CN104193637B - A specific clenbuterol hapten derivative, clenbuterol artificial antigen and its preparation method and application - Google Patents

Abstract

The invention discloses a specific clenbuterol hapten derivative, a clenbuterol artificial antigen, a preparation method and application thereof. The structural formula of the specific clenbuterol hapten derivatives synthesized according to the structure of clenbuterol is that a carboxyl active functional group structure is introduced into the three methyl ends of clenbuterol to maximize exposure The specific benzene ring accessory structure of clenbuterol, and the functional group that can be coupled with the carrier protein. A clenbuterol artificial antigen , is a conjugate obtained by coupling a clenbuterol hapten derivative with a carrier protein, wherein the carrier protein protein is bovine serum albumin (BSA) or ovalbumin (OVA); obtained by immunizing animals with an artificial antigen Highly specific monoclonal antibodies and polyclonal antibodies, artificial antigens and prepared high specific monoclonal antibodies can be applied to the detection of clenbuterol by colloidal gold method or fluorescent immunochromatography.

Description

A specific clenbuterol hapten derivative, clenbuterol artificial antigen and its preparation method and application

technical field

The invention relates to the technical field of biochemical and biological detection, in particular to a method for synthesizing a clenbuterol hapten and an artificial antigen and its application in detecting clenbuterol.

Background technique

With the development and progress of society, people's living standards are constantly improving, and people pay more and more attention to healthy diet. Food safety issues, especially the problem of veterinary drug residues in food have attracted more and more people's attention. In order to improve the safety of poultry and livestock products, in addition to strict control and effective management, the monitoring of toxic chemical substance residues such as additives and veterinary drugs is crucial. No important guarantee.

Clenbuterol (Clen) is a synthetic β ₂ -adrenergic receptor agonist, often used as a bronchodilator to prevent and treat bronchial asthma, asthmatic chronic bronchitis, emphysema and other respiratory diseases. induced bronchospasm. When its dosage reaches 5-10 times of the therapeutic amount, Clen can enhance muscle development and reduce fat deposition, so it is also called Clenbuterol. Therefore, Clen is often illegally used as a feed additive in the production of meat animals. More importantly, because Clen absorbs quickly in animals, distributes widely, and has high fat solubility, it has the characteristics of residual accumulation and long half-life. Poisoning incidents caused by people eating meat with Clen residues continue to occur. Therefore, governments of various countries prohibit Clen from being used as a feed additive in the production of meat animals. The Chinese government also stipulates that it is strictly forbidden to add Clen to the feed, and has formulated the Clen residue standard. Therefore, it is necessary to strengthen the detection and supervision of the residues of such drugs in food.

At present, HPLC and GC-MS methods are often used for the detection of clenbuterol residues in feed and animal-derived foods in national and industrial standards. Although this method has strong specificity, high sensitivity, and accuracy, the sample pretreatment steps are cumbersome and costly, coupled with expensive equipment, high cost, and high operational skill requirements, it is not suitable for screening and testing of large batches of samples. Immunoassay (such as enzyme-linked immunoassay, colloidal gold method, and fluorescent immunochromatography), due to its unique advantages in the qualitative and quantitative aspects of antigens and antibodies, and the advantages of easy operation and large sample size, it makes up for the shortcomings of physical and chemical analysis. In β- Doping residue detection plays an increasingly important role. Enzyme-linked immunosorbent assay can detect multiple samples at the same time, and has the advantages of sensitivity and specificity, but it is not suitable for on-site detection. The colloidal gold method and fluorescence immunochromatography are simple and fast to operate, do not require large-scale detection equipment, and are more suitable for on-site timely detection, which is the development trend of rapid detection.

The fundamental reason affecting the detection quality of immunoassays is the specificity and affinity of antibodies. These properties depend on the molecular structure of immune haptens. Therefore, the design and synthesis of immune hapten molecules are the key to producing specific antibodies and establishing small molecule drug residues. The most basic and critical steps of rapid detection technology research.

CN103159852A (2013-6-19) discloses a synthetic method of a specific clenbuterol artificial antigen, however, the molecular structure of the immune hapten used in this method and the preparation of the artificial antigen still need to be improved.

Contents of the invention

One of the objects of the present invention is to provide a specific clenbuterol hapten derivative.

The second object of the present invention is to provide a preparation method of specific clenbuterol hapten derivatives.

The third object of the present invention is to provide a specific clenbuterol antigen.

