CN113341134A - Method for detecting tiamulin by quantum dot fluorescence immunoassay - Google Patents

Abstract

The invention discloses a method for detecting tiamulin by using a quantum dot fluorescence immunoassay method, which comprises the following steps: preparing a detection solution; preparing and purifying tiamulin monoclonal antibody (TMLmab) in large quantity; establishing an indirect competitive enzyme-linked immunosorbent assay (ICELISA); further comprising step S4, coupling of QDs with IgG; the coupling effect of QDs and IgG is identified; establishment of Indirect competitive fluorescence immunoassay (icFLISA). Has the advantages that: in the research, a ZnCdSe/ZnS (nuclear shell) quantum dot and goat anti-mouse IgG are combined to serve as a novel fluorescent probe, and a fluorescence immunoassay method (icFLISA) is established and used for quantitative detection of tiamulin residues in animal edible tissues. Is more sensitive than the traditional indirect competitive enzyme-linked immunosorbent assay (icFLISA).

Description

Method for detecting tiamulin by quantum dot fluorescence immunoassay

Technical Field

The invention relates to the technical field of biomedical detection, in particular to a method for detecting tiamulin by using a quantum dot fluorescence immunoassay method.

Background

Tiamulin (Tiamulin) Tiamulin is prepared by fermenting Pleurotus mutilus of Pleurotus of higher fungus Basidiomycetes to obtain pleuromutilin, and then chemically synthesizing to obtain hydrogenated fumarate, which is a diterpene antibiotic special for livestock and poultry. The antibiotic is first proposed by Kavangh in Australia in 1951, is mainly used for preventing and treating respiratory diseases of livestock and poultry and promoting animal growth, and is one of ten veterinary antibiotics in the world. It can be used for preventing and treating diseases caused by mycoplasma infection and Escherichia coli, salmonella, spirochete, etc., has special effects on swine mycoplasmal pneumonia, hemophilus pleuropneumonia, enzootic pneumonia (asthma) and swine dysentery, and has good therapeutic effects on mycoplasma arthritis, actinobacillus pleuropneumonia, ileitis, colitis, edema, etc. Tiamulin is widely applied all over the world and is recommended by experts as the first choice drug for controlling the infection of swine and chicken mycoplasma in the United states. The traditional method for detecting tiamulin is an indirect competitive enzyme-linked immunosorbent assay (icFLISA), and the traditional detection method is not sensitive enough.

The quantum dot is a semiconductor nano-crystal material, mainly consists of II-VI or III-V group elements, and has the advantages of wide ultraviolet absorption range, narrow fluorescence emission spectrum, adjustable fluorescence size, good stability, photobleaching resistance and the like. Therefore, the quantum dots are widely applied to the fields of biological and medical immunodetection and provide a more powerful tool for immunoassay. In the research, a ZnCdSe/ZnS (nuclear shell) quantum dot and goat anti-mouse IgG are combined to serve as a novel fluorescent probe, and a fluorescence immunoassay method (icFLISA) is established and used for quantitative detection of tiamulin residues in animal edible tissues.

Disclosure of Invention

The invention aims to overcome the technical defects and provide a method for detecting tiamulin by using a quantum dot fluorescence immunoassay method, which is simple and sensitive in detection.

In order to solve the technical problems, the technical scheme provided by the invention is as follows: a method for detecting tiamulin by using a quantum dot fluorescence immunoassay method comprises the following steps:

s1, preparation of detection solution:

(1) PB buffer (0.01mol/L, pH8.0): a solution, weighing Na₂HPO₄·12H₂O3.58g; dissolving in deionized water, uniformly mixing, and fixing the volume to 100 mL; b, weighing NaH₂PO₄·2H₂O1.56g, dissolving in deionized water, uniformly mixing, and fixing the volume to 100 mL; weighing 93.2mL of solution A and 6.8mL of solution B, mixing uniformly, adjusting the pH to 8.0 to obtain PB buffer solution, and storing at room temperature;

(2) EDC solution: preparing EDC solution with the concentration of 1mg/mL, namely weighing 1mg carbodiimide, adding into 1mL PB buffer solution, and using the solution as it is;

