A kit for detecting the content of acrylamide in fried food and a detection method thereof

Abstract

The application relates to a kit for detecting the content of acrylamide in fried food and a detection method thereof, and belongs to the technical field of food safety detection. The kit comprises an enzyme-labeled plate, a standard solution and an anti-acrylamide monoclonal antibody. The anti-acrylamide monoclonal antibody can specifically combine with a hapten (AA-4MBA) synthesized by acrylamide and 4-mercaptobenzoic acid. The enzyme-labeled plate is coated with an antigen (AA-BSA) coupled by AA-4MBA and BSA. The kit has the advantages of simple operation, rapid detection, low cost, high sensitivity and good repeatability.

Description

Technical Field

[0001] This application relates to the field of food safety testing technology, and in particular to a kit for detecting acrylamide content in fried foods and a method thereof. Background Technology

[0002] Acrylamide is the core monomer in the industrial production of polyacrylamide, used in dye synthesis and wastewater purification. It is also naturally generated during the Maillard reaction in starchy foods when grilled, baked, or fried at temperatures exceeding 120°C. The neurotoxicity, reproductive and developmental toxicity, and genotoxicity of this substance have been authoritatively confirmed, and it is classified as a Group 2 carcinogen. Given its threat to human health, many countries have set stringent limits on acrylamide concentrations in drinking water, and acrylamide contamination in food has also attracted global attention. Therefore, organizations such as the WHO have explicitly recommended the development of simple, low-cost acrylamide detection methods to support quality control in related fields, which has become the core driving force behind the development of reagent kits.

[0003] Currently, the national standard GB5009.204-2014 specifies methods for the determination of acrylamide, including liquid chromatography-mass spectrometry (LC-MS) and gas chromatography-mass spectrometry (GC-MS), which require solid-phase extraction columns and chromatography columns for sample collection, respectively. While these methods offer high accuracy, they suffer from cumbersome procedures, expensive equipment, long detection cycles, and reliance on complex pretreatment steps such as solid-phase extraction, making them unsuitable for rapid on-site testing. Furthermore, existing ELISA kits on the market (such as those from Abraxis, Inc.) still require users to perform derivatization and extraction purification steps themselves, increasing operational complexity and time costs, thus limiting their widespread application.

[0004] Therefore, there is an urgent need in this field to develop a simple and efficient reagent kit and method for detecting acrylamide content in fried foods. Summary of the Invention

[0005] This application relates to a kit for detecting acrylamide content in fried foods and a detection method thereof; the standard of the kit is pre-derivatized with 4-mercaptobenzoic acid and extracted with ethyl acetate to obtain a stable standard that can be directly used for quantitative comparison; the food sample extraction method of the kit uses 4-mercaptobenzoic acid derivatization and extraction with ethyl acetate to obtain the sample working solution, eliminating the need for a solid-phase extraction column purification step; the above application can achieve simplified experimental operation and reduce usage costs.

[0006] In a first aspect, this application provides a kit for detecting acrylamide content in fried foods, comprising an enzyme-linked immunosorbent assay (ELISA) plate, a standard solution, and an anti-acrylamide monoclonal antibody; wherein the anti-acrylamide monoclonal antibody can specifically bind to a hapten (AA-4MBA) derived from acrylamide and 4-mercaptobenzoic acid; and the ELISA plate is coated with an acrylamide-conjugated antigen.

[0007] Optionally, the standard solution is prepared by including the following steps: S1: Provides a series of standard aqueous solutions of acrylamide at different concentrations; S2: Add 4-mercaptobenzoic acid working solution and alkaline solution to each standard aqueous solution to carry out derivatization reaction; S3: Neutralize the reaction system with acid; S4: Acrylamide derivatives produced by extraction using organic solvents; S5: Combine the extracts, concentrate and dry them, then redissolve them with sample diluent to obtain a ready-to-use standard solution. Optionally, the organic solvent is ethyl acetate.

