Monoclonal antibody resisting medroxyprogesterone acetate, application of monoclonal antibody and method for detecting MPA based on quantum dot labeled fluorescence-linked immunosorbent assay

Abstract

The invention relates to the field of biological detection, in particular to an anti-medroxyprogesterone monoclonal antibody, application of the monoclonal antibody and a method for detecting MPA based on a quantum dot labeled fluorescence-linked immunosorbent assay. The invention provides an anti-medroxyprogesterone monoclonal antibody. The antibody can accurately detect the content of medroxyprogesterone in a sample to be detected. Meanwhile, ZnCdSe / ZnS (core-shell) quantum dots are used as markers and are respectively coupled with an anti-medroxyprogesterone monoclonal antibody and rabbit anti-mouse IgG to prepare fluorescent probes, and a direct competitive fluorescent enzyme-linked immunosorbent assay method and an indirect competitive fluorescent enzyme-linked immunosorbent assay method are established by utilizing the prepared fluorescent probes; the method can be used for detecting medroxyprogesterone acetate residues in milk, and medroxyprogesterone acetate in milk can be rapidly and sensitively detected. The method has the advantages of rapidness, simplicity and convenience, and is more sensitive compared with an existing detection method.

Description

Technical Field

[0001] The invention relates to the field of biological detection, in particular to an anti-medroxyprogesterone acetate monoclonal antibody and application thereof and a method for detecting MPA by fluorescence-linked immunosorbent assay based on quantum dot labeling. Background Art

[0002] Medroxyprogesterone acetate (MPA) is a progestogen. Its chemical name is 6α-methyl-17α-hydroxypregnane-4-ene-3,20-dione acetate, and its chemical formula is C 24 H 34 O4, with a molecular weight of 386.52, was developed as the first generation of contraceptives in the mid-1960s. It is also used for assisted reproduction and the treatment of miscarriage, dysmenorrhea, uterine bleeding and endometriosis. As people have explored the function of MPA more deeply, they have found that MPA can interact with progesterone receptors and exert its effects in the body through lipid metabolism, sugar metabolism and the blood system. Clinically, MPA hormone replacement therapy is often used to assist in the fight against cancer. It is reported that progesterone is the most widely used progesterone in medicine, followed by MPA. MPA is relatively stable and not easy to degrade, so it will pollute the livestock production environment, thereby remaining in animal-derived foods and eventually enriching in the human body through the food chain. In order to protect the health of consumers, medroxyprogesterone acetate and its preparations are included in the banned list and shall not be used in the breeding process of food animals.

[0003] At present, the main methods for MPA detection are instrumental detection methods and immunological detection methods. Instrumental detection methods are accurate and sensitive, but require professional technicians to operate expensive instruments and produce a large amount of organic solvent waste; immunological methods such as ELISA and immunochromatography have the advantages of being fast, simple, and low-cost, but their detection limits are still high. Therefore, there is an urgent need for a method that can accurately, with a low detection limit, be universal, and pollution-free detect MPA. Summary of the invention

[0004] The purpose of the present invention is to provide an anti-medroxyprogesterone acetate monoclonal antibody and its application and a method for detecting MPA based on quantum dot labeling fluorescence-linked immunosorbent assay to solve the problems existing in the above-mentioned prior art. The anti-medroxyprogesterone acetate monoclonal antibody provided by the present invention can accurately detect the content of medroxyprogesterone acetate in the sample to be tested, and there is no requirement for professional technology and no pollution; at the same time, the detection limit is low.

[0005] To achieve the above object, the present invention provides the following solutions:

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

[0007] The heavy chain variable region includes CDR-H1 with an amino acid sequence as shown in SEQ ID NO.2, CDR-H2 with an amino acid sequence as shown in SEQ ID NO.4, and CDR-H3 with an amino acid sequence as shown in SEQ ID NO.6;

[0008] The light chain variable region includes CDR-L1 with an amino acid sequence as shown in SEQ ID NO.8, CDR-L2 with an amino acid sequence as shown in SEQ ID NO.10, and CDR-L3 with an amino acid sequence as shown in SEQ ID NO.12.

[0009] Preferably, the heavy chain variable region further comprises a heavy chain variable region framework region; the heavy chain variable region framework region comprises FR-H1 with an amino acid sequence as shown in SEQ ID NO.1, FR-H2 with an amino acid sequence as shown in SEQ ID NO.3, and FR-H3 with an amino acid sequence as shown in SEQ ID NO.5;

[0010] The light chain variable region also includes a light chain variable region framework region; the light chain variable region framework region includes FR-L1 as shown in the amino acid sequence of SEQ ID NO.7, FR-L2 as shown in the amino acid sequence of SEQ ID NO.9, and FR-L3 as shown in the amino acid sequence of SEQ ID NO.11.

[0011] The present invention provides the use of the above-mentioned anti-medroxyprogesterone acetate monoclonal antibody in the preparation of a product for detecting medroxyprogesterone acetate.

