A nanobody with broad-spectrum recognition of quinolone drugs and its preparation method and application

Abstract

This invention relates to the field of biotechnology, and more particularly to a nanobody with broad-spectrum recognition of quinolone drugs, its preparation method, and its applications. This invention utilizes artificial antigens of quinolone drugs to immunize camelids, constructing a phage-display nanobody library. A highly sensitive nanobody capable of broadly recognizing quinolone drugs is obtained through a conformational superposition screening method. This nanobody can recognize at least 39 quinolone drugs. Furthermore, this antibody exhibits strong environmental adaptability, tolerating alkaline solutions at pH 12, 80% methanol, and 40% acetonitrile solutions. Therefore, the quinolone drug nanobody prepared by this invention, with its broad recognition spectrum, high affinity, and good tolerability, is of great significance for the establishment of multi-residue immunoassay technology for quinolone drugs and for product development.

Description

Technical Field

[0001] This invention relates to the field of biotechnology, and in particular to a nanobody that broadly recognizes quinolone drugs, its preparation method, and its application. Background Technology

[0002] Quinolones (QNs) are a class of broad-spectrum antibacterial drugs with a 4-quinolone ring. Due to their low cost, broad antibacterial spectrum, strong antibacterial activity, and high bioavailability, they are widely used in animal husbandry, aquaculture, and the treatment of human diseases. However, once these drugs accumulate in the human body beyond safe limits, they can cause central nervous system toxicity, skin toxicity, and phototoxicity. Furthermore, low concentrations of quinolone drug residues in animal-derived foods can easily induce drug resistance in various pathogens, seriously threatening public health safety. Therefore, establishing a highly sensitive and rapid detection technology that can broadly and uniformly identify quinolones is crucial for timely and accurate monitoring of their residues.

[0003] Currently, the main technologies for detecting quinolone drug residues are instrumental analysis and immunoassay. Instrumental analysis has the advantage of high accuracy, but the instruments are expensive and difficult to operate, making it unsuitable for rapid screening of large-scale samples on-site. Immunoassay, based on antigen-antibody specific reactions, has the advantages of low cost, simplicity, speed, and high sensitivity, and is widely used in the rapid detection of hazardous compounds. Antibodies are the core reagents in immunoassays, determining the sensitivity, specificity, and stability of the analytical method. However, existing antibodies suffer from drawbacks such as heterogeneous affinity, low stability, difficulty in modification, and high production costs, necessitating the development of novel antibody materials. In the existing technology, the paper "Characterization, specific recognition, and the performance in fish matrix of a shark-derived single-domain antibody against enrofloxacin" discloses a shark-derived quinolone drug nanobody; however, this nanobody can only recognize enrofloxacin and does not possess broad-spectrum recognition.

[0004] Therefore, there is an urgent need to develop a nanobody for quinolone drugs with broad-spectrum recognition. Summary of the Invention

[0005] This invention involves isolating lymphocytes from peripheral blood of camels immunized with quinolone drugs, extracting RNA, constructing a quinolone nanobody phage display library, and obtaining a highly stable, highly sensitive nanobody capable of uniformly recognizing multiple quinolone drugs through a screening method based on the conformational classification and superposition of quinolone drugs. The antibody of this invention is derived from camels and also possesses advantages such as high tolerability, simple structure, and ease of modification.

[0006] Based on this, in a first aspect, the present invention provides a nanobody of a quinolone drug with broad-spectrum recognition or a quinolone drug-specific recognition fragment, comprising the following complementary determination regions:

[0007] CDR-H1: NYPMT;

[0008] CDR-H2: TIDSGGSSTYYADAVKG;

[0009] CDR-H3:DRYIGDDYRGNP.

[0010] Preferably, the amino acid sequence of the quinolone drug nanobody or the quinolone drug-specific recognition fragment is as follows:

[0011] (1) As shown in SEQ ID NO.1; or

[0012] (2) An amino acid sequence obtained by linking a tag peptide to the N-terminus and / or C-terminus of the sequence shown in SEQ ID NO.1.

[0013] In a second aspect, the present invention provides a nanobody encoding the quinolone drug or a nucleic acid molecule encoding a quinolone drug-specific recognition fragment.

[0014] Because of the degeneracy of genetic codons, the nucleic acid molecules can vary depending on the application.

