Fluorescence immunochromatography detection test strip for OVA-D in surimi product and application of fluorescence immunochromatography detection test strip
By designing a fluorescent immunochromatographic test strip for OVA-D in surimi products and using fluorescent quantum dot-labeled antibodies for detection, the problem of complex and expensive detection in existing technologies has been solved, achieving simple, rapid and sensitive OVA-D detection.
Patent Information
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2024-09-02
- Publication Date
- 2026-03-10
AI Technical Summary
Existing technologies are difficult to use quickly, easily and accurately to detect duck egg ovalbumin (OVA-D) in surimi products, especially in on-site testing where there are problems such as expensive instruments and complicated sample pretreatment.
A fluorescent immunochromatographic test strip for detecting OVA-D in surimi products was designed. It utilizes fluorescent quantum dot-labeled rabbit anti-OVA-D polyclonal antibody and goat IgG, combined with quantitative detection methods, to achieve detection through fluorescence signal analysis.
It enables simple, rapid and sensitive OVA-D detection, reduces the occurrence of false positive results, and ensures the accuracy and reliability of the test.
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Figure CN121633468A_ABST
Abstract
Description
TECHNICAL FIELD
[0001] The application belongs to the technical field of food allergen detection, and particularly relates to an OVA-D fluorescent immunochromatographic detection test strip for surimi products and application thereof. BACKGROUND
[0002] Duck egg ovalbumin (OVA-D) is the most abundant protein in duck egg white, composed of 386 amino acid residues, with a molecular weight of about 45 kDa. OVA-D molecules contain disulfide bonds and free sulfhydryl groups, which give it good gelation, emulsification and water retention properties, so it is widely used in the food industry. However, OVA-D is also one of the main allergens in duck egg white, which is stable in nature and not easily destroyed. After being ingested by the human body, it can cause immediate allergic reactions mediated by IgE. This allergic reaction can cause allergic reactions in the respiratory tract, digestive tract and skin, and even life-threatening in severe cases. Therefore, it is necessary to detect and correctly identify foods or raw materials containing ovalbumin.
[0003] Surimi products are gelatinous foods with certain elasticity made from raw or frozen surimi through a series of processing processes, which are deeply loved by consumers and have become an indispensable part of daily diet. However, some vendors add egg white and other non-fish meat proteins to low-value fish slurry to increase the gel strength of surimi, which damages the rights and interests of consumers. In addition, with the growing demand for salted egg yolk market and the development of desalination technology, salted egg white, as a byproduct of processing, is widely used in the production process of frozen surimi or raw fish slurry, but due to the lack of clear identification, it increases the risk of allergy.
[0004] Currently, the methods for detecting OVA-D in surimi mainly include mass spectrometry and immunoassay. Mass spectrometry, including high-performance liquid chromatography and tandem mass spectrometry, separates OVA-D from complex matrix by chromatographic separation principle, and then uses mass spectrometry detector to determine its structure information, thereby achieving detection. The advantage of this method is that the detection result is reliable and can quantify OVA-D, but the disadvantage is that it requires expensive instruments and professional operation, and the sample pretreatment process is complicated and time-consuming, which is not suitable for on-site rapid detection. SUMMARY
[0005] The purpose of the present application is to provide an OVA-D fluorescent immunochromatographic detection test strip for surimi products.
[0006] Another purpose of the present application is to provide a method for detecting OVA-D in surimi products.
[0007] The technical solution of the present application is as follows:
[0008] The OVA-D fluorescent immunochromatographic test strip for detecting surimi products comprises a substrate and a sample pad, a binding pad, a reaction membrane and a water absorption pad arranged on the substrate, the reaction membrane is arranged at the middle part of the substrate, one end of the sample pad is connected to the other end of the binding pad, one end of the binding pad is connected to the other end of the reaction membrane, and one end of the reaction membrane is connected to the other end of the water absorption pad.
[0009] The binding pad contains fluorescent quantum dot labeled rabbit anti-OVA-D polyclonal antibody and fluorescent quantum dot labeled goat IgG.
