Anti-clinafloxacin monoclonal antibody, fluorescence immunoadsorption method for detecting clinafloxacin based on anti-clinafloxacin monoclonal antibody and related application of anti-clinafloxacin monoclonal antibody

Abstract

The invention discloses a method for detecting clinafloxacin by using a quantum dot fluorescence immunosorbent assay. The method comprises the following steps: designing and synthesizing an artificial complete antigen of clinafloxacin (CLI); preparing a monoclonal antibody capable of identifying clinafloxacin; quantum dots (QDs) are coupled with an antibody to prepare two fluorescent probes; and establishing direct and indirect fluorescence immunoadsorption methods (dc-FLISA and ic-FLISA). The method has the advantages that the quantum dots are coupled with the anti-clinafloxacin monoclonal antibody and rabbit anti-mouse IgG to serve as a novel fluorescent probe, and a fluorescence immunoassay method is established and used for quantitative detection of clinafloxacin in animal-derived food. Compared with the traditional indirect competitive enzyme-linked immunosorbent assay (ic-ELISA), the method provided by the invention is more sensitive.

Description

Technical Field

[0001] The invention belongs to the technical field of gene engineering, and in particular relates to an anti-clinafloxacin monoclonal antibody and a fluorescent immunosorbent method for detecting clinafloxacin based on the anti-clinafloxacin monoclonal antibody and related applications. Background Art

[0002] Clinafloxacin (CLI), a fourth-generation fluoroquinolone (FQs) antibacterial drug, is often used as a veterinary drug additive due to its broad antibacterial spectrum and strong antibacterial activity. Irrational use of CLI not only leads to bacterial resistance, but also makes it easy for it to remain in animals and accumulate in the human body through the food chain, causing harm to human health, such as hypoglycemia, rash, and photosensitivity toxicity.

[0003] Fluoroquinolones are synthetic antibacterial drugs with a basic skeleton of a nitrogen (hetero) bicyclic structure. They have unique chemical and physical properties, including high fat solubility, strong cell penetration, easy absorption, and better anti-infection effect. Fluoroquinolones are highly sensitive to Gram-positive bacteria, Gram-negative bacteria, mycoplasma and anaerobic bacteria, so they are widely used in clinical and animal husbandry. However, in animal breeding and feed production, the abuse of antibiotics is becoming more and more serious, which accelerates the development of bacterial resistance and leads to the spread of fluoroquinolone-resistant strains. In addition, due to the enrichment of the food chain, fluoroquinolone antibiotics gradually accumulate in the human body through various channels, which may cause cancer, mutagenesis and drug tolerance to human health, posing a severe challenge to public health and safety. Therefore, establishing a method for detecting fluoroquinolone antibiotic residues in animal-derived foods is of great significance to improving food safety and protecting human health. Summary of the invention

[0004] In view of this, the purpose of the present invention is to provide a simple, fast, low-cost, easy-to-operate and highly sensitive method for detecting CLI. The present invention combines quantum dot labeling technology with immunological detection methods to establish a fluorescent-linked immunosorbent assay (FLISA), which can achieve rapid and sensitive detection of CLI.

[0005] In order to achieve the above-mentioned object of the invention, the present invention provides the following technical solutions:

[0006] The present invention provides an anti-clinafloxacin monoclonal antibody, wherein the anti-clinafloxacin monoclonal antibody comprises a heavy chain variable region and a light chain variable region;

[0007] The heavy chain variable region includes the nucleotide sequence shown in SEQ ID NO.1, and the heavy chain variable region includes the amino acid sequence shown in SEQ ID NO.2;

[0008] The light chain variable region comprises the nucleotide sequence shown in SEQ ID NO.9, and the light chain variable region comprises the amino acid sequence shown in SEQ ID NO.10.

[0009] In the present invention, the heavy chain variable region further comprises: an amino acid sequence as shown in SEQ ID NOs. 3 to 8;

[0010] The light chain variable region also includes: an amino acid sequence as shown in SEQ ID NOs.11-16.

