Quantum dot labeled testosterone propionate direct competitive fluorescence immunoassay detection method

Abstract

The application belongs to the technical field of immune detection, and particularly relates to a quantum dot labeled testosterone propionate direct competition fluorescence immune detection method. The application prepares a monoclonal antibody, and couples CdSe / ZnS (core / shell) quantum dots with the prepared anti-TP monoclonal antibody to synthesize a quantum dot (QDs) labeled anti-TP-mAb-QDs immune fluorescence probe, and construct a quantum dot labeled testosterone propionate direct competition fluorescence immune detection system for detecting testosterone propionate. The method for detecting testosterone propionate by the quantum dot labeled direct competition fluorescence immune detection method is simple in operation, and can detect the residual content of testosterone propionate in a sample to be detected without adding a chromogenic substance. The operation and reaction can be completed in one step. The method is simple, efficient, sensitive, low in detection cost, and can be applied to rapid detection of TP residues in feed, and has good accuracy.

Description

Technical Field

[0001] This invention belongs to the field of immunoassay technology, specifically relating to a direct competitive fluorescence immunoassay method for quantum dot-labeled testosterone propionate. Background Technology

[0002] Testosterone propionate is an esterified derivative of the natural androgen testosterone and belongs to the anabolic steroid drug category. my country's Ministry of Agriculture Announcement No. 824 lists testosterone propionate as a prohibited drug. Anabolic steroids include the natural sex hormone testosterone and a series of steroidal compounds derived from it; they are also known as anabolic steroid hormones. They not only possess the sex hormone functions of testosterone but also enhance protein assimilation efficiency. The abuse of anabolic steroids is mainly manifested in the illegal intake by athletes and the illegal addition by farmers. Excessive intake of anabolic steroid drugs can lead to physiological dysfunction in animals and even induce irreversible lesions.

[0003] Currently, the main methods for detecting anabolic hormone residues are physicochemical analysis methods such as LC-MS / MS, GC-MS, and SERS. Physicochemical analysis has the advantages of high precision and sensitivity, providing accurate qualitative and quantitative results. However, it relies on sophisticated instruments and equipment, is costly, and is complex to operate, requiring a specialized environment, which significantly limits its effectiveness.

[0004] Immunological testing plays a crucial role in the detection of prohibited substances due to its affordability, ease of operation, and rapid diagnostic capabilities. For example, Chinese patent CN106443021B discloses a quantum dot-labeled immunochromatographic test strip for detecting anabolic hormones and its preparation method. This method utilizes antigen preparation and an activated ester method to detect the antibody-quantum dot label of the analyte, thereby achieving rapid detection of five anabolic hormones. Furthermore, existing technologies have reported the use of immunological detection methods to detect structurally stable anabolic hormones such as testosterone, 19-demethyltestosterone, and methyltestosterone. However, reports on the detection of residual testosterone propionate are relatively scarce.

[0005] Therefore, there is an urgent need for a method that can detect trace amounts of testosterone propionate. Summary of the Invention

[0006] This invention constructs a quantum dot-labeled testosterone propionate direct competitive fluorescence immunoassay system by preparing monoclonal antibodies and synthesizing immunofluorescent probes, for the detection of testosterone propionate.

[0007] This invention also establishes a direct competitive fluorescence immunoassay method for the quantitative detection of testosterone propionate residues, and provides a rapid and feasible detection tool for the trace detection of testosterone propionate.

[0008] To achieve the above objectives, the technical solution created by this invention is implemented as follows:

[0009] An anti-testosterone propionate monoclonal antibody, comprising a heavy chain variable region and a light chain variable region, wherein the amino acid sequence of the heavy chain variable region is shown in SEQ ID NO.2 and the amino acid sequence of the light chain variable region is shown in SEQ ID NO.4.

[0010] Specifically, the anti-testosterone propionate TP monoclonal antibody is secreted by the hybridoma cell line 3F9, which has good sensitivity and stability.

[0011] Furthermore, the present invention also provides a gene encoding the monoclonal antibody. Specifically, the gene sequence encoding the heavy chain variable region of the monoclonal antibody is shown in SEQ ID NO.1, and the gene sequence encoding the light chain variable region of the monoclonal antibody is shown in SEQ ID NO.3.

[0012] Based on a general inventive concept, the present invention also provides a quantum dot-labeled immunofluorescent probe, which is prepared by conjugating CdSe / ZnS quantum dots with the anti-testosterone propionate monoclonal antibody.

[0013] Specifically, the mass ratio of quantum dots to monoclonal antibodies in the preparation of the immunofluorescent probe is 1:(5-10), and more preferably 1:5 or 1:10.

[0014] Based on a general inventive concept, the present invention also provides a fluorescent immunoassay kit for detecting testosterone propionate, the kit comprising:

[0015] (a) Coated titration plate: a titration plate coated with testosterone propionate coated antigen; (b) anti-testosterone propionate monoclonal antibody-quantum dot immunofluorescence probe; (c) testosterone propionate standard; wherein the optimal coating concentration of the testosterone propionate coated antigen is 2 μg / mL.

[0016] Based on a general inventive concept, the present invention also provides the application of the quantum dot-labeled immunofluorescent probe in the quantitative detection of testosterone propionate.

