Maltol hapten, antigen, antibody and preparation method and application thereof

Abstract

This invention discloses a maltol hapten, antigen, antibody, and their preparation methods and applications, relating to the maltol hapten, antigen, antibody, their preparation methods, and their application in detecting maltol compound residues in food. This invention prepares a maltol hapten, a maltol coating antigen, and an immunoassay antigen, and prepares a maltol monoclonal antibody, constructing an enzyme-linked immunosorbent assay (ELISA) method for directly detecting maltol compounds in food, including the IC50 assay for ethyl maltol. 50 The concentration reached 5.01 ng / mL, with a linear range of 1.47 ng / mL–16.96 ng / mL. The IC50 of p-methylmaltol was [not specified]. 50 The concentration reached 4.15 ng / mL; the cross-reactivity with other maltol structural analogs was very low, all less than 0.02%. This method has the advantages of high detection sensitivity, high specificity and high throughput, and establishes an immunoassay method for the rapid and accurate detection of ethyl maltol and methyl maltol.

Description

Technical Field

[0001] This invention relates to the field of food safety testing technology, and more specifically, to a maltol hapten, antigen, antibody, and their preparation methods and applications. Background Technology

[0002] Maltol, including ethyl maltol and methyl maltol, is widely used in the food industry as a highly effective flavor modifier and aroma enhancer. When using maltol as a food additive, relevant regulations and standards must be strictly followed to ensure its use within safe limits and to protect public health.

[0003] Currently, there are few methods for detecting maltol. Traditional methods include high-performance liquid chromatography (HPLC), liquid chromatography-mass spectrometry (LC-MS / MS), and gas chromatography-mass spectrometry (GC-MS / MS). While instrumental detection techniques offer high accuracy and good repeatability, they require expensive equipment, complex sample pretreatment, and long detection times, limiting their application in on-site testing. Enzyme-linked immunosorbent assay (ELISA), utilizing the specific binding ability of antigens and antibodies, offers advantages such as high sensitivity, speed, and high throughput, and is widely used in the detection of maltol residues.

[0004] There are few reports on the preparation of monoclonal antibodies against maltol, mainly because maltol has many similar structures, which is not conducive to the preparation of highly selective monoclonal antibodies. Therefore, there is an urgent need to prepare a specific hapten structure against ethyl maltol and methyl maltol to prepare high-quality monoclonal antibodies for the sensitive, rapid, and convenient detection of maltol compounds. Summary of the Invention

[0005] The purpose of this invention is to provide a maltol hapten, antigen, antibody, and their preparation methods and applications.

[0006] According to one aspect of the present invention, a maltol hapten is provided, the structure of which is shown in formula (I):

[0007]

[0008] According to another aspect of the present invention, a method for preparing maltol hapten is provided, comprising the following steps:

[0009] S1,4-(2-furanyl)-1-buten-4-ol was dissolved in anhydrous tetrahydrofuran, and a borane tetrahydrofuran complex solution was added dropwise. The mixture was stirred at room temperature, and sodium hydroxide solution and H2O2 solution were added. The mixture was stirred overnight. After the reaction was completed, the tetrahydrofuran was evaporated under reduced pressure, and the mixture was extracted with ethyl acetate, washed with saturated brine, dried, and purified by column chromatography to obtain intermediate 1. The reaction formula for this step is shown in formula (II):

[0010]

[0011] S2. Intermediate 1 was dissolved in a mixture of methanol and water, and reacted after passing chlorine gas through it. The mixture was then refluxed, and the pH was adjusted to slightly acidic using a 10% NaOH solution. The mixture was extracted with ethyl acetate, and the combined ethyl acetate phases were evaporated to dryness and purified by column chromatography to obtain intermediate 2. The reaction formula for this step is shown in formula (Ⅲ).

[0012]

[0013] S3. Take intermediate 2, add succinic anhydride, add pyridine to dissolve, and heat the reaction in an oil bath under nitrogen protection. After the reaction is complete, directly evaporate the organic phase to dryness, purify by column chromatography, and obtain maltol hapten. The reaction formula for this step is shown in formula (Ⅳ):

[0014]

[0015] In some embodiments, the molar concentration of the boranetetrahydrofuran complex solution in step S1 is 1 mol / L, the molar concentration of the sodium hydroxide solution in step S1 is 2 mol / L, and the concentration of the H2O2 solution is 30%.

