Hapten, complete antigen for detecting ethoxyquin and method of use thereof

Abstract

The application relates to a hapten for detecting ethoxyquin, a complete antigen and a use method thereof. The application discloses a hapten for detecting ethoxyquin, and then a complete antigen obtained by coupling ethoxyquin hapten with hemocyanin is used as an immunogen, and a complete antigen obtained by coupling ethoxyquin hapten with chicken egg white albumin is used as a coating agent. An indirect detection enzyme-linked immunoassay method for ethoxyquin is established. The half-inhibition concentration of the method for ethoxyquin is 0.96 ug / mL, the detection limit is 0.6 ug / mL, the linear detection range is 0.72 ug / mL-1.28 ug / mL, and the curve correlation coefficient is 0.996. The method is high in specificity and sensitivity, simple and fast, and has a wide application prospect in the detection of ethoxyquin residues in food. The method is very suitable for realizing rapid qualitative or quantitative detection of ethoxyquin in food by primary site personnel.

Description

Technical Field

[0001] This application relates to the field of immunoassay technology for ethoxyquinoline, and in particular to a hapten, a complete antigen for detecting ethoxyquinoline, and a method for using them. Background Technology

[0002] Ethoxyquin (EQ), also known as ethoxyquinoline, tiger skin disease, etc., with the chemical name 6-ethoxy-2,2,4-trimethyl-1,2-dihydroquinoline, is a commonly used preservative. It appears as a light brown viscous liquid, easily oxidized under natural light, and darkens in color upon exposure to air, but its effectiveness remains unaffected. Ethoxyquinoline is mainly used on the surface of fruits to prevent tiger skin disease, and in feed, poultry products, and aquatic products to prevent the oxidation and spoilage of nutrients such as proteins, fats, and vitamins. It is widely used due to its good antioxidant effect and low cost.

[0003] After use, most ethoxyquinoline residues remain on the surface of fruit and have a certain penetrating ability. Some people may experience allergic dermatitis and other symptoms after contact with ethoxyquinoline. Furthermore, excessive ethoxyquinoline residues can lead to poisoning of organs such as the heart and kidneys and have potential carcinogenic risks, seriously affecting human health. Considering the harm caused by the abuse of ethoxyquinoline, many countries and organizations have established strict residue standards for ethoxyquinoline. my country's GB 2760-2024 stipulates that the maximum residue limit for ethoxyquinoline in surface-treated fresh fruit is 1 mg / kg; GB 2763-2021 stipulates that the maximum residue limit for ethoxyquinoline in pears is 3 mg / kg.

[0004] Currently, the main methods for detecting ethoxyquinoline residues are instrumental analyses such as chromatography and chromatography-mass spectrometry. While these methods offer high accuracy and reproducibility, they are time-consuming, involve complex procedures, require expensive equipment, and necessitate operation by relatively specialized personnel, thus failing to meet the market's demand for rapid and convenient methods. In contrast, immunoassay methods, developed based on antigen-antibody specific recognition, offer advantages such as convenient and rapid detection, making them suitable for large-scale sample testing and playing an increasingly important role in the field of rapid detection. The foundation of immunological assays lies in the availability of high-quality, highly specific antibodies; therefore, the design of haptens and the synthesis of complete antigens are particularly crucial. Summary of the Invention

[0005] The purpose of this invention is to overcome the shortcomings of the prior art and provide a hapten, a complete antigen for detecting ethoxyquinoline and a method for using them.

[0006] The first objective of this invention is to provide an ethoxyquinoline hapten.

[0007] A second objective of this invention is to provide the application of the ethoxyquinoline hapten.

[0008] A third objective of this invention is to provide an ethoxyquinoline complete antigen.

[0009] A fourth object of the present invention is to provide the application of the ethoxyquinoline complete antigen.

[0010] A fifth object of the present invention is to provide a composition of an immunogen and a coating antigen.

[0011] A sixth object of the present invention is to provide a composition of antibody and coating agent.

[0012] A seventh object of the present invention is to provide applications of the composition.

[0013] The eighth objective of this invention is to provide a non-diagnostic immunoassay method for detecting ethoxyquinoline.

[0014] The ninth objective of this invention is to provide an immunoassay kit for ethoxyquinoline.

[0015] To achieve the above objectives, the present invention is implemented through the following technical solution: An ethoxyquinoline hapten, the structural formula of which is shown in formula (I). .

