A rapid detection method for fluoroquinolone residues based on a two-antibody sandwich

Abstract

The application discloses a rapid detection method for fluoroquinolone drug residues based on a one-antigen two-monoclonal antibody. The application discloses a monoclonal antibody NOR McAb and a monoclonal antibody SAR McAb respectively against the fluoroquinolone drugs norfloxacin NOR and sarafloxacin SAR, a kit containing the monoclonal antibody NOR McAb and the monoclonal antibody SAR McAb, a method for detecting the fluoroquinolone drugs, and application of the monoclonal antibody NOR McAb and the monoclonal antibody SAR McAb and the kit in detection of the fluoroquinolone drugs. The application is based on a coating agent (NOR-1-BSA) and the monoclonal antibody against sarafloxacin and norfloxacin, establishes an indirect competitive ELISA method and a competitive lanthanide fluorescence microsphere lateral flow immunochromatography test strip (LFM-ICS) rapid detection technology for simultaneously detecting at least 13 kinds of fluoroquinolone drugs, and is simple in production and simple, convenient and efficient in detection.

Description

TECHNICAL FIELD

[0001] The application belongs to the field of drug detection, and particularly relates to a rapid detection method for fluoroquinolone drug residues based on a one-antigen two-monoclonal antibody. BACKGROUND

[0002] Fluoroquinolones (FQs) are a class of synthetic broad-spectrum antibacterial drugs, which have been widely used in livestock and poultry, aquaculture and other breeding industries. They mainly target the synthesis of bacterial DNA gyrase and topoisomerase IV, hinder the synthesis and replication of bacterial DNA, and thus play a role in killing bacteria. Six FQs drugs have been approved for use in animals in the United States, including enrofloxacin, difloxacin, danofloxacin, marbofloxacin, orbifloxacin and sarafloxacin. Fluoroquinolones (FQs) have been applied to the prevention and treatment of aquatic diseases in China since the 1970s.

[0003] FQs drugs all have a 4-quinolone nucleus structure, a fluorine atom connected to C-6, and a piperazine ring at C-7. From the perspective of molecular structure, FQs can be divided into two subcategories according to whether there is a para-fluorophenyl group at N1 position, i.e. sarafloxacin and difloxacin with benzene ring structure, and norfloxacin, perfloxacin, ofloxacin, ciprofloxacin without benzene ring structure.

[0004] Fluoroquinolone residues caused by the irrational use or misuse of veterinary drugs can directly harm human health through the food chain, produce gastrointestinal adverse reactions such as nausea, vomiting, diarrhea and eye toxicity, have the risk of causing cancer, teratogenicity and mutagenicity, induce pathogenic bacteria in humans to develop drug resistance, cause cross-drug resistance, affect clinical drug use and new drug development, cause environmental pollution, etc. Many countries and regions around the world pay great attention to the detection and monitoring of quinolone drug residues in animal-derived foods, and have issued relevant regulations to prohibit or set maximum residue limits (MRLs). In 2002, China stipulated that the MRLs of ciprofloxacin, norfloxacin, enrofloxacin, sarafloxacin, difloxacin, oxolinic acid and flumequine in animal muscle tissue were 10-500 μg / kg, and in 2015, the Ministry of Agriculture of China issued Announcement No. 2292 to stop using lomefloxacin, perfloxacin, ofloxacin and norfloxacin as veterinary drugs in food animals.

[0005] In recent years, the detection techniques of veterinary drug residues mainly include microbial method, instrument detection method and immune analysis method. The microbial method has low sensitivity and poor specificity; the instrument detection method mainly includes chromatography, mass spectrometry-based high performance liquid chromatography, high performance liquid chromatography-tandem mass spectrometry and other instrument analysis methods, which can accurately detect whether the drug to be detected is contained and has high sensitivity, but the sample pretreatment is complex, the equipment is expensive, and professional personnel are needed for operation, which is not suitable for rapid detection of a large number of samples on site; the immune analysis method has the advantages of high specificity and no long sample pretreatment, so that rapid, simple and high-throughput detection of samples can be realized, and is widely used for screening of a large number of samples to be detected. Commonly used methods include enzyme-linked immunosorbent assay (ELISA), chemiluminescent enzyme immunoassay (CLEIA), radioimmunoassay (RIA), immunochromatography assay (ICS) and the like.

[0006] Among them, the immunochromatography assay is established on the basis of antigen-antibody specific reaction and chromatography technology, and is the most commonly used on-site testing technology at present. The colloidal gold test strip (GICA) using colloidal gold as a marker has relatively low sensitivity and cannot be quantified, and is only suitable for qualitative detection of high-concentration target compounds. The lanthanide fluorescence microsphere immunochromatography test strip (LFM-ICS) using polystyrene microspheres coated with lanthanide ion complex as a marker has higher sensitivity and can realize quantitative detection, has the advantages of long fluorescence lifetime and large Stocks displacement, and the method has been widely used for drug residue detection.

[0007] In addition, the method for detecting fluoroquinolones in the prior art cannot simultaneously detect the two subcategories of drugs with benzene ring structure and drugs without benzene ring structure.

[0008] Therefore, in order to protect the last line of defense of food safety, it is necessary to develop a rapid, sensitive and practical detection technology. SUMMARY

[0009] In order to overcome the defects of the prior art that the operation of detecting fluoroquinolones is complex, the sensitivity is low, and both sub-classes of drugs cannot be considered, considering the similarity of the structure of fluoroquinolones and the cross reaction of antigen and antibody, the application formulates a quantitative calculation rule of mixed samples of SAR and NOR by using a coating original (NOR-1-BSA) and two kinds of monoclonal antibodies (SAR McAb and NOR McAb), establishes an ELISA method and an LFM-ICS method for semi-quantitative detection of 13 kinds of fluoroquinolones, and provides a new idea for simultaneous detection of multiple fluoroquinolone residues in animal-derived food.

[0010] To solve the above technical problems, one of the technical solutions provided by the application is a monoclonal antibody NOR McAb against fluoroquinolone NOR, which comprises a heavy chain variable region and a light chain variable region, the heavy chain variable region comprises HCDR1, HCDR2 and HCDR3, and the light chain variable region comprises LCDR1, LCDR2 and LCDR3; the HCDR1 amino acid sequence of the heavy chain variable region of the monoclonal antibody NOR McAb is shown in SEQ ID NO: 5, the HCDR2 amino acid sequence is shown in SEQ ID NO: 6, and the HCDR3 amino acid sequence is shown in SEQ ID NO: 7; and the LCDR1 amino acid sequence of the light chain variable region of the monoclonal antibody NOR McAb is shown in SEQ ID NO: 8, the LCDR2 amino acid sequence is shown in SEQ ID NO: 9, and the LCDR3 amino acid sequence is shown in SEQ ID NO: 10.

[0011] In a preferred embodiment of the application, the amino acid sequence of the heavy chain variable region of the monoclonal antibody NOR McAb is shown in SEQ ID NO: 11, and / or the amino acid sequence of the light chain variable region of the monoclonal antibody NOR McAb is shown in SEQ ID NO: 12.

[0012] In a specific embodiment of the application, the amino acid sequence of the heavy chain variable region of the monoclonal antibody NOR McAb is shown in SEQ ID NO: 3, and / or the amino acid sequence of the light chain variable region of the monoclonal antibody NOR McAb is shown in SEQ ID NO: 4.

[0013] In a more preferred embodiment of the application, the heavy chain constant region of the monoclonal antibody NOR McAb is derived from the gamma chain of a murine antibody, and / or the light chain constant region of the monoclonal antibody NOR McAb is derived from the lambda chain of a murine antibody.

[0014] To solve the above technical problems, the second technical scheme of the present application provides a monoclonal antibody SARMcAb against SAR of fluoroquinolone drugs, which comprises a heavy chain variable region and a light chain variable region, the heavy chain variable region comprises HCDR1, HCDR2 and HCDR3, and the light chain variable region comprises LCDR1, LCDR2 and LCDR3; the HCDR1 amino acid sequence of the heavy chain variable region of the monoclonal antibody SARMcAb is shown in SEQ ID NO: 17, the HCDR2 amino acid sequence is shown in SEQ ID NO: 18, and the HCDR3 amino acid sequence is shown in SEQ ID NO: 19, and the LCDR1 amino acid sequence of the light chain variable region of the monoclonal antibody SARMcAb is shown in SEQ ID NO: 20, the LCDR2 amino acid sequence is shown in SEQ ID NO: 9, and the LCDR3 amino acid sequence is shown in SEQ ID NO: 21.

[0015] In a preferred embodiment of the present application, the amino acid sequence of the heavy chain variable region of the monoclonal antibody SARMcAb is shown in SEQ ID NO: 22, and / or the amino acid sequence of the light chain variable region of the monoclonal antibody SARMcAb is shown in SEQ ID NO: 23.

[0016] In a specific embodiment of the present application, the amino acid sequence comprising the heavy chain variable region of the monoclonal antibody NORMcAb is shown in SEQ ID NO: 15, and / or the amino acid sequence comprising the light chain variable region of the monoclonal antibody NORMcAb is shown in SEQ ID NO: 16.

[0017] In a more preferred embodiment of the present application, the heavy chain constant region of the monoclonal antibody SARMcAb is derived from the gamma chain of a murine antibody, and / or the light chain constant region of the monoclonal antibody SARMcAb is derived from the lambda chain of a murine antibody.

[0018] In the present application, the amino acid sequences of the CDRs listed above are shown according to the Kabat definition. However, it is well known in the art that the CDRs of an antibody can be defined in various ways in the art, such as Chothia based on the three-dimensional structure of the antibody and the topology of the CDR loops (Chothia et al. (1989) Nature 342: 877-883, Al-Lazikani et al., "Standard conformations for the canonical structures of immunoglobulins", Journal of Molecular Biology, 273, 927-948 (1997)), Kabat based on the variability of the antibody sequence (Kabat et al., Sequences of Proteins of Immunological Interest, 4th Ed., U.S. Department of Health and Human Services, National Institutes of Health (1987)), AbM (University of Bath), Contact (University College London), the international ImMunoGeneTics database (IMGT) (World Wide Web at imgt.cines.fr / ), and North CDR definition based on affinity propagation clustering using a large number of crystal structures. It will be understood by those skilled in the art that, unless otherwise specified, the terms "CDR" and "complementarity determining region" of a given antibody or region thereof (e.g., a variable region) are to be understood as encompassing complementarity determining regions as defined by any of the above known approaches described in the present application. Although the scope of the present application is based on the sequences shown according to the Kabat definition, the amino acid sequences corresponding to the other CDR definitions should also fall within the scope of the present application.

[0019] To solve the above technical problems, the third technical solution of the present application provides an isolated nucleic acid, which encodes the monoclonal antibody NOR McAb according to the first technical solution or the monoclonal antibody SARMcAb according to the second technical solution.

[0020] As known in the art, "nucleic acid" in the present application refers to a chain of nucleotides of any length and includes DNA and RNA. The nucleotides can be deoxyribonucleotides, ribonucleotides, modified nucleotides or bases, and / or their analogs, or any substrate that is capable of being incorporated into a chain by DNA or RNA polymerase.

[0021] In a preferred embodiment of the present application, the sequence of the nucleic acid encoding the heavy chain variable region of the monoclonal antibody NOR McAb is as set forth in SEQ ID NO: 1, and / or, the sequence of the nucleic acid encoding the light chain variable region of the monoclonal antibody NOR McAb is as set forth in SEQ ID NO: 2; or, the sequence of the nucleic acid encoding the heavy chain variable region of the monoclonal antibody S AR McAb is as set forth in SEQ ID NO: 13, and / or, the sequence of the nucleic acid encoding the light chain variable region of the monoclonal antibody S AR McAb is as set forth in SEQ ID NO: 14.

[0022] To solve the above technical problems, the technical solution four provided by the present application is a recombinant expression vector, wherein the recombinant expression vector comprises the nucleic acid according to the technical solution three.

[0023] As known in the art, the recombinant expression vector of the present application can be any suitable recombinant expression vector that is capable of being used to transform or transfect to deliver one or more genes or sequences of interest into any suitable host cell and preferably express the genes or sequences in the host cell. Suitable vectors include those designed for expansion and amplification or for expression or both, examples of vectors include but are not limited to viral vectors, naked DNA or RNA expression vectors, plasmids, cosmids or bacteriophage vectors, DNA or RNA expression vectors associated with cationic condensing agents, DNA or RNA expression vectors encapsulated in liposomes and certain eukaryotic cells, such as producer cells.

