A soft hair penicillic acid monoclonal antibody, a rapid detection test strip and a preparation method thereof

Abstract

The present application relates to the field of monoclonal antibody and in vitro detection technology, and specifically discloses a specific monoclonal antibody for binding to penicillic acid, wherein the heavy chain variable region of the monoclonal antibody has an amino acid sequence as shown in SEQ ID NO:1, and the light chain variable region has an amino acid sequence as shown in SEQ ID NO:2. A rapid test strip for penicillic acid is also disclosed, which comprises a base plate, a sample pad, a label pad, a nitrocellulose membrane and an absorbent paper sequentially connected to the base plate; the nitrocellulose membrane is provided with a detection line and a quality control line, the detection line is coated with a penicillic acid-carrier protein conjugate, and the quality control line is coated with an anti-mouse IgG antibody; the label pad is coated with a labeled monoclonal antibody. The present application fills the gap of the rapid immunodetection technology for penicillic acid, and provides a simple, rapid and economical detection method for the daily monitoring, risk warning and emergency disposal of penicillic acid in red koji and related products.

Description

Technical Field

[0001] This invention relates to the field of monoclonal antibody and in vitro detection technology, and in particular to a penicillic acid monoclonal antibody, a rapid test strip, and a method for preparing the same. Background Technology

[0002] In 2024, Kobayashi Pharmaceutical Co., Ltd. of Japan reported to the Japanese Ministry of Health, Labour and Welfare that the problematic component in its red yeast rice products might be "puberulic acid." Puberulic acid is a natural toxin produced by Penicillium mold during fermentation. Currently, there is no standard detection method for puberulic acid (PA), and instrumental methods are generally used for detection, such as high-performance liquid chromatography (HPLC), gas chromatography (GC), and liquid chromatography-mass spectrometry (LC-MS / MS). These methods rely on large instruments, are complex to operate, require specialized technicians, are time-consuming, and costly. There is currently no convenient and rapid detection method available; therefore, existing technologies require further improvement. Summary of the Invention

[0003] This invention aims to solve the above problems and provides the following technical solution, as detailed below: In a first aspect, the present invention provides a monoclonal antibody that specifically binds to penicillic acid, wherein the heavy chain variable region of the monoclonal antibody has an amino acid sequence as shown in SEQ ID NO:1, and the light chain variable region has an amino acid sequence as shown in SEQ ID NO:2.

[0004] Furthermore, the monoclonal antibody was produced by hybridoma cell line 3H8, which was deposited on June 25, 2026 at the China Center for Type Culture Collection (CCTCC) with accession number CCTCC NO: C2026137 and address: Wuhan University, Wuhan, Hubei Province, China.

[0005] In a second aspect, the present invention provides the application of a monoclonal antibody that specifically binds to penicillic acid as described in the first aspect in the detection of penicillic acid.

[0006] Thirdly, the present invention provides a rapid test strip for penicillic acid, comprising a base plate and a sample pad, a marking pad, a nitrocellulose membrane and absorbent paper sequentially overlapped on the base plate; The nitrocellulose membrane is provided with a detection line and a control line. The detection line is coated with a penicillic acid-carrier protein conjugate, and the control line is coated with an anti-mouse IgG antibody. The label pad is coated with the monoclonal antibody of claim 1, which is labeled with a marker.

[0007] Furthermore, the marker is colloidal gold particles, time-resolved fluorescent microspheres, carboxylated colored microspheres, or fluorescent quantum dot microspheres.

[0008] Fourthly, the present invention provides a method for preparing a rapid test strip for penicillic acid according to the third aspect, comprising the following steps: S1: Spray the penicillic acid-carrier protein conjugate and anti-mouse IgG antibody onto a nitrocellulose membrane to form a detection line and a control line. After drying, the coated membrane is obtained. S2: The monoclonal antibody described in the first aspect is conjugated with a label to obtain a labeled antibody, and the labeled antibody is sprayed or immersed on a labeling pad and dried. S3: The sample pad, marking pad, coating film and absorbent paper are sequentially overlapped and pasted onto the base plate, and then cut into shape to obtain a rapid test strip.

[0009] Furthermore, in step S1, the concentration of the penicillic acid-carrier protein conjugate is 0.01–0.1 mg / mL, and the concentration of the anti-mouse IgG antibody is 0.1–0.5 mg / mL.