The fourth object of the present invention is to provide a preparation method of specific clenbuterol antigen.

The fifth object of the present invention is to provide the application of the clenbuterol hapten and artificial antigen in clenbuterol immunoassay.

The present invention provides a specific clenbuterol hapten derivative compound, the chemical structural formula of which is:

.

The hapten derivative compound of the present invention is a derivative synthesized according to the structure orientation of clenbuterol, considering that clenbuterol with albuterol Structurally similar, so choose the end design similar to salbutamol structure to introduce carboxyl active functional group structure, retain and expose the difference and large immunogenic part of clenbuterol and salbutamol to the greatest extent, and at the same time have the same carrier protein Coupling reactive groups.

The present invention also protects the preparation method of the clenbuterol hapten derivative compound, which is characterized in that the existing compound 4-amino-3,5-dichlorobromoacetophenone (CAS: 37148-47-3 ) as a raw material, prepared by a two-step reaction, and its preparation route is as follows:

As a preference, the reaction conditions specifically include adding isopropanol as a solvent to 0.015-0.028 mol of 4-amino-3,5-dichlorobromoacetophenone solid, and adding 0.01-0.03 mol of 4-Amino-4methyl-pentanoic acid, react at room temperature for 0.8-1.2h, after the raw material is completely converted, crystallize, filter, wash and dry to obtain the first white solid;

Dissolve the first white solid in ethanol, add 0.001-0.003mol 92-98% KBH ₄ 0.002-0.003mol ZnCl ₂ successively, react at room temperature for 12-18h, filter; concentrate the filtrate, wash with isopropanol to obtain The second white solid is the specific clenbuterol hapten derivative.

The first white solid is 4-{[2-(4-amino-3,5-dichlorophenyl)-2-oxoethyl]amino}-4-methylpentanoic acid; the second white The solid is the clenbuterol hapten-derived compound.

A specific clenbuterol artificial antigen is characterized in that the clenbuterol artificial antigen is prepared from the specific clenbuterol hapten derivative compound, and its chemical structural formula is:

The protein, namely the carrier protein, is bovine serum albumin or ovalbumin.

A method for preparing clenbuterol artificial antigen by using the clenbuterol hapten derivative compound described in the present invention is characterized in that the clenbuterol hapten derivative compound is coupled with a carrier protein by the carbodiimide method to obtain , including the following steps:

P1. Dissolving the clenbuterol hapten derivative compound in DMSO or DMF to obtain a derivative compound solution;

P2. Add EDC (1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride) to P1, then add NHS (N-hydroxysuccinimide) at room temperature and mix evenly under stirring After reacting for 15-60 minutes; the molar ratio of EDC, NHS and clenbuterol hapten derivative compound is 1-2:1-1.5:1-2;

P3, adding the reaction solution obtained in P2 to a solution containing a carrier protein at 25-30 degrees and stirring, and performing a coupling reaction for 1-5 hours, wherein the carrier protein is bovine serum albumin or ovalbumin;

When the carrier protein is bovine serum albumin, the molar ratio of the derivative compound to bovine serum albumin is 40-60:1, and the synthetic artificial antigen is clenbuterol immunogen; when the carrier protein is ovalbumin, the derivative The molar ratio of the compound to ovalbumin is 30-50:1, and the synthetic artificial antigen is the coating source;

P4. The reaction solution described in P3 is dialyzed, and the dialysate is collected as the clenbuterol artificial antigen.

Preferably, the buffer of the carrier protein solution in P3 is 0.1 mol/L NaHCO ₃ buffer; the dialysate in P4 is 0.010.1 mol/L phosphate buffer (pH 7.2-7.9).

NaHCO ₃ buffer solution: weigh 8.4g NaHCO ₃ , dissolve in 1000ml ultrapure water, adjust the pH to 7.4; phosphate buffer solution: weigh NaH ₂ PO ₄ ·H ₂ O: 0.3g, NaCL: 9g, Na ₂ HPO ₄ ·12H ₂ O : Dilute 0.3g to 1000ml and adjust the pH to 7.1~7.8.

The present invention also protects the application of the clenbuterol artificial antigen mentioned above in the preparation of clenbuterol-specific antibodies; the antibodies are monoclonal antibodies or polyclonal antibodies.

The present invention also protects the use of clenbuterol artificial antigen as a raw material in the detection of clenbuterol immunoassay, and the immunoassay is colloidal gold method or fluorescent immunochromatography.

