An N-dimethylnitrosamine hapten, antigen and antibody, their preparation methods and applications

Abstract

This invention provides a method for preparing N-dimethylnitrosamine hapten, antigen, and antibody. The method involves condensing unsymmetrical dimethylhydrazine hydrochloride with 4-formylbenzoic acid to obtain an unsymmetrical dimethylhydrazine derivative hapten. This hapten has the same functional groups as N-dimethylnitrosamine and a similar structure, highlighting the specific antigenic determinant of this molecule. The antigen is then prepared by coupling a carrier protein using an active ester method. Finally, immunizing experimental animals with this antigen yields high-titer, highly specific antibodies against N-dimethylnitrosamine. The prepared antibody, when used in an indirect competitive ELISA method to detect NDMA, exhibits good stability and detection limits, and shows high consistency with gas chromatography-tandem mass spectrometry analysis results. This provides a rapid, sensitive, and cost-effective tool for high-throughput screening of NDMA in dried aquatic products.

Description

Technical Field

[0001] This invention belongs to the field of N-nitrosodimethylamine detection technology, specifically relating to an N-dimethylnitrosamine hapten, antigen and antibody, their preparation methods and applications. Background Technology

[0002] N-nitrosodimethylamine (NDMA) is carcinogenic and mutagenic. It is activated through oxidation and subsequent formation of carbocations, promoting DNA alkylation and is a potent carcinogen. GB2762—2022, the National Food Safety Standard for Maximum Levels of Contaminants in Food, stipulates that the NDMA content in aquatic products must not exceed 4 μg / kg, and in meat products, it must not exceed 3 μg / kg. The main pathways for NDMA generation in aquatic products are: nitrites and nitrates are typically added during the pickling process for color fixation, preservation, and to enhance the pickled flavor. Nitrites react adversely with amines or amino derivatives (such as chloramines) to produce nitrosamines. Currently, the detection methods reported domestically and internationally mainly involve instrumental analysis, employing sample pretreatment methods such as steam distillation and QuEChERs, and using instruments such as gas chromatography-mass spectrometry / mass spectrometry (GC-MS / MS) and gas chromatography-thermal analysis (GC-TEA) for determination. Instrumental methods are precise and stable confirmatory methods, but they suffer from drawbacks such as high cost, low sample throughput, cumbersome pretreatment, and complex operation, making them unsuitable for rapid on-site screening. Indirect competitive enzyme-linked immunosorbent assay (ic-ELISA), on the other hand, offers advantages such as simple operation, high sample throughput, low cost, ease of automation, and rapid on-site screening, providing a new method for the rapid screening of N-dimethylnitrosamine in large batches of aquatic product samples.

[0003] However, establishing an ic-ELISA method first requires the preparation of specific antibodies against the target analyte. Nitrosamines have molecular weights less than 1000 Da. Unlike large molecules such as proteins and nucleic acids, small molecules are reactive but lack immunogenicity; that is, they lack T-cell epitopes and cannot directly induce the body to produce corresponding specific antibodies. Therefore, small molecule compounds can only be called haptens (incomplete antigens). Based on immunological principles, they need to be coupled with carrier macromolecules to form artificial antigens, thereby acquiring immunogenicity. These artificial antigens then indirectly induce B cell proliferation and differentiation through T-cell epitopes, ultimately producing specific antibodies. Many small molecules to be detected cannot be directly used as haptens due to stability and other issues. Summary of the Invention

[0004] In view of this, the present invention provides an N-dimethylnitrosamine hapten, and further provides an artificial antigen prepared by coupling the hapten with a carrier protein, and an experimental animal immunized to produce specific antibodies against N-dimethylnitrosamine, and finally uses the obtained antibody to detect NDMA by ic-ELISA method.

[0005] One objective of this invention is to provide an N-dimethylnitrosamine hapten, the structure of which is shown in the following formula: .