(3) preparing PBS, CBS and PBST buffer solution;

s2, large-scale preparation and purification of tiamulin monoclonal antibody (TMLmAb): carrying out resuscitation and expanded culture on the TMLmAb hybridoma cell strain 4A 7; after about 3-5 days, when the growth state of the cells is good and the number of the cells is enough, injecting the cells into the abdominal cavity of a mouse, and preparing the monoclonal antibody ascites and purifying the monoclonal antibody ascites by an ammonium caprylate sulfate method; the concentration and purity of the purified TMLmAb are measured;

s3, establishment of indirect competitive enzyme-linked immunosorbent assay (ICELISA):

(1) the optimal concentration of the envelope antigen and the antibody for the ICELISA is measured by a chessboard method;

(2) diluting TML-OVA to appropriate concentration with CBS, adding 100 μ L of TML-OVA into 96-well transparent enzyme-labeled plate, incubating at 37 deg.C for 2 hr, washing with PBST for 3 times, and patting to dry; adding 200 mu L of blocking solution into each well, and incubating for 2 hours at 37 ℃; PBST is also used for washing for 3 times, and the solution is patted dry for standby;

(3) the iclelisa procedure was as follows: adding TML standard substances with different dilution concentrations into a 96-well plate, adding TMLmab solution with proper dilution concentration, incubating for 1h at 37 ℃ with 100 mu L of each well, and performing P incubationWashing with BST for 3 times, drying, adding diluted solution of goat anti-mouse IgG (1:5000) labeled with HRP (horse radish peroxidase) 100 μ L per well, incubating at 37 deg.C for 1h, washing for 4 times, and drying; adding 100 mu of LTMB developing solution into each hole, standing for 7 minutes at room temperature, and adding 100 mu of termination solution into each hole to carry out termination reaction; finally, the OD of each sample well is read by a microplate reader_450nmA value; establishing a standard curve by taking B/B0 as a vertical coordinate and taking the logarithm of TML concentration as a horizontal coordinate; b0: OD when TML concentration is zero_450nmA value; b: OD containing different concentrations of TML_450nmA value; the median inhibitory concentration (IC50) and limit of detection (LOD) were then calculated from the standard curve.

Further, the coupling of QDs to IgG is also included in step S4:

adding 5 μ L of QDs-COOH (ZnCdSe/ZnS, 8.0 μ M) and 15.3 μ L of EDC (1.0mg/mL) into a reaction vessel, and reacting in a shaking incubator at 25 ℃ for 30 min; then 5.56. mu.L of goat anti-mouse IgG (10.8mg/mL) and 74.14. mu.L of LPB buffer were added to make the total volume 100. mu.L; the mixture is put into a shaking incubator, reacted for 3 hours at the temperature of 25 ℃, and stored for standby at the temperature of 4 ℃.

Further, identification of the effect of coupling QDs to IgG:

(1) and (3) fluorescence spectrum characterization: in iMARK^TMSetting the excitation wavelength to be 450nm and the emission wavelength to be 610nm on an enzyme-labeling instrument, and respectively measuring the fluorescence spectra of QDs and QDs-IgG;

(2) and (3) agarose gel electrophoresis identification: weighing Agarose1g, dissolving in 100mL1 × TAE, and heating to dissolve completely (without Gelred); pouring the solution into an assembled glue groove, slightly shaking the glue groove to uniformly spread the liquid, placing the solution into an electrophoresis tank after solidification, and respectively adding QDs and QDs-IgG into a sample hole; setting the voltage to be 100V for 20min, and after electrophoresis is finished, placing the colloid sample under an ultraviolet lamp for observation and taking a picture;

(3) and (3) immunochromatography identification: firstly, dot coating Staphylococcal Protein A (SPA) on an NC membrane quality control line (C line) of two test strips, dot coating TMLmab on a detection line (T line), drying, and respectively adding diluted QDs and QDs-IgG on a sample pad; moving the sample from the sample pad to the NC film due to capillary action, and observing whether the T line and the C line have fluorescence or not under the irradiation of an ultraviolet lamp after 10 min; successful coupling was demonstrated and biological activity was retained if both the C-and T-lines on the QDs-IgG loaded strip were fluorescent.