[0010] Furthermore, the marker is a time-resolved fluorescent microsphere, and the coupling in step S2 includes the following operations: after activating the microsphere with EDC and NHS, a monoclonal antibody is added for reaction, and then bovine serum albumin is added for blocking.

[0011] Fifthly, the present invention also provides a method for detecting penicillic acid, comprising the step of using the monoclonal antibody described in the first aspect or the test strip described in the third aspect to detect the sample to be tested.

[0012] Furthermore, the detection is either qualitative or quantitative; the qualitative detection uses colloidal gold-labeled test strips, and the color development of the test line is observed by the naked eye for judgment; the quantitative detection uses fluorescently labeled test strips, and the signal value is read in conjunction with a fluorescence detector.

[0013] The present invention has the following beneficial effects: 1. This invention provides for the first time a highly sensitive monoclonal antibody specific to penicillic acid. A hybridoma cell line 3H8 that stably secretes antibodies against penicillic acid was obtained through hybridoma technology screening. The half-maximal inhibitory concentration (IC50) of the secreted antibody against penicillic acid is as low as 1.6 ng / mL, which has extremely high affinity and specificity, providing a core raw material for the immunological detection of penicillic acid.

[0014] 2. The colloidal gold qualitative test strip developed based on the antibody of this invention is simple to operate, requires no instruments or equipment, and the results can be read by the naked eye within 10 minutes after sample addition. It is suitable for rapid on-site screening and overcomes the shortcomings of traditional liquid chromatography-mass spectrometry (LC-MS / MS) which relies on large instruments, is complicated to operate, has a long detection time, and is costly.

[0015] 3. This invention utilizes a time-resolved fluorescent microsphere-labeled quantitative test strip, which, when used with a portable fluorescence detector, can complete quantitative detection within 8 minutes. Actual sample spiked recovery experiments show that when the amount of penicillic acid added to red yeast rice samples is 3~100 ng / g, the recovery rate of the quantitative test strip is 91.0%~108.1%, highly consistent with the recovery rate (80.3%~116.2%) of LC-MS / MS detection results, indicating that the quantitative detection results of this invention are accurate and reliable.

[0016] 4. This invention addresses the lack of detection methods for penicillic acid, filling the gap in rapid immunoassay technology for this toxin. It provides a simple, rapid, and economical detection method for daily monitoring, risk warning, and emergency response of penicillic acid in red yeast rice and related products. Attached Figure Description

[0017] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the description of the embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are only one embodiment of the present invention. For those skilled in the art, other embodiments can be derived from the provided drawings without creative effort.

[0018] Figure 1 : Schematic diagram of rapid test strip. Detailed Implementation

[0019] The technical solutions of the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only a part of the embodiments of the present invention, not all of them. The following embodiments are only for illustrative purposes and are not intended to limit the scope of the present invention in any way. All other embodiments obtained by those skilled in the art based on the embodiments of the present invention without creative effort are within the scope of protection of the present invention. Unless otherwise specified, the experimental methods used in the present invention are conventional methods. Unless otherwise specified, the materials and reagents used in the present invention are commercially available. Furthermore, other terms used in the present invention, unless otherwise specified, generally have the meanings commonly understood by those skilled in the art.

[0020] Example 1: Synthesis of penicillic acid (PA) antigen Weigh 1 mg of penicillic acid (prepared according to method CN120700067A) into a reaction flask, add 5-20 mg of BSA (bovine serum albumin), then add acetic acid / sodium acetate buffer at pH 4.2 and stir for 10 minutes to dissolve. Subsequently, add 0.5 mL of formaldehyde solution (37%) dropwise under magnetic stirring. After sample addition, stir the reaction mixture at room temperature for 6-12 hours. Place the reaction solution in a dialysis bag and dialyze it in 0.01 M, pH 7.2 PBS at 4°C with stirring. Change the dialysate every 8 hours for a total of 4 times. After dialysis, PA antigen PA-BSA is obtained.

[0021] The same method was used to conjugate PA with OVA (ovalbumin) to synthesize PA-OVA antigen.

[0022] Example 2: Preparation of penicillic acid (PA) antibody 2.1 BALB / c mice were immunized with PA-BSA. A total of 5 mice were immunized by subcutaneous injection of 0.1 mL of antigen in the back and intraperitoneal injection of 0.1 mL of antigen.

[0023] The immunization schedule is shown in the table below:

[0024] Mouse serum titer assay: PA-OVA antigen was diluted to 1 μg / mL with pH 9.6 carbonate buffer. 100 μL of the diluted antigen was added to each well of the ELISA plate, and the plates were coated at 4°C for 16 h to prepare the ELISA plates. The plates were then used to detect mouse serum titers. The results are shown in the table below.

