CN108181463B - A kind of beta-agonist hapten and artificial antibody and its preparation method and application - Google Patents

Abstract

The invention discloses a β-agonist hapten, the molecular structure of which is shown in the following formula (I):

. The present invention uses R‑(‑)‑salbutamol as a raw material, and successfully synthesizes the β‑agonist hapten through one-step chemical reaction. The hapten is then coupled with a carrier protein to prepare an artificial antigen, and a broad-spectrum specific β-agonist artificial antibody is obtained after immunization. The antibody can recognize 31 β-agonist drugs and their analogs, the half-inhibitory concentration of R-(-)-salbutamol is 0.5ng/mL, the linear range is 0.11-40.86ng/mL, and the minimum detection limit is 0.04 ng/mL. The antibody can simultaneously detect multiple β-agonist drugs, can be used for on-site rapid detection of food safety, and has broad application prospects.

Description

A kind of beta-agonist hapten and artificial antibody and its preparation method and application

technical field

The present invention relates to the technical field of food safety detection, in particular to a beta-agonist hapten and artificial antibody, and a preparation method and application thereof.

Background technique

β-agonist (β-agonist) is the abbreviation of β-adrenoceptor agonist (β-adrenoceptor agonist), which belongs to a kind of phenethylamine drugs. Because of its ability to change energy distribution, reduce fat production, promote fat decomposition, promote protein synthesis and reduce its degradation, beta-agonists have been added to feeds in the 1980s to improve cattle, sheep, pigs, etc. A report on the growth rate of lean meat in livestock. When the cumulative ingested dose of the human body exceeds the threshold or ingests high-residue animal visceral tissues, β-agonists will produce obvious physiological toxicity, resulting in palpitations, tremors, dizziness, nausea and vomiting, vasodilation, nervousness, rapid heartbeat, etc. Symptoms can even lead to death in severe cases, so countries around the world have banned the use of such substances in food animals.

However, β-agonists used as "clenbuterol" have appeared successively, such as clenbuterol, salbutamol, ractopamine, brobuterol and other drugs are used by unscrupulous merchants for profit. Among them, salbutamol has a chiral structure, and under physiological conditions, the levorotatory isomer can better bind to receptors and show physiological activity. Therefore, R-(-)-salbutamol is mostly used as a clinical drug for the treatment of respiratory diseases such as asthma. R-(-)-salbutamol, as the active structure of the salbutamol racemate, should be the object of key monitoring and testing. It is very necessary to strengthen the monitoring and testing of such drugs in food processing, storage and sales. However, there is no detection method for the chiral structure and broad-spectrum specificity of β-agonists.

At present, the detection methods of β-agonists mainly include high performance liquid chromatography (HPLC), liquid chromatography-tandem mass spectrometry (LC-MS or LC-MS/MS), gas chromatography (GC-MS), capillary electrophoresis chromatography (CE) )Wait. Although these methods have high accuracy, their equipment is expensive, the sample pretreatment is complicated and cumbersome, the detection cost is high, and professional operation is required, etc., which cannot achieve the purpose of high-throughput rapid detection. The immunodetection method has the characteristics of rapidity, sensitivity, accuracy, and simple operation, and does not require high sample purity, so it is suitable for on-site rapid detection of large quantities of samples. Although most of the current antibodies for the detection of β-agonists have high specificity, it is not possible to detect multiple β-agonists that may be added in the same experiment. Due to the great harm of abuse of β-agonists, there is an urgent need for immunoassays that can simultaneously identify and detect multiple β-agonists, especially antibodies that can simultaneously recognize multiple β-agonists.

SUMMARY OF THE INVENTION

The technical problem to be solved by the present invention is that there is no antibody capable of recognizing multiple β-agonists simultaneously in the prior art. In order to overcome the above-mentioned deficiencies of the prior art, a β-agonist hapten is provided, which is combined with the hapten and the β-agonist. The artificial antigen prepared by coupling with the carrier protein is an immunogen, and the prepared artificial antibody of broad-spectrum specific β-agonist can detect a variety of β-agonist drugs at the same time, and can be used for on-site rapid detection of food safety.

The object of the present invention is to provide a beta-agonist hapten.

Another object of the present invention is to provide the application of the β-agonist hapten in the preparation of β-agonist artificial antigen or antibody.

Another object of the present invention is to provide a method for preparing the β-agonist hapten.

Another object of the present invention is to provide a β-agonist artificial antigen.

Another object of the present invention is to provide a method for preparing the β-agonist artificial antigen.

