Maltol hapten, antigen and antibody as well as preparation method and application of maltol hapten, antigen and antibody

Abstract

The invention discloses a maltol hapten, an antigen, an antibody as well as a preparation method and application thereof, and relates to the maltol hapten, the antigen, the antibody as well as the preparation method thereof and the application of detecting residues of maltol compounds in food. According to the invention, a maltol hapten, a maltol coating antigen and an immunizing antigen are prepared, a maltol monoclonal antibody is prepared, an enzyme-linked immunosorbent assay method for directly detecting maltol compounds in food is constructed, the IC50 of ethyl maltol reaches 5.01 ng / mL, the linear range is 1.47 ng / mL to 16.96 ng / mL, and the IC50 of methyl maltol reaches 4.15 ng / mL; compared with the prior art, the method has the advantages that the detection sensitivity is high, the specificity is high, the detection flux is high and the like, and an immunodetection method is established for rapid and accurate detection of ethyl maltol and methyl maltol.

Description

Technical Field

[0001] The present invention relates to the technical field of food safety detection, and more specifically, to a maltol hapten, antigen, antibody, and a preparation method and application thereof. Background Art

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

[0003] At present, there are few methods for detecting maltol. Traditional detection methods include high performance liquid chromatography (HPLC), liquid chromatography and mass spectrometry (LC-MS / MS), gas chromatography and mass spectrometry (GC-MS / MS), etc. Although instrument detection technology has high accuracy and good repeatability, the required instruments are expensive, sample pretreatment is complicated, and the detection time is long, which has limitations in on-site detection. Enzyme-linked immunosorbent assay technology utilizes the specific binding ability of antigen and antibody, and has the advantages of high sensitivity, rapidity, 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, it is urgent to prepare a specific hapten structure for ethyl maltol and methyl maltol, and to prepare high-quality monoclonal antibodies for sensitive, rapid and simple detection of maltol compounds. Summary of the invention

[0005] The purpose of the present invention is to provide a maltol hapten, antigen, antibody and a preparation method and application thereof.

[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, there is provided a method for preparing a maltol hapten, comprising the following steps:

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

[0010]

[0011] S2. The intermediate 1 is dissolved in a mixture of methanol and water, chlorine is introduced into the mixture, the mixture is refluxed, the pH value is adjusted to be slightly acidic, the mixture is extracted with ethyl acetate, the ethyl acetate phases are combined and evaporated to dryness, and the mixture is purified by column to obtain the intermediate 2. The reaction formula of this step is shown in formula (III):

[0012]

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

[0014]

[0015] In some embodiments, the molar concentration of the borane tetrahydrofuran 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 mixture of methanol and water in step S2 is 1:1, and the concentration of the sodium hydroxide solution in step S2 is 10%.

[0017] According to another aspect of the present invention, there is provided a maltol antigen which is a conjugate of a maltol hapten and a carrier protein, and its structural formula 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, there is provided the use of maltol haptens and maltol antigens in immunological detection of maltols.

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

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

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

[0024] The beneficial effects of the present invention are as follows: the present invention prepares maltol haptens, applies maltol haptens to couple carrier proteins to obtain maltol coating antigens and maltol immunization antigens, and prepares specific monoclonal antibodies for directly detecting maltol compounds, the antibodies have good sensitivity and high specificity for maltol compounds; the antibodies are used to construct an enzyme-linked immunosorbent assay method for directly detecting methyl maltol and ethyl maltol in food, and the IC value for ethyl maltol is 50 reached 5.01ng / mL, the linear range was 1.47ng / mL-16.96ng / mL, and the IC of p-methyl maltol 50 Reach 4.15ng / mL; the cross-reaction to other maltol structural analogs is 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 (the detection limit can reach ppb level), high specificity, high detection flux, etc., and has broad application prospects. An immunoassay method has been established for the rapid and accurate detection of ethyl maltol and methyl maltol. BRIEF DESCRIPTION OF THE DRAWINGS

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

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

[0027] Figure 3 This is an ultraviolet scanning diagram of a maltol coating antigen and its hapten, and bovine serum albumin according to an embodiment of the present invention.