[0025] The test results showed that mouse No. 3 had the highest titer. When the serum was diluted 81,000 times, the detection signal was 1.454. Finally, mouse No. 3 was used for cell fusion.

[0026] 2.2 Cell Fusion 1) Take one SP2 / 0 tube, passage it after revival, count the cells, and then passage it proportionally for 3 days. 2) Gently pipette healthy mouse myeloma SP2 / 0 cells from the culture dish. Transfer to 50mL centrifuge tubes: After euthanizing mice by enucleating their eyeballs to collect blood, juvenile them by pulling their necks and immersing them in 75% alcohol for 5 minutes. 3) Pour a small amount of serum-free DMEM medium into a petri dish, and place the cell sieve and syringe core into the dish: 4) Under aseptic conditions, remove the mouse spleen, place it on a cell sieve, and gently crush the spleen thoroughly with the syringe plunger. Then, transfer the crushed cells into a centrifuge tube containing SP2 / 0 cells and centrifuge at 1200 rpm for 5 minutes at room temperature. 5) Take the thymus of the mouse and crush it, transfer it to a 15mL centrifuge tube, add 1mL of HAT medium, and incubate in an incubator (37℃ 5%CO2); 6) Discard the supernatant after centrifugation, carefully and gently rehydrate the cells with serum-free DMEM medium, and centrifuge again at 1200 rpm for 5 min at room temperature. 7) Discard the supernatant after centrifugation, gently tap the bottom of the centrifuge tube to fully suspend the cells, then place the centrifuge tube in a 37°C water bath, and slowly add 1 mL of polyethylene glycol (PEG4000) over 1 min. After standing for 1 min, slowly add 2 mL of serum-free DMEM medium over 2 min, followed by slowly adding 8 mL of serum-free DMEM medium over 2 min. Centrifuge at 1000 rpm for 5 min at room temperature. 8) Discard the supernatant, add 10 mL of serum, and carefully blow the cells evenly. Add the prepared thymocytes, and then add 25 mL of sterile semi-solid culture medium (MC). Mix thoroughly and pour into 20 cell culture dishes, 2 mL / dish. Place in a humidified box and incubate in an incubator (37°C, 5% CO2).

[0027] 9) Around day 10, begin selecting clones. At this point, the cell clusters are visible to the naked eye and easily distinguishable under a microscope (be careful to prevent the culture medium from turning yellow, which could lead to insufficient nutrients and cell death. If there are many confluent cell clusters, clones can be selected earlier). Select 10 plates * 92 cell clones and culture them in 96-well cell culture plates (pre-coated with 25% HT medium containing mouse thymocytes, 50 μL / well). Change the medium once on day 2. By day 4, the cells will have filled the 96-well plate. At this point, use indirect ELISA to detect antibodies in the culture medium supernatant.

[0028] 2.3 Screening of positive clones PA-BSA was diluted with coating buffer (CBS) to a concentration of 1 μg / mL. 100 μL / well was added to a 96-well microplate for coating. After coating overnight at 4°C, the plate was washed three times with PBST washing buffer.

[0029] Block with blocking solution (5% skim milk powder / PBS), 200 μL / well, incubate at 37°C for 2 h, and then wash 3 times with washing solution.

[0030] Add 100 μL of cell culture supernatant from a 96-well plate to each well, including negative control (SP2 / 0 culture supernatant), blank control (PBS solution), and positive control (PBS diluted 1000 times with positive serum). Incubate at 37°C for 45 min and wash three times with washing buffer.

[0031] Add 100 μL / well of enzyme-labeled secondary antibody (goat anti-mouse IgG-HRP) diluted 1000 times with PBS, incubate at 37°C for 45 min, and then wash 3 times with washing buffer.

[0032] Add 100 μL of TMB (tracebium bicarbonate) per well and develop the color for about 10-15 minutes.

[0033] Add 50 μL of stop solution (2M concentrated sulfuric acid) per well to terminate the reaction.

[0034] The absorbance at 450 nm was measured using an ELISA reader, and the data was recorded and saved.

