Polymyxin b and colistin highly specific haptens, artificial antigens, and methods of making and using the same

Abstract

This invention relates to the field of biochemical technology, and particularly to highly specific haptens of polymyxin B and colistin, artificial antigens, their preparation methods, and applications. The haptens have structures as shown in Formula I or Formula II. The applications include: (1) preparing specific antibodies against polymyxin B or colistin; (2) detecting specific antibodies against polymyxin B or colistin; and (3) preparing reagents for detecting specific antibodies against polymyxin B or colistin. This invention further provides artificial antigens obtained by conjugating the haptens with carrier proteins. Using the artificial antigens provided by this invention as immunogens to immunize experimental animals, highly specific and sensitive antiserum can be prepared, which can then be used to extract and prepare polyclonal antibodies. These methods can be used to establish highly specific, highly sensitive, simple, and rapid detection methods for polymyxin B and colistin, and have significant application value.

Description

Technical Field

[0001] This invention relates to the field of biochemical technology, and in particular to polymyxin B and colistin high-specificity haptens, artificial antigens, their preparation methods and applications. Background Technology

[0002] Polymyxins are polypeptide antibiotics, including polymyxin B (PMB) and colistin (or polymyxin E, PME). They are structurally very similar, both consisting of an acyl tail, a linear tripeptide, and a cyclic heptacapeptide, differing only in the 6th amino acid residue of the cyclic heptacapeptide: polymyxin B uses D-phenylalanine, while colistin uses D-leucine. Polymyxins are effective against Gram-negative bacteria and are therefore widely used in clinical practice and animal husbandry.

[0003] However, the biosafety and resistance risks of polymyxins have always been a major concern in the industry. With the discovery of related resistance genes and in-depth research into their transmission mechanisms, the control of resistance and residue monitoring of these drugs has become particularly important. To ensure food safety, relevant food safety standards have set strict thresholds for polymyxin residue limits in animal-derived foods (such as cow's milk and goat's milk). Therefore, establishing rapid and effective detection methods for polymyxin B and colistin is of great significance for ensuring food safety and the rational use of drugs.

[0004] The main methods for detecting polymyxins include microbiological methods and instrumental methods based on liquid chromatography and mass spectrometry. However, microbiological methods are time-consuming and have poor specificity; instrumental methods require expensive large-scale equipment and specialized operators, which limits their application in rapid detection at the production line. Immunoassays based on antigen-antibody recognition have the advantages of high sensitivity, speed, ease of operation, and low cost, and have great application potential in polymyxin detection.

[0005] In current technologies, artificial antigens are typically prepared by directly conjugating polymyxin B or colistin technical grade to a carrier protein. This method leads to heterogeneity of conjugation sites, resulting in insufficient antibody specificity. When the established immunoassay method is used to detect real samples, false positive results are easily generated due to the cross-presence of colistin or polymyxin B.

[0006] Antibodies, as the core recognition element in immunoassays, determine the specificity of detection methods, while hapten design is key to preparing highly specific antibodies. To accurately assess the illegal addition of polymyxin B and the residual amount of colistin in animal-derived foods, it is urgent to design specific haptens for polymyxin B and colistin respectively, and then prepare their respective specific antibodies, thereby providing highly specific recognition elements for the accurate detection of polymyxin B and colistin. Summary of the Invention

[0007] To address the problems existing in the prior art, this invention provides polymyxin B and colistin highly specific haptens, artificial antigens, their preparation methods, and applications.

[0008] In a first aspect, the present invention provides a hapten having a structure as shown in Formula I or Formula II: Formula I, Formula II.

[0009] In a second aspect, the present invention provides an artificial antigen, comprising: the aforementioned hapten, and a carrier protein coupled to the hapten.

[0010] Furthermore, the carrier protein includes one or more of bovine serum albumin, ovalbumin, keyhole hemocyanin, bovine thyroglobulin, or human serum albumin; Preferably, the carrier protein is bovine serum albumin or keyhole hemocyanin; More preferably, the coupling method of the carrier protein includes one or more of the following: active ester method, carbodiimide method, glutaraldehyde method, maleimide method, mixed anhydride method, or click chemical reaction.