Further, the establishment of indirect competitive fluorescence immunoassay (icfliisa):

the principle of icFLISA is similar to the icELISA except that HRP-labeled goat anti-mouse IgG is replaced with the more sensitive QDs-IgG;

(1) diluting TML-OVA to appropriate concentration with CBS, adding 100 μ L of TML-OVA into 96-well black enzyme label plate, incubating at 37 deg.C for 2 hr, washing with PBST for 3 times, and patting to dry; adding 200 mu L of blocking solution into each well, and incubating for 2 hours at 37 ℃; the same PBST was washed 3 times, patted dry, and reserved

(2) The concrete steps of the icFLISA: adding standard substance containing TML with different concentrations, adding TMLmAb (100 μ L) with appropriate dilution concentration, incubating at 37 deg.C for 1h, washing with PBST for 3 times, and drying; then adding diluted QDs-IgG (1:100), incubating for 1h at 37 ℃ with 100. mu.L of each well, washing for 3 times with PBST, and patting dry; finally, 100. mu.L of PBS buffer was added to each well and the fluorescence intensity (F) of each well was measured using an iMARKTM microplate reader, according to the formula:

F/F0＝(Fsample-Fblank)/(Fcontrol-Fblank)

establishing a standard curve by using the relative fluorescence intensity values, and calculating an inhibition rate F/F0, wherein the F0 value is the fluorescence intensity value without adding the TML standard substance, the F value is the fluorescence intensity value containing the TML standard substance at different concentrations, the F/F0 is used as a vertical coordinate, and the concentration of the TML is used as a horizontal coordinate; calculating IC from linear regression equation of curve₅₀And LOD.

Further, comparison of the iciELISA and the icFLISA in actual sample recovery: pre-processing a pork sample by using a methanol-water extraction method: firstly, freeze-drying a pork sample, weighing 0.3g of the dried sample, putting the sample into a centrifuge tube, and adding 10mL of a water-methanol mixed solution (in a volume ratio of 1: 1); placing the mixture in an ultrasonic water bath for ultrasonic treatment for 40 min; after centrifugation, the supernatant was retained; then adding 10mL of mixed solution of water and methanol into the centrifuge tube to resuspend the sediment, and carrying out ultrasonic treatment for 40 min; after centrifugation, the supernatant was retained; repeating for three timesThe extraction process, placing all supernatants in a beaker; placing the beaker on a hot plate heated to 60 ℃ and allowing the liquid to evaporate until about 0.5mL of solution is maintained; diluting the concentrated extract to 5 mL; adding TML with different concentrations into the treated pork diluent, wherein the content is set to be 4 concentration gradients: 0.5, 1, 2, 5 ng/mL; each set of experiments was set to 3 replicates and their OD was determined according to established detection methods for icELISA and icFLISA_450nmSubstituting the obtained result into the established standard curve to calculate the concentration of TML, calculating the recovery rate and the coefficient of variation of the sample, and evaluating the feasibility of the method; the sample recovery rate and the coefficient of variation are calculated as follows:

the sample recovery rate is measured concentration/sample addition concentration x 100%

Coefficient of variation (CV%) (standard deviation SD/mean) × 100%.

Compared with the prior art, the invention has the advantages that: in the research, a ZnCdSe/ZnS (nuclear shell) quantum dot and goat anti-mouse IgG are combined to serve as a novel fluorescent probe, and a fluorescence immunoassay method (icFLISA) is established and used for quantitative detection of tiamulin residues in animal edible tissues. Is more sensitive than the traditional indirect competitive enzyme-linked immunosorbent assay (icFLISA).

Drawings

FIG. 1 is a schematic reaction diagram of the icFLISA.

FIG. 2 shows SDS-PAGE results before and after TML mAb purification (1: ascites before purification; 2: ascites after purification).

FIG. 3 shows fluorescence spectra of QDs and QDs-IgG (1: QDs, 2: QDs-IgG).

FIG. 4 is agarose gel electrophoresis imaging.

FIG. 5 shows the results of immunochromatography on an NC membrane under ultraviolet irradiation.

FIG. 6 is a standard curve for determining TML by the ICELISA method.

FIG. 7 is a TML standard curve measured by the icFLISA method.