[0017] Based on a general inventive concept, the present invention also provides a method for direct competitive fluorescence immunoassay of testosterone propionate based on the aforementioned immunofluorescent probe, comprising the following steps:

[0018] (1) Synthesis of immunogens: Testosterone propionate (TP) immunogen TP-BSA and testosterone propionate coated antigen TP-OVA were synthesized by oxime method and carbodiimide method, respectively.

[0019] (2) Preparation of monoclonal antibodies: 6-8 week old mice were immunized with testosterone propionate immunogen TP-BSA, and the serum titer and sensitivity of the polyclonal antibody serum of the immunized mice were determined by indirect ELISA and icELISA methods, respectively.

[0020] Select the most sensitive immunized mice and perform super immunization. 3-5 days after super immunization, use spleen cells from immunized mice to perform cell fusion. Screen the cell fusion results, prepare positive hybridoma cell lines through subcloning, and prepare high-purity anti-TP monoclonal antibodies.

[0021] (3) Synthesis of immunofluorescent probe: The CdSe / ZnS quantum dots were coupled with the anti-TP monoclonal antibody prepared in step (2) using the carbodiimide method to obtain quantum dot-labeled anti-TP-mAb-QDs immunofluorescent probe; the prepared anti-TP-mAb-QDs fluorescent probe was diluted to obtain anti-TP-mAb-QDs fluorescent probe dilution.

[0022] (4) The testosterone propionate-coated original TP-OVA was coated using a titration plate to obtain the complete testosterone propionate antigen coated in the titration plate; TP standard solutions of different concentrations were prepared.

[0023] (5) Add the anti-TP-mAb-QDs fluorescent probe dilution solution and TP standard solutions of different concentrations to the titration plate coated with testosterone propionate-coated antigen TP-OVA. By competitively binding with TP-OVA coated in the titration plate, an antigen-antibody luminescent immune complex is formed.

[0024] (6) Detect the fluorescence intensity of the formed antigen-antibody luminescent immune complex, and plot the standard curve with the logarithm of the concentration of TP in the TP concentration gradient standard solution as the abscissa and the obtained fluorescence intensity ratio as the ordinate.

[0025] (7) Replace the TP concentration gradient standard solution with the sample solution to be tested, and repeat steps (5) and (6) to obtain the fluorescence intensity value of the sample solution to be tested. By comparing it with the standard curve, the concentration of TP in the sample to be tested can be obtained.

[0026] Specifically, in step (2), during the first immunization, the immunogen is mixed and emulsified with Freund's complete adjuvant; during subsequent booster immunizations, the immunogen is mixed and emulsified with Freund's incomplete adjuvant.

[0027] Specifically, in step (3), the prepared anti-TP-mAb-QDs fluorescent probe is diluted at a ratio of 1:400 to obtain the anti-TP-mAb-QDs fluorescent probe dilution solution.

[0028] Specifically, in step (3), the mass ratio of quantum dots to monoclonal antibodies during the preparation of the immunofluorescence probe is 1:(5-10).

[0029] Specifically, in step (4), the concentrations of the TP concentration gradient standard solutions are 20 ng / mL, 10 ng / mL, 5 ng / mL, 2.5 ng / mL, 1.25 ng / mL, 0.625 ng / mL, and 0.3125 ng / mL, respectively.

[0030] Specifically, in step (4), the testosterone propionate-coated antigen TP-OVA coated on the titration plate is prepared by the following steps: TP-OVA is diluted to a final concentration of 2 μg / mL using CBS carbonate buffer, and added to the titration plate at a rate of 100 μL / well. The plate is then coated at 37°C for 3 h. After the antigen coating is completed, the plate is washed three times with phosphate buffer containing 0.5% Tween.

[0031] Specifically, the fluorescence intensity of the antigen-antibody luminescent complex is measured under the conditions of an emission wavelength of 450 nm and an excitation wavelength of 610 nm.

[0032] Compared with the prior art, the beneficial effects of the present invention are as follows:

[0033] 1. The quantum dot-labeled testosterone propionate direct competitive fluorescence immunoassay system designed and constructed in this invention can achieve quantitative and highly sensitive detection of testosterone propionate, with a wide detection range and a low detection limit, and can be applied to the rapid detection of testosterone propionate residues.

[0034] 2. The quantum dot-labeled direct competitive fluorescence immunoassay method for detecting testosterone propionate described in this invention is simple to operate and can detect the residual content of testosterone propionate in the sample without adding chromogenic substances. That is, the concentration of testosterone propionate in the sample can be directly detected by the fluorescence intensity of the antigen-antibody immune complex. Both the operation and the reaction can be completed in one step, which is simple and convenient and has good accuracy. Attached Figure Description

[0035] Figure 1 This is an identification diagram of the TP complete antigen TP-OVA in Example 1; wherein, Figure 1 A represents the results of ultraviolet scanning spectroscopy identification; Figure 1 B represents the SDS-PAGE identification result;

[0036] Figure 2 The results of the determination of mouse serum titer and sensitivity in Example 2 are shown; among them, Figure 2 A represents the determination of polyclonal antibody serum titer using ELISA. Figure 2 In A, NC serves as a blank control. Figure 2B represents the determination of polyclonal antibody serum sensitivity using icELISA.

[0037] Figure 3 IC50 of the supernatant of four testosterone propionate-resistant hybridoma cells after three freeze-thaw cycles in Example 2 50 value;

[0038] Figure 4 The image shows the SDS-PAGE identification results of the anti-TP monoclonal antibody in Example 2; in the figure, Line M is the standard molecular weight marker; Line 1 is the ascites fluid before purification; Line 2 is the anti-TP-mAb after purification.