[0008] Optionally, the anti-acrylamide monoclonal antibody comprises CDR1, CDR2, and CDR3 as shown in SEQ ID NO:1.

[0009] Optionally, CDR1, CDR2, or CDR3 may be determined according to the Kabat numbering scheme, IMGT numbering scheme, AbM numbering scheme, Chothia numbering scheme, Contact numbering scheme, or any combination thereof.

[0010] Optionally, the kit may further include one or more of the following components: horseradish peroxidase-labeled secondary antibody, derivatization reagent, sodium hydroxide solution, hydrochloric acid solution, washing buffer, sample dilution solution, substrate chromogenic solution, and stop solution.

[0011] Secondly, this application provides a method for detecting acrylamide content in fried foods using the kit described in the first aspect, comprising the following steps: (1) Sample pretreatment: After homogenizing the sample to be tested, extract it with salt solution, defatt it with n-hexane, take the lower aqueous phase and carry out derivatization reaction with derivatization reagent, extract the derivative with organic solvent after reaction, concentrate and dry it and then reconstitute to obtain the sample working solution. (2) Competitive ELISA detection: The sample working solution and the anti-acrylamide monoclonal antibody are added to the ELISA plate for a competitive immune reaction; the ELISA plate is coated with acrylamide conjugated antigen; (3) Color development and detection: Add the horseradish peroxidase-labeled secondary antibody to the enzyme-labeled plate after the reaction in step (2), wash, add the substrate for color development, add the stop solution to terminate the reaction, and measure the absorbance value. (4) Quantitative analysis: Plot a standard curve based on the absorbance values ​​of the standard solution, and calculate the acrylamide content in the sample based on the standard curve.

[0012] Optionally, in the sample pretreatment, the derivatization reagent is 4-mercaptobenzoic acid, the derivatization reaction is a Michael addition reaction, and the reaction conditions are: reaction at 50°C in the dark for 1.5-2 hours.

[0013] In summary, this application includes at least one of the following beneficial technical effects: This application achieves quantitative analysis of small molecule acrylamide by detecting derivatives of acrylamide (AA-4MBA) in samples, with high detection sensitivity, and can also detect samples with low content levels; This application provides a pre-derivative standard product, which eliminates the need for further derivatization steps during use, thus saving time. Compared to the high requirements for sample processing and the low throughput of mainstream chromatographic methods, the sample derivatization, extraction and concentration steps provided in this application can effectively detect acrylamide in samples without the need for additional solid phase extraction column purification steps, thereby improving efficiency and reducing costs. This application is widely applicable to food companies and testing institutions for monitoring and controlling the acrylamide content of samples, and has significant promotional value. Attached Figure Description

[0014] Figure 1 This is the standard curve for this application. Detailed Implementation

[0015] To further clarify the objectives, technical solutions, and advantages of this invention, the invention will be described in more detail below with reference to specific implementation examples, but the invention is not limited thereto.

[0016] I. Definition To explain this specification, the following definitions will be used, and terms used in the singular may also include plural forms, where appropriate. It should be understood that the terminology used herein is for the purpose of describing particular embodiments only and is not intended to be restrictive.

[0017] The terms "monoclonal antibody," "monoclonal antibody," or "monoclonal antibody composition" refer to antibody molecules that consist of a single molecule. A monoclonal antibody composition exhibits specific binding specificity and affinity for a particular epitope.

[0018] The term "single-domain antibody (sdAb)" generally refers to an antibody in which a single variable domain (e.g., a heavy chain variable domain (VH) or light chain variable domain (VL), a heavy chain variable domain derived from camelid heavy chain antibodies, or a VH-like single domain (v-NAR) derived from fish IgNARs) is sufficient to confer antigen binding. That is, the single variable domain does not need to interact with another variable domain to recognize the target antigen. Examples of single-domain antibodies include a single-domain antibody (WO2005035572A2) derived from camelids (lambs and camels) and cartilaginous fish (e.g., nurse sharks). The camelid-derived single-domain antibody, also referred to as VHH in this application, consists of only one heavy chain variable region, consisting of only one chain from the C-terminus to the N-terminus: FR4-CDR3-FR3-CDR2-FR2-CDR1-FR1; it is also known as a "nanobody." Single-domain antibodies are the smallest known units that can bind to a target antigen.

