阅读说明：本技术 一种多粘菌素b和粘菌素半抗原、人工抗原及其制备方法与应用 (Polymyxin B, colistin hapten and artificial antigen as well as preparation method and application thereof ) 是由 王战辉 温凯 沈建忠 余文博 于雪芝 江海洋 张英杰 唐盈盈 何敏 郝晨淇 于 2021-08-13 设计创作，主要内容包括：本发明涉及生物化学技术领域,具体公开了一种多粘菌素B和粘菌素半抗原、人工抗原及其制备方法与应用。本发明的多粘菌素B和粘菌素半抗原,其结构如式I所示：将由上述半抗原制备得到的多粘菌素B和粘菌素人工抗原免疫实验动物,可得到效价高,灵敏度高的多粘菌素B和粘菌素的抗体。本发明提供的多粘菌素B和粘菌素半抗原、人工抗原及其制备的抗体为建立快速、简便、价廉、灵敏、特异的多粘菌素B和粘菌素检测方法提供了新的物质材料基础。(The invention relates to the technical field of biochemistry, and particularly discloses polymyxin B, a colistin hapten, an artificial antigen, and a preparation method and application thereof. The structures of polymyxin B and colistin hapten of the invention are shown as formula I:)

1. A polymyxin B and colistin hapten, having the structure shown in formula I:

2. a process for the preparation of polymyxin B and colistin haptens as claimed in claim 1 comprising: and (2) sequentially coupling cysteine, lysine, threonine, lysine and 6-methylheptanoic acid by a polypeptide solid phase synthesis method.

3. Polymyxin B and colistin artificial antigens, which are obtained by coupling the polymyxin B and colistin haptens of claim 1 with a carrier protein;

wherein the carrier protein is selected from bovine serum albumin, ovalbumin, keyhole limpet hemocyanin, bovine thyroglobulin or human serum albumin, preferably bovine serum albumin or ovalbumin.

4. The polymyxin B and colistin artificial antigen of claim 3, wherein the molar ratio of polymyxin B and colistin hapten to carrier protein is (8-9): 1.

5. The method for preparing polymyxin B and colistin artificial antigens as claimed in claim 3 or 4, wherein maleimidocaproic acid is first coupled to the amino group of the carrier protein by active ester method, and then the maleimide group of the carrier protein is coupled to the thiol group of the polymyxin B and colistin hapten by active ester method.

6. Use of a polymyxin B and colistin hapten as defined in claim 1 or a polymyxin B and colistin artificial antigen as defined in claim 3 or 4 in any one of the following:

(1) the application in preparing polymyxin B and colistin specific antibody;

(2) the application of the polypeptide in detecting polymyxin B and colistin specific antibodies.

7. Polymyxin B and colistin-specific antibodies, which are prepared by immunizing an animal with the polymyxin B and colistin artificial antigen of claim 3 or 4 as an immunogen; the polymyxin B and the colistin specific antibody are monoclonal antibodies or polyclonal antibodies.

8. Use of polymyxin B and colistin-specific antibodies of claim 7 in any one of:

(1) the application in detecting polymyxin B and colistin;

(2) the application in the preparation of the polymyxin B and the immunochromatographic test strip of the colistin;

(3) the application in preparing an ELISA kit of polymyxin B and colistin;

(4) the application in the preparation of polymyxin B and colistin chemiluminescence kits.

9. A detection reagent or test strip comprising polymyxin B of claim 7 and a colistin-specific antibody.

10. A kit comprising polymyxin B of claim 7 and a colistin-specific antibody.

Technical Field

The invention relates to the technical field of biochemistry, in particular to a polymyxin B and colistin (polymyxin E) hapten and artificial antigen as well as a preparation method and application thereof.

