals , Promote protein deposition in animals and inhibit fat formation, thereby significantly increasing the lean meat rate of animal carcasses. However, ractopamine metabolizes slowly in the body, is easy to accumulate, and has a high drug residue rate. When humans eat animal foods with residues of such drugs, they will be poisoned and cause considerable harm to human health, usually manifested as muscle tremors, palpitations, fever, Nausea, vomiting, etc., severe coma or even death. Therefore, many countries have banned the addition of β ₂ -stimulants in animal feed, but food safety incidents about β ₂ -stimulants occur from time to time. Announcement No. 1025 of the Ministry of Agriculture of my country stipulates that the minimum standard for clenbuterol hydrochloride, ractopamine, salbutamol and cimaterol is 3 ng/mL.

Due to the advantages of fast, accurate and convenient, immunochromatography has been greatly promoted in the detection and application of clenbuterol hydrochloride and its substitutes. It is a research method for qualitative or quantitative detection of a certain substance. At present, the markers commonly used in the detection of ractopamine hydrochloride by immunochromatography include colloidal gold and fluorescent microspheres. After the antigen reaction, the markers in the complex can be directly measured, and the qualitative or even quantitative analysis of the target substance can be directly performed.

At present, most of the labels and antibodies are directly coupled through covalent bonds or physical adsorption, especially the labeling of β-receptor agonist antibodies. This coupling method has the advantages of short coupling time and simple operation, but in this coupling method, some antigen-binding sites are shielded due to the random binding of antibodies to the label, which is the so-called steric hindrance, which leads to the efficiency of antibody labeling. and marker dispersion is reduced.

Contents of the invention

The object of the present invention is to provide a method for labeling ractopamine monoclonal antibody with fluorescent microspheres in view of the defects of the above-mentioned prior art. The conjugate can exhibit good colloidal stability and binding properties.

The technical scheme that the present invention takes in order to realize the above object is:

The method for labeling ractopamine monoclonal antibody with fluorescent microspheres: (1) Coupling reaction of amino-modified fluorescent microspheres with active esterified biotin in PBS solution for 2 h to obtain fluorescent microsphere-biotin conjugates, and then centrifuged , Wash to remove unbound biotin; (2) Mix streptavidin and fluorescent microsphere-biotin conjugate for 2 h, centrifuge, wash with PBS solution to remove uncoupled streptavidin avidin to obtain fluorescent microsphere-biotin-streptavidin conjugate; (3) adding biotinylated ractopamine monoclonal antibody to fluorescent microsphere-biotin-streptavidin conjugate After gently shaking and reacting in the conjugate solution for 1 h, wash and centrifuge to remove unconjugated biotinylated ractopamine monoclonal antibody, and obtain fluorescent microsphere-labeled ractopamine monoclonal antibody, that is, fluorescent microsphere-biotin-chain Mycoavidin-biotinylated ractopamine monoclonal antibody; the active esterified biotin is to dissolve the biotin-PEG ₅₀₀₀ complex in DMF solution, add dicyclohexylcarboimide and N-hydroxysuccinyl After the imine was stirred and reacted at room temperature for 24 h, the precipitate was centrifuged to obtain active esterified biotin; the preparation method of the amino-modified fluorescent microspheres: 10 mg of fluorescent microspheres were washed and dissolved in 1 mL of distilled water by ultrasonic. After adding 20 μL of glacial acetic acid and 20 μL of (3-trimethoxysilylpropyl)-diethylethylenediamine for ultrasonic dissolution, stir magnetically for 3-4 h, wash and resuspend with 10 mM MES buffer at pH 5.5; The particle size of the fluorescent microspheres is 175 nm.

The preparation method of the biotinylated ractopamine monoclonal antibody: dissolve biotin N-hydroxysuccinimide ester NHSB in 1 mL DMSO solution to prepare a solution with a concentration of 1 mg/mL, add 120 μL to 1 mL ractopamine In the dopamine monoclonal antibody solution, keep stirring at room temperature for 2-4 h; then add 9.6 μL of 1 mol/L NH ₄ Cl solution and stir at room temperature for 10 min, then fully dialyze with PBS solution at 4°C to remove free Biotin; put the dialyzed sample on a 1 ml molecular sieve column, slowly elute with PBS solution, wash down the antibody between 1 and 3 ml, then add sodium azide and 1.0 g of the target product to the collected /L of BSA solution and stored at 4°C in the dark for future use.

The molar ratio of the streptavidin to the fluorescent microsphere-biotin conjugate mixed reaction is 1000:1.

The molar ratio of the fluorescent microsphere-labeled biotinylated ractopamine monoclonal antibody to the fluorescent microsphere-biotin-streptavidin conjugate is 1:1000.