[0019] The term "complementarity-determining region" or "CDR region" or "CDR" refers to a region within the variable domain of an antibody that is highly variable in sequence and forms a structurally defined loop ("hypervariant loop") and / or contains antigen contact residues ("antigen contact sites"). The CDR is primarily responsible for binding to antigen epitopes. Heavy chain CDRs are typically referred to as CDR1, CDR2, and CDR3, numbered sequentially starting from the N-terminus. In a given heavy chain variable region amino acid sequence, the precise amino acid sequence boundaries of each CDR can be determined using any of a number of known antibody CDR assignment systems or combinations thereof, including, for example: Chothia (Chothia et al. (1989) Nature 342: 877-883, Al-Lazikani et al., “Standard conformations for the canonical structures of immunoglobulins”, Journal of Molecular Biology, 273, 927-948 (1997)) based on antibody sequence variability; Kabat (Kabat et al., Sequences of Proteins of Immunological Interest, 4th ed., Department of Health and Human Services, National Institutes of Health (1987)); AbM (University of Bath); Contact (University College London); and the International ImMunoGeneTics database (IMGT). (http: / / imgt.cines.fr / ), and the North CDR definition based on affinity propagation clustering utilizing a large number of crystal structures. Unless otherwise stated, in this invention, the term "CDR" or "CDR sequence" encompasses a CDR sequence determined in any of the foregoing methods. A CDR may also be determined based on having the same AbM numbering position as a reference CDR sequence (e.g., any of the CDR sequences in the examples of this invention). In one embodiment, the CDR of the single-domain antibody of this invention is positioned according to the AbM numbering scheme. Unless otherwise stated, in this invention, when referring to the positions of residues in the antibody variable region and the CDR (including heavy chain variable region residues), it refers to the numbering position according to the AbM numbering system.

[0020] II. A kit for detecting acrylamide content in fried foods according to this application. This application provides a kit for detecting acrylamide content in fried foods. The kit consists of an enzyme-linked immunosorbent assay (ELISA) plate coated with antigen AA-BSA, a standard solution (pretreated standard), an anti-acrylamide antibody, an HRP-labeled secondary antibody, and a sample diluent. In one specific embodiment, the kit includes a 96-well ELISA plate (coated with antigen AA-BSA), six vials of standards (1 mL each, concentrations of 0 ppb, 4 ppb, 20 ppb, 80 ppb, 400 ppb, and 1600 ppb, respectively), an anti-acrylamide antibody solution (6 mL, concentration 0.36 ug / mL), an HRP-labeled secondary antibody solution (6 mL, concentration 1 ug / mL), a derivatization reagent (120 mL), reagent A (60 mL, 0.1 mol / L sodium hydroxide), reagent B (2 mL, 1 mol / L hydrochloric acid), 20× wash buffer (25 mL, 0.2 mol / L PBST), sample dilution buffer (60 mL, 10% ethanol, solvent: 0.01 mol / L PBS), substrate chromogenic solution (12 mL, 3,3',5,5'-tetramethylbenzidine + H2O2), stop solution (12 mL, 1 mol / L H2SO4), and a procedure sheet. The kit described in this application is suitable for the detection of fried or baked potato and cereal food samples.