Background

Polymyxin B (PMB) and colistin (colistin or Polymyxin E, PME) are cationic polypeptide antibiotics obtained from culture fluid of polymyxa bacillus, and are clinically applied in the last 50 th century, but are gradually replaced by other safer antibacterial drugs after being marketed for 20 years due to toxic and side effects such as nephrotoxicity. However, with the severe condition of bacterial drug resistance and the delay of research and development of new antibacterial drugs, polymyxin is reused clinically in about 2000 years after disappearance of nearly 30 years, and the polymyxin is used as a drug for an intensive care unit, is mainly suitable for multiple drug-resistant bacterial infection and is known as the last line of defense against the multiple drug-resistant bacterial infection. Meanwhile, polymyxin B and colistin are widely used in livestock and poultry breeding activities for resisting bacteria and promoting growth.

However, the toxic side effects of polymyxin B and colistin have been of increasing concern and concern for a long time. Particularly, after the polymyxin drug resistance gene MCR-1 was reported for the first time in 2016, the control of the drug resistance of polymyxin drugs is more and more important. The residual limit of colistin in animal tissues is clearly specified in GB31650-2019, wherein the residual limit of cow milk and sheep milk is 50 mug/kg. There is therefore a need for a rapid and efficient assay for polymyxin B and colistin.

At present, the detection means of polymyxin B and colistin mainly comprises Liquid Chromatography (LC) and liquid chromatography-mass spectrometry (LC-MS/MS), but large instruments are high in price, poor in operability and not easy to carry, rapid detection in the production line is difficult to realize, and effective supervision on the use of polymyxin B and colistin is severely restricted. The immunoassay technology based on antigen-antibody specific reaction has the advantages of high speed, easy operation and low cost. Therefore, the development of a simple and quick monoclonal antibody for detecting polymyxin B and colistin is important.

The antibody is the core reagent of immunoassay, and the design and synthesis of hapten and artificial antigen are key technologies. At present, polymyxin B and colistin reported in documents and patents are both of original drug structures and are directly coupled with carrier protein to prepare artificial antigens, the problems of unfixed coupling sites, low sensitivity of antibody preparation, unbalanced cross reaction and the like exist, and the polymyxin B and colistin can not be well used for detecting actual samples of the polymyxin B and the colistin.

Disclosure of Invention

Aiming at the problems in the prior art, the invention aims to provide a polymyxin B and colistin hapten and an artificial antigen which can realize the rapid, sensitive and specific detection of the polymyxin B and colistin, a preparation method and application thereof, and the preparation and application of a monoclonal antibody thereof.

In order to achieve the object, the invention provides a polymyxin B and a colistin hapten in a first aspect, wherein the structures of the polymyxin B and the colistin hapten are shown as a formula I:

the invention selects the branched chain part of the drug as the hapten in the design of the hapten, simultaneously creatively introduces cysteine into the branched chain polypeptide parts of polymyxin B and colistin (by utilizing a polypeptide solid phase synthesis mode), and generates a specific click chemical reaction through the cysteine and the carrier protein modified by maleimide, so that the coupling site is single, the space conformation displayed by the hapten on the carrier protein is kept consistent, and finally the antibody with high titer and high affinity is obtained.

In a second aspect, the present invention provides a process for the preparation of polymyxin B and colistin haptens as described above, which comprises: and (2) sequentially coupling cysteine, lysine, threonine, lysine and 6-methylheptanoic acid by a polypeptide solid phase synthesis method.

As a specific embodiment, the present invention provides a method for preparing a hapten represented by the formula (I), comprising the steps of:

(1) firstly, amino and cysteine resin with protected side chain (FMOC-S-tributyl-L-cysteine 4-benzyl ester resin) are reacted for 20min at room temperature in 20% pyridine, 9-Fluorenylmethoxycarbonyl (FMOC) protected N-terminal amino is removed, and ninhydrin reagent tests color development;

(2) adding N, N-Diisopropylethylamine (DIEA), O-benzotriazole-tetramethyluronium Hexafluorophosphate (HBTU) and (S) -4- (BOC-amino) -2- (FMOC-amino) butyric acid into the coupling resin, reacting for 1h at room temperature, detecting with ninhydrin reagent to obtain colorless solution, and performing the next step;