[0012] Preferably, the product includes a detection kit and a detection reagent;

[0013] The detection kit comprises a direct competitive fluorescent enzyme-linked immunosorbent assay kit and an indirect competitive fluorescent enzyme-linked immunosorbent assay kit.

[0014] The present invention provides a product for detecting medroxyprogesterone acetate, and the product comprises the above-mentioned anti-medroxyprogesterone acetate monoclonal antibody.

[0015] Preferably, the product further comprises HRP-labeled rabbit anti-mouse IgG;

[0016] The medroxyprogesterone acetate monoclonal antibody is a quantum dot-modified anti-medroxyprogesterone acetate monoclonal antibody; the rabbit anti-mouse IgG is a quantum dot-modified rabbit anti-mouse IgG;

[0017] The quantum dots are ZnCdSe / ZnS.

[0018] Preferably, the product includes a detection kit and a detection reagent;

[0019] The detection kit comprises a direct competitive fluorescent enzyme-linked immunosorbent assay kit and an indirect competitive fluorescent enzyme-linked immunosorbent assay kit.

[0020] The present invention provides the use of the above-mentioned anti-medroxyprogesterone acetate monoclonal antibody or the above-mentioned product in detecting medroxyprogesterone acetate.

[0021] The present invention provides a method for detecting medroxyprogesterone acetate, comprising the following steps:

[0022] After coating with medroxyprogesterone acetate, adding the sample to be tested and the anti-medroxyprogesterone acetate monoclonal antibody modified with quantum dots, detecting the fluorescence value, and bringing the fluorescence value into the medroxyprogesterone acetate standard curve to obtain the concentration of medroxyprogesterone acetate in the sample to be tested;

[0023] Or, after coating with medroxyprogesterone acetate, add the sample to be tested, the anti-medroxyprogesterone acetate monoclonal antibody modified with quantum dots, and the HRP-labeled rabbit anti-mouse IgG modified with quantum dots, detect the fluorescence value, and bring the fluorescence value into the medroxyprogesterone acetate standard curve to obtain the concentration of medroxyprogesterone acetate in the sample to be tested;

[0024] The anti-medroxyprogesterone acetate monoclonal antibody comprises a heavy chain variable region and a light chain variable region;

[0025] The heavy chain variable region includes CDR-H1 with an amino acid sequence as shown in SEQ ID NO.2, CDR-H2 with an amino acid sequence as shown in SEQ ID NO.4, and CDR-H3 with an amino acid sequence as shown in SEQ ID NO.6;

[0026] The light chain variable region includes CDR-L1 with an amino acid sequence as shown in SEQ ID NO.8, CDR-L2 with an amino acid sequence as shown in SEQ ID NO.10, and CDR-L3 with an amino acid sequence as shown in SEQ ID NO.12.

[0027] Preferably, the quantum dots are ZnCdSe / ZnS;

[0028] The sample to be tested is milk.

[0029] The present invention discloses the following technical effects:

[0030] The invention provides an anti-medroxyprogesterone acetate monoclonal antibody, which can accurately detect the content of medroxyprogesterone acetate in a sample to be tested; and a direct competitive fluorescent enzyme-linked immunosorbent assay kit and an indirect competitive fluorescent enzyme-linked immunosorbent assay kit can be prepared by using the monoclonal antibody, the detection method of the kit is simple, no technical requirements are placed on operators, a large amount of organic solvent waste liquid is not generated, and the detection limit is lower than other methods in the prior art.

[0031] The invention is based on the monoclonal antibody, takes ZnCdSe / ZnS (core-shell) quantum dots as markers, couples them with anti-medroxyprogesterone acetate monoclonal antibodies and rabbit anti-mouse IgG respectively to prepare fluorescent probes, and establishes a direct competitive fluorescent enzyme-linked immunosorbent assay (dc-FLISA) and an indirect competitive fluorescent enzyme-linked immunosorbent assay (ic-FLISA) by using the prepared fluorescent probes. The method can be used for sensitive detection of medroxyprogesterone acetate residues in milk, realizes rapid detection of medroxyprogesterone acetate in milk, has the advantages of being rapid and simple, and is more sensitive than existing detection methods. The experimental results show that the detection limit of medroxyprogesterone acetate in the sample by direct competitive fluorescent enzyme-linked immunosorbent assay is 1.41 ng / mL; the detection limit of medroxyprogesterone acetate in the sample by indirect competitive fluorescent enzyme-linked immunosorbent assay is 1.20 ng / mL; the recovery rate of the direct competitive fluorescent enzyme-linked immunosorbent assay in milk is 99.38%-105.11%, and the recovery rate of the indirect competitive fluorescent enzyme-linked immunosorbent assay is 96.02%-101.64%, and the coefficient of variation of the two methods is between 3.92%-7.08%, both less than 8%. It can be seen that the two fluorescent immunoassay methods established based on the monoclonal antibody provided by the present invention have high accuracy, precision and feasibility, and can be used to detect MPA in milk samples. BRIEF DESCRIPTION OF THE DRAWINGS

[0032] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings required for use in the embodiments will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying creative work.