[0011] The present invention also provides a nucleic acid molecule, which comprises the nucleic acid molecule of the anti-clinafloxacin monoclonal antibody.

[0012] The present invention also provides an expression vector, which comprises the above-mentioned nucleic acid molecule.

[0013] The present invention also provides a host cell, which comprises the above nucleic acid molecule or the above vector.

[0014] The present invention also provides a kit for detecting clinafloxacin, wherein the kit comprises the above-mentioned anti-clinafloxacin monoclonal antibody.

[0015] The present invention also provides a fluorescent immunosorbent assay for clinafloxacin, which comprises using the above-mentioned anti-clinafloxacin monoclonal antibody for treatment.

[0016] The present invention also provides the use of the above anti-clinafloxacin monoclonal antibody or the above fluorescent immunosorbent assay in detecting clinafloxacin in animal-derived food.

[0017] In the present invention, the titer of the anti-clinafloxacin monoclonal antibody is greater than or equal to 1:2.56×10 5 , the IC of the anti-clinafloxacin monoclonal antibody 50 The affinity of the monoclonal antibody is greater than or equal to 2.76×10 8 L / mol.

[0018] The present invention uses the carbodiimide method to couple the hapten clinafloxacin with the carrier protein to synthesize the complete antigen, and uses the immunogen to immunize mice. The best mouse titer is 1:2.56×10 4 ,IC 50The mouse was subsequently subjected to cell fusion, and a hybridoma cell line 6B8 that can stably secrete anti-clinafloxacin monoclonal antibodies was screened through multiple subcloning. Ascites was prepared and purified to obtain the 6B8 monoclonal antibody, with a titer of 1:2.56×10 5 ,IC 50 The affinity constant is 5.48 ng / mL and 2.76×10 8 L / mol, and the cross-reaction rate with other structural analogs is less than 9%. The present invention obtains an anti-clinafloxacin monoclonal antibody with high titer, high sensitivity and high affinity.

[0019] The present invention uses ZnCdSe / ZnS (core-shell) quantum dots coupled with 6B8 monoclonal antibody and goat anti-mouse IgG as novel fluorescent probes to establish direct and indirect fluorescent immunosorbent assays (dc-FLISA and ic-FLISA) for quantitative detection of clinafloxacin residues in animal-derived foods. The linear regression equation of dc-FLISA is y=-0.3219x+0.5773, R 2 is 0.9934, IC 50 The LOD and LOD were 1.74 ng / mL and 0.10 ng / mL, respectively, and the linear detection range was 0.20-14.86 ng / mL. The linear regression equation of ic-FLISA was y=-0.3093x+0.4771, R 2 is 0.9945, IC 50 The LOD and LOD were 0.84 ng / mL and 0.04 ng / mL, and the linear detection range was 0.09-7.87 ng / mL. It is more sensitive than the traditional indirect competitive enzyme-linked immunosorbent assay (ic-ELISA). BRIEF DESCRIPTION OF THE DRAWINGS

[0020] Figure 1 Schematic diagram of the principle of complete antigen synthesis in Example 1;

[0021] Figure 2 The UV-VI characterization diagrams of the complete antigens in Example 1; wherein Figure a is CLI-BSA, and Figure b is CLI-OVA;

[0022] Figure 3 FTIR characterization diagrams of the complete antigen in Example 1; wherein Figure a is CLI-BSA, and Figure b is CLI-OVA;

[0023] Figure 4 The MALDI-TOF MS characterization diagrams of the complete antigen in Example 1; wherein Figure a is BSA, Figure b is OVA, Figure c is CLI-BSA, and Figure d is CLI-OVA;

[0024] Figure 5The graph is a graph of the titer and sensitivity of the mouse polyclonal antibody serum in Example 2; wherein graph a is the titer of the polyclonal antibody serum, and graph b is the standard curve of the sensitivity of the polyclonal antibody serum;

[0025] Figure 6 ELISA identification of hybridoma cells in Example 3;

[0026] Figure 7 The purification diagram of 6B8 monoclonal antibody in Example 3; M in the figure refers to: Marker; Mark 1 in the figure is: before ascites purification; Mark 2 in the figure is: after ascites purification;