[0006] The second objective of this invention is to provide a method for preparing the above-mentioned hapten, the method comprising the following steps: preparing a solution of unsymmetrical dimethylhydrazine hydrochloride, and adding dropwise a DMF solution containing 4-formylbenzoic acid under stirring with a glass rod until the turbidity disappears and the addition is stopped, stirring continuously and then placing the solution in a constant temperature oven at 37°C and shaking overnight, finally centrifuging the reaction solution and freeze-drying it under vacuum to obtain the hapten.

[0007] Furthermore, the mass ratio of unsymmetrical dimethylhydrazine hydrochloride to 4-formylbenzoic acid is 1:(1.5~2.5). Centrifugation conditions: speed 4000~5000 r / min, time 10~20 min.

[0008] The third objective of this invention is to provide an N-dimethylnitrosamine antigen, which is obtained by chemically coupling the above-mentioned N-dimethylnitrosamine hapten to a carrier protein, and the specific structural formula is as follows: .

[0009] The fourth objective of this invention is to provide a method for preparing the above-mentioned antigen, comprising the following steps: S1. Dissolve the above hapten, N,N-dicyclohexylcarbodiimide and N-hydroxythiosuccinimide in DMF, stir and react to obtain reaction solution III; S2. Dissolve BSA in PBS buffer, then add it dropwise to reaction solution III, stir overnight at 4°C to obtain reaction solution IV; S3. Centrifuge reaction solution IV, collect the supernatant, dialyze to obtain antigen solution.

[0010] Furthermore, in step S21, the mass ratio of the hapten, N,N-dicyclohexylcarbodiimide, and N-hydroxythiosuccinimide is 1:(0.8~1.0):(1.4~1.8). Stirring reaction conditions: 450~550 rpm, 10~14 h; In step S22, the mass ratio of hapten to BSA is 1:6.5~7.0; the PBS buffer concentration is 0.1 M and the pH is 7.4.

[0011] Centrifugation conditions in step S23: 4500~6000 rpm, time 12~17 min; dialysis conditions: dialysis buffer is 0.01 M PBS buffer with pH 7.4, dialysis for 3 days, and change the dialysis buffer 3 times a day.

[0012] The fifth objective of this invention is to provide a hybridoma cell line NDMA / 6E5, which is prepared using the above-mentioned N-dimethylnitrosamine antigen as an immunogen, and the hybridoma cell line NDMA / 6E5 has the accession number CCTCC NO: C202653.

[0013] The sixth objective of this invention is to provide a monoclonal antibody, which is secreted by the aforementioned hybridoma cell line.

[0014] The seventh objective of this invention is to provide the application of the above-mentioned N-dimethylnitrosamine hapten, N-dimethylnitrosamine antigen, hybridoma cell line NDMA / 6E5, and monoclonal antibody in the detection of N-dimethylnitrosamine.

[0015] Preferably, the application specifically involves detection using the ic-ELISA method.

[0016] Compared with the prior art, the beneficial effects of the present invention are as follows: The hapten of the paper cup of this invention has the same group as N-dimethylnitrosamine and has a similar structure, highlighting the specific antigenic determinant of this molecule. The antigen can be prepared by coupling the carrier protein with the active ester method. Immunizing experimental animals with this antigen can obtain specific antibodies against N-dimethylnitrosamine with high titer and good specificity.

[0017] After optimization, the linear range of ic-ELISA is 1~15μg / L, IC50 50 =4.78 μg / L, with extremely low cross-reactivity (<4.89%). Furthermore, the method was validated using five dried aquatic products, with recoveries ranging from 67.54% to 82.12%, limits of detection ranging from 0.8 to 1.42 μg / kg, and coefficients of variation ranging from 3.54% to 11.71%. These results were highly consistent with gas chromatography-tandem mass spectrometry (GC-MS / MS) analysis (R... 2 =0.989). This derivatization-based immunochemical indirect competitive enzyme-linked immunosorbent assay provides a rapid, sensitive, and cost-effective tool for high-throughput screening of NDMA in dried aquatic products, suitable for routine food safety monitoring. Attached Figure Description

[0018] Figure 1 This is the concentration standard curve of the UDMH standard in Example 1 of the present invention.