The beneficial effects of the present invention are:

1. High labeling efficiency: Compared with traditional fluorescent microspheres directly coupled to antibodies, biotin-streptavidin as a coupling arm reduces the steric hindrance effect of the binding between fluorescent microspheres and ractopamine monoclonal antibodies. Small, and a fluorescent microsphere-PEG ₅₀₀₀ -biotin-streptavidin can be coupled to a biotinylated antibody 1:4, resulting in a corresponding increase in labeling efficiency.

2. Good dispersibility and solubility: the biotin combined with fluorescent microspheres is a complex modified with hydrophilic PEG ₅₀₀₀ , and the embedding of hydrophilic PEG ₅₀₀₀ improves the complexation of fluorescent microspheres-antibody conjugates. Solubility in the solution, and because of the longer coupling arm, the conjugate can be well dispersed in the solution, basically no flocculation and precipitation will occur, showing good colloidal stability.

3. Strong binding: After the PEG ₅₀₀₀ -biotin-streptavidin coupling arm is combined with the biotin on the ractopamine monoclonal antibody, it ensures the directional distribution of the antibody binding sites on the surface of the fluorescent microspheres and enhances the fluorescence The binding performance of the microsphere-ractopamine monoclonal antibody conjugate to the target substance is mainly reflected in the improved sensitivity and stability of the fluorescent microsphere test strip.

Description of drawings

The above-mentioned and/or additional aspects and advantages of the present invention will become apparent and easy to understand from the description of the embodiments in conjunction with the following drawings:

Fig. 1 Fluorescent microspheres labeled ractopamine monoclonal antibody prepared by the present invention.

Detailed ways

This example provides detailed implementation and specific operation process, but the scope of protection of the present invention is not limited to the following examples.

Biotin-PEG ₅₀₀₀ complex was purchased from Shanghai Yanyi Biological Co., Ltd.;

Fluorescent microspheres were purchased from Merck, Germany, with a particle size of 175 nm.

Example 1: Preparation method of novel conjugates of fluorescent microsphere-labeled ractopamine monoclonal antibody in the present invention

1) Preparation method of amino-modified fluorescent microspheres

Wash 10 mg of fluorescent microspheres and ultrasonically dissolve them in 1 mL of distilled water, add 20 μL of glacial acetic acid and 20 μL of (3-trimethoxysilylpropyl)-diethylethylenediamine for ultrasonic dissolution, and stir magnetically for 3 to 4 h, washed with 10 mM MES buffer (pH 5.5) and resuspended.

2) Preparation method of active esterified biotin

Accurately weigh 4.5 mg of biotin-PEG ₅₀₀₀ complex and dissolve it in 2 mL of DMF solution, add 20.5 mg of dicyclohexylcarboimide and 10.5 mg of N-hydroxysuccinimide, and react with magnetic stirring at room temperature for 24 h, then Centrifuge at 4000 rpm for 5 min, discard the precipitate, and obtain active esterified biotin, which is set aside.

3) A method for labeling ractopamine monoclonal antibody with fluorescent microspheres, characterized in that: the preparation method of the biotinylated ractopamine monoclonal antibody: dissolve biotin N-hydroxysuccinimide ester ( NHSB) to prepare a solution with a concentration of 1 mg/mL, take 120 μL and add it to 1 mL of ractopamine monoclonal antibody solution with a concentration of 1.9 mg/mL, keep stirring at room temperature, and keep warm for 2~4h; then add 9.6 μL of NH ₄ Cl (1 mol/L) solution was stirred at room temperature for 10 min, then fully dialyzed with PBS solution at 4 °C to remove free biotin; put the dialyzed sample on a 1 ml molecular sieve column, and slowly elute with PBS solution , the antibody was washed between 1-3 ml, and then sodium azide and 1.0 g/L BSA solution were added to the collected target product, and stored at 4 °C in the dark for future use.

4) Label 35 μg/mg (antibody/microsphere) immune microspheres with 5 mL system, specific method: 2.0 mg amino-modified fluorescent microspheres and 3.5 mg active esterified biotin in 5 mL PBS solution (pH 7.4 or so) in the coupling reaction for 2 h, then centrifuged and washed to remove unbound biotin to obtain the fluorescent microsphere-biotin conjugate; add the fluorescent microsphere-biotin conjugate prepared above to 10 mg /mL streptavidin solution, the molar ratio of streptavidin and fluorescent microspheres-biotin conjugate mixed reaction is 1000:1, the final volume is guaranteed to be 5 mL, and the reaction is gently shaken for 2 h Afterwards, centrifuge and wash with PBS buffer solution to remove unconjugated streptavidin to obtain fluorescent microspheres-biotin-streptavidin conjugates; then correspondingly labeled biotinylated ractopamine Monoclonal antibody (diluted 10 times and added slowly) was added to the above-mentioned conjugate solution to a final concentration of 35 μg/mg, and after shaking for 1 h, washed and centrifuged to remove unconjugated biotinylated antibody, that is A novel conjugate of ractopamine monoclonal antibody labeled with fluorescent microspheres was obtained and reconstituted with 500 μL of reconstitution solution.