[0033] Figure 1 This is a purity identification diagram of anti-medroxyprogesterone acetate monoclonal antibody; where M is a marker, lane 1 is ascites (before purification), and lane 2 is monoclonal antibody (after purification);

[0034] Figure 2 Agarose gel electrophoresis diagram of QDs, fluorescent probe QDs-mAb and QDs-IgG; wherein lane 1 is QDs, lane 2 is QDs-mAb, lane 3 is QDs, and lane 4 is QDs-IgG;

[0035] Figure 3 The immunochromatographic identification results of QDs, fluorescent probe QDs-mAb and QDs-IgG;

[0036] Figure 4 Standard curves for MPA determination for dc-FLISA and ic-FLISA;

[0037] Figure 5 Schematic diagram of the process of dc-FLISA;

[0038] Figure 6 Schematic diagram of the ic-FLISA process. DETAILED DESCRIPTION

[0039] Various exemplary embodiments of the present invention will now be described in detail. This detailed description should not be considered as limiting the present invention, but should be understood as a more detailed description of certain aspects, features, and embodiments of the present invention.

[0040] It should be understood that the terms described in the present invention are only for describing a particular embodiment and are not intended to limit the present invention. In addition, for the numerical range in the present invention, it should be understood that each intermediate value between the upper and lower limits of the scope is also specifically disclosed. The intermediate value in any stated value or stated range, and each smaller range between any other stated value or intermediate value in the described range is also included in the present invention. The upper and lower limits of these smaller ranges can be independently included or excluded in the scope.

[0041] Unless otherwise indicated, all technical and scientific terms used herein have the same meanings as those generally understood by those skilled in the art. Although the present invention describes only preferred methods and materials, any methods and materials similar or equivalent to those described herein may also be used in the implementation or testing of the present invention. All documents mentioned in this specification are incorporated by reference to disclose and describe the methods and / or materials associated with the documents. In the event of a conflict with any incorporated document, the content of this specification shall prevail.

[0042] It will be apparent to those skilled in the art that various modifications and variations may be made to the specific embodiments of the present invention description without departing from the scope or spirit of the present invention. Other embodiments derived from the present invention description will be apparent to those skilled in the art. The present invention description and examples are exemplary only.

[0043] The words “include,” “including,” “have,” “contain,” etc. used in this document are open-ended terms, meaning including but not limited to.

[0044] Example 1 Preparation of detection solution, anti-medroxyprogesterone acetate monoclonal antibody (anti-MPAmAb), and preparation of immunogen MPA-BSA and coating antigen MPA-OVA

[0045] 1. Preparation of detection solution:

[0046] (1) BBS buffer (0.2 M, pH 8.0): Solution A is a 0.05 M borax solution (weigh 1.907 g of borax and dilute to 100 mL with ultra-light water (ddw); Solution B is a 0.2 M boric acid solution (weigh 1.237 g of boric acid and dilute to 100 mL with ddw); Solution A and Solution B are mixed in a volume ratio of 3:7, and the pH is accurately adjusted to 8.0 and stored at room temperature;

[0047] (2) EDC solution: Weigh an appropriate amount of carbodiimide hydrochloride and add BBS buffer to prepare an EDC solution with a concentration of 1 mg / mL. The solution should be prepared and used immediately.

[0048] (3) PBS buffer: 8 g sodium chloride, 0.2 g potassium chloride, 1.44 g disodium hydrogen phosphate, and 0.24 g potassium dihydrogen phosphate were dissolved in 900 mL double distilled water, adjusted to pH 7.4 with hydrochloric acid, and made up to 1 L with water;

[0049] (4) CBS buffer: 1.59 g sodium phosphate; 2.93 g sodium hydrogen phosphate, add double distilled water to 900 ml, adjust the pH to 9.6, and adjust the volume to 1000 mL;

[0050] (5) PBST solution: 0.01 mol / L phosphate buffered saline (PBS), pH 7.4, containing 0.05% Tween 20.

[0051] 2. Preparation and purification of anti-medroxyprogesterone acetate monoclonal antibody (anti-MPAmAb or anti-MPA monoclonal antibody 6E2) in large quantities, the specific steps are as follows:

[0052] About 7-10 days before the preparation of anti-medroxyprogesterone acetate monoclonal antibodies, sterilized clarified paraffin was injected into the multiparous mice at a rate of 500 μL / mouse. The hybridoma cell line 6E2 cells, which were in good condition and had good secretion of anti-medroxyprogesterone acetate monoclonal antibodies, were inoculated at a rate of 1.5×10 6 The hybridoma cell line 6E2 cells were injected into the abdominal cavity of the parturient mice at a density of 100 / mL. The ascites production in the abdomen of the parturient mice was observed. The ascites was collected for about a week and purified by the octanoic acid-ammonium sulfate method. The concentration, purity and performance of the purified monoclonal antibody were evaluated, and finally the anti-medroxyprogesterone acetate monoclonal antibody was obtained. The purity identification chart of the anti-medroxyprogesterone acetate monoclonal antibody is shown in the figure Figure 1 As shown, the results showed that there were many miscellaneous bands before ascites purification, but only two obvious bands, heavy chain and light chain, were shown after purification, indicating that the purification result was good.