[0016] As a specific implementation method, the preparation method of the ethoxyquinoline hapten includes the following steps: S1, anhydrous acetone, zinc trifluoromethanesulfonate, and methyl 4-aminobenzoate are fully dissolved in an organic solvent and reacted completely to obtain the intermediate ester. S2, intermediate ester, methanol and NaOH aqueous solution react fully, acid precipitation, take the precipitate, and that is the product.

[0017] The preparation method references existing technologies: Kundu D, Kundu SK, Majee A et al. . A facilesynthesis of 2,2,4-trisubstituted-1,2-dihydroquinolines catalyzed by zinctriflate under solvent-free conditions[J]. Journal of the Chinese ChemicalSociety, 2008, with slight modifications.

[0018] This invention also claims protection for the use of the ethoxyquinoline hapten, wherein the use is one or more of the following: Preparation of ethoxyquinoline complete antigen; Establish a non-diagnostic method for detecting ethoxyquinoline; Prepare an ethoxyquinoline immunoassay kit.

[0019] The present invention also claims protection for an ethoxyquinoline complete antigen, which is the ethoxyquinoline hapten coupled with a carrier protein, the structural formula of which is shown in formula (II). .

[0020] Preferably, the carrier protein is ovalbumin (OVA) or hemocyanin (KLH).

[0021] Preferably, the ethoxyquinoline complete antigen is prepared by the N,N'-carbonyldiimidazole method, whereby the ethoxyquinoline hapten is conjugated with a carrier protein.

[0022] This invention further claims protection for the use of the ethoxyquinoline complete antigen, wherein the use is one or both of the following: Establish a non-diagnostic method for detecting ethoxyquinoline; Prepare an ethoxyquinoline immunoassay kit.

[0023] The present invention also claims a composition of an immunogen and a coating antigen, wherein the immunogen and the coating antigen are both ethoxyquinoline haptens of claim 1 conjugated with a carrier protein, the structural formula of which is shown in formula (II). ; The carrier protein of the coating antigen is chicken ovalbumin, and the carrier protein of the immunogen is hemocyanin.

[0024] This invention also claims protection for a composition of an antibody and a coating antigen, wherein the antibody is prepared by immunizing animals with an immunogen. Both the immunogen and the coating antigen are ethoxyquinoline haptens coupled with a carrier protein, and their structural formulas are shown in formula (II). ; The carrier protein of the coating antigen is chicken ovalbumin, and the carrier protein of the immunogen is hemocyanin.

[0025] Preferably, the antibody is a polyclonal antibody.

[0026] The present invention also claims the use of the composition, wherein the use is one or more of the following: Establish a non-diagnostic method for detecting ethoxyquinoline; Prepare an ethoxyquinoline immunoassay kit.

[0027] And, a non-diagnostic immunoassay method for detecting ethoxyquinoline, utilizing the composition.

[0028] Preferably, the immunoassay method for detecting ethoxyquinoline is an indirect enzyme-linked immunosorbent assay (ELISA).

[0029] As a specific implementation method, the method includes the following steps: S1. Coat the microplate wells with the coating agent, incubate, wash, and block. S2. Add the standard solution of ethoxyquinoline to the corresponding wells of the ELISA plate; S3. Add the antibody to the corresponding well of the ELISA plate, incubate, and wash the plate. S4. Add the enzyme-labeled secondary antibody solution to all wells of the ELISA plate, incubate, and wash the plate. S5. Add the colorimetric solution to the ELISA plate and develop the color; S6. Add the stop solution to the microplate to stop the reaction, and read the absorbance value of each well using a microplate reader. S7. Use the four-parameter fitting module of Origin 8.5 to plot the standard curve and calculate the corresponding detection parameters of the inhibition curve; S8, converted to the actual content of ethoxyquinoline in the sample to be tested.

[0030] More preferably, the colorimetric solution in step S5 is the substrate solution corresponding to the enzyme in the enzyme-labeled secondary antibody in step S4.

[0031] More preferably, the terminating solution in step S6 is a concentrated sulfuric acid solution.

[0032] More preferably, the terminating solution in step S6 is a concentrated sulfuric acid solution with a concentration of 2 mol / L.

[0033] And, an ethoxyquinoline immunoassay kit containing the composition.

[0034] Preferably, the coating material is coated in the microwells of the ELISA plate.

