A rabbit monoclonal antibody of ractopamine and its preparation method and application

Abstract

The present application relates to the technical field of bioengineering, and particularly relates to a rabbit monoclonal antibody of ractopamine and a preparation method and application thereof.The rabbit monoclonal antibody comprises an antibody heavy chain and an antibody light chain, the heavy chain variable region of the rabbit monoclonal antibody comprises an amino acid sequence as shown in SEQ ID NO.1 or SEQ ID NO.2, and the light chain variable region of the rabbit monoclonal antibody comprises an amino acid sequence as shown in SEQ ID NO.3 or SEQ ID NO.4.A rabbit monoclonal antibody for detecting ractopamine is obtained by the present application, the rabbit monoclonal antibody has high tolerance to urea, and has important significance in the field of detecting ractopamine in animal urine.

Description

TECHNICAL FIELD

[0001] The present application relates to the field of bioengineering technology, and particularly relates to a rabbit monoclonal antibody of ractopamine and a preparation method and application thereof. BACKGROUND

[0002] Ractopamine (RAC) is one of the main beta-adrenergic agonists, which can be used to promote animal growth and improve lean / fat ratio. However, in recent years, RAC has been frequently misused. Excessive intake of beta-adrenergic agonists through animal-derived products can cause serious physiological side effects, such as muscle pain, dizziness, tachycardia, tension, and even death. Efficient detection of RAC residues in animal urine is an effective method to regulate illegal use of RAC.

[0003] Immunoassay is based on the antigen-antibody reaction. Due to its low cost, easy use, and high throughput, it has become one of the most important tools for detecting chemical pollutants. Monoclonal antibodies (mAbs) are the core reagents of immunoassay, which determine the sensitivity, analysis time, accuracy, and precision of the analysis method. Improving detection sensitivity, simplifying detection procedures, and shortening detection time are the primary tasks of developing high-throughput trace screening methods for RAC. Most of the core reagents for RAC immunoassay methods are mouse monoclonal antibodies (MmAbs), but the harsh urine detection environment can severely damage the detection performance of immunoassay. Urea, as one of the main components in urine, studies have shown that it can significantly denature the inherent structure of MmAbs and interfere with their biological functions. This makes MmAbs have poor application effect in immunoassay methods for complex urine samples containing a large amount of urea. Previous studies have shown that simply diluting urine cannot effectively eliminate the influence of urea. In recent years, some immunoassay methods based on MmAbs, such as lateral flow immunoassay or indirect competitive enzyme-linked immunosorbent assay (icELISA), have been used to detect RAC in urine. These methods not only lack sufficient sensitivity to meet the requirements of trace RAC detection, but also require multiple sample pretreatments. SUMMARY

[0004] In order to solve the problems existing in the prior art, the present application provides a rabbit monoclonal antibody of ractopamine and a preparation method and application thereof.

[0005] In a first aspect, the present application provides a rabbit monoclonal antibody of ractopamine, wherein the rabbit monoclonal antibody comprises an antibody heavy chain and an antibody light chain,

[0006] The heavy chain variable region of the rabbit monoclonal antibody comprises an amino acid sequence as shown in SEQ ID NO. 1 or SEQ ID NO. 2; and the light chain variable region of the rabbit monoclonal antibody comprises an amino acid sequence as shown in SEQ ID NO. 3 or SEQ ID NO. 4.

[0007] The present application screens antibody-secreting cells secreting RAC-specific antibodies by a competitive screening method, and prepares a rabbit monoclonal antibody (RmAbs) with surprising tolerance to high urea environment and high affinity to RAC, and accordingly establishes a stable and rapid icELISA method based on RmAbs, which can directly detect RAC in urine without any sample pretreatment, and the detection performance is significantly improved, which lays a foundation for preparing other urea-tolerant antibodies in vitro.

[0008] The chemical formula of ractopamine is as follows:

[0009]

[0010] The present application further provides a nucleic acid for encoding the rabbit monoclonal antibody.

[0011] Further, the nucleic acid comprises one or more of the nucleotide sequences as shown in SEQ ID NO. 5-8.

[0012] The present application further provides a biological material comprising the nucleic acid, which is an expression cassette, a vector or a transgenic cell.

[0013] The present application further provides a kit comprising the rabbit monoclonal antibody, or the nucleic acid, or the biological material.