[0021] III. A method for quantitative detection of acrylamide using enzyme-linked immunosorbent assay (ELISA) as described in this application. Due to the small molecular weight and simple structure of acrylamide (AA), it is difficult to prepare specific antibodies against it. This application uses a hapten (AA-4MBA) derived from acrylamide and 4-mercaptobenzoic acid, conjugated with KLH protein, as the antigen to prepare a specific antibody against acrylamide derivatives. This establishes a competitive enzyme-linked immunosorbent assay (ELISA) method for the quantitative analysis of AA by detecting acrylamide derivatives. A highly specific and sensitive ELISA method is established using an acrylamide derivative antigen (AA-4MBA) conjugated with bovine serum albumin (BSA) coating antigen AA-BSA and a monoclonal antibody that can bind to this coating antigen. The enzyme-linked immunosorbent assay (ELISA) method for quantitative detection of acrylamide of the present invention includes the following steps: (1) coating the coating antigen AA-BSA onto an enzyme-labeled microplate; (2) blocking the microplate with BSA blocking solution; (3) adding the sample working solution and acrylamide monoclonal antibody to the microplate for competitive reaction, and simultaneously setting up the reaction wells of the reference standard; (4) adding HRP enzyme-labeled secondary antibody conjugated with the monoclonal antibody that has been well bound to the antigen in the microplate; (5) adding TMB substrate for colorimetric reaction; (6) adding stop solution to terminate the reaction, and reading the absorbance values ​​of the standard and the sample at a wavelength of 450 nm, plotting a standard curve to quantitatively detect the content of acrylamide in the sample. The acrylamide enzyme-linked immunosorbent assay (ELISA) kit utilizes a competitive assay principle and the specific binding reaction between antigen and antibody. Acrylamide-conjugated antigens are pre-coated onto microplate strips. These, along with the derivatized sample or standard, are added to the wells along with enzyme-labeled secondary antibody and specific monoclonal antibody. If the sample or standard contains residual target material, it will compete with the antibody added to the wells, inhibiting antibody binding to the antigen coating at the bottom of the well. Simultaneously, the enzyme-labeled secondary antibody binds to the primary antibody, forming an antigen-antibody-enzyme-labeled secondary antibody complex. Washing the plate removes the antibody-enzyme-labeled secondary antibody complex that has not bound to the antigen. Adding TMB substrate causes the complex to bind to the wells, resulting in a blue reaction. After adding stop solution, the color turns yellow. The absorbance is read at 450 nm. The absorbance of the sample or standard is negatively correlated with its acrylamide content.

[0022] Example 1: Screening for specific monoclonal antibodies against acrylamide derivatives Alpaca were immunized with a high-titer antiserum obtained by using a hapten (AA-4MBA) derived from acrylamide and 4-mercaptobenzoic acid and conjugated with KLH protein, provided by Guangzhou Wanfei Biotechnology. After animal immunization, 50 mL of fresh alpaca blood was collected, and peripheral blood mononuclear cells (PBMCs) were separated using Ficoll-Paque density gradient separation medium. RNA was extracted, reverse transcribed, amplified using universal primers, cloned into phage particles, transformed into TG1 strain, and a phage library was established for screening monoclonal antibodies. Nanobodies with high specificity and affinity (precisely recognizing acrylamide) were obtained and named Nb. AA -14.

[0023] Analysis of Nb AA The sequence of -14 showed that the antibody Nb AA The amino acid sequence of VHH in -14 is QVQLVESGGGLVEAGGSLKLSCAASGNIYDINAMGWYRQAPGEERELVAAIITDGSTRYADSVKGRFTISRENAKNTVTLQMGSLKPEDTAVYYCAAGPHYGLGVVRFWGQGTQVTVSS (SEQ ID NO:1). The amino acid sequences of CDR1, CDR2 and CDR3 defined by AbM are GNIYDINAMG (SEQ ID NO:2), AIITDGSTR (SEQ ID NO:3) and GPHYGLGVVRF (SEQ ID NO:4), respectively.