[0019] Figure 2 This is the proton NMR spectrum of the hapten CP-NDMA in Example 1 of the present invention.

[0020] Figure 3 This is the carbon NMR spectrum of the hapten CP-NDMA in Example 1 of the present invention.

[0021] Figure 4 The Fourier transform infrared spectroscopy results of the hapten CP-NDMA in Example 1 of this invention are shown.

[0022] Figure 5 The actual reaction result after replacing 4-formylbenzoic acid with 2-formylbenzoic acid in Example 1 of this invention.

[0023] Figure 6 This is a photograph of the hapten CP-NDMA product in Example 1 of the present invention.

[0024] Figure 7 This is the ultraviolet scan spectrum of antigen CP-NDMA-BSA in Example 1 of the present invention.

[0025] Figure 8 This is the UV-Vis spectrum of the original CEPSEM-OVA coating in Example 1 of the present invention.

[0026] Figure 9 This is a standard curve diagram of ic-ELISA detection in Example 3 of the present invention. Detailed Implementation

[0027] The present invention will be further described in detail below with reference to specific embodiments, so that those skilled in the art can more clearly understand the present invention. Unless otherwise specified, the technical means used in the following embodiments are all conventional means well known to those skilled in the art, and all reagents and consumables are commercially available products.

[0028] Example 1 This embodiment provides a method for preparing N-dimethylnitrosamine hapten, antigen, and coating agent, as detailed below: 1.1 Reduction of N-dimethylnitrosamine Add 100 mL of 1 g / L NDMA solution to a 100 mL stoppered three-necked flask, and dilute with high-purity nitrogen at a concentration of 1 h·200 mL / min. -1The sample was deoxygenated at a rapid rate, and a certain amount of zinc powder was quickly added. The rubber stopper was then tightened, and the sample was placed in a constant-temperature shaker and shaken in the dark. After 12 hours of reaction, the unreacted zinc powder was separated by vacuum filtration through a 0.22 μm membrane, thereby terminating the reaction (reaction formula as follows), yielding unsymmetrical dimethylhydrazine (UDMH).

[0029]

[0030] The purification steps for UDMH are as follows: (1) pH adjustment to remove ammonium ions (NH4+) 4+ ) and dimethylamine (DMA): Add excess NaOH (1M) to the mixed solution to adjust the pH to above 12. NH 4+ It is converted to NH3, and dimethylamine (DMA) is converted to a neutral molecule; both can be removed by volatilization upon heating. UDMH still exists partially as a salt in solution under strong alkaline conditions.

[0031] (2) Distillation separation of UDMH and NDMA: The boiling point of UDMH is 63℃, while the boiling points of NDMA and DMA are 152℃ and 6.9℃, respectively. Therefore, impurities can be separated by vacuum distillation. The alkali-treated solution is transferred to a vacuum distillation apparatus and distilled at 40-50℃ under reduced pressure (e.g., 20-30 mmHg) with nitrogen protection throughout the process. The UDMH fraction is collected.

[0032] Furthermore, the conversion rate of the purified UDMH was determined, as follows: UDMH standard was diluted with ultrapure water to prepare a series of concentrations (0, 25, 50, 100, 250, 500, 1000 μg / mL). 0.5 mL of each concentration standard solution was added to 2 mL of p-dimethylaminobenzaldehyde solution (PDAB solution) and 1 mL of 1M HCl. The mixture was vortexed and allowed to stand at room temperature in the dark for 10 min (until complete color development). The absorbance (A) was measured at 460 nm. The graph was plotted with UDMH concentration as the x-axis. x ), absorbance is the ordinate ( y ), fitting linear equations (such as y = kx + b , R ²≥0.99).

[0033] Sample pretreatment is similar to that of standards. Take 0.5 mL of sample solution (which can be diluted appropriately), add PDAB solution and HCl according to the standard curve procedure, and determine A. 460 Substitute the values ​​into the standard curve equation to calculate the UDMH concentration (C0) in the sample. x ).