Another object of the present invention is to provide a broad-spectrum specific β-agonist artificial antibody.

Another object of the present invention is to provide the application of the broad-spectrum specific β-agonist artificial antibody in the preparation of a kit for detecting β-agonist drugs and/or analogs thereof.

Another object of the present invention is to provide a kit for detecting β-agonist drugs and/or their analogs.

In order to achieve the above object, the present invention is achieved through the following schemes:

A beta-agonist hapten whose molecular structure is shown in the following formula (I):

。

.

The present invention claims the application of the β-agonist hapten in the preparation of β-agonist artificial antigen or antibody.

The preparation method of the β-agonist hapten comprises the following steps:

S1. Dissolve R-(-)-salbutamol in methanol, fill with nitrogen and add succinic anhydride, and fully stir the reaction;

S2. Centrifuge the reaction product obtained in S1 and discard the supernatant, wash the precipitate with absolute ethanol, and obtain the target product after vacuum drying.

Preferably, the mass-to-volume ratio of R-(-)-salbutamol to methanol in S1 is 4-7 mg/mL, and the substance-to-substance ratio of R-(-)-salbutamol to succinic anhydride is 1 to 1.5:1 .

More preferably, the mass volume ratio of R-(-)-salbutamol to methanol in S1 is 5.5 mg/mL, and the substance ratio of R-(-)-salbutamol to succinic anhydride is 1.2:1.

Preferably, the time of the stirring reaction in S1 is 6-15 h. More preferably, the time of the stirring reaction in S1 is 12h.

The present invention also claims to protect a β-agonist artificial antigen, which is obtained by coupling the above-mentioned β-agonist hapten with a carrier protein.

Preferably, the carrier protein is bovine serum albumin (BSA) or ovalbumin (OVA).

The preparation method of the β-agonist artificial antigen comprises the following steps:

S1. Dissolve triethylamine in dimethylformamide (DMF) solution, stir at 4 °C for 10-20 min, then add β-agonist hapten and isobutyl chloroformate, and stir at 4 °C for 15 min. ~60min, to obtain beta-agonist hapten solution;

S2. Dissolve the carrier protein in the carbonate buffer, add the β-agonist hapten solution obtained from S1 dropwise while stirring, and stir the reaction at 4°C for 6-15 hours. PBS solution was dialyzed to obtain β-agonist artificial antigen.

The above-mentioned preparation method is an active ester method, and preferably, the preparation method is a mixed acid anhydride method.

Preferably, the time of the stirring reaction in S1 is 30min, and the time of the stirring reaction in S2 is 12h.

Preferably, the concentration of the PBS solution in S2 is 0.01 mol/L, and the pH is 7.4.

The present invention also claims to protect a broad-spectrum specific β-agonist artificial antibody, which is prepared from the above-mentioned β-agonist artificial antigen as an immunogen and purified by the octanoic acid-ammonium sulfate method.

As a preferred embodiment, the preparation method of the broad-spectrum specific β-agonist artificial antibody specifically includes the following steps:

The above β-agonist artificial antigen solution was mixed and emulsified with an equal amount of adjuvant. The initial immunization was mixed and emulsified with Freund's complete adjuvant and then immunized. The booster immunization interval was 3 weeks, and blood was collected from the 7th day after the third immunization to detect the antiserum titer until the antiserum titer no longer increased. , and centrifuged to take the upper serum. The artificial antibody of broad-spectrum specific β-agonist was purified by octanoic acid-ammonium sulfate method.

The present invention also claims the application of the above-mentioned broad-spectrum specific β-agonist artificial antibody in the preparation of a kit for detecting β-agonist drugs and/or their analogs.

The present invention also claims a kit for detecting β-agonist drugs and/or their analogs, comprising the above-mentioned β-agonist hapten, the above-mentioned β-agonist artificial antigen or the above-mentioned broad-spectrum specific β-agonist artificial antigen. Antibody.

Compared with the prior art, the present invention has the following beneficial effects:

The present invention provides a β-agonist hapten, an artificial antigen prepared by coupling the hapten and a carrier protein as an immunogen, and the prepared artificial antibody of a broad-spectrum specific β-agonist has high sensitivity and good broad-spectrum specificity , 31 β-agonist drugs and their analogs can be identified. Among them, the half inhibitory concentration of R-(-)-salbutamol was 0.5ng/mL, the linear range was 0.11-40.86ng/mL, and the minimum detection limit was 0.04ng/mL. The antibody can simultaneously detect a variety of β-agonist drugs, can be used for on-site rapid detection of food safety, and has broad application prospects.