[0027] Figure 8 Figure 3 is a graph showing the titer and sensitivity of the 6B8 monoclonal antibody; Figure a is the titer of the 6B8 monoclonal antibody, and Figure b is the standard curve of the sensitivity of the 6B8 monoclonal antibody;

[0028] Fig. 9 is the affinity determination curve of the 6B8 monoclonal antibody in Example 3;

[0029] Fig.10 Schematic diagram of the preparation of fluorescent probes in Example 4; Figure a is QDs-mAb, and Figure b is QDs-IgG;

[0030] Fig.11 Agarose gel electrophoresis imaging of the fluorescent probe in Example 4; in the figure, 1: QDs, 2: QDs-mAb, 3: QDs, 4: QDs-IgG;

[0031] Fig.12 The fluorescence spectrum of the fluorescent probe in Example 4; the mark 1 in the figure refers to: QDs; the mark 2 in the figure refers to: QDs-mAb; the mark 3 in the figure refers to: QDs; the mark 4 in the figure refers to: QDs-IgG;

[0032] Fig.13 The immunochromatographic results of the fluorescent probe in Example 4; the label 1 in the figure refers to: QDs; the label 2 in the figure refers to: QDs-mAb; the label 3 in the figure refers to: QDs-IgG;

[0033] Fig.14 Schematic diagram of the reaction of dc-FLISA and ic-FLISA in Example 4; Figure a is dc-FLISA, and Figure b is ic-FLISA;

[0034] Fig.15 The standard curves of clinafloxacin determined by dc-FLISA and ic-FLISA methods in Example 4; Figure a is dc-FLISA, and Figure b is ic-FLISA. DETAILED DESCRIPTION

[0035] The technical solutions provided by the present invention are described in detail below in conjunction with the embodiments, but they should not be construed as limiting the protection scope of the present invention.

[0036] Example 1

[0037] Synthesis of complete antigen

[0038] 5 mg BSA (bovine serum albumin) was weighed and dissolved in 1 mL 0.1 M MES buffer [2 (-N-morpholino) ethanesulfonic acid, pH 5.5], 5.5 mg EDC [1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride] and 4 mg NHS (N-hydroxysuccinimide) were added, and the mixture was stirred for 1 h in the dark after being fully mixed. The reaction mixture was liquid A; 3 mg CLI was weighed and dissolved in 200 μL 0.1 M HCl, and then 500 μL 0.05 M CBS (pH 9.6), the reaction mixture is liquid B; liquid B is slowly added to liquid A, and stirred overnight at 4°C in the dark; the reaction liquid is collected, transferred to a dialysis bag, and dialyzed in a large volume of PBS buffer at 4°C for 3 days. During the dialysis process, the dialysate is replaced 3 to 4 times a day; after the dialysis is completed, the dialyzed reaction liquid is centrifuged, the supernatant is collected, and stored at -20°C to obtain the complete antigen CLI-BSA.

[0039] The preparation method of CLI-OVA is the same as above, and OVA is used instead of BSA to continue to synthesize the complete antigen CLI-OVA.

[0040] The above antigen synthesis process refers to Figure 1 shown.

[0041] The complete antigen synthesized by the present invention is purified by UV-vi ( Figure 2 )、FTIR( Figure 3 ) and MALDI-TOF MS( Figure 4 ) is characterized by Figure 2 It was shown that the characteristic peaks of CLI-BSA and CLI-OVA were at 282 nm, which were shifted compared with the carrier protein and hapten. Figure 3 It was shown that CLI-BSA and CLI-OVA contained both the characteristic peaks of the carrier protein and the characteristic peaks of the hapten; Figure 4 It shows that the molecular weight of the complete antigen is greater than that of the carrier protein. The coupling ratio of CLI-BSA is 5.07:1 and the coupling ratio of CLI-OVA is 15.12:1 calculated by the following formula. The above together indicate that the complete antigen coupling is successful.

[0042] Coupling ratio = (M 完全抗原 -M 载体蛋白 ) / M 半抗原

[0043] M represents the relative molecular mass.