[0024] Example 2: Preparation of enzyme-labeled plates coated with antigen AA-BSA The specific steps for preparing an AA-BSA-coated ELISA plate are as follows: (1) Add 100 μL of acrylamide-conjugated antigen (AA-BSA solution, which is a coating antigen of BSA conjugated with acrylamide derivative antigen (AA-4MBA) and purchased from Guangzhou Wanfei Biotechnology) to each well of the 96T microplate. The solvent is 0.05 mol / L carbonate-bicarbonate buffer (CB solution). (2) After sealing with film, place at 4°C for 14 hours; (3) Discard the CB solution in the microwells, wash twice with 0.01 mol / L PBST washing solution, add 180 uL of blocking solution (2% BSA + 5% sucrose + 5% trehalose) to the microwells, with 0.01 mol / L PBST as the solvent, and place at 37°C for 1 hour; (4) Discard the sealing liquid in the micropores and vacuum dry at room temperature for 30 minutes.

[0025] Example 3: Preparation of Standard Solution This embodiment provides a standard that has been pre-derivatized (Michael addition reaction) and extracted and concentrated with organic reagents, which can be used directly by the user during operation without the need for further derivatization and extraction steps, saving time and simplifying the operation. The specific method is as follows: (1) Prepare a set of acrylamide standards in 50 ml PP centrifuge tubes with distilled water, with concentrations of 0 ppb, 4 ppb, 20 ppb, 80 ppb, and 400 ppb, respectively. 1600 ppb, each tube is 10 mL / tube. The standard used is Macklin acrylamide standard, CAS: 79-06-1; (2) Prepare 4-mercaptobenzoic acid working solution with a concentration of 2 mg / mL and anhydrous methanol as the solvent; (3) Add 2 mL of 4-mercaptobenzoic acid working solution and 685 μL of 0.1 mol / L sodium hydroxide solution to each tube of standard in (1), vortex mix for 15 seconds, and then incubate at 50°C in the dark for 2 hours; (4) Add 25 μL of 0.1 mol / L hydrochloric acid solution to each tube, vortex mix for 15 seconds; (5) Add 4 mL of ethyl acetate reagent to each tube, mix (6) Extract the acrylamide derivative for 15 seconds; centrifuge at 4000 rpm for 3 minutes, and transfer the collected supernatant (ethyl acetate) to a new 15 mL PP centrifuge tube; (7) add 4 mL of ethyl acetate reagent to the 50 mL centrifuge tube from step (6) above and repeat the extraction, centrifuge for 3 minutes, and transfer the collected supernatant to the same 15 mL PP centrifuge tube with the corresponding concentration from step (6) above; (8) dry the collected supernatant ethyl acetate twice using a nitrogen blower at 45°C; (9) add 1 mL of 10% ethanol solution (solvent is 0.01 Mol / L PBS, pH 7.8) to each tube and vortex for 30 seconds to reconstitute. Thus, a set of derivatized, more stable, and directly detectable standard solutions (concentration gradient 0 ppb, 4 ppb, 20 ppb, 80 ppb, 400 ppb, 1600 ppb) can be obtained.