Detailed Description

The following further illustrates embodiments of the present invention.

A method for detecting tiamulin by using a quantum dot fluorescence immunoassay method comprises the following steps:

s1, preparation of detection solution:

(1) PB buffer (0.01mol/L, pH8.0): a solution, weighing Na₂HPO₄·12H₂O3.58g; dissolving in deionized water, uniformly mixing, and fixing the volume to 100 mL; b, weighing NaH₂PO₄·2H₂O1.56g, dissolving in deionized water, uniformly mixing, and fixing the volume to 100 mL; weighing 93.2mL of solution A and 6.8mL of solution B, mixing uniformly, adjusting the pH to 8.0 to obtain PB buffer solution, and storing at room temperature;

(2) EDC solution: preparing EDC solution with the concentration of 1mg/mL, namely weighing 1mg carbodiimide, adding into 1mL PB buffer solution, and using the solution as it is;

(3) preparing PBS, CBS and PBST buffer solution;

s2, large-scale preparation and purification of tiamulin monoclonal antibody (TMLmAb): carrying out resuscitation and expanded culture on the TMLmAb hybridoma cell strain 4A 7; after about 3-5 days, when the growth state of the cells is good and the number of the cells is enough, injecting the cells into the abdominal cavity of a mouse, and preparing the monoclonal antibody ascites and purifying the monoclonal antibody ascites by an ammonium caprylate sulfate method; the concentration and purity of the purified TMLmAb are measured;

s3, establishment of indirect competitive enzyme-linked immunosorbent assay (ICELISA):

(1) the optimal concentration of the envelope antigen and the antibody for the ICELISA is measured by a chessboard method;

(2) diluting TML-OVA to appropriate concentration with CBS, adding 100 μ L of TML-OVA into 96-well transparent enzyme-labeled plate, incubating at 37 deg.C for 2 hr, washing with PBST for 3 times, and patting to dry; adding 200 mu L of blocking solution into each well, and incubating for 2 hours at 37 ℃; PBST is also used for washing for 3 times, and the solution is patted dry for standby;

(3) the iclelisa procedure was as follows: adding TML standard substances with different dilution concentrations into a 96-well plate, adding a TMLmab solution with a proper dilution concentration, incubating for 1h at 37 ℃ with 100 muL of each well, washing for 3 times by PBST, drying, adding a diluted solution of goat anti-mouse IgG (1:5000) marked by HRP (horse radish peroxidase), incubating for 1h at 37 ℃, washing for 4 times, and drying by beating; adding 100 mu of LTMB developing solution into each hole, standing for 7 minutes at room temperature, adding 100 mu of stopping solution into each hole, and stopping reaction(ii) a Finally, the OD of each sample well is read by a microplate reader_450nmA value; establishing a standard curve by taking B/B0 as a vertical coordinate and taking the logarithm of TML concentration as a horizontal coordinate; b0: OD when TML concentration is zero_450nmA value; b: OD containing different concentrations of TML_450nmA value; the median inhibitory concentration (IC50) and limit of detection (LOD) were then calculated from the standard curve.

Also included is the step of S4, coupling of QDs to IgG:

adding 5 μ L of QDs-COOH (ZnCdSe/ZnS, 8.0 μ M) and 15.3 μ L of EDC (1.0mg/mL) into a reaction vessel, and reacting in a shaking incubator at 25 ℃ for 30 min; then 5.56. mu.L of goat anti-mouse IgG (10.8mg/mL) and 74.14. mu.L of LPB buffer were added to make the total volume 100. mu.L; the mixture is put into a shaking incubator, reacted for 3 hours at the temperature of 25 ℃, and stored for standby at the temperature of 4 ℃.