[0011] The artificial antigen described in this invention can be either a coating antigen or an immunogen.

[0012] Thirdly, the present invention provides a hybridoma cell line, which is prepared by using the aforementioned artificial antigen as an immunogen.

[0013] Fourthly, the present invention provides an antibody, which is prepared by using the aforementioned artificial antigen as an immunogen; preferably, the antibody is a monoclonal antibody or a polyclonal antibody.

[0014] Fifthly, the present invention provides a kit comprising: the aforementioned hapten, or the aforementioned artificial antigen, or the aforementioned hybridoma cell line, or the aforementioned antibody.

[0015] Sixthly, the present invention provides a method for preparing the aforementioned hapten, wherein the method for preparing the hapten as shown in Formula I includes: Using a solid-phase polypeptide synthesis method, threonine, 2,4-diaminobutyric acid, 2,4-diaminobutyric acid, leucine, D-phenylalanine, 2,4-diaminobutyric acid, 2,4-diaminobutyric acid, and cysteine ​​are sequentially coupled and cyclized; and / or, The preparation method of the hapten as shown in Formula II includes: Threonine, 2,4-diaminobutyric acid, 2,4-diaminobutyric acid, leucine, D-leucine, 2,4-diaminobutyric acid, 2,4-diaminobutyric acid, and cysteine ​​were sequentially coupled and cyclized using a solid-phase polypeptide synthesis method.

[0016] As a preferred embodiment, the method for preparing the hapten as shown in Formula I includes: (1) The N-terminal amino group protected by 9-fluorenyloxycarbonyl-O-tert-butyl-S-triphenylmethyl-2-chlorothreonine (Fmoc-Thr(tBu)-2Cl(Trt)) resin is reacted in pyridine at 22~27℃ for 15~25 minutes to remove the N-terminal amino group protected by 9-fluorenyloxycarbonyl (Fmoc).

[0017] (2) Add N,N-diisopropylethylamine (DIEA), O-benzotriazole-tetramethylurea hexafluorophosphate (HBTU) and N-fluorenylmethoxycarbonyl-N'-(tert-butyloxycarbonyl)-2,4-diaminobutyric acid (Fmoc-Dab(Boc)-OH) to the coupling resin, react, and proceed to the next step after the reaction is colorless as detected by ninhydrin reagent; (3) The coupling resin treated in (2) is reacted in pyridine at 22~27℃ for 15~25 minutes to remove the N-terminal amino group protected by Fmoc; (4) Add DIEA, HBTU and Fmoc-Dab(Boc)-OH to the coupling resin after (3) treatment, react, and repeat steps (2) and (3) after the color is detected by ninhydrin reagent. Connect Fmoc-Dab(Boc)-OH, N-fluorenylmethoxycarbonyl-leucine (Fmoc-Leu-OH), N-fluorenylmethoxycarbonyl-D-phenylalanine (Fmoc-D-Phe-OH), N-fluorenylmethoxycarbonyl-N'-(1-(4,4-dimethyl-2,6-dioxocyclohexylene)-3-methylbutyl)-2,4-diaminobutyric acid (Fmoc-Dab(Dde)-OH), and N-tert-butoxycarbonyl-S-triphenylmethyl-cysteine ​​(Boc-Cys(trt)-OH) to the coupling resin in sequence. (5) The coupling resin treated in (4) was treated in 1-3% hydrazine hydrate + 96-99% dichloromethane (DCM) to remove 1-(4,4-dimethyl-2,6-dioxocyclohexylene)-3-methylbutyl (Dde) from the Dab residue. (6) After thoroughly washing the resin treated in (5) with DCM and methanol, react it in 1-3% trifluoroacetic acid (TFA) + 96-99% DCM at 22-27℃ for 6-10h, filter it, add N,N'-diisopropylcarbodiimide (DIC) and 1-hydroxybenzotriazole (HOBT) and react it under weakly alkaline conditions for 10-14h to cyclize the peptide. After the ninhydrin reagent test shows no color, cut the resin to obtain the crude peptide. (7) After the crude peptides are purified by high performance liquid chromatography, they can be further freeze-dried under vacuum.