[0016] In some embodiments, the volume ratio of methanol to water in the methanol-water mixture in step S2 is 1:1.

[0017] According to another aspect of the present invention, a conjugate of maltol antigen as a maltol hapten and a carrier protein is provided, the structural formula of which is shown in formula (V):

[0018]

[0019] In some embodiments, the carrier protein is any one of lactoferrin, bovine serum albumin, ovalbumin, or hemocyanin.

[0020] According to a fourth aspect of the present invention, the application of maltol hapten and maltol antigen in the immunological detection of maltol is provided.

[0021] According to a fifth aspect of the present invention, a maltol antibody is provided, which is prepared by animal immunization with maltol antigen, and the maltol antibody is a maltol monoclonal antibody.

[0022] According to a sixth aspect of the present invention, an immunoassay kit for detecting maltol compounds is provided, comprising maltol antigen and maltol antibody as coating agents.

[0023] According to a seventh aspect of the present invention, a method for detecting maltol compounds is provided, the method comprising using maltol antigens and maltol antibodies to detect maltol compound residues in food, the maltol compounds including methyl maltol and ethyl maltol.

[0024] The beneficial effects of this invention are as follows: This invention prepares a maltol hapten, and uses the maltol hapten to conjugate a carrier protein to obtain maltol coating antigen and maltol immunoassay antigen. A specific monoclonal antibody for the direct detection of maltol compounds is also prepared, which exhibits good sensitivity and high specificity for maltol compounds. This antibody is used to construct an enzyme-linked immunosorbent assay (ELISA) method for the direct detection of methylmaltol and ethylmaltol in food. The IC50 value for ethylmaltol is [not specified in the original text]. 50 The concentration reached 5.01 ng / mL, with a linear range of 1.47 ng / mL–16.96 ng / mL. The IC50 of p-methylmaltol was [not specified]. 50 The concentration reached 4.15 ng / mL; the cross-reactivity with other maltol structural analogs was very low, all less than 0.02%, which can effectively eliminate the interference of other analogs. This method has the advantages of high detection sensitivity (detection limit can reach ppb level), high specificity, and high detection throughput, and has broad application prospects. It has established an immunoassay method for the rapid and accurate detection of ethyl maltol and methyl maltol. Attached Figure Description

[0025] Figure 1 This is a mass spectrum of a maltol hapten according to one embodiment of the present invention.

[0026] Figure 2 This is an ultraviolet scan of maltol immunogenic antigen and its hapten, lactoferrin, according to one embodiment of the present invention.

[0027] Figure 3 This is an ultraviolet scan of maltol-coated antigen and its hapten, and bovine serum albumin, according to one embodiment of the present invention.

[0028] Figure 4 This is a standard curve of an indirect competitive ELISA based on a maltol monoclonal antibody, representing one embodiment of the present invention.

[0029] Figure 5 This is a synthetic route diagram of a maltol hapten according to one embodiment of the present invention. Detailed Implementation

[0030] The present invention is further described in detail through specific implementation examples. It should be understood that these embodiments are for illustrative purposes only and are not intended to limit the scope of protection of the present invention. After reading this invention, any modifications of the present invention in various equivalent forms by those skilled in the art fall within the scope defined by the appended claims. Unless otherwise specified, all raw materials and reagents of the present invention are commercially available.

[0031] Example 1: Preparation of maltol hapten

[0032] The preparation method of maltol hapten includes the following steps:

[0033] S1. Add 5g of 4-(2-furanyl)-1-buten-4-ol and 30mL of anhydrous tetrahydrofuran to a 250mL reaction flask. Then slowly add 50mL of 1M borane tetrahydrofuran complex solution to the reaction flask. Stir at room temperature for 2h. Then add 30mL of 2M sodium hydroxide solution and 12mL of 30% H2O2 solution. Stir overnight. After the reaction is completed, evaporate the tetrahydrofuran under reduced pressure. Extract with ethyl acetate, wash with saturated brine, dry with anhydrous sodium sulfate, and purify by column chromatography to obtain 3.5g of intermediate 1.