[0035] Generally, a positive hybridoma cell line is defined as a cell supernatant absorbance value (A450) greater than 2.1 times that of the negative control. Based on this criterion, clones that tested positive in the first screening were further cultured and then screened a second time using the same method, resulting in five positive hybridoma cell lines: clones 3A6, 3H8, 5B6, 10G3, and 11D7. After testing the sensitivity of the cell supernatant, clone 3H8, with the highest sensitivity, was ultimately used for ascites preparation and antibody purification.

[0036] The sensitivity data for fixed-line cell detection are shown in the table below:

[0037] The data shows that the antibody produced by cells of clone 3H8 has the highest sensitivity (IC50) at 1.6 ng / mL.

[0038] The hybridoma cell line 3H8 was deposited at the China Center for Type Culture Collection (CCTCC) on June 25, 2026, with accession number CCTCC NO: C2026137, at Wuhan University, Wuhan, Hubei Province, China.

[0039] 2.4 Ascites preparation 1) Adjust the temperature of the constant temperature water bath to 37℃-40℃.

[0040] 2) Take the frozen cell line 3H8 out of the liquid nitrogen tank and immediately place it in warm water at 37℃-40℃ and shake it rapidly until it is completely thawed.

[0041] 3) Transfer the cell cryopreservation suspension to a centrifuge tube, add 10 ml of culture medium (DMEM + 20% newborn calf serum), and gently pipette to mix.

[0042] 4) Centrifuge the cell suspension at 1000 rpm / min for 5 min at room temperature and discard the supernatant.

[0043] 5) Add culture medium to the cell pellet, gently pipette to mix, and then transfer to a cell culture dish. Incubate at 37°C in a cell culture incubator.

[0044] 6) Culture the cells to the logarithmic growth phase, wash with PBS solution and count the cells.

[0045] 7) One week before inoculation, mice were injected with wax oil via intraperitoneal injection, 500 μL / Balb / c mice.

[0046] 8) Cells were inoculated via intraperitoneal injection, with each mouse receiving 1 ml (5*10⁶ cells). 5 -10*10 5 (pcs / ml).

[0047] 9) One week later, collect the ascites fluid, centrifuge at 5000 rpm for 10 min, collect the supernatant, and store at -20 degrees Celsius.

[0048] 2.5 Antibody Purification 1) Pretreatment of ascites samples: The supernatant of the thawed ascites was diluted 3 times with coupling buffer (20mM sodium phosphate solution, pH7.0), centrifuged at 1200rpm and 4℃ for 10min, and then filtered through a 0.22μm filter membrane to remove fat, cell debris and small particulate matter. 2) Equilibration: Equilibrate the column with 10 times the volume of the antibody affinity pre-packed column of coupling buffer, maintaining a flow rate of 1 mL / min; 3) Sample loading: Add the sample to the column, collect the eluent, and maintain a flow rate of 1 mL / min; 4) Washing: Pass the column through 5 column volumes of coupling buffer at a flow rate of 1 mL / min. 5) Elution: Elute the antibody with 5 column volumes of elution buffer (0.1M glycine-hydrochloric acid solution, pH 3.0), collect it in a centrifuge tube, maintain a flow rate of 1 mL / min, and then immediately adjust the pH of the collected solution to 7.0 with 1M pH 9.0 Tris-HCl buffer. 6) Dialysis: Dialyze the antibody overnight using 0.01M PBS buffer, changing the buffer 3 times to obtain purified penicillic acid monoclonal antibody.

[0049] The amino acid sequence of the heavy chain variable region of the penicillic acid monoclonal antibody is shown in SEQ ID NO:1, and the amino acid sequence of the light chain variable region is shown in SEQ ID NO:2.

[0050] SEQ ID NO:1 EVKLVESGGGLVKPGGSLKLSCAPSGFTFSGYAMSWVRQTPEKRLEWVASISNGGGTYYPDSVKGRFTISRDNTRNILYLQMSSLRSEDTAMYYCSRHRYYSWHFDVWGAGTTVTVSS SEQ ID NO:2 YIVLTQSPAILSVSPGERVSFSCRASQNIGTSVHWFQQKRNGFPRLLIKFVSESISGVPPRFSGSSGTDFTLSINSVETEDIADYYCQQTYGWPWTFGGGTKLEIK Example 3: Establishment of a rapid test strip method for detecting penicillic acid (PA) 3.1 Colloidal gold-labeled antibodies (1) Preparation of colloidal gold Measure 200 mL of ultrapure water and add it to a clean 250 mL Erlenmeyer flask. Place the flask on the heating plate of a magnetic stirrer, add the stir bar, and turn on the stirring knob to start stirring. Use a pipette to add 2 mL of 1% chloroauric acid solution to the 100 mL of ultrapure water. Turn on the heating knob and heat to boiling. Quickly add 1-3 mL of 2% trisodium citrate aqueous solution. The solution will change from gray to black within 2 minutes, and finally turn red. Heat and stir for 10 minutes, then turn off the heating knob and stir until room temperature. Add ultrapure water to a final volume of 200 mL and store away from light.