[0018] The preparation method of the hapten shown in Formula II is the same as that of the hapten shown in Formula I, except that Fmoc-D-Phe-OH in the preparation process is replaced with N-fluorenylmethoxycarbonyl-D-leucine (Fmoc-D-Leu-OH).

[0019] In a seventh aspect, the present invention provides a method for preparing the aforementioned artificial antigen, comprising: Maleimide hexanoic acid was coupled to the amino group of the carrier protein using the active ester method; The maleimide group on the carrier protein and the thiol group on the hapten are coupled by a click chemical reaction.

[0020] As a preferred embodiment, the aforementioned method for preparing the artificial antigen includes: (1) Ethyl dimethylamino carbodiimide (EDC), N-hydroxysuccinimide (NHS) and maleimide hexanoic acid were mixed in N,N-dimethylformamide (DMF) solvent and reacted at 22~27℃ for 16~30 hours; the resulting maleimide NHS activated ester was purified by silica gel column chromatography and then obtained solution A by rotary evaporation.

[0021] (2) After dissolving solution A in DMF, add it to the carrier protein dissolved in carbonate buffer (CB) and stir at 22~27℃ for 16~30h. Dialyze the reaction product with phosphate buffer (PBS) for 3 days, changing the dialysate every 6~10h to obtain solution B.

[0022] (3) Dissolve the hapten shown in Formula I or Formula II and slowly add it dropwise into solution B. Stir and react at 22-27°C for 16-30 hours. Dialyze the resulting conjugate product with PBS for 2-4 days, changing the dialysis solution every 6-10 hours during this period.

[0023] Eighthly, the present invention provides the use of the aforementioned hapten or the aforementioned artificial antigen in any of the following: (1) Prepare specific antibodies against polymyxin B or colistin; (2) Detect specific antibodies against polymyxin B or colistin; (3) Prepare reagents for detecting polymyxin B or colistin-specific antibodies; preferably, the reagents are enzyme-linked immunosorbent assay (ELISA) reagents, immunochromatographic reagents, Western blotting reagents, immunohistochemical reagents or immunofluorescence reagents.

[0024] Ninthly, the present invention provides the use of the aforementioned antibody in any of the following: (1) Detect polymyxin B or colistin; (2) Prepare test strips, reagents or kits for detecting polymyxin B or colistin; Preferably, the test strip is an immunochromatographic test strip; and / or, the kit is an ELISA kit or a chemiluminescence kit.

[0025] The present invention has the following beneficial effects: This invention discloses for the first time novel polymyxin B and colistin haptens, artificial antigens, and their preparation methods. Immunizing animals with the aforementioned polymyxin B and colistin artificial antigens yields highly potent and sensitive specific antibodies (obtained through antiserum purification). These specific antibodies can be used to establish rapid, simple, inexpensive, sensitive, and specific methods for detecting polymyxin B and colistin.

[0026] The polymyxin B and colistin haptens provided by this invention have fixed coupling sites, and the prepared artificial antigens have the advantages of good uniformity. The prepared antibodies have the advantages of high titer and high sensitivity, and can be used to establish rapid detection methods for polymyxin B and colistin. The polymyxin B and colistin haptens, artificial antigens, and the antibodies prepared by this invention are suitable for blood drug concentration monitoring and veterinary drug residue analysis, and have good application prospects. Attached Figure Description

[0027] To more clearly illustrate the technical solutions in this invention or the prior art, the drawings used in the description of the embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are some embodiments of this invention. For those skilled in the art, other drawings can be obtained from these drawings without creative effort.

