Hybridoma cell strain 1G10, monoclonal antibody thereof and application

Abstract

The application belongs to the technical field of bioengineering, and particularly relates to a hybridoma cell strain 1G10, a monoclonal antibody thereof and application. The hybridoma cell strain with high sensitivity to lead ions and no obvious cross to other metal ions is cultured through a screening mechanism, and then the monoclonal antibody is separated and cultured, so that the monoclonal antibody can be used for detection of lead ions in edible oil, and rapid and efficient detection of lead ions in edible oil is realized.

Description

Technical Field

[0001] This invention belongs to the field of bioengineering technology, and particularly relates to a hybridoma cell line 1G10, its monoclonal antibody, and its applications. Background Technology

[0002] Edible oil, an indispensable part of our daily diet, directly impacts public health. However, during production and processing, it can be contaminated with heavy metals. This can originate from the raw materials themselves, such as oil crops grown in polluted soil, or from contamination during refining and storage due to contact with equipment and containers containing heavy metals. Once heavy metals enter the human body through edible oil, they accumulate in large quantities, severely damaging vital organs such as the liver, kidneys, and nervous system, leading to various health problems. Therefore, testing edible oil for heavy metals is crucial for ensuring food safety and is essential for regulating the edible oil market and promoting the healthy development of the industry. To effectively protect consumer health, my country has established strict limits on lead content in edible oil, stipulating that it must not exceed 0.1 mg / kg.

[0003] Traditional analytical techniques for detecting lead ions, such as atomic absorption spectrometry (AAS), inductively coupled plasma optical emission spectrometry (ICP-OES), and inductively coupled plasma mass spectrometry (ICP-MS), while exhibiting excellent precision and accuracy, have numerous limitations. These methods place extremely demanding requirements on equipment, resulting in high purchase costs and complex maintenance, necessitating operation and upkeep by specialized technicians. Furthermore, their sample pretreatment processes are cumbersome, time-consuming, and labor-intensive, typically requiring multiple steps such as digestion and extraction, leading to a lengthy overall detection cycle and hindering rapid on-site detection.

[0004] In recent years, with the development of bioengineering technology, new technical means have been provided for the detection of lead ions. For example, Chinese patent CN106872708A discloses a kit for detecting lead ions based on direct competitive enzyme-linked immunosorbent assay (ELISA) and its application. Designed based on the principle of direct competitive immunochromatography between antigen and antibody, the kit uses goat anti-mouse IgG secondary antibody pre-coated on the detection plate. This allows for the simultaneous addition of three substances: anti-lead ion monoclonal antibody, the sample or lead ion standard solution, and enzyme-labeled hapten (Pb-ITCBE-HRP). The anti-lead ion monoclonal antibody binds to the pre-coated goat anti-mouse IgG secondary antibody to form a complex. Lead ions in the sample or standard and the enzyme-labeled hapten competitively bind to the anti-lead ion monoclonal antibody. When the substrate chromogenic solution is added, the absorbance value of the sample is negatively correlated with the residual lead ion content. By comparing with the standard curve and multiplying by the corresponding dilution factor, the lead ion content in the sample can be obtained.

[0005] However, the aforementioned existing technologies still rely on instrument readings, and the monoclonal antibodies and kits they produce can only be applied to water samples. Therefore, developing a cell line and its monoclonal antibody capable of detecting lead ions in edible oils, overcoming the shortcomings of traditional detection technologies, and meeting the urgent need for lead ion detection across the entire edible oil industry chain, has significant economic, social, and food safety implications. It also provides a crucial opportunity to promote innovative development in agricultural and food testing technologies. Summary of the Invention

[0006] To address the lack of cell lines and monoclonal antibodies for the detection of lead ions in edible oils in existing technologies, this invention provides a hybridoma cell line 1G10, its monoclonal antibody, and its application. Through a screening mechanism, a hybridoma cell line with high sensitivity to lead ions and no significant cross-contamination with other metal ions is cultured, and then a monoclonal antibody is isolated and cultured, which can be used for the detection of lead ions in edible oils.

[0007] The specific solution of the present invention is as follows:

[0008] A hybridoma cell line 1G10 was deposited at the China General Microbiological Culture Collection Center (CGMCC) on October 31, 2024, and is classified as a monoclonal cell line with accession number CGMCC No. 46241.

[0009] Application of a hybridoma cell line 1G10 in the preparation of mouse-derived monoclonal antibodies against lead ions.