(3) reacting the coupling resin in 20% pyridine at room temperature for 20min, removing the N-terminal amino protected by 9-Fluorenylmethoxycarbonyl (FMOC), and displaying blue color by ninhydrin reagent;

(4) adding DIEA, HBTU and FMOC-O-tert-butyl-L-threonine into the coupling resin, reacting for 1h at room temperature, detecting with ninhydrin reagent to obtain colorless solution, and performing the next operation;

(5) reacting the coupling resin in 20% pyridine at room temperature for 20min, removing the N-terminal amino protected by 9-Fluorenylmethoxycarbonyl (FMOC), and displaying blue color by ninhydrin reagent;

(6) adding DIEA, HBTU and (S) -4- (BOC-amino) -2- (FMOC-amino) butyric acid into the coupling resin, reacting for 1h at room temperature, detecting with ninhydrin reagent to obtain colorless solution, and performing the next step;

(7) reacting the coupling resin in 20% pyridine at room temperature for 20min, removing the N-terminal amino protected by 9-Fluorenylmethoxycarbonyl (FMOC), and displaying blue color by ninhydrin reagent;

(8) adding DIEA, HBTU and 6-methylheptanoic acid into the coupling resin, reacting for 1h at room temperature, detecting with ninhydrin reagent to obtain colorless, and cutting the resin to obtain crude peptide;

(9) and (3) after the crude peptide is purified by HPLC, vacuum freeze drying is carried out, and the hapten shown in the formula (I) is obtained.

In a third aspect, the present invention provides polymyxin B and colistin artificial antigens, which are obtained by coupling the above-mentioned polymyxin B and colistin haptens with a carrier protein;

wherein the carrier protein is selected from Bovine Serum Albumin (BSA), Ovalbumin (OVA), Keyhole Limpet Hemocyanin (KLH), Bovine Thyroglobulin (BTG), and Human Serum Albumin (HSA), preferably bovine serum albumin or ovalbumin.

The polymyxin B and colistin artificial antigen can be used as immunogen and also can be used as coating antigen.

In the invention, in the polymyxin B and colistin artificial antigens, the molar ratio of the polymyxin B and colistin haptens to the carrier protein is (8-9):1, and preferably 8.4: 1.

In a fourth aspect, the present invention provides the preparation method of the polymyxin B and the colistin artificial antigen, firstly, maleimide caproic acid is coupled to the amino group of the carrier protein by an active ester method, and then, maleimide group on the carrier protein is coupled to the sulfhydryl group of the polymyxin B and the colistin hapten by an active ester method.

As a specific embodiment, the present invention provides a method for preparing the artificial antigen, comprising the steps of:

(1) mixing maleimidocaproic acid, Ethyldimethylaminocarbodiimide (EDC) and N-hydroxysuccinimide (NHS), dissolving in N, N-Dimethylformamide (DMF), and stirring at room temperature for 24 h. The obtained maleimide NHS activated ester is obtained by rotary evaporation after column chromatography purification.

(2) Dissolving maleimide NHS activated ester with DMF, adding into carrier protein dissolved in CB solution, and stirring at room temperature for reaction for 24 h. The resulting product was dialyzed against 10mM PBS for 3 days with 3-4 changes per day.

(3) Dissolving the hapten shown in the formula (I), dropwise adding the hapten into a maleimide-coupled carrier protein solution, and stirring at room temperature for reacting for 24 hours. The resulting product was dialyzed against 10mM PBS for 3 days with 3-4 changes per day.

In a fifth aspect, the present invention provides the use of a polymyxin B and colistin hapten or a polymyxin B and colistin artificial antigen as described above in any one of:

(1) the application in preparing polymyxin B and colistin specific antibody;

(2) the application of the polypeptide in detecting polymyxin B and colistin specific antibodies.

In a sixth aspect, the invention provides polymyxin B and colistin-specific antibodies prepared by immunizing an animal with the above-described polymyxin B and colistin artificial antigens as immunogens; the polymyxin B and the colistin specific antibody are monoclonal antibodies or polyclonal antibodies.