In addition, the present invention uses R-(-)-salbutamol as raw material, and successfully synthesizes beta-agonist hapten through one-step chemical reaction. The hapten is then coupled with a carrier protein to prepare an artificial antigen, and after immunization, a broad-spectrum specific β-agonist artificial antibody is obtained. The preparation method of the β-agonist hapten, artificial antigen and artificial antibody is simple, and is easy for large-scale production and application.

Description of drawings

FIG. 1 is the synthetic route diagram of the β-agonist hapten in Example 1. FIG.

Fig. 2 is the UV scanning spectrum of the β-agonist artificial antigen in Example 2; A is BSA, B is OVA.

FIG. 3 is the standard curve of the indirect competition ELISA of broad-spectrum specific β-agonist antibody against R-(-)-salbutamol in Example 4. FIG.

Detailed ways

The present invention will be further elaborated below in conjunction with the accompanying drawings and specific embodiments of the description, and the embodiments are only used to explain the present invention, but not to limit the scope of the present invention. The test methods used in the following examples are conventional methods unless otherwise specified; the materials, reagents, etc. used are commercially available reagents and materials unless otherwise specified.

Example 1 Synthesis and identification of beta-agonist hapten

1. The synthetic route is shown in Figure 1, 220 mg of R-(-)-salbutamol was weighed and dissolved in 40 mL of methanol, 77 mg of succinic anhydride was added, and the mixture was mixed under nitrogen protection. The mixture was stirred at room temperature for 12 h and a white suspension appeared. The reaction solution was placed in a 50 mL centrifuge tube and centrifuged at 3000 r/min for 15 min, the supernatant was discarded, the precipitate was washed with 100 mL absolute ethanol for 3 times, and dried in vacuum for 24 h to obtain a white solid, and the final product was subjected to ¹ H-NMR Hydrogen and mass spectrometry identification.

2. The result is as follows:

Hydrogen spectrum results:

¹H-NMR (DMSO, 400 MHz): δ (ppm) 1.12 (9H, s), 2.38-2.41 (2H, m), 2.53-2.56 (2H, m), 2.76-2.78 (2H, m), 4.61- 4.62 (1H, m), 5.05 (2H, d,J= 2.0Hz ), 6.77 (1H, d,J= 8.0 Hz ), 7.10 (1H, dd,J= 8.4, 2.0 Hz), 7.27 (1H, d, J= 2.0 Hz).

Mass spectrum: m/z (+ESI): 340.1 [M+H] ⁺ .

Both results indicated that the derivatization site was correct, and the β-agonist hapten was successfully synthesized.

Example 2 Synthesis and identification of β-agonist artificial antigen

1. Synthesis of β-agonist artificial antigen

(1) Dissolve 7.5 μL of triethylamine in 0.15 mL of DMF, stir at 4 °C for 15 min, add 3.4 mg of the β-agonist hapten obtained in Example 1 and 1 μL of isobutyl chloroformate, and stir at 4 °C After 30min of reaction, the β-agonist hapten solution was obtained;

(2) Dissolve 8 mg of BSA in 1 mL of carbonate buffer to obtain a carrier protein solution;

(3) After stirring evenly, the carrier protein solution obtained in step (2) was added dropwise to the β-agonist hapten solution obtained in step (1), and the reaction was stirred at 4°C for 12 hours;

(4) Put the reacted solution in a dialysis bag, and dialyze it with 0.01mol/L PBS solution (pH=7.4) at 4°C for 3 days, during which the dialysate is changed twice a day, and the β-agonist is obtained after the dialysis. artificial antigens.

In addition, the carrier protein BSA was replaced by OVA and the coupling method was the same as above.

2. Identification

The above synthetic β-agonist artificial antigen was taken and subjected to ultraviolet scanning. The results are shown in Figure 2.

Specifically, the UV scanning spectra of carrier proteins (BSA and OVA), β-agonist hapten and β-agonist artificial antigen were compared and analyzed, and the maximum absorption wavelength of the L-coupling product (ie, β-agonist artificial antigen) was They are 230nm and 229nm, respectively, which are different from the maximum absorption wavelength (212nm) of the L-hapten (ie, β-agonist hapten), while the maximum absorption wavelength of the carrier protein is 278nm, which is different from that of the conjugate. Therefore, it is proved that The conjugation product is a substance different from both the hapten and the carrier protein. The situation for racemic haptens and conjugates is similar to that for the levoform. Since there are no small molecules such as haptens in the dialysis product, the absorption peak cannot be generated by the mixture of carrier protein and hapten, but by the coupled product. Therefore, it can be proved that the β-agonist hapten is successfully coupled to the carrier protein.