[0028] Figure 4 The standard curve of the indirect competitive ELISA established based on the maltol monoclonal antibody according to one embodiment of the present invention.

[0029] Figure 5 The present invention is a synthetic route of maltol hapten according to one embodiment of the present invention. DETAILED DESCRIPTION

[0030] The present invention is further described in detail through specific implementation cases, and it should be understood that these embodiments are only used to illustrate the present invention and are not used to limit the scope of protection of the present invention. After reading the present invention, the modifications of various equivalent forms of the present invention by those skilled in the art are all limited by the claims attached to this application. Unless otherwise specified, all raw materials and reagents of the present invention are raw materials and reagents that can be purchased in the conventional market.

[0031] Example 1 Preparation of maltol haptens

[0032] The method for preparing maltol hapten comprises the following steps:

[0033] S1. Add 5 g of 4-(2-furyl)-1-butene-4-ol and 30 mL of anhydrous tetrahydrofuran to a 250 mL reaction bottle, then slowly add 50 mL of 1 M borane tetrahydrofuran complex solution to the reaction bottle, stir at room temperature for 2 h, then add 30 mL of 2 M sodium hydroxide solution and 12 mL of 30% H2O2 solution, and then stir overnight. After the reaction is completed, the tetrahydrofuran is evaporated under reduced pressure, ethyl acetate is added for extraction, washed with saturated brine, dried over anhydrous sodium sulfate, and purified by column chromatography to obtain 3.5 g of intermediate 1;

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

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

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

[0037] Example 2 Preparation of maltol immunization antigen and coating antigen

[0038] 2.1 Preparation of maltol antigens for immunization

[0039] 21.4 mg of the maltol hapten prepared in Example 1 was dissolved in 600 μL of 0.1 M MES solution, and 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, and the mixture was reacted for 4 h at room temperature in the dark to obtain a hapten activation solution; 20 mg of lactoferrin (LF) was dissolved in 5 mL of CBS buffer solution (Na2CO3 1.69 g, NaHCO3 2.95 g were diluted to 1 L with primary water) with a pH value of 9.6, and 290 μL of the hapten activation solution was added with stirring in an ice bath, and after stirring evenly, the mixture was coupled at 4°C in the dark for 2 h. The coupling mixture was dialyzed with 0.01M PBS buffer solution at 4°C for 3 days, and the dialyzate was replaced twice a day to obtain the maltol immunization antigen, i.e., maltol hapten-LF. The maltol immunization antigen was dispensed at a concentration of 1 mg / mL and stored in a -20°C refrigerator.

[0040] Identification: UV scanning was performed on the carrier protein (LF), maltol hapten and maltol immune antigen (maltol hapten-LF). Figure 2 As shown, the ultraviolet absorption peak of the maltol immunization antigen has an obvious blue shift compared with the ultraviolet absorption peak of the maltol hapten, and the maltol immunization antigen has the characteristic absorption peaks of both malt classification hapten and LF, indicating that the maltol immunization antigen was successfully coupled and prepared.

[0041] 2.2 Preparation of maltol coating antigens

[0042] 21.4 mg of the maltol hapten prepared in Example 1 was dissolved in 600 μL of 0.1 M MES solution, and 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, and the mixture was stirred at room temperature in the dark for 4 h to obtain a hapten activation solution; 20 mg of bovine serum albumin (BSA) was dissolved in 5 mL of CBS buffer solution (Na2CO3 1.69 g, NaHCO3 2.95 g, diluted to 1 L with primary water) at pH 9.6, and 290 μL of the hapten activation solution was added with stirring in an ice bath, and after stirring evenly, the mixture was coupled at 4° C. in the dark for 2 h; the coupling mixture was heated at 4° C. with 0.01 M The PBS buffer solution was dialyzed for 3 days, and the dialyzate was replaced twice a day to obtain the maltol coating antigen, namely the maltol hapten-BSA. The maltol coating antigen was dispensed at a concentration of 1 mg / mL and frozen in a -20°C refrigerator.