[0028] Figure 1 This is the mass spectrum of the polymyxin B high-specificity hapten shown in Formula I provided in the embodiments of the present invention.

[0029] Figure 2 This is the mass spectrum of the colistin high-specificity hapten shown in Formula II provided in the embodiments of the present invention.

[0030] Figure 3 The curves showing the inhibition of polymyxin B and colistin by the antiserum induced by polymyxin B immunogen provided in this embodiment of the invention.

[0031] Figure 4 The curves showing the inhibition of colistin and polymyxin B by the antiserum induced by colistin immunogen provided in this embodiment of the invention are shown. Detailed Implementation

[0032] To make the objectives, technical solutions, and advantages of this invention clearer, the technical solutions of this invention will be clearly and completely described below. Obviously, the described embodiments are only some embodiments of this invention, not all embodiments. Based on the embodiments of this invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of this invention.

[0033] Unless otherwise specified, the experimental methods involved in the following embodiments are conventional methods in the art. For example, you can refer to the experimental manual in the art or follow the conditions recommended in the manufacturer's instructions.

[0034] Unless otherwise specified, all experimental materials and reagents used in the following examples are commercially available.

[0035] In the quantitative experiments described below, all experiments were performed in triplicate, and the results were averaged. The PBS buffer used in the examples was pH 7.4, 0.01M. The carbonate buffer used in the examples was pH 9.6, 0.05mol / L sodium carbonate buffer.

[0036] In the following embodiments: BSA: Bovine serum albumin.

[0037] OVA: Ovalbumin.

[0038] DMF: N,N-dimethylformamide.

[0039] EDC: 1-(3-Dimethylaminopropyl)-3-ethyl-carbodiimide.

[0040] NHS: N-hydroxysuccinimide.

[0041] DIEA: N,N-diisopropylethylamine.

[0042] HBTU: O-benzotriazole-tetramethylurea hexafluorophosphate.

[0043] Fmoc: N-fluorenemethyloxycarbonyl.

[0044] Boc: tert-Butoxycarbonyl.

[0045] Dde: 1-(4,4-dimethyl-2,6-dioxocyclohexylene)-3-methylbutyl.

[0046] DCM: Dichloromethane.

[0047] TFA: Trifluoroacetic acid.

[0048] DIC: N,N'-Diisopropylcarbodiimide.

[0049] HOBT: 1-Hydroxybenzotriazole.

[0050] Fmoc-Thr(tBu)-2Cl(Trt): N-fluorenylmethoxycarbonyl-O-tert-butyl-S-triphenylmethyl-2-chlorothreonine.

[0051] Fmoc-Dab(Boc)-OH: N-fluorenylmethoxycarbonyl-N'-(tert-butyloxycarbonyl)-2,4-diaminobutyric acid.

[0052] Fmoc-Leu-OH: N-fluorenylmethoxycarbonyl-leucine.

[0053] Fmoc-D-Phe-OH: N-fluorenylmethoxycarbonyl-D-phenylalanine.

[0054] Fmoc-Dab(Dde)-OH: N-fluorenylmethoxycarbonyl-N'-(1-(4,4-dimethyl-2,6-dioxocyclohexylene)-3-methylbutyl)-2,4-diaminobutyric acid.

[0055] Boc-Cys(Trt)-OH: N-tert-Butoxycarbonyl-S-triphenylmethyl-cysteine.

[0056] Fmoc-D-Leu-OH: N-fluorenylmethoxycarbonyl-D-leucine.