[0024] To solve the above technical problems, the technical solution five provided by the present application is a transformant, wherein the transformant comprises the nucleic acid according to the technical solution three or the recombinant expression vector according to the technical solution four, and the host cell of the transformant is a prokaryotic cell or a eukaryotic cell.

[0025] As known in the art, the term "host cell" refers to any type of cell that can contain the nucleic acid or vector described in the present application. In exemplary aspects, the host cell is a eukaryotic cell, such as a plant, animal, fungus or seaweed; or can be a prokaryotic cell, such as a bacterium or protozoan.

[0026] To solve the above technical problems, the sixth technical solution of the present application provides a kit for detecting fluoroquinolones, wherein the kit comprises a first antibody, and the first antibody is the monoclonal antibody NOR McAb according to the first technical solution and / or the monoclonal antibody SAR McAb according to the second technical solution.

[0027] The kit according to the sixth technical solution further comprises a coating antigen, wherein the coating antigen is a complete antigen prepared by coupling a hapten NOR and a carrier protein; and / or the kit further comprises a second antibody, wherein the second antibody can specifically bind to the first antibody.

[0028] In a preferred embodiment of the present application, the carrier protein is bovine serum albumin.

[0029] In a more preferred embodiment of the present application, the coating antigen is NOR-1-BSA, wherein the NOR-1-BSA is an aminoethyl connected to a nitrogen atom of a piperazine ring of the hapten NOR and coupled with the bovine serum albumin.

[0030] The kit according to the sixth technical solution, wherein the second antibody is labeled with an enzyme, and the kit further comprises an ELISA coating solution, an ELISA blocking solution, an ELISA substrate developing solution and / or an ELISA termination solution, and the ELISA substrate developing solution comprises ELISA substrate developing solution A and ELISA substrate developing solution B in a volume ratio of 1:1.

[0031] In a preferred embodiment of the present application, the second antibody is labeled with horseradish peroxidase, and / or the mother liquor concentration of the second antibody is 0.8 mg / mL to 1.2 mg / mL, and the dilution ratio is 1:18000 to 1:22000, wherein the dilution ratio is a volume ratio of the mother liquor to 1xPBST containing FBS, and / or the ELISA coating solution is a carbonate buffer solution with a pH of 9.4 to 9.8 and a concentration of 0.04 mol / L to 0.06 mol / L, and / or the ELISA blocking solution is gelatin with a mass percentage of 0.8% to 1.2%, and / or the ELISA substrate developing solution A comprises 25 g / L to 30 g / L sodium acetate, 3.0 g / L to 3.5 g / L citric acid and 0.5 mL / L to 0.7 mL / L 30% H2O2, and / or the ELISA substrate developing solution B comprises 0.25 g / L to 0.35 g / L TMB, 0.3 g / L to 0.5 g / L EDTA-2Na, 1.8 g / L to 2.0 g / L citric acid and 90 mL / L to 110 mL / L glycerol, and / or the ELISA termination solution is 1.8 mol / L to 2.2 mol / L sulfuric acid.

[0032] In a more preferable embodiment of the present application, the second antibody is goat anti-mouse IgG-HRP, and / or the dilution ratio of the second antibody is 1:20000, and / or the volume percentage of the FBS is 5%, and / or the ELISA coating solution is a carbonate buffer with a pH of 9.6 and a concentration of 0.05 mol / L, and / or the ELISA blocking solution is 1% gelatin by mass percentage, and / or the ELISA substrate developing solution A contains 27.2 g / L sodium acetate, 3.2 g / L citric acid and 0.3 mL / L 30% H2O2, and / or the ELISA substrate developing solution B contains 0.3 g / L TMB, 0.4 g / L EDTA-2Na, 1.9 g / L citric acid and 100 mL / L glycerol, and / or the ELISA termination solution is 2 mol / L sulfuric acid.

[0033] The kit according to the sixth aspect, wherein the concentration of the coating antigen is 0.25 μg / mL to 0.10 μg / mL; and / or, the concentration of the antibody stock solution of the NOR McAb or the SAR McAb is 5.0 mg / mL to 8.0 mg / mL, the dilution ratio of the antibody of the SAR McAb is 1:800000 to 1:1200000, and / or the dilution ratio of the antibody of the NOR McAb is 1:600000 to 1:1000000; the dilution ratio is the volume ratio of the antibody stock solution to 1xPBST containing FBS.

[0034] In a preferred embodiment of the present application, the concentration of the coating antigen is 0.125 μg / mL; and / or, the dilution ratio of the antibody of the SAR McAb is 1:1200000, the dilution ratio of the antibody of the NOR McAb is 1:600000, and / or the volume percentage of the FBS is 5%.

[0035] The kit according to the sixth aspect, wherein the first antibody is coupled with a label, and the coating antigen and the second antibody are respectively fixed on a test strip, the coating antigen is fixed on the T line of the test strip, and the second antibody is fixed on the C line of the test strip; in a preferable embodiment of the present application:

[0036] The test strip is a test strip in which an NC membrane, a water absorption pad and a sample pad are sequentially attached to a PVC base plate.

[0037] The first antibody is coupled with lanthanide fluorescent microspheres to obtain an antibody microsphere conjugate LFM-mAb.

[0038] The second antibody is goat anti-mouse IgG, and the concentration thereof is preferably 0.4 mg / mL to 0.6 mg / mL, and more preferably 0.5 mg / mL; and / or,

[0039] The coating agent is NOR-1-BSA, preferably at a concentration of 0.05-0.4 mg / mL, more preferably 0.2 mg / mL.

[0040] The kit as claimed in the sixth aspect, wherein the preparation method of the LFM-mAb comprises the following steps:

[0041] (1) dilute the lanthanide fluorescent microspheres with MES buffer and ultrasonic dispersion;

[0042] (2) after adding NHS and EDC for activation, continue to activate by adding MES buffer, ultrasonic dispersion, centrifugal separation to discard the supernatant, and collect the precipitate;

[0043] (3) resuspend the precipitate in step (2) with a boric acid buffer containing a H3BO3 solution and a borax solution;

[0044] (4) add the NOR McAb or the SAR McAb for coupling, mix, centrifugal separation to discard the supernatant, and collect the precipitate;

[0045] (5) resuspend the precipitate in step (4) with a microsphere blocking solution containing 6-7 g / L BSA, 1.5-2.5 g / L NaN3 and 5.8-6.2 g / L Tris, pH 7.5-8.5, for blocking, then mix, centrifugal separation to discard the supernatant, and collect the precipitate;

[0046] (6) add a microsphere diluent to the precipitate in step (5), ultrasonic mixing to obtain LFM-SAR mAb or LFM-NOR mAb, the microsphere diluent containing 6-7 g / L BSA, 1.5-2.5 g / L NaN3, 5.8-6.2 g / L Tris and 6-7 g / L PVP, pH 7.5-8.5.

[0047] In the preferred technical solution of the present application, the preparation method comprises the following steps:

[0048] (1) dilute the lanthanide fluorescent microspheres with 23-27 mmol / L MES buffer and ultrasonic dispersion, the mass / volume ratio being 0.8-1.2%.

[0049] (2) after adding 15-17 mg / mL NHS and 7-9 mg / mL EDC for activation, continue to activate by adding 23-27 mmol / L MES buffer, ultrasonic dispersion, centrifugal separation to discard the supernatant, and collect the precipitate.

[0050] (3) resuspend the precipitate in step (2) with a boric acid buffer containing a 0.08-0.12 mol / L H3BO3 solution and a 23-27 mmol / L borax solution.

[0051] (4) adding the NOR McAb or the SAR McAb with a concentration of 0.8 mg / mL to 1.2 mg / mL, mixing, centrifuging, discarding the supernatant, and collecting the precipitate.

[0052] (5) resuspending the precipitate in step (4) with a microsphere blocking solution with a pH of 7.5 to 8.5, blocking, mixing, centrifuging, discarding the supernatant, and collecting the precipitate, wherein the microsphere blocking solution contains 6 to 7 g / L BSA, 1.5 to 2.5 g / L NaN3, and 5.8 to 6.2 g / L Tris.

[0053] (6) adding a microsphere dilution solution with a pH of 7.5 to 8.5 to the precipitate in step (5), mixing, and obtaining LFM-SAR mAb or LFM-NOR mAb, wherein the microsphere dilution solution contains 6 to 7 g / L BSA, 1.5 to 2.5 g / L NaN3, 5.8 to 6.2 g / L Tris, and 6 to 7 g / L PVP.

[0054] Preferably, the concentration of the MES buffer is 25 mmol / L; the mass-volume ratio of the lanthanide fluorescent microspheres is 1%; the concentration of the NHS is 16 mg / mL; the concentration of the EDC is 8 mg / mL; the concentration of the H3BO3 solution is 0.1 mol / L; the concentration of the borax solution is 25 mmol / L; the volume ratio of the H3BO3 solution to the borax solution in the boric acid buffer is 2:3; the concentration of the NOR McAb or the SAR McAb is 1.0 mg / mL; the pH of the microsphere blocking solution is 8.0; the microsphere blocking solution contains 5 g / L BSA, 2 g / L NaN3, and 6.05 g / L Tris; the pH of the microsphere dilution solution is 8.0; and the microsphere dilution solution contains 5 g / L BSA, 2 g / L NaN3, 6.05 g / L Tris, and 5 g / L PVP.

[0055] To solve the above technical problems, a seventh technical solution of the present application provides a method for detecting fluoroquinolones, which comprises using the monoclonal antibody NOR McAb according to the first technical solution and / or the monoclonal antibody SAR McAb according to the second technical solution; or the method comprises using the kit according to the sixth technical solution.

[0056] The method according to the seventh technical solution comprises the following steps:

[0057] (1) the coating agent is diluted with ELISA coating solution, coated in an enzyme-labeled plate, and the plate is washed with sample diluent, which is 1xPBS containing 0.4mL / L-0.6mL / L Tween-20, pH 7.2-7.6; preferably, the sample diluent is 1xPBS containing 0.5mL / L Tween-20, pH 7.4;

[0058] (2) ELISA blocking solution is added, incubated, and then the plate is washed with sample diluent;

[0059] (3) the sample to be tested and the diluted monoclonal antibody NOR McAb or the monoclonal antibody SARMcAb are added, incubated, and then the plate is washed with sample diluent;

[0060] (4) a second antibody labeled with an enzyme is added, incubated, and then the plate is washed with sample diluent; ELISA color developing solution is added for color development; ELISA termination solution is added for termination;

[0061] (5) detection is performed with an enzyme-labeled instrument, and the absorbance value is read; the read absorbance value is substituted into the SAR standard curve and the NOR standard curve, respectively, to calculate the theoretical concentration S and the theoretical concentration N, and the total theoretical concentration of fluoroquinolone drugs in the sample to be tested is S+90%N.

[0062] The method as claimed in claim 7, comprising the following steps:

[0063] (1) the LFM-SAR mAb or LFM-NOR mAb and the sample to be tested are mixed in a volume ratio of 0.6:100-1.2:100; preferably, the volume ratio of the LFM-SAR mAb and the sample to be tested is 0.8:100, and the volume ratio of the LFM-NOR mAb and the sample to be tested is 1:100.

[0064] (2) the mixture in step (1) is reacted and then added to the sample pad of the test strip.

[0065] (3) after light shielding reaction, the fluorescence is observed under ultraviolet light, and the fluorescence values of the T line and the C line are read with a high-sensitivity fluorescence analyzer.

[0066] (4) the measured fluorescence values are substituted into the SAR inhibition standard curve and the NOR inhibition standard curve, respectively, to calculate the theoretical concentration S and the theoretical concentration N, and the total theoretical concentration of fluoroquinolone drugs in the sample to be tested is S+90%N.

[0067] To solve the above technical problems, the eighth technical solution of the present application provides: application of the monoclonal antibody NOR McAb of the first technical solution, the monoclonal antibody SAR McAb of the second technical solution, the nucleic acid of the third technical solution, the recombinant expression vector of the fourth technical solution, the transformant of the fifth technical solution or the kit of the sixth technical solution in detection of fluoroquinolones.

[0068] On the basis of common knowledge in the art, the above-mentioned preferred conditions can be combined arbitrarily, i.e. to obtain each preferred example of the present application.