[0034] The conversion rate of UDMH is calculated using the following formula:

[0035] Finally, according to the standard curve (see...) Figure 1 The conversion rate was calculated to be 83%, indicating that this experimental method can achieve the reduction of N-dimethylnitrosamine.

[0036] The above experiments verified the feasibility of NDMA to UDMH and calculated the conversion rate between NDMA and UDMH.

[0037] 1.2 Preparation of N-dimethylnitrosamine hapten (CP-NDMA) Dissolve 0.3 g of unsymmetrical dimethylhydrazine hydrochloride (UDMH·HCl) in 5 mL of ultrapure water and stir on a magnetic stirrer to obtain liquid A. Dissolve 0.6 g of 4-formylbenzoic acid in 5 mL of DMF to obtain liquid B. While stirring magnetically, slowly add liquid B dropwise to liquid A, followed by adding DMF dropwise until the turbidity disappears. After stirring magnetically at room temperature for 4 h, place the solution in a 37 °C incubator and shake overnight. Centrifuge the reaction mixture at 4500 r / min for 15 min, discard the supernatant, and collect the pale yellow precipitate. Freeze-dry the hapten precipitate under vacuum and store it at -20 °C protected from light. The prepared CP-NDMA has the following structure: .

[0038] A comprehensive analysis of the obtained hapten was conducted by combining the results of proton NMR, carbon NMR, and Fourier transform infrared spectroscopy. hapten CP-NDMA 1 H NMR (600 MHz, DMSO, TMS) δ 10.10(s,1H), 7.87 (d, J =8.5 Hz, 2H), 7.59 (d, J = 8.5 Hz, 2H), 7.28 (s, 1H), 2.97(s, 6H); 13 C NMR (151MHz, DMSO, TMS) δ 167.69, 141.68, 130.16, 130.12, 128.92, 125.24, 42.85 (see...) Figure 2 , 3 ).

[0039] The hapten CP-NDMA was at 1668.8 cm⁻¹ -1 A strong absorption peak appeared nearby, which is the stretching vibration peak of C=O, indicating that the reaction product has a free -COOH structure; at 1544.2 cm⁻¹ -1A stretching vibration peak of the C=N bond appeared nearby, overlapping somewhat with the strong absorption peak of the benzene ring; at 942.3 cm⁻¹ -1 The characteristic absorption peak of NN in the hydrazone group (C=NN) appeared nearby; at 1039.6 cm⁻¹. -1 The characteristic absorption peak of CN in the hydrazone group (C=NN) is present (see Figure 4 ).

[0040] The above results indicate that the hapten was successfully synthesized.

[0041] Furthermore, this application also attempted to replace 4-formylbenzoic acid with 2-formylbenzoic acid to prepare the hapten, and found that almost no precipitation occurred during the entire process, and the yield was extremely low (see...). Figure 5 This method is not suitable for preparing haptens. However, the synthesis of haptens using unsymmetrical dimethylhydrazine hydrochloride and 4-formylbenzoic acid is rapid and yields high output (see...). Figure 6 ).

[0042] 1.3 Preparation of N-dimethylnitrosamine antigen (CP-NDMA-BSA) The hapten (CP-NDMA, 19.2 mg) was dissolved together with NHS (17.5 mg) and DCC (30.0 mg) in DMF (2 mL) and stirred at room temperature for 12 h. Then, BSA (134 mg) was dissolved in phosphate-buffered saline (PBS, 0.1 M, pH 7.4, 10 mL) and added dropwise to the mixture, and stirred overnight at 4 °C. The supernatant was then centrifuged at 5000 rpm for 15 min. Finally, the solution was dialyzed against PBS (0.01 M, pH 7.4) for 3 days to remove the solute, changing the dialysate 3 times daily to obtain a purified solution, which was then stored at -20 °C for immunization. The prepared CP-NDMA-BSA has the following structure: .