[0015] Preferably, the nucleotide sequence of the nucleic acid molecule is shown in SEQ ID NO.2.

[0016] The nucleic acid molecules of this invention can be expressed using suitable expression systems to obtain corresponding proteins or polypeptides. These expression systems include, but are not limited to, bacterial, yeast, filamentous fungi, animal cells, insect cells, plant cells, or cell-free expression systems.

[0017] Thirdly, the present invention provides a biomaterial containing a nanobody of the quinolone drug or a quinolone drug-specific recognition fragment, or the nucleic acid molecule; the biomaterial includes at least one of recombinant DNA, expression cassette, plasmid vector, viral vector, engineered bacteria, and transgenic cell line.

[0018] Fourthly, the present invention provides a quinolone drug detection reagent or kit, wherein the detection reagent or kit contains a nanobody or quinolone drug-specific recognition fragment of the quinolone drug, or the nucleic acid molecule, or the biological material.

[0019] Fifthly, the present invention provides a pharmaceutical product containing a nanobody of the quinolone drug or a quinolone drug-specific recognition fragment, or the nucleic acid molecule, or the biological material.

[0020] In a sixth aspect, the present invention provides an ELISA detection kit for the analysis of quinolone drug residues. The ELISA detection kit includes a box, a removable enzyme-labeled plate disposed within the box, and reagents disposed within the box. Each well of the enzyme-labeled plate is coated with an artificial antigen of a quinolone drug. The reagents include nanobodies of the quinolone drug or quinolone drug-specific recognition fragments, as well as quinolone drug standard solutions, enzyme-labeled secondary antibodies, buffer solutions (e.g., PBS), washing solutions (e.g., PBST), chromogenic solutions, and reaction termination solutions.

[0021] In a seventh aspect, the present invention provides the use of the nanobody or quinolone drug-specific recognition fragment of the quinolone drug, the nucleic acid molecule, the biomaterial, the reagent or kit, the drug, or the ELISA detection kit in at least one of the following aspects:

[0022] (1) Detection of quinolone drugs;

[0023] (2) Preparation of quinolone drug detection reagents or kits;

[0024] (3) Enrichment and purification of quinolone drugs;

[0025] (4) Preparation of enrichment and / or purification reagents for quinolone drugs.

[0026] Eighthly, the present invention provides a method for preparing nanobodies or quinolone-specific recognition fragments of the aforementioned quinolone drugs, characterized in that it includes: preparing a phage antibody library and then performing five rounds of panning sequentially, wherein the first round of panning uses acid elution; the second round of panning uses elution standards I and II, wherein standard I is norfloxacin and sarafloxacin, and standard II is ofloxacin and enrofloxacin; the third round of panning uses standard III, wherein standard III is dafluxacin, moxifloxacin, pazufloxacin, and garazoxin; the fourth round of panning uses standard IV, wherein standard IV is oxaquinic acid, flumethylquine, nalidixic acid, and pipemidic acid; and the fifth round of panning uses standards I to IV.

[0027] When using the nanobodies or quinolone-specific recognition fragments of the present invention to detect the specific binding ability with quinolone drugs, methods include, but are not limited to, enzyme-linked immunosorbent assay (ELISA), chemiluminescence immunoassay (CLIA), fluorescence immunoassay (FIA), lateral flow immunoassay (LFIA), immunoarray method, and affinity chromatography.

[0028] In specific implementation, the nanobody of the quinolone drug shown in SEQ ID NO.1 can also be used as a precursor and modified by random or site-directed mutagenesis techniques to obtain mutants with better properties (water solubility, stability, affinity and specificity, etc.). These mutants are all within the scope of protection of this invention.

[0029] Compared with the prior art, the beneficial effects of the present invention are as follows:

[0030] The quinolone drug nanobodies provided by this invention can accurately and sensitively detect multiple quinolone drug residues in samples, IC50. 50 The concentration ranges from 1.1 to 102.1 ng / mL. This antibody possesses characteristics such as simple structure, resistance to alkaline solutions and organic solvents, and ease of production, making it significant for low-cost, large-volume on-site detection of quinolone drug residues. Attached Figure Description

[0031] Figure 1 This is a structural diagram of a quinolone drug hapten.

[0032] Figure 2 Structural analysis of nanobodies for quinolone drugs.