[0010] A mouse-derived monoclonal antibody against lead ions, wherein the mouse-derived monoclonal antibody against lead ions is obtained by immunizing mice with the hybridoma cell line 1G10 described in claim 1.

[0011] Furthermore, the monoclonal antibody includes a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region includes the amino acid sequence shown in SEQ ID NO.1, and the light chain variable region includes the amino acid sequence shown in SEQ ID NO.2.

[0012] Application of a mouse-derived monoclonal antibody against lead ions in the detection of lead ions in edible oils.

[0013] A colloidal gold test strip for detecting lead ions in edible oil, the test strip comprising a mouse-derived monoclonal antibody against lead ions as described in any one of claims 3 or 4.

[0014] Furthermore, the colloidal gold test strip also includes a PVC board, a sample pad, a conjugate pad, a nitrocellulose membrane, and an absorbent pad, which are sequentially attached to the PVC board. The conjugate pad is prepared by spraying colloidal gold-labeled monoclonal antibodies onto a glass fiber membrane. The nitrocellulose membrane has a control line and a detection line. The control line is sprayed with goat anti-mouse secondary antibody, and the detection line is sprayed with Pb-BSA solution.

[0015] A kit for detecting lead ions in edible oil, the kit comprising the colloidal gold test strip as described in any one of claims 6 or 7.

[0016] Furthermore, the kit also includes a lead heavy metal ion extraction solution, a diluent, and a pH adjuster. The lead heavy metal ion extraction solution comprises n-hexane and dilute nitric acid solution, the diluent is a tris(hydroxymethyl)propanesulfonic acid solution containing EDTA, and the pH adjuster is a sodium hydroxide solution.

[0017] The beneficial effects of this invention are:

[0018] 1. In practical work, the inventors discovered that compared to detecting lead ions in water samples, detecting lead ions in edible oils requires more potent monoclonal antibodies with stronger binding capacity to lead ions due to the higher viscosity of oil and the stronger intermolecular forces. This invention, in screening hybridoma cell lines, adds 20% edible oil to the buffer solution and uses this screening mechanism to cultivate hybridoma cell lines with high sensitivity to lead ions and no significant cross-linking to other metal ions. Monoclonal antibodies are then isolated and cultured from these cells. These antibodies possess a specific gene sequence that exhibits a stronger binding capacity to lead ions in oil samples, thus providing higher detection sensitivity.

[0019] 2. Addressing the technical problem that current methods for detecting lead ions in edible oils often require large-scale testing instruments, resulting in low detection efficiency and high costs, the inventors of this application have screened out specific monoclonal antibodies with extremely strong binding ability to lead ions. These monoclonal antibodies are labeled with colloidal gold. Using a competitive method, when the sample to be tested is dropped onto the sample pad, the sample moves along the test strip towards the absorbent pad under capillary action. Various components in the sample diffuse forward with the liquid flow. When the sample reaches the binding pad, it interacts with the colloidal gold-labeled monoclonal antibody on the pad. If the lead ion content in the sample reaches the detection limit, the lead ions will bind to the colloidal gold-labeled monoclonal antibody on the binding pad. Thus, when the sample continues to flow to the detection line, no free colloidal gold-labeled monoclonal antibody specifically binds to the antigen on the detection line (T line), and no color change occurs on the T line. If the sample contains no lead ions or its content is below the detection limit, there will be a large amount of colloidal gold-labeled monoclonal antibodies that have not bound to lead ions. When these antibodies flow to the detection line, they specifically bind to the antigen on the detection line (T line), resulting in a color change on the T line. In other words, the intensity of the T line color is inversely proportional to the concentration of the small molecule to be detected in the sample. Regardless of whether the sample contains the small molecule to be detected, the colloidal gold labeling that has not bound to the antigen on the T line will eventually reach the control line (C line). The substance immobilized on the C line will non-specifically bind to the colloidal gold labeling, forming a red band at the C line, indicating that the detection process is proceeding normally. If no band appears on the C line, it indicates a problem with the detection process, and the result is unreliable. This colloidal gold immunochromatography technique based on special monoclonal antibodies is simpler and faster to operate than traditional instrument detection methods. It requires no professional operators or complex equipment, and while ensuring high sensitivity, it can meet the needs of rapid on-site detection of lead ion content in edible oils. Attached Figure Description

[0020] Figure 1 This is a schematic diagram of the structure of a colloidal gold test strip for detecting lead ions in edible oil according to the present invention;

[0021] Figure 2 This is a curve showing the fitting relationship between lead ion concentration and absorbance value in Experimental Example 1 of this invention.