[0010] The reaction membrane is provided with a detection zone, the detection zone is provided with a detection line and a control line from the other end to one end of the reaction membrane, the detection line is coated with OVA-D antigen, and the control line is coated with rabbit anti-goat IgG antibody.
[0011] In a preferred embodiment of the present application, the reaction membrane is a nitrocellulose membrane.
[0012] In a preferred embodiment of the present application, the substrate is a PVC plate.
[0013] In a preferred embodiment of the present application, the reaction membrane is a nitrocellulose membrane, and the substrate is a PVC plate.
[0014] The method for detecting OVA-D in surimi products comprises the following steps: dissolving a surimi sample with a sample treatment buffer, standing to obtain supernatant, then adding the supernatant dropwise to the sample pad of the OVA-D fluorescent immunochromatographic test strip for detecting surimi products to react, and detecting the fluorescence signal by a dry-type fluorescence detector after the reaction is completed.
[0015] In a preferred embodiment of the present application, the sample treatment buffer is PBS with a pH of 7.4 and containing Tween-20.
[0016] Further preferably, the content of Tween-20 in the PBS is 0.5%.
[0017] More preferably, the concentration of the PBS is 10 mmol / L.
[0018] In a preferred embodiment of the present application, the detection is qualitative detection.
[0019] In a preferred embodiment of the present application, the detection is quantitative detection.
[0020] The present application has the following beneficial effects:
[0021] 1、The fluorescent signal of the application is stable, the fluorescent intensity of T line and C line is not affected by complex matrix in the detected sample, the detection sensitivity is higher, and the reliability of quantitative detection of duck egg albumin is also guaranteed.
[0022] 2、In the detection process of the application, the pretreatment process is simpler and faster, the solvent consumption is less, and the processing time is short. It can be quantitative detection and qualitative detection, has good detection accuracy and sensitivity, and is not easy to appear false positive results. BRIEF DESCRIPTION OF DRAWINGS
[0023] Figure 1 It is a structure schematic diagram of OVA-D fluorescent immunochromatographic test strip in the specific embodiment of the application.
[0024] Figure 2 It is a reduced SDS-PAGE map of OVA-D separated and purified in the specific embodiment of the application. Wherein: M, standard protein; 1, OVA-D.
[0025] Figure 3 It is a mass spectrum identification result graph of two protein bands of OVA-D in the specific embodiment of the application. Wherein: A, amino acid sequence alignment result of OVA-D-up; B, amino acid sequence alignment result of OVA-D-down.
[0026] Figure 4 It is an SDS-PAGE map of rabbit anti-OVA-D polyclonal antibody separated and purified by Protein A affinity chromatography column in the specific embodiment of the application. Wherein: M, standard protein.
[0027] Figure 5 It is a Western Blot result graph of verifying the specificity and affinity of rabbit anti-OVA-D polyclonal antibody in the specific embodiment of the application. Wherein: M, standard protein; 1, whole egg white protein of chicken egg; 2, whole egg white protein of duck egg; 3, rough protein of abalone; 4, rough protein of sea bass; 5, rough protein of scallop; 6, rough protein of South American white prawn.
[0028] Figure 6 It is a competitive inhibition curve and a standard curve in the specific embodiment of the application. DETAILED DESCRIPTION
[0029] The technical solutions of the application are further described and explained in the following specific embodiments combined with the drawings.
[0030] As Figure 1 and 2As shown, an OVA-D fluorescent immunochromatographic test strip for detecting surimi products comprises a substrate 1 (PVC plate) and a sample pad 2, a binding pad 3 (glass fiber pad), a reaction membrane 4 (nitrocellulose membrane) and a water absorption pad 5 arranged on the substrate 1. The reaction membrane 4 is arranged at the middle part of the substrate 1, and the other end of the binding pad 3 is overlapped below one end of the sample pad 2. The other end of the water absorption pad 5 is overlapped above one end of the reaction membrane 4. The other end of the water absorption pad 5 is overlapped above one end of the reaction membrane 4.
