Gluten specific alpaca polyclonal antibody and double antibody-streptavidin enzyme-linked immunosorbent assay (ELISA) bran quality inspection test kit thereof

Abstract

The invention discloses a gluten specific alpaca polyclonal antibody and a double antibody-streptavidin enzyme-linked immunosorbent assay (ELISA) bran quality inspection test kit thereof. The detection kit provided by the invention takes an alpaca polyclonal antibody pAb as a capture antibody and biotinylated pAb as a detection antibody to carry out ELISA detection on gluten in a food matrix, and a novel double antibody-streptavidin ELISA detection method is constructed based on the detection kit. The detection method has good sensitivity and specificity, the EC50 value of alcohol-soluble protein is 51.21 ng / mL, the detection limit is as low as 0.07 ng / mL, the detection limit of the method is 4000 times lower than that of an existing R5 antibody method, and the sensitivity is better; when the alpaca polyclonal antibody pAb prepared by the invention is used for detection, the detection cost is reduced, reliable technical support is provided for gluten allergen detection, and the alpaca polyclonal antibody pAb is suitable for clinical and scientific research application of immunoassay kits, immune test strips and the like.

Description

Technical Field

[0001] The present application relates to the technical field of food safety detection, and in particular to a gluten-specific alpaca polyclonal antibody and a double antibody-streptavidin ELISA gluten detection kit thereof. Background Art

[0002] Gluten is a protein complex found in cereal grains such as barley, wheat, and rye, primarily composed of glutenin and gliadin. Gluten is the primary cause of gluten-related diseases, and currently the only way to manage them is to adhere to a strict gluten-free diet. With increasing awareness of food safety and health, people are paying more and more attention to food ingredients and their effects on the human body. Therefore, accurate gluten detection methods and strict regulation of "gluten-free" labels are crucial to ensuring food safety. As the primary component of gluten, gliadin is a key factor in triggering gluten-related diseases and an important criterion for quantitative gluten detection. Currently, immunoassays are the preferred method for detecting gliadin, but this method faces challenges in its specificity for gliadin. Therefore, developing highly specific, sensitive, and high-affinity detection methods is crucial to improving detection accuracy.

[0003] Enzyme-linked immunosorbent assay (ELISA) is currently widely used for the detection of gluten in food. ELISA methods primarily utilize specific antibodies against gluten proteins (such as gliadin and glutenin). For example, the publicly available R5 ELISA method (based on the R5 monoclonal antibody) is an official method recognized by the Codex Alimentarius Commission and the AOAC (e.g., AOAC 120.001). It targets the repeating sequence (QPFPQPQ) of ω-gliadin in wheat, rye, and barley gluten, with a detection limit of 1.5–5 mg gluten / kg (ppm). The G12 ELISA (based on the G12 monoclonal antibody) recognizes a toxic epitope (33-mer peptide present in α-gliadin) in gluten and is suitable for testing patients with celiac disease, with a detection limit of approximately 1–3 ppm. The Skerritt antibody method (targeting glutenin) has lower specificity and has been replaced by the R5 / G12 method.

[0004] The existing R5 antibody is sensitive to gluten from wheat, rye, and barley, but may miss oats (some varieties) or hydrolyzed / fermented gluten. The G12 antibody is sensitive to α-gliadin but may underestimate other gluten components (such as γ-gliadin). Furthermore, in actual testing, there is interference from the food matrix. For example, high-temperature processing, acid hydrolysis, or fermentation can destroy gluten epitopes, leading to false negatives. High-fat, high-sugar, or high-protein samples may nonspecifically bind to the antibody, affecting accuracy. Some currently available gluten-detecting antibodies may cross-react with non-target proteins (such as corn and rice), requiring validation to eliminate. Furthermore, the minimum detection limit of existing methods is typically 1 ppm, which is insufficient to meet the requirements of gluten-free foods with extremely low gluten levels (e.g., <1 ppm) (Codex standard: <20 ppm). Immunoassays, such as ELISA, offer advantages such as simplicity, low cost, and rapid detection, making them suitable for rapid, on-site, large-scale testing and meeting the testing needs of consumers, businesses, and regulators. The sensitivity and specificity of existing gluten detection reagents need to be improved. In view of this, the present invention is proposed. Summary of the Invention

[0005] In response to the defects and shortcomings of existing gluten detection, the present invention provides the preparation and application of polyclonal antibodies for gluten allergens in food matrices and a highly sensitive gluten allergen detection kit in food matrices established based on camel-derived polyclonal antibodies, which has high sensitivity and low cost.