[0039] Figure 5 The identification results of the immunofluorescent probe anti-TP-mAb-QDs in Example 3 are shown; wherein, Figure 5 A represents the fluorescence spectroscopy identification result; Figure 5 B represents the results of agarose gel electrophoresis. Figure 5 C represents the SDS-PAGE identification result; Figure 5 D represents the immunochromatographic identification results; Line 1 represents anti-TP-mAb-QDs; Line 2 represents orange-red QDs.

[0040] Figure 6 The standard curve of TP was determined using the fluorescence immunoassay method of this invention; wherein, Figure 6 A is the TP standard curve plotted using the indirect ELISA method; Figure 6 B is the TP standard curve plotted using the dc-FLISA method;

[0041] Figure 7 This is a schematic flowchart of the fluorescence immunoassay method in Example 5. Detailed Implementation

[0042] Various exemplary embodiments of the present invention will now be described in detail. This detailed description should not be considered as a limitation of the present invention, but rather as a more detailed description of certain aspects, features, and embodiments of the present invention.

[0043] It should be understood that the terminology used in this invention is merely for describing particular embodiments and is not intended to limit the invention. Furthermore, with respect to numerical ranges in this invention, it should be understood that each intermediate value between the upper and lower limits of the range is also specifically disclosed. Every smaller range between any stated value or intermediate value within a stated range, and any other stated value or intermediate value within said range, is also included in this invention. The upper and lower limits of these smaller ranges may be independently included or excluded from the range.

[0044] Unless otherwise stated, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art. While only preferred methods and materials have been described herein, any methods and materials similar or equivalent to those described herein may be used in the implementation or testing of this invention. All references to this specification are incorporated by way of citation to disclose and describe methods and / or materials associated with those references. In the event of any conflict with any incorporated reference, the content of this specification shall prevail.

[0045] The carbonate (CBS) buffer used in the following examples is 0.05 mol / L, pH 9.6. The washing buffer PBST used in the following examples is PBS (0.01 mol / L, pH 7.4) + 0.05% Tween-20.

[0046] In the following examples, the TMB reagent was used for colorimetric reaction during ELISA assay. The TMB reagent includes solution A and solution B. Solution A is prepared by dissolving 0.5g of urea peroxide in 1000mL of 0.1mol / L pH5.0 sodium acetate / citric acid buffer (approximately 150mL of 0.1mol citric acid was added to 0.1mol 850mL of sodium acetate), then adding phenacetin solution (0.08g preheated and dissolved in 15mL of ddw), mixing well, and storing at 4°C.

[0047] Solution B is prepared by dissolving 1.27 g of TMB (3,3',5,5'-tetramethylbenzidine) in 500 mL of methanol by heating, then mixing it with 500 mL of glycerol and storing at 4 °C.

[0048] Example 1: Synthesis of Complete Antigen

[0049] The C-3 carbonyl group was modified by oxime reaction to introduce a carboxyl group (TP-CMO). This carboxyl group was then cross-linked with macromolecular proteins via amide bonds. Immunogens were synthesized using a mixed anhydride method, and coating antigens were synthesized using a carbodiimide method. The testosterone propionate (TP) used was of analytical grade with a purity of 99.8%. The specific steps are as follows:

[0050] (1) Testosterone propionate oxime reaction: 10 mg of testosterone propionate (TP) was dissolved in 1 mL of anhydrous pyridine, and 7.4 mg of carboxymethoxyamine hemihydrochloride (CMO) was added. The mixture was reacted at room temperature for 24 hours and then evaporated to dryness. The mixture was reconstituted with 1 mL of deionized water and then extracted three times with 3 mL of ethyl acetate. The aqueous phase was discarded and the mixture was dried under nitrogen. The residue was testosterone propionate-carboxymethyl oxime (TP-CMO).

[0051] (2) Synthesis of immunogen (TP-BSA) by mixed anhydride method: Dissolve the residue TP-CMO obtained in step (1) with 1 mL DMF and nitrogen blowing, add 7 μL triethylamine, cool to 4℃, add 30 μL isobutyl chloroformate, react at 4℃ for 30 minutes to obtain solution A; weigh 20 mg BSA and dissolve it with 3 mL PBS solution (containing 20% ​​DMF) to obtain solution B; add solution A dropwise to solution B and mix well, react at 4℃ overnight, dialyze with deionized water for one day, and then dialyze with 1×PBS buffer for three days. After dialysis, centrifuge and collect the supernatant to obtain the immunogen TP-CMO-MA-BSA (hereinafter referred to as TP-BSA), aliquot and store at -20℃;

[0052] (3) Synthesis of coating antigen (TP-OVA) by carbodiimide method: Dissolve the residue TP-CMO obtained in step (1) by nitrogen blowing with 1 mL DMF, then add 6 mg of 1-ethyl-(3-dimethylaminopropyl)-carbodiimide hydrochloride (EDC) and 8 mg of N-hydroxysuccinimide (NHS), stir at room temperature and react for four hours to obtain solution C; weigh 12 mg of chicken ovalbumin (OVA) and dissolve it in 3 mL of PBS solution (containing 20% ​​DMF) to obtain solution D; add solution C dropwise to solution D and mix well, stir overnight at 4 °C, and then dialyze with 1×PBS buffer for three days; after dialysis, centrifuge and collect the supernatant to obtain coating antigen TP-CMO-EDC-OVA (hereinafter referred to as TP-OVA), aliquot and store at -20 °C.