[0044] Example 2

[0045] Preparation of monoclonal hybridoma cell line against clinafloxacin

[0046] 1. Immunization of Mice

[0047] Three 6-8 week old female BALB / c mice were selected for immunization, and the complete antigen CLI-BSA synthesized in Example 1 was appropriately diluted with PBS, and then mixed with adjuvants in equal proportions, and emulsified using a homogenizer. The immunization dose for each mouse was 50 μg, 200 μL, and the injection method was subcutaneous multi-point injection. Each immunization was separated by two weeks, and Freund's complete adjuvant was used for the first immunization, and Freund's incomplete adjuvant was used for subsequent immunizations. One week after the three immunizations, the tail venous blood of the mice was taken, and the titer of the polyantibody serum was determined by i-ELISA, and the sensitivity of the polyantibody serum was determined by ic-ELISA.

[0048] 2. Determination of the titer of polyclonal antiserum

[0049] 2.1 Indirect ELISA (i-ELISA) to measure the titer of polyclonal antiserum

[0050] (1) Coating: Dilute CLI-OVA with CBS to a final concentration of 1 μg / mL, 100 μL / well, and coat overnight at 4°C. After coating, wash three times with PBST, 300 μL / well, and pat dry;

[0051] (2) Blocking: 5% porcine serum was selected as the blocking solution, 200 μL / well, incubated at 37°C for 2 h. After blocking, the cells were washed three times and patted dry.

[0052] (3) Primary antibody: Dilute mouse polyclonal antibody serum with PBS, add 200 μL to the first well, and then dilute in multiples. Incubate at 37°C for 1 h, wash 5 times, and pat dry.

[0053] (4) Secondary antibody: HRP-goat anti-mouse was mixed with blocking solution at a ratio of 1:5000, 100 μL / well, reacted at 37°C for 1 h, washed 5 times and patted dry;

[0054] (5) Color development: Add TMB color development solution into each well at 100 μL / well and react for 5–10 min in the dark;

[0055] (6) Stop: Add stop solution (2 mol / L H2SO4) into each well at 100 μL / well;

[0056] (7) Reading: Use a multifunctional microplate reader, set the reading wavelength to 450 nm, and record the absorbance value.

[0057] (8) Result determination: When the OD of the hole to be tested is 450nm / Negative well OD 450nm ≥2.1, and the OD of the hole to be tested 450nm When the OD value is ≥0.2, the corresponding serum dilution factor is the polyclonal antibody serum titer. At the same time, the dilution factor with an OD value near 1.0 is selected as the serum dilution factor for polyclonal antibody serum sensitivity identification. Figure 5 The polyclonal antibody serum of mouse No. 1 with the highest titer can reach 1:2.56×10 4 .

[0058] 2.2 Indirect competitive ELISA (ic-ELISA) to measure the sensitivity of polyclonal antiserum

[0059] Steps (1), (2), (4), (5), (6), and (7) are the same as 2.1 above;

[0060] (3) Primary antibody: Add 200 μL of 500 ng / mL CLI standard solution to the first well, dilute it to the second-to-last well, and do not add CLI standard to the last well as B0. Dilute the polyclonal antibody serum according to the optimal ratio and add 100 μL / well. Incubate at 37°C for 1 h, wash 5 times and pat dry.

[0061] (8) Result determination: Draw a standard curve with the logarithmic value of the CLI standard concentration as the horizontal axis and B / B0 as the vertical axis. Figure 5 Middle b shows that the polyclonal antibody serum of mouse No. 1 has the best sensitivity, IC 50 =45.42ng / mL.

