Colloidal gold test strip for detecting nonylphenol polyoxyethylene ether in food contact material and hybridoma cell strain

Abstract

The invention relates to a colloidal gold test strip for detecting nonylphenol polyoxyethylene ether in a food contact material and a hybridoma cell strain. The colloidal gold test strip comprises a PVC (Polyvinyl Chloride) plate, a sample pad, a gold label pad, a nitrocellulose membrane and a water absorption pad, the colloidal gold pad is prepared by spraying an anti-nonylphenol polyoxyethylene ether monoclonal antibody on a glass fiber membrane, the nitrocellulose membrane is provided with a quality control line and a detection line, the quality control line is formed by spraying a goat-anti-mouse secondary antibody, the detection line is formed by spraying a nonylphenol polyoxyethylene ether-BSA solution, and the quality control line is formed by spraying a goat-anti-mouse secondary antibody. The sample pad, the gold label pad, the nitrocellulose membrane and the water absorption pad are sequentially adhered to the PVC plate, and the anti-nonylphenol polyoxyethylene ether monoclonal antibody is a mouse anti-nonylphenol polyoxyethylene ether monoclonal antibody obtained by mouse immunization of a hybridoma cell strain LDP with the preservation number of CGMCC No.46228. The colloidal gold test strip disclosed by the invention is low in detection cost in detection of nonylphenol polyoxyethylene ether in a food contact material; the detection operation is simple; the detection efficiency is high.

Description

Technical Field

[0001] The invention relates to the technical field of analytical chemistry, in particular to a colloidal gold test strip and a hybridoma cell strain for detecting nonylphenol polyoxyethylene ether in food contact materials. Background Art

[0002] Nonylphenol polyoxyethylene ether is a nonionic surfactant commonly used in pesticide production. As an emulsifier or dispersant, it is widely used in pesticide emulsifiable concentrates, microemulsions, water emulsions, suspensions or water dispersible granules. It is an environmental hormone, that is, an endocrine disruptor. In 2001, the European Union listed it as a "priority management hazardous substance". At present, nonylphenol polyoxyethylene ether is still widely used in China's plastics, printing and dyeing, textile and other chemical industries. In recent years, there have been many reports on its migration and potential health risks in plastic products, especially food packaging and food contact materials. Nonylphenol polyoxyethylene ether may degrade into toxic substances such as nonylphenol in the environment, which have toxic effects on aquatic organisms and have a certain bioaccumulation capacity. This may cause damage to the balance of aquatic ecosystems. Long-term contact or ingestion of nonylphenol polyoxyethylene ether may be harmful to human health. It can bind to estrogen receptors and thus affect the reproductive, immune, nervous and cardiovascular functions of humans and animals. Due to its environmental impact and health risks, it is of great significance to establish a rapid detection method for nonylphenol polyoxyethylene ether in food contact materials.

[0003] The detection methods of nonylphenol polyoxyethylene ether mainly include high performance liquid chromatography (HPLC), gas chromatography (GC) and mass spectrometry (MS). However, these methods have high equipment cost, high maintenance and operation cost, high requirements for sample pretreatment, complex operation, long analysis time, and are not suitable for rapid detection. Summary of the invention

[0004] In order to solve the above technical problems, the present invention provides a colloidal gold test strip and a hybridoma cell line for detecting nonylphenol polyoxyethylene ether in food contact materials, that is, a colloidal gold test strip and a detection method for quantitatively detecting nonylphenol polyoxyethylene ether in food contact materials with low cost, simple operation and high efficiency.

[0005] The present invention is achieved through the following technical solutions:

[0006] The first object of the present invention is to provide a hybridoma cell line that secretes anti-nonylphenol polyoxyethylene ether monoclonal antibodies. The hybridoma cell line was deposited in the General Microbiology Center of China National Microbiological Culture Collection Committee on October 31, 2024, with the deposit address at No. 3, Yard No. 1, Beichen West Road, Chaoyang District, Beijing, with the deposit number CGMCC No. 46228, and is classified as a monoclonal cell line.