[0053] This embodiment synthesizes the coated antigen (TP-OVA) via oxime synthesis combined with carbodiimide synthesis, and then analyzes it using ultraviolet scanning (UV scanning). Figure 1 A) and SDS-PAGE electrophoresis ( Figure 1 B) The results of the identification showed that the complete antigen TP-OVA was successfully conjugated, and the identification results showed that the complete testosterone propionate antigen (TP-OVA) was obtained.

[0054] Example 2: Preparation of Monoclonal Antibodies

[0055] 1. Animal immunization

[0056] (1) The immunogen (TP-BSA) synthesized in Example 1 was used to immunize 6-8 week old Balb / c female mice using the back multi-point injection method. In this example, two mice, No. 1 and No. 2, were selected. For the first immunization (i.e., the initial immunization), Freund's complete adjuvant was used, and each mouse was immunized with 100 μg of TP-BSA. For subsequent booster immunizations, the adjuvant was changed to Freund's incomplete adjuvant, and the immunization dose was reduced to 50 μg / mouse. During the immunization process, the antigen solution and adjuvant were mixed and emulsified with equal volumes (100 μL each), and the volume of antigen drawn was calculated. The remainder was replenished with 1×PBS buffer.

[0057] (2) During the immunization process, a booster immunization was performed every two weeks as one cycle, following the same procedure as the initial immunization; two weeks after the third immunization, the titer of the polyclonal antibody serum was determined using an indirect ELISA method, and the sensitivity (IC50) of the polyclonal antibody serum of the immunized mice was determined using an icELISA method. 50 ).

[0058] The indirect ELISA method is operated as follows:

[0059] 1) Coating: Adjust the concentration of the original TP-OVA to the optimal coating concentration (2 μg / mL) using CBS carbonate buffer, add 100 μL / well to a 96-well plate, incubate at 37°C for 2 h, wash 3 times with PBST washing buffer, and pat dry.

[0060] 2) Blocking: Add 5% porcine serum to a 96-well plate. It is best to prepare and use it immediately. The amount added is 200 μL / well. Incubate overnight at 4°C. Wash 3 times with PBST. Pat dry and store at 4°C for later use.

[0061] 3) Add polyantiserum: Add 100 μL of 1:100 polyantiserum directly to the first well. For the second well onwards, serially dilute the polyantiserum with PBS. Set up NC group and BC group at the same time. Incubate at 37℃ for 1 h. Wash 5 times with PBST and pat dry.

[0062] 4) Add enzyme-labeled secondary antibody: Dilute goat anti-mouse Goat-IgG-HRP with 5% porcine serum at a ratio of 1:5000, then add 100 μL / well to a 96-well plate, incubate at 37°C for 1 h, and wash the plate 5 times with PBST.

[0063] 5) Color development: Use TMB reagent for color development reaction. Mix equal volumes of solution A and solution B in the TMB color development reagent, add 100 μL / well to a 96-well plate, and react at room temperature for 5-7 min.

[0064] 6) Termination: Add 2 mol / L H2SO4 to each of the 96-well plates to be tested, at a volume of 100 μL / well;

[0065] 7) Reading: OD is measured using an ELISA reader. 450 absorbance value in nm;

[0066] 8) Result determination: OD of the test hole 450 nm / negative pore OD 450 If the nm value is ≥2.1, it is considered a positive well. The maximum dilution of the serum in a positive well is the titer of the sample to be tested.

[0067] The indirect competitive ELISA (icELISA) method is as follows:

[0068] 1) The coating and blocking processes are the same as those for the indirect ELISA method described above;

[0069] 2) Select mouse number 2 and use OD 450 The sensitivity of polyclonal antibody serum with a concentration close to 1.0 was determined by serial dilution. The specific steps were as follows: using a certain concentration of TP standard solution as an inhibitor, serial dilutions were performed until the third-to-last well was reached. 100 μL of this solution was aspirated, and 100 μL of OD200 solution was added sequentially from the second-to-last blank well to the wells with the highest standard concentration. 450 Polyclonal antibody serum with an nm reading of approximately 1.0 was incubated at 37°C for 1 hour, washed 5 times with PBST, and patted dry.

[0070] 3) Add enzyme-labeled secondary antibody: Dilute goat anti-mouse Goat-IgG-HRP with 5% porcine serum at a ratio of 1:5000, then add 100 μL / well to a 96-well plate, incubate at 37°C for 1 h, and wash the plate 5 times with PBST.

[0071] 4) Color development: Use TMB reagent for color development reaction. Mix equal volumes of solution A and solution B in the TMB color development reagent, add 100 μL / well to a 96-well plate, and react at room temperature for 7 min.

[0072] 5) Termination: Add 2 mol / L H2SO4 to each of the 96-well plates to be tested, at a volume of 100 μL / well;

[0073] 6) Reading: Measure the absorbance value at OD450nm using an ELISA reader;

[0074] 7) Result determination: If the OD of the suppression pore is... 450 A smaller value indicates that mice have produced specific antibodies. Then, an inhibition curve of TP against the monoclonal antibody was plotted with B / B0 as the ordinate and the logarithm of TP concentration (ng / mL) as the abscissa. Based on the plotted standard curve, a regression equation was derived, and the 50% IC50 of TP mAb against TP was calculated. 50 Value (where B0: OD when TP concentration is 0) 450 nm value; B: OD of other TP concentrations 450 The value of nm is used to measure the sensitivity of TP mAb, IC 50 The smaller the value, the stronger its sensitivity.