[0057] Example 1: Preparation and characterization of polymyxin B and colistin high-specificity haptens I. Preparation of Polymyxin B High-Specificity Hapten and Colistin High-Specificity Hapten 1. Preparation of the polymyxin B high-specificity hapten shown in Formula I (1) First, the amino and side-chain protected Fmoc-Thr(tBu)-2Cl(Trt) resin was reacted in 20% pyridine at 25 °C for 20 min to remove the N-terminal amino group protected by Fmoc, and the color was tested by ninhydrin reagent. (2) Add DIEA, HBTU and Fmoc-Dab(Boc)-OH to the coupling resin, react, and after the ninhydrin reagent test shows no color, proceed to the next step; (3) The above coupling resin was reacted in 20% pyridine at 25 °C for 20 min to remove the N-terminal amino group protected by Fmoc. The ninhydrin reagent showed a blue color. (4) Add DIEA, HBTU and Fmoc-Dab(Boc)-OH to the coupling resin, react, and after the ninhydrin reagent test is colorless, repeat steps (2) and (3) to sequentially connect Fmoc-Dab(Boc)-OH, Fmoc-Leu-OH, Fmoc-D-Phe-OH, Fmoc-Dab(Dde)-OH and Boc-Cys(trt)-OH to the coupling resin; (5) The above coupling resin was treated in 2% hydrazine hydrate + 98% DCM to remove Dde from the Dab residues; (6) After thoroughly washing the resin with DCM and methanol, react it in 2% TFA + 98% DCM at 25 °C for 8 h and then filter it. Add DIC and HOBT and react under weakly alkaline conditions for 12 h to cyclize the peptide. After the ninhydrin reagent test shows no color, cut the resin to obtain the crude peptide. (7) The crude peptide was purified by high performance liquid chromatography and then freeze-dried under vacuum to obtain the hapten shown in Formula I, named H2-cys.

[0058] 2. Preparation of the highly specific colistin hapten shown in Formula II The preparation method of the colistin hapten is the same as that of the polymyxin B hapten, except that Fmoc-D-Phe-OH is replaced with Fmoc-D-Leu-OH. It is named H3-cys.

[0059] II. Characterization of polymyxin B high-specificity hapten and colistin high-specificity hapten 1. Mass spectrometry identification of polymyxin B high-specificity hapten The polymyxin B high-specificity hapten shown in Formula I (molecular formula: C) 38 H 66 N 12 Mass spectrometry identification results of O9S: MS m / z [M+H]+ theoretical value: 865.23; measured value: 865.6, which is consistent with the molecular weight of the target product. The mass spectrum is shown below. Figure 1 .

[0060] 2. Mass spectrometry identification of colistin high-specificity hapten Formula II shows a highly specific colistin hapten (molecular formula: C). 35 H 68 N 12 Mass spectrometry identification results of O9S: MS m / z [M+H]+ theoretical value: 831.04; measured value: 831.6, which is consistent with the molecular weight of the target product. See the mass spectrum below. Figure 2 .

[0061] Example 2: Preparation and characterization of polymyxin B artificial antigen and colistin artificial antigen The difference between the preparation methods of the immunogen and the coating antigen described in this embodiment lies in the type of carrier protein used: the immunogen carrier protein is BSA, and the coating antigen carrier protein is OVA.

[0062] I. Synthesis of Polymyxin B Immunogen (1) Weigh 100 mg of maleimide hexanoic acid, 136 mg of EDC and 82 mg of NHS and add them to 1 mL of DMF. Stir at 25 °C and 300 r / min for 24 h. After purification by column chromatography and rotary evaporation, solution A is obtained.

[0063] (2) Add solution A to 500 μL DMF, mix, and then slowly add 5 mL of 20 mg BSA protein dissolved in 5 mL CB solution. Stir at 25 °C and 300 r / min for 24 h. Dialyze the reaction product using 5 L 10 mM PBS for 3 days, changing the dialysis buffer every 8 h to obtain solution B.

[0064] (3) Weigh 15.1 mg of the hapten of Formula I prepared in Example 1, dissolve it in 500 μL of DMF, and add it dropwise to solution B. Stir at 25 °C and 300 r / min for 24 h. Dialyze the resulting reaction solution in 10 mM PBS for 3 days, changing the dialysate every 8 h. The resulting antigen is named H2-cys-BSA.