[0007] The second object of the present invention is to provide an anti-nonylphenol polyoxyethylene ether monoclonal antibody secreted and produced by the hybridoma cell line.

[0008] The third object of the present invention is to provide a composition comprising the anti-nonylphenol polyoxyethylene ether monoclonal antibody.

[0009] The fourth object of the present invention is to provide a colloidal gold test strip for quantitatively detecting nonylphenol polyoxyethylene ether in food contact materials, comprising the anti-nonylphenol polyoxyethylene ether monoclonal antibody, or the composition.

[0010] In one embodiment of the present invention, the colloidal gold test strip comprises a PVC plate, a sample pad, a gold label pad, a nitrocellulose membrane and a water absorbent pad;

[0011] The gold label pad is prepared by spraying the anti-nonylphenol polyoxyethylene ether mouse monoclonal antibody on a glass fiber membrane;

[0012] The nitrocellulose membrane is provided with a detection line and a quality control line in sequence.

[0013] In one embodiment of the present invention, the quality control line is obtained by spraying goat anti-mouse secondary antibody;

[0014] And / or, the detection line is obtained by spraying a nonylphenol polyoxyethylene ether-BSA solution.

[0015] In one embodiment of the present invention, the food contact material comprises one or more of wood pulp paper, steamer paper, kraft paper and waxed paper.

[0016] The fifth object of the present invention is to provide a kit for quantitatively detecting nonylphenol polyoxyethylene ether in food contact materials, comprising at least one of the anti-nonylphenol polyoxyethylene ether monoclonal antibody, the composition and the colloidal gold test strip.

[0017] The sixth object of the present invention is to provide the use of the hybridoma cell line, the anti-nonylphenol polyoxyethylene ether monoclonal antibody, the composition, the colloidal gold test strip or the kit in detecting nonylphenol polyoxyethylene ether.

[0018] The seventh object of the present invention is to provide a method for quantitatively detecting nonylphenol polyoxyethylene ether in food contact materials, comprising the following steps:

[0019] (1) Select food contact material samples and cut them into 2-4 cm pieces 2 small fragments, mixing the sample fragments with methanol, performing ultrasonic extraction, filtering the obtained extract to obtain a test solution;

[0020] (2) The test solution obtained in step (1) is tested using the colloidal gold test strip or the kit described in claims 8-9.

[0021] In step (1), the added amount of the sample fragments and the methanol is 1 g-2 g: 15 mL-20 mL;

[0022] In step (2), the detection time is 8 min-11 min.

[0023] The colloidal gold test strip for quantitatively detecting nonylphenol polyoxyethylene ether in food contact materials of the present invention comprises a PVC board, a sample pad, a gold label pad, a nitrocellulose membrane and a water-absorbing pad; the gold label pad is prepared by spraying a mouse monoclonal antibody against nonylphenol polyoxyethylene ether on a glass fiber membrane, a quality control line and a detection line are arranged on the nitrocellulose membrane, the quality control line is sprayed with a sheep anti-mouse secondary antibody, the detection line is sprayed with a nonylphenol polyoxyethylene ether-BSA solution, the sample pad, the gold label pad, the nitrocellulose membrane and the water-absorbing pad are sequentially pasted on the PVC board, the mouse monoclonal antibody against nonylphenol polyoxyethylene ether is a mouse monoclonal antibody against nonylphenol polyoxyethylene ether ions obtained by immunizing a hybridoma cell line LDP with a deposit number of CGMCC No. 46228 through mice. The food contact material refers to wood pulp paper (paper cup), steamer paper, kraft paper, and waxed paper.

[0024] The above technical solution of the present invention has the following advantages compared with the prior art:

[0025] The invention provides a colloidal gold test strip and a hybridoma cell strain for detecting nonylphenol polyoxyethylene ether in food contact materials. Compared with the prior art, the invention has low detection cost, simple detection operation and high detection efficiency.