[0022] Figure 3 The figure shows the experimental results of Experiment Example 4 of this invention. Detailed Implementation

[0023] The technical solutions of the present invention will be clearly and completely described below with reference to the embodiments of the present invention. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those of ordinary skill in the art without creative effort are within the scope of protection of the present invention.

[0024] Example 1

[0025] Screening of a hybridoma cell line 1G10 includes the following steps:

[0026] lead ions (Pb) 2+ The antigen was coupled to the carrier protein KLH (hemocyanin) to obtain a complete antigen, which was then used to immunize mice. Unlike existing technologies, this embodiment added 0.01M phosphate buffer as a screening matrix during the serum screening stage. The phosphate buffer contained 20% edible oil, specifically rapeseed oil and soybean oil, to evaluate the serum of the experimental group mice. Simultaneously, this screening matrix was used to detect the mouse serum's responsiveness to other metal ions (Cd). 2+ Zn 2+ Cu 2+ Mg 2+ Ca 2 + The cross-linking was performed. A screening matrix without metal ions was used as the control group. The inhibition rate of mouse serum was calculated to select the matrix that inhibited Pb. 2 + Cell fusion was performed on mice with high sensitivity and no significant cross-linking to other metal ions. In this example, five mice underwent serum screening, and the results are shown in Table 1 below. The experimental groups were supplemented with 20 ng / mL Pb. 2+ 200ng / mCd2 + 200 ng / mL Zn 2+ 200 ng / mL Cu 2+ 200 ng / mL Mg 2+ 200 ng / mL Ca 2+ The inhibition rate of mouse serum was calculated using the following formula:

[0027]

[0028] Table 1. Results of mouse serum screening

[0029]

[0030] The antibody produced by mouse #3 was against Pb. 2+The inhibition rate was 63% at a concentration of 2 ng / mL. The best effect was observed against other metal ions at a concentration of 20 ng / mL, where the inhibition rate was less than 10%. This effect was also observed against Pb. 2+ The mouse was selected for cell fusion due to its high sensitivity and lack of significant cross-contamination with other metal ions. After fusion, the cells were screened again using the aforementioned 0.01M phosphate-buffered saline matrix containing 20% ​​edible oil, resulting in a hybridoma cell line capable of producing monoclonal antibodies tolerant to this matrix, named hybridoma cell line 1G10. The applicant deposited hybridoma cell line 1G10 at the China General Microbiological Culture Collection Center (CGMCC), classifying it as a monoclonal cell line with accession number CGMCC No. 46241, and the deposit date was October 31, 2024.

[0031] The preparation of monoclonal antibodies includes the following steps: Hybridoma cell line 1G10 is expanded and cultured, then injected into mice to induce ascites; 1 mL of ascites is taken, and an equal volume of sodium acetate buffer (1 mL) is added. 33 μL of caprylic acid is added dropwise while stirring at room temperature, vortexed for 30 min, and centrifuged at 8000 r / min for 5 min to obtain the supernatant; 2 mL of saturated ammonium sulfate is added, and the mixture is allowed to stand at 4℃ for 2 h; after centrifugation at 8000 r / min for 5 min, the supernatant is discarded, and the precipitate is dissolved in PBS solution and dialyzed for 2-3 days to obtain an anti-lead ion monoclonal antibody. The monoclonal antibody includes a heavy chain variable region and a light chain variable region. The heavy chain variable region includes the amino acid sequence shown in SEQ ID NO.1, and the light chain variable region includes the amino acid sequence shown in SEQ ID NO.2.

[0032] IC50 for detecting monoclonal antibodies in various edible oil sample matrices 50 The IC50 value is 3.2–5.9 ng / mL. It is applicable to the determination of lead (Pb) in edible oils. 2+ ) detection.