[0033] Figure 3 This is the standard curve established for norfloxacin detection using the indirect competitive ELISA method in Example 4 of this invention.

[0034] Figure 4 The results of the pH tolerance test of the quinolone drug nanobody in Example 5 of this invention are shown.

[0035] Figure 5 The results of the methanol tolerance test of the quinolone drug nanobody in Example 5 of this invention are shown.

[0036] Figure 6 The results of the acetonitrile tolerance test of the quinolone drug nanobody in Example 5 of this invention are shown. Detailed Implementation

[0037] To make the objectives, technical solutions, and advantages of this invention clearer, the technical solutions of this invention will be clearly and completely described below. Obviously, the described embodiments are only some embodiments of this invention, not all embodiments. Based on the embodiments of this invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of this invention.

[0038] Unless otherwise specified, all techniques or conditions used in the examples were performed using conventional methods or in accordance with techniques or conditions described in the literature in this field, or according to the product instructions. Reagents and instruments used without specified manufacturers were all conventional products that could be purchased from legitimate channels. The conventional methods referred to Sambrook et al.'s Molecular Cloning: A Laboratory Manual (Sambrook J & Russell DW, 2001) or the conditions recommended in the manufacturer's instructions.

[0039] Example 1: Construction of an anti-quinolone drug nanobody immune library

[0040] 1. Norfloxacin (NOR) Figure 1 Artificial antigens NOR-OVA and NOR-KLH were obtained by conjugating them with ovalbumin (OVA) and keyhole limpet hemocyanin (KLH) via the active ester method.

[0041] 2. Emulsify 300 μg of NOR-KLH with an equal volume of Freund's complete adjuvant and administer subcutaneous injections at multiple sites in the neck of Bactrian camels. Booster immunizations are performed every 3 weeks using 150 μg of NOR-KLH mixed with an equal volume of Freund's incomplete adjuvant. Seven days after each immunization, venous blood is collected, and heavy chain antibodies and conventional antibodies are separated in the serum. The specificity and sensitivity of the heavy chain antibodies are determined using an indirect competitive ELISA method. The blood sample with the highest sensitivity is selected for lymphocyte separation.

[0042] 3. Total RNA was extracted from lymphocytes using the Trizol method, and the first strand of cDNA was synthesized according to the Invitrogen reverse transcription kit instructions.

[0043] 4. The variable region encoding gene of the heavy chain antibody was obtained by two rounds of PCR using Taq Mix DNA polymerase. The upstream primer sequence for the first round of PCR was F1: 5′-GTCCTGGCTGCTCTTCTACAAGG-3′ (SEQ ID NO.3), and the downstream primer sequence was R1: 5′-GGTACGTGCTGTTGAACTGTTCC-3′ (SEQ ID NO.4). The PCR reaction conditions were: 95°C for 5 min; 95°C for 30 s, 55°C for 30 s, 72°C for 1 min, 30 cycles; 72°C for 10 min. The PCR products were subjected to 1% agarose gel electrophoresis, and the target fragment of 650bp-750bp was recovered using a DNA fragment recovery kit. The recovered target fragment was used as a template for the second round of PCR. The upstream primer sequence for the second round of PCR was F1: 5′-CATGCCATGACTGTGGCCCAGGCGGCCGAGTCTGGGGGAGR-3′ (SEQ ID NO. 5, R indicates a degenerate primer, either A or G), and the downstream primer sequence was R1: 5′-CATGCCATGACTCGCGGCCGGCCTGGCCTGAGGAGACGGTGACCTGGGT-3′ (SEQ ID NO. 6). The PCR products were subjected to 1% agarose gel electrophoresis, and the target fragment (400-500 bp) was recovered using a DNA fragment recovery kit. After quantification, the fragment was stored at -20 °C for later use. The phage plasmid pcomb3x and the amplified products from the second round of PCR were digested with sfiI restriction enzyme at 50 °C for 3 hours. After identification by 1% agarose gel electrophoresis, the target fragment was excised and recovered. The target fragment was ligated using T4 DNA ligase at 16 °C overnight. The ligation product was dialyzed to remove salt ions, and after concentration determination, it was stored at -20 °C for later use.