[0026] Biomaterial Deposit

[0027] A hybridoma cell line LDP that secretes nonylphenol polyoxyethylene ether monoclonal antibody has been deposited in the General Microbiology Center CGMCC of the China Microbiological Culture Collection Administration. The deposit address is the Institute of Microbiology, Chinese Academy of Sciences, No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing. It is classified and named as a monoclonal cell line. The deposit date is October 31, 2024, and the deposit number is CGMCC No.46228. BRIEF DESCRIPTION OF THE DRAWINGS

[0028] In order to make the content of the present invention more clearly understood, the present invention is further described in detail below according to specific embodiments of the present invention in conjunction with the accompanying drawings, wherein:

[0029] Figure 1 It is the principle diagram of the colloidal gold test strip in the present invention;

[0030] Figure 2 It is a sensitivity test result diagram of the antibody of the present invention in PBS buffer. DETAILED DESCRIPTION

[0031] The present invention is further described below in conjunction with the accompanying drawings and specific embodiments so that those skilled in the art can better understand the present invention and implement it, but the embodiments are not intended to limit the present invention.

[0032] Unless otherwise specified, the experimental methods used in the following examples are all conventional methods, and the materials, reagents, etc. used are all available from commercial sources unless otherwise specified.

[0033] Example 1 Test strip detection of nonylphenol polyoxyethylene ether in food contact materials

[0034] 1. Preparation of colloidal gold test strips for quantitative detection of nonylphenol polyoxyethylene ether

[0035] The colloidal gold test strip of this embodiment includes a PVC plate, a sample pad, a gold label pad, a nitrocellulose membrane and a water absorbent pad (see Figure 1 ), wherein the gold label pad is prepared by spraying anti-nonylphenol polyoxyethylene ether mouse monoclonal antibody on a glass fiber membrane, a quality control line and a detection line are arranged on the nitrocellulose membrane, the quality control line is sprayed by goat anti-mouse secondary antibody, and the detection line is sprayed by nonylphenol polyoxyethylene ether-BSA (bovine serum albumin) solution, and then the sample pad, gold label pad, nitrocellulose membrane and absorbent pad are sequentially pasted on the PVC board.

[0036] In this embodiment, the sample pad, gold label pad, nitrocellulose membrane and absorbent pad are sequentially pasted on the PVC board in order from top to bottom, wherein the overlapping length of the sample pad and the gold label pad is 15 mm, the overlapping length of the gold label pad and the nitrocellulose membrane is 3 mm, the overlapping length of the nitrocellulose membrane and the absorbent pad is 15 mm, and the sample pad and the absorbent pad are aligned with the upper and lower edges of the PVC board.

[0037] This embodiment also provides a method for preparing the colloidal gold test strip, comprising the following specific steps:

[0038] (1) Monoclonal antibody preparation

[0039] After coupling nonylphenol polyoxyethylene ether with carrier protein KLH (hemocyanin), mice were immunized after obtaining complete antigen. Different from the prior art, in this example, 0.01M phosphate buffer containing 10% methanol was added as the screening mechanism matrix during the serum screening stage, and the serum of 5 mice in the experimental group had the highest titer. Therefore, this ratio was used as the screening matrix to screen out mice that could produce antibodies that tolerated the matrix, and the screening matrix was used to detect the cross-talk of mouse serum to other structural analogs.

[0040] Table 1 Mouse serum screening results

[0041]

[0042] Among them, the antibody produced by 4# mouse has an inhibition rate of 68% when the concentration of nonylphenol polyoxyethylene ether is 500ng / mL, and an inhibition rate of less than 10% when the concentration of other structural analogs is 5000ng / mL, with the best effect. It has high sensitivity to nonylphenol polyoxyethylene ether and has no obvious cross-reaction with other structural analogs. Therefore, this mouse is selected for cell fusion; after fusion, the above-mentioned 0.01M phosphate buffer matrix containing 10% methanol is used again to screen cells to obtain a hybridoma cell line that can produce monoclonal antibodies that tolerate the matrix.