[0069] The reagents and raw materials used in the present application are commercially available.

[0070] The positive progress effect of the present application is that:

[0071] The present application is based on a coating agent (NOR-1-BSA) and a monoclonal antibody against sarafloxacin and norfloxacin, and establishes an indirect competitive ELISA method and a competitive lanthanide fluorescence microsphere lateral flow immunochromatography test strip (LFM-ICS) rapid detection technology for simultaneously detecting at least 13 kinds of fluoroquinolones. Since the same ELISA plate or the same test strip is matched with different antibodies or immunoprobes (lanthanide microspheres) labeled with the same antibody, two subgroups of drugs can be detected, which is simple in production and convenient, efficient in detection.

[0072] Using the ELISA method, it is measured that the half inhibitory concentration (IC 50 ) of SARMcAb to SAR is 4.56 ng / mL under the coating of 0.125 μg / mL NOR-1-BSA, and the IC 50 of NOR McAb to NOR is 1.30 ng / mL. For the mixed samples of SAR and NOR with the same concentration in the range of 1-32 ng / mL, the average cross-recovery rate can reach 96.78%; using the LFM-ICS method, it is measured that the IC 50 of LFM-SAR mAb to SAR is 1.93 ng / mL under the coating of 0.2 mg / mL NOR-1-BSA T-line antigen, and the IC 50 of LFM-NOR mAb to NOR is 0.21 ng / mL. The average cross-recovery rate of the mixed samples of SAR and NOR with the same concentration under three concentration gradients of 5, 2.5 and 1 ng / mL is 102.90%.

[0073] According to the established calculation rules, the cross-recovery rates of 12 fluoroquinolones are more than 30% by using ELISA method and LFM-ICS method, and the cross-recovery rate of daflloxacin is more than 15%, i.e. using NOR-1-BSA as the coating agent, and using SAR McAb and NOR McAb for detection, the determination of fluoroquinolone subclass and the semi-quantitative detection of at least 13 fluoroquinolones can be realized, and when multiple FQs exist in the sample, the detection is more accurate, and the preliminary screening of fluoroquinolone residues can be realized. BRIEF DESCRIPTION OF DRAWINGS

[0074] Figure 1 For the cross-reaction LFM-ICS test of the coating agent and the antibody, the T line of the test strip is coated from left to right with 3 mg / mL SAR-1-OVA, 3 mg / mL SAR-1-OVA, 3 mg / mL NOR-1-BSA, and 3 mg / mL NOR-1-BSA; and the chromatography liquid is 1 μL of 1×PBST diluent of LFM-SAR mAb, 1 μL of 1×PBST diluent of LFM-NOR mAb, 1 μL of 1×PBST diluent of LFM-SAR mAb, and 1 μL of 1×PBST diluent of LFM-NOR mAb from left to right.

[0075] Figure 2 For the selection and optimization of the homologous ELISA reaction system conditions of the coating concentration of SAR-1-OVA and the dilution degree of SAR McAb antibody.

[0076] Figure 3 For the exploration of the heterologous ELISA reaction system conditions of the coating concentration of NOR-1-BSA and the dilution degree of SAR McAb antibody.

[0077] Figure 4 For the exploration of the homologous reaction system of the coating concentration of NOR-1-BSA and the dilution degree of NOR McAb antibody.

[0078] Figure 5A The SAR standard competitive inhibition curve established by the ELISA method.

[0079] Figure 5B The theoretical concentration and cross-recovery rate of NOR at each actual concentration.

[0080] Figure 6A The NOR standard competitive inhibition curve established by the ELISA method.

[0081] Figure 6B The theoretical concentration and cross-recovery rate of SAR at each actual concentration.

[0082] Figure 7 The fitting curve of the theoretical total concentration and the actual total concentration of SAR and NOR.

[0083] Figure 8 LFM-ICS test strips for LFM-SAR mAb optimal T-line antigen coating concentration exploration, from left to right are 0.4, 0.2, 0.1 and 0.05 mg / mL NOR-1-BSA T-line coated negative samples and 20 ng / mL SAR positive samples.

[0084] Figure 9 LFM-ICS test strips for LFM-SAR mAb optimal T-line antigen coating concentration exploration.

[0085] Figure 10 LFM-ICS test strips for LFM-SAR mAb optimal microsphere dosage exploration, from left to right are 1.2, 1.0, 0.8 and 0.6 μL LFM-SAR McAb negative samples and 20 ng / mL SAR positive samples.

[0086] Figure 11 LFM-ICS test strips for LFM-SAR mAb optimal microsphere dosage exploration.

[0087] Figure 12 LFM-ICS test strips for LFM-NOR mAb optimal T-line antigen coating concentration exploration, from left to right are 0.4, 0.2, 0.1 and 0.05 mg / mL NOR-1-BSA T-line coated negative samples and 20 ng / mL NOR positive samples.

[0088] Figure 13 LFM-ICS test strips for LFM-NOR mAb optimal T-line antigen coating concentration exploration.

[0089] Figure 14 LFM-ICS test strips for LFM-NOR mAb optimal microsphere dosage exploration, from left to right are 1.2, 1.0, 0.8 and 0.6 μL LFM-NOR McAb negative samples and 20 ng / mL NOR positive samples.

[0090] Figure 15 LFM-ICS test strips for LFM-NOR mAb optimal microsphere dosage exploration.

[0091] Figure 16 LFM-ICS test strips for SAR inhibition standard curve, from left to right are 0 ng / mL SAR negative samples and 0.625, 1.25, 2.5, 5, 10, 20 ng / mL SAR positive samples.

[0092] Figure 17A SAR inhibition standard curve established by LFM-ICS method.

[0093] Figure 17B Theoretical concentration and cross-recovery rate of NOR at each actual concentration.

[0094] Figure 18 NOR inhibition standard curve of LFM-ICS test strip, from left to right, 0 ng / mL negative sample and 0.078125, 0.15625, 0.3125, 0.625, 2.5, 10 ng / mL NOR positive sample.

[0095] Figure 19A NOR inhibition standard curve established by LFM-ICS method.

[0096] Figure 19B Theoretical concentration and cross-recovery rate of SAR at each actual concentration. DETAILED DESCRIPTION

[0097] The present application will be further described by way of examples, but the present application is not limited to the examples described. The experimental methods in the following examples, if no specific conditions are indicated, are selected according to conventional methods and conditions, or according to the instructions of the commercial products.

[0098] Materials and reagents

[0099] 1. Main reagents

[0100] The anti-SARfloxacin monoclonal antibody (SAR McAb, VH amino acid sequence as shown in SEQ ID NO: 3, VL amino acid sequence as shown in SEQ ID NO: 4) and the anti-norfloxacin monoclonal antibody (NOR McAb, VH amino acid sequence as shown in SEQ ID NO: 1, VL amino acid sequence as shown in SEQ ID NO: 2) used in the present application are prepared by the laboratory.

[0101] The complete antigen (e.g. SAR-1-OVA and NOR-1-BSA) used in the present application can be any complete antigen synthesized by coupling hapten and carrier protein by methods well known to those skilled in the art. The monoclonal antibody is only directed against the drug antigenic determinant, thereby recognizing and binding to the drug regardless of the type of carrier protein, so when a detection method is established using the antibody of the present application, a carrier protein commonly used in the art can be selected, such as bovine serum albumin (BSA) or chicken egg white albumin (OVA) and the like.

[0102] The chemicals used in the present application are all of analytical purity, as shown in Table 1 below.

[0103] Table 1 Main reagents

[0104]

[0105]

[0106] 2. Preparation of primary reagent solution

[0107] (1) ELISA coating solution (CBS, pH = 9.6): Na2CO3 1.696 g, NaHCO3 2.856 g, dd H2O 1000 mL.

[0108] (2) 1% gelatin (ELISA blocking solution): gelatin 20 g, dd H2O (preheated to 50°C) 2000 mL.

[0109] (3) ELISA substrate developing solution:

[0110] A solution: sodium acetate 13.6 g, citric acid 1.6 g, 30% H2O2 0.3 mL, dd H2O 500 mL.

[0111] B solution: TMB 0.15 g (dissolved in 3 mL DMSO), EDTA-2Na 0.2 g, citric acid 0.95 g, glycerol 50 mL, dd H2O to 500 mL.

[0112] Prepare for use according to A:B = 1:1.

[0113] (4) 2 mol / L sulfuric acid (ELISA termination solution): concentrated sulfuric acid (98%) 21.7 mL, dd H2O 178.3 mL.

[0114] (5) 25 mmol / L MES buffer (pH = 7.2): MES 0.244 g, dd H2O 50 mL. Adjust pH to 7.2 with 0.1 mol / L NaOH solution.

[0115] (6) 16 mg / mL NHS solution: NHS 16 mg, dd H2O 1 mL.

[0116] (7) 8 mg / mL EDC solution: EDC 8 mg, dd H2O 1 mL.

[0117] (8) 2x boric acid buffer solution (pH = 8.5):

[0118] A solution (0.1 mol / L H3BO3): H3BO3 0.618 g, dd H2O 100 mL.

[0119] B solution (25 mmol / L borax): Na2B7O4·10H2O 0.954 g, dd H2O 100 mL.

[0120] Prepare for use according to A:B = 2:3.

[0121] (9) Microsphere blocking solution (pH = 8.0): BSA 500 mg, NaN3 200 mg, Tris 605 mg, dd H2O 100 mL. Adjust pH to 8.0 with 0.1 mol / L HC1 solution.

[0122] (10) Microsphere dilution solution (pH = 8.0): BSA 500 mg, NaN3 200 mg, Tris 605 mg, PVP 500 mg, dd H2O 100 mL. Adjust pH to 8.0 with 0.1 mol / L HC1 solution.

[0123] (11) 1 x PBS sample dilution solution (pH = 7.4): NaCl 8 g, KCl 0.2 g, KH2PO4 0.2 g, Na2HPO4-12H2O 2.86 g, dd H2O 1 L. To prepare 1 x PBST, add 0.5 mL Tween-20 to the above solution.

[0124] 3. Main instruments

[0125] The main instruments used in the experiment are shown in Table 2 below.

[0126] Table 2 Main instruments

[0127]

[0128] Preparation and verification of coated antigen and monoclonal antibody of the present application

[0129] To establish a method for detecting fluoroquinolone drug residues, the present application considers that the benzene ring has a greater influence on the immunogenicity and immunoreactivity of small molecule antigens. Two kinds of FQs monoclonal antibodies were developed according to the above two subgroups.

[0130] The FQs without benzene ring structure take norfloxacin (NOR) hapten with the most common structure of this subgroup as a lead compound, which is modified by different groups, coupled with carrier protein to prepare different complete antigens, and then the monoclonal antibody with broad-spectrum specificity and high affinity for fluoroquinolone drugs is developed. One method is to connect an aminoethyl group to the N of the piperazine ring of NOR, named NOR-1, and then NOR-1 is connected to BSA by glutaraldehyde method to synthesize NOR-1-BSA; another method is to directly couple the carboxyl group at C-3 of NOR with the amino group of BSA to form NOR-BSA by carbodiimide method.

[0131] The inventors found that the antibody (NOR McAb) produced by immunizing mice with NOR-1-BSA has higher sensitivity and better broad-spectrum specificity, with a titer of 1:4 x 10 4 ~ 1:8 x 10 5The half-inhibitory quantity (IC 50 ) of 11 kinds of FQs is below 11 ng / mL, and the cross-reaction rate of 11 kinds of FQs is 16.2%~104.2%.