Identification of the coupling effect of QDs to IgG:

(1) and (3) fluorescence spectrum characterization: in iMARK^TMSetting the excitation wavelength to be 450nm and the emission wavelength to be 610nm on an enzyme-labeling instrument, and respectively measuring the fluorescence spectra of QDs and QDs-IgG;

(2) and (3) agarose gel electrophoresis identification: weighing Agarose1g, dissolving in 100mL1 × TAE, and heating to dissolve completely (without Gelred); pouring the solution into an assembled glue groove, slightly shaking the glue groove to uniformly spread the liquid, placing the solution into an electrophoresis tank after solidification, and respectively adding QDs and QDs-IgG into a sample hole; setting the voltage to be 100V for 20min, and after electrophoresis is finished, placing the colloid sample under an ultraviolet lamp for observation and taking a picture;

(3) and (3) immunochromatography identification: firstly, dot coating Staphylococcal Protein A (SPA) on an NC membrane quality control line (C line) of two test strips, dot coating TMLmab on a detection line (T line), drying, and respectively adding diluted QDs and QDs-IgG on a sample pad; moving the sample from the sample pad to the NC film due to capillary action, and observing whether the T line and the C line have fluorescence or not under the irradiation of an ultraviolet lamp after 10 min; successful coupling was demonstrated and biological activity was retained if both the C-and T-lines on the QDs-IgG loaded strip were fluorescent.

Establishment of indirect competitive fluorescence immunoassay (icFLISA):

the principle of icFLISA is similar to the icELISA except that HRP-labeled goat anti-mouse IgG is replaced with the more sensitive QDs-IgG;

(1) diluting TML-OVA to appropriate concentration with CBS, adding 100 μ L of TML-OVA into 96-well black enzyme label plate, incubating at 37 deg.C for 2 hr, washing with PBST for 3 times, and patting to dry; adding 200 mu L of blocking solution into each well, and incubating for 2 hours at 37 ℃; the same PBST was washed 3 times, patted dry, and reserved

(2) The concrete steps of the icFLISA: adding standard substance containing TML with different concentrations, adding TMLmAb (100 μ L) with appropriate dilution concentration, incubating at 37 deg.C for 1h, washing with PBST for 3 times, and drying; then adding diluted QDs-IgG (1:100), incubating for 1h at 37 ℃ with 100. mu.L of each well, washing for 3 times with PBST, and patting dry; finally, 100. mu.L of PBS buffer was added to each well and the fluorescence intensity (F) of each well was measured using an iMARKTM microplate reader, as shown in FIG. 1, with reference to the equation:

F/F0＝(Fsample-Fblank)/(Fcontrol-Fblank)

establishing a standard curve by using the relative fluorescence intensity values, and calculating an inhibition rate F/F0, wherein the F0 value is the fluorescence intensity value without adding the TML standard substance, the F value is the fluorescence intensity value containing the TML standard substance at different concentrations, the F/F0 is used as a vertical coordinate, and the concentration of the TML is used as a horizontal coordinate; calculating IC from linear regression equation of curve₅₀And LOD.

Comparison of iclelisa and icfliisa in actual sample recovery: pre-processing a pork sample by using a methanol-water extraction method: firstly, freeze-drying a pork sample, weighing 0.3g of the dried sample, putting the sample into a centrifuge tube, and adding 10mL of a water-methanol mixed solution (in a volume ratio of 1: 1); placing the mixture in an ultrasonic water bath for ultrasonic treatment for 40 min; after centrifugation, the supernatant was retained; then adding 10mL of mixed solution of water and methanol into the centrifuge tube to resuspend the sediment, and carrying out ultrasonic treatment for 40 min; after centrifugation, the supernatant was retained; repeating the extraction process three times, and placing all supernatants in a beaker; placing the beaker on a hot plate heated to 60 ℃ and allowing the liquid to evaporate until about 0.5mL of solution is maintained; diluting the concentrated extract to 5 mL; adding TML with different concentrations into the treated pork diluent, wherein the content is set to be 4 concentration gradients: 0.5, 1, 2, 5ngPer mL; each set of experiments was set to 3 replicates and their OD was determined according to established detection methods for icELISA and icFLISA_450nmSubstituting the obtained result into the established standard curve to calculate the concentration of TML, calculating the recovery rate and the coefficient of variation of the sample, and evaluating the feasibility of the method; the sample recovery rate and the coefficient of variation are calculated as follows:

the sample recovery rate is measured concentration/sample addition concentration x 100%

Coefficient of variation (CV%) (standard deviation SD/mean) × 100%.