[0043] Identification: UV scanning was performed on the carrier protein (BSA), maltol hapten and maltol coating antigen. The results were as follows: Figure 3 As shown, the ultraviolet absorption peak of the maltol coating antigen has an obvious blue shift compared with the ultraviolet absorption peak of the maltol hapten, and the maltol coating antigen has the characteristic absorption peaks of both maltol hapten and BSA, indicating that the maltol coating antigen is successfully coupled.

[0044] Example 3 Preparation and identification of maltol antibodies

[0045] 3.1 Preparation of maltol antibodies

[0046] 1. Antibody Preparation

[0047] The maltol immunization antigen prepared in Example 2 was emulsified evenly with an equal amount of immunization adjuvant (Freund's complete adjuvant for the first immunization, and Freund's incomplete adjuvant for subsequent booster immunizations) to immunize mice. Balb / C mice aged 6 to 7 weeks were immunized by multiple injections in the back, subcutaneous tissues of various parts, intraperitoneal cavity and feet, and the second immunization was performed 2 weeks later, and booster immunization was performed every 2 weeks thereafter. Blood was collected from the tail of the mice 1 week after the fourth booster immunization, and the serum titer was determined by indirect competitive ELISA. When the titer no longer increased, intraperitoneal injection was used for booster immunization. Blood was collected from the heart 3 days later, and the water bath was 0.5 to 1h, and centrifuged at 4°C and 10,000 for 15min. The supernatant was taken as antiserum, that is, polyclonal antibody.

[0048] Splenocytes were mixed with SP2 / 0 myeloma cells using PEG for cell fusion. After subcloning and four limiting dilutions, a portion of the positive hybridoma was cryopreserved and the other portion was injected into the peritoneal cavity of mice to produce ascites. The ascites was collected seven days later and purified using a protein G immunoaffinity column to obtain monoclonal antibodies.

[0049] 3.2 Antibody identification

[0050] Use carbonate buffer (CBS, pH = 9.6) as the diluent for maltol coating antigen, phosphate buffer (PBS, 0.01M, pH = 7.4) as the diluent for antiserum and standard, and phosphate Tween buffer (PBST, 0.01M) as the diluent for horseradish peroxidase labeled goat anti-mouse solution. Dilute the maltol coating antigen to 1μg / mL, add 100μL to each well of a 96-well ELISA plate, and incubate overnight at 4°C. After washing the plate twice with PBST, add 120μL of 5% bovine serum albumin to each well, incubate in a 37°C water bath for 3h, spin dry and dry at 37°C for 1h. Dilute the antiserum 1000, 2000, 4000, 8000, 16000, 32000, 64000 times, and 1μg / mL maltol standard. The above solution was added to a 96-well plate coated with maltol coating antigen according to the general indirect competitive ELISA method, incubated in a 37°C water bath for 40 minutes, washed 5 times with PBST, 100 μL of 5000-fold diluted horseradish peroxidase-labeled goat anti-mouse solution was added, incubated in a 37°C water bath for 30 minutes, washed 5 times with PBST, 100 μL of 3,3′,5,5′-tetramethylbenzidine (TMB) substrate colorimetric solution was added, incubated in a 37°C water bath for 10 minutes again, 10% concentrated sulfuric acid was added to terminate the reaction, and the data was read on an ELISA reader. The results of the indirect competitive ELISA are shown in Table 1.

[0051] The antiserum dilution multiple with an absorbance value (OD) of 1 to 1.5 at a wavelength of 450 nm was selected as the titer of the antiserum, and the inhibition rate was calculated using the following formula.