[0031] The binding pad 3 contains fluorescent quantum dot labeled rabbit anti-OVA-D polyclonal antibody and fluorescent quantum dot labeled goat IgG.
[0032] The reaction membrane 4 is provided with a detection zone, which is provided with a detection line 41 and a control line 42 from the other end to one end of the reaction membrane 4. The detection line 41 is coated with OVA-D antigen, and the control line 42 is coated with rabbit anti-goat IgG antibody.
[0033] The preparation of the above OVA-D antigen comprises: based on the prior art (Geng, Xie, Wang, et al. Large-scale purification of ovalbumin using polyethylene glycol precipitation and isoelectric precipitation [J]. Poultry Science, 2019, 98(3): 1545-1550. DOI: 10.3382 / ps / pey402. and Zhao Yongjuan, Weng Ling, Yan Longjie, et al. Competitive ELISA for detecting egg white content in surimi products [J]. Food Science, 2017, 38(14): 284-289. DOI: 10.7506 / spkx1002-6630-201714044.), duck egg white is pretreated by polyethylene glycol aqueous two-phase extraction method, and the protein is eluted and collected by anion exchange chromatography to obtain OVA-D antigen.
[0034] Preferably, the method comprises: duck egg white is diluted with 50 mmol / L NaCl solution at a ratio of 1:3 (v:v), and stirred at room temperature for 2 h. The pH value of the solution is adjusted to pH 5.0, and the supernatant is obtained after centrifugation for 0.5 h. Polyethylene glycol 8000 (PEG 8000) is added to the egg white solution in three times, and the final PEG 8000 addition amount is 15% (v:w) of the solution volume. The supernatant is obtained after centrifugation for 0.5 h. The supernatant is dialyzed in 20 mmol / L Tris-HCl buffer to equilibrium, and then loaded on a DEAE-anion exchange chromatography column. The purified OVA-D antigen is collected from the third protein elution peak separated by 20 mmol / L Tris-HCl buffer containing 0-0.2 mol / L NaCl.
[0035] The third protein elution peak is collected and analyzed by reducing SDS-PAGE, and the final result is shown in Figure 2 The results show that there are only two protein bands (named OVA-D-up and OVA-D-down, respectively) at the position of about 45 kDa, which is speculated to be the result of glycosylation of OVA-D. In order to verify the two protein bands, the gel is cut and subjected to mass spectrometry identification, and the detection results are shown in Figure 3 The results show that the two protein bands at the position of about 45 kDa are the same protein OVA-D, and the purity is high.
[0036] The preparation of the above fluorescent quantum dot labeled rabbit anti-OVA-D polyclonal antibody comprises:
[0037] (1) According to the prior art (Hong Qian. Molecular cloning, in vitro expression and antibody preparation of Litopenaeus vannamei polyphenol oxidase [D]. Jimei University, 2023. DOI: 10.27720 / d.cnki.gjmdx.2023.000209.), OVA-D is used as an antigen to immunize New Zealand male rabbits. After immunization, the serum is obtained by centrifugation of whole blood, and the rabbit anti-OVA-D polyclonal antibody is obtained by Protein A affinity chromatography;
[0038] Preferably, the step comprises: immunizing New Zealand male rabbits with the above OVA-D antigen at a concentration of 2 mg / mL, a total of 4 times, and collecting the serum after immunization and purifying it by Protein A affinity chromatography. The elution peak collected is the rabbit anti-OVA-D polyclonal antibody;
[0039] After immunizing New Zealand male rabbits with OVA-D antigen, the rabbit serum is separated and purified by Protein A affinity chromatography, and the single elution peak after elution is analyzed by SDS-PAGE. The results are shown in Figure 4As shown, protein bands were observed only at 55 kDa and 25 kDa, corresponding to the heavy and light chains of IgG. Western blotting was used to verify the specificity and affinity of the antibody for the OVA-D antigen. The results are as follows... Figure 5 As shown, the results indicate that the OVA-D polyclonal antibody only reacts with OVA-D and does not have any cross-reaction with other proteins in duck egg white or fish paste, indicating that the OVA-D polyclonal antibody has good specificity and affinity.