[0026] Example 4: Construction of Standard Curve The steps for creating the standard curve provided in this embodiment are as follows: (1) Add 50 mL of the standard solution prepared in Example 3 (standard concentrations of 0 ppb, 4 ppb, 20 ppb, 80 ppb, 400 ppb, and 1600 ppb, respectively) to each well of the AA-BSA coated antigen microplate prepared in Example 2, with three parallel wells for each concentration; (2) Add 50 mL of the anti-acrylamide monoclonal antibody from Example 1 (concentration 0.36 ug / mL, solvent 0.01 mol / L Tris solution, pH 8.5) to each well and mix gently for 1 minute; (3) Then add 50 μL of enzyme-labeled secondary antibody (HRP-rabbit anti-alpaca IgG solution, concentration 1 ug / mL, solvent 0.01 mol / L Tris solution, pH 8.5) to each well; incubate at room temperature (25 ± 2.5) °C in the dark for 30 minutes; (4) Wash the plate 4 times, adding 250 μL each time. 1× washing buffer (0.01 mol / L PBST solution), shake the plate as dry as possible for the last time and blot dry on absorbent paper; (5) Add 100 μL of TMB substrate (3,3',5,5'-tetramethylbenzidine and H2O2 mixture) to each well, and incubate at room temperature (25±2.5) °C in the dark for 15 minutes (cover the microplate appropriately during incubation); (6) Add 100 μL of stop solution (1 mol / L H2SO4) to each well to stop the reaction, read the OD value of each microwell at 450 nm wavelength using an enzyme-linked immunosorbent assay reader, and perform linear regression on the standard determination results using the logit-log method. The linear results of the standard curve are as follows. Figure 1 As shown, the correlation coefficient of the standard curve is 0.9979.

[0027] Example 5 Sample Pretreatment This embodiment relates to a method for the derivatization and extraction collection of samples (such as fried potato chips) for enzyme-linked immunosorbent assay (ELISA) of acrylamide. When performing such sample detection, acrylamide in the sample needs to be extracted into an aqueous sodium chloride solution, defatted with n-hexane, then derivatized with 4-mercaptobenzoic acid at a suitable pH (Michael addition reaction), and then extracted with ethyl acetate and concentrated and enriched with nitrogen blowing equipment for detection. The specific steps of the sample pretreatment method are as follows: (1) Weigh 2g of homogenized potato chip sample and place it in a clean 50mL centrifuge tube. Add 20mL of sample extraction solution (5mol / L NaCl aqueous solution) and vortex for 3min. Centrifuge at 6000 rpm for 5min at room temperature (25±2.5)°C. (2) Transfer 12mL of supernatant to a new 50mL centrifuge tube, add 10mL of n-hexane, vortex at maximum speed for 1min, and centrifuge at 6000 rpm for 2min at room temperature (25±2.5)°C. (3) Remove the upper n-hexane, take 10mL of the lower sample solution to a new 50mL centrifuge tube, and add 2mL of derivatization reagent (2mg / mL). (4) Add 1.1 mL of reagent A (0.1 mol / L sodium hydroxide solution) and 1.1 mL of reagent A (0.1 mol / L sodium hydroxide solution), vortex for 30 s; (5) Add 4 mL of ethyl acetate, vortex for 1 min, centrifuge at 6000 rpm for 2 min at room temperature (25±2.5) °C, and transfer the upper ethyl acetate to a clean 15 mL centrifuge tube; (6) Add another 4 mL of ethyl acetate to the remaining liquid, repeat step (5) to extract the derivative again, and transfer all the upper ethyl acetate to the same 15 mL centrifuge tube from step (5); (7) Dry the upper ethyl acetate collected twice with nitrogen at 45 °C using a nitrogen blower; add 1 mL of sample diluent (10% ethanol solution, solvent is 0.01 mol / L PBS, pH 7.8), rapidly vortex-oscillate for 1 min to reconstitute the dried substance, and take 50 μL of the reconstituted solution for testing.

[0028] Example 6: Limit of Detection (LOD) The limit of detection (LOD) refers to the lowest concentration of the target analyte that the kit can reliably detect, i.e., the lowest concentration at which the signal value is significantly higher than that of the blank sample. The blank sample is a matrix that does not contain the target analyte. In this invention, sample dilutions are used as blank samples to determine the LOD: 10 aliquots of sample dilutions are derivatized and extracted (refer to Example 5), processed, and then detected (refer to Example 4). The detection results are shown in Table 1. The LOD is 2.35 ppb.