Preferably, the artificial antigen is obtained by coupling the polymyxin B and colistin hapten with bovine serum albumin.

The monoclonal antibody provided by the invention is a broad-spectrum antibody and can be used for simultaneously detecting polymyxin B and colistin.

The invention also provides a hybridoma cell, namely an anti-polymyxin B and colistin monoclonal antibody hybridoma cell 5B 10. The monoclonal antibody secreted by the hybridoma cell 5B10 also belongs to the protection scope of the invention.

In a seventh aspect, the invention provides the use of polymyxin B and colistin-specific antibodies in any one of:

(1) the application in detecting polymyxin B and colistin;

(2) the application in the preparation of the polymyxin B and the immunochromatographic test strip of the colistin;

(3) the application in preparing an ELISA kit of polymyxin B and colistin;

(4) the application in the preparation of polymyxin B and colistin chemiluminescence kits.

In an eighth aspect, the present invention provides a detection reagent or a detection test strip comprising the above-mentioned polymyxin B and a colistin-specific antibody.

In a ninth aspect, the present invention provides a kit comprising polymyxin B as described above and an antibody specific for colistin.

The invention also provides a rapid, sensitive and broad-spectrum polymyxin B and colistin immunoassay method established based on the monoclonal antibody and the coating antigen.

Preferably, the coating antigen is obtained by coupling the polymyxin B and colistin hapten with ovalbumin.

The invention has the beneficial effects that:

the invention discloses a novel polymyxin B and colistin hapten, artificial antigen and a preparation method thereof for the first time. The polymyxin B, colistin hapten and artificial antigen provided by the invention and the antibody prepared by the polymyxin B and colistin hapten provide new material for establishing a rapid, simple, convenient, cheap, sensitive and specific method for detecting polymyxin B and colistin.

The polymyxin B and colistin hapten provided by the invention has a fixed coupling site, the prepared artificial antigen has the advantage of good uniformity, and the prepared monoclonal antibody has the advantages of high titer and high sensitivity, and can be used for establishing a rapid detection method for polymyxin B and colistin. The polymyxin B, the colistin hapten and the artificial antigen provided by the invention and the antibody prepared by the artificial antigen are suitable for blood concentration monitoring and veterinary drug residue analysis and detection, and have good application prospects.

Drawings

FIG. 1 is a mass spectrum of polymyxin B and colistin haptens of formula I.

FIG. 2 is a MALDI-TOF plot of polymyxin B and colistin immunogens of formula I.

FIG. 3 is a standard graph of polymyxin B and colistin antibodies.

FIG. 4 is a MALDI-TOF plot of polymyxin B and colistin immunogens of formula II.

Detailed Description

Preferred embodiments of the present invention will be described in detail with reference to the following examples. It is to be understood that the following examples are given for illustrative purposes only and are not intended to limit the scope of the present invention. Various modifications and alterations of this invention will become apparent to those skilled in the art without departing from the spirit and scope of this invention.

The experimental procedures used in the following examples are all conventional procedures unless otherwise specified. The materials, reagents and the like used in the following examples are commercially available (conventional biochemical reagents Co., Ltd.) unless otherwise specified.

The quantitative tests in the following examples, all set up three replicates and the results averaged. All PBS buffers used in the examples were 0.01M PBS buffer, pH 7.4. The carbonate buffers used in the examples were all 0.05mol/L sodium carbonate buffer at pH 9.6.

NHS is an abbreviation for N-hydroxysuccinimide. EDC is an abbreviation for 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride. N-hydroxysuccinimide, 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride were obtained from sigma. Bovine Serum Albumin (BSA) was purchased from sigma. Ovalbumin (Ovalbumin, OVA) was purchased from sigma. Freund's complete adjuvant and Freund's incomplete adjuvant were purchased from sigma.