[0006] The first purpose of the present invention is to provide a gluten-specific alpaca polyclonal antibody with high detection sensitivity and good specificity, which provides a core raw material for establishing a high-sensitivity immunoassay method for gluten allergens in food matrices.

[0007] The second object of the present invention is to provide the use of gluten-specific alpaca polyclonal antibodies.

[0008] The third object of the present invention is to provide a double antibody-streptavidin ELISA gluten detection kit.

[0009] A fourth object of the present invention is to provide a method for detecting gluten allergens in food matrices, which solves the technical problem in the prior art of lacking antibodies that can detect gluten allergens in food matrices with high sensitivity and high specificity.

[0010] In order to achieve the above-mentioned purpose of the present invention, it is achieved through the following technical solutions: The present invention provides a gluten-specific alpaca polyclonal antibody. Alpacas are immunized with wheat prolamin as an immunogen to obtain antiserum containing gluten that specifically recognizes gluten. The alpaca polyclonal antibody is then purified and separated based on an octanoic acid-ammonium sulfate precipitation method to prepare the alpaca polyclonal antibody.

[0011] The alpaca polyclonal antibody provided by the present invention uses wheat prolamin as an immunogen to immunize alpacas to obtain antiserum that can specifically recognize gluten. The antiserum is purified to separate and obtain the highest titer alpaca polyclonal antibody pAb with complete antigen recognition ability (about 55 kDa). Compared with traditional antibodies, the polyclonal antibody prepared by the present invention has a yield of 4.29 mg (pAb yield per milliliter of alpaca serum), and EC 50 The value is 2.06 μmol. The gluten-specific alpaca polyclonal antibody prepared by the present invention has high expression, high affinity and high sensitivity, and can be used to prepare more gluten detection products. Based on this, pAb is used as a capture antibody and biotinylation is performed to prepare a detection antibody. The present invention establishes a double antibody-streptavidin ELISA detection method system, which can not only significantly improve the sensitivity and specificity of the detection, but also the EC of alcohol-soluble protein is 50 The value was 51.21 ng / mL, and the detection limit was as low as 0.07 ng / mL. Through the dual recognition of specific capture antibodies and detection antibodies, different epitopes of the antigen were targeted, which significantly reduced cross-reactions and nonspecificity, providing reliable technical support for the rapid detection of gluten components in food. By optimizing the ELISA reaction conditions, the detection sensitivity and specificity were precisely controlled to avoid false positive results caused by nonspecific binding, providing a reference for the development of improving the accuracy of food allergen detection.

[0012] Furthermore, the steps for immunizing alpacas are as follows: wheat alcohol-soluble protein is mixed with Freund's complete adjuvant and then the alpacas are immunized three times, with Freund's incomplete adjuvant used for the second and third immunizations, and the three immunizations are separated by three weeks; after the third immunization, blood is collected, centrifuged, and serum is separated to obtain antiserum containing specific gluten recognition.

[0013] Furthermore, the purification method based on the caprylic acid-ammonium sulfate precipitation method is as follows: the antiserum is diluted with acetic acid-sodium acetate buffer and the pH is adjusted to 4~5, caprylic acid is added to the serum diluent at a volume ratio of 1:20~30, and the supernatant is collected by centrifugation; then ammonium sulfate is added to the supernatant to a final saturation of 40~50%, and dialyzed to obtain an alpaca polyclonal antibody solution.

[0014] As a more preferred embodiment, the present invention provides a method for preparing wheat prolamin, comprising dissolving wheat prolamin powder in a 0.15 M (pH 7.0) NaCl solution, stirring at room temperature for 0.5 to 1 hour, and then centrifuging to discard the supernatant; washing the precipitate with a 0.15 M NaCl solution, repeating the centrifugation; and redissolving the precipitate in a 50% to 70% ethanol aqueous solution and stirring to dissolve at room temperature.

[0015] The method for immunizing alpacas is as follows: alcohol-soluble protein and Freund's complete adjuvant are mixed in a volume ratio of 1:1 and the alpacas are immunized three times. The second and third immunizations use Freund's incomplete adjuvant, and the three immunizations are separated by three weeks. After the third immunization, blood is collected, centrifuged, and serum is separated and stored at -80°C for future use.

[0016] The method for separating serum proteins was as follows: the antiserum was diluted with acetic acid-sodium acetate buffer and the pH was adjusted to 4.5. Caprylic acid was added to the serum diluent at a volume ratio of 1:25, and the supernatant was collected by centrifugation. Ammonium sulfate was then added to the supernatant to a final saturation of 45%, and the pAb solution was dialyzed to obtain the solution, which was then stored at -20°C.