[0029] Table 1

[0030] Example 7 Recovery Rate The recovery rate determination of the method of this invention uses fried potato chip samples as an example. Three different fried potato chip samples and three different concentrations (100 ug / kg, 500 ug / kg, 1500 ug / kg) of acrylamide addition were designed for each sample. The sample extraction, derivatization, and extraction procedures in Example 5 and the ELISA plate-dotting procedure in Example 4 were then followed. Three replicates were designed for each sample and the added recovery concentration. The background concentration of the potato chips and the added recovery sample concentration were calculated using the logit-log method, and the recovery rate for each added concentration was calculated. The results are shown in Table 2. The acrylamide content of all samples was lower than the EU regulation limit of 750 ug / kg, and the recovery rates of the three added concentrations for different samples reached over 80%.

[0031] Table 2

[0032] Of course, the above description is only a specific embodiment of this application and is not intended to limit the scope of the invention. All equivalent changes or modifications made in accordance with the features and principles described in the claims of this invention should be included in the scope of the claims of this invention.

Claims

1. A kit for detecting acrylamide content in fried foods, characterized in that, The enzyme-labeled plate includes a standard solution and an anti-acrylamide monoclonal antibody; wherein the anti-acrylamide monoclonal antibody can specifically bind to a hapten (AA-4MBA) derived from acrylamide and 4-mercaptobenzoic acid; the enzyme-labeled plate is coated with an acrylamide-conjugated antigen.

2. The reagent kit according to claim 1, characterized in that, The method for preparing the standard solution includes the following steps: S1: Provides a series of standard aqueous solutions of acrylamide at different concentrations; S2: Add 4-mercaptobenzoic acid working solution and alkaline solution to each standard aqueous solution to carry out derivatization reaction; S3: Neutralize the reaction system with acid; S4: Acrylamide derivatives produced by extraction using organic solvents; S5: Combine the extracts, concentrate and dry them, then redissolve them with the sample diluent to obtain the ready-to-use standard solution.

3. The reagent kit according to claim 2, characterized in that, The organic solvent is ethyl acetate.

4. The reagent kit according to claim 1, characterized in that, The anti-acrylamide monoclonal antibody comprises CDR1, CDR2 and CDR3 as shown in SEQ ID NO:

1.

5. The kit according to claim 4, characterized in that, The CDR1, CDR2, or CDR3 is determined according to the Kabat numbering scheme, the IMGT numbering scheme, the AbM numbering scheme, the Chothia numbering scheme, the Contact numbering scheme, or any combination thereof.

6. The reagent kit according to claim 1, characterized in that, The kit also includes one or more of the following components: horseradish peroxidase-labeled secondary antibody, derivatization reagent, sodium hydroxide solution, hydrochloric acid solution, washing buffer, sample dilution solution, substrate chromogenic solution, and stop solution.

7. A method for detecting acrylamide content in fried foods using the kit described in any one of claims 1-6, characterized in that, Includes the following steps: (1) Sample pretreatment: After homogenizing the sample to be tested, extract it with salt solution, defatt it with n-hexane, take the lower aqueous phase and carry out derivatization reaction with derivatization reagent, extract the derivative with organic solvent after reaction, concentrate and dry it and then reconstitute to obtain the sample working solution. (2) Competitive ELISA detection: The sample working solution and the anti-acrylamide monoclonal antibody are added to the ELISA plate for a competitive immune reaction; the ELISA plate is coated with acrylamide conjugated antigen; (3) Color development and detection: Add the horseradish peroxidase-labeled secondary antibody to the enzyme-labeled plate after the reaction in step (2), wash, add the substrate for color development, add the stop solution to terminate the reaction, and measure the absorbance value. (4) Quantitative analysis: Plot a standard curve based on the absorbance values ​​of the standard solution, and calculate the acrylamide content in the sample based on the standard curve.

8. The detection method according to claim 7, characterized in that, In the sample pretreatment, the derivatization reagent is 4-mercaptobenzoic acid, the derivatization reaction is a Michael addition reaction, and the reaction conditions are: reaction at 50°C in the dark for 1.5-2 hours.

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