[0075] Results of indirect ELISA and indirect competitive ELISA (icELISA) are as follows Figure 2 As shown in Figure A, it can be seen that the serum titers of both immunized mice reached 1:2.048×10⁵, but mouse No. 1 was superior; the OD of mouse No. 1, which had the higher titer, was selected. 450 The sensitivity of polyclonal antibody serum with a value close to 1.0 was determined by dilution factor. Figure 2As shown in Figure B, the polyclonal antibody serum of mouse 1 has an inhibitory effect on TP, and the corresponding linear regression equation for the standard inhibition curve is: y = -0.2378x + 0.762R. 2 =0.98, calculate its IC 50 The concentration was 12.64 ng / mL.

[0076] 2. Cell fusion and monoclonal antibody preparation

[0077] (1) Select the No. 1 mouse with better sensitivity as the experimental subject and administer super-immunization by intraperitoneal injection. The immunization dose is 100 μg / mouse. 3-5 days after superimmunization, isolate mouse spleen cells aseptically and mix them with myeloma cells SP2 / 0 in the logarithmic growth phase at a ratio of 10:1. Use 1 ml of 45% PEG4000 for cell fusion. (2) 7-10 days after cell fusion, use indirect ELISA and indirect competitive ELISA (icELISA) to screen the culture supernatant of hybridoma cells in 96-well plates. Then, prepare testosterone propionate monoclonal hybridoma cell lines through 3-5 subclonings.

[0078] Following cell fusion and subsequent counting and detection steps, the cell fusion rate reached 80%, and the positive rate reached 15%. After three subcloning processes, four hybridoma cell lines stably secreting anti-testosterone propionate antibodies were screened out and named 3F9, 6D3, 7E12, and 7B2, respectively. The titers and sensitivities (IC50) of the cell supernatant for each cell line were analyzed. 50 The assays were performed (specific assay methods are described in the aforementioned indirect ELISA and indirect competitive ELISA (icELISA) methods), and the results are shown in Table 1. Table 1 shows that the 3F9 and 6D3 monoclonal antibodies had high sensitivity, at 5.918 and 6.37 ng / mL, respectively.

[0079] Table 1. Titer and sensitivity (IC50) of supernatant from four anti-TP monoclonal antibody hybridoma cell lines 50 )

[0080]

[0081]

[0082] Cell line stability was determined by indirect competitive ELISA (icELISA) using three cycles of cryopreservation and thawing. 50 Value changes as follows Figure 3 As shown, the 3F9 and 6D3 cell lines exhibited the best stability. Therefore, considering both sensitivity and stability results, the 3F9 cell line was selected for preparing monoclonal antibodies, and the performance of the monoclonal antibodies was evaluated in various aspects.

[0083] (3) The testosterone propionate monoclonal hybridoma cell line 3F9 was resuscitated and expanded. Ascites fluid was induced in vivo and purified using the ammonium octanoate sulfate method to obtain anti-TP monoclonal antibodies. These antibodies were then identified and purified by SDS-PAGE electrophoresis to obtain highly pure anti-testosterone propionate (TP) monoclonal antibodies. The results are as follows: Figure 4 As shown.

[0084] This invention also sequenced the monoclonal antibody from the 3F9 cell line, and the results are as follows:

[0085] Heavy chain variable region V H The gene sequence is shown in SEQ ID NO.1, specifically:

[0086] GGGAAGGGATCATCAACTCTCTGTGTTGCCTCTGGATTCACTTTCAGTAACTCCTGGATGAACTGGGTCCCGCCAGTCTCCAGAGAAGGGGCTTGAGTGGATTGCTGAAATTAGATTGAACTCTAATAATTATGCAACACATTATGCGGAGTCTGTGAAAG GGAGGTTCACCATTTCAAGAGATGATTCCAAAAGTAGTGTCTACCTGCAAATGAACAATTTAAGAGTTGAAGACACTGGCATTTATTACTGTAAGGGCTGGGACGTTTACTATGCTATGGACTACTGGGGCCAAGGGACCACGGTCACCGTCTCCTCAAAA

[0087] Heavy chain variable region V H The amino acid sequence is shown in SEQ ID NO.2, specifically as follows:

[0088] GKGSSTLCVASGFTFSNSWMNWVRQSPEKGLEWIAEIRLNSNNYATHYAESVKGRFTISRDDSKSSVYLQMNNLRVEDTGIYYCKGWDVYYAMDYWGQGTTVTVSSK

[0089] Specifically, V H The amino acid sequences of each site in the variable region are shown in Table 2 below.