Compared with the traditional fluorescent microsphere-antibody conjugates, the new conjugates prepared above have significantly better dispersion under the electron microscope. After being placed at 4 °C for 6 months, they are still very transparent and will not occur. Flocculation and precipitation phenomenon, showing good colloidal stability. Its strong binding ability is reflected by the increase in the sensitivity of the fluorescent microsphere test strip in the following examples.

Example 2: Application of novel conjugates of fluorescent microsphere-labeled ractopamine monoclonal antibody on fluorescent microsphere test strips

1) Preparation of fluorescent microsphere immunochromatographic test strips

Preparation of fluorescent microsphere pads: After re-dissolving the conjugate prepared in Example 1 with 0.01 M PNPB (which contains 5% sucrose and 0.05% Tween-20) to the initial volume, use BIODOT Dispensing System, according to 4 μL The amount per cm is sprayed onto the glass fiber membrane, dried in vacuum at 25 ℃ for 1-2 hours, and placed in a dry environment for later use.

Coat the ractopamine-BSA conjugate and the donkey anti-mouse secondary antibody on the NC membrane: use 0.01 M PBS buffer to adjust the coating concentration to 0.4 mg/mL and 0.4 mg/mL respectively, and spray the membrane volume to 0.74 μL /cm, the detection line is coated with ractopamine-BSA conjugate, and the quality control line is coated with donkey anti-mouse secondary antibody. 9 mm, dried overnight at 37°C, and stored in a dry environment at room temperature for later use.

Preparation of fluorescent microsphere immunochromatography test strips: Paste filter paper, sample pad, fluorescent microsphere pad, NC membrane and absorbent paper on the bottom plate successively, and cut the pasted test strips into test strips with a cutting machine. Paper strips, assembled into the prepared test strip box, put into an aluminum foil bag, add a desiccant, and seal it for storage. It can be stored for at least one year in a dry environment at room temperature.

2) Quantitative detection of ractopamine in pork with fluorescent microsphere test strips

Sample pretreatment: Take 2.5 g of pork sample in a 50 mL centrifuge tube, add 5 mL of distilled water, heat and extract in boiling water for 15 min, take out all the supernatant in a new 10 mL centrifuge tube, add 1 mL of n-hexane and shake gently Let it stand for 1 min, remove the upper fat layer, and reserve the lower supernatant.

Detection steps: (1) Add the negative sample and the sample extract with a series of concentrations (0.5, 1, 2, 3, 4, 5, 6, 8, 10, 15 ng/mL) into the sample hole of the test strip, and react After 10 minutes, put the test strip into the detection window of the fluorescent microsphere reader;

(2) Fluorescent microspheres emit strong fluorescence under the excitation of LED light source;

(3) After the emitted fluorescence is converged by the CCD scanning system, it is sent to the photomultiplier tube through the photoelectric focusing tube, and the optical signal is enhanced. After passing through the signal conversion element and software processing, the intensity of the fluorescence is input in the form of numerical values. ;

(4) During the experiment, the values corresponding to the fluorescence intensity were measured with a series of concentrations, so as to establish a standard curve based on the series of values and the corresponding concentrations of ractopamine standard substances. Finally, according to the output value of the test sample, the content of ractopamine in the test sample is calculated;

(5) Draw 100 μL of the extract for detection. Finally, according to the data of the test sample, the output values are 1.95, and the residual amount of ractopamine in the test sample is found to be 0.25 ng/mL by checking the standard curve.

Example 3: Detection of residual ractopamine in pig liver on a fluorescent microsphere test strip prepared by a novel conjugate of ractopamine monoclonal antibody labeled with fluorescent microspheres

The sample pretreatment method and test strip production are the same as in Example 2.

Qualitative and quantitative detection methods are the same as in Example 2, and a standard curve is drawn by adding a series of concentrations (0.5, 1, 2, 3, 4, 5, 6, 8, 10, 15 ng/mL) and corresponding fluorescence intensity values. Draw 100 μL of the extract and add it to the sample well. After 10 minutes, use a reader to detect. According to the data output of the test sample, the output values are 2.80. Check the standard curve to find that the residual amount of ractopamine in the test sample is 0.

Example 4: Detection of Ractopamine Residue in Feed on Fluorescent Microsphere Test Strips Prepared by New Conjugate of Fluorescent Microsphere-labeled Ractopamine Monoclonal Antibody

Sample pretreatment: Weigh 0.5 g of feed sample, add 1 mL of sample extract (pH7.0 50 mmol/L Tris-HCl), stir in the test tube for 1 min, let it stand for 10 min, and take the supernatant to be tested sample.