[0043] CP-NDMA-BSA identification: Ultraviolet scanning (200–400 nm) was performed on the carrier protein BSA, the hapten CP-NDMA, and the immunogen CP-NDMA-BSA. The absorption curves of the immunogen CP-NDMA-BSA showed significant changes compared to BSA and UDMH. Figure 7 This indicates that the hapten CEPSEM was successfully conjugated to BSA. The conjugation ratio of hapten IV to BSA, as determined by the Coomassie Brilliant Blue assay, was 25:1.

[0044] 1.4 Preparation method of coating agent (CEPSEM-OVA) The hapten (CP-NDMA, 19.2 mg) was dissolved together with NHS (17.5 mg) and DCC (30.0 mg) in DMF (2 mL) and stirred at room temperature for 12 h. Then, OVA (89 mg) was dissolved in phosphate-buffered saline (PBS, 0.1 M, pH 7.4, 10 mL), added dropwise to the mixture, and stirred overnight at 4 °C. The supernatant was then centrifuged at 5000 rpm for 15 min. Finally, the solution was dialyzed against PBS (0.01 M, pH 7.4) for 3 days to remove the solute, changing the dialysate 3 times daily to obtain a purified solution. This purified solution was stored at -20 °C for coating enzyme-linked immunosorbent assay (ELISA) plates for the N-dimethylnitrosamine immunoassay. The structure of the prepared coated CP-NDMA-OVA is shown below: .

[0045] Identification of CEPSEM-OVA: Ultraviolet (UV) scanning (200–400 nm) was performed on the carrier protein OVA, hapten CP-NDMA, and coating precursor CP-NDMA-OVA. The absorption curves of the coating precursor CP-NDMA-OVA showed significant changes compared to the carrier protein OVA and CP-NDMA. Figure 8 This indicates that the hapten CP-NDMA and the carrier protein OVA were successfully conjugated. The conjugation ratio of the hapten CP-NDMA to the carrier protein OVA was determined to be 23:1 using the Coomassie Brilliant Blue assay.

[0046] Example 2 This embodiment uses the CP-NDMA-BSA prepared in Example 1 to obtain hybridoma cell lines, and further obtains antibodies, as detailed below: 2.1 Determination of serum titer and specificity in mice The immunogen was slowly thawed, and then an equal volume of adjuvant was added (French complete adjuvant was used for the first immunization, and Freund's incomplete adjuvant was used for subsequent booster immunizations). After complete emulsification, six healthy Balb / c mice weighing 15–30 g were immunized using various subcutaneous injection methods, including subcutaneous injection at the back and other sites. One week after the second booster immunization, blood was collected from the orbital venous plexus, and serum titers were determined using an indirect competitive ELISA.

[0047] Antibody titer and specificity detection: The antibody positive titer was determined by indirect competitive ELISA with a value 2.1 times that of negative serum, and the positive titer of the antibody was 1:8000.

[0048] 2.2 Preparation of Monoclonal Antibodies After five consecutive immunizations, one BALB / c mouse that had received the final booster immunization was euthanized by orbital bleeding (serum was collected, which is the positive serum). The mouse was then disinfected by immersion in 75% alcohol for 5 minutes. 3~5×107 Add SP2 / 0 myeloma cells and spleen cells from immunized mice to a 50 mL centrifuge tube, mix well, and centrifuge at 1500 rpm for 5 min. Discard the supernatant and blot dry with sterile filter paper. Gently tap the bottom of the tube to loosen the cells. Place the centrifuge tube in a 37°C water bath and slowly add 0.8 mL of 50% PEG pre-warmed to 37°C over 1 min, stirring gently with the tip of a pipette while adding. Continue stirring for 30 s after adding all the PEG, and let stand for 1 min.

[0049] Slowly add 10 mL of pre-warmed RPMI-1640 basal solution (37°C). Specifically, add 1 mL dropwise over the first minute, 2 mL over the second minute, and then slowly add the remaining RPMI-1640 basal solution, gently shaking the centrifuge tube as you add. Slowly add 40 mL of RPMI-1640 basal solution, and after adding all, gently invert the tube to mix. Centrifuge at 1500 rpm for 5 minutes. Discard the supernatant. Using a dropper, slowly add 10 mL of HAT medium containing feeder cells along the tube wall, stirring gently with the dropper. Slowly aspirate confluent cells and add them dropwise close to the feeder cell surface, stirring mechanically to mix. Seed into six 96-well plates, approximately 150 µL / well, and incubate at 37°C in a 5% CO2 cell culture incubator.