[0014] In a second aspect, the present application provides a preparation method of a rabbit monoclonal antibody, comprising:

[0015] Obtaining lymphocytes of rabbits immunized by a ractopamine immunogen, and obtaining cells specifically secreting rabbit monoclonal antibodies by competitive screening;

[0016] The competitive screening comprises:

[0017] Coating ractopamine coating agents on a nanowell plate, adding the lymphocytes and a fluorescently labeled secondary antibody into the wells of the nanowell plate after blocking, adding ractopamine into the wells of the nanowell plate after the lymphocytes secrete ractopamine-specific antibodies, and screening cells specifically secreting rabbit monoclonal antibodies against ractopamine according to the fluorescence intensity of the wells of the nanowell plate.

[0018] Further, the cell secreting the specific rabbit monoclonal antibody against ractopamine is screened according to the fluorescence intensity of the hole of the nanopore plate, and the cell is obtained by the following steps:

[0019] The cell is screened to meet the following conditions:

[0020] i) the fluorescence intensity increases during the process of waiting for the lymphocyte to secrete the ractopamine specific antibody;

[0021] ii) the fluorescence intensity decreases after the ractopamine is added into the hole of the nanopore plate.

[0022] Further, the ractopamine immunogen is obtained by connecting hemocyanin with ractopamine; and / or, the ractopamine coating agent is obtained by connecting bovine serum albumin with ractopamine.

[0023] Further, the lymphocyte of the rabbit immunized by the ractopamine immunogen comprises the following steps:

[0024] The rabbit is immunized four times by using the ractopamine immunogen, the first immunization is performed by using the amount of 0.4-0.8 mg per rabbit, and the second to fourth immunization is performed every 21-27 days after the first immunization, and the amount is 1-1.2 mg per rabbit.

[0025] The application further provides the application of the rabbit monoclonal antibody, the nucleic acid, the biological material, the kit or the rabbit monoclonal antibody prepared by the preparation method in detecting ractopamine; preferably, the application in detecting ractopamine in urine.

[0026] The application has the following beneficial effects:

[0027] The application first prepares a RAC specific RmAb1, the affinity of which is 0.007 ng / mL, and the RmAb1 can resist 3M urea, which is beneficial to establish a stable RAC immune analysis method in the urine without sample pretreatment.

[0028] The detection limit of the indirect competitive enzyme-linked immunosorbent method based on the RmAb1 for RAC is 0.0042-0.014 μg / L, the variation coefficient of the urine of pigs, sheep and cattle is less than 11.7%, and the sensitivity is significantly improved by 10-100 times. Therefore, the super-sensitive rabbit monoclonal antibody with high urea resistance provided by the application has a relatively broad prospect in scientific research and practical application. BRIEF DESCRIPTION OF DRAWINGS

[0029] In order to more clearly illustrate the technical solutions of the present application or the prior art, the drawings needed to be used in the following embodiments or prior art description will be briefly introduced. Obviously, the drawings described below are some embodiments of the present application, and for those skilled in the art, other drawings can also be obtained without creative labor on the basis of these drawings.

[0030] Figure 1 is a schematic diagram of the ractopamine immunogen and coating antigen provided by embodiment 1 of the present application; wherein A is a ractopamine immunogen, and B is a ractopamine coating antigen.

[0031] Figure 2 is a fluorescence intensity analysis result of single antibody secreting cells 0-8h provided by embodiment 1 of the present application; wherein A is a RAC-specific antibody secreting cell, B is a BSA-specific antibody secreting cell, and C is a non-RAC / BSA-specific antibody secreting cell.

[0032] Figure 3 is a nucleic acid electrophoresis map and a protein gel electrophoresis map of RmAb1 provided by embodiment 1 of the present application; wherein A is a nucleic acid electrophoresis map, B is a non-denatured protein electrophoresis map, and C is a denatured protein electrophoresis map.

[0033] Figure 4 is an affinity analysis result of RmAb1 provided by embodiment 1 of the present application; wherein A is an antibody titer analysis result, and B is an antibody IC 50 analysis result.

[0034] Figure 5 is a urea tolerance analysis result of RmAb1 provided by embodiment 2 of the present application; wherein A is the change of antibody affinity in different concentrations of urea, and B is the change of antibody thermal stability related parameters Tagg and Tm in different concentrations of urea.