Example 3 Preparation of broad-spectrum specific β-agonist artificial antibody

1. Immunity

The above β-agonist artificial antigen solution was mixed and emulsified with an equal amount of adjuvant. The initial immunization was mixed and emulsified with Freund's complete adjuvant and then immunized. The interval of booster immunization was 3 weeks, and the dose of each immunization was 6mL/only. On the 7th day after the third immunization, blood was collected to detect the antiserum titer until the antiserum titer no longer increased. After blood collection, the antiserum was separated by natural coagulation at room temperature, and placed at 4 °C overnight, at 4000 r/min. Centrifuge for 30 min, and take the upper serum.

2. Purification (octanoic acid-ammonium sulfate method):

(1) Take 3mL of antiserum, add 2 times the volume of 0.06mol/L sodium acetate buffer with pH 4.8;

(2) Add 225 μL of n-octanoic acid dropwise to the sample solution while stirring, continue stirring for 30 minutes after adding, and let stand at 4°C for 2 hours;

(3) Centrifuge at 4°C and 10,000 r/min for 10 min, take the supernatant, add 1/10 volume of the supernatant with 0.1 mol/L PBS solution with a pH of 7.4, and adjust the pH to 7.4 with 1 mol/L NaOH solution ;

(4) Under ice bath conditions, add 0.277 g/mL ammonium sulfate (to make the final saturation 45%), stir for 1 h, centrifuge at 4°C and 10000 r/min for 10 min, and discard the supernatant;

(5) Dissolve the precipitate with 2 mL of PBS solution, dialyze with 0.01 mol/L PBS solution with a pH of 7.4 overnight, and change the medium 7 times to obtain the purified broad-spectrum specific β-agonist artificial antibody.

Example 4 ELISA detection of broad-spectrum specific β-agonist artificial antibody

1. ELISA detection method

(1) Dilute the β-agonist artificial antigen with carbonate buffer, add 100 μL/well to the well of the ELISA plate, and place it in a 37°C water bath overnight;

(2) After taking out, wash the plate twice with an automatic plate washer, pat dry, add 120 μL of blocking solution (5% nonfat milk powder) to each well, incubate in a 37°C water bath for 3 hours, spin dry the liquid in the well, and use the enzyme label plate. Dry in a 37°C oven for later use.

(3) The artificial antibody obtained in Example 3 was diluted 1:32000 times with PBST, and the R-(-)-salbutamol drug was diluted to 1000, 100, 10, 1, 0.1, 0.01, 0.001 ng/mL;

(4) Add 50 µL of R-(-)-salbutamol drug dilution solution to each row (three groups in parallel), plus 50 µL of antibody dilution solution, incubate at 37°C for 40 min, and wash 5 times;

(5) Add 100 μL of goat anti-rabbit enzyme-labeled secondary antibody diluted 5000 times to each well, incubate at 37°C for 30 min, and wash the plate 5 times;

(6) Add 100 μL of color developing solution to each well, place in a water bath at 37°C for 10 min, and then add 50 μL of stop solution to each well to stop the reaction;

(7) Measure the absorbance value of each well at 450nm with ELISA detector.

In addition, the above-mentioned R-(-)-salbutamol drug was replaced with a β-agonist drug and its analogs, and the above test was carried out at the same dilution ratio to determine the cross-reaction of the antibody to the β-agonist drug and its analogs. Rate.

2. Results

According to the above optimal coating original and immunogen dilution combination, the standard curve of antibody to R-(-)-salbutamol was drawn, as shown in Figure 3. The half-inhibitory concentration (IC ₅₀ ) of artificial antibody to R-(-)-salbutamol was 0.5ng/mL, the linear range (IC ₂₀ ～IC ₈₀ ) was 0.11-40.86 ng/mL, and the minimum detection limit was 0.04 ng/mL. The cross-reactivity of artificial antibodies to other β-agonist drugs and their analogs is shown in Table 1.

Table 1 Cross-reaction of broad-spectrum specific β-agonist artificial antibodies with β-agonist drugs and their analogs

The above results show that the artificial antibody obtained in Example 3 can recognize 31 β-agonists and their analogs with broad-spectrum specificity, has good sensitivity, meets the detection requirements, and can be applied to broad-spectrum specific β-agonist immunodetection.