[0053] The amino acid sequence of the heavy chain variable region of the monoclonal antibody includes CDR-H1, CDR-H2 and CDR-H3; the heavy chain variable region framework region includes FR-H1, FR-H2 and FR-H3; the light chain variable region amino acid sequence includes CDR-L1, CDR-L2 and CDR-L3; the light chain variable region framework region includes FR-L1, FR-L2 and FR-L3, and the specific sequences are as follows:

[0054] FR-H1:MLMTSRDGLPSLWKPLPALPICRST, SEQ ID NO.1;

[0055] CDR-H1: TSKMRTRL, SEQ ID NO.2;

[0056] FR-H2: HISVQETLGSMLWTTGA, SEQ ID NO.3;

[0057] CDR-H2: QSAQAQIQ, SEQ ID NO.4;

[0058] FR-H3: LVQSGPELKKPGETVKISCKASGVKEPQSPSPQPKQQP, SEQ ID NO.5;

[0059] CDR-H3: HRSIHWTL, SEQ ID NO.6;

[0060] FR-L1: GGSAALGSGSTGDIVLTQSPASLAV, SEQ ID NO.7;

[0061] CDR-L1: SLGQRATISY, SEQ ID NO.8;

[0062] FR-L2:RASKSVSTSGYSYMHWNQ, SEQ ID NO.9;

[0063] CDR-L2: QKPGQPP, SEQ ID NO.10;

[0064] FR-L3: RLLIYLVSNLESGVPARFSGSGSGTDFTLNIH, SEQ ID NO.11;

[0065] CDR-L3: PVEEDAAT, SEQ ID NO. 12.

[0066] 3. Preparation of immunogen MPA-BSA (used for immunizing mice before preparing anti-medroxyprogesterone acetate monoclonal antibodies) and coating antigen MPA-OVA. The medroxyprogesterone acetate (MPA) molecule carries a carbonyl group, which is oximated to a carboxyl group under the action of carboxymethylhydroxylamine hydrochloride (CMO). The carboxyl group is activated by the activator carbodiimide hydrochloride (EDC), so that it combines with the amino group on the carrier protein to form an amide bond, thereby synthesizing a complete antigen. The detailed operation steps are as follows:

[0067] (1) Weigh 3.8 mg of MPA and dissolve it in 600 μL of methanol and 1.4 mL of pyridine. Then weigh 1.8 mg of CMO and add it to the above solution. Stir it at room temperature for 24 h. Blow dry the resulting solution with nitrogen gas, then add 600 μL of ddw, add H2SO4 to adjust the pH to 3-4, and measure the pH with a wide range pH test paper.

[0068] (2) Afterwards, the solution obtained in step (1) was extracted with 1 mL of ethyl acetate, the upper organic phase was drawn by a syringe, and the extraction was performed 4 times; anhydrous Na2SO4 was used to absorb the water remaining in the ethyl acetate, and the remaining liquid was blown dry with nitrogen to obtain an oximation product, and 600 μL of dimethylformamide (DMF) was added to dissolve the oximation product;

[0069] (3) Weigh 3.5 mg EDC and 2.0 mg N-hydroxysuccinimide (NHS), dissolve them in 200 μL 2-morpholineethanesulfonic acid (MES), add them to the solution obtained in step (2), and stir for 4 h in a dark environment;

[0070] (4) Weigh 7.8 mg of bovine serum albumin (BSA) and 4.6 mg of chicken ovalbumin (OVA) and dissolve them in 1 mL of CBS respectively. Divide the solution obtained in step (3) into two equal parts and add them dropwise to the BSA and OVA solutions respectively. Stir at 4°C overnight.

[0071] (5) dialyzing the solution obtained in step (4) at 4°C for three days (PBS, 0.01 mol / L, pH 7.4), replacing the dialysate every 8 hours;

[0072] (6) After dialysis is completed, the supernatant is collected by centrifugation and stored at -20°C to obtain the immunogen MPA-BSA and the coating antigen MPA-OVA.