The experimental results are as follows:

concentration and purity determination of TML mAb

The concentration of ascites before purification was 35.5mg/mL and the concentration of TMLmAb after purification was 5.8mg/mL as determined by ultramicro UV spectrophotometer. The SDS-PAGE results are shown in FIG. 2, which shows that the purified TML mAb is of higher purity.

Identification of QDs-IgG coupling effect

Referring to FIGS. 3 to 5, the fluorescence emission spectra of QDs and QDs-IgG are shown in FIG. 3, and both QDs and QDs-IgG have a maximum absorption peak at an emission wavelength of 610nm at an excitation wavelength of 450 nm. The fluorescence intensity of QDs-IgG is lower compared to QDs, probably due to depletion and dilution of QDs during coupling. Agarose gel electrophoresis FIG. 4 also shows that the molecular weight of coupled QDs-IgG is large, and the migration speed is slow compared with that of QDs, which also shows that the coupling is successful. In addition, immunochromatography further confirmed the successful coupling, as shown in FIG. 5, QDs-IgG on test strip No. 2 could bind to TML mAb on T line and SPA on C line under the irradiation of ultraviolet lamp, and both showed fluorescence, while QDs on test strip No. 1 could not bind to T, C line and showed no fluorescence. Therefore, based on the above characterization, successful IgG coupling was successfully demonstrated, and the original biological activity was retained.

Standard curves for the ICELISA and the icFLISA

The standard curve established by the iclelisa is shown in 6, the linear regression equation is that y is-0.5263 x +0.236, R2 is 0.9859, and the IC50 is calculated to be 0.33ng/mL, and the LOD is calculated to be 0.054 ng/mL; with F/F0 as ordinate and TML concentration as abscissa, linear regression equations for icFLISA (fig. 7) were established with y-0.1705 x +0.3014 and R2-0.9819, measuring IC50 at 0.069ng/mL and LOD at 0.309pg/mL, with the LOD of icFLISA being lower and more sensitive than that of iclisa, and lower levels of TML could be detected.

Adding a mark to recover a result

TML standards with the concentrations of 0.5ng/mL, 1.0ng/mL and 2.0ng/mL are added in sequence to the treated pork sample diluent. Meanwhile, the results of verification by using the ICELISA and the icFLISA show that the recovery rate of the ICELISA is 68.2-106.8%, and the coefficient of variation is less than 11.9%; the recovery rate of the icFLISA is 85.6-96.8%, and the coefficient of variation is less than 9.4% (Table 1). The results show that the accuracy of the icFLISA detection method is higher than that of the icELISA detection method.

TABLE 1 detection of TML in tagged pork samples by ICELISA and ICFLISA

Quantum dot labeled antibody

The quantum dots can be combined with different biomolecules such as proteins, antibodies, aptamers, oligonucleotides, etc., thereby obtaining a unique nanoparticle having the characteristics of two materials, i.e., the optical/electrochemical characteristics of the quantum dots and the biological functions of the biomolecules.

Antibodies may be the first biomolecule to bind to quantum dots in view of their specificity, affinity and versatility in different immunoassay methods. The quantum dots used should be modified beforehand to obtain water solubility and the desired functional groups, preferably carboxylic acids or amino groups. The experiment adopts a simple carbodiimide method to activate the carboxyl of the quantum dot, and then the quantum dot is coupled with the amino terminal of the antibody, and the method is simple, convenient, quick and low in cost. However, in the experimental process, attention needs to be paid to the adding proportion of each solution, the reaction temperature and the reaction pH, and if the quantum dot amount and the antibody amount are not appropriate, insufficient coupling and a large amount of byproducts are generated, so that the experimental effect is influenced.

The present invention and the embodiments thereof have been described above, and the description is not restrictive, and the embodiments shown in the detailed description are only a part of the embodiments of the present invention, not all embodiments, and the actual configuration is not limited thereto. In summary, those skilled in the art should appreciate that they can readily use the disclosed conception and specific embodiments as a basis for designing or modifying other structures for carrying out the same purposes of the present invention without departing from the spirit and scope of the invention as defined by the appended claims.