EXAMPLE 1 preparation of polymyxin B and colistin haptens

Preparation of mono-, polymyxin B and colistin haptens

1. Preparation of polymyxin B and colistin haptens of formula I

(1) Firstly, amino and cysteine resin with protected side chain (FMOC-S-tributyl-L-cysteine 4-benzyl ester resin) are reacted for 20min at room temperature in 20% pyridine, 9-Fluorenylmethoxycarbonyl (FMOC) protected N-terminal amino is removed, and ninhydrin reagent tests color development;

(2) adding N, N-Diisopropylethylamine (DIEA), O-benzotriazole-tetramethyluronium Hexafluorophosphate (HBTU) and (S) -4- (BOC-amino) -2- (FMOC-amino) butyric acid into the coupling resin, reacting for 1h at room temperature, detecting with ninhydrin reagent to obtain colorless solution, and performing the next step;

(3) reacting the coupling resin in 20% pyridine at room temperature for 20min, removing the N-terminal amino protected by 9-Fluorenylmethoxycarbonyl (FMOC), and displaying blue color by ninhydrin reagent;

(4) adding DIEA, HBTU and FMOC-O-tert-butyl-L-threonine into the coupling resin, reacting for 1h at room temperature, detecting with ninhydrin reagent to obtain colorless solution, and performing the next operation;

(5) reacting the coupling resin in 20% pyridine at room temperature for 20min, removing the N-terminal amino protected by 9-Fluorenylmethoxycarbonyl (FMOC), and displaying blue color by ninhydrin reagent;

(6) adding DIEA, HBTU and (S) -4- (BOC-amino) -2- (FMOC-amino) butyric acid into the coupling resin, reacting for 1h at room temperature, detecting with ninhydrin reagent to obtain colorless solution, and performing the next step;

(7) reacting the coupling resin in 20% pyridine at room temperature for 20min, removing the N-terminal amino protected by 9-Fluorenylmethoxycarbonyl (FMOC), and displaying blue color by ninhydrin reagent;

(8) adding DIEA, HBTU and 6-methylheptanoic acid into the coupling resin, reacting for 1h at room temperature, detecting with ninhydrin reagent to obtain colorless, and cutting the resin to obtain crude peptide;

(9) after the crude peptide is purified by HPLC, the hapten shown in the formula (I) is obtained by vacuum freeze drying and is named as PMB-A.

Characterization of the polymyxin B and colistin haptens

1. Identification by mass spectrometry

Step one polymyxa shown as formula IA colistin B and colistin hapten (molecular formula: C)₂₃H₄₄N₆O₇S) mass spectrum identification result: MS M/z [ M + H ]]⁺Theoretical value: 549.3; measured value: 549.4, which is matched with the molecular weight of the target product, and the mass spectrum is shown in figure 1.

Example 2 preparation and characterization of polymyxin B and colistin Artificial antigens

The immunogen and the coating antigen described in this example were prepared by methods that differ in the type of carrier protein used, BSA was used as the carrier protein for the immunogen, and OVA was used as the carrier protein for the coating antigen.

Synthesis and characterization of Mono-, polymyxin B and colistin immunogens

1. Preparation of polymyxin B and colistin immunogens

(1) 200mg of maleimidocaproic acid, 272mg of EDC and 163mg of NHS are weighed, mixed and dissolved in 2mL of DMF solvent, stirred at room temperature at 300r/min and reacted for 24 h. The obtained maleimide NHS activated ester is obtained by rotary evaporation after column chromatography purification.

(2) 5.5mg of maleimide NHS activated ester was weighed, dissolved in 500. mu.L of DMF, and added to 20mg of BSA protein dissolved in 5mL of CB solution, stirred at room temperature at 300r/min, and reacted for 24 hours. The resulting reaction solution was dialyzed against 5L of 10mM PBS for 3 days, and the solution was changed 3 to 4 times a day.