[0017] The present invention provides use of gluten-specific alpaca polyclonal antibodies in detecting gluten.

[0018] The present invention provides a double antibody-streptavidin ELISA gluten detection kit, which comprises a capture antibody and a detection antibody; the capture antibody is the above-mentioned alpaca polyclonal antibody, and the detection antibody is a biotinylated alpaca polyclonal antibody; The preparation method of the biotinylated alpaca polyclonal antibody comprises the following steps: dissolving sulfo-NHS-LC-biotin in dimethylaminoformamide, mixing the biotinylated alpaca polyclonal antibody with the alpaca polyclonal antibody at a molar ratio of 5 to 15:1, and then allowing the biotinylated alpaca polyclonal antibody to stand on ice to obtain the biotinylated alpaca polyclonal antibody.

[0019] Preferably, the kit further contains a buffer, an ELISA plate, a blocking solution, a secondary antibody, and a color developing solution.

[0020] More preferably, the detection method of the kit is as follows: diluting the capture antibody with a buffer solution, fixing it in an ELISA plate, and incubating; taking out the ELISA plate, washing it, and patting the liquid dry, adding a blocking solution, incubating, and drying the blocking solution, and inverting to dry; adding the analyte and incubating, then adding the detection antibody and incubating, and then adding the secondary antibody, and after incubation, adding the color developing solution, and terminating the reaction after color development.

[0021] The present invention also provides use of the above kit in detecting gluten allergens in food matrices.

[0022] The present invention provides a method for detecting gluten allergens in a food matrix, using the above-mentioned kit for detection.

[0023] Preferably, the capture antibody concentration used in the kit is 0.5~8 μg / mL, the detection antibody concentration is 0.5~8 μg / mL, the blocking solution is 1% gelatin, the secondary antibody is enzyme-labeled streptavidin diluted 1:2000~5000, and the buffer is PBS.

[0024] More preferably, the capture antibody concentration used in the kit is 4 μg / mL, the detection antibody concentration is 4 μg / mL, the blocking solution is 1% gelatin, the secondary antibody is enzyme-labeled streptavidin diluted 1:3000, and the buffer is 5 mM pH 7.5 PBS.

[0025] Preferably, the coating concentration of the coating agent is 4 μg / mL.

[0026] The present invention has the following beneficial effects: The present invention provides a gluten-specific alpaca polyclonal antibody and a double antibody-streptavidin ELISA gluten detection kit. The alpaca-derived gluten-specific polyclonal antibody prepared by the present invention has high expression levels, high affinity, and high sensitivity, can specifically identify gluten, and can be used to detect gluten allergens in food matrices. Based on the gluten-specific alpaca polyclonal antibody, a double antibody-streptavidin (SA-ELISA) detection method and kit are established. The SA-ELISA uses the alpaca polyclonal antibody as the capture antibody and a biotinylated alpaca polyclonal antibody as the detection antibody. The antibody is biotinylated using an alpaca anti-gliadin polyclonal antibody pAb. The method is simple, rapid, convenient, and low-cost to operate, making it suitable for replacing expensive secondary antibodies. This method overcomes the limitations of traditional methods for detecting gluten in food, improving detection efficiency while reducing experimental cost and complexity.

[0027] The detection method provided by the present invention can not only significantly improve the sensitivity and specificity of detection, but also 50 The value was 51.21 ng / mL, with a detection limit as low as 0.07 ng / mL. Through dual recognition by capture and detection antibodies, targeting different epitopes of the antigen significantly reduces cross-reactivity and nonspecificity. This method achieves a detection limit 4000-fold lower than the R5 antibody method specified in the Food Codex Alimentarius and 7000-fold lower than the sensitivity of the Cat. No. EKT-GL30 kit. This superior sensitivity provides reliable technical support for the rapid detection of gluten allergens in food matrices and is suitable for clinical and scientific applications such as immunoassay kits and immunoassay strips. BRIEF DESCRIPTION OF THE DRAWINGS

[0028] Figure 1 This is a graph showing the alpaca serum titer after the third immunization.

[0029] Figure 2 Figure 2 is the SDS-PAGE analysis and purification results of pAb.

[0030] Figure 3 This is the result diagram of the blocking buffer optimization for the double antibody-streptavidin ELISA.

[0031] Figure 4This is the buffer optimization result diagram for the double antibody-streptavidin ELISA.

[0032] Figure 5 Graph showing the results of optimizing the working concentration of antibodies for the double antibody-streptavidin ELISA.

[0033] Figure 6 This is the optimization result diagram of the double antibody-streptavidin ELISA system at different ionic strengths.