[0065] II. Synthesis of Polymyxin B Coating Origin Replace BSA with OVA, and follow the same steps as in step one of this embodiment. The resulting packet is originally named H2-cys-OVA.

[0066] III. Synthesis of colistin immunogen The difference between the preparation methods of the immunogen and the coating antigen described in this embodiment lies in the type of carrier protein used: the immunogen carrier protein is BSA, and the coating antigen carrier protein is OVA.

[0067] Except for replacing the hapten of Formula I prepared in Example 1 (15.1 mg) with the hapten of Formula II prepared in Example 1 (14.8 mg), the synthesis of polymyxin B immunogen in step one of this example was identical. The resulting antigen was named H3-cys-BSA.

[0068] IV. Synthesis of Colistin Coating Origin Replace BSA with OVA, and follow the same procedure as step three in this embodiment. The resulting coating is originally named H3-cys-OVA.

[0069] Example 3: Preparation of polymyxin B and colistin antiserum The polymyxin B immunogen and colistin immunogen prepared in Example 2 were emulsified with an equal volume of Freund's adjuvant and then used to immunize 6-8 week old female Balb / c mice weighing 20-23 g. The initial immunization used Freund's complete adjuvant (purchased from Sigma), and booster immunizations used Freund's incomplete adjuvant (purchased from Sigma). Immunization was performed via subcutaneous injection at multiple sites on the nape of the neck, with a dose of 100 μg per mouse, immunized at 4-week intervals, for a total of four immunizations. On day 7 after each immunization, blood was collected from the tail vein, centrifuged at 4000 rpm for 10 min, and the supernatant was collected as antiserum.

[0070] Example 4: Identification of the affinity and specificity of polymyxin B antiserum The polymyxin B antiserum obtained after the fourth immunization in Example 3 was identified using an indirect competitive ELISA method. The specific operational steps are as follows: (1) Coating: Dilute the original H3-cys-OVA to 1.5 μg / mL with CB solution, add 100 μL to each well of the microplate, and incubate at 37°C for 2 hours; (2) Washing: Pour out the liquid in the hole, wash with washing solution 3 times, 1 minute each time, and pat dry on absorbent paper; (3) Blocking: Add 155 μL of blocking solution to each well, incubate at 37°C for 1 hour, and wash; (4) Sample addition: After adding 50 μL of polymyxin B standard diluted with PBS and 50 μL of colistin standard diluted with PBS to each well, add 50 μL of working concentration antiserum to each well containing the standard, incubate at 37 ℃ for 30 min, and wash. (5) Add secondary antibody: Add 100 μL of HRP-goat anti-mouse IgG to each well, incubate at 37 ℃ for 30 min, and wash; (6) Color development: Add 100 μL of freshly prepared TMB solution to each well and develop color at 37 °C in the dark for 15 min; (7) Termination: Add 50 μL of 2 mol / L H2SO4 solution to each well to terminate the reaction; (8) Reading: Use an ELISA reader to read the OD450nm value of each well; (9) Establishing a standard curve: Plot a standard inhibition curve with the logarithmic values ​​of polymyxin B standard and colistin concentrations as the x-axis and the OD values ​​corresponding to each concentration as the y-axis. Then, using Origin 2019 Pro software, perform a four-parameter equation fitting with the logarithmic values ​​of the standard concentrations as the x-axis and the OD values ​​as the y-axis to calculate the IC50 of the antiserum against polymyxin B and colistin. 50 value.

[0071] (10) Specificity evaluation: Calculate the cross-reactivity (CR) of the antiserum to colistin: CR={IC 50 (Polymycin B) / IC 50 (Colistin)}×100%.

[0072] The results are as follows Figure 3 As shown, the IC50 of antiserum against polymyxin B 50 5.04 ng / mL -1 IC50 of colistin 50 160.68 ng / mL -1 The CR was 3.13%, indicating that the antiserum had a high affinity for and high specificity for polymyxin B.