[0132] The nucleotide sequences of the heavy chain variable region (VH) and the light chain variable region (VL) of the NOR McAb antibody prepared by the application are as follows after sequencing:

[0133] NOR McAb VH nucleotide (SEQ ID NO: 1)

[0134] GACGGTGATATCAAGCTGCAGGAGTCAGGGGCTGAGCTTGTGAAGCCTGGGGCTTCAGTGAAGCTGTCCTGCAAGGCTTCTGGCTACACCTTCACCAGCTACTGGATGCACTGGGTGAAGCAGAGGCCTGGACAGGGACTTGAGTGGATTGGATGGATTTATCCTAGGGATGGTAGTACTAAGTACAATGAGAAGTTCAAGGGCAAGGCCACATTGACTGTAGACACATCCTCCAGCACAGCGTACATGGAGCTCCACAGCCTGACATCTGAGGACTCTGCGGTCTATTTCTGTGCAAGAGGGGGATATAGGTACAGAGACTATGCTATGGACTACTGGGGTCAAGGAACCTCGGTCACCGTCTCCTCAGGATCCCTTATC

[0135] NOR McAb VL nucleotide (SEQ ID NO: 2)

[0136] GGTGGCGGATCGGACATTGAGCTCACCCAGTCTCCTGCTTCCTTAGCTGTATCTCTGGGGCAGAGGGCCACCATCTCATACAGGGCCAGCAAAAGTGTCAGTACATCTGGCTATAGTTATATGCACTGGAACCAACAGAAACCAGGACAGCCACCCAGACTCCTCATCTATCTTGTATCCAACCTAGAATCTGGGGTCCCTGCCAGGTTCAGTGGCAGTGGGTCTGGGACAGACTTCACCCTCAACATCCATCCTGTGGAGGAGGAGGATGCTGCAACCTATTACTGTCAGCACATTAGGGAGCTTACACGTTCGGAGGGGGCACAAAGTTGGAACTAAAACGG

[0137] The amino acid sequence obtained after translation of the above nucleotide sequence is as follows:

[0138] Amino acid comprising NOR McAb VH (SEQ ID NO: 3):

[0139] DGDIKLQESGAELVKPGASVKLSCKASGYTFTSYWMHWVKQRPGQGLEWIGWIYPRDGSTKYNEKFKGKATLTVDTSSSTAYMELHSLTSEDSAVYFCARGGYRYRDYAMDYWGQGTSVTVSSGSLI

[0140] Amino acid comprising NOR McAb VL (SEQ ID NO: 4):

[0141] GGGSDIELTQSPASLAVSLGQRATISYRASKSVSTSGYSYMHWNQQKPGQPPRLLIYLVSNLESGVPARFSGSGSGTDFTLNIHPVEEEDAATYYCQHIRELTRSEGAQSWN

[0142] The CDR regions of the amino acid sequences comprising the variable region of the antibody are defined using the Kabat rule, and the amino acid sequences of the CDR regions of the NOR McAb antibody prepared in the present application are as follows:

[0143] HCDR1 (SEQ ID NO: 5): SYWMH

[0144] HCDR2 (SEQ ID NO: 6): WIYPRDGSTKYNEKFKG

[0145] HCDR3 (SEQ ID NO: 7): GGYRYRDYAMDY

[0146] LCDR1 (SEQ ID NO: 8): RASKSVSTSGYSYMH

[0147] LCDR2 (SEQ ID NO: 9): LVSNLES

[0148] LCDR3 (SEQ ID NO: 10): QHIR

[0149] NOR McAb VH amino acid sequence (SEQ ID NO: 11):

[0150] DIKLQESGAELVKPGASVKLSCKASGYTFTSYWMHWVKQRPGQGLEWIGWIYPRDGSTKYNEKFKGKATLTVDTSSSTAYMELHSLTSEDSAVYFCARGGYRYRDYAMDYWGQGTSVTVSS

[0151] NOR McAb VL amino acid sequence (SEQ ID NO: 12):

[0152] DIELTQSPASLAVSLGQRATISYRASKSVSTSGYSYMHWNQQKPGQPPRLLIYLVSNLESGVPARFSGSGSGTDFTLNIHPVEEEDAATYYCQHIRELTRSEGAQSWN

[0153] The heavy chain constant region of the NOR McAb of the present application is derived from the gamma chain of a murine antibody, and the light chain constant region is derived from the lambda chain of a murine antibody.

[0154] The monoclonal antibodies for sarafloxacin and difloxacin with benzene ring structure are prepared by using SAR-1-BSA as immunogen, and the preparation method is the same as that of NOR-1-BSA. Both of them form a spacer of -(CH2)2-N=C-(CH2)3-CH=N- structure between the hapten and BSA, so that the hapten mother nucleus is not changed in spatial structure, thereby ensuring the production of antibodies against the hapten and the effective recognition of antigen-antibody. The titer of the anti-SAR monoclonal antibody (SAR McAb) prepared by the present application can reach 1:4x10 6 , the IC 50 is 4.75 ng / mL, the cross-reactivity rate to DIF is nearly 100%, and the cross-reactivity rate to other 12 FQs drugs is lower than 10%.

[0155] The heavy chain variable region (VH) and light chain variable region (VL) nucleotide sequences of the SAR McAb antibody prepared in the present application are as follows after sequencing:

[0156] SAR McAb VH nucleotide (SEQ ID NO: 13)

[0157] GACGGTGATATCAAGCTGCAGGAGTCAGGACCTGAGTTGGTGAAGCCTGGGGCTTCAGTGAAGATGTCCTGCAAGGCTCCTGGCTACACATTCACTGACTACTACATGCACTGGGTGAAGCAGAGCCATGGAAAGAGCCTTGAGTGGATTGGATATATTTATCCTAACAATGGTGATACTTTCTACAACCAGAAGTTCAAGGGCAAGGCCACATTGACTGTAGACAAATCCTCTAGCACAGCCCACATGGAGCTCCGGAGCCTGACATCTGAGGAGTCTGCAGTCTATTATTGTGCAATTACTACGGTAGTAGCTATGGACTACTGGGGTCAAGGAACCTCGGTCACCGTCTCCTCAGGATCCCTTATC

[0158] SAR McAb VL nucleotide (SEQ ID NO: 14)

[0159] GGTGGCGGATCGGACATTGAGCTCACTCAGTCTCCTGCTTCCTTAGCTGTATCTCTGGGGCAGAGGGCCACCATCTCATACAGGACCAGCAAAAGTGTCAGTACATCTGGCTATAGTTATATGCACTGGAACCAACAGAAACCAGGACAGCCACCCAGACTCCTCATCTATCTTGTATCCAACCTAGAATCTAGGGTCCCTGCCAGGTTCAGTGGCAGTGGGTCTGGGACAGACTTCACCCTCAACATCCATCCTGTGGAGGAGGAGGATGCTGCAACCTATTACTGTCAGCACATTAGGGAGCTTACACGTTCGGAGGGGGGCACAAAGTTGGAAATGAAACGG

[0160] The amino acid sequence obtained after translation of the above nucleotide sequence is as follows:

[0161] Amino acids comprising the VH of the SAR McAb (SEQ ID NO: 15):

[0162] DGDIKLQESGPELVKPGASVKMSCKAPGYTFTDYYMHWVKQSHGKSLEWIGYIYPNNGDTFYNQKFKGKATLTVDKSSSTAHMELRSLTSEESAVYYCAITTVVAMDYWGQGTSVTVSSGSLI

[0163] Amino acids comprising the VL of the SAR McAb (SEQ ID NO: 16):

[0164] GGGSDIELTQSPASLAVSLGQRATISYRTSKSVSTSGYSYMHWNQQKPGQPPRLLIYLVSNLESRVPARFSGSGSGTDFTLNIHPVEEEDAATYYCQHIRELTRSEGGTKLEMKR

[0165] The CDR regions of the above-described amino acid sequences comprising the variable regions of the antibody were defined using the Kabat convention, and the amino acid sequences of the CDR regions of the SAR McAb antibody prepared in the present application are as follows:

[0166] HCDR1 (SEQ ID NO: 17): DYYMH

[0167] HCDR2 (SEQ ID NO: 18): YIYPNNGDTFYNQKFKG

[0168] HCDR3 (SEQ ID NO: 19): TTVVAMDY

[0169] LCDR1 (SEQ ID NO: 20): RTSKSVSTSGYSYMH

[0170] LCDR2 (SEQ ID NO: 9): LVSNLES

[0171] LCDR3 (SEQ ID NO: 21): QHIRELTR

[0172] Amino acid sequence of the VH of the SAR McAb (SEQ ID NO: 22):

[0173] DIKLQESGPELVKPGASVKMSCKAPGYTFTDYYMHWVKQSHGKSLEWIGYIYPNNGDTFYNQKFKGKATLTVDKSSSTAHMELRSLTSEESAVYYCAITTVVAMDYWGQGTSVTVSS

[0174] SAR McAb VL amino acid sequence (SEQ ID NO: 23):

[0175] DIELTQSPASLAVSLGQRATISYRTSKSVSTSGYSYMHWNQQKPGQPPRLLIYLVSNLESRVPARFSGSGSGTDFTLNIHPVEEEDAATYYCQHIRELTRSEGGTKLEMK

[0176] The heavy chain constant region of the SAR McAb of the present application is derived from the gamma chain of a murine antibody, and the light chain constant region is derived from the lambda chain of a murine antibody.

[0177] In the investigation of the cross-reaction between the coating agent and the antibody, the T line of the test strip was coated with 3 mg / mL SAR-1-OVA and 3 mg / mL NOR-1-BSA respectively, and the C line was coated with 0.5 mg / mL goat anti-mouse IgG. The LFM-SAR mAb or LFM-NOR mAb dilution [1 μL LFM-mAb (antibody microsphere conjugate), diluted with 1 x PBST to 100 μL] was reacted with the two kinds of coated test strips respectively, as shown in the following table, the fluorescence intensity produced by the reaction of LFM-NOR mAb with SAR-1-OVA was extremely weak, but the reaction of LFM-SAR mAb with NOR-1-BSA showed a bright and obvious fluorescence spot, indicating that the cross-reaction between LFM-NOR mAb and SAR-1-OVA was extremely low, and the cross-reaction between LFM-SAR mAb and NOR-1-BSA was relatively strong, which was similar to the cross-reaction between the hapten and the antibody, that is, both LFM-SAR mAb and LFM-NOR mAb could detect fluoroquinolones with NOR-1-BSA as the coating agent. Figure 1

[0178] Establishment of indirect competitive ELISA method for detecting fluoroquinolone residues:

[0179] Example 1: Indirect competitive ELISA detection steps

[0180] ​Coating: The coating solution was prepared by diluting the previously synthesized complete antigen SAR-1-OVA or NOR-1-BSA in carbonate buffer (0.05 mol / L, pH 9.6, CBS) to the desired concentration, and adding 100 μL / well to the enzyme-labeled plate, which was then coated overnight at 4°C in the refrigerator. The plate was washed 3 times with 1 × PBST at an interval of 5 min.

[0181] Blocking: The blocking solution was added at 200 μL / well, and incubated at 37°C for 2 h. The plate was washed 3 times with 1 × PBST at an interval of 5 min.

[0182] Primary antibody incubation: Fluoroquinolone standard and monoclonal antibody were each added at 50 μL / well, diluted with 1 × PBST containing 5% FBS (fetal bovine serum), and incubated at 37°C for 1.5 h. The plate was washed 3 times with 1 × PBST at an interval of 5 min.

[0183] Secondary antibody incubation: Goat anti-mouse IgG-HRP (the concentration of goat anti-mouse IgG-HRP stock solution was 0.8 mg / mL-1.2 mg / mL) was diluted 1:20,000 with 1 × PBST containing 5% FBS, and added at 100 μL / well. The plate was incubated at 37°C for 1 h, and washed 3 times with 1 × PBST at an interval of 5 min.

[0184] Color development: TMB color developing solution (A solution:B solution = 1:1) was added at 100 μL / well, and color developed at 37°C for 15 min.

[0185] Termination: The termination solution was added at 50 μL / well.

[0186] Detection: The plate was immediately detected with an enzyme-labeled instrument, and the absorbance value (OD value) was read.

[0187] Example 2 Optimization of ELISA optimal conditions

[0188] Following the detection steps in Example 1, the concentration of the coating agent and the antibody dilution were optimized using the checkerboard method. The concentration of the stock antibody solution of the NOR McAb or SAR McAb to be diluted was 5.0 mg / mL to 8.0 mg / mL. For the homologous ELISA reaction system using SAR-1-OVA coating agent and SAR McAb as primary antibodies, four coating agent concentrations were set up: 0.25 μg / mL, 0.20 μg / mL, 0.125 μg / mL, and 0.10 μg / mL, with antibody dilution ratios of 1:600000, 1:800000, and 1:1000000. SAR standards at concentrations of 32, 16, 8, 4, 2, 1, 0.5, and 0 ng / mL were used as competitors for competitive reactions. For a heterologous ELISA reaction system using NOR-1-BSA coating antigen and SAR McAb as primary antibodies, four coating antigen concentrations were set up: 0.25 μg / mL, 0.20 μg / mL, 0.125 μg / mL, and 0.10 μg / mL, with antibody dilution ratios of 1:800000, 1:1000000, and 1:1200000. SAR standards at concentrations of 32, 16, 8, 4, 2, 1, 0.5, and 0 ng / mL were used as competitors for competitive reactions.