[0034] S2. Take a 100 mL reaction flask, add 2.5 g of intermediate 1, add 30 mL of a mixture of methanol and water (where the volume ratio of methanol to water is 1:1), dissolve intermediate 1, pass chlorine gas through at -20 °C for 30 min, react for 3 h, reflux at 100 °C for 3.5 h, adjust the pH of the system to slightly acidic with 10% NaOH solution, extract with ethyl acetate multiple times, combine the ethyl acetate phases, evaporate to dryness, and purify by column chromatography to obtain 1.2 g of intermediate 2;

[0035] S3. Take a 100 mL reaction flask, add 0.5 g of intermediate 2, 0.5 g of succinic anhydride, and 10 mL of pyridine to dissolve intermediate 2 and succinic anhydride. Then, under nitrogen protection, heat the mixture in an oil bath at 40 °C for 3 h. After the reaction, evaporate the organic phase directly to dryness and purify by column chromatography to obtain 0.67 g of maltol hapten, with a yield of 84.8%. Its structural formula is shown in formula (I). For the specific preparation and synthesis route, see [link to specific preparation route]. Figure 5 .

[0036] The maltol hapten was identified by mass spectrometry, and the obtained mass spectrum is shown in the figure. Figure 1 .from Figure 1 It can be seen that the molecular ion peak of the maltol hapten is ESI-[M+H]. + 271.21 and [M+Na] + The value was 293.17, which was the highest value, and it was consistent with the molecular weight of 270.24 of the maltol hapten, indicating that the maltol hapten shown in formula (Ⅰ) was successfully synthesized.

[0037] Example 2: Preparation of maltol-based immunomodulatory antigens and coating antigens

[0038] 2.1 Preparation of maltol-based immunogenic antigens

[0039] 21.4 mg of the maltol hapten prepared in Example 1 was dissolved in 600 μL of 0.1 M MES solution. 390 μL of 100 mg / mL 1-ethyl-(3-dimethylaminopropyl)carbodiimide (EDC) solution and 460 μL of 100 mg / mL N-hydroxysuccinimide (NHS) solution were added with stirring. The mixture was stirred at room temperature in the dark for 4 h to obtain the hapten activation solution. 20 mg of lactoferrin (LF) was dissolved in 5 mL of CBS buffer solution with a pH of 9.6 (1.69 g of Na2CO3 and 2.95 g of NaHCO3 were diluted to 1 L with primary water). 290 μL of the hapten activation solution was added with stirring in an ice bath. After stirring evenly, the mixture was coupled at 4 °C in the dark for 2 h. The conjugated mixture was dialyzed against 0.01M PBS buffer at 4°C for 3 days, with the dialysate changed twice daily, to obtain maltol hapten-LF, an antigen for immunotherapy. The maltol hapten-LF was aliquoted at a concentration of 1 mg / mL and stored at -20°C.

[0040] Identification: Ultraviolet scanning was performed to determine the carrier protein (LF), maltol hapten, and maltol immunoassay antigen (maltol hapten-LF). The results are as follows: Figure 2 As shown, the UV absorption peak of the maltol immunogen shows a significant blue shift compared to that of the maltol hapten, and the maltol immunogen also possesses the characteristic absorption peaks of both the maltol hapten and LF, indicating that the maltol immunogen was successfully prepared by conjugation.

[0041] 2.2 Preparation of maltol-coated antigens

[0042] 21.4 mg of the maltol hapten prepared in Example 1 was dissolved in 600 μL of 0.1 M MES solution. 390 μL of 100 mg / mL 1-ethyl-(3-dimethylaminopropyl)carbodiimide (EDC) solution and 460 μL of 100 mg / mL N-hydroxysuccinimide (NHS) solution were added with stirring. The mixture was stirred at room temperature in the dark for 4 h to obtain the hapten activation solution. 20 mg of bovine serum albumin (BSA) was dissolved in 5 mL of pH 9.6 CBS buffer solution (1.69 g Na₂CO₃ and 2.95 g NaHCO₃ diluted to 1 L with primary water). 290 μL of the hapten activation solution was added with stirring in an ice bath. After thorough mixing, the mixture was coupled at 4 °C in the dark for 2 h. The coupling mixture was then incubated at 4 °C with 0.01 M M M acetic acid solution. Dialysis with PBS buffer for 3 days, changing the dialysate twice daily, yielded the maltol-coated antigen, i.e., maltol hapten-BSA. The maltol-coated antigen was aliquoted at a concentration of 1 mg / mL and stored at -20°C.