[0050] Day 0 was recorded from the day of fusion. After 3 days, one drop of HAT medium was added to each well. After 5 days, half the volume of medium was aspirated every 2 days and replaced with an equal volume of HT medium. Four days after fusion, the fused cells were monitored, and wells showing hybridoma cell growth were marked and recorded, with the fusion rate calculated. Six to seven days after fusion, when the cells in the wells reached 1 / 10 to 1 / 5 of the bottom, the culture supernatant was collected, and positive cell wells were screened using an indirect competitive ELISA method. The original coating concentration was 10 mg / L. Each cell culture well had two wells: a 0 well and a 200 µg / L drug well. 50 µL of culture supernatant was added to each well for indirect competitive ELISA detection.

[0051] Select 4-6 wells with strong positive results in the supernatant test and only 1-2 well-morphologically formed colonies for subcloning using the limiting dilution method. After 3-4 cloning cycles, finally select monoclonal hybridoma cell lines that secrete NDMA-specific antibodies.

[0052] Preparation and identification of ascites monoclonal antibodies: Seven days prior to inoculation, several Balb / c mice were pretreated by intraperitoneal injection of 0.5 mL of Freund's incomplete adjuvant. Hybridoma cell lines were resuspended in RPMI-1640 basal medium to expand cell culture, and the cell number was adjusted to 1 × 10⁻⁶ cells / mL. 6The concentration was 0.5 mL / mL, and each mouse was intraperitoneally inoculated. Ascites fluid was collected and purified when the mice's abdomens became significantly distended, their mental state deteriorated, and they became immobile and near death. The monoclonal antibody obtained in this invention was identified as mouse IgG2a using a mouse mab Isotyping Test Kit purchased from Lablead. The result showed it to be mouse IgG2a, possessing a κ light chain.

[0053] Example 3 This embodiment provides a method for detecting NDMA using ELISA, as detailed below: IC-ELISA was performed on 96-well polystyrene microplates. The wells were coated with 100 μL of hapten-OVA (1 μg / mL, dissolved in 0.05 mol / L carbonate buffer, pH 9.6, 100 μL / well) for 2 h at 37 °C. The plates were washed three times with 0.01 mol / L PBST solution (1000 mL phosphate buffer containing 0.5 mL Tween-20), and blocked with 1% skim milk powder in PBST solution (200 μL / well). After 2 h of incubation, the plates were washed three times with PBST solution. Subsequently, analytes diluted to specific concentrations and diluted antibodies (50 μL / well) were added to the wells. After 1 h of incubation, the plates were washed and further incubated for 30 min with goat anti-mouse IgG-HRP (100 μL / well diluted 1:3000 in PBST containing 1% bovine serum albumin). After washing five times, add colorimetric reagents A and B in a 5:1 volume ratio (total volume 100 μL / well). Incubate at 37°C for 10 min, then terminate the reaction by adding sulfuric acid solution (2 mol / L, 50 μL / well). Measure the absorbance at 450 nm using a microplate reader.

[0054] Furthermore, to improve the performance of ic-ELISA, the competitive incubation parameters were further optimized, including the coating antigen concentration (0.25, 0.5, 1.0, 2.0 μg / mL), primary antibody dilution factor (1 / 8000, 1 / 16000, 1 / 32000, 1 / 64000), the competition time between the antibody and CP-NDMA and the coating antigen (30, 60, 90, 120 min), and the dilution factor of hydrogen peroxide-labeled goat anti-mouse secondary antibody (1 / 2000, 1 / 3000, 1 / 4000, 1 / 5000), to obtain optimal detection performance. Immunoassay performance was measured by B0 and IC50. 50 Determined; Maximum B0 and Minimum IC 50 This will be the best choice for ic-ELISA (see Table 1). A standard curve was obtained under optimal conditions by plotting the relative absorbance of B / B0 against the logarithm of CP-NDMA concentration. Figure 9The regression equation for the standard curve is: y = -0.4985x + 0.8399, IC0 50 The value is 4.78 μg / L, and the working range is 1-15 μg / L.