[0189] Plot a competition standard curve at different standard concentrations, with the logarithm of the final standard concentration (lgC) on the x-axis and the binding rate (B / B0, where B is the OD value of the standard at a certain concentration and B0 is the OD value of the blank sample) on the y-axis. Calculate the half-maximal inhibitory concentration (IC50) based on the standard curve. 50 (i.e., the standard concentration corresponding to 50% inhibition rate) and the ratio of maximum absorbance to half-maximum inhibitory concentration (ODmax / IC). 50 According to IC 50 Smaller, ODmax / IC 50 The correlation coefficient R with the standard curve 2 The optimal concentration of the original coating agent and the antibody dilution ratio are selected based on the principle of prioritizing these two factors. The two optimal conditions—original coating agent concentration and antibody dilution—are plotted on the x-axis, with IC50 as the metric. 50 and Odmax / IC 50 Combination graphs of primary and secondary ordinates, comprehensively comparing IC 50 Odmax / IC 50 R 2 The changing trends of the three factors. The homologous ELISA results for SAR-1-OVA coating antigen and SAR McAb as primary antibodies are as follows: Figure 2 As shown, when the original SAR-1-OVA coating concentration is 0.20 μg / mL and the SAR McAb dilution is 1:600000, the IC50 of SAR is... 50 The lowest value was 4.83 ng / mL, while the ODmax / IC50 ratio was also low.50 The value is large, so the homologous coating selects 0.20 μg / mL as the best coating concentration, and 1:600000 as the best antibody dilution ratio. The results of the heterologous ELISA using NOR-1-BSA coating and SAR McAb as the primary antibody are shown in Table 1. Figure 3 As shown in Table 1, when the NOR-1-BSA coating concentration is 0.125 μg / mL and the antibody dilution is 1:1200000, the IC 50 The minimum is 4.60 ng / mL and the ODmax / IC 50 The value is maximum, and the IC 50 is smaller than that under the best conditions of homologous coating, and the sensitivity is slightly higher. Both are less than the maximum residue limit of the veterinary drug sarafloxacin, indicating that heterologous ELISA can be used to replace homologous ELISA to detect SAR.

[0190] For the ELISA reaction system using NOR-1-BSA coating and NOR McAb as the primary antibody, four coating concentrations of 0.25 μg / mL, 0.20 μg / mL, 0.125 μg / mL, and 0.10 μg / mL, and antibody dilution ratios of 1:600000, 1:800000, 1:1000000 were set, and 16, 8, 4, 2, 1, 0.5, 0.25, and 0 ng / mL NOR standard as the competitor for competition reaction.

[0191] The results are shown in Table 2. Figure 4 As shown in Table 2, when the coating concentration is 0.10 μg / mL and the antibody dilution is 1:1000000, the IC 50 The minimum is 0.69 ng / mL, and the ODmax / IC 50 The value is maximum, but the OD 450nm value of the blank sample is <0.5, and the data error is large, so 0.125 μg / mL is selected as the best coating concentration, and 1:600000 as the best antibody dilution ratio (R 2 =0.9701), and the IC 50 is 1.04 ng / mL.

[0192] Example 3: Establishment and cross-analysis of the ELISA method for detecting SAR / NOR

[0193] The ELISA method for detecting SAR / NOR is constructed according to the following technical route:

[0194] 1. NOR-1-BSA as coating, SAR McAb as primary antibody, establish ELISA method for detecting SAR.

[0195] 2. NOR-1-BSA as coating, NOR McAb as primary antibody, establish ELISA method for detecting NOR.

[0196] The original NOR-1-BSA coating concentration was 0.125 μg / mL, and the SAR monoclonal antibody dilution was 1:1,200,000. Competitive reactions were conducted using SAR at concentrations of 32, 16, 8, 4, 2, 1, 0.5, 0.25, and 0 ng / mL as competing standards to establish an ELISA standard curve for SAR detection. The SAR standard curve is a linear equation: B / B0 = A1 × lgc(SAR) + D1, where A1 is the slope of the SAR standard curve, B is the absorbance of the test sample, B0 is the absorbance of the blank sample, c is the final concentration of the standard, and D1 is the intercept of the SAR standard curve.

[0197] like Figure 5A The linear equation is B / B0 = -0.4089 × lg c(SAR) + 0.7696, R 2 =0.9867, IC 50 The concentration was 4.56 ng / mL, with a linear range of 0.25–32 ng / mL, measured at IC50. 20 The limit of detection (LOD) was calculated to be 0.84 ng / mL. Cross-reactivity assays were then performed, with NOR standards serially diluted to 128, 64, 32, 16, 8, 4, 1, and 0 ng / mL, and measured using the established ELISA method for SAR detection. Following the steps in Example 1, the IC50 of NOR against SAR McAb was determined. 50 The concentration was 58.24 ng / mL. According to the formula: Cross-reactivity rate (CR%) = [IC50] / mL 50 (SAR Standard) / IC 50 The cross-reactivity was calculated by multiplying the (NOR standard) by 100%, i.e., CR% = (4.56 ng / mL / 58.24 ng / mL) × 100% = 7.83%.

[0198] Substituting the B / B0 ratio measured at each NOR standard concentration into the SAR standard curve, the corresponding theoretical recovery concentration and cross-recovery rate (i.e., the ratio of the theoretical concentration to the actual NOR standard concentration) were calculated. Figure 5B As can be seen, when the NOR standard concentration was 1 and 4 ng / mL, the calculated theoretical recovery concentration was lower than the limit of detection of the established ELISA method for detecting SAR. Therefore, these two samples were determined to be negative. When the NOR standard concentration was 8–128 ng / mL, the cross-recovery rates were close at each concentration, with an average cross-recovery rate of 9.49%. The cross-reactivity of SAR monoclonal antibodies to NOR cannot be ignored. Furthermore, due to the cross-reactivity, the presence of non-benzene ring structures such as NOR in the sample will increase the SAR value in the result calculation.

[0199] The NOR-1-BSA coating concentration was 0.125 μg / mL, the NOR monoclonal antibody dilution was 1:600000, and 16, 8, 4, 2, 1, 0.5, 0.25 and 0 ng / mL NOR standard was used as the competitor for the competition reaction. The NOR ELISA standard curve was established as a linear equation B / B0=A2xlgc(NOR)+D2, wherein A2 is the slope of the NOR standard curve linear equation, B is the absorbance value of the sample to be tested, B0 is the absorbance value of the blank sample, c is the final concentration of the standard, and D2 is the intercept of the NOR standard curve linear equation.

[0200] As Figure 6A , the linear equation is B / B0=-0.3037xlgc(NOR)+0.5345, R 2 =0.9884, the IC 50 is 1.30 ng / mL, the linear range is 0.25-16 ng / mL, and the lowest detection limit is 0.13 ng / mL. The SAR standard was diluted to 128, 64, 32, 16, 8, 4, 1, 0.5 and 0 ng / mL, and the established ELISA method for detecting NOR was used for determination, and the IC 50 of SAR to NOR McAb was greater than 1000 ng / mL, and the cross-reactivity was less than (1.30 ng / mL / 1000 ng / mL)x100%=0.13%. As Figure 6B , the theoretical recovery concentration of the SAR standard in the six gradients of 0.5-64 ng / mL was less than the above-mentioned lowest detection limit of NOR 0.13 ng / mL, and the samples in these SAR concentration ranges were also determined to be negative samples, and the cross-recovery rate of the 128 ng / mL SAR standard sample was only 0.10%, indicating that the cross-reaction of NOR monoclonal antibody to SAR was negligible.

[0201] Example 4 Establishment of a combined ELISA method for simultaneous detection of SAR and NOR based on a primary antigen and a secondary monoclonal antibody

[0202] Under the optimal coating concentration of NOR-1-BSA of 0.125 μg / mL and the optimal dilution ratio of 1:1200000 of SAR McAb and 1:600000 of NOR McAb, a combined ELISA was established as follows: NOR-1-BSA was used as the coating antigen of the ELISA plate, two holes were used for each sample, one hole was added with SAR McAb and the other hole was added with NOR McAb, and the remaining steps were the same as the indirect competitive ELISA of the present study to form a combined ELISA for simultaneously detecting mixed samples of SAR and NOR. The cross-reaction of SAR McAb to NOR cannot be ignored, and the cross-reaction of NOR McAb to SAR can be ignored. Therefore, the theoretical concentration calculated by using SAR McAb to detect mixed samples of SAR and NOR with the same concentration includes a certain concentration of SAR and part of NOR. When the mixed samples are quantitatively detected by using the combined ELISA, the concentration of the cross-reacted NOR of about 10% should be subtracted to obtain the correct theoretical total concentration.

[0203] The B / B0 obtained by using SAR McAb to detect mixed samples of SAR and NOR with the same concentration was brought into the standard curve of SAR to calculate the theoretical concentration of SAR, which is represented by S. Similarly, the theoretical concentration of NOR detected by using NOR McAb is represented by N. According to the above cross-reaction research results, when the concentration of NOR is ≥8 ng / mL, the theoretical total concentration should be S+90%N; when the concentration of NOR is <8 ng / mL, SAR McAb cannot be used for detection, and the theoretical total concentration is S+N. Therefore, the calculation method of "the theoretical total concentration of the sample that can be detected as positive by SAR McAb and NOR McAb is S+90%N" is set. The mixed liquid of SAR and NOR with the same concentration of 16, 8, 4, 2, 1 and 0 ng / mL was used as the competitive standard, and the combined ELISA was used for detection to calculate the theoretical total concentration of each added concentration sample. The percentage of the theoretical total concentration to the actual total concentration is shown in Table 3, and the percentage of the theoretical total concentration to the actual total concentration is 96.78%. See Figure 7 Combined with chi-square test (p value of 0.99) and K-S test, it is confirmed that the theoretical total concentration of mixed samples of SAR and NOR calculated by using the rule is very close to the actual total concentration, and the ELISA method and the calculation rule are relatively reliable.

[0204] Table 3 Quantitative calculation rule of ELISA method for detecting mixed samples of SAR and NOR with the same concentration

[0205]

[0206] Example 5 Semi-quantitative detection of various fluoroquinolones by combined ELISA

[0207] Since the SAR McAb and NOR McAb are prepared by immunizing mice with the complete immunogen formed by coupling the carrier protein to the lead compounds SAR and NOR, respectively, which are the most structurally conservative core drugs in the two subgroups of FQs, and by using the hybridoma technology. According to the results of Example 4, when detecting the mixed sample of SAR and NOR at the same concentration, i.e. the ratio of SAR and NOR is 1:1, the quantification of SAR depends on the detection results of SAR McAb as the primary antibody (i.e. the total theoretical concentration S measured by SAR McAb in Table 3), and the quantification of NOR depends on the detection results of both SAR McAb as the primary antibody and NOR McAb as the primary antibody (i.e. the total theoretical concentration N measured by NOR McAb in Table 3), and the above-mentioned quantitative calculation rule of "S+90%N" is also applicable to the detection of the mixed sample of SAR and NOR at unknown ratio and the single sample of SAR or NOR. In addition, since the other 11 fluoroquinolones have different degrees of cross-reactions with the SAR McAb and NOR McAb, the semi-quantitative detection of the other 11 fluoroquinolones can be theoretically achieved by means of the two monoclonal antibodies and the quantitative rule.

[0208] According to the method of Example 1, the 13 fluoroquinolones were detected by using SAR McAb and NOR McAb as the primary antibody, respectively, to obtain the theoretical concentration substituted into the SAR standard curve and the theoretical concentration substituted into the NOR standard curve, and the total theoretical concentration was calculated by adding the two values using the rule of "S+90%N", and the ratio of the total theoretical concentration to the actual concentration was defined as the total cross-recovery rate. As shown in Table 4, by comparing the theoretical concentration results of each FQs detected by SAR McAb and NOR McAb, it can be simply judged which subgroup the drug belongs to; the total cross-recovery rates of SAR, DIF, NOR, PEF, CIP, ENR, LML, FLU, NAD, ENO, OFL and MAR at the actual concentration of 10 ng / mL and 20 ng / mL are within the range of 30% to 100%, while the maximum residue limit of danofloxacin (DAN) in animal-derived food is 10 ng / g, and the total cross-recovery rate is more than 15%, and the method established can detect the residue at the addition level of 10 ng / mL, and the actual sample detection is useful, i.e. the ELISA method for detecting fluoroquinolones established can achieve the preliminary classification of subgroups and the semi-quantitative screening of at least 13 FQs drugs with the same parent core structure.