(3) 9.8mg of the hapten of the formula (I) prepared in example 1 was weighed, dissolved in 500. mu.L of DMF, added dropwise to the solution of maleimide-coupled BSA protein, stirred at 300r/min at room temperature and reacted for 24 h. The reaction solution was dialyzed against 10mM PBS for 3 days, and the solution was changed 3 to 4 times a day. The resulting antigen was named PMB-A-BSA.

2. Identification of polymyxin B and colistin immunogens

The binding ratio of BSA to hapten in PMB-A-BSA solution was determined by Matrix-Assisted Laser Desorption/Ionization Time of Flight Mass Spectrometry (MALDI-TOF-MS). The results are shown in FIG. 2.

Binding ratio { M (conjugate) -M (protein) }/M (hapten)

The molecular weight of BSA was 64963.3, the molecular weight of hapten of formula I was 548.4, and the molecular weight of the conjugate was 69577.6, the binding ratio of BSA to hapten was calculated to be 8.4, i.e., 8.4 haptens were coupled on average on one BSA molecule in PMB-A-BSA. The resulting coating antigen was named PMB-A-BSA.

Synthesis of di-and polymyxin B and colistin coatingen

1. Preparation of polymyxin B and colistin coatingen

OVA was used instead of BSA, as in step one of this example. The resulting immunogen was designated PMB-A-OVA.

EXAMPLE 3 preparation of polymyxin B and colistin monoclonal antibodies

First, animal immunization

The PMB-A-BSA solution (concentration of 2.3mg/mL) prepared in example 2 was used to immunize 8 Balb/c female mice, each of which was 100. mu.g each, in a single immunization, 4 times with three-week intervals, in a manner of subcutaneous multi-point injection at the back of the neck, and an additional intraperitoneal immunization was performed 4 days before the fusion.

Second, cell fusion and cloning

1. The mice with the best antiserum performance were selected for cell fusion experiments.

2. After 4 days of intraperitoneal immunization, splenocytes are taken and fused with SP2/0 myeloma cells according to the ratio of 10:1 (quantitative ratio), cell supernatants are measured by an ICELISA, and positive holes and competitive holes are screened.

3. And cloning the cell hole with the best rank by using a limiting dilution method to obtain a hybridoma cell strain capable of secreting polymyxin B and colistin monoclonal antibodies. A hybridoma cell strain is named as anti-polymyxin B and colistin monoclonal antibody hybridoma cell 5B10 respectively.

Thirdly, freezing and recovering cells

Making hybridoma cell 5B10 into 1 × 10 with frozen stock solution⁶Cell suspension per mL, preserved for long period in liquid nitrogen. During recovery, the cryopreservation tube preserved in liquid nitrogen is taken out, immediately placed into a water bath at 37 ℃ for unfreezing, centrifuged to remove the cryopreservation liquid, and then transferred into a culture dish for culture.

Preparation and purification of monoclonal antibody

1. Preparation of ascites

4 12-week-old Balb/c mice were injected intraperitoneally with sterile paraffin oil (0.4 mL/mouse). Hybridoma cell 5B10 (5X 10) was intraperitoneally injected 7 days later⁵One/only). Ascites was collected 7 days later.

2. Antibody purification

Purifying by Protein A immunoaffinity column method, and storing the purified antibody at-20 deg.C.

Fifth, identification of monoclonal antibody

And (3) respectively identifying the monoclonal antibody solution obtained in the step (2) as follows:

1. determination of the potency of monoclonal antibodies by indirect ELISA

(1) PMB-A-OVA is used as a coating antigen to coat the ELISA plate

PMB-A-OVA solutions prepared in example 2 (diluted with carbonate buffer) were used for coating, diluted in a series of concentrations, one row per concentration, 100. mu.L/well.

(2) Incubate at 37 ℃ for 2 h.

(3) Add 100. mu.L/well blocking solution and incubate at 37 ℃ for 1 h. And (5) washing the plate.

(4) mu.L of monoclonal antibody solution (diluted with PBS buffer) was added to each well at a series of concentrations.

(5) Incubate for 30min at room temperature and wash the plate.