[0073] (7) The animal immunization scheme is as follows: 6-8 week old female BALB / c mice are selected and subcutaneously immunized with the immunogen MPA-BSA. The immunization steps are as follows: 100 μL of adjuvant is drawn, the immunogen MPA-BSA immunization dose is 50 μg / mouse, the immunization volume is 200 μL / mouse, the remaining volume is filled with PBS, and the emulsifier is used to mix and emulsify it. When the sample is white foamy and there is no diffusion in the water, the emulsification can be stopped. The sample is sucked into the syringe and injected into the subcutaneous part of the back of the mouse by multi-point injection. A total of 4 immunizations are performed, each with an interval of 14 days. The first immunization uses Freund's complete adjuvant, and the rest use Freund's incomplete adjuvant. The booster immunization time is 3-5 days before fusion, the immunization dose is 100 μg / mouse, and the immunogen MPA-BSA is diluted to a volume of 100 μL with sterile PBS and injected intraperitoneally.

[0074] The detection solution, anti-medroxyprogesterone acetate monoclonal antibody, immunogen MPA-BSA and coating antigen MPA-OVA prepared in this example were used in subsequent experiments.

[0075] Example 2 Preparation and Identification of Coupling Products QDs-mAb and QDs-IgG (Rabbit-anti-Mouse QDs-IgG)

[0076] 1. Preparation of coupling products QDs-mAb and QDs-IgG:

[0077] QDs and anti-medroxyprogesterone acetate monoclonal antibody were coupled. The specific steps were as follows: 6.25 μL (1 / 16 of the system) of QDs-COOH (ZnCdSe / ZnS, 8.0 μM, purchased from Wuhan Jiayuan Quantum Dots, item number Q2605) was taken and mixed with 19.15 μL of 1 mg / mL EDC solution (EDC was diluted with pH 5.0) and 10 μL of ... 8.0 BBS was dissolved and prepared as needed), and activated at 25°C for 30 min under light-shielding conditions; after the QDs surface carboxyl groups were activated, 12 μL of anti-medroxyprogesterone acetate monoclonal antibody (concentration of 8 mg / mL) was added according to the molar ratio of anti-medroxyprogesterone acetate monoclonal antibody to quantum dots (ZnCdSe / ZnS) of 1:10, and the rest was made up with BBS buffer, and the reaction was carried out at 25°C and 220 r / min under light-shielding conditions for 3 h; 8000 r / min, centrifuged for 3 min to remove the precipitate, and the supernatant was stored at 4°C to obtain the QDs-anti-MPA-mAb fluorescent probe (hereinafter referred to as QDs-mAb);

[0078] QDs and HRP-labeled rabbit anti-mouse IgG were coupled. The specific steps were as follows: 6.25 μL (1 / 16 of the system) of QDs-COOH (ZnCdSe / ZnS, 8.0 μM) was taken and mixed with 19.15 μL of 1 mg / mL EDC solution (EDC was diluted with pH 5.0). 8.0 BBS was dissolved and prepared before use), and activated at 25°C for 30 min under light-shielding conditions; after the carboxyl groups on the QDs surface were activated, 12 μL of HRP-labeled rabbit anti-mouse IgG (ZnCdSe / ZnS) was added at a molar ratio of 1:10 between HRP-labeled rabbit anti-mouse IgG (ZnCdSe / ZnS) and quantum dots, and the rest was made up with BBS buffer, and the reaction was carried out at 25°C and 220 r / min under light-shielding conditions for 3 h; the precipitate was removed by centrifugation at 8000 r / min for 3 min, and the supernatant was stored at 4°C to obtain the QDs-anti-MPA-mAb fluorescent probe (hereinafter referred to as QDs-mAb).

[0079] 2. Identification of the coupling products QDs-mAb and QDs-IgG:

[0080] (1) Agarose gel electrophoresis identification: 1% Agarose was completely melted and the two fluorescent probes were poured into the gel preparation tank respectively, and cooled to solidify; QDs (ZnCdSe / ZnS) and the two fluorescent probes were diluted 1:20 with BBS buffer respectively; 4 μL of sample was loaded into each well, 100 V, 20 min; UV was used to observe the gel, the sample order was marked, and the results were photographed and stored. Figure 2 As shown. Figure 2 It can be seen that when electrophoresed under the same conditions, QDs-mAb and QDs-IgG lagged behind free quantum dots. This is because after the quantum dots were coupled with proteins to synthesize probes, the molecular weight increased and the migration speed slowed down. This preliminarily indicates that the two fluorescent probes were successfully coupled.