[0209] Table 4 The theoretical concentration and cross-recovery rate of 13 fluoroquinolones detected by ELISA method using SAR McAb and NOR McAb, respectively

[0210]

[0211]

[0212] *:“<” means the theoretical concentration calculated after the substitution of the calibration curve is lower than the LOD, and is judged as a negative sample. The total theoretical concentration is directly expressed by S or N.

[0213] Establishment of competitive LFM-ICS analysis method for detecting fluoroquinolones

[0214] Example 6 Competitive LFM-ICS analysis procedure

[0215] 1. Coupling procedure of SAR and NOR monoclonal antibodies with lanthanide fluorescent microspheres

[0216] a) Take 100 μL of 1% lanthanide fluorescent microspheres, dilute with 400 μL of MES buffer, and disperse the microspheres by ultrasonication at 10% power for 10 min;

[0217] b) Add 50 μL of NHS and EDC solution to the microspheres, activate for 15 min at room temperature with shaking, and continue to activate for 15 min after adding 400 μL of MES buffer;

[0218] c) Disperse the agglomerated long-life fluorescent microspheres by ultrasonication at 4% power for 15 min;

[0219] d) Centrifuge at 12000 rpm / min for 25 min at 4°C, and discard the supernatant;

[0220] e) Resuspend the precipitate with 200 μL of boric acid buffer, and ultrasonicate at 6% power for 5 min;

[0221] f) Add 100 μL of SAR or NOR monoclonal antibody at a concentration of 1.0 mg / mL, and couple at 4°C for 24 h with shaking;

[0222] g) After coupling, ultrasonicate at 4% power for 5 min, and centrifuge at 12000 rpm / min for 25 min at 4°C, and discard the supernatant;

[0223] h) Resuspend with 500 μL of blocking solution, and block at 4°C for 6 h;

[0224] i) After blocking, ultrasonicate at 4% power for 5 min, and centrifuge at 12000 rpm / min for 25 min at 4°C, and discard the supernatant;

[0225] j) Add 300 μL of diluent, ultrasonicate at 6% power for 5 min, and obtain the antibody microsphere conjugate (LFMs-mAb), which is stored at 4°C in the dark.

[0226] 2. Test strip preparation and LFM-ICS detection procedure

[0227] The NC membrane, water absorption pad and treated sample pad were pasted on the PVC base plate in sequence, and the water absorption pad and sample pad overlapped the NC membrane by about 2 mm. The test strip was sprayed with 0.5 mg / mL quality control C line (goat anti-mouse IgG) and a certain concentration of detection T line (NOR-1-BSA) using XYZ3050 test strip line spraying system, and then cut into a test strip with a width of 3.8 mm, and placed in an oven at 37°C for drying.

[0228] During LFM-ICS detection, 100 μL of the sample to be detected (1×PBS dilution) was mixed with a suitable amount of microspheres coupled with antibodies, and after 5 min of reaction, added to the sample pad, and after 15 min of reaction in the dark, the fluorescence was observed under ultraviolet light, and the fluorescence values of the T line and the C line were read using a high-sensitivity fluorescence analyzer. If neither the C line (quality control line) nor the T line (detection line) is bright, it may be that the microspheres are not successfully coupled with the antibodies, in which case it is determined that the test strip preparation is invalid; for negative samples containing no drug to be detected, when the LFM-SAR mAb is chromatographed to the T line, it will bind to the antigen coated on the T line, and the remaining LFM-SAR mAb continues to be chromatographed to the C line, which is recognized by goat anti-mouse IgG, so both the C line and the T line are bright; for positive samples containing a certain concentration of drug to be detected, since the drug has been pre-bound to the LFM-SAR mAb to form an antigen-antibody complex LFM-SAR mAb-Ag, the LFM-SAR mAb binding to the T line antigen is reduced, and the LFM-SAR mAb-Ag continues to be chromatographed, which is captured by goat anti-mouse IgG, and finally the C line is bright and the T line is not bright or has a relatively dark fluorescence.

[0229] Example 7 Optimization of the optimal reaction conditions of LFM-ICS

[0230] For the heterologous LFM-ICS reaction system with NOR-1-BSA as the T line coating antigen and LFM-SAR mAb as the labeled monoclonal antibody, the optimal T line coating concentration and the amount of microspheres were explored. According to the principle of controlling a single variable, the amount of LFM-SAR mAb microspheres was set to 0.8 μL, and NOR-1-BSA with concentrations of 0.4, 0.2, 0.1 and 0.05 mg / mL was sprayed on the T line position, respectively. The blank diluent was set as the negative sample, and the 20 ng / mL SAR standard diluent was set as the positive sample. The test strip was prepared and detected according to the steps in Example 6, and the detection was repeated three times in parallel, as shown in Table 1. Figure 8 The ratio of the fluorescence values of the T line of the positive sample to the negative sample was the binding rate (FIs / FIb, FIs was the T line fluorescence value of the positive sample of a certain concentration of standard, and FIb was the T line fluorescence value of the negative sample of the blank diluent). According to the formula: inhibition rate = (1-FIs / FIb) × 100%, the inhibition rate under each coating concentration was calculated, and the optimal T line antigen coating concentration was determined by combining the inhibition rate and the fluorescence intensity.

[0231] The combination chart as shown in Figure 9 was drawn with the T-line coating concentration as the abscissa, the T-line fluorescence values of the blank diluent negative sample and the positive sample as the primary ordinate, and the inhibition rate as the secondary ordinate. The results showed that the T-line fluorescence value decreased with the decrease of the T-line coating concentration, and the fluorescence value of the negative sample was visible to the naked eye. The inhibition rate changed in a broken line, and when the coating concentration was 0.2 mg / mL NOR-1-BSA, the inhibition rate was the highest, being 95.93%, and the concentration was selected as the optimal T-line antigen coating concentration for detecting SAR.

[0232] Under the optimal T-line antigen coating concentration of 0.2 mg / mL NOR-1-BSA, the microsphere dosage was set to be 1.2, 1, 0.8 and 0.6 μL, the blank diluent was set as the negative sample, and the 20 ng / mL SAR standard diluent was set as the positive sample, and the inhibition rate under each microsphere dosage was calculated. The optimal microsphere dosage was determined according to the inhibition rate and the fluorescence intensity. As shown in Figure 10 , Figure 11 when the LFM-SAR mAb dosage was 1.2, 1.0 and 0.8 μL, the inhibition rates of the three were not much different, and the inhibition rate of 0.8 μL was the highest, being 91.56%, and the T-line fluorescence value of the negative sample was also higher. Therefore, 0.8 μL was selected as the optimal microsphere dosage for detecting SAR.

[0233] For the homologous LFM-ICS reaction system with NOR-1-BSA as the T-line coating antigen and LFM-NOR mAb as the labeled monoclonal antibody, the LFM-NOR mAb microsphere dosage was controlled to be 1.0 μL, the test strip T-line was coated with 0.4, 0.2, 0.1 and 0.05 mg / mL NOR-1-BSA, the blank diluent was set as the negative sample, and the 20 ng / mL NOR standard diluent was set as the positive sample. The T-line fluorescence intensity was observed, and the inhibition rate under different T-line coating concentrations was calculated. Figure 12 , Figure 13 The results showed that when the T-line was coated with 0.1 and 0.05 mg / mL NOR-1-BSA, the fluorescence value of the negative sample was low. When the T-line was coated with 0.4 and 0.2 mg / mL NOR-1-BSA, the T-line and the C-line of the negative sample were visible to the naked eye, and the inhibition rates were close, and the inhibition rate of the latter was slightly higher, being 82.76%. Therefore, 0.2 mg / mL NOR-1-BSA was selected as the optimal T-line antigen coating concentration for detecting NOR.

[0234] Then under the optimal T-line coating concentration of 0.2 mg / mL NOR-1-BSA, the LFM-NOR mAb microsphere dosage was set to be 1.2, 1.0, 0.8 and 0.6 μL, the blank diluent was set as the negative sample, and the 20 ng / mL NOR standard diluent was set as the positive sample. The inhibition rates were compared, as shown in Figure 14 . As shown in Figure 15The inhibition rate was highest (87.90%) when the LFMs-SAR mAb dosage was 1.0 μL, and the data difference was not significant. Therefore, 1.0 μL was selected as the optimal microsphere dosage for NOR detection.

[0235] Example 8: Establishment and Cross-analysis of the LFM-ICS method for detecting SAR / NOR

[0236] Under the conditions of the optimal T-line antigen coating concentration of 0.2 mg / mL NOR-1-BSA and the optimal LFMs-SARmAb microsphere dosage of 0.8 μL obtained above, SAR standards were diluted to 20, 10, 5, 2.5, 1.25, 0.625, and 0 ng / mL, and the T-line fluorescence values ​​of samples at each concentration were detected. Figure 16 As shown. A SAR inhibition standard curve was plotted with the logarithm of the SAR standard concentration (lg C) on the x-axis and the binding rate (FIs / FIb) on the y-axis. The SAR inhibition standard curve is a linear equation: FIs / FIb = A3 × lgc(SAR) + D3, where A3 is the slope of the linear equation of the SAR inhibition standard curve, FIs is the T-line fluorescence value of the test sample, FIb is the T-line fluorescence value of the blank sample, c is the SAR standard concentration, and D3 is the intercept of the linear equation of the SAR inhibition standard curve.

[0237] like Figure 17A As shown, within the linear range of 0.625–20 ng / mL, the equation is FIs / FIb = -0.461 × lg c(SAR) + 0.631, R 2 =0.9835, its sensitivity IC 50 =1.93 ng / mL, which is similar to the IC50 of the SAR previously measured in our laboratory using SAR-1-OVA as the T-line envelope. 50 (1.37 ng / mL) is close, and the limit of detection is 0.43 ng / mL.

[0238] NOR standards were serially diluted to 256, 128, 64, 32, 16, 8, 4, 2, 1, 0.5, and 0 ng / mL, and detected using the established LFM-ICS method for detecting SAR. Inhibition curves were fitted, and the half-maximal inhibitory concentration (IC50) was calculated. 50 The cross-reactivity of LFMs-SAR mAb with NOR [IC] 50 (SAR Standard) / IC 50 [(NOR standard)]×100%; then substitute the FIs / FIb corresponding to each NOR concentration into the SAR suppression standard curve to calculate the theoretical concentration and cross-recovery rate (theoretical concentration / actual NOR standard concentration). The results show the IC50 of NOR against LFMs-SAR mAb. 50The cross-reactivity of the LFM-SAR mAb to NOR was 13.78 ng / mL, and the cross-reactivity of the LFM-SAR mAb to NOR was 13.97%. Figure 17B When the actual concentration of NOR was in the range of 4-256 ng / mL, the percentage of the theoretical concentration to the actual concentration was close, and the average was 11.88%; with the decrease of the concentration, the theoretical recovery concentration calculated by substituting FIs / FIb into the inhibition standard curve of SAR was less than the minimum detection limit 0.43 ng / mL, that is, the LFM-ICS method for detecting SAR could not detect NOR standard sample lower than 4 ng / mL. In general, the cross-reactivity of the LFM-SAR mAb to NOR could not be ignored for the LFM-ICS method.

[0239] Similarly, under the optimal T-line antigen coating concentration of 0.2 mg / mL NOR-1-BSA and the optimal microsphere dosage of 1.0 μL LFM-SAR mAb, the NOR standard was diluted to 10, 2.5, 0.625, 0.3125, 0.15625, 0.078125 and 0 ng / mL, and the inhibition standard curve of NOR was drawn, which was a straight line equation FIs / FIb=A4×lgc(NOR)+D4, wherein A4 was the slope of the straight line equation of the NOR inhibition standard curve, FIs was the T-line fluorescence value of the sample to be tested, FIb was the T-line fluorescence value of the blank sample, c was the NOR standard concentration, and D4 was the intercept of the straight line equation of the NOR inhibition standard curve.

[0240] Similarly, Figure 18 and Figure 19A In the linear range of 0.078125-10 ng / mL, the NOR inhibition standard curve was FIs / FIb=-0.2936×lg c(NOR)+0.2983 (R 2 =0.9823), the IC 50 was 0.21 ng / mL, and the LOD was 0.02 ng / mL.