Further, the application of the clenbuterol artificial antigen in the detection of clenbuterol by fluorescent immunochromatography, the steps are as follows:

P1. Using the clenbuterol artificial antigen to immunize animals to prepare antibodies;

P2. Cell fusion and screening;

P3. Ascites preparation and antibody purification;

P4. The antibody obtained in P3 and the anti-rabbit IgG secondary antibody were labeled with fluorescent dyes respectively, and prepared into a detection analysis solution;

P5. Spray the clenbuterol coating agent and rabbit IgG antibody on the front and back ends of the nitrocellulose membrane strip (the front end is close to the absorbent paper end, and the back end is close to the sample pad) to prepare the test line and quality control Wire;

P6. the glass fiber sample pad, the nitrocellulose membrane containing detection line and quality control line described in P5, absorbent paper and PVC black floor are assembled into immunochromatography test strips;

P7. Cut the test strip described in P6 to an appropriate width and put it into a plastic card box, and use it together with the detection and analysis solution described in P4 on a fluorescent immunochromatography detector for detection.

The advantages and characteristics of the present invention;

The present invention provides a clenbuterol derivative compound synthesized in a directional way. According to the structural characteristics of clenbuterol and albuterol shown in FIG. 3, an active functional group structure ( Carboxyl group), the maximum degree of retention exposes the specific structure of the two and the structure with strong immunity (ie, the structural part of the benzene ring end); based on the preparation method of the artificial antigen of the present invention, it is prepared by using the above-mentioned clenbuterol derivative compound A specific clenbuterol artificial antigen is obtained; the specific artificial antigen is prepared into a specific antibody, which is successfully used in clenbuterol fluorescence immunochromatography detection.

The antigen and antibody raw materials of the present invention are all self-made, the cost of raw materials is greatly reduced, and the antigen and antibody have strong specificity. Combining with fluorescent immunochromatography technology, a high-specificity and high-sensitivity rapid detection method for clenbuterol is established.

Description of drawings

Fig. 1 is the structural formula of clenbuterol derivative compound;

Figure 2 is the structural formula of clenbuterol artificial antigen;

Figure 3 is a schematic diagram of the comparison of the structural formulas of clenbuterol and salbutamol;

Fig. 4 is the ¹ H-NMR spectrum of clenbuterol derivative compound;

Fig. 5 is the SDS-PAGE picture of clenbuterol artificial antigen (immunogen and coating former);

Fig. 6 is the structure of the clenbuterol fluorescence immunochromatography detection card without plastic card;

Fig. 7 is the structure of the clenbuterol fluorescence immunochromatography detection card with a plastic cartridge;

Fig. 8 is a fluorescence immunochromatography detection clenbuterol standard inhibition curve;

Figure 9 is a negative result spectrum of clenbuterol fluorescence immunochromatography detection;

Fig. 10 is an atlas of positive results detected by clenbuterol fluorescence immunochromatography.

Detailed ways

Below is to further describe the present invention in conjunction with accompanying drawing. Unless otherwise specified, the raw materials of the kits used in the examples of the present invention are conventional reagents.

Example 1

The first directed synthesis of clenbuterol hapten-derived compounds:

S1. Weigh 4-amino-3,5-dichlorobromoacetophenone (domestic CAS: 37148-47-3)

Put 5.65g (0.02mol) of solid in a 50ml three-necked flask, add 20ml of isopropanol as a solvent, and add 2.63g (0.02mol) of 4-amino-4methyl-pentanoic acid (imported brand, CAS: 3235 -46-9), reacted at room temperature for 1h (TLC followed the reaction, the raw material had been completely converted), crystallized, filtered, washed and dried to obtain a white solid, namely: 4 - {[ 2 -(4 -amino-3,5 -dichloro phenyl)-2-oxoethyl]amino}-4-methylpentanoic acid.

S2. Dissolve 0.66g (0.002mol) of the white solid obtained in S1 in 20ml of ethanol, add 0.11g (0.002mol) 95% KBH ₄ , 0.27g (0.002mol) ZnCl ₂ successively, react at room temperature for 15 hours, and filter . The filtrate was concentrated and washed with isopropanol to obtain 0.58 g of the white solid shown in Figure 1, namely: a clenbuterol derivative compound. The synthesized derivative compound was identified by ¹ H-NMR , and the result is shown in FIG. 4 .