[0073] Example 5: Identification of Colistin Antiserum Affinity and Specificity The colistin antiserum obtained after the fourth immunization in Example 3 was identified using an indirect competitive ELISA method. Except for changing the coating antigen to H2-cys-OVA, the CR calculation formula in step (10) was changed to: CR={IC 50 (Colistin) / IC 50 (Polymyxin B) × 100%.

[0074] The rest is the same as in Example 4.

[0075] The results are as follows Figure 4 As shown, the IC50 of antiserum against colistin 50 5.52 ng / mL -1 IC50 of polymyxin B 50 206.39 ng / mL -1 The CR was 2.69%, indicating that the antiserum had a high affinity for and high specificity for colistin.

[0076] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, and not to limit them; although the present invention has been described in detail with reference to the foregoing embodiments, those skilled in the art should understand that modifications can still be made to the technical solutions described in the foregoing embodiments, or equivalent substitutions can be made to some of the technical features; and these modifications or substitutions 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 invention.

Claims

1. A hapten, characterized in that, It has a structure as shown in Equation I or Equation II: Formula I, Formula II.

2. An artificial antigen, characterized in that, include: The hapten of claim 1, and the carrier protein coupled to the hapten.

3. The artificial antigen according to claim 2, characterized in that, The carrier protein includes one or more of bovine serum albumin, ovalbumin, keyhole hemocyanin, bovine thyroglobulin, or human serum albumin. Preferably, the carrier protein is bovine serum albumin or keyhole hemocyanin; More preferably, the coupling method of the carrier protein includes one or more of the following: active ester method, carbodiimide method, glutaraldehyde method, maleimide method, mixed anhydride method, or click chemical reaction.

4. A hybridoma cell line, characterized in that, The hybridoma cell line is prepared using the artificial antigen described in claim 2 or 3 as an immunogen.

5. An antibody, characterized in that, The antibody is prepared using the artificial antigen described in claim 2 or 3 as an immunogen; Preferably, the antibody is a monoclonal antibody or a polyclonal antibody.

6. A reagent kit, characterized in that, include: The hapten of claim 1, or the artificial antigen of claim 2 or 3, or the hybridoma cell line of claim 4, or the antibody of claim 5.

7. The method for preparing the hapten according to claim 1, characterized in that, The preparation method of the hapten shown in Formula I includes: Using a solid-phase polypeptide synthesis method, threonine, 2,4-diaminobutyric acid, 2,4-diaminobutyric acid, leucine, D-phenylalanine, 2,4-diaminobutyric acid, 2,4-diaminobutyric acid, and cysteine ​​are sequentially coupled and cyclized; and / or, The preparation method of the hapten as shown in Formula II includes: Threonine, 2,4-diaminobutyric acid, 2,4-diaminobutyric acid, leucine, D-leucine, 2,4-diaminobutyric acid, 2,4-diaminobutyric acid, and cysteine ​​were sequentially coupled and cyclized using a solid-phase polypeptide synthesis method.

8. The method for preparing the artificial antigen according to claim 2, characterized in that, include: Maleimide hexanoic acid was coupled to the amino group of the carrier protein using the active ester method; The maleimide group on the carrier protein and the thiol group on the hapten are coupled by a click chemical reaction.

9. The use of the hapten of claim 1, or the artificial antigen of claim 2 or 3, in any of the following: (1) Prepare specific antibodies against polymyxin B or colistin; (2) Detect specific antibodies against polymyxin B or colistin; (3) Prepare reagents for detecting polymyxin B or colistin-specific antibodies; Preferably, the reagent is an enzyme-linked immunosorbent assay (ELISA) reagent, an immunochromatographic reagent, a Western blotting reagent, an immunohistochemical reagent, or an immunofluorescence reagent.

10. The use of the antibody according to claim 5 in any of the following: (1) Detect polymyxin B or colistin; (2) Prepare test strips, reagents or kits for detecting polymyxin B or colistin; Preferably, the test strip is an immunochromatographic test strip; and / or, The kit is an ELISA kit or a chemiluminescence kit.

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