[0062] 3. Preparation and identification of positive monoclonal hybridoma cell lines

[0063] (1) Superimmunization: Superimmunization was performed 3 to 5 days before cell fusion, and mice with the best immune effect were selected. The immune dose was 100 μg, 200 μL, and the immunization method was intraperitoneal injection;

[0064] (2) Preparation of feeder layer cells: One day before cell fusion, Kunming mice were killed by cervical dislocation, and 10 mL of HAT medium was drawn into the mouse peritoneal cavity with a syringe. The mouse abdomen was slowly shaken and then the medium was sucked out. The medium was diluted and mixed with new HAT medium, and 100 μL / well was added to a 96-well cell culture plate;

[0065] (3) Preparation of myeloma SP2 / 0 cells: Select SP2 / 0 cells with good growth status, centrifuge at 1000 rpm for 10 min after blowing off, resuspend in 20 mL of GNK solution, and take a small amount of cell resuspension for counting;

[0066] (4) Preparation of splenocytes: The spleen of the mouse was removed and ground, transferred to a sterile centrifuge tube, centrifuged at 1000 rpm for 10 min, resuspended in 20 mL of GNK solution, and counted;

[0067] (5) Cell fusion: SP2 / 0 cells and mouse spleen cells were mixed at a ratio of 1:8, centrifuged at 1000 rpm for 10 min, the supernatant was discarded, the cells were resuspended and transferred to a 37°C water bath; 1 mL of PEG1500 was drawn and added at a constant speed within 90 s. After the addition was completed, the tube was incubated at 37°C in a water bath for 90 s; 15 mL of GNK solution was drawn and the centrifuge tube was slowly rotated. 1 mL was added at a constant speed in the first 30 s, 3 mL was added at a constant speed in the middle 30 s, and the remaining GNK was added at a constant speed in the last 30 s to terminate cell fusion. The tube was incubated at 37°C in a water bath for 5 min. After the incubation was completed, GNK was added to a total volume of 40 mL and the tube was centrifuged at 1000 rpm for 10 min; the supernatant was discarded, the cells were resuspended in HAT medium, mixed evenly, and added to the culture plate with feeder layer cells at 100 μL / well and placed in an incubator.

[0068] (6) Screening of positive hybridoma cell lines: 7 to 10 days after cell fusion, high-throughput screening of positive cell clusters was performed. After multiple subcloning, three hybridoma cell lines that can stably secrete anti-CLI monoclonal antibodies were selected, namely 1B9, 3G4 and 6B8. Figure 6 The results showed that the supernatant titer and sensitivity of 6B8 cell line were the best, so 6B8 cell line was selected for subsequent experiments.

[0069] Example 3

[0070] Preparation and identification of anti-clinafloxacin monoclonal antibody

[0071] 1. Purification of monoclonal antibodies and determination of their titer and sensitivity

[0072] The 6B8 cell line was expanded and cultured. About 1×10 6 The cells were injected intraperitoneally into parous Balb / c mice. After about 7 to 10 days, when the abdomen of the mice was obviously swollen and their movements were slow, the ascites was collected and purified by the caprylic acid-ammonium sulfate precipitation method to obtain a high-purity monoclonal antibody ( Figure 7 The monoclonal antibody titer was 1:2.56×10 5 ( Figure 8 a), IC 50 5.48 ng / mL ( Figure 8 b).

[0073] 2. Monoclonal Antibody Affinity Determination

[0074] Select 0.5 and 1 μg / mL CLI-OVA coated plates, add 1:1000 diluted monoclonal antibody to the first well, and then dilute it in turn, and measure its absorbance at 450nm. Use the antibody concentration as the horizontal axis and the absorbance at 450nm as the vertical axis to make a standard curve, determine the 100% absorbance value, and draw a double reciprocal curve with the reciprocal of the monoclonal antibody concentration as the horizontal axis and the reciprocal of the absorbance as the vertical axis. Calculate the monoclonal antibody concentration corresponding to 50% absorbance, convert it to molar concentration, and substitute it into the following formula to obtain the affinity constant Kaff of the monoclonal antibody as 2.76×10 8 L / mol( Fig. 9 ).

[0075] Kaff=(n-1) / 2(n[Ab']t-[Abt])

[0076] n=[Ag]t / [Ag']t

[0077] Where [Ag]t and [Ag']t represent high and low coating concentrations, respectively;

[0078] [Ab] and [Abt'] represent the corresponding monoclonal antibody molar concentrations.