A second directed synthesis of clenbuterol hapten-derived compounds:

Add 15 ml of isopropanol as a solvent to 0.015 mol of 4-amino-3,5-dichlorobromoacetophenone solid, add 0.01 mol of 4-amino-4-methyl-pentanoic acid under ice-water bath conditions, and After reacting for 0.8h, crystallize, filter, wash and dry the raw material after complete conversion to obtain the first white solid;

Dissolve the first white solid in 15ml of ethanol, add 0.001mol 92% KBH ₄ 0.002mol ZnCl ₂ successively, react at room temperature for 12 hours, then filter; the filtrate is concentrated, washed with isopropanol, and the second white solid is obtained as specific gram Renbuterol hapten derivative.

The third directed synthesis method of clenbuterol hapten-derived compounds:

Add 30 ml of isopropanol as a solvent to 0.028 mol of 4-amino-3,5-dichlorobromoacetophenone solid, add 0.03 mol of 4-amino-4-methyl-pentanoic acid under ice-water bath conditions, and After reacting for 1.2h, crystallize, filter, wash and dry the raw materials to obtain the first white solid after complete conversion;

Dissolve the first white solid in 30ml of ethanol, add 0.003mol 98% KBH ₄ 0.003mol ZnCl ₂ successively, react at room temperature for 18 hours, and filter; the filtrate is concentrated, washed with isopropanol, and the second white solid is obtained. Renbuterol hapten derivative.

Example 2

The first synthesis method of clenbuterol artificial antigen (immunogen and coater):

Weigh 40mg of clenbuterol derivative compound, dissolve it in 2ml DMF to form a final concentration of 20mg/ml, take 90μL of it and mix it with 6μL EDC (100mg/ml), then add 10ul NHS (50mg/ml), and mix evenly React under stirring conditions for 15~60min.

Centrifuge the above reaction mixture (1600rmp) and add it to 1ml 6mg/ml BSA solution (or add it to 1ml 4.5mg/ml OVA solution). After stirring at room temperature for 1~5h, dialyze 4 times with phosphate buffer. Change the fluid every 12 hours. The dialysate was collected, and the concentrations of the immunogen and the coating agent were determined to be 5.4 mg/ml and 3.8 mg/ml, respectively, using the Quick Start Bradford Protein Assay Kit from BIO-RAD. The structural formula of the obtained clenbuterol artificial antigen (immunogen and coater) is shown in Figure 2. The protein in Figure 2 is BSA (bovine serum albumin) or OVA (ovalbumin).

The BSA solution (or OVA solution) preparation method: Weigh 60mg BSA (or 45mg OVA) and dissolve it in 10ml of 0.1mol/L NaHCO ₃ buffer.

The preparation method of the NaHCO ₃ buffer solution: weigh 8.4g NaHCO ₃ , dissolve it in 1000ml ultrapure water, and adjust the pH to 7.1-7.9;

The preparation method of the phosphate buffer solution: Weigh NaH ₂ PO ₄ ·2H ₂ O: 0.3g, NaCL: 9g, Na ₂ HPO ₄ ·12H ₂ O: 0.3g to 1000ml, and adjust the pH to 7.3-7.8.

The SDS-PAGE images of Clenbuterol immunogen (Clen-BSA) and BSA are shown in Figure 5, M: marker; 1: BSA; 2: Clen-BSA; 3: Clen-OVA; 4: OVA, by It can be seen from the accompanying drawings that the molecular weight of BSA is 66kDa, and the molecular weight of some Clen-BSA is larger than that of BSA, so the electrophoretic mobility is smaller than that of BSA, indicating that the coupling is successful. Similarly, the molecular weight of Clen-OVA is also larger than that of OVA, and the coupling is successful.

The second synthesis method of clenbuterol artificial antigen (immunogen and coater):

P1, dissolving the clenbuterol hapten derivative compound in DMSO to obtain a derivative compound solution;

P2. Add EDC (1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride) to P1, then add NHS (N-hydroxysuccinimide) at room temperature and mix evenly under stirring After reacting for 15 minutes; the molar ratio of EDC, NHS and clenbuterol hapten derivative compound is 1:1~1:1;

P3. Add the reaction solution obtained in P2 to a solution containing a carrier protein at 25 degrees and stir, and perform a coupling reaction for 1 hour, wherein the carrier protein is bovine serum albumin or ovalbumin;

When the carrier protein is bovine serum albumin, the molar ratio of the derivative compound to bovine serum albumin is 60:1, and the synthetic artificial antigen is clenbuterol immunogen; when the carrier protein is ovalbumin, the derivative compound and The molar ratio of ovalbumin is 30:1, and the synthetic artificial antigen is the coating source;

P4. The reaction solution described in P3 is dialyzed, and the dialysate is collected as the clenbuterol artificial antigen.