[0044] 5. Take 100 ng of the ligation product and perform electroporation transformation in *E. coli* ER2738 electroporation competent cells (10 tubes in total). Serially dilute the transformed bacterial culture and plate it on ampicillin-containing plates. Incubate overnight at 37 °C and count colonies to calculate the library size. Randomly select single colonies from the plates and send them to a biotechnology company for sequencing to assess library diversity. Centrifuge the remaining bacterial culture, discard the supernatant, and plate the precipitate on ampicillin-containing plates. Incubate overnight at 37 °C. Scrape the bacterial colony on the culture plates with 2×YTG medium. The resulting quinolone drug nanobody gene library is then aliquoted with 20% glycerol and stored at -80 °C for later use.

[0045] 6. Take 1 mL of the nanobody gene library and place it in 200 mL of 2×YTG liquid medium containing ampicillin. Incubate at 37 °C and 240 rpm until OD reaches 100%.600 ≈0.6, add helper phage M13KO7 at a multiplicity of infection (MOI) of 20:1, incubate at 37 °C for 30 min, shake at 240 rpm for 30 min, centrifuge at 5000 rpm for 10 min, discard the supernatant, resuspend the cells in 2×YT medium containing kanamycin and ampicillin, and incubate overnight at 37 °C and 240 rpm. Centrifuge the overnight cells at 4000 rpm for 20 min, add 1 / 5 volume of 20% PEG-NaCl solution to the supernatant, and incubate at 4 °C overnight. Centrifuge at 12000 rpm for 20 min, resuspend the precipitate in 1 mL PBS, and filter through a 0.45 μm membrane to obtain the quinolone drug phage-displaying nanobody library. Take 10 μL of the phage-displaying nanobody library and perform serial dilutions, incubate at 37 °C for 30 min to infect logarithmic-phase E. coli, and calculate the library capacity by colony counting.

[0046] Example 2: Panning and Identification of Antiquinolone Drug Nanobodies

[0047] Quinolone drug nanobodies were selected from the phage display nanobody library prepared in Example 1 using a conformational classification and superposition screening method. The specific screening scheme is shown in Table 1.

[0048] 1. Add 100 μL of the prepared coated original NOR-OVA to each well of a microplate and coat at 37 °C for 2 h. Discard the liquid in the wells, wash 3 times with PBST, and spin dry. Add 300 μL of 5% skim milk to each well and block at 37 °C for 1.5 h. Discard the liquid in the wells, wash 3 times with PBST, and spin dry for later use.

[0049] 2. First round of panning: Add 5% OVA solution to the prepared phage antibody library and block at 37 °C for 1 h. Add 100 μL / well of the blocked phage antibody library to the coated plate (original coating concentration 10 μg / mL) and incubate at 37 °C for 1 h; discard the liquid in the wells, wash 10 times with PBST, then wash 5 times with PBS, and spin dry. Add 100 μL of eluent (0.2M glycine, pH 2.2) to each well, place on a shaker, react at 25 °C for 8 min, and add 30 μL of Tris-HCl (1 M, pH 8.0). Take 10 μL for titer determination, and use the remaining eluent to infect logarithmic-phase Escherichia coli ER2738 to prepare the next round of phage library according to the method in Example 1.

[0050] Rounds 2-5 of selection: To obtain nanobodies capable of broadly recognizing quinolone drugs, 39 quinolone drugs were divided into four classes based on their conformations. Representative quinolone drugs were selected for competitive elution. The prepared phage antibody library was blocked using the method described in the first round. 100 μL / well of the blocked phage antibody library was added to the pre-coated plate (original concentration 1 μg / mL), and incubated at 37°C for 1 h. The liquid in the wells was discarded, and the plate was washed 10 times with PBST, then 5 times with PBS, and dried. 100 ng / mL of quinolone drug standards I (norfloxacin and sarafloxacin) and II (ofloxacin and enrofloxacin) were added to each well, and the plate was incubated at 37°C for 1 h for competitive elution. 10 μL was used for titer determination, and the remaining eluent was used to infect logarithmic-phase *E. coli* ER2738 to prepare the next round of phage libraries according to the method described in Example 1. The third and fourth rounds of selection were the same as the second round. The third round used quinolone drug standard III (dafloxacin, moxifloxacin, pazufloxacin, and galafloxacin), the fourth round used quinolone drug standard IV (oxaquinic acid, flumethin, nalidixic acid, and pipemidic acid), and the fifth round reduced the original coating concentration to 0.1 μg / mL and used quinolone drug standards I-IV at 1 ng / mL.