[0034] Figure 7 This is the pH optimization result of double antibody-streptavidin ELISA.

[0035] Figure 8 This is the optimization result of enzyme-labeled streptavidin at different dilution multiples in the double antibody-streptavidin ELISA.

[0036] Figure 9 This is the sensitivity result diagram of double antibody-streptavidin ELISA.

[0037] Figure 10 This is the matrix effect result of double antibody-streptavidin ELISA.

[0038] Figure 11 This is a graph showing the cross-reactivity results of the double antibody-streptavidin ELISA.

[0039] Figure 12 The graph shows the results of the spike recovery test for four food samples. DETAILED DESCRIPTION

[0040] The use of the detection kit for gluten allergens in food matrices provided by the present invention addresses the technical problem of the lack of highly sensitive and rapid methods in the prior art. The present invention is further illustrated below with reference to the accompanying drawings and specific examples, which are not intended to limit the present invention in any way. Unless otherwise specified, the reagents, methods, and equipment used in the present invention are conventional in the art.

[0041] Unless otherwise specified, the reagents and materials used in the following examples were commercially available.

[0042] Example 1 Preparation of polyclonal antibodies pAb specific for alpaca gluten recognition 1. Preparation of polyclonal antibodies This example dissolves wheat prolamin and uses it as an immunogen to immunize alpacas, obtaining alpaca antiserum that specifically recognizes gluten, and then isolating alpaca polyclonal antibodies from it. Specifically, the following steps are involved: (1) Pretreatment of wheat alcohol-soluble protein: Weigh 5 g of wheat alcohol-soluble protein and add 50 mL of 0.15 M NaCl solution (pH 7.0). Stir at room temperature for 1 hour to fully dissolve the albumin and globulin. Centrifuge at 5000 rpm for 15 minutes, collect the precipitate, and discard the supernatant. Wash the precipitate 1-2 times with a small amount of 0.15 M NaCl solution, using the same centrifugation conditions as above, to ensure that the albumin and globulin are completely removed. Add 50 mL of 60% (v / v) ethanol aqueous solution to the precipitate and stir at room temperature for 1 hour to fully dissolve the alcohol-soluble protein.

[0043] (2) Preparation of alpaca polyclonal antibodies: Alpaca immunization was performed with 4 mg of alcohol-soluble protein emulsified with the same volume of Freund's Complete Adjuvant, FCA (1:1, v / v) for the first immunization. Three weeks after the first immunization, the alpaca was boosted with alcohol-soluble protein mixed with Freund's Incomplete Adjuvant, FIA (4 mg, 1:1, v / v) and injected into the alpaca. After the third immunization, 50 mL of blood was collected and centrifuged at 3500 rpm for 10 min at 4°C to separate the serum, which was then stored at -80°C for later use.

[0044] (3) Serum protein separation: dilute the antiserum with acetic acid-sodium acetate buffer and adjust the pH to 4.5. Slowly add caprylic acid (25 mL / L serum diluent) while stirring. Stir for 30 minutes and then centrifuge. Collect the supernatant, then adjust the pH of the supernatant to 7.4 with PBS buffer. Add ammonium sulfate powder to a final concentration of 45% saturation at 4°C. Stir for 30 minutes and then centrifuge. Collect the precipitate, dissolve the precipitate with a small amount of dialysate, dialyze to remove excess salt, and store the resulting pAb solution at -20°C.

[0045] (4) Detection of antibody titer: The titer of alpaca serum was detected by indirect enzyme-linked immunosorbent assay (ID-ELISA). The specific method is as follows: dilute the alcohol-soluble protein standard with refrigerated dilution buffer, place it in a 96-well plate, and place 100 μL per well at 37°C for 12 hours. Wash twice with washing solution, then coat with blocking solution, 200 μL per well, incubate at 37°C for 3 hours, add serial dilutions of antibodies to the wells, and incubate at 37°C for 1 hour. After washing three times, add goat anti-camel pAb (H&L) labeled with horseradish peroxidase (HRP) to the plate, 100 μL per well, and incubate at 37°C for 30 minutes. After washing five times, add color development solution to the plate, 100 μL per well, incubate at 37°C for 10 minutes, and finally add stop solution (50 μL per well) to stop the reaction. Measure the absorbance at 450 nm.

[0046] The results of alpaca serum titer determination are as follows Figure 1 As shown, the green curve is the alpaca serum titer before immunization, the purple curve is the alpaca serum titer measured after the second immunization, and the blue curve is the alpaca serum titer measured after the third immunization. The highest alpaca serum titer measured after the third immunization is 1.28×10 7 .