Example 5 Beta-agonist addition and recovery experiment in samples

1. Sample pretreatment

1ng, 5ng, 10ng of salbutamol, clenbuterol, brombuterol and cibuterol standards were added to pig urine samples without beta-agonist, respectively. The pig urine samples were centrifuged at 13000 r/min for 10 min, and the supernatant was diluted 5 times with PBST for detection.

2. The results are shown in Table 2. The results show that all four drugs can be detected in this method. The intra-batch experimental recovery rate is between 81.8 and 118.3%, and the inter-batch experimental recovery rate is between 87.6 and 114.0%. The coefficient of variation ( CV%) are less than 15%, indicating that the ELISA detection method adopted in Example 4 can fully meet the requirements of β-agonist multi-residue detection.

Table 2 Recovery results of β-agonists added to pig urine samples

Example 6 Comparison of ELISA method and HPLC-MS method of β-agonist

According to the multi-residues of β-receptor agonists in pig urine by liquid chromatography-tandem mass spectrometry (Ministry of Agriculture Announcement No. 1025-11-2008), the HPLC-MS method was verified. In the detection of spiked urine samples, the corresponding concentrations of the four β-agonists at three spiked levels (1, 5, and 10 ng/mL) and blank samples were detected. The correlations between the results of ELISA and HPLC-MS for salbutamol, clenbuterol, brobuterol and cibuterol were Y = 0.939 x + 0.085 (R ² =0.981), Y = 1.280 x + 0.088 ( R ² =0.999), Y = 1.303 x + 0.138 (R ² =0.994), Y = 1.142x + 0.122 (R ² =0.971).

The above results show that the measurement results of the ELISA method and HPLC-MS method adopted in Example 4 have good consistency, and the detection recovery results are all within a reasonable range. Therefore, the ELISA method used in Example 4 to detect artificial antibodies of broad-spectrum specific β-agonists is a broad-spectrum detection method with strong reliability, which can be used to detect multiple β-agonists and their analogs simultaneously.

Claims (3)

1. a broad-spectrum specificity β-agonist artificial antibody, is characterized in that, is prepared by β-agonist artificial antigen as immunogen and purifies after preparation, comprises the steps: 1. Immunity: The β-agonist artificial antigen solution was mixed and emulsified with equal amount of adjuvant. The initial immunization was mixed and emulsified with Freund's complete adjuvant and then immunized. After 4 weeks, the immunogen was emulsified with incomplete Freund's adjuvant and then boosted. The immunization interval was 3 weeks, and the dose of each immunization was 6 mL/only; the blood was collected on the 7th day after the third immunization to detect the antiserum titer, until the antiserum titer was no longer long, and the blood was collected by natural coagulation at room temperature and then separated. Serum, placed overnight at 4 °C, centrifuged at 4000 r/min for 30 min, and the upper serum was taken; 2. Purification: (1) Take 3 mL of antiserum, add 2 times the volume of 0.06mol/L sodium acetate buffer with a pH of 4.8; (2) Add 225 μL of n-octanoic acid dropwise to the sample solution while stirring, continue stirring for 30 minutes after adding, and let stand at 4°C for 2 hours; (3) Centrifuge at 4°C and 10000r/min for 10min, take the supernatant, add 1/10 volume of the supernatant with 0.1mol/L PBS solution with pH of 7.4, adjust the pH to 7.4 with 1mol/L NaOH solution ; (4) Under ice bath conditions, add 0.277 g/mL ammonium sulfate (to make the final saturation 45%), stir for 1 h, centrifuge at 4°C and 10000 r/min for 10 min, and discard the supernatant; (5) Dissolve the precipitate with 2 mL of PBS solution, dialyze with 0.01 mol/L PBS solution with a pH of 7.4 overnight, and change the medium 7 times to obtain a purified broad-spectrum specific β-agonist artificial antibody; The β-agonist artificial antigen is obtained by coupling a β-agonist hapten with a carrier protein whose molecular structure is shown in the following formula (I);

2. Application of the broad-spectrum specific β-agonist artificial antibody of claim 1 in the preparation of a kit for detecting β-agonist drugs and/or analogs thereof. 3. A test kit for the detection of β-agonist drugs and/or its analogs, characterized in that, containing the β-agonist hapten described in claim 1, a β-agonist artificial antigen or a broad-spectrum specific β- Agonist artificial antibodies.

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