[0090] Table 2

[0091] name sequence FR-H1 TLCVASGFTFS CDR-H1 NSWMN FR-H2 WVRQSPEKGLEWIA CDR-H2 EIRLNSNNYATHYAESVKG FR-H3 RFTISRDDSKSSVYLQMNNLRVEDTGIYYCKG CDR-H3 WDVYYAMDY FR-H4 WGQGTTVTVSS

[0092] Light chain variable region V L The gene sequence is shown in SEQ ID NO.3, specifically:

[0093] TTGACCTGGCTCCTGAGGTTCCAGGTTCCTCTGAGTGACATTGTGCTGACACAGTCTCCTGCTTCCTTAGCTGTATCTCTGGGGCAGAGGGCCACCATCTCATACAGGGCCAGCAAAAGTGTCAGTACATCTGGCTATAGTTATATGCACTGGAACCAACAGAAACCAGGACAGCCACCCAGACTCCTCATCTATCTTGTATCCAACCTAG AATCTGGGGTCCCTGCCAGGTTCAGTGGCAGTGGGTCTGGGACAGACTTCACCCTCAACATCCATCCTGTGGAGGAGGAGGATGCTGCAACCTATTACTGTCAGCACATTAGGGAGCTTACACGTTCGGAGGGGGGACCAAGCTGGAAATTAAACGGGCTGATGCTGCACCAACTGTATCCATCTTCCCACCATCCAGTAAGCTTGGGAAAA

[0094] Light chain variable region V L The amino acid sequence is shown in SEQ ID NO.4, specifically:

[0095] LTWLLRFQVPLSDIVLTQSPASLAVSLGQRATISYRASKSVSTSGYSYMHWNQQKPGQPPRLLIYLVSNLESGVPARFSGSGSGTDFTLNIHPVEEEDAATYYCQHIRELTRSEGGPSWKLNGLMLHQLYPSSHHPVSLGK

[0096] Specifically, V L The amino acid sequences of each site in the variable region are shown in Table 3 below.

[0097] Table 3

[0098] name sequence FR-L1 DIVLTQSPASLAVSLGQRATISY CDR-L1 RASKSVSTSGYSYMH FR-L2 WNQQKPGQPPRLLIY CDR-L2 LVSNLES FR-L3 GVPARFSGSGSGTDFTLNIHPVEEEDAATYYC CDR-L3 QHIR

[0099] Example 3 Synthesis of Immunofluorescent Probes

[0100] 1. Using the EDC method, quantum dot (QDs) labeled immunofluorescent probes are synthesized using orange CdSe / ZnS (core / shell) quantum dots. This example uses a monoclonal antibody from the 3F9 cell line as an example to synthesize the immunofluorescent probe. Those skilled in the art will know that monoclonal antibodies from 6D3, 7E12, or 7B2 cell lines can also synthesize immunofluorescent probes with high specificity and sensitivity, which will not be elaborated here. The specific steps for synthesizing the immunofluorescent probe are as follows:

[0101] (1) Mix 5 μL of orange QDs (8 μM) with 15.3 μL of EDC solution (1 mg / mL) to make orange QDs: EDC = 1:2000, and react in the dark at 25℃ and 220 r / min for 30 min.

[0102] (2) Add anti-TP monoclonal antibody according to the ratio of quantum dots (QDs): monoclonal antibody (mAb) = 1:10 (mass ratio), and supplement with PB buffer to make the total reaction volume 100μL. React in the dark at 25℃ and 220r / min for 3h.

[0103] (3) Add BSA as blocking solution and make its concentration 3 mg / mL. React at 25℃ and 220 r / min in the dark for 30 min to synthesize anti-TP-mAb-QDs immunofluorescence probe. Store at 4℃ in the dark.

[0104] The results were identified using methods such as fluorescence spectroscopy, agarose gel electrophoresis, SDS-PAGE electrophoresis, and immunochromatography. The identification results are as follows: Figure 5 As shown, from Figure 5 The results show that the synthesized immunofluorescent probe still retains its original biological activity, thus confirming that the anti-TP-mAb-QDs immunofluorescent probe was successfully obtained using this method.

[0105] 2. The secondary antibody probe (goat anti-mouse IgG-QDs fluorescent probe) was synthesized using the same method, except that the binding ratio was QDs:mAb = 1:5.

[0106] Example 4: Establishment of an Indirect Competitive ic-ELISA Method

[0107] 1. Determination of optimal coating concentration and fluorescent probe dilution factor

[0108] A bifunctional checkerboard method was used to explore the optimal working concentrations of the fluorescent probe and coating antigen. Unlike the traditional checkerboard method, a certain amount of TP standard was added simultaneously with the titer determination, and the well with the best inhibition was selected as the optimal working concentration of the fluorescent probe and coating antigen. The specific steps are as follows:

[0109] (1) Coating: Set the coating concentration gradient to 4 μg / mL, 2 μg / mL, 1 μg / mL and 0.5 μg / mL. Dilute TP-OVA to the corresponding concentration with CBS carbonate buffer and add 100 μL / well to the black microplate. Coat overnight at 4℃, discard TP-OVA, wash the plate three times with PBST washing solution and pat dry.

[0110] (2) Blocking: Add blocking solution (5% porcine serum) to the black microplate at a rate of 200 μL / well, place it in an oven at 37°C for two hours, then discard the porcine serum, wash the plate three times with PBST washing solution, and pat dry.

[0111] (3) Primary antibody: First, prepare a stock solution of TP standard at 1 mg / mL. When using, dilute it to a suitable concentration (specifically 10 ng / mL). Then, dilute the anti-TP-mAb-QDs fluorescent probe prepared in Example 3 with fluorescent antibody diluent (purchased from Beijing Solarbio Science & Technology Co., Ltd., catalog number A1840) at different gradients of 1:50, 1:100, 1:200, and 1:400. Add the primary antibody according to the layout shown in Table 4. Place the plate in a constant temperature oven at 37°C for 1 hour. After the reaction, remove the liquid from the wells, wash the plate three times, and pat it dry.