[0047] Take 20 mL of serum after the third immunization to purify the pAb band and analyze it by SDS-PAGE. The results are as follows: Figure 2 As shown, a clear single band is observed at 55 kDa, consistent with the theoretical molecular weight. Calculations indicate a pAb yield of 4.29 mg per ml of alpaca serum.

[0048] 2. Biotinylated pAb To simplify the process and reduce costs, the carboxyl group of biotin was chemically linked to the S-amine group of the pAb for detection, based on the biotin-streptavidin system, the strongest known non-covalent interaction system. First, an appropriate amount of sulfo-NHS-LC-biotin was dissolved in dimethylformamide (DMF) to prepare a 10 mM solution. This 10 mM solution and the polyclonal antibody were then added at a 10:1 molar ratio. The reaction was incubated on ice for 2 hours and stored at -20°C until use.

[0049] Example 2 Establishment and Optimization of Double Antibody-Streptavidin ELISA 1. Establishment of a novel double antibody-streptavidin ELISA method According to the method for preparing alpaca-specific polyclonal antibodies recognizing gluten determined in Example 1, 50 mL of blood was collected after three immunizations, and the serum was separated by centrifugation at 3500 rpm for 10 min at 4°C. The serum was diluted with acetic acid-sodium acetate buffer and the pH was adjusted to 4.5. Caprylic acid was added at a ratio of (25 mL / L serum diluent), and the supernatant was collected by centrifugation. Ammonium sulfate was then added to the supernatant to a final saturation of 45%, and the supernatant was stored at -80°C until use.

[0050] To prepare alpaca biotinylated pAb, dissolve an appropriate amount of sulfo-NHS-LC-biotin in dimethylaminoformamide to prepare a 10 mM stock solution. Chemically link the carboxyl group of biotin to the S-amine group of the pAb. Add the 10 mM solution and purified polyclonal antibody at a 10:1 molar ratio. Incubate on ice for 2 hours. After dialysis, store the product at -20°C until further use. Optimize ELISA conditions.

[0051] ELISA solution preparation: 1.5 L 10 mM PBS solution (pH 7.4): Weigh 13.7 g NaCl, 0.2 g KCl, 1.44 g Na2HPO4, and 0.24 g KH2PO4 and dissolve in approximately 1.4 L deionized water. Stir to dissolve, then dilute to 1.5 L. Adjust the pH to 7.4 using a pH meter. 1 L 50 mM CBS solution (pH 9.6): Weigh 5.3 g Na2CO3 and 4.2 g NaHCO3, dissolve in 1 L deionized water, dilute to volume, and adjust the pH to 9.6 with dilute HCl or NaOH; 1 L 10 mM TBS solution (pH 8.0): Weigh 1.21 g Tris and dissolve in 800 mL deionized water. Adjust the pH to 8.0 with dilute HCl and make up to 1 L. 1.5 L coating buffer (pH 9.6): weigh 2.5 g Na2CO3 and 4.4 g NaHCO3 and dissolve them in pure water to make up to 1.5 L; 150 mL blocking solution: Weigh 4.5 g skim milk powder and dissolve it in PBS (pH 7.4) to make up to 150 mL. 20 mL 3% BSA: Weigh 0.6 g BSA and dissolve it in 20 mL deionized water; 20 mL 3% skim milk powder: weigh 0.6 g skim milk powder and dissolve it in 20 mL deionized water; 20 mL 1% casein: Weigh 0.2 g casein and dissolve it in 20 mL deionized water; 20 mL 1% gelatin: Weigh 0.2 g gelatin and dissolve it in 20 mL deionized water; Color development solution: Use a pipette to draw two-component TMB color development solution A and two-component TMB color development solution B in a 1:1 ratio; 1 L stop solution (10% concentrated sulfuric acid): Dissolve 100 mL of concentrated sulfuric acid in pure water and dilute to 1 L.