[0241] The SAR standard was further diluted to 200, 50, 12.5, 3.125, 0.78125 and 0 ng / mL, and the FIs / FIb of each concentration was determined by the LFM-ICS method for detecting SAR, and the IC 50 , the cross-reactivity of the LFM-SAR mAb to NOR, and the cross-reactivity of SAR at each concentration were calculated. The IC 50 of SAR to the LFM-SAR mAb was greater than 1000 ng / mL, that is, the cross-reactivity of the LFM-SAR mAb to SAR was less than 0.021%; similarly, Figure 19BThe cross-recovery rate of the SAR standard sample was 0.05% at 50 and 200 ng / mL, and the other three concentrations of the SAR sample could not be detected, being negative samples. Therefore, the cross-reaction of the LFM-NOR mAb to SAR was negligible.

[0242] Example 9: Establishment of a combined LFM-ICS method for simultaneous detection of SAR and NOR based on two antigens

[0243] Under the optimal T-line antigen coating concentration of 0.2 mg / mL NOR-1-BSA and the optimal microsphere dosage of 0.8 μL LFM-SAR mAb and 1.0 μL LFM-NOR mAb, a combined LFM-ICS method for simultaneous detection of SAR and NOR was established. That is, according to the test strip preparation and LFM-ICS detection steps in Example 6, the T-line of the test strip was coated with 0.2 mg / mL NOR-1-BSA, the C-line was coated with 0.5 mg / mL goat anti-mouse IgG, and the mixed diluent of SAR and NOR standard samples at the same concentration was detected by LFM-SAR mAb and LFM-NOR mAb to form a combined LFM-ICS. Since the LFM-SAR mAb also has a non-negligible cross-reaction to NOR, and the cross-reaction of the LFM-NOR mAb to SAR is minimal, for the combined LFM-ICS method, about 10% of the NOR concentration involved in the cross-reaction should also be deducted when quantifying the mixed sample of SAR and NOR.

[0244] The SAR and NOR were diluted to 10, 5, 2.5, 1 and 0 ng / mL of the same concentration mixture, as shown in Table 7. The FIs / FIb obtained by detecting the SAR and NOR mixed sample at the same concentration by LFM-SAR mAb were introduced into the inhibition standard curve of SAR to calculate the total theoretical concentration, denoted by S. Similarly, the total theoretical concentration obtained by LFM-NOR mAb was denoted by N. Based on the above cross-reaction results, when the NOR concentration was ≥4 ng / mL, the total theoretical concentration was S+90%N; when the NOR concentration was <4 ng / mL, the total theoretical concentration was S+N. The calculation rule was established that "the sample that can be detected as positive by both LFM-SAR mAb and LFM-NOR mAb has a total theoretical concentration of S+90%N". Finally, the percentage of the corresponding theoretical total concentration to the actual total concentration at each concentration was obtained, with an average percentage of 102.90%. It was confirmed that the total theoretical concentration of the mixed sample of SAR and NOR calculated by the rule was very close to the actual total concentration, and similarly, reliable detection results could also be obtained by using the LFM-ICS method.

[0245] Table 7 Calculation rule for detecting SAR and NOR mixed sample at the same concentration by LFM-ICS method

[0246]

[0247] Example 10 Semi-quantitative detection of multiple fluoroquinolones by combined LFM-ICS

[0248] Thirteen FQs standards were diluted to 10 and 20 ng / mL, respectively, and the T-line fluorescence values of each positive and negative sample were determined by combined LFM-ICS under the optimal conditions for detecting SAR and NOR, as shown in Table 8. The measured FIs / FIb were substituted into the inhibition standard curve of SAR or NOR to obtain the theoretical concentrations S and N. The total theoretical concentration and total cross-recovery were calculated using the quantitative rules proposed in Example 9. The LFM-ICS method can also determine which subcategory the detected drug belongs to by comparing the results of LFM-SAR McAb and LFM-NOR McAb detection. The total cross-recovery of SAR, DIF, NOR, PEF, CIP, ENR, LML, FLU, NAD, ENO, OFL, and MAR at actual concentrations of 10 and 20 ng / mL was within the range of 30% to 110%, while the maximum residue limit of danofloxacin (DAN) in animal-derived food was 10 ng / g, and the total cross-recovery was more than 15%. The method established can detect residues at the 10 ng / mL level, and it is useful for actual sample detection. The LFM-ICS method for detecting fluoroquinolones can also achieve semi-quantitative screening of at least 13 FQs drugs. Ciprofloxacin, lomefloxacin, pefloxacin, ofloxacin, and norfloxacin, which are prohibited for use in food animals in China, are all included in this method.

[0249] Table 8 Theoretical concentrations and cross-recoveries of 13 fluoroquinolones detected by LFM-ICS using SAR McAb and NOR McAb, respectively

[0250]

[0251] * “\” indicates that the theoretical concentration calculated after substituting the standard curve is lower than the LOD, and it is judged as a negative sample. The total theoretical concentration is directly represented by S or N.

[0252] Discussion

[0253] 1. Heterologous coating and homologous coating

[0254] Based on the cross-reaction of NOR-1-BSA with SARMcAb, the NOR-1-BSA as a coating antigen, SARMcAb and NOR McAb as two monoclonal antibodies, and two kinds of lanthanide fluorescent microspheres coupled with the two monoclonal antibodies, the ELISA method and LFM-ICS method for simultaneous detection of SARMcAb and NOR McAb and semi-quantitative detection of at least 9 kinds of fluoroquinolones were established. NOR-1-BSA is a coating antigen prepared by modifying NOR hapten and coupling with BSA, and SARMcAb is a monoclonal antibody prepared by hybridoma technology using SARMcAb as an immunogen. Therefore, NOR-1-BSA is a heterologous coating for SARMcAb and the microspheres coupled with SARMcAb.

[0255] Current studies have shown that heterologous coating is related to the sensitivity and specificity of detection. Zhu Guo Nian et al. (Zhu Guo Nian, Mao Lijuan, Shi Haiyan, Huang Yali, Cheng Jingli, Synthesis and identification of artificial antigen of dichloroquinolinic acid, Chinese Agricultural Science 2005, 38(1): 86-90.) compared the sensitivity of homologous and heterologous coating using dichloroquinolinic acid as an example. When the spacer arm of heterologous coating is longer, it is more conducive to the binding of antigenic determinant and antibody. The strong affinity of antibody to heterologous coating reduces the binding of antibody to homologous hapten, reduces the competition ability of homologous hapten in the competition process, and thus reduces the sensitivity of detection. It is also pointed out that the stronger the affinity of antigen-antibody reaction, the higher the sensitivity. The competition ability of hapten and coating antigen in the competition reaction plays a key role. The study of Hammock et al. (Yoo Jung Kim, Young Ae Cho, Hye-Sung Lee, Yong Tae Lee, Shirley J. Gee, Bruce D. Hammock, Synthesis of haptens for immunoassay of organophosphorus pesticides and effect of heterology in hapten spacer arm length on immunoassay sensitivity, Analytica Chimica Acta 475 (2003) 85-96) showed that the heterology of the spacer arm in the heterologous coating and the structure of the hapten had a great influence on the sensitivity, while the length of the spacer arm had no significant effect.

[0256] From the perspective of spacer arm, SAR-1-OVA and NOR-1-BSA were prepared by connecting an aminoethyl on the N of piperazine ring through nucleophilic substitution reaction, and then connecting with OVA or BSA carrier through glutaraldehyde method. The spacer arms of the two are the same, both of which are -(CH2)2-N=C-(CH2)3-CH=N- structure. From the perspective of hapten structure, SAR and NOR belong to fluoroquinolones, which have 4-quinolone nucleus structure. Compared with NOR, SAR has a larger para-fluorophenyl at position 1. Therefore, SAR McAb and LFM-SAR mAb can bind to NOR-1-BSA, but NOR McAb and LFM-NOR mAb are difficult to bind to SAR-1-OVA due to the steric hindrance of SAR.

[0257] In this study, there is little difference between homologous and heterologous coating sensitivity. The titer of SAR McAb is between 1:80w-1:160w when coated with SAR-1-OVA and NOR-1-BSA, and the latter has a slightly higher titer. Therefore, the sensitivity of SAR McAb is close when coated with homologous and heterologous, and the working concentration of antigen and antibody is reduced when coated with NOR-1-BSA. Since the optimal coating concentration of SAR McAb and NOR McAb (LFM-SAR McAb and LFM-NOR McAb) is consistent when the optimal conditions are explored, it lays the foundation for detection with one coating.

[0258] 2. Cross reaction and simultaneous detection

[0259] In the production process of food animals, different types of drugs may be used to prevent and treat animal diseases, so multiple veterinary drug components may exist in animal-derived foods at the same time, and in veterinary drug residue detection, it is often necessary to detect multiple different types of veterinary drug residues in the same sample at the same time to determine whether it exceeds the national maximum residue limit. For different types of drugs, due to large structural differences, the cross reaction between antigen and antibody is small, and the simultaneous detection using specific antibodies of each type of drug has high accuracy; while for similar drugs of the same type, such as sulfonamides and quinolones, the cross reaction rate is relatively large, so a broad-spectrum monoclonal antibody can be used for semi-quantitative detection of a class of drugs.

[0260] The study firstly quantitatively detected the mixed samples of S AR and NOR, and then extended to semi-quantitatively detect a variety of fluoroquinolones. When the ratio of S AR and NOR in the mixed sample was 1:1, the recovery result of the mixed sample detected by NOR McAb was close to the actual amount of NOR standard added, because the cross-reactivity of NOR McAb to S AR was very small. The cross-reactivity of S AR McAb to NOR could not be ignored, and the recovery result of the mixed sample detected by S AR McAb contained the actual amount of S AR standard added and part of the amount of NOR standard. Therefore, the calculation rule of detecting the mixed sample by two kinds of McAbs was established. Since the content of NOR standard was more than 8 ng / mL, it could be detected by S AR McAb, and the cross-reactivity of S AR McAb to NOR was close to 10%. The content of NOR standard was more than 4 ng / mL, it could be detected by LFM-SAR mAb, and the cross-reactivity of LFM-SAR mAb to NOR was also close to 10%. Therefore, no matter what the ratio of S AR and NOR in the mixed sample was, as long as the sample could be detected by S AR McAb and NOR McAb (or LFM-SAR mAb and LFM-NOR mAb) as positive, the calculation rule could be used for quantitative detection.

[0261] The cross-reactivity of NOR McAb to DIF in S AR sub-class was still very small, and the cross-reactivity of S AR McAb to other 10 fluoroquinolones in NOR sub-class was different. That is, using S AR McAb and NOR McAb to detect one of the 13 FQs respectively, the results could determine which sub-class the drug belonged to. The combined ELISA method and combined LFM-ICS method established by the two kinds of McAbs and the quantitative calculation rule could also semi-quantitatively detect at least 13 kinds of fluoroquinolones. Especially when multiple FQs existed in the sample at the same time, a kind of superposition effect could be formed, so the detection of drug residues in combined drugs was more accurate.

Claims

1. A monoclonal antibody, NOR McAb, against the fluoroquinolone drug NOR, characterized in that, The monoclonal antibody NOR McAb comprises a heavy chain variable region and a light chain variable region. The heavy chain variable region comprises HCDR1, HCDR2, and HCDR3, and the light chain variable region comprises LCDR1, LCDR2, and LCDR3. The amino acid sequences of HCDR1, HCDR2, and HCDR3 in the heavy chain variable region of the monoclonal antibody NOR McAb are shown in SEQ ID NO: 5, SEQ ID NO: 6, and SEQ ID NO: 7, respectively. The amino acid sequences of LCDR1, LCDR2, and LCDR3 in the light chain variable region of the monoclonal antibody NOR McAb are shown in SEQ ID NO: 8, SEQ ID NO: 9, and SEQ ID NO: 10, respectively.

2. The monoclonal antibody NOR McAb as described in claim 1, characterized in that, The amino acid sequence of the heavy chain variable region of the monoclonal antibody NOR McAb is shown in SEQ ID NO: 11, and / or the amino acid sequence of the light chain variable region of the monoclonal antibody NOR McAb is shown in SEQ ID NO:

12.