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

[0053] Table 1 Indirect competitive ELISA test results

[0054] Antiserum Packing original Potency Inhibition rate Maltol hapten-LF Maltol hapten-BSA 1∶64000 84.36％

[0055] The best artificial antigen combination was selected based on the titer and inhibition rate of the comprehensive antiserum. From the results in Table 1, it can be seen that the highest antibody titer is 64000, and the corresponding inhibition is 84.36%, which has high inhibition and high titer. Therefore, the artificial antigen with lactoferrin as the carrier protein was used as the maltol immunization antigen (maltol hapten-LF), and the artificial antigen with bovine serum albumin as the carrier protein was used as the maltol coating antigen (maltol hapten-BSA) for immunodetection.

[0056] Example 4: Coating concentration of maltol coating antigen and antibody dilution multiple

[0057] Use carbonate buffer (CBS, pH = 9.6) as the diluent for maltol coating antigen, phosphate buffer (PBS, 0.01M, pH = 7.4) as the diluent for maltol polyclonal antibody and standard, and phosphate Tween buffer (PBST, 0.01M) as the diluent for the goat anti-mouse solution labeled with horseradish peroxidase. Dilute the maltol coating antigen to 2, 1, 0.5, 0.25, 0.125, 0.0625 μg / mL, add 100 μL to each well of a 96-well ELISA plate, and incubate overnight at 4°C. After washing the plate twice with PBST, add 120 μL of 5% bovine serum albumin to each well, incubate in a 37°C water bath for 3 hours, spin dry, and dry at 37°C for 1 hour. The maltol polyclonal antibody was diluted 1000, 2000, 4000, 8000, 16000, 32000, and 64000 times, respectively, and a 1 μg / mL maltol standard was used; the above solution was added to a 96-well plate coated with the corresponding coating material according to a general indirect competitive ELISA method, incubated in a 37°C water bath for 40 minutes, washed 5 times with PBST, 100 μL of a 5000-fold diluted horseradish peroxidase-labeled goat anti-mouse solution was added, incubated in a 37°C water bath for 30 minutes, washed 5 times with PBST, 100 μL of a 3,3′,5,5′-tetramethylbenzidine (TMB) substrate colorimetric solution was added, and incubated in a 37°C water bath for 10 minutes again, 10% concentrated sulfuric acid was added to terminate the reaction, and the absorbance (OD) was measured by an enzyme label analyzer. The inhibition rate was calculated by selecting the coating concentration and antibody dilution multiple when the absorbance value was 1 to 1.5 at a wavelength of 450 nm. The test results are shown in Table 2.

[0058] Table 2 Results of chessboard detection method

[0059] OD value Coating concentration Antibody dilution factor Inhibition rate 1.430 2 μg / mL 1∶8000 79.94％ 1.293 1 μg / 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 coating antigen is 1 μg / mL, and the dilution factor of maltol polyclonal antibody is 4000 times.

[0061] Example 5 Sensitivity evaluation of maltol monoclonal antibodies

[0062] According to the optimal coating concentration of Example 4, the maltol monoclonal antibody prepared in Example 3 was used to draw an enzyme-linked immunosorbent assay (ELISA) standard curve.

[0063] Use carbonate buffer (CBS, pH = 9.6) as the diluent for maltol coating antigen, phosphate buffer (PBS, 0.01M, pH = 7.4) as the diluent for maltol monoclonal antibody and standard, and phosphate Tween buffer (PBST, 0.01M) as the diluent for horseradish peroxidase labeled goat anti-mouse solution. Dilute the maltol coating antigen to 1 μg / mL, add 100 μL to each well of a 96-well ELISA plate, and incubate at 4°C overnight. After washing the plate twice with PBST, add 120 μL of 5% bovine serum albumin to each well, incubate in a 37°C water bath for 3 hours, spin dry, and dry at 37°C for 1 hour. The maltol standard (ethyl maltol) was prepared into solutions with concentrations of 1000, 100, 10, 1, 0.1, 0.01, and 0.001 μg / mL, respectively, and then added to a 96-well plate coated with a maltol-based antigen according to a general indirect competitive ELISA method with a maltol-based monoclonal antibody, incubated in a 37°C water bath for 40 min, washed 5 times with PBST, added with 100 μL of a 5000-fold diluted horseradish peroxidase-labeled goat anti-mouse solution, incubated in a 37°C water bath for 30 min, washed 5 times with PBST, added with 100 μL of a 3,3′,5,5′-tetramethylbenzidine (TMB) substrate colorimetric solution, incubated again in a 37°C water bath for 10 min, added with 10% concentrated sulfuric acid to terminate the reaction, and measured the absorbance (OD) using an enzyme labeled analyzer.