[0035] Figure 6 is a salt ion, pH, methanol and acetonitrile tolerance analysis result of RmAb1 provided by embodiment 2 of the present application; wherein A is a salt ion tolerance analysis result, B is a methanol tolerance analysis result, C is a pH value tolerance analysis result, and D is an acetonitrile tolerance analysis result. DETAILED DESCRIPTION

[0036] In order to make the purpose, technical solutions and advantages of the present application more clear, the technical solutions in the present application will be described clearly and completely below in combination with the drawings in the present application. Obviously, the described embodiments are some embodiments of the present application, and not all embodiments. Based on the embodiments in the present application, all other embodiments obtained by those skilled in the art without creative labor belong to the scope of protection of the present application.

[0037] Example 1

[0038] 1. Immunization of New Zealand White Rabbits with Ractopamine Immunogen

[0039] The concentration of the Ractopamine immunogen (as shown in Figure A of Figure 1 ) was adjusted to 1 mg / mL with PBS buffer, and the concentration of the immunogen was determined by the Bicinchoninic Acid method;

[0040] (1) Six New Zealand White rabbits were numbered rabbit #1, rabbit #2, rabbit #3, rabbit #4, rabbit #5, and rabbit #6, respectively, and were immunized a total of 6 times by multi-point injection in the nape of the neck;

[0041] (2) First immunization: Each rabbit was immunized with 0.6 mL of the primary immunization preparation (containing 1 mg of Ractopamine immunogen per 1 mL of the primary immunization preparation), using Freund's complete adjuvant as the adjuvant;

[0042] (3) Second to fourth immunization: Starting from the day of the first immunization, each rabbit was immunized once every 3 weeks for a total of 3 times, with each immunization being 1 mg of Ractopamine immunogen.

[0043] 2. Affinity determination of rabbit antisera

[0044] The titer and sensitivity of the antisera obtained one week after immunization were determined. The determination method was icELISA.

[0045] Specifically, 50 μL of different concentrations of small molecule competitors (RAC or its structural analogs) and diluted antiserum solution were added to the coated ELISA plate, which was then incubated in a 37°C incubator for 30 min, followed by 3 washes with PBST solution and blotting. The enzyme-labeled secondary antibody was incubated in a 37°C incubator for 30 min, followed by 3 washes with PBST solution and blotting. TMB was used for color development for 15 min. The reaction was stopped with 2% concentrated sulfuric acid. The IC 50 of the antisera of rabbit #4 was 0.04 ng / mL –1 (Table 1). Therefore, rabbit #4 was selected for spleen lymphocyte preparation.

[0046] Table 1. Performance determination of serum of immunized New Zealand White rabbits

[0047]

[0048] 3. Method for preparing rabbit spleen lymphocytes

[0049] (1) After 3 weeks of the sixth immunization, the rabbit ear vein pulse booster immunization was carried out, and the pulse dose was 4 mg. 3-4 days after the pulse, the rabbit spleen was removed under sterile conditions and quickly placed in a centrifuge tube containing 50 mL of RPMI 1640 medium, soaked for about 5 min;

[0050] (2) Prepare 3 9 cm disposable culture dishes, two of which are added with 20 mL of RPMI 1640 medium, and the other dish is placed in 3 sterile 45 μm filter screens;

[0051] (3) Place the spleen in the first dish containing RPMI 1640 medium, remove the excess fat and fibrous tissue on it, and place it in the second dish. Use scissors to cut it into three parts and place them in three dishes, respectively;

[0052] (4) Add 1 mL of red blood cell lysate to each filter screen, grind the spleen with a sterile glass pestle for 3 min, add 2 mL of RPMI 1640 medium to the sterile filter screen, and wash the filter screen. The ground and filtered cells are placed in a 50 mL centrifuge tube and centrifuged at 1000 rpm for 10 min;

[0053] (5) The spleen cells are frozen at a cell density of 10 9 / mL;

[0054] (6) The frozen spleen cells in liquid nitrogen are thawed in a 37°C water bath, and RPMI 1640 medium is added. Centrifuge at 1000 rpm for 5 min;

[0055] (7) The cell pellet after centrifugation is resuspended with 5 mL of RPMI 1640 medium (cell concentration is 10 8 -10 9 mL –1 ), carefully added on the separation liquid (ratio 1:1), centrifuged at 800g, 20°C±2°C for 20 min;

[0056] (8) The centrifuge tube is divided into four layers from top to bottom, which are diluent layer, lymphocyte layer, separation liquid layer and red blood cell layer. Collect the lymphocytes into a centrifuge tube containing 5 mL of PBS, mix well, and centrifuge at 500g for 20 min;