[0051] (2) Preparation of gold-labeled antibodies ① Take two clean 1.5mL centrifuge tubes and add 1mL of colloidal gold to each.

[0052] ② Add 5-10 µL of 0.2 M potassium carbonate and mix well.

[0053] ③ Add 4-15ug of PA antibody, mix well, and react at room temperature for 10-30min.

[0054] ④ Add 10µL of 20% BSA, mix well, and react at room temperature for 5-30 minutes.

[0055] ⑤ Centrifuge at 13000 rpm for 10-40 min, discard the supernatant, and reconstitute the precipitate with 1 / 10 volume of reconstitution solution for later use.

[0056] 3.2 Microsphere-labeled antibody (options include carboxylated colored microspheres, time-resolved fluorescent microspheres, fluorescent quantum dot microspheres, etc.; this case uses time-resolved fluorescent microspheres). (1) Prepare 10 mg / mL NHS and 10 mg / mL EDC immediately before use: Solution A: 10 mg / ml N-hydroxysuccinimide (NHS) / MES solution Solution B: 10 mg / ml of 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (EDC) / MES solution (2) Add 100 μL of microspheres to a 2 mL centrifuge tube, then add 900 μL of 50 mM pH 6.0 MES buffer, and vortex for 5 s to mix evenly; (3) Add 10 μL of solution A to the microsphere suspension, mix for 5 s, then add 10 μL of solution B, mix again for 5 s, and shake at room temperature for 5-60 min. (4) Centrifuge at 14000r / min for 5-30min, discard the supernatant, add 1ml of borate buffer, sonicate to disperse evenly, and set aside for use.

[0057] (5) After the above 1 ml of activated microsphere suspension is ultrasonically dispersed, add 5-25 μg of PA antibody, vortex for 5 s to mix evenly, and shake at room temperature for 10-60 min to react. (6) Add 100 μL of 10% BSA solution, shake well at room temperature and block the reaction for 10-60 min; (7) Centrifuge at 13000 r / min for 15 min. Add 1 ml of microsphere preservation solution to the centrifuged microspheres, mix well, and store in the dark for later use.

[0058] 3.3 Scrapping and Gold / Microsphere Spraying (1) Scratching membrane Cut the nitrocellulose membrane into 30cm strips, attach them to a PVC board, and equilibrate for at least 30 minutes at 25°C and 50% humidity.

[0059] ① Line C: Goat anti-mouse IgG was diluted with 10mM, pH 7.4 PBS to a concentration of 0.2mg / mL, and sprayed onto a nitrocellulose membrane using a membrane scrubbing instrument at a distance of 5mm from the absorbent paper. This is the quality control line.

[0060] ② T line: The PA-BSA antigen was diluted with 10mM, pH 7.4 PBS to a concentration of 0.05mg / mL. It was then sprayed onto a nitrocellulose membrane using a membrane scrubbing instrument at a distance of 5mm from the C line. This is the detection line.

[0061] Place the cut film into a forced-air drying oven and dry at 37℃ for 16-24 hours.

[0062] (2) Gold spraying / microsphere spraying ① Gold spraying instrument parameter settings: spray volume 8ul / cm, spray film length 300mm; ② Place the sprayed colloidal gold or microsphere pads in a 37℃ oven and dry for 24 hours.

[0063] 3.4 Test strip assembly (1) Apply absorbent paper: Remove the film on the PVC board, apply absorbent paper to the top, and press the absorbent paper 2mm over the NC film; (2) Gold-plated pad / microsphere pad: Gold pad or microsphere pad is pressed against NC film 2mm; (3) Apply sample pad: Remove the bottom film, apply the sample pad to the bottom, and press the gold pad or microsphere pad 2mm under the sample pad; (4) Cutting into strips: Cutting with a strip cutting machine to a width of 3.5mm; (5) Inserting the test strips into the cartridge: Place the cut test strips into the cartridge, then seal and store them in an aluminum foil bag. A diagram of the test strips is shown below. Figure 1 As shown.