The test strip is made with embodiment 2.

Qualitative and quantitative detection methods are the same as in Example 2, and a standard curve is drawn by adding a series of concentrations (0.1, 0.5, 1, 2, 2.5, 3, 5, 7, 10, 15 ng/mL) and corresponding fluorescence intensity values. Draw 100 μL of the extract and add it to the sample well, and use a reader to detect after 10 minutes. According to the data output of the test sample, the output values are 1.43, and the residual amount of ractopamine in the test sample is found to be 0.8 ng/mL by checking the standard curve. .

Example 5: Detection of residual ractopamine in pig urine on a fluorescent microsphere test strip prepared by a novel conjugate of ractopamine monoclonal antibody labeled with fluorescent microspheres

Urine samples can be tested directly without pretreatment. Qualitative and quantitative detection methods are the same as in Example 2, and a standard curve is drawn by adding a series of concentrations (0.1, 0.5, 1, 1.5, 2, 2.5, 3, 5, 7, 10 ng/mL) and corresponding fluorescence intensity values. Draw 100 μL of urine sample into the sample hole, and use the reader to detect after 10 minutes. According to the data output of the test sample, the output values are 1.55. Checking the standard curve shows that the residual amount of ractopamine in the test sample is 0.57 ng / mL.

Claims (4)

1. the method for fluorescent microsphere mark Ractopamine monoclonal antibody, it is characterized in that: (1) amido modified fluorescent microsphere obtains fluorescent microsphere-biotin conjugates with biotin coupling reaction 2 h in PBS solution of active esterification, more unconjugated biotin is removed in centrifugal, washing; (2) by Streptavidin with after fluorescent microsphere-biotin conjugates mixing jog reacts 2 h, Streptavidin centrifugal, remove not coupling with the PBS solution washing, obtain fluorescent microsphere-biotin-Streptavidin conjugate; (3) biotinylated Ractopamine monoclonal antibody is joined in the conjugate solution of fluorescent microsphere-biotin-Streptavidin after jog reacts 1 h, washing, centrifugal to remove the biotinylation Ractopamine monoclonal antibody in not coupling, obtaining fluorescent microsphere mark Ractopamine monoclonal antibody is fluorescent microsphere-biotin-Streptavidin-biotinylation Ractopamine monoclonal antibody; The biotin of described active esterification is by biotin-PEG ₅₀₀₀compound is dissolved in DMF solution, add dicyclohexyl carbon imines and N-hydroxy-succinamide after stirring at room react 24 h, the centrifugal precipitation of abandoning then, obtain the biotin of active esterification; The preparation method of described amido modified fluorescent microsphere: 10 mg fluorescent microspheres clean after ultrasonic dissolution in 1 mL distilled water, after adding 20 μ L glacial acetic acids and 20 μ L (3-trimethoxy silicon propyl group)-diethyl ethylenediamine ultrasonic dissolution, magnetic agitation 3～4 h, also resuspended with the MES damping fluid washing of 10 mM pH5.5; Described fluorescent microsphere particle diameter is 175 nm.

2. the method for fluorescent microsphere mark Ractopamine monoclonal antibody according to claim 1, it is characterized in that: described biotinylation Ractopamine monoclonal antibody the preparation method: dissolve biotin N-hydroxy-succinamide ester NHSB with 1 mL DMSO solution and be mixed with the solution that concentration is 1 mg/mL, getting 120 μ L joins in the Ractopamine monoclonal antibody solution that 1mL concentration is 1.9 mg/mL, at room temperature continue to stir, be incubated 2 ~ 4 h; Then the NH that adds 9.6 μ L 1 mol/L ₄cl solution at room temperature stirs 10 min, with PBS solution, under 4 ℃, fully dialyses and removes free biotin; By the molecular sieve column of 1 ml on the sample after having dialysed, with the slow wash-out of PBS solution, antibody, under washing between 1 ~ 3 ml, then adds the BSA solution of Sodium azide and 1.0 g/L in the target product of collecting, and under 4 ℃, keeps in Dark Place standby.

3. the method for fluorescent microsphere mark Ractopamine monoclonal antibody according to claim 1, it is characterized in that: the molar ratio of described Streptavidin and fluorescent microsphere-biotin conjugates hybrid reaction is 1000:1.

4. the method for fluorescent microsphere mark Ractopamine monoclonal antibody according to claim 1, it is characterized in that: the molar ratio of the conjugate of described fluorescent microsphere mark biotinylation Ractopamine monoclonal antibody and fluorescent microsphere-biotin-Streptavidin is 1:1000.