[0052] Using pAb as the capture antibody and biotinylated pAb as the detection antibody, the working concentration of antigen and antibody was determined by the checkerboard method. The specific method is: (1) Coating: Dilute the alpaca polyclonal antibody pAb that specifically recognizes gluten with coating buffer to a concentration of 0.5-8 μg / mL and immobilize it in a microtiter plate. Add 100 μL to each well and incubate at 4°C for 12 h. (2) Blocking: Take out the ELISA plate, wash it twice and pat dry the liquid. Add 120 μL of 1% gelatin (PBS) to each well and incubate it in a 37°C constant temperature water bath for 3 h. Then, spin dry the blocking solution and invert it in a 37°C oven to dry for 1 h. (3) Add the protein to be tested: dilute the alcohol-soluble protein standard 4-fold with refrigerated dilution buffer PBS solution to 4 ng / mL, add 100 μL to each well, use three columns for each antibody concentration, the first two columns are set as antibody detection wells, and the last column is set as a negative control well. Incubate in a constant temperature water bath at 37 °C for 40 min; (4) Add biotinylated antibody pAb: dilute its concentration to 0.5-8 μg / mL, add 100 μL to each well, and incubate in a 37°C constant temperature water bath for 30 min; (5) Add secondary antibody: horseradish peroxidase (HRP)-labeled goat anti-camel IgG (H&L) secondary antibody, diluted 1:3000 in 5 mM pH 7.5 PBS with enzyme-labeled streptavidin. Add 100 μL to each well and incubate in a 37°C constant temperature water bath for 30 min. (6) Add color development solution: Add 100 μL of color development solution to each well, develop at 37°C for 10 min, and then add 50 μL of color development solution to each well to terminate the reaction. (7) Read the results: Use an enzyme-labeled instrument to measure the absorbance at 450 nm, record the data and calculate the P / N. The optimal condition is when the ratio is the largest.

[0053] 2. Optimization of conditions Based on the double antibody-streptavidin ELISA method established above, the experimental conditions were optimized, specifically optimizing the concentration of paired antibodies, blocking solution type, buffer type, salt ion concentration, pH value, and enzyme-labeled streptavidin dilution ratio.

[0054] For sandwich ELISA assays, the type of blocking buffer will affect the OD value of the negative control on the microplate. 450 The absorbance at mm was first optimized with different blocking solutions and buffers, and then measured with different blocking solutions and buffers. Figure 3 and Figure 4 As shown in the figure, when the blocking solution was 1% gelatin and the buffer was 10 mM pH 7.4 PBS, the highest P / N values ​​were 2.9 and 2.7, which can be used for subsequent ELISA experimental condition optimization.

[0055] Since the concentration of paired antibodies will affect the minimum detection limit of the method, thus leading to false positives in actual sample detection, the working concentration of the antibodies was optimized. Figure 5 As shown in the checkerboard plot, the P / N value was the highest when the capture antibody pAb concentration was 4 μg / mL and the detection antibody pAb concentration was 4 μg / mL.

[0056] In the ELISA system, ionic strength can affect the specific binding interaction between antigens and antibodies, which may affect the sensitivity of the test. Therefore, the effects of different ionic strengths (5, 10, 20, 30 and 40 mM) were investigated. The results are as follows: Figure 6 As shown in the figure, when the ionic strength was 5 mM, the limit of detection (LOD) was 0.96 ng / mL.

[0057] The influence range of pH value (pH 6.5-8.5) was then investigated, and the results were as follows: Figure 7 As shown, the LOD was as low as 0.76 ng / mL at pH 7.0.

[0058] Finally, the experiment was carried out using enzyme-labeled streptavidin with different dilutions. The results were as follows: Figure 8 As shown, a LOD as low as 0.07 ng / mL was obtained when enzyme-labeled streptavidin was diluted 1:3000.

[0059] In summary, the optimization results showed that the entire detection system with 1% gelatin as blocking solution, 1:3000 dilution of enzyme-labeled streptavidin, and 5 mM pH 7.5 PBS as buffer had better detection effect.

[0060] A standard curve of double antibody-streptavidin ELISA based on alpaca pAb polyclonal antibody was constructed in the optimized reaction system, such as Figure 9 As shown in the figure, the results showed that the EC50 value of prolamin was 51.21 ng / mL and the limit of detection (LOD) was 0.07 ng / mL.

[0061] 3. Determination of detection limit and specificity The absorbance mean and standard deviation were calculated by repeating the measurement of blank matrix samples 10 times, and the precision of the experimental system was verified by relative standard deviation (RSD = standard deviation / mean × 100%). The optimized double antibody-streptavidin ELISA method was used to detect cross-reactivity of 10 non-target food allergens (zein, folliculin, ovalbumin, α-casein, β-casein, β-lactoglobulin, pea protein concentrate, chickpea protein concentrate, soy protein concentrate, and peanut crude extract).

[0062] The results are as follows Figure 10 As shown in the figure, the pAb showed high binding activity to hordein or ergosterin, which is the same as the existing antibody R5 and meets the requirements for detecting gluten in barley and rye. In addition, the pAb did not show cross-reactivity to alcohol-soluble proteins that were not required for detection, such as avenin, quinoa and zein. At the same time, in order to prevent false positive results in actual applications, the cross-reactivity of the pAb to unrelated common allergens was also evaluated, and the results are shown in the figure. Figure 11As shown, cross-reactivity to ovomucin, ovalbumin, α-casein, β-casein, β-lactoglobulin, pea protein, chickpea protein, soy protein and peanut crude extract was very low.