[0051] 3. After five rounds of selection, single clones were randomly selected and their titer, sensitivity, and cross-reactivity were determined using indirect competitive phage-ELISA. Positive clones with broad sensitivity and cross-reactivity in the phage-ELISA results were sent to a biotechnology service company for sequencing, yielding the base sequence of the quinolone drug nanobody (SEQ ID NO. 2), whose encoded amino acid sequence is shown in SEQ ID NO. 1. The structural analysis of this quinolone drug nanobody is as follows: Figure 2 As shown, its amino acid sequence includes four framework regions (FRs) and three complementarity-determining regions (CDRs).

[0052] Table 1. Five rounds of phage selection conditions for camels and corresponding phage input / output amounts and enrichment results.

[0053]

[0054] Example 3: Soluble Expression, Purification, and Identification of Quinolone Drug Nanobodies

[0055] 1. The obtained quinolone drug nanobody sequence was sent to a biotechnology service company for synthesis. The synthesized nanobody and expression vector pJB33 were digested with the restriction endonuclease sfiI. The quinolone drug nanobody gene and pJB33 vector gene were recovered by 1% agarose gel electrophoresis. The obtained quinolone drug nanobody gene fragment was ligated to the expression vector pJB33 using T4 DNA ligase to obtain a recombinant expression plasmid, which was then transformed into BL21(DE3) competent cells. The transformed bacterial culture was plated on 2×YT plates containing ampicillin and incubated upside down at 37 °C for 16 h. Single colonies were picked, cultured with shaking at 240 rpm for 12 h, and subjected to bacterial PCR. Positive clones with the correct band size were sent to the biotechnology service company for sequencing.

[0056] 2. Inoculate the correctly sequenced positive clones into 2×TY medium containing ampicillin and incubate at 37 °C with shaking until OD. 600 ≈0.6-0.8, add IPTG (0.1 mM), and induce expression at 150 rpm and 25 °C for 16 h. After induction, centrifuge the bacterial culture at 4000 rpm for 15 min and discard the supernatant. Extract periplasmic protein using ultrasonic disruption. Then purify by nickel column chromatography using gradient elution with different concentrations of imidazole (100 mM, 150 mM, 200 mM, 250 mM, 300 mM). The purified nanobodies were identified using SDS-PAGE and indirect competitive ELISA.

[0057] Example 4: Quinolone Drug Nanobodies for the Detection of Quinolone Drugs

[0058] 1. Determine the optimal antigen-antibody concentration using the checkerboard method. Serially dilute the original NOR-OVA to different concentrations for coating. After blocking, add 50 μL of PBS to each well, followed by 50 μL of serially diluted quinolone drug nanobody. After reacting for 30 min, wash and add enzyme-labeled secondary antibody. After incubation, add TMB chromogenic buffer, react for 10 min, and then add stop solution. Read the OD value using a microplate reader. 450 The optimal working concentrations of antigen and antibody for coating are determined by selecting wells with OD values ​​of 1.5-2.0.

[0059] 2. Based on the determined initial coating concentration, 100 μL / well was used for coating at 37°C for 2 hours. The plates were washed three times with PBST and then dried. 150 μL of 5% skim milk (w / v) was added to each well, and the plates were blocked at 37°C for 1 hour. After washing three times with PBST, the plates were dried. 50 μL of serially diluted quinolone drug standard solution was added to each well, along with 50 μL of the quinolone drug nanobody prepared in Example 2. The reaction was carried out at 37°C for 30 minutes. After washing three times with PBST and drying, 100 μL of horseradish peroxidase-labeled rabbit anti-C-MYC tag antibody was added to each well, and the plates were incubated at 37°C for 30 minutes. After washing three times with PBST and drying, 100 μL of TMB chromogenic solution was added to each well, and the plates were developed at 37°C in the dark for 10 minutes. 50 μL of stop solution (2 M H₂SO₄) was added to each well, and the absorbance was read at 450 nm. Using the logarithm of each quinolone drug concentration as the x-axis and the OD value corresponding to each quinolone drug concentration as the y-axis, a standard curve was plotted using Origin 8.5 software with four-parameter logarithmic fitting.