[0035] Compared with the prior art, the present invention has the following beneficial effects: This invention discloses an enzyme-linked immunosorbent assay (ELISA) method for the indirect detection of ethoxyquinoline hapten. The method utilizes a complete antigen obtained by conjugating hemocyanin with the ethoxyquinoline hapten as an immunogen and a complete antigen obtained by conjugating chicken oocyte albumin with the ethoxyquinoline hapten as a coating antigen. The method establishes an indirect enzyme-linked immunosorbent assay (ELISA) method for the detection of ethoxyquinoline. The half-maximum inhibitory concentration (WMC) for ethoxyquinoline is 0.96 μg / mL, the limit of detection (LOD) is 0.6 μg / mL, the linear detection range is 0.72 μg / mL to 1.28 μg / mL, and the correlation coefficient is 0.996. This method is highly specific, sensitive, simple, and rapid, and has broad application prospects in the detection of ethoxyquinoline residues in food. It is particularly suitable for field personnel at the grassroots level to achieve rapid qualitative or quantitative detection of ethoxyquinoline in food. Attached Figure Description

[0036] Figure 1 This is a synthetic route diagram for the ethoxyquinoline hapten EQ-1.

[0037] Figure 2 This is a mass spectrometry result of EQ-1, a half-antibiotic containing ethoxyquinoline.

[0038] Figure 3 Ultraviolet scans of ethoxyquinoline hapten (EQ-1), the complete antigen of hemocyanin carrier (EQ-1-KLH), and hemocyanin (KLH).

[0039] Figure 4 Ultraviolet scan images of ethoxyquinoline hapten (EQ-1), complete antigen of chicken ovalbumin carrier (EQ-1-OVA), and chicken ovalbumin (OVA).

[0040] Figure 5 The standard curve of the inhibition of ethoxyquinoline by the antibody prepared for the hapten (EQ-1). Detailed Implementation

[0041] To make the objectives, technical solutions, and advantages of this application clearer, the following detailed description is provided in conjunction with the accompanying drawings and embodiments. It should be understood that the specific embodiments described herein are merely illustrative and not intended to limit the scope of this application. Unless otherwise specified, the experimental methods used in the following embodiments are conventional methods; the materials and reagents used, unless otherwise specified, are commercially available.

[0042] Example 1: Preparation of ethoxyquinoline hapten I. Experimental Methods The synthetic route for ethoxyquinoline hapten is shown below. Figure 1 The specific synthesis method is as follows: 3.3 g of methyl 4-aminobenzoate and 730 mg of zinc trifluoromethanesulfonate were dissolved in 6 mL of anhydrous acetone and reacted at 70 °C for 12 h. The intermediate ester was obtained by column chromatography. The solvent was removed by concentration under reduced pressure, followed by hydrolysis and acidification. The product was extracted with dichloromethane and saturated brine, retaining the organic phase. The dichloromethane was removed by concentration under reduced pressure, and the product EQ-1 was obtained by column chromatography.

[0043] II. Experimental Results The obtained product was analyzed by mass spectrometry, and the results are shown in the figure. Figure 2 The results showed that the molecular weight was 217.8, which was consistent with the target analyte, and the compound (ethoxyquinoline hapten EQ-1) with the structural formula shown in formula (Ⅰ) was successfully prepared. .

[0044] Example 2 Preparation of ethoxyquinoline complete antigen I. Experimental Methods Complete antigens were prepared by coupling carrier proteins using the active ester method.

[0045] The specific steps are as follows: Dissolve 10.8 mg (0.05 mmol) ethoxyquinoline hapten EQ-1, 14.4 mg (0.075 mmol) EDC and 8.65 mg (0.075 mmol) NHS in 1 mL DMF, react overnight at 4℃, and take the supernatant as solution A.

[0046] Take 87.5 mg (0.00025 mmol) of the carrier protein keyhole hemocyanin (KLH) or 35.45 mg (0.0008 mmol) of chicken ovalbumin carrier protein (OVA), and dissolve them in 3 mL of CBS buffer. This solution is referred to as solution B.

[0047] Solution A was added dropwise to solution B, and the reaction was carried out at 4°C for 12 h. After the reaction, the supernatant was collected by centrifugation and dialyzed in PBS (0.01 M, pH 7.4) at 4°C for 3 days. Subsequently, the complete antigen of hemocyanin conjugate (EQ-1-KLH) and the complete antigen of ovalbumin conjugate (EQ-1-OVA) were diluted with PBS (0.01 M, pH 7.4) and stored at -20°C for later use.

[0048] II. Experimental Results The ethoxyquinoline hapten (EQ-1), the complete antigen conjugated with hemocyanin (EQ-1-KLH), the complete antigen conjugated with ovalbumin (EQ-1-OVA), hemocyanin (KLH), and ovalbumin (OVA) were measured by ultraviolet scanning (190~400 nm).