[0081] (2) Immunochromatographic identification: The coated antigen MPA-OVA or anti-medroxyprogesterone acetate monoclonal antibody (MPA-OVA was used in this example) was evenly applied to the detection line of the test strip, and after drying, it was installed in the groove. The quality control line of the test strip was sprayed with SPA in advance; QDs and fluorescent probes (QDs-mAb or QDs-IgG) were diluted with PBS in advance and mixed evenly; the mixed solution (QDs diluted 20 times, the concentration was 0.4 μM, QDs-mAb or QDs-IgG diluted 20 times) was dripped into the sample well, and the test strip chromatography was completed; in the dark, the test strip was irradiated with ultraviolet light. If both the T line and the C line were colored, it means that the probe coupling was successful. The results are as follows: Figure 3As shown. Immunochromatography is used to identify whether the fluorescent probe still retains biological activity. Quantum dots will not be captured by the coated antigen or antibody on the T line, nor by the SPA on the C line; probes that have lost their biological activity will not be captured by the T line, but will only bind to the C line; only probes that are successfully coupled and have biological activity will emit fluorescence on the T line and the C line. Figure 3 It can be seen that under ultraviolet light, the test strip with free QDs added showed no fluorescence on the T line and C line, while the test strip with fluorescent probe added showed orange fluorescence on the C line and T line. The results show that the coupled fluorescent probe has biological activity.

[0082] The QDs-mAb and QDs-IgG prepared in this example were used in subsequent examples.

[0083] Example 3 Establishment of direct competitive fluorescent enzyme-linked immunosorbent assay (dc-FLISA)

[0084] The principle of direct competitive FLISA is the same as that of dc-ELISA, where the coated antigen at the bottom of the plate competes with the MPA standard solution for the primary antibody. The difference is that ELISA uses colorimetric reading, that is, the enzyme-labeled antibody reacts with the enzyme substrate; while dc-FLISA converts the marker from enzyme to quantum dots and uses excitation light-emission light to read the fluorescence value. The process diagram of dc-FLISA is shown below: Figure 5 As shown, the specific operations are as follows:

[0085] (1) Coating and blocking: CBS buffer (0.05M, pH 9.6) was used as the coating buffer; the coating antigen MPA-OVA was diluted to 1 μg / mL, and added to the wells of the ELISA plate at a volume of 100 μL / well, and coated at 4°C overnight. The next day, the coating solution in the wells was discarded, and the antigens that were not firmly coated were washed with PBST solution, and added to the wells at a volume of 300 μL / well, and the excess washing solution was patted dry to remove; 5% porcine serum was selected as the blocking solution, 200 μL / well, placed at 37°C for 2 hours, washed 3 times, and patted dry for use;

[0086] (2) Primary antibody: According to the sensitivity of the antibody, determine the concentration of MPA standard to be added to the first well (10 ng / mL), and dilute it to the 7th well, and add PBS to the 8th well; add 100 μL of diluted QDs-mAb to each well (the dilution ratio of QDs-mAb is 1:50), bind at 37°C for 1 h, wash and pat dry;

[0087] (3) Result analysis: Set up three groups of blank controls, add PBS at 100 μL / well, and read the fluorescence value. Calculate F / F0 according to the formula as follows:

[0088] F / F0=(F sample -F blank ) / (Fcontrol -F blank );

[0089] Among them, F sample F is the fluorescence value of the wells with MPA standard solution added; control is the fluorescence value of the well without adding MPA standard solution; F blank is the fluorescence value of the blank control well.

[0090] Establish the standard curve of MPA concentration logarithm (abscissa) - F / F0 (ordinate) and obtain the fitting equation. The standard curve is as follows: Figure 4 As shown, y = -0.2184x + 0.2619, R 2 is 0.9705. According to the equation, IC 50 IC 10 、IC 20 ~IC 80 Evaluation, calculation results: half inhibitory concentration (IC 50 ) and LOD were 81.3 pg / mL and 1.41 ng / mL, respectively.

[0091] Example 4 Preliminary screening of indirect competitive fluorescent enzyme-linked immunosorbent assay (ic-ELISA)

[0092] (1) The most suitable concentration of coating antigen MPA-OVA and dilution multiple of anti-medroxyprogesterone acetate monoclonal antibody for ic-ELISA were determined by chessboard method. The most suitable concentration of coating antigen MPA-OVA was 2 μg / mL and dilution multiple of anti-medroxyprogesterone acetate monoclonal antibody was 1:80000.

[0093] (2) Dilute the coated antigen MPA-OVA with CBS solution to 2 μg / mL of the coated antigen MPA-OVA diluent, add the coated antigen MPA-OVA diluent to a 96-well transparent ELISA plate at a dosage of 100 μL / well, incubate at 37°C for 2 h, wash three times with PBST, and pat dry; add blocking solution at a dosage of 200 μL / well, incubate at 37°C for 2 h, wash three times with PBST solution, pat dry, and set aside;

[0094] (3) The steps of ic-ELISA are as follows:

[0095] The initial screening concentration of MPA standard solution was set to 10 ng / mL, and the two-fold dilution method was used to dilute the solution to the second-to-last well, with 100 μL per well. A control well (B0) without adding a standard was set.

[0096] After anti-medroxyprogesterone acetate monoclonal antibody was diluted to an appropriate concentration at a dilution ratio of 1:80,000, 100 μL per well was added to a 96-well ELISA plate, incubated at 37°C for 1 h, washed 5 times, and patted dry;

[0097] HRP-labeled rabbit anti-mouse IgG was diluted at a dilution factor of 1:5000 and added to the wells at a volume of 100 μL / well. The wells were incubated at 37°C for 1 h, washed 7 times, and patted dry.