[0060] The results are as follows Figure 3 As shown, the antibody has an IC50 response to norfloxacin. 50 With a concentration of 1.2 ng / mL, it has a broad recognition spectrum and can identify at least 39 quinolone drugs, including ofloxacin, norfloxacin, lomefloxacin, pefloxacin, enrofloxacin, ciprofloxacin, sarafloxacin, diflufloxacin, and sparfloxacin (Table 2).

[0061] Table 2 shows the IC50 values ​​of nanobodies against 39 QNs. 50 value

[0062]

[0063] Example 5: Tolerability Analysis of Quinolone Drug Nanobodies

[0064] 1. The procedure for determining the pH tolerance and organic solvent tolerance of quinolone drug nanobodies is the same as that for establishing the indirect competitive ELISA standard curve. The antibodies were reacted in different pH solutions (5, 6, 7, 8, 9, 10, 11, and 12), different concentrations of methanol solution (80%, 40%, 20%, 10%, 5%, and 2.5%), and different concentrations of acetonitrile solution (40%, 20%, 10%, 5%, and 2.5%), and their IC50 values ​​were measured under different conditions. 50 value.

[0065] 2. Results are as follows Figure 4 , Figure 5 and Figure 6 As shown, the antibody can still function normally in PB solution at pH 12, 80% methanol, and 40% acetonitrile, indicating that it has strong tolerance to alkaline solutions and organic solvents.

[0066] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, and not to limit them; although the present invention has been described in detail with reference to the foregoing embodiments, those skilled in the art should understand that modifications can still be made to the technical solutions described in the foregoing embodiments, or equivalent substitutions can be made to some of the technical features; and these modifications or substitutions do not cause the essence of the corresponding technical solutions to deviate from the spirit and scope of the technical solutions of the embodiments of the present invention.

Claims

1. A nanobody for a quinolone drug, characterized in that, Including the following complementary determinant regions: CDR-H1: NYPMT; CDR-H2: TIDSGGSSTYYADAVKG; CDR-H3:DRYIGDDYRGNP.

2. The nanobody of a quinolone drug according to claim 1, characterized in that, The amino acid sequence is as follows: (1) As shown in SEQ ID NO.1; or (2) An amino acid sequence obtained by linking a tag peptide to the N-terminus and / or C-terminus of the sequence shown in SEQ ID NO.

1.

3. A nucleic acid molecule encoding a nanobody of a quinolone drug as described in claim 1 or 2.

4. The nucleic acid molecule according to claim 3, characterized in that, The nucleotide sequence is shown in SEQ ID NO.

2.

5. A biomaterial, characterized in that, The biomaterial contains a nanobody of a quinolone drug as described in claim 1 or 2, or a nucleic acid molecule as described in claim 3 or 4; the biomaterial includes at least one of recombinant DNA, expression cassette, plasmid vector, viral vector, engineered bacteria, and transgenic cell line.

6. A quinolone drug detection reagent or kit, characterized in that, The detection reagent or kit contains a nanobody of a quinolone drug as described in claim 1 or 2, or a nucleic acid molecule as described in claim 3 or 4, or a biomaterial as described in claim 5.

7. A medicine, characterized in that, The drug contains a nanobody of a quinolone drug as described in claim 1 or 2, or a nucleic acid molecule as described in claim 3 or 4, or a biomaterial as described in claim 5.

8. An ELISA detection kit for the analysis of quinolone drug residues, characterized in that, The ELISA test kit includes a box, a removable enzyme-labeled plate disposed within the box, and reagents disposed within the box; each well of the enzyme-labeled plate is coated with a quinolone drug artificial antigen, and the reagents include nanobodies of the quinolone drug as described in claim 1 or 2, as well as quinolone drug standard solutions, enzyme-labeled secondary antibodies, buffer solutions, washing solutions, chromogenic solutions, and reaction termination solutions.

9. The use of the nanobody of the quinolone drug of claim 1 or 2, the nucleic acid molecule of claim 3 or 4, the biomaterial of claim 5, the reagent or kit of claim 6, the pharmaceutical product of claim 7, or the ELISA detection kit of claim 8 in at least one of the following aspects: (1) Detection of quinolone drugs for non-disease diagnosis and treatment purposes; (2) Preparation of quinolone drug detection reagents or kits; (3) Enrichment and purification of quinolone drugs; (4) Preparation of enrichment and / or purification reagents for quinolone drugs.

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