[0063] In summary, the dual-antibody-streptavidin ELISA method established in this example uses capture and detection antibodies to specifically dually recognize different epitopes of the antigen, effectively reducing cross-reactions and nonspecific signals. Condition optimization further suppresses background interference. The method's minimum detection limit is significantly lower than the maximum residual gluten-free protein level of 20 mg / kg. Furthermore, compared to existing studies using rabbit polyclonal antibodies that require highly diluted serum, the alpaca polyclonal antibody pAb strategy employed in this invention simplifies the experimental process and reduces detection costs while maintaining high sensitivity, overcoming the operational complexity drawbacks of existing technologies.

[0064] Example 3 Analysis and determination verification of spiked samples Milk, gluten-free almond milk, gluten-free rice flour, and gluten-free soy sauce samples that had been tested negative for gluten using a commercial gluten detection kit were purchased from local farmers' markets. Three concentrations of alcohol-soluble proteins were added to each sample for recovery testing. Subsequently, 10 mL of refrigerated milk or almond milk was placed in a 15 mL centrifuge tube and centrifuged at 4500 rpm for 10 minutes. One gram of rice flour or 1 mL of soy sauce was mixed with 1 g of skim milk powder and 10 mL of 60% ethanol. The mixture was centrifuged at 4500 rpm for 10 minutes. After discarding the upper fat layer, the solution was diluted several times with PBS for testing.

[0065] At the same time, the domestic commercial kit Cat.No.EKT-GL30 was used for verification. The coefficient of determination (R 2 ), and use it to characterize correlation.

[0066] The results of the additive recovery test of four food samples are shown in Table 1. Figure 12 As shown, the recovery of milk extract decreased significantly after dilution, indicating that the calibration curve for undiluted milk extract can be applied to the detection of alcohol-soluble proteins. Furthermore, the recoveries of almond milk extract after 0- and 8-fold dilutions were within a reasonable range of 80-120%, which is acceptable. However, considering that higher dilutions can lead to reduced sensitivity, a 0-fold dilution was selected for further analysis. Similarly, calibration curves constructed using 10-fold diluted rice flour and gluten-free soy sauce extracts enabled the quantitative detection of alcohol-soluble proteins, demonstrating that this 10-fold dilution effectively eliminates matrix interference.

[0067] Table 1 Addition recovery test results

[0068] The test results showed that the limits of detection (LOD) and limits of quantification (LOQ) of the four food samples were as follows: LOD and LOQ in milk were 0.94 ng / mL and 2.06 ng / mL, respectively; LOD and LOQ in almond milk were 0.33 ng / mL and 0.86 ng / mL, respectively; LOD and LOQ in rice milk were 4.28 ng / g and 42.23 ng / g, respectively; and LOD and LOQ in soy sauce were 34.5 ng / g and 60.08 ng / g, respectively.

[0069] The double antibody-streptavidin ELISA method gave an acceptable average recovery range of 80.66% to 104.37%, and the coefficient of variation (CV) was less than 11%.

[0070] In addition, the accuracy of sandwich ELISA was evaluated using the existing domestic commercial kit Cat.No.EKT-GL30 kit. The results showed that the correlation coefficient (R 2 ) is greater than 0.9751, demonstrating that the dual-antibody-streptavidin ELISA method can be used to detect gluten allergens in food matrices with acceptable accuracy and reproducibility. However, the use of the existing commercial R5 antibody kit requires a 500-fold dilution of the sample, while the use of the Cat. No. EKT-GL30 kit requires a 50-fold dilution, which can lead to inaccurate test results. Furthermore, the detection limit of the currently available R5 antibody kit is 0.3 ppm (i.e., 300 ng / mL), and the detection limit of the Cat. No. EKT-GL30 kit is 0.5 ppm (i.e., 500 ng / mL). However, the detection limit of the dual-antibody-streptavidin ELISA method provided by the present invention is as low as 0.07 ng / mL, which is 4000-fold lower than the detection limit of the official R5 antibody method specified in the Food Code and 7000-fold less sensitive than the Cat. No. EKT-GL30 kit, demonstrating superior sensitivity and specificity.