[0112] Table 4. Optimal working concentrations of fluorescent probes and coating antigens using the bifunctional checkerboard method.

[0113]

[0114] (4) Results determination: Readings were performed using a multi-functional microplate reader. Two blank-coated wells were set up as controls. 1×PBS buffer was added to the blank controls at a volume of 100 μL / well. The excitation wavelength of the microplate reader was set to 450 nm and the emission wavelength to 610 nm to determine the optimal working concentrations of the fluorescent probe and the coated antigen. The results are shown in Table 5.

[0115] Table 5 Results of optimal working concentrations of fluorescent probes and coating antigens

[0116]

[0117] The results in Table 5 show that the optimal working concentration of the fluorescent probe is 1 μg / mL when the coating concentration is 1 μg / mL and the dilution ratio is 1:400.

[0118] 2. Determination of specificity and sensitivity

[0119] Quantitative detection can be achieved by replacing goat anti-mouse IgG-HRP with quantum dot-labeled secondary antibody probes (goat anti-mouse IgG-QDs), reading fluorescence intensity values, and establishing a standard curve. The specific steps are as follows:

[0120] (1) Coating: Dilute TP-OVA to 2 μg / mL with CBS carbonate buffer, add 100 μL / well to the black microplate, coat overnight at 4℃, discard TP-OVA, wash the plate three times and pat dry;

[0121] (2) Blocking: Add blocking solution (5% porcine serum) to the black microplate at a rate of 200 μL / well, place it in a 37°C oven for 2 hours to block the reaction, discard the porcine serum, wash the plate three times and pat it dry.

[0122] (3) Primary antibody: Add 200 μL of diluted TP standard solution to the first well, then serially dilute and add it to the first to eleventh columns respectively. The twelfth column is a blank control well (numbered B0) without TP standard solution. Then add 100 μL of positive polyclonal antibody serum diluted 1:6400 to each well, incubate at 37℃ for 1 h, discard the primary antibody, wash the plate five times and pat dry.

[0123] (4) Secondary antibody: Dilute the enzyme-labeled secondary antibody (goat anti-mouse IgG-QDs) (1:400) with blocking buffer (5% porcine serum) at an appropriate ratio, add 100 μL / well to the black ELISA plate, incubate at 37℃ for 1 h, discard the secondary antibody, wash the plate five times with PBST washing buffer and pat dry.

[0124] (5) Color development: Use TMB reagent for color development reaction. Mix equal volumes of solution A and solution B in TMB color development reagent, add 100 μL / well to the black microplate, and develop color in the dark for 5-10 min.

[0125] (6) Termination: After the color development is complete, add an equal volume of stop solution (2 mol / L H2SO4) to the color development solution. Immediately after termination, read the OD of each well of the microplate using a microplate reader. 450nm value.

[0126] The measurement results are as follows Figure 6 As shown in A, Figure 6 A is the TP standard curve plotted using the indirect ic-ELISA method. Figure 6 From A, we can conclude that the sensitivity (ICP-ELISA) of detecting TP content using the ic-ELISA method is... 50 The concentration was 5.83 ng / mL.

[0127] Furthermore, this invention also uses other structural analogs of TP and prepares solutions with the same concentration as the TP standard solution to replace the TP standard solution in step (3), and detects the cross-reactivity of the TP monoclonal antibody with other structural analogs, thereby evaluating the specificity of the monoclonal antibody. The results in Table 6 show that the cross-reactivity rate of this monoclonal antibody with the other five structural analogs is less than 0.01%, obtaining a highly sensitive and specific testosterone propionate monoclonal antibody.

[0128] Table 6 Cross-reactivity of TP monoclonal antibodies with other structural analogs

[0129]

[0130] Example 5: Establishment of a direct competitive fluorescence immunosorbent assay (dc-FLISA)

[0131] The specific steps are as follows:

[0132] (1) Dilute TP-OVA to a final concentration of 2 μg / mL using CBS carbonate buffer, add 100 μL / well to a black microtiter plate, coat at 37°C for 3 h, and wash three times with phosphate buffer containing 0.5% Tween after the antigen coating is completed.

[0133] (2) The anti-TP-mAb-QDs fluorescent probe prepared in Example 3 was diluted with fluorescent antibody diluent at a ratio of 1:400 to obtain anti-TP mAb-QDs fluorescent probe diluent;

[0134] (3) Prepare TP standard solutions of different concentrations, specifically 20 ng / mL, 10 ng / mL, 5 ng / mL, 2.5 ng / mL, 1.25 ng / mL, 0.625 ng / mL, 0.3125 ng / mL and 0 ng / mL, to obtain TP concentration gradient standard solutions;

[0135] (4) The anti-TP-mAb-QDs fluorescent probe dilution solution and TP concentration gradient standard solution were added to the black microtiter plate and reacted at 37°C for 1 h. The anti-TP-mAb-QDs fluorescent probe competitively binds to TP-OVA coated in the black microtiter plate. After the competitive binding is completed, the anti-TP-mAb-QDs fluorescent probe is washed three times with phosphate buffer containing 0.5% Tween to obtain the antigen-antibody luminescent immune complex.