[0057] (9) The separated lymphocyte pellet is washed twice, and the cell pellet is resuspended with RPMI 1640 (5% serum) medium to adjust the cell concentration to 10 5 mL –1 . Uniformly spread in a 6-well plate cell culture dish, and put it in a cell culture box for later use

[0058] 4. Sorting of single rabbit antibody secreting cells

[0059] (1) Pretreatment of H25-25 Nanowell plate: inject 1 mL of absolute ethanol into each macro well, inject slowly along the well wall bottom, and make sure to inject slowly. Place the nanowell plate on the ALS special fixture, and centrifuge the nanowell plate at a high speed (500 g, 2 min) at room temperature to remove the small amount of air bubbles in the nanowell;

[0060] (2) Washing of H25-25 Nanowell plate: aspirate 0.5 mL of absolute ethanol, add 2 mL of CBS, and then aspirate again (each time cover the lid), repeat the washing with CBS for 5 times, (note: do not let the nanowell be empty of liquid (at least leave 0.5 mL –1 ), and do not tilt when moving;

[0061] (3) Coating of H25-25 Nanowell plate: add 0.5 mL of ractopamine coating solution (as shown in B of Figure 1 ) to each macro well, at a concentration of 10-20 μg mL –1 , and place at 4°C overnight;

[0062] (4) Blocking of H25-25 Nanowell plate: after discarding the coating solution, wash with CBS for 3 times, add 0.5 mL of 2% BSA to each well, and block at 37°C for 1 h;

[0063] (5) Plating of rabbit spleen lymphocytes: add 0.5 mL of cell suspension (density is suggested to be 10 5 mL –1 ) and fluorescently labeled secondary antibody (suggested to be 1-2 μg mL –1 ) mixture to each macro well, plate by evenly adding the solution in a "Z" shape, and let stand at room temperature for 10 min, and centrifuge at 300 g for 3 min.

[0064] (6) Collection of fluorescent signal of antigen-specific antibody secreting cells: on the Cellcelector™, scan using a 10X objective, scan the fluorescence and bright field once at 0 h, and then scan every 2 h in turn (make sure that the position of the cells does not change each time);

[0065] (7) Competitive screening of RAC-specific antibody secreting cells: after 4 h of antibody secretion, add RAC (1 μg mL –1 ) to the nanowell to compete with the ractopamine coating solution for binding to the RAC-specific antibody secreted from the single RAC-specific antibody secreting cell, continue to scan for 4 h, and observe the change in fluorescence intensity, the results are shown in Figure 2 , A is the RAC-specific antibody secreting cell, B is the BSA-specific antibody secreting cell, and C is the non-RAC / BSA-specific antibody secreting cell.

[0066] (8) Capture of antigen-specific antibody-secreting cells. On the Cellcelector™, select a 30-μm capillary needle, adjust the appropriate height and position, and select cells that have enhanced nanowell wall fluorescence within 0-4 h but diminished nanowell wall fluorescence due to competitive binding of RAC within 4-8 h. Collect the RAC-specific antibody-secreting cells in a PCR tube containing lysis buffer and immediately store at -80 °C.

[0067] 5. Preparation of cDNA from single antibody-secreting cells in vitro

[0068] (1) Transfer the single antigen-specific ASC cells from -80 °C to a 70 °C metal bath and lyse for 10 min;

[0069] (2) Add DNase I (1 U μL –1 ) 5 μL and 1 μL of DNase I buffer to the PCR tube, mix the PCR tube up and down, and gently centrifuge. Let stand at room temperature for 5 min (note that the standing time should not exceed 10 min, otherwise the entire nucleic acid yield will be affected);

[0070] (3) Add 1.2 μL of 25 mM EDTA, invert the PCR tube, and gently centrifuge. Let stand at 70 °C in a metal bath for 5 min, and the RNA extraction is complete;

[0071] (4) Continue to add 2 μL of Oligo(dT) 20 (50 mM) and 1 μL of 10 mM dNTP Mix to the above PCR tube, invert the PCR tube, and gently centrifuge. Let stand at 70 °C in a metal bath for 5 min;

[0072] (5) After standing on ice for 2 min, sequentially add 6 μL of 5x RT Buffer, 1 μL of RNaseOUT™ (40 U μL –1 ), 1 μL of SuperScript™ III RT (200 U μL –1 ), and 1 μL of 0.1 M DTT, invert the PCR tube, and gently centrifuge. Place the PCR tube in a PCR instrument, and incubate at 50 °C for 50 min and at 85 °C for 5 min;

[0073] (6) Add 1 μL of RNase H (2 U μL –1 ), and let stand at 37 °C for 20 min. The product is the cDNA of the single cell, which is stored at -20 °C.