Claims (5)

1. A method for detecting tiamulin by using a quantum dot fluorescence immunoassay method is characterized by comprising the following steps:

s1, preparation of detection solution:

(1) PB buffer (0.01mol/L, pH8.0): a solution, weighing Na₂HPO₄·12H₂O3.58g; dissolving in deionized water, uniformly mixing, and fixing the volume to 100 mL; b, weighing NaH₂PO₄·2H₂O1.56g, dissolving in deionized water, uniformly mixing, and fixing the volume to 100 mL; weighing 93.2mL of solution A and 6.8mL of solution B, mixing uniformly, adjusting the pH to 8.0 to obtain PB buffer solution, and storing at room temperature;

(2) EDC solution: preparing EDC solution with the concentration of 1mg/mL, namely weighing 1mg carbodiimide, adding into 1mL PB buffer solution, and using the solution as it is;

(3) preparing PBS, CBS and PBST buffer solution;

s2, large-scale preparation and purification of tiamulin monoclonal antibody (TMLmAb): carrying out resuscitation and expanded culture on the TMLmAb hybridoma cell strain 4A 7; after about 3-5 days, when the growth state of the cells is good and the number of the cells is enough, injecting the cells into the abdominal cavity of a mouse, and preparing the monoclonal antibody ascites and purifying the monoclonal antibody ascites by an ammonium caprylate sulfate method; the concentration and purity of the purified TMLmAb are measured;

s3, establishment of indirect competitive enzyme-linked immunosorbent assay (ICELISA):

(1) the optimal concentration of the envelope antigen and the antibody for the ICELISA is measured by a chessboard method;

(2) diluting TML-OVA to appropriate concentration with CBS, adding 100 μ L of TML-OVA into 96-well transparent enzyme-labeled plate, incubating at 37 deg.C for 2 hr, washing with PBST for 3 times, and patting to dry; adding 200 mu L of blocking solution into each well, and incubating for 2 hours at 37 ℃; PBST is also used for washing for 3 times, and the solution is patted dry for standby;

(3) the iclelisa procedure was as follows: adding TML standard substances with different dilution concentrations into a 96-well plate, adding a TMLmab solution with a proper dilution concentration, incubating for 1h at 37 ℃ with 100 muL of each well, washing for 3 times by PBST, drying, adding a diluted solution of goat anti-mouse IgG (1:5000) marked by HRP (horse radish peroxidase), incubating for 1h at 37 ℃, washing for 4 times, and drying by beating; adding 100 mu of LTMB developing solution into each hole, standing for 7 minutes at room temperature, and adding 100 mu of termination solution into each hole to carry out termination reaction; finally, the OD of each sample well is read by a microplate reader_450nmA value; establishing a standard curve by taking B/B0 as a vertical coordinate and taking the logarithm of TML concentration as a horizontal coordinate; b0: OD when TML concentration is zero_450nmA value; b: OD containing different concentrations of TML_450nmA value; the median inhibitory concentration (IC50) and limit of detection (LOD) were then calculated from the standard curve.

2. The method for detecting tiamulin by quantum dot fluorescence immunoassay according to claim 1, further comprising the steps of S4, coupling QDs with IgG:

adding 5 μ L of QDs-COOH (ZnCdSe/ZnS, 8.0 μ M) and 15.3 μ L of EDC (1.0mg/mL) into a reaction vessel, and reacting in a shaking incubator at 25 ℃ for 30 min; then 5.56. mu.L of goat anti-mouse IgG (10.8mg/mL) and 74.14. mu.L of LPB buffer were added to make the total volume 100. mu.L; the mixture is put into a shaking incubator, reacted for 3 hours at the temperature of 25 ℃, and stored for standby at the temperature of 4 ℃.

3. The method for detecting tiamulin by using quantum dot fluorescence immunoassay according to claim 2, wherein the identification of the coupling effect of QDs and IgG is as follows:

(1) and (3) fluorescence spectrum characterization: in iMARK^TMSetting the excitation wavelength to be 450nm and the emission wavelength to be 610nm on an enzyme-labeling instrument, and respectively measuring the fluorescence spectra of QDs and QDs-IgG;