[0040] (2) According to the existing technology (Yuhao H, Ranran L, Wenye Z, et al. Development of afluorescent multiplexed lateral flow immunoassay for the simultaneous detection of crustacean allergen tropomyosin, sarcoplasmic calcium bindingprotein and egg allergen ovalbumin in different matrices and commercial foods [J]. FoodChemistry, 2024, 440: 138275-.
[0041] DOI:10.1016 / j.foodchem.2023.138275.), fluorescent quantum dots are activated using a chemical cross-linking agent, and then coupled with rabbit anti-OVA-D polyclonal antibody to obtain the fluorescent quantum dots. The fluorescent quantum dots are preferably cadmium selenide quantum dots (CdSe QDs) with a particle size of about 5 nm, an excitation wavelength of 620 nm-630 nm, and activated carboxyl groups on the surface. The chemical cross-linking agent is preferably a mixture of 0.35% NHS (N-Hydroxysuccinimide) and 0.2% EDC (1-Ethyl-3-(3-dimethylaminopropyl)carbodiimide) in a volume ratio of 1:1.
[0042] Preferably, this step includes:
[0043] ① Activation of fluorescent quantum dots: Add 100 μL of 40 mmol / L PBS (pH 6.0) containing 0.4 mg / mL fluorescent quantum dots to two 0.5 mL EP tubes respectively, then add 2.5 μL of 0.2% EDC and 2.5 μL of 0.35% NHS respectively, mix well, and place in a shaker at 37℃, 220 rpm, and react in the dark for 30 min to obtain the activated fluorescent quantum dot solution;
[0044] ② Coupled labeling: Add 0.2 mol / L PB (pH 6.5) buffer to the activated fluorescent quantum dot solution at a ratio of 20:1 (v:v), mix well, then add rabbit anti-OVA-D polyclonal antibody to a final concentration of 0.15 mg / mL, vortex mix, and then react in a shaker at 37℃ and 220 rpm in the dark for 2-4 h to obtain a fluorescent quantum dot-labeled rabbit anti-OVA-D polyclonal antibody solution;
[0045] The preparation of the above-mentioned fluorescent quantum dot-labeled goat IgG is basically the same as the preparation of fluorescent quantum dot-labeled rabbit anti-OVA-D polyclonal antibody. The difference is that: after the activated fluorescent quantum dot solution is mixed with 0.2 mol / L BB (pH 8.2) buffer at a ratio of 20:1 (v:v), goat IgG (purchased from Shanghai Beyotime Biotechnology Co., Ltd., catalog number: A7007) to a final concentration of 0.5 mg / mL, a fluorescent quantum dot-labeled goat IgG solution is obtained.
[0046] The specific processing of the aforementioned conjugate pad 3 includes: based on existing technology (Chen Guoxin. Quantitative detection of fungal toxins based on aggregation-induced emission microspheres fluorescence immunochromatography [D]. Nanchang University, 2023. DOI:10.27232 / d.cnki.gnchu.2023.003878.), the above-mentioned fluorescent quantum dot-labeled rabbit anti-OVA-D polyclonal antibody solution, fluorescent quantum dot-labeled goat IgG solution, and resuspension are mixed evenly at a volume ratio of 3:1:1, and then sprayed onto the conjugate pad 3 using a gold sprayer, and finally freeze-dried under vacuum. The resuspension is a buffer solution of 50 mmol / L PBS (pH 7.4) containing 0.1% Tween-20 and 5% trehalose.