[0079] 4. Specificity of monoclonal antibodies

[0080] Clinfloxacin, gatifloxacin, moxifloxacin, marbofloxacin and sarafloxacin are all fluoroquinolone antibiotics with similar structures. Cross-reactivity was determined by IC-ELISA and IC was calculated. 50 , cross-reaction rate = (CLIIC 50 / Structural analogs IC 50 )×100%, the results are shown in Table 1.

[0081] Table 1 Cross-reactivity of CLI monoclonal antibodies

[0082]

[0083] 5. Amplification of variable region sequences of monoclonal antibodies

[0084] The present invention amplifies and sequences the variable region sequence of the monoclonal antibody, and the sequencing result is:

[0085] The heavy chain variable region sequence is shown in SEQ ID NO.1:

[0086] GATGTGCAGCTGGTGGAGTCTGGGGGAGGCTTAGTGCAGCCTGGAGGGTCCCGGAAACTCTCCTGTGCAGCCTCTGGATTCACTTTCAGTAGCTTTGGAATGCACTGGGTTCGTCAGGCTCCAGAGAAGGGGCTGGAGTGGGTCGCATACATTAGTAGTGGCGGTAGTACCATCTACTAT GCAGACAAAGTGAAGGGCCGATTCACCATCTCCAGAGACAATCCCAAGAACACCCTGTTCCTGCAAATGGCCAGTCTACGGTCTGAGGACACGGCCATGTATTACTGTGTCAGAGAGGACAATAGGTACGACAGCTGGTTCTTCGAAGTCTGGGGCGCAGGGACCACGGTCACTGTCTCC

[0087] The amino acid sequence of the heavy chain variable region is shown in SEQ ID NO.2:

[0088] DVQLVESGGGLVOPGGSRKLSCAASGETESSFGMHWVRQAPEKGLEWVAYISSEGSTIYYADKVKGRETISRDNPKNTLFLQMASLRSEDTAMYYCVREDNRYDSWFFEVWGAGTTWTVS

[0089] Further analysis revealed the amino acid sequences of the FR and CDR of the heavy chain variable region of the monoclonal antibody as shown in the following table:

[0090] Table 2 Amino acid sequences of FR and CDR in the heavy chain variable region of monoclonal antibodies

[0091]

[0092] The light chain variable region sequence is shown in SEQ ID NO.9:

[0093] GACATTTGTGCTGACACAGTCTCCTGCTTCCTTAGCTGTATCTCTGGGGCAGAGGGCCACCATCTCATACAGGGCCAGCAAAAGTGTCAGTACATCTGGCCTATAGTTATATGCACTGGAACCAACAAAAACCACGACAACCACCCACACTCCTCATCTATCTTGT ATCCAACCTATAATCTGGGGTCCCTGCCAAGTTCAATGGCAGTGGGTCTGGGACAGACTTCACCCTCAACATCCATCCTGTGGAGGAGGAAGATGCTGCAACCTATTACTGTCAGCACATTAGGGAGCTTACACGTTCGGAGGGGGGACCAAGCTGGAAATAAAAC

[0094] The amino acid sequence of the light chain variable region is shown in SEQ ID NO.10:

[0095] DIVLTQSPASLAVSLGQRATISYRASKSVSTSGLLYALEPTKTTTTTHTPHLSCIQPIIWGP CQVQWQWVWDRLHPQHPSCGGGRCCNLLLSAHGAYT

[0096] Further analysis revealed the amino acid sequences of the monoclonal antibody light chain variable region FR and CDR as shown in the following table:

[0097] Table 3 Amino acid sequences of FR and CDR of monoclonal antibody light chain variable region

[0098]

[0099] The nucleotide and amino acid sequences of the heavy chain and light chain variable regions of the monoclonal antibody disclosed in the present invention can be used to obtain the monoclonal antibody of the present invention through techniques such as genetic engineering or protein engineering, and can further be modified by adding, deleting, replacing, or the like one or more amino acids to obtain active fragments or conservative variants thereof, thereby enhancing their affinity and thus being applied to the field of antibiotic residue detection.