[0071] In summary, the camel-derived polyclonal antibody for food-specific gluten recognition provided by the present invention can meet the detection requirements and has good recognition ability for gluten allergens in food matrices, and is an essential material for establishing a method for detecting gluten allergens in food matrices. The highly sensitive detection kit and detection method for gluten allergens in food matrices established based on the camel-derived polyclonal antibody, using alpaca polyclonal antibody pAb as the capture antibody and biotinylated pAb as the detection antibody, is used to detect food gluten by double antibody-streptavidin ELISA, which has good sensitivity and specificity. 50The value was 51.21 ng / mL, with a detection limit as low as 0.07 ng / mL, which is 7,000 times lower than the detection limit of the official R5 antibody method specified by the Food Codex. The method and detection kit provided by this invention not only reduce testing costs but also provide reliable technical support for gluten allergen detection. They are suitable for clinical and scientific applications such as immunoassay kits and immunoassay strips, and have broad development prospects.

[0072] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, rather than to limit them. Although the present invention has been described in detail with reference to the above embodiments, those skilled in the art should understand that they can still modify the technical solutions described in the above embodiments, or replace some or all of the technical features therein with equivalents. These modifications or replacements do not deviate from the essence of the corresponding technical solutions within the scope of the technical solutions of the embodiments of the present invention. Any other changes, modifications, substitutions, combinations, and simplifications that do not deviate from the spirit and principles of the present invention shall be considered equivalent replacement methods and are included in the scope of protection of the present invention.

Claims

1. A gluten-specific alpaca polyclonal antibody, characterized in that The polyclonal antibody uses wheat alcohol-soluble protein as an immunogen, immunizes alpacas to obtain antiserum containing gluten that specifically recognizes gluten, and then purifies and separates based on an octanoic acid-ammonium sulfate precipitation method to prepare the alpaca polyclonal antibody.

2. The polyclonal antibody according to claim 1, characterized in that The steps for immunizing alpacas are as follows: wheat alcohol-soluble protein is mixed with Freund's complete adjuvant and the alpacas are immunized three times respectively. The second and third immunizations use Freund's incomplete adjuvant, and the three immunizations are separated by three weeks; after the third immunization, blood is collected, centrifuged, and serum is separated to obtain antiserum containing specific gluten recognition.

3. The polyclonal antibody according to claim 1, characterized in that The purification method based on the caprylic acid-ammonium sulfate precipitation method is as follows: the antiserum is diluted with acetic acid-sodium acetate buffer and the pH is adjusted to 4-5, caprylic acid is added to the serum diluent at a volume ratio of 1:20-30, and the supernatant is collected by centrifugation; then ammonium sulfate is added to the supernatant to a final saturation of 40-50%, and dialyzed to obtain an alpaca polyclonal antibody solution.

4. Use of the polyclonal antibody according to any one of claims 1 to 3 in detecting gluten.

5. A double antibody-streptavidin ELISA gluten detection kit, characterized in that: The kit comprises a capture antibody and a detection antibody; The capture antibody is the alpaca polyclonal antibody according to any one of claims 1 to 3, and the detection antibody is the biotinylated alpaca polyclonal antibody according to any one of claims 1 to 3; The preparation method of the biotinylated alpaca polyclonal antibody comprises the following steps: dissolving sulfo-NHS-LC-biotin in dimethylaminoformamide, mixing the mixture with the alpaca polyclonal antibody according to claim 1 at a molar ratio of 5 to 15:1, and then allowing the mixture to stand on ice to obtain the biotinylated alpaca polyclonal antibody.

6. The kit according to claim 5, characterized in that The kit also contains a buffer solution, an ELISA plate, a blocking solution, a secondary antibody, and a color developing solution.

7. The kit according to claim 6, characterized in that The detection method of the kit is as follows: diluting the capture antibody with a buffer solution, fixing it in an ELISA plate, and incubating; taking out the ELISA plate, washing it, patting the liquid dry, adding a blocking solution, incubating, and then drying the blocking solution by shaking, and inverting to dry; adding the analyte and incubating, then adding the detection antibody and incubating, then adding the secondary antibody, and after incubation, adding the color developing solution, and terminating the reaction after color development.

8. Use of the kit according to claim 5 or 6 in the detection of gluten allergens in food matrices.

9. A method for detecting gluten allergens in a food matrix, characterized in that: The kit according to claim 6 is used for only detection.

10. The method according to claim 9, characterized in that: The capture antibody concentration used in the kit is 0.5~8 μg / mL, the detection antibody concentration is 0.5~8 μg / mL, the blocking solution is 1% gelatin, the secondary antibody is enzyme-labeled streptavidin diluted 1:2000~5000, and the buffer is PBS.

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