[0098] After mixing the TMB colorimetric solution in equal proportions, add 100 μL / well to the wells and color develop at room temperature for 8 min; add 100 μL of stop solution to each well to stop the color development reaction;

[0099] Finally, the OD of each well was read using a microplate reader. 450nm The value of

[0100] The logarithm of MPA concentration was used as the horizontal axis and F / F0 as the vertical axis to establish a standard curve (F is the OD corresponding to the wells with different concentrations of MPA standard added). 450nm value; F0 is the OD corresponding to the well with MPA standard concentration of 0 450nm The half inhibitory concentration (IC 50 ) was 0.26 ng / mL and the limit of detection (LOD) was 0.024 ng / mL.

[0101] Example 5 Establishment of indirect competitive fluorescent enzyme-linked immunosorbent assay (ic-FLISA)

[0102] The principle of indirect competitive FLISA is the same as that of ic-ELISA, except that quantum dot markers are used instead of traditional HRP markers. The MPA standard and the antigen coated on the bottom of the plate compete for binding to the anti-MPA monoclonal antibody, and the secondary antibody QDs-IgG labeled with quantum dots is used to bind to the primary antibody to achieve the purpose of signal amplification. Like dc-FLISA, the fluorescence value is read by excitation light-emission light. The process diagram of ic-FLISA is shown in the figure below. Figure 6 As shown, the specific steps are as follows:

[0103] (1) The coating, blocking and primary antibody addition steps were the same as those in the dc-FLISA method in Example 3, and the primary antibody was an appropriately diluted monoclonal antibody (1:35000);

[0104] (2) Secondary antibody: This step is the same as the dc-FLISA method in Example 3, except that: unbound excess anti-medroxyprogesterone acetate monoclonal antibody is washed away, and QDs-IgG diluted in an appropriate ratio (1:200) is added and reacted at 37° C. for 1 h;

[0105] (3) Wash to remove excess probes, set up three groups of blank controls, and add PBS at a volume of 100 μL / well;

[0106] (4) Analysis of results: The analysis method is the same as that of dc-FLISA above, and the standard curve is as follows: Figure 4 As shown, the fitting equation y = -0.2810x + 0.0984, R 2 =0.97838; then calculate the IC of this method 50 value, detection limit (IC 10 ) and linear detection range (IC 20 ~IC 80 ) The calculation result is: IC 50 It is 38.0pg / mL and the LOD is 1.20pg / mL.

[0107] Example 6 Spiked Recovery Experiment

[0108] The sample spike recovery experiment was carried out using the dc-FLISA and ic-FLISA methods. The specific steps are as follows:

[0109] Use dc-FLISA (the experimental steps are the same as in Example 3, except that the MPA standard is replaced by the sample to be tested) and ic-ELISA (the experimental steps are the same as in Example 5) to verify whether the milk sample is negative. Pre-treat the negative sample. First, boil the milk for 5-10 minutes, filter with gauze to remove the solids, and then centrifuge at high speed (10000rpm, 10min) to obtain skimmed milk. Under a water bath of 40°C, adjust the pH of the supernatant to 4.7-4.8 with 10% acetic acid, stand for 30 minutes, and then centrifuge at high speed (10000rpm, 10min). Adjust the pH to 7.2-7.4, centrifuge, and obtain whey. Add different amounts of MPA standard to the whey to make the final concentrations of 0.5, 0.8, and 1ng / mL (ic-FLISA only sets three concentrations of 0.1, 0.2, and 0.4ng / mL according to its linear range of detection), and repeat 6 groups each. According to the matrix standard curve, the actual recovery concentration, average recovery rate, coefficient of variation, and standard deviation are calculated. The formulas for spike recovery and coefficient of variation are as follows:

[0110] Recovery rate = actual detection concentration of sample / concentration of added MPA × 100%;

[0111] Coefficient of variation (CV%) = (standard deviation SD / mean value Mean) × 100%.

[0112] Different concentrations of MPA were added to the treated negative milk samples. When dc-FLISA was detected, the concentrations of MPA were 0.5, 0.8, and 1 ng / mL; when ic-FLISA was detected, the concentrations of MPA were 0.1, 0.2, and 0.4 ng / mL. The established method was used for verification. The recovery results of addition are shown in Table 1. The recovery rates of dc-FLISA in milk were 99.38%-105.11%, and the recovery rates of ic-FLISA were 96.02%-101.64%. The coefficients of variation of the two methods were between 3.92% and 7.08%, both less than 8%. The data show that the two fluorescent immunoassay methods established by the present invention have high accuracy, precision, and feasibility, and can be used to detect MPA in milk samples.