[0043] Identification: Ultraviolet scanning was performed on the carrier protein (BSA), maltol hapten, and maltol-coated antigen. The results are as follows: Figure 3 As shown, the UV absorption peak of the maltol-coated antigen shows a significant blue shift compared to that of the maltol hapten, and the maltol-coated antigen possesses the characteristic absorption peaks of both the maltol hapten and BSA, indicating that the maltol-coated antigen was successfully conjugated.

[0044] Example 3: Preparation and Identification of Maltol Antibodies

[0045] 3.1 Preparation of maltol antibodies

[0046] 1. Antibody preparation

[0047] The maltol-based immunizing antigen prepared in Example 2 was emulsified with an equal volume of immunizing adjuvant (French complete adjuvant for the first immunization, and Freund's incomplete adjuvant for subsequent booster immunizations) and then used to immunize mice. Balb / C mice aged 6-7 weeks were immunized via various injection methods, including subcutaneous injection in the back, subcutaneous injection at various sites, intraperitoneal injection, and paw injection. A second immunization was administered 2 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 serum titers were determined using an indirect competitive ELISA. When the titer no longer increased, a booster immunization was administered via intraperitoneal injection. Three days later, blood was collected from the heart, incubated in water for 0.5-1 hour, centrifuged at 4°C and 10,000 rpm for 15 minutes, and the supernatant was collected as antiserum, which is the polyclonal antibody.

[0048] Spleen cells were fused with SP2 / 0 myeloma cells using PEG. After subcloning and four limiting dilutions, a portion of the positive hybridoma was cryopreserved, while the other portion was injected into the peritoneal cavity of mice to generate ascites. The ascites was collected seven days later and purified using a protein G immunoaffinity column to yield the monoclonal antibody.

[0049] 3.2 Identification of Antibodies

[0050] Carbonate buffer (CBS, pH 9.6) was used as the diluent for the maltol-coating antigen, phosphate buffer (PBS, 0.01 M, pH 7.4) was used as the diluent for the antiserum and standards, and Tween phosphate buffer (PBST, 0.01 M) was used as the diluent for the horseradish peroxidase-labeled goat anti-mouse solution. The maltol-coating antigen was diluted to 1 μg / mL and added to each well of a 96-well ELISA plate (100 μL per well), and incubated overnight at 4°C. After washing twice with PBST, 120 μL of 5% bovine serum albumin was added to each well, and the plate was incubated at 37°C for 3 h, centrifuged, and then dried at 37°C for 1 h. The antiserum was diluted 1000, 2000, 4000, 8000, 16000, 32000, and 64000 times, respectively, and 1 μg / mL maltol standards were prepared. The above solution was added to a 96-well plate coated with maltol-coated antigen using the standard indirect competitive ELISA method. The plate was incubated at 37°C for 40 min, washed 5 times with PBST, and then 100 μL of horseradish peroxidase-labeled goat anti-mouse solution (diluted 5000 times) was added. The plate was incubated at 37°C for 30 min, washed 5 times with PBST, and then 100 μL of 3,3',5,5'-tetramethylbenzidine (TMB) substrate chromogenic solution was added. The plate was incubated again at 37°C for 10 min, and the reaction was terminated with 10% concentrated sulfuric acid. Data were read using a microplate reader. The results of the indirect competitive ELISA assay are shown in Table 1.

[0051] The antiserum titer is determined by selecting an antiserum dilution factor with an absorbance (OD) of 1–1.5 at a wavelength of 450 nm. The inhibition rate is calculated using the following formula.

[0052] Inhibition rate = 100 - (OD inhibition / OD titer) * 100

[0053] Table 1. Results of Indirect Competitive ELISA Measurement

[0054] antiserum Envelope valence Inhibition rate Maltol hapten-LF Maltol Hapten-BSA 1:64000 84.36％

[0055] Based on the combined titer and inhibition rate of the antiserum, the optimal combination of artificial antigens was selected. As shown in Table 1, the highest antibody titer was 64,000, with a corresponding inhibition rate of 84.36%, indicating high inhibition and high titer. Therefore, artificial antigens with lactoferrin as the carrier protein were used as maltol hapten-LF for immunoassay, and artificial antigens with bovine serum albumin as the carrier protein were used as maltol coating antigens (maltol hapten-BSA) for immunoassay.