[0100] Example 4

[0101] Establishment of direct and indirect fluorescence immunosorbent assay for clinafloxacin

[0102] 1. Synthesis of Immunofluorescence Probes ( Fig.10 )

[0103] (1) Prepare a 1 mg / mL LEDC solution (prepared on demand), mix 6.25 μL QDs with 19.15 μL LEDC, and shake at 25°C in the dark for 30 min;

[0104] (2) Add 25 μL of anti-CLI monoclonal antibody at a molar ratio of 10:1 with quantum dots, and then make up to 100 μL with BBS, and shake at 25°C in the dark for 3 h;

[0105] (3) Centrifuge at 8000 rpm for 3 min, take the supernatant and store it at 4°C to obtain QDs-anti-CLI-mAb (QDs-mAb) fluorescent probe.

[0106] The steps for synthesizing QDs-Rabbit-anti-Mouse-IgG (QDs-IgG) were similar to those for QDs-mAb, except that the ratio of antibody addition needed to be changed.

[0107] The fluorescent probe synthesized by the present invention was analyzed by agarose gel electrophoresis ( Fig.11 ), fluorescence spectrum ( Fig.12 ) and immunochromatography ( Fig.13 ) were characterized. As shown in Figure 0, the fluorescent probe lags behind QDs because the molecular weight, charge, etc. change during the coupling process; as shown in Figure 0, the maximum emission wavelength of QDs and fluorescent probes are both 610nm; as shown in Figure 0, both C-line and T-line of the two fluorescent probes show fluorescence, which together indicate that the fluorescent probe was successfully synthesized.

[0108] 2. Establishment of direct competitive fluorescence immunoassay (dc-FLISA) Fig.14 a)

[0109] (1) Coating and blocking: Same as i-ELISA;

[0110] (2) Primary antibody: add 200 μL of 20 ng / mL standard solution to the first well, dilute it to the second to last well, and add 100 μL of PBS to the last well; dilute the fluorescent probe QDs-mAb to a certain ratio with PBS, add 100 μL / well, incubate at 37°C for 1 h, wash and pat dry;

[0111] (3) Set up blank control: add 100 μL of PBS to each well;

[0112] (4) Results: Fluorescence intensity values ​​were read using excitation light-emission light. F / F0 was calculated according to formula 4.1:

[0113] F / F0=(F 添加CLI -F 空白 ) / (F 未加CLI -F 空白 )

[0114] Wherein, F represents the fluorescence intensity value.

[0115] The logarithm of CLI concentration is used as the horizontal axis, and the F / F0 value is used as the vertical axis. According to the equation, IC 50 , LOD and linear detection range.

[0116] 3. Establishment of indirect competitive fluorescence immunosorbent assay (ic-FLISA) Fig.14 b)

[0117] (1) Coating, blocking and primary antibody addition were the same as those for dc-FLISA. The primary antibody was a monoclonal antibody diluted at a certain ratio. The cells were incubated at 37°C for 1 h, washed and patted dry.

[0118] (2) Secondary antibody: QDs-IgG was diluted at a ratio of 1:200, 100 μL was added to each well, incubated at 37 °C for 1 h, washed and patted dry;

[0119] (3) Set up blank control: add 100 μL of PBS to each well;

[0120] (4) Results: The analysis method was the same as that of dc-FLISA.

[0121] Depend on Fig.15 As shown in a, the linear regression equation of dc-FLISA is y = -0.3219x + 0.5773, R 2 is 0.9934, IC 50 and LOD were 1.74 ng / mL and 0.10 ng / mL, and the linear detection range was 0.20-14.86 ng / mL.

[0122] Depend on Fig.15 As shown in b, the linear regression equation of ic-FLISA is y = -0.3093x + 0.4771, R 2 is 0.9945, IC 50 and LOD were 0.84 ng / mL and 0.04 ng / mL, and the linear detection range was 0.09-7.87 ng / mL.