[0074] 6. Preparation of antibody variable region genes from single antibody-secreting cells

[0075] (1) Take the cDNA of single antibody secreting cell as template, optimize the primers of variable region of heavy chain and light chain of rabbit, pre-denature at 95℃ for 3 min; 95℃ for 30 s, 55℃ for 30 s, 72℃ for 2 min, 30 cycles; 72℃ for 10 min. After the PCR reaction, the product is identified by agarose gel electrophoresis, and the target fragment is recovered. Figure 3

[0076] (2) After the target fragment is recovered, add plasmid and ligase to a sterile centrifuge tube, gently shake the centrifuge tube to mix the contents, and centrifuge for 3-5 s. Place the mixed reaction solution at room temperature for 5 min. After the reaction is completed, place the centrifuge tube on ice for subsequent transformation reaction;

[0077] (3) Take part of the ligation product and add it to 50-100 μL of DH5α competent cells (the competent cells should be taken out from the -70℃ refrigerator and placed in an ice bath, and the ligation product should be added when it is just thawed, and the amount of ligation product added should not exceed one-tenth of the volume of competent cells), gently mix, ice bath for 30 min, 42℃ water bath for 90 s, ice bath for 2-3 min, do not shake the centrifuge tube during this period. Add 350 μL of preheated LB (without antibiotics) medium at 37℃, shake culture at 180 rpm and 37℃ for 45-60 min. Mix the bacterial solution in the centrifuge tube, take 200 μL and add it to LB solid agar medium containing ampicillin, and gently spread the cells evenly with a sterile bent glass rod or glass beads. After the surface of the plate is dry, invert the plate and culture at 37℃ for 12-16 h;

[0078] (4) Pick a single colony and inoculate it in 1-5 mL of 2×YT medium containing ampicillin at a final concentration of 50-100 μg / mL, shake culture at 37℃ overnight, and send the bacterial solution for sequencing to determine the sequence information (Table 2). –1

[0079] Table 2 Variable region sequence of RmAb1

[0080]

[0081] 7. In vitro preparation and performance determination of rabbit monoclonal antibody

[0082] (1) According to the preference of mammalian cell expression system, optimize the codons of amplified VH and VL, synthesize the full gene of variable region, and clone it into the mammalian cell expression vector pFUSE-rabbit Fc containing the constant region of rabbit monoclonal antibody;

[0083] (2) Place the HEK-293 cells in a 5% CO2 constant temperature shaker, and shake culture at 37℃ and 120 rpm; ​​

[0084] (3) To ensure the transfection effect, cells in the exponential phase (density about 2-4 x 10 6 cells mL -1 ) with a survival rate of more than 98% were transfected;

[0085] (4) Two 15 mL sterile centrifuge tubes were prepared, 5 mL Opti-MEM and 100 μg sterile plasmid DNA were added to one of the tubes and mixed gently by blowing; another centrifuge tube was taken, 5 mL Opti-MEM and 500 μL Lipofectamine 2000 transfection reagent were added and mixed gently by blowing;

[0086] (5) All the liquid in the centrifuge tube containing the transfection reagent was transferred to the centrifuge tube containing the plasmid, mixed gently by blowing, the cells were taken out from the constant temperature shaker, the prepared plasmid carrier compound was added while shaking, and then put back into the CO2 constant temperature shaker for shaking culture. 3 h later, appropriate amount of antibiotic was added as needed;

[0087] (6) The expression amount of the product was determined 6 days after transfection, and the appropriate harvest time was selected according to the cell state and the expression amount. The harvested antibody was purified by Protein A (B) in the above. Figure 3

[0088] (7) The binding performance and inhibition performance were analyzed by ELISA and icELISA. The binding performance titer of the antibody was 73338.4, and the IC 50 of the antibody was 0.007 ng mL -1 ( Figure 4 )

[0089] (8) The specificity of the antibody was evaluated by analyzing the cross-reactivity with other β-adrenergic agonists, and the results showed that the antibodies prepared by the two methods had almost no cross-reactivity with other β-adrenergic agonists (Table 3);