(2) and (3) agarose gel electrophoresis identification: weighing Agarose1g, dissolving in 100mL1 × TAE, and heating to dissolve completely (without Gelred); pouring the solution into an assembled glue groove, slightly shaking the glue groove to uniformly spread the liquid, placing the solution into an electrophoresis tank after solidification, and respectively adding QDs and QDs-IgG into a sample hole; setting the voltage to be 100V for 20min, and after electrophoresis is finished, placing the colloid sample under an ultraviolet lamp for observation and taking a picture;

(3) and (3) immunochromatography identification: firstly, dot coating Staphylococcal Protein A (SPA) on an NC membrane quality control line (C line) of two test strips, dot coating TMLmab on a detection line (T line), drying, and respectively adding diluted QDs and QDs-IgG on a sample pad; moving the sample from the sample pad to the NC film due to capillary action, and observing whether the T line and the C line have fluorescence or not under the irradiation of an ultraviolet lamp after 10 min; successful coupling was demonstrated and biological activity was retained if both the C-and T-lines on the QDs-IgG loaded strip were fluorescent.

4. The method for detecting tiamulin by using quantum dot fluorescence immunoassay method according to claim 3, wherein the establishment of indirect competition fluorescence immunoassay (icFLISA):

the principle of icFLISA is similar to the icELISA except that HRP-labeled goat anti-mouse IgG is replaced with the more sensitive QDs-IgG;

(1) diluting TML-OVA to appropriate concentration with CBS, adding 100 μ L of TML-OVA into 96-well black enzyme label plate, incubating at 37 deg.C for 2 hr, washing with PBST for 3 times, and patting to dry; adding 200 mu L of blocking solution into each well, and incubating for 2 hours at 37 ℃; the same PBST was washed 3 times, patted dry, and reserved

(2) The concrete steps of the icFLISA: adding standard substance containing TML with different concentrations, adding TMLmAb (100 μ L) with appropriate dilution concentration, incubating at 37 deg.C for 1h, washing with PBST for 3 times, and drying; then adding diluted QDs-IgG (1:100), incubating for 1h at 37 ℃ with 100. mu.L of each well, washing for 3 times with PBST, and patting dry; finally, 100. mu.L of PBS buffer was added to each well and the fluorescence intensity (F) of each well was measured using an iMARKTM microplate reader, according to the formula:

F/F0＝(Fsample-Fblank)/(Fcontrol-Fblank)

establishing a standard curve by using the relative fluorescence intensity value, and calculating the inhibition rate F/F0, wherein the F0 value isThe fluorescence intensity value of the TML standard is not added, the F value is the fluorescence intensity value of the TML standard with different concentrations, the F/F0 is used as the ordinate, and the concentration of the TML is used as the abscissa; calculating IC from linear regression equation of curve₅₀And LOD.

5. The method for detecting tiamulin by using quantum dot fluorescence immunoassay according to claim 1, wherein the comparison between the iciELISA and the icFLISA in actual sample recovery: pre-processing a pork sample by using a methanol-water extraction method: firstly, freeze-drying a pork sample, weighing 0.3g of the dried sample, putting the sample into a centrifuge tube, and adding 10mL of a water-methanol mixed solution (in a volume ratio of 1: 1); placing the mixture in an ultrasonic water bath for ultrasonic treatment for 40 min; after centrifugation, the supernatant was retained; then adding 10mL of mixed solution of water and methanol into the centrifuge tube to resuspend the sediment, and carrying out ultrasonic treatment for 40 min; after centrifugation, the supernatant was retained; repeating the extraction process three times, and placing all supernatants in a beaker; placing the beaker on a hot plate heated to 60 ℃ and allowing the liquid to evaporate until about 0.5mL of solution is maintained; diluting the concentrated extract to 5 mL; adding TML with different concentrations into the treated pork diluent, wherein the content is set to be 4 concentration gradients: 0.5, 1, 2, 5 ng/mL; each set of experiments was set to 3 replicates and their OD was determined according to established detection methods for icELISA and icFLISA_450nmSubstituting the obtained result into the established standard curve to calculate the concentration of TML, calculating the recovery rate and the coefficient of variation of the sample, and evaluating the feasibility of the method; the sample recovery rate and the coefficient of variation are calculated as follows:

the sample recovery rate is measured concentration/sample addition concentration x 100%

Coefficient of variation (CV%) (standard deviation SD/mean) × 100%.

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