[0055] Table 1 Optimization of various conditions for ic-ELISA

[0056] Furthermore, the specificity was assessed using a cross-reactivity ratio method: Nine structural analogs of CP-NDMA (N-dimethylnitrosamine, N-diethylnitrosamine, CP-NDEA, N-dimethylformamide, unsymmetrical dimethylhydrazine, benzaldehyde, benzoic acid, 4-formylbenzoic acid, and o-carboxybenzaldehyde) were serially diluted to gradient concentrations, and an indirect competitive ELISA was performed. A standard curve was plotted, and the IC50 was calculated. 50 Value. The IC for CP-NDMA. 50 The cross-reactivity of the competitors is obtained by comparing the IC50 value with that of the competitors (see Table 2).

[0057] Table 2 Results of cross-reactivity test

[0058] The results showed that the monoclonal antibody prepared in this study had high specificity for the NDMA derivative CP-NDMA and no cross-reactivity with other drugs of the same class or other drug classes. The results also showed that mAb 6E5 exhibited cross-reactivity with NDMA (4.89%), NDEA (4.26%), and 4-FBA (4.57%). This is because the hapten CP-NDMA is structurally similar to these compounds, but the NDMA content differs significantly from that of other analogues. Furthermore, the NDMA content in dried aquatic products was significantly higher than that of other analogues. Therefore, when detecting NDMA after derivatization, other analogues did not significantly affect the results.

[0059] Example 4 To verify the reliability of the above detection method, this embodiment also tested its sensitivity, accuracy, and precision, as detailed below: Twenty dried fish products purchased from the market were tested for NDMA using GC-MS / MS, and the results showed that they did not contain NDMA. Each sample was tested using ic-ELISA to determine the limit of detection (LOD) and limit of quantitation (LOQ), where: LOD was determined based on the mean of 20 blank samples + 3 times the standard deviation (SD); LOQ values ​​were determined from 20 blank samples + 10 times the SD.

[0060] The accuracy and precision of this method are expressed as average recovery (%) and coefficient of variation (CV), respectively.

[0061] The average recovery (%) was calculated using five spiked replicate blanks at 1, 2, and 4 times the LOQ of NDMA for three different analyses, using the following formula: (measured concentration / spiked concentration) × 100% (results are in NDMA equivalents).

[0062] The coefficient of variation (CV%) was determined by performing five repeated experiments on the above samples, spiking NDMA at three different levels, and was calculated as: (standard deviation / mean) × 100%.

[0063] Samples including grilled squid strips, squid ear strips, grilled cod fillets, yellow croaker crisps, and dried shrimp were selected for study. Representative samples were crushed after removing the edible parts, prepared into homogeneous samples, and 5g was placed in a 50mL centrifuge tube. 5mL of water was added, and the mixture was vortexed. 10mL of acetonitrile was accurately added, and the mixture was vortexed for 2 minutes. The mixture was then incubated at -20℃ for 20 minutes. Ceramic homogenants, magnesium sulfate, and sodium chloride were added, and the mixture was vortexed for 2 minutes. The mixture was then centrifuged at 9000 rpm for 5 minutes. The supernatant was diluted 10-fold with PBST buffer and derivatized. The spiked samples were analyzed using an optimized ELISA protocol to estimate the recovery rate, and then subjected to ic-ELISA detection (see Table 3).