[0033] Example 2

[0034] This embodiment provides a colloidal gold test strip for detecting lead ions in edible oil, as shown in the attached image. Figure 1 As shown, it includes a sample pad, a conjugate pad, a nitrocellulose membrane, and an absorbent pad. The conjugate pad is made of lead-resistant (Pb) material. 2+Mouse-derived monoclonal antibodies were prepared by spraying them onto a glass fiber membrane. The nitrocellulose membrane had control and detection lines. The control lines were formed by spraying goat anti-mouse secondary antibody, and the detection lines were formed by spraying Pb-BSA (bovine serum albumin) solution. Subsequently, the sample pad, conjugate pad, nitrocellulose membrane, and absorbent pad were sequentially adhered to a PVC board. The overlap length between the sample pad and the conjugate pad was 15 mm, the overlap length between the conjugate pad and the nitrocellulose membrane was 3 mm, and the overlap length between the nitrocellulose membrane and the absorbent pad was 17 mm. Both the sample pad and the absorbent pad were aligned with the top and bottom edges of the PVC board.

[0035] The preparation method of this colloidal gold test strip includes the following steps:

[0036] (1) Use the monoclonal antibody prepared in Example 1.

[0037] (2) Preparation of colloidal gold pads: Using trisodium citrate as a reducing agent, chloroauric acid was reduced to prepare colloidal gold particles with a particle size of 20 nm: First, 1000 mL of 0.01% chloroauric acid aqueous solution was taken and heated to boiling with a constant temperature electromagnetic stirrer. 2 mL of 20% trisodium citrate aqueous solution was added while stirring continuously. Stirring and heating continued for 10 min. When the above mixed solution was stably bright wine red, it was cooled to room temperature to obtain gold nanoparticles with a particle size of 20 nm.

[0038] In this embodiment, the pH of the colloidal gold solution was first adjusted to 9 using K2CO3 solution. Then, a monoclonal antibody with a pH of 7.4 was added and allowed to react fully. Lead (Pb) was then added. 2+ The concentration of the monoclonal antibody was 100 μg / mL. BSA solution was added to the above mixture to block excess sites, and the precipitate was collected by centrifugation to obtain the monoclonal antibody-colloidal gold label. The monoclonal antibody-colloidal gold label was then sprayed onto a glass fiber membrane.

[0039] (3) Spray control lines and test lines onto the nitrocellulose membrane.

[0040] In this embodiment, the goat anti-mouse secondary antibody and Pb-BSA solution were first sprayed onto a nitrocellulose membrane to form three lines, which served as the quality control line and the detection line (two lines), respectively. Then, the membrane was dried at 45°C for 72 hours.

[0041] (4) Assemble the PVC board, sample pad, conjugate pad, nitrocellulose membrane and absorbent pad.

[0042] (5) Cut the colloidal gold test strip assembled in step S4 into strips to obtain colloidal gold test strips. In this embodiment, the specifications of the colloidal gold test strips are 8cm long × 4mm wide.

[0043] This embodiment also provides a kit for detecting lead ions in edible oils. The kit includes a lead heavy metal ion extraction solution, a diluent, a pH adjuster, and the colloidal gold test strip prepared above. The lead heavy metal ion extraction solution includes n-hexane and 0.75M dilute nitric acid solution, the diluent is a trimethylolpropanesulfonic acid solution containing EDTA, and the pH adjuster is a 5M sodium hydroxide solution.

[0044] This embodiment also provides a method for using a kit for detecting lead ions in edible oils, comprising the following steps: Take 0.5g of the sample to be tested into a 50mL centrifuge tube, add n-hexane and 0.75M dilute nitric acid, and shake to obtain mixture A. Centrifuge mixture A at 3000r / min for 5min, take 100μL of the supernatant, add 270μL of 5M sodium hydroxide solution, and shake to obtain mixture B. Add tris(hydroxymethyl)aminopropanesulfonic acid diluent containing 0.1M EDTA to mixture B to obtain mixture C, and let stand for 3min. Take 100μL of mixture C and add it to the sample pad of a colloidal gold test strip preheated to 37℃ for detection, and time for 8min. After the timer expires, remove the test strip and read the result within 20s.

[0045] Experimental Example 1

[0046] The monoclonal antibody obtained by incubating the hybridoma cell line 1G10 (accession number CGMCC No. 46241) used in this invention was incubated with the disclosed monoclonal antibody 1E5 (obtained by incubating the hybridoma cell line with accession number CGMCC No. 17387, Chinese Patent CN102426239A). Following the method described in the examples, the two monoclonal antibodies were tested for IC50 in lead ions in edible oil. 50 The values ​​and experimental results are shown in Table 2 and Appendix 2 below. Figure 2 As shown:

[0047] Table 2. Sensitivity test results of different antibodies in edible oils

[0048]

[0049]

[0050] Analysis of experimental results: The IC50 value of the monoclonal antibody 1G10 used in this invention is 3.2-5.9 ng / mL, which is 7-9 times higher than the IC50 value of the monoclonal antibody 1E5 in the prior art, which is 21.3-58.2 ng / mL.