(6) mu.L of horseradish peroxidase-labeled goat anti-mouse IgG was added to each well and incubated at room temperature for 30 min. And (5) washing the plate.

(7) Adding 100 μ L of TMB color developing solution, and developing in dark for 15 min.

(8) Adding 50 mu L of 2mol/L sulfuric acid to stop the reaction; read OD₄₅₀The value is obtained.

And (4) judging the result: by OD₄₅₀The corresponding antibody dilution at a value of 1.6 was initially taken as the titer of the antibody.

As a result: the titer of the antibody detected by an indirect ELISA method is 1: 160000.

3. Calculation of monoclonal antibody sensitivity

(1) PMB-A-OVA solution prepared in example 2 (diluted with carbonate buffer, 2.1mg/mL) was used for coating at 100. mu.L/well.

(2) Incubate at 37 ℃ for 2 h.

(3) Add 100. mu.L/well blocking solution and incubate at 37 ℃ for 1 h. And (5) washing the plate.

(4) mu.L of PMB or PME standard solution (consisting of PMB or PME and PBS buffer; concentration of PMB or PME is 0ng/mL, 0.3ng/mL, 0.9ng/mL, 2.7ng/mL, 8.1ng/mL, 24.3ng/mL, respectively; wells to which only PBS buffer was added are control wells) was added to each well, 3 replicate wells were set for each concentration.

(5) mu.L of monoclonal antibody solution (20 ng/mL) was added to each well. Incubate for 30min at room temperature and wash the plate.

(6) Add 100. mu.L of goat anti-mouse IgG labeled with horseradish peroxidase to each well, wash the plate for 30 min.

(7) Adding TMB color development solution, and developing in dark for 15 min.

(8) Adding 50 mu L of 2mol/L sulfuric acid into each hole to stop reaction; read OD₄₅₀The value is obtained.

Using a-log 10 (standard substance concentration) value as an abscissa and an OD value as an ordinate, fitting by using a four-parameter equation of Origin 8.5, and establishing a standard curve to obtain IC₅₀Values, as shown in fig. 3. Sensitivity of monoclonal antibodies to detect polymyxin B (IC)₅₀Value) of 2.2 ng/mL, sensitivity for colistin detection (IC)₅₀Value) was 2.8 ng/mL.

Comparative experiment 1

The control experiment was performed first using the hapten of formula II below for antigen preparation.

The hapten shown in the formula II is polymyxin B sulfate, has the CAS number of 1405-20-5 and is purchased from Shanghai-sourced leaf biotechnology limited.

The preparation process of the immunogen and the coating antigen is as follows:

after 39.7mg of the hapten shown in the formula II, 50mg of BSA or OVA and 1mL of ultrapure water were dissolved, 68. mu.L of a 5% glutaraldehyde solution was added, and the mixture was stirred at 300r/min at room temperature and reacted for 20 min. The reaction solution was dialyzed against 10mM PBS for 3 days, and the solution was changed 3 to 4 times a day. The resulting antigen was named PMB-GA-BSA/OVA.

The identification result of the immunogen MALDI-TOF-MS is shown in figure 4, and the calculated coupling ratio is as follows: r ═ 10.5.

The molecular weight of BSA was 64963.3, the molecular weight of hapten of formula II was 1169.5, and the molecular weight of the conjugate was 77199.6, calculated as the binding ratio of BSA to hapten was 10.5.

8 Balb/c female mice with the age of 6-8 weeks were immunized with 100. mu.g each mouse at a single time, 4 times in total, three weeks apart, by subcutaneous multi-point injection at the back of the neck, 7 days after the fourth immunization, and the tail veins of the mice were sampled, and the serum titer and the inhibitory effect were measured by indirect ELISA method. The specific method comprises the following steps:

1. mouse serum sample treatment

Respectively collecting tail vein serum of mice, mixing the mouse serum collected by 8 mice, collecting the mixture into a 1.5mL EP tube, centrifuging the mixture at 4 ℃ for 15min at 1000r/min, and taking out supernate, namely the mouse polyclonal antibody, wherein PMB-GA-BSA and PMB-A-BSA immunized mice are respectively named as pAb-GA and pAb-A.