[0064] Table 3 Recovery rates and coefficients of variation for each actual sample

[0065] Table 3 shows that the method validation using five types of dried aquatic products yielded recoveries ranging from 67.54% to 82.12%, limits of detection ranging from 0.8 to 1.42 μg / kg, and coefficients of variation ranging from 3.54% to 11.71%. The detection results were highly consistent with those obtained by gas chromatography-tandem mass spectrometry (GC-MS / MS). 2 =0.989). This derivatization-based immunochemical indirect competitive enzyme-linked immunosorbent assay provides a rapid, sensitive, and cost-effective tool for high-throughput screening of NDMA in dried aquatic products, suitable for routine food safety monitoring.

[0066] In summary, this application successfully prepared N-dimethylnitrosamine hapten and antigen, and further used the antigen to prepare specific antibodies. The prepared antibodies were then used to detect NDMA by ic-ELISA, achieving advantages such as simple operation steps, high sample throughput, low cost, easy automation control, and rapid on-site screening.

[0067] Unless otherwise specified, all raw materials used in this invention are existing substances that can be purchased directly from the market.

[0068] The above are merely preferred embodiments of the present invention and are not intended to limit the scope of protection of the present invention. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the scope of protection of the present invention.

Claims

1. An N-dimethylnitrosamine hapten, characterized in that, The structure of the hapten is shown in the following formula: 。 2. A method for preparing the hapten according to claim 1, characterized in that, The preparation method is as follows: unsymmetrical dimethylhydrazine hydrochloride is prepared into a solution, and DMF solution containing 4-formylbenzoic acid is added dropwise under stirring with a glass rod until the turbidity disappears and the addition is stopped. After continuous stirring, the solution is placed in a constant temperature oven at 37°C and shaken overnight. Finally, the reaction solution is centrifuged and freeze-dried under vacuum to obtain the hapten.

3. The preparation method according to claim 2, characterized in that, The mass ratio of unsymmetrical dimethylhydrazine hydrochloride to 4-formylbenzoic acid is 1:(1.5~2.5). Centrifugation conditions: speed 4000~5000 r / min, time 10~20 min.

4. An N-dimethylnitrosamine antigen, characterized in that, The antigen is obtained by chemically coupling the N-dimethylnitrosamine hapten of claim 1 to a carrier protein, and its specific structural formula is as follows: 。 5. A method for preparing the antigen according to claim 4, characterized in that, Includes the following steps: S1. Dissolve the hapten, N,N-dicyclohexylcarbodiimide and N-hydroxythiosuccinimide described in claim 1 in DMF, stir and react to obtain reaction solution I; S2. Dissolve BSA in PBS buffer, then add it dropwise to reaction solution I, stir overnight at 4°C to obtain reaction solution II; S3. Centrifuge reaction solution II, collect the supernatant, dialyze to obtain antigen solution.

6. The preparation method according to claim 5, characterized in that, In step S1, the mass ratio of hapten, N,N-dicyclohexylcarbodiimide and N-hydroxythiosuccinimide is 1:(0.8~1.0):(1.4~1.8); the stirring reaction conditions are: 450~550 rpm and 10~14 h. In step S2, the mass ratio of hapten to BSA is 1:6.5~7.0; the PBS buffer concentration is 0.1M and the pH is 7.

4. Centrifugation conditions in step S3: 4500~6000 rpm, time 12~17 min; dialysis conditions: dialysis buffer is 0.01M PBS buffer with pH 7.4, dialysis for 3 days, and change the dialysis buffer 3 times a day.

7. A hybridoma cell line NDMA / 6E5, characterized in that, The hybridoma cell line NDMA / 6E5 was prepared using the N-dimethylnitrosamine antigen described in claim 5 as an immunogen, and the hybridoma cell line NDMA / 6E5 has the accession number CCTCCNO: C202653.

8. A monoclonal antibody, characterized in that, The monoclonal antibody is secreted by the hybridoma cell line described in claim 7.

9. The use of the N-dimethylnitrosamine hapten of claim 1, the N-dimethylnitrosamine antigen of claim 5, the hybridoma cell line NDMA / 6E5 of claim 7, and the monoclonal antibody of claim 8 in the detection of N-dimethylnitrosamine.

10. The application according to claim 9, characterized in that, The specific application involves using the indirect competitive ELISA method for detection.

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