[0064] With OD value as the ordinate and the corresponding logarithmic value of standard concentration as the abscissa, origin8.5 software was used to fit the function with four parameters: y = (AD) / [1+(X / C)B]+D, where A and D represent the minimum and maximum absorbance values ​​(OD) of drug concentration, respectively, and C is the midpoint concentration. When the standard concentration is equal to C, the OD value is (A+D) / 2, and the half-maximal inhibitory concentration at the inflection point of the curve is IC 50 , B represents the steepness of the curve, called the slope factor: IC 10 The detection limit is IC 20 ~IC 80 The detection range is 1.5. The standard curve of ELISA was established with ethyl maltol as the standard. Figure 4 The correlation coefficients and parameters are shown in Table 3.

[0065] Table 3 Detection parameters of maltol monoclonal antibodies to ethyl maltol

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

[0067] Combination Figure 4 As shown in Table 3, the standard curve established with ethyl maltol standard has a typical S-shaped curve, good detection sensitivity, and IC 50 Reached 5.01ng / mL, the linear range was 1.47ng / mL-16.96ng / mL.

[0068] Example 6 Specificity evaluation of maltol monoclonal antibodies

[0069] Using the maltol monoclonal antibody prepared in Example 3, methyl maltol, ethyl maltol and common additives were subjected to enzyme-linked immunosorbent assay (ELISA), and their corresponding IC 50 The cross-reaction rate was calculated and the results are shown in Table 4.

[0070] Table 4 Cross-reaction rate of maltol monoclonal antibodies to common additives

[0071]

[0072] As can be seen from Table 4, the cross-reaction rate of the maltol monoclonal antibody to maltol analogs and common additives is less than 0.02%, indicating that the maltol monoclonal antibody prepared by the present invention has a strong specificity for detecting maltol compounds.

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

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

[0075] Extract and concentrate the maltol compounds in the sample to be tested to prepare the test solution, then add the maltol standard and maltol monoclonal antibody to the 96-well transparent polystyrene ELISA plate coated with the coating antigen, incubate in a 37°C water bath for 40 minutes, wash the plate 5 times with PBST, add 100 μL of 5000-fold diluted horseradish peroxidase-labeled goat anti-mouse solution, incubate in a 37°C water bath for 30 minutes, wash the plate 5 times with PBST, add 100 μL of 3,3',5,5'-tetramethylbenzidine (TMB) substrate colorimetric solution, incubate in a 37°C water bath for 10 minutes again, and add 10% concentrated sulfuric acid to terminate the reaction. Use an ELISA instrument to test the absorbance value. By comparing the absorbance values ​​of the test solution and the maltol standard, the content of maltol compounds in the sample can be quantitatively analyzed.

[0076] Example 8 Accuracy test of immunoassay kit for detecting malt phenol compounds

[0077] Take 400 blank oil samples (200 each of sesame oil and edible vegetable oil). First, collect oil samples according to the principle of randomness and representativeness to ensure that the samples can truly reflect the quality of the overall oil (for solid or semi-solid oil samples, pretreatment such as peeling and impurity removal is required to ensure the smooth progress of subsequent steps). Add ethyl maltol standard to 100 blank samples of sesame oil and 100 blank samples of edible vegetable oil to a final concentration of 5 μg / L, dissolve all oil samples in n-hexane and heat and shake to mix, and soak and extract oil at room temperature. After extraction, centrifuge at 4000r / min for 2min to remove impurities, and properly store under appropriate conditions to ensure the representativeness of the oil sample and the accuracy of the test. Take the immunoassay kit for detecting malt phenol compounds prepared in Example 7, and repeat the measurement three times for each sample. The test results are shown in Table 5.