3. The monoclonal antibody NOR McAb as described in claim 2, characterized in that, The heavy chain constant region of the monoclonal antibody NOR McAb is derived from the γ chain of the murine antibody, and / or the light chain constant region of the monoclonal antibody NOR McAb is derived from the λ chain of the murine antibody.

4. A monoclonal antibody SAR McAb against the fluoroquinolone drug SAR, characterized in that, The monoclonal antibody SAR McAb comprises a heavy chain variable region and a light chain variable region. The heavy chain variable region comprises HCDR1, HCDR2, and HCDR3, and the light chain variable region comprises LCDR1, LCDR2, and LCDR3. The amino acid sequences of HCDR1, HCDR2, and HCDR3 in the heavy chain variable region of the monoclonal antibody SAR McAb are shown in SEQ ID NO: 17, SEQ ID NO: 18, and SEQ ID NO: 19, respectively. The amino acid sequences of LCDR1, LCDR2, and LCDR3 in the light chain variable region of the monoclonal antibody SAR McAb are shown in SEQ ID NO: 20, SEQ ID NO: 9, and SEQ ID NO: 21, respectively.

5. The monoclonal antibody SAR McAb as described in claim 4, characterized in that, The amino acid sequence of the heavy chain variable region of the monoclonal antibody SAR McAb is shown in SEQ ID NO: 22, and / or the amino acid sequence of the light chain variable region of the monoclonal antibody SAR McAb is shown in SEQ ID NO:

23.

6. The monoclonal antibody SAR McAb as described in claim 5, characterized in that, The heavy chain constant region of the monoclonal antibody SAR McAb is derived from the γ chain of the murine antibody, and / or the light chain constant region of the monoclonal antibody SAR McAb is derived from the λ chain of the murine antibody.

7. An isolated nucleic acid, characterized in that, The nucleic acid encodes the monoclonal antibody NORMcAb as described in claim 1 or the monoclonal antibody SAR McAb as described in claim 4.

8. The nucleic acid as described in claim 7, characterized in that, The sequence of the nucleic acid encoding the heavy chain variable region of the monoclonal antibody NOR McAb is as shown in SEQ ID NO: 1, and / or, the sequence of the nucleic acid encoding the light chain variable region of the monoclonal antibody NOR McAb is as shown in SEQ ID NO: 2; or, The sequence of the nucleic acid encoding the heavy chain variable region of the monoclonal antibody SAR McAb is shown in SEQ ID NO: 13, and / or the sequence of the nucleic acid encoding the light chain variable region of the monoclonal antibody SAR McAb is shown in SEQ ID NO:

14.

9. A recombinant expression vector, characterized in that, The recombinant expression vector comprises the nucleic acid as described in claim 7 or 8.

10. A transformant, characterized in that, The transformant comprises the nucleic acid as described in claim 7 or 8, or the recombinant expression vector as described in claim 9, wherein the host cell of the transformant is a prokaryotic cell or a eukaryotic cell.

11. A kit for detecting fluoroquinolone drugs, characterized in that, The kit contains a first antibody, which is the monoclonal antibody NOR McAb as described in claim 1 and / or the monoclonal antibody SAR McAb as described in claim 4.

12. The kit according to claim 11, characterized in that, The kit further comprises a coating antigen, which is a complete antigen prepared by conjugating a hapten NOR with a carrier protein; and / or, the kit further comprises a second antibody that specifically binds to the first antibody.

13. The kit according to claim 12, characterized in that, The carrier protein is bovine serum albumin.

14. The kit according to claim 13, characterized in that, The coating material is NOR-1-BSA, which is an aminoethyl group attached to the nitrogen atom of the piperazine ring of the hapten NOR and coupled with bovine serum albumin.

15. The kit according to claim 12, characterized in that, The second antibody is enzyme-labeled, and the kit further includes ELISA coating solution, ELISA blocking solution, ELISA substrate chromogenic solution and / or ELISA stop solution, wherein the ELISA substrate chromogenic solution comprises ELISA substrate chromogenic solution A and ELISA substrate chromogenic solution B in a volume ratio of 1:

1.

16. The kit according to claim 15, characterized in that, The second antibody is labeled with horseradish peroxidase, and / or the stock solution concentration of the second antibody is 0.8 mg / mL to 1.2 mg / mL, with a dilution ratio of 1:18000 to 1:22000, where the dilution ratio is the volume ratio of the stock solution to 1×PBST containing FBS, and / or the ELISA coating solution is a carbonate buffer with a pH of 9.4 to 9.8 and a concentration of 0.04 mol / L to 0.06 mol / L, and / or the ELISA blocking solution is 0.8% to 1.2% gelatin by weight, and / or the ELISA substrate chromogenic solution A contains 25 g / L to 30 g / L sodium acetate, 3.0 g / L to 3.5 g / L citric acid, and 0.5 mL / L to 0.7 mL / L 30% H2O2, and / or the ELISA substrate chromogenic solution B contains 0.25 g / L to 0.35 g / L TMB, 0.3 g / L sodium acetate, and 0.3 g / L citric acid. The ELISA stop solution consists of 0.5 g / L EDTA-2Na, 1.8 g / L citric acid, and 90 mL / L glycerol, and / or 1.8 mol / L 2.2 mol / L sulfuric acid.

17. The kit as claimed in claim 16, characterized in that, The second antibody is goat anti-mouse IgG-HRP.

18. The kit according to claim 15, characterized in that, The concentration of the coating antigen is 0.25 μg / mL to 0.10 μg / mL; and / or, the concentration of the antibody stock solution of the NOR McAb or the SAR McAb is 5.0 mg / mL to 8.0 mg / mL, the antibody dilution ratio of the SAR McAb is 1:800000 to 1:1200000, and / or the antibody dilution ratio of the NOR McAb is 1:600000 to 1:1000000; the dilution ratio is the volume ratio of the antibody stock solution to 1×PBST containing FBS.

19. The kit according to claim 12, characterized in that, The first antibody is conjugated with a marker, and the coating antigen and the second antibody are respectively immobilized on the test strip. The coating antigen is immobilized on the T line of the test strip, and the second antibody is immobilized on the C line of the test strip.

20. The kit according to claim 19, characterized in that, The test strip is a test strip with an NC film, an absorbent pad, and a sample pad sequentially attached to a PVC base plate; The first antibody was conjugated with lanthanide fluorescent microspheres to obtain antibody-microsphere conjugates LFMs-mAb; The second antibody is goat anti-mouse IgG; and / or, the coating agent is NOR-1-BSA.

21. The kit according to claim 20, characterized in that, The concentration of the goat anti-mouse IgG is 0.4 mg / mL to 0.6 mg / mL; and / or the concentration of the NOR-1-BSA is 0.05 to 0.4 mg / mL.

22. The kit according to claim 21, characterized in that, The concentration of the goat anti-mouse IgG is 0.5 mg / mL; and / or the concentration of the NOR-1-BSA is 0.2 mg / mL.

23. The kit according to claim 20, characterized in that, The preparation method of the LFMs-mAb includes the following steps: (1) Dilute lanthanide fluorescent microspheres with MES buffer and sonicate them; (2) After adding NHS and EDC for activation, add MES buffer for further activation, sonicate to disperse, centrifuge to discard the supernatant, and collect the precipitate; (3) Resuspend the precipitate in step (2) in a borate buffer solution containing H3BO3 and borax solution; (4) Add the NOR McAb or the SAR McAb, mix well, centrifuge and discard the supernatant, and collect the precipitate; (5) Resuspend the precipitate in step (4) with microsphere blocking solution, block it, then mix and centrifuge to discard the supernatant and collect the precipitate. The microsphere blocking solution contains 6~7 g / L BSA, 1.5~2.5 g / L NaN3 and 5.8~6.2 g / L Tris, and the pH is 7.5~8.

5. (6) Add microsphere diluent to the precipitate in step (5), and mix by ultrasonication to obtain LFM-SAR mAb or LFM-NORmAb. The microsphere diluent contains 6~7 g / L BSA, 1.5~2.5 g / L NaN3, 5.8~6.2 g / L Tris and 6~7 g / L PVP, and the pH is 7.5~8.

5.

24. The kit according to claim 23, characterized in that, The preparation method includes the following steps: (1) Dilute lanthanide fluorescent microspheres with 23-27 mmol / L MES buffer to a volume ratio of 0.8-1.2% and then sonicate them. (2) After activation with 15-17 mg / mL NHS and 7-9 mg / mL EDC, add 23-27 mmol / L MES buffer for further activation, sonicate to disperse, centrifuge to discard the supernatant, and collect the precipitate; (3) Resuspend the precipitate in step (2) in a borate buffer solution containing 0.08~0.12 mol / L H3BO3 solution and 23~27 mmol / L borax solution; (4) Add the NOR McAb or SAR McAb at a concentration of 0.8 mg / mL to 1.2 mg / mL, mix well, centrifuge and discard the supernatant, and collect the precipitate; (5) Resuspend the precipitate in step (4) with a microsphere blocking solution with pH 7.5~8.5, block it, then mix and centrifuge to discard the supernatant and collect the precipitate. The microsphere blocking solution contains 6~7 g / L BSA, 1.5~2.5 g / L NaN3 and 5.8~6.2 g / L Tris. (6) Add a microsphere diluent with a pH of 7.5 to 8.5 to the precipitate in step (5), and mix it by ultrasonication to obtain LFM-SAR mAb or LFM-NOR mAb. The microsphere diluent contains 6 to 7 g / L BSA, 1.5 to 2.5 g / L NaN3, 5.8 to 6.2 g / L Tris and 6 to 7 g / L PVP.

25. A method for detecting fluoroquinolone drugs, characterized in that, The method includes using the monoclonal antibody NOR McAb as described in any one of claims 1 to 3 and / or the monoclonal antibody SARMcAb as described in any one of claims 4 to 6; or the method includes using the kit as described in any one of claims 11 to 24.

26. The method as described in claim 25, characterized in that, The method includes the following steps: (1) Dilute the coating agent with ELISA coating solution, add it to the microplate for coating, and wash the plate with sample diluent, wherein the sample diluent is 1×PBS containing 0.4 mL / L~0.6 mL / L Tween-20 and pH 7.2~7.6; (2) Add ELISA blocking solution, incubate, and then wash the plate with sample dilution solution; (3) Add the sample to be tested, and the diluted monoclonal antibody NOR McAb or the monoclonal antibody SARMcAb, incubate, and then wash the plate with sample dilution buffer. (4) Add the enzyme-labeled second antibody, incubate, and wash the plate with sample dilution buffer; add ELISA colorimetric solution for color development; add ELISA stop solution to terminate the reaction; (5) Use an enzyme-linked immunosorbent assay (ELISA) reader to detect and read the absorbance value; substitute the read absorbance value into the SAR standard curve and the NOR standard curve respectively, and calculate the theoretical concentration S and the theoretical concentration N respectively. The total theoretical concentration of fluoroquinolone drugs in the sample to be tested = S + 90%N.

27. The method as described in claim 26, characterized in that, The sample diluent described in step (1) contains 0.5 mL / L Tween-20 1×PBS with a pH of 7.

4.

28. The method as described in claim 25, characterized in that, The method includes the following steps: (1) Mix LFM-SAR mAb or LFM-NOR mAb with the sample to be tested at a volume ratio of 0.6:100 to 1.2:100; (2) After the mixture in step (1) reacts, it is added to the sample pad of the test strip; (3) After the reaction in the dark, observe the fluorescence under a UV lamp and read the fluorescence values ​​of the T line and C line using a high-sensitivity fluorescence analyzer; (4) Substitute the measured fluorescence values ​​into the SAR inhibition standard curve and the NOR inhibition standard curve respectively, and calculate the theoretical concentration S and theoretical concentration N respectively. The total theoretical concentration of fluoroquinolone drugs in the sample to be tested = S + 90%N.

29. The method as described in claim 28, characterized in that, The volume ratio of the LFM-SAR mAb to the sample to be tested in step (1) is 0.8:100; and / or the volume ratio of the LFM-NOR mAb to the sample to be tested is 1:

100.

30. The use of the monoclonal antibody NOR McAb as described in any one of claims 1 to 3, the monoclonal antibody SAR McAb as described in any one of claims 4 to 6, the nucleic acid as described in claim 7 or 8, the recombinant expression vector as described in claim 9, the transformant as described in claim 10, or the kit as described in any one of claims 11 to 24 in the detection of fluoroquinolone drugs.

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