[0123] 3. Comparison of ic-ELISA, dc-FLISA and ic-FLISA in actual sample recovery

[0124] Milk was selected for addition recovery, the milk was boiled for 5-10 minutes, the solid was filtered out with gauze, the filtered milk was centrifuged at 10000rpm for 10 minutes to obtain skim milk; the skim milk was transferred to a 40℃ water bath, the pH was adjusted to 4.7 with acetic acid, after a water bath for 30 minutes, it was centrifuged at 11000rpm for 10 minutes; the supernatant was adjusted to pH 7.2-7.4 with 5M NaOH, and stored at -20℃ for use when testing. Different amounts of CLI standard solution were added to the blank milk sample, and the concentration of CLI added was set according to the linear detection range of different methods, 1, 2 and 4 ng / mL for ic-ELISA and dc-FLISA, and 0.5, 1, 2 ng / mL for ic-FLISA. According to the standard curve, the CLI content was calculated, and the recovery rate and coefficient of variation (CV) were calculated according to the following formula to evaluate the feasibility of the method. The results showed that the recovery rate of ic-ELISA was 94.00% to 97.42%, the recovery rate of dc-FLISA was 94.50% to 101.38%, and the recovery rate of ic-FLISA was 97.25% to 108.33%, and the coefficient of variation was less than 9%. (Table 4)

[0125] Sample recovery rate = measured concentration / sample addition concentration × 100%

[0126] Coefficient of variation (CV%) = (standard deviation SD / mean value mean) × 100%.

[0127] Table 4 Detection of CLI in spiked milk samples by ic-ELISA, dc-FLISA and ic-FLISA

[0128]

[0129] Note: a means the data are Mean±SD; b means 6 parallel experiments were set for each group of data.

[0130] The present invention and its implementation methods are described above, and such description is not restrictive. The specific implementation methods are only part of the embodiments of the present invention, not all of the embodiments, and the actual structure is not limited thereto. In short, if ordinary technicians in the field are inspired by it, without departing from the purpose of the invention, they can design a structure and implementation method similar to the technical solution without creativity, which should fall within the protection scope of the present invention.

Claims

1. An anti-clinafloxacin monoclonal antibody, characterized in that: The anti-clinafloxacin monoclonal antibody comprises a heavy chain variable region and a light chain variable region; The heavy chain variable region includes the nucleotide sequence shown in SEQ ID NO.1, and the heavy chain variable region includes the amino acid sequence shown in SEQ ID NO.2; The light chain variable region comprises the nucleotide sequence shown in SEQ ID NO.9, and the light chain variable region comprises the amino acid sequence shown in SEQ ID NO.

10.

2. The anti-clinafloxacin monoclonal antibody according to claim 1, characterized in that: The heavy chain variable region further comprises: an amino acid sequence as shown in SEQ ID NOs. 3 to 8; The light chain variable region also includes: an amino acid sequence as shown in SEQ ID NOs.11-16.

3. A nucleic acid molecule, characterized in that The nucleic acid molecule comprises the nucleic acid molecule of the anti-clinafloxacin monoclonal antibody according to any one of claims 1 or 2.

4. An expression vector, characterized in that: The expression vector comprises the nucleic acid molecule of claim 3.

5. A host cell, characterized in that The host cell comprises the nucleic acid molecule of claim 3 or the vector of claim 4.

6. A kit for detecting clinafloxacin, characterized in that: The kit comprises the anti-clinafloxacin monoclonal antibody according to any one of claims 1 or 2.

7. A fluorescent immunosorbent assay for detecting clinafloxacin, characterized in that: The fluorescent immunosorbent assay comprises treating with the anti-clinafloxacin monoclonal antibody according to any one of claims 1 or 2.

8. Use of the anti-clinafloxacin monoclonal antibody according to any one of claims 1 or 2 or the fluorescent immunosorbent assay according to claim 7 in detecting clinafloxacin in animal-derived food.

9. The use according to claim 8, characterized in that: The titer of the anti-clinafloxacin monoclonal antibody is greater than or equal to 1:2.56×10 5 , the IC of the anti-clinafloxacin monoclonal antibody 50 5.48 ng / mL; The affinity of the monoclonal antibody is greater than or equal to 2.76×10 8 L / mol.

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