The third synthesis method of clenbuterol artificial antigen (immunogen and coater):

P1, dissolving the clenbuterol hapten derivative compound in DMF to obtain a derivative compound solution;

P2. Add EDC (1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride) to P1, then add NHS (N-hydroxysuccinimide) at room temperature and mix evenly under stirring After reacting for 15 minutes; the molar ratio of EDC, NHS and clenbuterol hapten derivative compound is 2:1.5:1;

P3. Add the reaction solution obtained in P2 to a solution containing a carrier protein at 25 degrees and stir, and perform a coupling reaction for 1 hour, wherein the carrier protein is bovine serum albumin or ovalbumin;

When the carrier protein is bovine serum albumin, the molar ratio of the derivative compound to bovine serum albumin is 40:1, and the synthetic artificial antigen is clenbuterol immunogen; when the carrier protein is ovalbumin, the derivative compound and The molar ratio of ovalbumin is 50:1, and the synthetic artificial antigen is the coating source;

P4. The reaction solution described in P3 is dialyzed, and the dialysate is collected as the clenbuterol artificial antigen.

Example 3

Application of clenbuterol artificial antigen in the detection of clenbuterol by fluorescence immunochromatography:

P1. Immunized animals

Dilute the clenbuterol immunogen (Clen-BSA) obtained by the first synthesis method in Example 2 to 0.2mg/ml, mix 500ul of the immunogen with an equal volume of complete Freund's adjuvant, emulsify completely, and immunize BALB/c mice were injected subcutaneously on the back and feet of the mice for immunization. Complete Freund's adjuvant was used for the first immunization, followed by incomplete Freund's adjuvant. One week after the fourth immunization, blood was collected from the orbit, the serum was separated, and the titer of anti-clenbuterol antibody was measured. As detected by ELISA, the antibody titer of the mice after four immunizations was 1:322,000.

P2. Cell Fusion and Screening

After the four immunizations, the mice were immunized again by intraperitoneal injection of about 100 μg of immunogen, and 3 days later, the spleens of the mice were taken for fusion. Take SP2/0 cells and splenocytes, add serum-free culture medium, centrifuge (1500rpm, 3min), take sedimented cells, add 1ml 50% polyethylene glycol 4000 drop by drop, let stand for 90 seconds. Then 10ml of serum-free culture solution pre-warmed at 37°C was added dropwise and allowed to stand for 5 minutes. After fusion, the cell suspension was centrifuged (1000rpm, 3min), and the complete culture solution was used to inoculate the 96-well plate with feeder cells at 2×10 ⁴ /ml myeloma cells per well. Place them in a 37°C, 5% CO ₂ incubator for two days, and add 2×HAT complete culture solution to make the final concentration in the wells 1×HAT. When the hybridoma cell colony grows to 1/10-1/5 of the area of the bottom of the well, use the ELISA method to screen the fusion cell antibody-positive wells.

P3. Ascites preparation and antibody purification

BALB/c mice were taken, and 0.5ml of paraffin oil was injected intraperitoneally, and 0.5ml of 1×10 ⁶ positive hybridoma cells were injected intraperitoneally after 7 days. Observe the growth of the mice, and abdominal swelling can be seen in about 7 days, and the ascites should be collected in time. A high-purity monoclonal antibody was purified by affinity chromatography (Protein G Resin affinity purification), with a protein content of 4 mg.

P4. Detection and analysis solution preparation

a. Fluorescently label the monoclonal antibody and anti-rabbit IgG antibody obtained in P3 above.

b. Dilute the fluorescently labeled antibody with BSA-containing phosphate buffer to prepare a detection analysis solution.