[0078] Table 5 Detection results of malt phenol compounds by immunoassay kit

[0079]

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

[0081] Example 9 Crossover rate test of immunoassay kit for maltol compounds

[0082] Take 6 blank oil samples, add appropriate amount of ethyl maltol standard solution, methyl maltol standard solution, cinnamaldehyde standard solution, vanillin standard solution, methyl benzoate standard solution and isomalt standard solution, respectively, and prepare 6 test samples with a final concentration of 5 μg / L, take the immunoassay kit for detecting maltol compounds prepared in Example 7, and repeat the measurement three times for each test sample. The test results are shown in Table 6.

[0083] Table 6 Cross-reaction experiment results

[0084]

[0085] Note: “+” indicates positive, “-” indicates negative.

[0086] As can be seen from Table 6, only the oil samples with ethyl maltol and methyl maltol added were detected as positive, while the blank oil samples with cinnamaldehyde, vanillin, benzoic acid methyl and isomalt added were not detected as positive, indicating that the immunoassay kit for maltol compounds prepared by the present invention has good specificity for maltol compounds and a low cross-reaction rate.

[0087] The above are only some embodiments of the present invention. For those skilled in the art, several modifications and improvements may be made without departing from the inventive concept of the present invention, which all fall within the protection scope of the present invention.

Claims

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

2. The method for preparing the maltol hapten according to claim 1, characterized in that: The following steps are involved: S1.4-(2-Furyl)-1-butene-4-ol was dissolved in anhydrous tetrahydrofuran, and a borane tetrahydrofuran complex solution was added dropwise. The reaction was stirred at room temperature, and a sodium hydroxide solution and a H2O2 solution were added. The reaction was stirred overnight. After the reaction was completed, the tetrahydrofuran was evaporated under reduced pressure, and the mixture was extracted with ethyl acetate. The mixture was washed with saturated brine, dried, and purified by column chromatography to obtain intermediate 1. The reaction formula of this step is shown in formula (II): S2. The intermediate 1 is dissolved in a mixture of methanol and water, chlorine is introduced into the mixture for reaction, refluxed, the pH value is adjusted to be slightly acidic, extracted with ethyl acetate, the ethyl acetate phases are combined and evaporated to dryness, and purified by column to obtain the intermediate 2. The reaction formula of this step is shown in formula (III): S3. Take the intermediate 2, add succinic anhydride, add pyridine to dissolve, heat in an oil bath under nitrogen protection, and evaporate the organic phase directly after the reaction, and purify it by column to obtain maltol hapten. The reaction formula of this step is shown in formula (IV):

3. The method according to claim 2, characterized in that The molar concentration of the borane tetrahydrofuran 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%.

4. The method according to claim 2, characterized in that: The volume ratio of methanol to water in the mixed solution of methanol and water in step S2 is 1:1, and the concentration of the sodium hydroxide solution in step S2 is 10%.

5. Maltol antigen, characterized in that: The maltol antigen is a conjugate of the maltol hapten according to claim 1 and a carrier protein, 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. Use of the maltol hapten according to claim 1 and the maltol antigen according to claim 5 in immunological detection of maltols.

8. Maltol antibody, characterized in that The maltol antibody is prepared by immunizing an animal with the maltol antigen according to claim 5, and the maltol antibody is a maltol monoclonal antibody.

9. An immunoassay kit for detecting maltol compounds, characterized in that: The invention comprises the maltol antigen according to claim 5 and the maltol antibody according to claim 8 as coating agents.

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

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