[0056] Example 4: Coating concentration of maltol-based antigen and antibody dilution factor

[0057] Carbonate buffer (CBS, pH 9.6) was used as the dilution buffer for the maltol coating antigen, phosphate buffer (PBS, 0.01 M, pH 7.4) was used as the dilution buffer for the maltol polyclonal antibody and standards, and Tween phosphate buffer (PBST, 0.01 M) was used as the dilution buffer for the horseradish peroxidase-labeled goat anti-mouse solution. The maltol coating antigen was diluted to concentrations of 2, 1, 0.5, 0.25, 0.125, and 0.0625 μg / mL, respectively, and 100 μL was added to each well of a 96-well ELISA plate. The plates were incubated overnight at 4°C. After washing twice with PBST, 120 μL of 5% bovine serum albumin was added to each well, and the plates were incubated at 37°C for 3 hours. After spin-drying, the plates were dried at 37°C for 1 hour. Maltol polyclonal antibodies were diluted 1000, 2000, 4000, 8000, 16000, 32000, and 64000 times, respectively, with 1 μg / mL maltol standard. The above solutions were added to 96-well plates pre-coated with the corresponding antigens using a standard indirect competitive ELISA method. The plates were incubated at 37°C for 40 min, washed 5 times with PBST, and then 100 μL of horseradish peroxidase-labeled goat anti-mouse solution (5000-fold diluted) was added. The plates were incubated at 37°C for 30 min, washed 5 times with PBST, and then 100 μL of 3,3',5,5'-tetramethylbenzidine (TMB) substrate chromogenic solution was added. The plates were incubated again at 37°C for 10 min, and the reaction was terminated with 10% concentrated sulfuric acid. The absorbance (OD) was measured using an enzyme-linked immunosorbent assay (ELISA) analyzer. The inhibition rate was calculated by selecting the coating concentration and antibody dilution factor at an absorbance of 1 to 1.5 at a wavelength of 450 nm. The detection results are shown in Table 2.

[0058] Table 2. Results of the chessboard detection method

[0059] OD value Coating concentration antibody dilution factor Inhibition rate 1.430 2μg / mL 1:8000 79.94％ 1.293 1μg / mL 1:4000 80.59％ 1.411 0.5 μg / mL 1:1000 80.3％

[0060] As shown in Table 2, the optimal coating concentration of maltol-based antigen is 1 μg / mL, and the dilution factor of maltol polyclonal antibody is 4000-fold.

[0061] Example 5: Sensitivity evaluation of maltol monoclonal antibodies

[0062] Based on the optimal coating concentration of Example 4, an enzyme-linked immunosorbent assay (ELISA) standard curve was plotted using the maltol monoclonal antibody prepared in Example 3.

[0063] Carbonate buffer (CBS, pH 9.6) was used as the dilution buffer for the maltol coating antigen, phosphate buffer (PBS, 0.01 M, pH 7.4) was used as the dilution buffer for the maltol monoclonal antibody and standards, and Tween phosphate buffer (PBST, 0.01 M) was used as the dilution buffer for the horseradish peroxidase-labeled goat anti-mouse solution. The maltol coating antigen was diluted to 1 μg / mL and added to each well of a 96-well ELISA plate (100 μL per well), and incubated overnight at 4°C. After washing twice with PBST, 120 μL of 5% bovine serum albumin was added to each well, and the plate was incubated at 37°C for 3 hours. After spin drying, the plate was dried at 37°C for 1 hour. Maltol standard (ethyl maltol) was prepared into solutions with concentrations of 1000, 100, 10, 1, 0.1, 0.01, and 0.001 μg / mL. These solutions, along with maltol monoclonal antibodies, were then added to 96-well plates coated with maltol-coated antigen using a standard indirect competitive ELISA method. The plates were incubated at 37°C for 40 min, washed 5 times with PBST, and then 100 μL of horseradish peroxidase-labeled goat anti-mouse solution (diluted 5000 times) was added. The plates were incubated at 37°C for 30 min, washed 5 times with PBST, and then 100 μL of 3,3',5,5'-tetramethylbenzidine (TMB) substrate chromogenic solution was added. The plates were incubated again at 37°C for 10 min, and the reaction was terminated with 10% concentrated sulfuric acid. The absorbance (OD) was measured using an ELISA reader.