[0051] Experimental Example 2

[0052] Optimization experiment of reagent kit sample content. Following the reagent preparation method of a reagent kit for detecting lead ions in edible oils described in the examples, the samples were tested according to the prepared amounts in Table 3 below. The detection method was the same as the method used for the aforementioned reagent kit for detecting lead ions in edible oils. Based on the ratio of the T line to the C line on the test strip, the optimal solution combination was selected as the one with the largest difference between the T / C value (2.0-4.0) when the Pb addition was 0 μg / kg and the T / C value (75 μg / kg). The experimental results are shown in Table 3 below:

[0053] Table 3. Reagent dosage and results analysis of the reagent kit

[0054]

[0055]

[0056] The experimental results above show that the optimal solution combination is 3 mL of n-hexane, 10 mL of nitric acid, and 3 mL of diluent, at which point the ratio of the T line to the C line on the test strip is the largest.

[0057] Experimental Example 3

[0058] 1) Sample testing steps

[0059] Take 0.5g of the edible oil sample to be tested, and take a sample free of lead ions (Pb). 2+ Edible oil samples, with added lead ions (Pb) 2+ To a concentration of 150 μg / kg, add 5 mL concentrated nitric acid, 5 mL hydrofluoric acid, and 3 mL perchloric acid. Continue heating until thick white fumes are emitted. Cover with a watch glass to decompose the black organic carbon compounds. Depending on the digestion progress, add another 3 mL concentrated nitric acid, 3 mL hydrofluoric acid, and 1 mL perchloric acid to repeat the digestion process. Remove from heat and cool. Add 2 mL concentrated nitric acid and heat to dissolve the soluble residue. After cooling, transfer to a 25 mL volumetric flask, filter dry, and use for inductively coupled plasma atomic emission spectrometry (ICP).

[0060] The experimental group used the detection kit and detection method provided by this invention for detection.

[0061] 2) Recovery rate detection

[0062] The oil sample to be tested, prepared in step (1), was analyzed using the method in (1) to obtain lead ions (Pb). 2+ The recovery rates are shown in Table 4:

[0063] Table 4. ICP-MS detection method for lead (Pb) ions in various oils and fats 2+ Recovery rate

[0064]

[0065] The experimental results show that ICP-MS is an effective method for detecting lead ions (Pb) in edible oils. 2+ The recovery rate was between 82.7% and 95.1%, lower than the experimental method provided by this invention. Furthermore, the ICP-MS detection results for soybean oil deviated from the actual content by up to 20%. Compared to the detection kit and method provided by this invention, the accuracy is significantly lower.

[0066] (2) Comparison with other detection methods

[0067] As shown in Table 5, compared with traditional instrumental methods (atomic absorption spectroscopy refers to HJ 786-2016, X-ray fluorescence spectroscopy refers to GB / T 5195.15-2017), the immunochromatographic method provided by this invention not only has similar accuracy and repeatability to instrumental methods, but also has a safer detection process, lower cost, and shorter time.

[0068] Table 5. Detection of lead (Pb) ions in oils using different detection methods. 2+ Comparison of )

[0069]

[0070] Experiment Example 4

[0071] Specific detection experiment for lead ions. For samples containing other divalent metal ions: take samples that do not contain lead (Pb). 2+ Edible oil samples were supplemented with cadmium (Cd). 2+ ), calcium (Oa) 2+ ), magnesium (Mg) 2+ ), Zinc (Zn) 2+ ), copper (Cu) 2+ ), nickel (Ni 2+ The concentration was increased to 750 μg / kg. Experimental results are as follows: Figure 3 As shown.

[0072] The test strip method was used for detection, and the results showed that the test strip did not recognize other divalent metal ions, cadmium (Cd). 2 + ), calcium (Ca 2+ ), magnesium (Mg) 2+ ), Zinc (Zn) 2+ ), copper (Cu) 2+ ), nickel (Ni 2+ This method provides specific detection results that are not affected by other metal ions.