P5. Preparation of clenbuterol fluorescent immunochromatography test paper card

a. Dilute the Clenbuterol coating agent (Clen-OVA) and rabbit antibody IgG obtained in Example 2 to an appropriate concentration (0.25 mg/ml) with coating buffer (phosphate buffer). At a temperature of 25±5°C, evenly spray the diluted Clen-OVA and rabbit antibody IgG on the nitrocellulose membrane (to form a test line and a quality control line, respectively), and dry at a humidity of 12%-30% About 5~8 hours, dry and store for later use;

b. Paste the coated nitrocellulose membrane obtained in step a, glass fiber paper and absorbent paper on the black PVC substrate in order to form a test card (Figure 6), and cut it into an appropriate width as required. In Figure 6, 1: black PVC substrate; 2: absorbent paper; 3: nitrocellulose membrane; 4: glass fiber; 5: quality control line (C line); 6: testing line (T line);

c. Put the test card obtained in step b into the lower cover of the cartridge, and close the upper cover to form a complete test card with a cartridge (Figure 7). In Figure 7, 1: lower cover; 2: upper cover; 3: sample loading window; 4: glass fiber; 5: detection window; 6: quality control line belt (C line); 7: test line belt (T line) ;8: Nitrocellulose membrane; 9: Clenbuterol project logo (Clen); 10: Barcode identification area;

P6. Detection

Take 100ul of sample and 100ul of detection and analysis solution to mix evenly, take 100ul and add it to the sampling window of the test card, react for 5-10min, and detect it on the FCR fluorescence immunoassay analyzer (manufactured by Huzhou Haichuang Biotechnology Co., Ltd.). According to the ratio of the T-line signal value to the C-line signal value (T/C value) of the sample and the Cutof value, the positive and negative results are judged.

P7. Sensitivity of Fluorescence Immunochromatography for Detection of Clenbuterol

Add clenbuterol standard (purchased from DR company in Germany) to the blank urine and configure it as 0ng/ml, 0.25ng/ml, 0.5ng/ml, 1ng/ml, 2ng/ml, 4ng/ml, 8ng/ml ml serial concentration samples. The above-mentioned series of concentration samples were tested according to the steps in P6, and each sample was repeated 3 times. The experimental results of the test are shown in Table 1. According to the data in Table 1, the Clen concentration is used as the abscissa and the T/C value is the ordinate to draw the standard The curve (regression by four-parameter regression method) is shown in Figure 8. The equation corresponding to the curve in Fig. 8 is shown in Table 2, and the calculated IC ₅₀ =0.86ng/ml.

The 0ng/ml sample was tested 6 times repeatedly, and the average value (X), standard deviation (SD) and precision (CV value) of the T/C values were calculated respectively. The calculation method of sensitivity is the Clen concentration value corresponding to the T/C value of X-2*SD in the standard curve in Figure (8).

Substitute the T/C value of X-2*SD in the data in Table 3 into the equation corresponding to the standard curve in Figure 8 as the y value (equation: y = (A - D) / [1 + (x/C) ^B ] + D) The obtained x value is 0.16ng/ml, that is, the sensitivity is 0.16ng/ml.

P8. Detection principle of clenbuterol fluorescence immunochromatography test paper card

 Using competition method detection, the clenbuterol antigen in the sample and the clenbuterol antigen (coating source) on the detection line (T line) compete for binding to the fluorescently labeled anti-clenbuterol antibody in the assay solution. When the antigen concentration in the sample is very low, the number of fluorescent antibodies bound to the detection line increases, and the fluorescence signal on the detection line becomes stronger. Therefore, the difference between the fluorescence signal on the detection line (T line) and the fluorescence signal on the quality control line (C line) The ratio (T/C value) is large; on the contrary, when the concentration of clenbuterol antigen in the sample is high, the T/C value is small. Therefore, the higher the clenbuterol content in the sample, the lower the T/C value. According to the comparison of the T/C value and the Cut of value, the positive or negative of the test result is judged.

As shown in Figures 9 and 10, they are the negative and positive results of clenbuterol fluorescence immunochromatography detection. It can be seen from the figure that compared with the negative sample test result spectrum, the T-line peak value in the positive sample test result spectrum is much smaller than the negative sample peak value. Negative and positive samples are clearly distinguished.

P9. Fluorescence immunochromatography to detect cross-reaction of clenbuterol

Common β2-receptor agonists were prepared into 30ng/ml samples with negative pig urine for fluorescence immunochromatography detection. Compared with normal negative urine samples and Cutof values, they all showed negative results. The test results are shown in Table 2:

Table 2 Fluorescence immunochromatographic detection results of common β2 receptor agonists

Note: The cut off value is the T/C value corresponding to 1ng/ml clenbuterol plus 2 times SD range. Namely: 0.47~0.86.