[0043] The hybridoma cell line of the monoclonal antibody is expanded and cultured, and then injected into mice to induce ascites; 1 mL of ascites is taken, and 1 mL of an equal volume of sodium acetate buffer is added, 33 μL of caprylic acid is added dropwise under stirring at room temperature, and the mixture is shaken for 30 minutes and centrifuged at 8000 r / min for 5 minutes to obtain a supernatant; 2 mL of saturated ammonium sulfate is added, and the mixture is allowed to stand at 4°C for 2 hours; the mixture is centrifuged at 8000 r / min for 5 minutes, the supernatant is discarded, and the precipitate is dissolved in a PBS solution and dialyzed for 2-3 days to obtain a monoclonal antibody against nonylphenol polyoxyethylene ether.

[0044] The following examples use the mouse monoclonal antibody against nonylphenol polyoxyethylene ether obtained from the hybridoma cell line LDP as the experimental object. The sensitivity of the antibody was measured in PBS buffer. Figure 2 IC in various food contact material sample matrices 50 The values ​​are shown in Table 2.

[0045] Table 2 Comparison of the sensitivity of antibody LDP in various food contact materials

[0046] Name of food contact material <![CDATA[Antibody IC 50 (ng / mL)]]> Paper cup (wood pulp paper) 223.6 Steamer paper 275.3 Kraft paper 296.7 Wax paper 286.6

[0047] (2) Preparation of colloidal gold pad

[0048] Before preparing the colloidal gold particles, the method further includes: preparing a colloidal gold solution, wherein the colloidal gold solution is a mixture of a chloroauric acid aqueous solution and a trisodium citrate aqueous solution.

[0049] In this embodiment, trisodium citrate is used as a reducing agent to reduce chloroauric acid to prepare colloidal gold particles with a particle size of 20 nm: first, 1000 mL of 0.01% chloroauric acid aqueous solution is taken and heated to boiling with a constant temperature electromagnetic stirrer, and 2 mL of 20% trisodium citrate aqueous solution is added under continuous stirring, and stirring and heating are continued for 10 minutes. When the above mixed solution is stably in a transparent wine red color, it is cooled to room temperature to obtain gold nanoparticles with a particle size of 20 nm.

[0050] In this embodiment, the pH value of the colloidal gold solution is first adjusted to 9 with a K2CO3 solution, and then a monoclonal antibody with a pH value of 7.4 is added for sufficient reaction, wherein the concentration of the nonylphenol polyoxyethylene ether monoclonal antibody is 100 μg / mL; a BSA solution is added to the above-mentioned mixed solution to block excess sites, and the precipitate is collected by centrifugation to obtain a monoclonal antibody-colloidal gold marker.

[0051] The monoclonal antibody-colloidal gold marker was then sprayed onto the glass fiber membrane.

[0052] (3) Spraying the quality control line and the test line on the nitrocellulose membrane

[0053] In this embodiment, goat anti-mouse secondary antibody and nonylphenol polyoxyethylene ether-BSA solution were first sprayed on a nitrocellulose membrane to form three lines, which were used as a quality control line and a detection line (two lines), and then dried at 40° C. for 48 hours.

[0054] (4) Assemble the PVC plate, sample pad, gold label pad, nitrocellulose membrane and absorbent pad.

[0055] (5) Cut the colloidal gold test paper assembled in step S4 into strips to obtain colloidal gold test paper strips. In this embodiment, the colloidal gold test paper strips have a size of 8 cm in length and 4 mm in width.

[0056] 2. Prepare the sample to be tested

[0057] (1) Test strip optimization experimental samples: Take a food contact material sample that does not contain nonylphenol polyoxyethylene ether and add nonylphenol polyoxyethylene ether to the concentration of 0 and 10 mg / kg respectively.

[0058] (2) Test strip standard curve samples: Take food contact material samples that do not contain nonylphenol ethoxylates and add nonylphenol ethoxylates to concentrations of 0, 0.2 mg / kg, 0.5 mg / kg, 1 mg / kg, 2.5 mg / kg, 5 mg / kg, 10 mg / kg and 20 mg / kg respectively.