[0064] Plotting the OD value on the ordinate and the corresponding logarithmic concentration of the standard on the abscissa, a curve was fitted to the function using four parameters in Origin 8.5 software: y = (AD) / [1 + (X / C)B] + D, where A and D represent the minimum and maximum absorbance (OD) values ​​of the drug concentration, respectively, C is the midpoint concentration, the OD value when the standard concentration equals C is (A + D) / 2, and the half-maximum inhibitory concentration (IC50) at the inflection point of the curve is 1 / 2. 50 B represents the steepness of the curve, called the slope factor: denoted by IC. 10 To determine the detection limit, using IC 20 ~IC 80 To define the detection range, a standard curve for ELISA was established using ethyl maltol as the standard. The standard curve is shown below. Figure 4 As shown, the correlation coefficients and parameters are shown in Table 3.

[0065] Table 3. Detection parameters for ethyl maltol by maltol monoclonal antibodies.

[0066] <![CDATA[IC 50 (ng / mL)]]> Linear range (ng / mL) Correlation coefficient Maltol monoclonal antibodies 5.01 1.47-16.96 0.99928

[0067] Combination Figure 4 As shown in Table 3, the standard curve established using ethyl maltol standard exhibits a typical S-shaped curve, demonstrating good detection sensitivity and IC50. 50 The concentration reached 5.01 ng / mL, with a linear range of 1.47 ng / mL to 16.96 ng / mL.

[0068] Example 6: Specificity evaluation of maltol monoclonal antibodies

[0069] Using the maltol monoclonal antibody prepared in Example 3, enzyme-linked immunosorbent assay (ELISA) was performed on methylmaltol, ethylmaltol, and common additives. The corresponding IC50 values ​​were obtained by fitting the antibody. 50 The cross-reactivity rate was calculated, and the results are shown in Table 4.

[0070] Table 4. Cross-reactivity rates of maltol monoclonal antibodies to common additives.

[0071]

[0072] As shown in Table 4, the cross-reactivity of the maltol monoclonal antibody with maltol analogs and common additives is less than 0.02%, indicating that the maltol monoclonal antibody prepared in this invention has strong specificity for the detection of maltol compounds.

[0073] Example 7: An immunoassay kit for detecting maltol compounds

[0074] An immunoassay kit for detecting maltol compounds includes a 96-well transparent polystyrene microplate coated with the maltol coating antigen (maltol hapten-BSA) prepared in Example 2, maltol standards, maltol monoclonal antibody prepared in Example 4, horseradish peroxidase-labeled goat anti-mouse solution, 3,3',5,5'-tetramethylbenzidine (TMB) substrate chromogenic solution, 10% concentrated sulfuric acid stop solution, concentrated dilution solution, and concentrated washing solution.

[0075] Maltol compounds were extracted and concentrated from the test samples to prepare the test solution. Then, maltol standards and maltol monoclonal antibodies were added to 96-well transparent polystyrene ELISA plates coated with the antigen. The plates were incubated at 37°C for 40 min, washed five times with PBST, and then 100 μL of horseradish peroxidase-labeled goat anti-mouse solution (diluted 5000 times) was added. The plates were incubated at 37°C for 30 min, washed five times with PBST, and then 100 μL of 3,3',5,5'-tetramethylbenzidine (TMB) substrate chromogenic solution was added. The plates were incubated again at 37°C for 10 min, and the reaction was terminated with 10% concentrated sulfuric acid. The absorbance was measured using an ELISA reader. The content of maltol compounds in the samples was quantitatively analyzed by comparing the absorbance values ​​of the test solution and the maltol standards.

[0076] Example 8: Accuracy test of the immunoassay kit for detecting maltol compounds.

[0077] Forty-one blank oil samples were collected (200 each of sesame oil and edible vegetable oil). First, oil samples were collected randomly and representatively to ensure they accurately reflected the overall quality of the oils. (For solid or semi-solid oil samples, pretreatment such as peeling and impurity removal was necessary to ensure smooth subsequent steps.) Ethyl maltol standard was added to 100 blank sesame oil samples and 100 blank edible vegetable oil samples to a final concentration of 5 μg / L. All oil samples were dissolved in n-hexane, heated, shaken, and mixed thoroughly. The oil was then extracted at room temperature. After extraction, impurities were removed by centrifugation at 4000 rpm for 2 minutes, and the samples were properly stored under suitable conditions to ensure the representativeness of the oil samples and the accuracy of the detection. The immunoassay kit for detecting maltol compounds prepared in Example 7 was used, and each sample was measured three times. The results are shown in Table 5.