[0049] The results are as follows Figure 3 and4 As shown: The UV absorption peak of the complete antigen conjugated with hemocyanin (EQ-1-KLH) is significantly shifted compared to the UV absorption peaks of the ethoxyquinoline hapten and hemocyanin, indicating that the complete antigen was successfully prepared; the UV absorption peak of the complete antigen conjugated with chicken ovalbumin (EQ-1-OVA) is also significantly shifted compared to the UV absorption peaks of the ethoxyquinoline hapten and chicken ovalbumin, indicating that the complete antigen was successfully prepared. Their structural formulas are shown in formulas (II-1) and (II-2), respectively. ; .

[0050] Example 3 Preparation of ethoxyquinoline polyclonal antibody I. Experimental Methods In Example 2, the complete antigen of hemocyanin conjugated with formula (II-1) (EQ-1-KLH) prepared according to formula (II-1) was emulsified with an equal volume of immunoadjuvant (Frederick's complete adjuvant was used for the first immunization, and Frederick's incomplete adjuvant was used for subsequent booster immunizations) and mice were immunized. Balb / c mice aged 6-7 weeks were immunized via subcutaneous injection in the back and intraperitoneal injection at multiple sites. A booster immunization was performed 3 weeks later, followed by booster immunizations every 2 weeks. One week after the fourth booster immunization, blood was collected from the tail of the mice, and the serum titer and inhibition rate were determined using an indirect competitive ELISA.

[0051] Phosphate-buffered saline (PBS, 0.1 mol / L, pH 7.4) was used as the dilution and reaction buffer for all samples; EQ-1-OVA was used as the coating agent; 50 μL of PBS buffer or 1 mg / mL ethoxyquinoline standard solution and 50 μL of polyclonal antibody diluted appropriately were added to a 96-well microplate. After the competitive reaction was completed, enzyme-labeled secondary antibody was added, and after the colorimetric reaction, the absorbance value was measured by microplate reader.

[0052] II. Experimental Results The results showed that the titer of the prepared immunogen-based polyclonal antiserum was 1:8000, with an inhibition rate of 42%.

[0053] Example 4: A method for indirect enzyme-linked immunosorbent assay (ELISA) detection of ethoxyquinoline. I. Experimental Methods An indirect competitive ELISA standard curve was established using the antiserum prepared in Example 4, wherein EQ-1-OVA was used as the coating agent; carbonate buffer (CBS buffer) was used as the coating agent dilution solution; phosphate buffer solution (PBS, 0.1 mol / L, pH 7.4) was used as the dilution solution and reaction buffer for all samples; and PBST (100 mL 0.1 mol / L PBS, 500 μL Tween-20, 2 g BSA) was used as the blocking solution. The specific method is as follows: (1) Dilute the coating agent to 1 μg / mL with the coating agent diluent, add 100 μL / well to the well of the microplate, and incubate in a water bath at 37°C for 12 h.

[0054] (2) After washing the machine twice, pat dry, add 140 μL of blocking solution to each well, incubate at 37°C for 3 h, shake off the blocking solution and pat dry, place in a 37°C oven for 30 min, and store at 4°C for later use.

[0055] (3) Add 50 μL of a series of concentrations (100, 200, 400, 600, 800, 1000, 1200, 1400, 1600, 1800, 2000 ng / mL) of ethoxyquinoline standard solution to the prepared coated plate.

[0056] (4) Add 50 μL of antibody diluted 1:8000 to each well, incubate at 37°C for 30 min, wash 5 times with washing buffer, and pat dry on absorbent paper; (5) Dilute goat anti-mouse IgG-HRP 5000 times with PBS, add 100 μL to each well, and incubate at 37°C for 30 min. Wash the plate 5 times and then pat dry.

[0057] (6) Mix the TMB colorimetric solution according to the ratio, add 100 μL to each well, and incubate at 37℃ for 10 min.

[0058] (7) Add 50 μL of stop solution to each well to terminate the reaction.

[0059] (8) The absorbance (OD) of each well was measured at a wavelength of 450 nm using an ELISA reader. The antiserum dilution factor corresponding to an absorbance value of around 1.0 is the antiserum titer. The inhibition rate was calculated as follows:

[0060] Note: B0 is valence orifice A. 450nm Value, B is the suppression pore A 450nm value.

[0061] (9) Using the B / B0 value as the ordinate and the corresponding logarithmic concentration of the standard as the abscissa, curve fitting was performed using the four-parameter logarithmic function of Origin 8.5 software. The IC50 value was calculated from the curve. 50The half-maximal inhibitory concentration (IC50) is the concentration at which the concentration of water is reduced to half-maximal inhibitory concentration (IC50). 10 To determine the detection limit, using IC 20 ~IC 80 This refers to the detection range.