Claims (9)

1. A specific clenbuterol hapten derivative is characterized in that said derivative is applicable to the preparation of clenbuterol artificial antigen, and its chemical structural formula is: . 2. A method for preparing specific clenbuterol hapten derivatives as claimed in claim 1, characterized in that, taking compound 4-amino-3,5-dichlorobromoacetophenone as raw material, through two Step reaction preparation, its preparation route is as follows: . 3. A method for preparing specific clenbuterol hapten derivatives as claimed in claim 2, wherein the reaction conditions are specifically 4-amino-3,5-dichloro at 0.015-0.028mol Add isopropanol to the solid bromoacetophenone as a solvent, add 0.01-0.03mol of 4-amino-4-methyl-valeric acid in an ice-water bath, react at room temperature for 0.8-1.2h, and crystallize after the raw materials are completely converted , filtering, washing and drying to obtain the first white solid; Dissolve the first white solid in ethanol, add 0.001-0.003mol 92-98% KBH ₄ and 0.002-0.003mol ZnCl ₂ successively, react at room temperature for 12-18h, and filter; the filtrate is concentrated, washed with isopropanol, The obtained second white solid is the specific clenbuterol hapten derivative. 4. A specific clenbuterol artificial antigen prepared by specific clenbuterol hapten derivatives according to claim 1, characterized in that its chemical structural formula is: The protein, namely the carrier protein, is bovine serum albumin or ovalbumin. 5. A method for preparing specific clenbuterol artificial antigen as claimed in claim 4, characterized in that the specific clenbuterol hapten derivative compound is coupled with carrier protein by carbodiimide method obtained, including the following steps: P1. Dissolving the clenbuterol hapten derivative compound in DMSO or DMF to obtain a derivative compound solution; P2. Add EDC·HCl (1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride) to P1, then add NHS (N-hydroxysuccinimide) at room temperature and stir Mix evenly and react for 15-60 minutes; the molar ratio of EDC, NHS and clenbuterol hapten derivative compound is 1-2:1-1.5:1-2; P3, adding the reaction solution obtained in P2 to a solution containing a carrier protein at 25-30 degrees and stirring, and performing a coupling reaction for 1-5 hours, wherein the carrier protein is bovine serum albumin or ovalbumin; When the carrier protein is bovine serum albumin, the molar ratio of the derivative compound to bovine serum albumin is 40-60:1, and the synthetic artificial antigen is clenbuterol immunogen; when the carrier protein is ovalbumin, the derivative The molar ratio of the compound to ovalbumin is 30-50:1, and the synthetic artificial antigen is the coating source; P4. The reaction solution described in P3 is dialyzed, and the dialysate is collected as the clenbuterol artificial antigen. 6. according to the preparation method of the described specific clenbuterol artificial antigen of claim 5, it is characterized in that, the damping fluid of carrier protein solution described in _P3 is the NaHCO buffering fluid of 0.1mol/L; Dialysis described in P4 The solution is 0.01mol/L phosphate buffer solution with pH of 7.2~7.9. 7. The specific clenbuterol artificial antigen according to claim 4, characterized in that it can be used to prepare clenbuterol antibody, and the antibody is a monoclonal antibody or a polyclonal antibody. 8. The application of specific clenbuterol artificial antigen according to claim 4, characterized in that it can be used in the detection of clenbuterol by colloidal gold method or fluorescent immunochromatography. 9. according to the application of the described specific clenbuterol artificial antigen of claim 8, it is characterized in that, the application of described clenbuterol artificial antigen in fluorescent immunochromatography detection clenbuterol, its steps are as follows: P1. Using the clenbuterol artificial antigen to immunize animals to prepare antibodies; P2. Cell fusion and screening; P3. Ascites preparation and antibody purification; P4. The antibody obtained in P3 and the anti-rabbit IgG secondary antibody were labeled with fluorescent dyes respectively, and prepared into a detection analysis solution; P5. Spray the clenbuterol coating agent and rabbit IgG antibody on the front and rear ends of the nitrocellulose membrane strip to prepare the test line and quality control line, wherein the end close to the absorbent paper is the front end, and the end close to the sample pad is the back end; P6. the glass fiber sample pad, the nitrocellulose membrane, absorbent paper and PVC black floor that contain detection line and quality control line described in P5 are assembled into immunochromatography test strips; P7. Cut the test strip described in P6 and put it into a plastic cartridge, and use it together with the detection and analysis solution described in P4 on a fluorescent immunochromatography detector for detection.