[0059] (3) LC-MS / MS standard curve samples: Take food contact material samples that do not contain nonylphenol ethoxylates and add nonylphenol ethoxylates to concentrations of 0.5 mg / kg, 1 mg / kg, 2 mg / kg and 5 mg / kg, respectively.

[0060] (4) Samples to be tested (comparison of recovery rate and test method): Take a food contact material sample that does not contain nonylphenol polyoxyethylene ether and add nonylphenol polyoxyethylene ether at a concentration of 10 mg / kg.

[0061] 3. Quantitative immunochromatographic detection of nonylphenol polyoxyethylene ether in food contact materials

[0062] (1) Sample testing steps

[0063] Select food contact material samples and cut into 2-4cm 2 Weigh 2.00g of sample fragments, place in a 50mL conical flask, add 30mL of methanol, ultrasonically extract for 30min, transfer 2mL of extract to pass through a 0.45μm organic phase filter, and wait for detection. Take 100μL of the treatment solution and add 1mL of 0.1M phosphate buffer solution to obtain a mixed solution, and let it stand for 3min. Take 100μL of the mixed solution and add it to the sample well (marked with S) of the test card that has been preheated to 37℃, cover the incubator lid, and time for 8min; after the timing ends, take out the test card and read the result within 20s (see Table 3).

[0064] The amount of methanol added is 30 mL, and the amount of 0.1M phosphate buffer solution added is 1 mL. According to the ratio of the T line to the C line of the test strip, the solution combination with a T / C value between 2.0 and 4.0 when the addition amount of nonylphenol polyoxyethylene ether is 0 mg / kg and the largest difference in T / C value when the addition amount of nonylphenol polyoxyethylene ether is 10 mg / kg is selected as the best solution combination. The preferred solution combination is 30 mL of methanol and 1 mL of 0.1M phosphate buffer solution.

[0065] Table 3 Optimization of detection solution ratio

[0066]

[0067]

[0068] (2) Establishing a standard curve

[0069] Take 2.00g sample fragments of the standard curve sample of the food contact material to be tested prepared in step 2(2), place them in a 50mL conical flask, add 30mL of methanol, ultrasonically extract for 30min, transfer 2mL of the extract to pass through a 0.45μm organic phase filter, and wait for detection. Take 100μL of the treated solution and add 1mL of 0.1M phosphate buffer solution to obtain a mixed solution, and let it stand for 3min. Take 100μL of the mixed solution and add it to the sample well (marked with S) of the test card that has been preheated to 37℃, cover the incubator lid, and time for 8min; after the timer ends, take out the test card and read the result within 20s. Obtain the standard curve, and the detection line is 2mg / kg.

[0070] (3) Recovery rate test

[0071] Take 2.00g sample fragments of the food contact material sample to be tested prepared in step 2 (4), place them in a 50mL conical flask, add 30mL of methanol, ultrasonically extract for 30min, transfer 2mL of the extract to pass through a 0.45μm organic phase filter, and wait for detection. Take 100μL of the treated solution and add 1mL of 0.1M phosphate buffer solution to obtain a mixed solution, and let it stand for 3min. Take 100μL of the mixed solution and add it to the sample well (marked with S) of the test card that has been preheated to 37°C, cover the incubator lid, and time for 8min; after the timing ends, take out the test card and read the result within 20s. The recovery rate of nonylphenol polyoxyethylene ether is shown in Table 4:

[0072] Table 4 Recovery rates of nonylphenol polyoxyethylene ethers in various food contact materials

[0073] Name of food contact material Addition concentration (mg / kg) Detection concentration (mg / kg) Recovery rate (%) Paper cups 10 9.68 96.8 Steamer paper 10 10.88 108.8 Kraft paper 10 11.21 112.1 Wax paper 10 9.35 93.5

[0074] As can be seen from the above table, the recovery rate of the test strip provided by the present invention in paper cups, steamer paper, kraft paper, and waxed paper is within an acceptable range, meeting the recovery rate requirement of the analytical method.