2. Measurement of the potency of polyclonal antibodies by indirect ELISA

(1) PMB-GA-OVA and PMB-A-OVA are used as coating antigens to coat the enzyme label plate, and pAb-GA and pAb-A are respectively detected.

The PMB-A-OVA solution prepared in example 2 (diluted with carbonate buffer) and the PMB-GA-OVA prepared above (diluted with carbonate buffer) were coated separately in a series of concentrations, one line per concentration, 100. mu.L/well.

(2) Incubate at 37 ℃ for 2 h.

(3) Add 100. mu.L/well blocking solution and incubate at 37 ℃ for 1 h. And (5) washing the plate.

(4) 100 μ L of polyclonal antibody solution (diluted with PBS buffer) was added to each well at serial concentrations.

(5) Incubate for 30min at room temperature and wash the plate.

(6) mu.L of horseradish peroxidase-labeled goat anti-mouse IgG was added to each well and incubated at room temperature for 30 min. And (5) washing the plate.

(7) Adding 100 μ L of TMB color developing solution, and developing in dark for 15 min.

(8) Adding 50 mu L of 2mol/L sulfuric acid to stop the reaction; read OD₄₅₀The value is obtained.

And (4) judging the result: by OD₄₅₀The corresponding antibody dilution at a value of 1.6 was initially taken as the titer of the antibody.

As a result: the titers of the polyclonal antibodies pAb-GA and pAb-A detected by an indirect ELISA method are 1:1000 and 1:20000 respectively.

3. Detection of inhibitory Effect of polyclonal antibody

(1) The above PMB-GA-OVA and PMB-A-OVA solutions (diluted with carbonate buffer to a final concentration of 2. mu.g/mL) were used for coating at 100. mu.L/well.

(2) Incubate at 37 ℃ for 2 h.

(3) Add 100. mu.L/well blocking solution and incubate at 37 ℃ for 1 h. And (5) washing the plate.

(4) mu.L of PMB or PME standard solution (consisting of PMB or PME and PBS buffer; concentration of PMB or PME is 0ng/mL, 3ng/mL, 9ng/mL, 27ng/mL, 81ng/mL, 243ng/mL, respectively; wells with PBS buffer added only are control wells) was added to each well, 3 duplicate wells were set for each concentration.

(5) Add 50. mu.L of polyclonal antibody solution to each well. Incubate for 30min at room temperature and wash the plate.

(6) Add 100. mu.L of goat anti-mouse IgG labeled with horseradish peroxidase to each well, wash the plate for 30 min.

(7) Adding TMB color development solution, and developing in dark for 15 min.

(8) Adding 50 mu L of 2mol/L sulfuric acid into each hole to stop reaction; read OD₄₅₀The value is obtained.

Using a-log 10 (standard substance concentration) value as an abscissa and an OD value as an ordinate, fitting by using a four-parameter equation of Origin 8.5, and establishing a standard curve to obtain IC₅₀The value is obtained. IC detection of polymyxin by polyclonal antibody pAb-GA₅₀pAb-A IC for polymyxin at a value of 95ng/mL₅₀The value was 21 ng/mL.

As can be seen from the above, the antiserum antibody titer obtained from the immunogen obtained from the hapten of formula I is 20 times (20000:1000) higher than that obtained from the immunogen immune antiserum obtained from the hapten coupling of formula II, and the immune antiserum antibody IC obtained from the immunogen obtained from the hapten coupling of formula I₅₀Less than 4.5 fold (21 ng/mL: 95ng/mL) of the immunogen immune antiserum from the hapten coupling of formula II.

Although the invention has been described in detail hereinabove with respect to a general description and specific embodiments thereof, it will be apparent to those skilled in the art that modifications or improvements may be made thereto based on the invention. Accordingly, such modifications and improvements are intended to be within the scope of the invention as claimed.