[0064] 3.4 Product Usage Instructions (1) Colloidal gold qualitative test strip ① Tear open the aluminum foil bag seal and take out the test card from the aluminum foil bag.

[0065] ② Add 100 μL of the sample to be tested into the sample well of the test card and react at room temperature for 10 min.

[0066] (2) Fluorescent quantitative test strips It needs to be used in conjunction with a quantitative fluorescence detector and an incubator. ① Insert the data card into the corresponding port on the analyzer.

[0067] ② Tear open the aluminum foil bag seal, remove the test card from the bag, and place it on the incubator heating plate. Please use it as soon as possible within 30 minutes.

[0068] ③ Add 100 μL of the sample to be tested into the sample well of the test card and react for 8 min.

[0069] ④ Insert the test card into the test slot and press the "Quick Test" button. The tester will automatically test the test card.

[0070] ⑤ Read / print the test results from the testing instrument.

[0071] Two red yeast rice samples were tested using the colloidal gold qualitative test strip, the fluorescent quantitative test strip, and liquid chromatography-tandem mass spectrometry of this invention, respectively. A certain concentration of penicillic acid was added to each sample. The test results are as follows:

[0072]

[0073] The test results show that the colloidal gold qualitative test strip, the fluorescent quantitative test strip, and the LC-MS / MS test results are highly consistent, and the test results are accurate and reliable.

[0074] It is understood that those skilled in the art can make equivalent substitutions or modifications to the technical solutions and concepts of this invention, and all such substitutions or modifications should fall within the protection scope of the appended claims.

Claims

1. A monoclonal antibody that specifically binds to penicillic acid, characterized in that, The heavy chain variable region of the monoclonal antibody has an amino acid sequence as shown in SEQ ID NO:1, and the light chain variable region has an amino acid sequence as shown in SEQ ID NO:

2.

2. The monoclonal antibody specifically binding to penicillic acid according to claim 1, characterized in that, The monoclonal antibody was produced by hybridoma cell line 3H8, whose accession number is CCTCC NO:C2026137.

3. The application of the monoclonal antibody specifically binding to penicillic acid as described in claim 1 in the detection of penicillic acid.

4. A rapid test strip for penicillic acid, characterized in that, Includes a base plate and a sample pad, a marking pad, a nitrocellulose membrane, and absorbent paper that are sequentially overlapped on the base plate; The nitrocellulose membrane is provided with a detection line and a control line. The detection line is coated with a penicillic acid-carrier protein conjugate, and the control line is coated with an anti-mouse IgG antibody. The label pad is coated with the monoclonal antibody of claim 1, which is labeled with a marker.

5. The rapid test strip for penicillic acid according to claim 4, characterized in that, The marker is colloidal gold particles, time-resolved fluorescent microspheres, carboxylated colored microspheres, or fluorescent quantum dot microspheres.

6. A method for preparing a rapid test strip for penicillic acid according to claim 4, characterized in that, Includes the following steps: S1: Spray the penicillic acid-carrier protein conjugate and anti-mouse IgG antibody onto a nitrocellulose membrane to form a detection line and a control line. After drying, the coated membrane is obtained. S2: The monoclonal antibody according to claim 1 or 2 is conjugated with a marker to obtain a labeled antibody, and the labeled antibody is sprayed or immersed on a labeling pad and dried. S3: The sample pad, marking pad, coating film and absorbent paper are sequentially overlapped and pasted onto the base plate, and then cut into shape to obtain a rapid test strip.

7. The preparation method according to claim 6, characterized in that, In step S1, the concentration of the penicillic acid-carrier protein conjugate is 0.01–0.1 mg / mL, and the concentration of the anti-mouse IgG antibody is 0.1–0.5 mg / mL.

8. The preparation method according to claim 6, characterized in that, The marker is a time-resolved fluorescent microsphere. The coupling in step S2 includes the following operations: after activating the microspheres with EDC and NHS, a monoclonal antibody is added for reaction, and then bovine serum albumin is added for blocking.

9. A method for detecting penicillic acid, characterized in that, The procedure includes the step of testing the sample using the monoclonal antibody of claim 1 or the test strip of claim 4.

10. The method according to claim 9, characterized in that, The detection can be qualitative or quantitative; the qualitative detection uses colloidal gold-labeled test strips, and the color of the test line is observed by the naked eye for judgment; the quantitative detection uses fluorescently labeled test strips, and the signal value is read in conjunction with a fluorescence detector.

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