[0075] 4. Comparison between rapid immune test method and traditional method

[0076] It can be seen from Table 5 that, compared with the traditional instrument method, the immunochromatography method provided by the present invention not only has similar accuracy and repeatability as the instrument method, but also has a safer detection process, low cost and short time.

[0077] Table 5 Comparison of different detection methods for nonylphenol polyoxyethylene ether in food contact materials

[0078]

[0079] 5. Specificity of rapid immunoassay

[0080] Samples of paper cups, steamer paper, kraft paper and waxed paper containing the structural analogs listed in Table 1 were taken and tested using the test strip method. The test results showed that the test strip did not recognize other structural analogs. The test results of this method were specific and would not be interfered with by other structural analogs.

[0081] Obviously, the above embodiments are merely examples for clear explanation and are not intended to limit the implementation methods. For those skilled in the art, other different forms of changes or modifications can be made based on the above description. It is not necessary and impossible to list all the implementation methods here. The obvious changes or modifications derived from these are still within the protection scope of the invention.

Claims

1. A hybridoma cell line secreting anti-nonylphenol polyoxyethylene ether monoclonal antibody, characterized in that: The hybridoma cell line was deposited in the General Microbiology Center of China Culture Collection Administration on October 31, 2024, with the deposit address at No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing, with the deposit number CGMCC No. 46228, and the classification name is monoclonal cell line.

2. An anti-nonylphenol polyoxyethylene ether monoclonal antibody, characterized in that: The hybridoma cell line according to claim 1 is secreted and produced.

3. A composition, characterized in that The invention comprises the anti-nonylphenol polyoxyethylene ether monoclonal antibody as claimed in claim 2.

4. A colloidal gold test strip for quantitatively detecting nonylphenol polyoxyethylene ether in food contact materials, characterized in that: It comprises the anti-nonylphenol polyoxyethylene ether monoclonal antibody according to claim 2, or the composition according to claim 3.

5. The colloidal gold test strip according to claim 4, characterized in that: The colloidal gold test strip comprises a PVC plate, a sample pad, a gold label pad, a nitrocellulose membrane and a water absorbent pad; The gold label pad is prepared by spraying the anti-nonylphenol polyoxyethylene ether mouse monoclonal antibody according to claim 2 on a glass fiber membrane; The nitrocellulose membrane is provided with a detection line and a quality control line in sequence.

6. The colloidal gold test strip according to claim 5, characterized in that: The quality control line is obtained by spraying goat anti-mouse secondary antibody; And / or, the detection line is obtained by spraying a nonylphenol polyoxyethylene ether-BSA solution.

7. The colloidal gold test strip according to claim 4, characterized in that: The food contact material includes one or more of wood pulp paper, steamer paper, kraft paper and waxed paper.

8. A kit for quantitatively detecting nonylphenol polyoxyethylene ether in food contact materials, characterized in that: The invention comprises at least one of the anti-nonylphenol polyoxyethylene ether monoclonal antibody according to claim 2, the composition according to claim 3 and the colloidal gold test strip according to any one of claims 4 to 7.

9. Use of the hybridoma cell line according to claim 1, the anti-nonylphenol polyoxyethylene ether monoclonal antibody according to claim 2, the composition according to claim 3, the colloidal gold test strip according to any one of claims 4 to 7, or the kit according to claims 8 to 9 in detecting nonylphenol polyoxyethylene ether.

10. A method for quantitatively detecting nonylphenol polyoxyethylene ether in food contact materials, characterized in that: The following steps are involved: (1) Select food contact material samples and cut them into 2-4 cm pieces 2 small fragments, mixing the sample fragments with methanol, performing ultrasonic extraction, filtering the obtained extract to obtain a test solution; (2) The test solution obtained in step (1) is tested using the colloidal gold test strip described in any one of claims 4 to 7 or the kit described in claims 8 to 9.

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