[0073] Experimental Example 5

[0074] Detection limit experiment. The detection limit includes detection in water samples and oil samples. For lead ion detection in water samples, the existing immunoassay method (patent CN106872708A) was followed. The detection of lead ions in environmental water samples was compared, and the 1G10 mouse-derived anti-lead ion method provided by this invention achieves a minimum detection limit (LOD) of 0.05 μg / L, which is more sensitive than the B6F6 mouse-derived anti-lead ion method in the existing immunoassay method (CN 106872708A). The detection results are shown in Table 6 below:

[0075] Table 6. Results of lead ion content in water samples detected by different detection methods.

[0076]

[0077] The detection limit test for oil samples, the sample preparation method is as follows: Take oil samples that do not contain lead (Pb) 2+ Edible oil samples were supplemented with lead ions (Pb). 2+ () up to concentrations of 0, 25, 75, 150, 225, 375, 600 and 1000 μg / kg.

[0078] Detection Method: Add 0.5g of the edible oil standard curve sample to a 50mL centrifuge tube, along with 3mL of n-hexane and 10mL of 0.75M dilute nitric acid to obtain mixture A. Centrifuge 1mL of mixture A at 3000r / min for 5min, and take 100μL of the supernatant. Adjust the pH to neutral by adding 5M sodium hydroxide solution to obtain mixture B. Add 3mL of diluent containing 0.1M EDTA to 100μL of mixture B to obtain mixture C, and let it stand for 3min. Add 100μL of mixture C to the sample pad of the test strip, which has been preheated to 37℃, and time for 8min. After timing, remove the test card and read the result within 20s. The standard curve is obtained, and the detection limit is 25μg / kg.

[0079] It will be apparent to those skilled in the art that the present invention is not limited to the details of the exemplary embodiments described above, and that the invention can be implemented in other specific forms without departing from its spirit or essential characteristics. Therefore, the embodiments should be considered exemplary and not restrictive in all respects. Furthermore, it should be understood that although this specification describes embodiments, it does not encompass only one technical solution. This descriptive method is merely for clarity, and those skilled in the art should consider the specification as a whole. The technical solutions in the embodiments can also be appropriately combined to form other embodiments that can be understood by those skilled in the art.

Claims

1. A hybridoma cell line 1G10, characterized in that, The hybridoma cell line 1G10 was deposited at the China General Microbiological Culture Collection Center (CGMCC) on October 31, 2024, and its classification name is monoclonal cell line, with accession number CGMCC No. 46241.

2. The use of the hybridoma cell line 1G10 according to claim 1 in the preparation of a mouse-derived monoclonal antibody against lead ions, wherein the monoclonal antibody comprises a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region is the amino acid sequence shown in SEQ ID NO.1 and the light chain variable region is the amino acid sequence shown in SEQ ID NO.

2.

3. A mouse-derived monoclonal antibody against lead ions, characterized in that, The mouse-derived anti-lead ion monoclonal antibody was obtained by immunizing mice with the hybridoma cell line 1G10 described in claim 1; the monoclonal antibody includes a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region is the amino acid sequence shown in SEQ ID NO.1 and the light chain variable region is the amino acid sequence shown in SEQ ID NO.

2.

4. The application of the mouse-derived anti-lead monoclonal antibody as described in claim 3 in the detection of lead ions in edible oils.

5. A colloidal gold test strip for detecting lead ions in edible oil, characterized in that, The test strip comprises the mouse-derived anti-lead ion monoclonal antibody as described in claim 3.

6. A colloidal gold test strip for detecting lead ions in edible oil according to claim 5, characterized in that, The colloidal gold test strip also includes a PVC plate, a sample pad, a conjugate pad, a nitrocellulose membrane, and an absorbent pad, which are sequentially attached to the PVC plate. The conjugate pad is prepared by spraying colloidal gold-labeled monoclonal antibody onto a glass fiber membrane. The nitrocellulose membrane has a control line and a detection line. The control line is sprayed with goat anti-mouse secondary antibody, and the detection line is sprayed with Pb-BSA solution.

7. A reagent kit for detecting lead ions in edible oils, characterized in that, The kit includes the colloidal gold test strip as described in any one of claims 5 or 6.

8. A reagent kit for detecting lead ions in edible oil according to claim 7, characterized in that, The kit also includes a lead heavy metal ion extraction solution, a diluent, and a pH adjuster. The lead heavy metal ion extraction solution comprises n-hexane and dilute nitric acid solution. The diluent is a trimethylolpropanesulfonic acid solution containing EDTA. The pH adjuster is a sodium hydroxide solution.

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