[0136] (5) The fluorescence intensity of the formed antigen-antibody luminescent immune complex was detected using a multifunctional microplate reader. The emission wavelength was 450 nm and the excitation wavelength was 610 nm. A standard curve was plotted with the logarithm of the TP concentration gradient in the standard solution as the abscissa and the obtained fluorescence intensity ratio as the ordinate. Figure 6 B).

[0137] The measurement results are as follows Figure 6 As shown in Figure B, the sensitivity (IC50) of the dc-FLISA method for detecting TP content can be observed. 50 The detection limit was 0.58 ng / mL, the detection range was 0.062 ng / mL, and the detection range was 0.062-5.455 ng / mL. This is comparable to the traditional ic-ELISA method (…). Figure 6A) Compared to the previous method, under the same antibody conditions, the dc-FLISA method established using quantum dot-labeled anti-TP-mAb-QDs fluorescent probes showed a 10-fold increase in sensitivity and a significant reduction in the detection limit. The flowchart of the direct competitive fluorescence immunosorbent assay (dc-FLISA) method established in Example 5 is shown below. Figure 7 As shown.

[0138] Specifically, the results of the standard curve analysis for ic-ELISA and dc-FLISA are shown in Table 7.

[0139] Table 7 shows the analytical results comparing ic-ELISA and dc-FLISA.

[0140]

[0141] Example 6: Recycling Verification Experiment

[0142] The indirect competitive ic-ELISA method and the direct competitive fluorescence immunosorbent assay (dc-FLISA) method established in this invention were used to detect TP in feed. The specific experimental steps are as follows:

[0143] (1) Sample pretreatment

[0144] The feed samples tested included grains such as wheat, barley, corn, soybeans, yellow soybeans, sorghum, peanuts, and oats, as well as finished feed or feed ingredients such as soybean meal. The samples to be tested were ground into powder, and 1.00±0.05g of homogenized sample was weighed into a 10mL centrifuge tube. The sample was extracted with 3ml of ethyl acetate by ultrasonication for 20min, and then centrifuged at 3500r / min for 10min. The extraction was repeated once. The two extracts were combined and evaporated to dryness at 45℃. The extracts were then reconstituted with phosphate buffer containing 5% methanol, filtered, and diluted 20 times before testing. A negative blank feed sample control group was also set up.

[0145] (2) Spike Recovery Experiment

[0146] TP standard was added to the negative blank feed sample matrix solution (same as pretreatment) to achieve final TP concentrations of 1, 5, 10, and 20 ng / mL. Each sample was then diluted 20-fold with phosphate buffer to obtain spiked test sample dilutions. Detection was performed using both ic-ELISA and dc-FLISA methods, with six replicates. The sample recovery rate and coefficient of variation were calculated using the following formulas.

[0147] Recovery rate = (detected concentration / spiking amount) × 100%;

[0148] Coefficient of variation (CV) = (Standard deviation SD / Mean) × 100%.

[0149] (3) Experimental Results

[0150] In the spiked recovery experiment, TP standard at concentrations of 1, 5, 10, and 20 ng / mL was added to the diluted negative blank feed sample, with six replicates for each spike concentration. This method was used to determine the content of artificially added TP in the feed and to validate the established dc-FLISA method. The results are shown in Table 8.

[0151] Table 8. Spiked recovery results of TP in feed detected by dc-FLISA / ic-ELISA

[0152]

[0153] The recoveries of TP in Table 8 ranged from 87.06% to 113.02%, with coefficients of variation all below 10%. These results indicate that the fluorescence immunoassay method established in this invention has high accuracy and precision.

[0154] The above embodiments are illustrative examples of the implementation of the present invention. The implementation of the present invention is not limited to the above embodiments. Any changes, modifications, substitutions, combinations, or simplifications made without departing from the spirit and principle of the present invention shall be considered equivalent substitutions and are included within the protection scope of the present invention.

Claims

1. A monoclonal antibody against testosterone propionate, characterized in that, It includes a heavy chain variable region and a light chain variable region, the amino acid sequence of the heavy chain variable region is shown in SEQ ID NO.2, and the amino acid sequence of the light chain variable region is shown in SEQ ID NO.

4.

2. The gene encoding the monoclonal antibody of claim 1, characterized in that, The gene sequence encoding the heavy chain variable region of the monoclonal antibody is shown in SEQ ID NO.1, and the gene sequence encoding the light chain variable region of the monoclonal antibody is shown in SEQ ID NO.

3.

3. A quantum dot-labeled immunofluorescent probe, characterized in that, The immunofluorescent probe is prepared by conjugating CdSe / ZnS quantum dots with the anti-testosterone propionate monoclonal antibody as described in claim 1.

4. The immunofluorescent probe as described in claim 3, characterized in that, The mass ratio of quantum dots to monoclonal antibodies during preparation is 1:(5-10).

5. A fluorescent immunoassay kit for detecting testosterone propionate, characterized in that, The kit includes: (a) Titration plate coated with testosterone propionate-coated antigen; (b) Anti-testosterone propionate monoclonal antibody-quantum dot immunofluorescent probe; (c) Testosterone propionate standard; The anti-testosterone propionate monoclonal antibody-quantum dot immunofluorescent probe is the quantum dot-labeled immunofluorescent probe as described in claim 3.

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