[0062] (10) Cross-reactivity was evaluated using an ic-ELISA assay for ethoxyquinoline to detect seven antioxidants and three structural analogs that may be used in combination with ethoxyquinoline. Standard curves were plotted, and the IC50 values ​​of each analog were calculated. 50 And calculate the cross-reactivity rate.

[0063] II. Experimental Results The results are as follows Figure 5 As shown in Table 1, the standard curve exhibits a typical S-shaped curve, with a correlation coefficient of 0.996 and a detection limit IC50. 10 The concentration was 0.6 μg / mL, and the IC50 was [value missing]. 50 The concentration was 0.96 μg / mL, with a linear range of IC50. 20 ~IC 80 The concentration ranges from 0.72 μg / mL to 1.28 μg / mL.

[0064] Table 1. Detection efficacy of antiserum against ethoxyquinoline

[0065] Example 5: Specificity of the indirect enzyme-linked immunosorbent assay (ELISA) for detecting ethoxyquinoline. I. Experimental Methods The method of Example 4 was used to detect ethoxyquinoline structural analogs (butylated hydroxyanisole, 2,6-di-tert-butyl-p-cresol, 2,5-di-tert-butylhydroquinone, epigallocatechin gallate, 2,4,5-trihydroxyphenylbutanone, vitamin E, D-isoascorbic acid, quinclorac, dichloroquinoline acid, and 8-hydroxyquinoline copper), where the cross-reactivity (CR%) = (IC50 of structural analogs) 50 / ethoxyquinoline IC 50 ) × 100% II. Experimental Results The cross-reactivity results are shown in Table 2, indicating that the method has high specificity and no cross-reactivity with other similar structures. Therefore, this method can be used to detect the content of ethoxyquinoline in food.

[0066] Table 2. Cross-reactivity rates of ethoxyquinoline with structural analogs in ic-ELISA

[0067] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention and are not intended to limit the scope of protection of the present invention. For those skilled in the art, other variations or modifications can be made based on the above description and ideas, and it is neither necessary nor possible to exhaustively describe all implementation methods here. Any modifications, equivalent substitutions, and improvements made within the spirit and principles of the present invention should be included within the scope of protection of the claims of the present invention.

Claims

1. An ethoxyquinoline hapten, characterized in that, Its structural formula is shown in formula (I). 。 2. The application of the ethoxyquinoline hapten according to claim 1, characterized in that, The application is one or more of the following: Preparation of ethoxyquinoline complete antigen; Establish a non-diagnostic method for detecting ethoxyquinoline; Prepare an ethoxyquinoline immunoassay kit.

3. An ethoxyquinoline complete antigen, characterized in that, It is the ethoxyquinoline hapten of claim 1 coupled with a carrier protein, and its structural formula is shown in formula (II). 。 4. The application of the ethoxyquinoline complete antigen according to claim 3, characterized in that, The application is one or both of the following: Establish a non-diagnostic method for detecting ethoxyquinoline; Prepare an ethoxyquinoline immunoassay kit.

5. A composition of an immunogen and a coating antigen, characterized in that, Both the immunogen and the coating antigen are ethoxyquinoline haptens as described in claim 1, coupled with a carrier protein, the structural formula of which is shown in formula (II). ； The carrier protein of the coating antigen is chicken ovalbumin, and the carrier protein of the immunogen is hemocyanin.

6. A composition of an antibody and a coating antigen, characterized in that, The antibody was prepared by immunizing animals with an immunogen. Both the immunogen and the coating antigen are ethoxyquinoline haptens as described in claim 1, coupled with a carrier protein, the structural formula of which is shown in formula (II). ； The carrier protein of the coating antigen is chicken ovalbumin, and the carrier protein of the immunogen is hemocyanin.

7. The use of the composition according to claim 5 or 6, characterized in that, The application is one or more of the following: Establish a non-diagnostic method for detecting ethoxyquinoline; Prepare an ethoxyquinoline immunoassay kit.

8. A non-diagnostic immunoassay method for detecting ethoxyquinoline, characterized in that, The composition according to claim 5 or 6 is used.

9. The immunoassay method for detecting ethoxyquinoline according to claim 8, characterized in that, The immunoassay method for detecting ethoxyquinoline is an indirect enzyme-linked immunosorbent assay (ELISA).

10. An immunoassay kit for ethoxyquinoline, characterized in that, Contains the composition of claim 5 or 6.

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