[0113] Table 1 Recovery of MPA in milk samples

[0114]

[0115] Note: a means the data are Mean±SD; b means 6 parallel experiments were set for each group of data.

[0116] The embodiments described above are only descriptions of the preferred modes of the present invention, and are not intended to limit the scope of the present invention. Without departing from the design spirit of the present invention, various modifications and improvements made to the technical solutions of the present invention by ordinary technicians in this field should all fall within the protection scope determined by the claims of the present invention.

Claims

1. An anti-medroxyprogesterone acetate monoclonal antibody, characterized in that: The anti-medroxyprogesterone acetate monoclonal antibody comprises a heavy chain variable region and a light chain variable region; The heavy chain variable region includes CDR-H1 with an amino acid sequence as shown in SEQ ID NO.2, CDR-H2 with an amino acid sequence as shown in SEQ ID NO.4, and CDR-H3 with an amino acid sequence as shown in SEQ ID NO.6; The light chain variable region includes CDR-L1 with an amino acid sequence as shown in SEQ ID NO.8, CDR-L2 with an amino acid sequence as shown in SEQ ID NO.10, and CDR-L3 with an amino acid sequence as shown in SEQ ID NO.

12.

2. The anti-medroxyprogesterone acetate monoclonal antibody according to claim 1, characterized in that The heavy chain variable region also includes a heavy chain variable region framework region; the heavy chain variable region framework region includes FR-H1 with an amino acid sequence as shown in SEQ ID NO.1, FR-H2 with an amino acid sequence as shown in SEQ ID NO.3, and FR-H3 with an amino acid sequence as shown in SEQ ID NO.5; The light chain variable region also includes a light chain variable region framework region; the light chain variable region framework region includes FR-L1 as shown in the amino acid sequence of SEQ ID NO.7, FR-L2 as shown in the amino acid sequence of SEQ ID NO.9, and FR-L3 as shown in the amino acid sequence of SEQ ID NO.

11.

3. Use of the anti-medroxyprogesterone acetate monoclonal antibody according to claim 1 or 2 in the preparation of a product for detecting medroxyprogesterone acetate.

4. The use according to claim 3, characterized in that: The products include test kits and test reagents; The detection kit comprises a direct competitive fluorescent enzyme-linked immunosorbent assay kit and an indirect competitive fluorescent enzyme-linked immunosorbent assay kit.

5. A product for detecting medroxyprogesterone acetate, characterized in that: The product comprises the anti-medroxyprogesterone acetate monoclonal antibody according to claim 1 or 2.

6. The product according to claim 5, characterized in that The product also includes HRP-labeled rabbit anti-mouse IgG; The medroxyprogesterone acetate monoclonal antibody is a quantum dot-modified anti-medroxyprogesterone acetate monoclonal antibody; the rabbit anti-mouse IgG is a quantum dot-modified rabbit anti-mouse IgG; The quantum dots are ZnCdSe / ZnS.

7. The product according to claim 5 or 6, characterized in that The products include test kits and test reagents; The detection kit comprises a direct competitive fluorescent enzyme-linked immunosorbent assay kit and an indirect competitive fluorescent enzyme-linked immunosorbent assay kit.

8. Use of the anti-medroxyprogesterone acetate monoclonal antibody according to claim 1 or 2 or the product according to any one of claims 5 to 7 in detecting medroxyprogesterone acetate.

9. A method for detecting medroxyprogesterone acetate, characterized in that: The following steps are involved: After coating with medroxyprogesterone acetate, adding the sample to be tested and the anti-medroxyprogesterone acetate monoclonal antibody modified with quantum dots, detecting the fluorescence value, and bringing the fluorescence value into the medroxyprogesterone acetate standard curve to obtain the concentration of medroxyprogesterone acetate in the sample to be tested; Or, after coating with medroxyprogesterone acetate, add the sample to be tested, the anti-medroxyprogesterone acetate monoclonal antibody modified with quantum dots, and the HRP-labeled rabbit anti-mouse IgG modified with quantum dots, detect the fluorescence value, and bring the fluorescence value into the medroxyprogesterone acetate standard curve to obtain the concentration of medroxyprogesterone acetate in the sample to be tested; The anti-medroxyprogesterone acetate monoclonal antibody comprises a heavy chain variable region and a light chain variable region; The heavy chain variable region includes CDR-H1 with an amino acid sequence as shown in SEQ ID NO.2, CDR-H2 with an amino acid sequence as shown in SEQ ID NO.4, and CDR-H3 with an amino acid sequence as shown in SEQ ID NO.6; The light chain variable region includes CDR-L1 with an amino acid sequence as shown in SEQ ID NO.8, CDR-L2 with an amino acid sequence as shown in SEQ ID NO.10, and CDR-L3 with an amino acid sequence as shown in SEQ ID NO.

12.

10. The method according to claim 9, characterized in that The quantum dots are ZnCdSe / ZnS; The sample to be tested is milk.

Images