[0078] Table 5. Detection results of maltol compounds using the immunoassay kit.

[0079]

[0080] As shown in Table 5, the immunoassay kit and rapid detection method for detecting maltol compounds of the present invention can accurately detect 5 μg / L of maltol compounds in edible oils with very low false positive and false negative rates, ensuring high accuracy of the detection results.

[0081] Example 9: Crossover assay of an immunoassay kit for maltol compounds

[0082] Six blank oil samples were taken, and appropriate amounts of ethyl maltol standard solution, methyl maltol standard solution, cinnamaldehyde standard solution, vanillin standard solution, methyl benzoate standard solution, and isomaltol standard solution were added to prepare six test samples with a final concentration of 5 μg / L. The immunoassay kit for detecting maltol compounds prepared in Example 7 was used, and each test sample was measured three times. The test results are shown in Table 6.

[0083] Table 6. Results of Cross-Reaction Experiment

[0084]

[0085] Note: "+" indicates positive, and "-" indicates negative.

[0086] As shown in Table 6, only the oil samples containing ethyl maltol and methyl maltol tested positive, while the blank oil samples containing cinnamaldehyde, vanillin, methyl benzoate and isomaltol did not test positive. This indicates that the immunoassay kit for maltol compounds prepared in this invention has good specificity for maltol compounds and a low cross-reactivity rate.

[0087] The above descriptions are merely some embodiments of the present invention. For those skilled in the art, various modifications and improvements can be made without departing from the inventive concept of the present invention, and these all fall within the protection scope of the present invention.

Claims

1. A maltol hapten, characterized in that, Its structure is shown in equation (Ⅰ):

2. The method for preparing the maltol hapten according to claim 1, characterized in that, Includes the following steps: S1,4-(2-furanyl)-1-buten-4-ol was dissolved in anhydrous tetrahydrofuran, and a borane tetrahydrofuran complex solution was added dropwise. The mixture was stirred at room temperature, and sodium hydroxide solution and H2O2 solution were added. The mixture was stirred overnight. After the reaction was completed, the tetrahydrofuran was evaporated under reduced pressure, and the mixture was extracted with ethyl acetate, washed with saturated brine, dried, and purified by column chromatography to obtain intermediate 1. The reaction formula for this step is shown in formula (II): S2. Dissolve intermediate 1 in a mixture of methanol and water, pass chlorine gas through it, reflux, adjust the pH to slightly acidic with 10% NaOH solution, extract with ethyl acetate, combine the ethyl acetate phases, evaporate to dryness, and purify by column chromatography to obtain intermediate 2. The reaction formula for this step is shown in formula (Ⅲ): S3. Take intermediate 2, add succinic anhydride, add pyridine to dissolve, and heat the reaction in an oil bath under nitrogen protection. After the reaction is complete, directly evaporate the organic phase to dryness, purify by column chromatography, and obtain maltol hapten. The reaction formula for this step is shown in formula (Ⅳ):

3. The method according to claim 2, characterized in that, In step S1, the molar concentration of the boranetetrahydrofuran complex solution is 1 mol / L, the molar concentration of the sodium hydroxide solution is 2 mol / L, and the concentration of the H2O2 solution is 30%.

4. The method according to claim 2, characterized in that, In step S2, the volume ratio of methanol to water in the methanol-water mixture is 1:

1.

5. Maltol antigen, characterized in that, The maltol antigen is a conjugate of the maltol hapten and carrier protein as described in claim 1, and its structural formula is shown in formula (V):

6. The maltol antigen according to claim 5, characterized in that, The carrier protein is any one of lactoferrin, bovine serum albumin, ovalbumin, or hemocyanin.

7. The application of the maltol hapten of claim 1 and the maltol antigen of claim 5 in the immunological detection of maltol.

8. Maltol antibodies, characterized in that, The maltol antibody is prepared by animal immunization with the maltol antigen described in claim 5, and the maltol antibody is a maltol monoclonal antibody.

9. An immunoassay kit for detecting maltol compounds, characterized in that, This includes the maltol antigen of claim 5 and the maltol antibody of claim 8, which serve as coating agents.

10. A method for detecting maltol compounds, characterized in that, The method involves using the maltol antigen of claim 5 and the maltol antibody of claim 8 to detect maltol compound residues in food, wherein the maltol compounds include methyl maltol and ethyl maltol.

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