[0090] CR = IC 50 (RAC) / IC 50 (other β-adrenergic agonists) Formula 1

[0091] Table 3 Cross-reactivity analysis of RmAb1

[0092]

[0093]

[0094]

[0095]

[0096] ​Example 2

[0097] Based on the above prepared rabbit monoclonal antibody, an icELISA method for detecting ractopamine residues in urine samples was provided in this example, and the specific process was as follows:

[0098] (1) Different concentrations of urea solution were configured to analyze the urea tolerance of the antibody. It was found that RmAb1 showed high tolerance in 3M urea, and the IC 50 was 0.007 to 0.009 ng mL -1 between A) in (B) of Figure 5 . The thermal stability and aggregation of RmAb1 were evaluated by Tm and Tagg, respectively. The Tm and Tagg values of RmAb1 did not change significantly in 0M-3M urea, indicating that RmAb1 could tolerate 3M urea (B) of Figure 5 .

[0099] (2) The physicochemical parameters of the buffer system (salt particle concentration, pH value, organic solvent concentration) had a great influence on the sensitivity of ELISA. Therefore, the NaCl concentration, pH value of the buffer solution, and the tolerance of the reaction system to methanol and acetonitrile were evaluated. The results showed that the tolerance of RmAb1 icELISA to salt ion strength was 580mM (A) of Figure 6 . The optimal pH value of RmAb1 was between 5-8 (C) of Figure 6 . In icELISA, the tolerance of RmAb1 to methanol and acetonitrile was as high as 20% and 40% (B and D) of Figure 6 . Figure 6

[0100] (3) Based on the prepared RmAb3 with high sensitivity, an icELISA method was established for the detection of CAP in actual samples. Based on the optimized icELISA method, the RAC added samples and actual positive samples were detected, and compared with UPLC-MS / MS, the established icELISA method was comprehensively evaluated from sensitivity, accuracy and precision. The recovery rate and coefficient of variation (CV) of the spiked samples evaluated the established icELISA method had high accuracy and precision (Table 4), and the actual positive pig samples confirmed by HPLC-MS / MS were re-detected by this icELISA method, and the results showed that the consistency of the two methods was good (Table 5).

[0101] In summary, the preparation and application technology of the extremely urea-tolerant super-sensitive rabbit monoclonal antibody based on the above has a shorter detection time and lower labor cost, which meets the requirements of screening rapid analysis methods.

[0102] Table 4 RmAb1 in actual samples of the added recovery analysis ​

[0103]

[0104] Table 5 Detection of RAC positive samples by icELISA and HPLC-MS / MS based on RmAb1

[0105]

[0106] Finally, it should be noted that the above examples are merely intended to illustrate the technical solutions of the present application, and are not intended to limit the same; even though the present application has been described in detail with reference to the foregoing examples, those of ordinary skill in the art should understand that they can still modify the technical solutions recorded in the foregoing examples, or make equivalent replacements for some of the technical features; and these modifications or replacements do not cause the essence of the corresponding technical solutions to deviate from the spirit and scope of the technical solutions of the embodiments of the present application.

Claims

1. A rabbit monoclonal antibody to ractopamine, characterized in that, The rabbit monoclonal antibody comprises an antibody heavy chain and an antibody light chain, The amino acid sequence of the heavy chain variable region of the rabbit monoclonal antibody is shown as SEQ ID NO. 1; and the amino acid sequence of the light chain variable region of the rabbit monoclonal antibody is shown as SEQ ID NO.

3.

2. A nucleic acid, characterized in that, The nucleic acid is used for encoding the rabbit monoclonal antibody of claim 1.

3. The nucleic acid of claim 2, wherein, The nucleotide sequence comprises SEQ ID NO. 5 and SEQ ID NO.

7.

4. A biomaterial, characterized by, The biological material comprises the nucleic acid of claim 2 or 3; and the biological material is an expression cassette, a vector or a transgenic cell.

5. A kit characterized in that, The rabbit monoclonal antibody of claim 1, or the nucleic acid of claim 2 or 3, or the biological material of claim 4.

6. Use of the rabbit monoclonal antibody of claim 1, or the nucleic acid of claim 2 or 3, or the biological material of claim 4, or the kit of claim 5 in detecting ractopamine.

7. Use according to claim 6, characterized in that, The detection of ractopamine is the detection of ractopamine in urine.

Images