[0047] The treatment of reaction membrane 4 specifically included: using a membrane scribing machine, 1 mg / mL of OVA-D antigen and 1 mg / mL of rabbit anti-goat IgG (purchased from Thermo Fisher Scientific, catalog number: A16144) were scribed onto the detection line 41 (T line) and control line 42 (C line) of reaction membrane 4, respectively. The scribing volume was 1 μL / cm, for a total of 30 cm. After scribing, reaction membrane 4 was dried in a constant temperature drying oven at 37℃ for 4-12 hours.
[0048] The specific method of using this invention is as follows:
[0049] (1) Dissolve the fish paste sample in sample processing buffer, let it stand for 5-10 min, obtain the supernatant, and dilute it appropriately to obtain the sample solution; the sample processing buffer is 10 mmol / L PBS (pH 7.4) buffer containing 0.5% Tween-20;
[0050] (2) The sample solution is dropped onto the sample pad 2 of the test strip. Due to the capillary force of the absorbent pad 5, the liquid first reaches the conjugate pad 3. The duck egg white protein in the sample solution binds to the rabbit anti-OVA-D polyclonal antibody labeled with fluorescent quantum dots in the conjugate pad 3 to form a complex, namely OVA-(rabbit anti-OVA-D polyclonal antibody)-fluorescent quantum dots. The sample solution also contains rabbit anti-OVA-D polyclonal antibodies that have not bound to the OVA-D antigen. When the liquid continues to move to the T line on the reaction membrane 4, the remaining rabbit anti-OVA-D polyclonal antibody in the liquid will form a complex with the OVA-D antigen here and be fixed on the T line. The goat IgG labeled with fluorescent quantum dots does not react with the OVA-D antigen and does not form a complex. It continues to move to the C line and can bind to the rabbit anti-goat IgG antibody.
[0051] It should be noted that since the amount of fluorescent quantum dot-labeled rabbit anti-OVA-D polyclonal antibody on the binding pad is fixed (derived from optimized experiments, and considering factors such as detection sensitivity and cost, too much or too little is detrimental to the detection effect), if the amount of OVA-D antigen in the sample solution exceeds the amount that the fluorescent quantum dot-labeled rabbit anti-OVA-D polyclonal antibody can bind, the remaining OVA-D antigen and antigen-antibody conjugate will continue to flow with the liquid. When it flows to the T line, the OVA-D antigen present on the T line cannot be recognized and bound by the antibody, and the band will not show a color reaction, which can be considered as positive. Conversely, if there is excess fluorescent quantum dot-labeled rabbit anti-OVA-D polyclonal antibody in the sample solution, it will continue to bind to the OVA-D antigen on the T line, at which point the T line will produce a band color. At the same time, the presence of the C line is to verify the effectiveness of the invention. It is immobilized with rabbit anti-goat IgG antibody that can bind to fluorescent quantum dot-labeled goat IgG, ensuring that it is not affected by the presence of positive samples in the sample regardless of the experimental conditions, and always shows a red line, thus proving that the experiment is proceeding normally. If the C line does not show red, it indicates that the invention is abnormal and has lost its detection function.
[0052] (3) Under the illumination of a 365nm ultraviolet lamp, the above C line and T line will emit fluorescence of different intensities. The fluorescence signal values on the T line and C line are detected, and the fluorescence signal values on the T line and C line are substituted into the standard curve to calculate the concentration of duck egg albumin in the sample to be tested.
[0053] Specifically: OVA-D antigen solutions containing different concentrations (0, 0.25, 0.1, 1, 5, 10, 25, 50, 100 μg / mL) are added dropwise to the sample pad 2 of the test strip of this invention, with an addition volume of 75 μL. The reaction time is 15 min. After the reaction, the fluorescence signal intensity is detected using a dry immunofluorescence detector, and a standard curve is plotted based on the detection results. The fish paste sample is dissolved in sample processing buffer, and after standing for 5-10 min, the supernatant is the sample solution. The sample solution is diluted 3-fold and added dropwise to the sample pad 2 of the test strip of this invention for reaction. After the reaction, the fluorescence signal is detected using a dry immunofluorescence detector, and the OVA-D content is calculated by combining the final measured fluorescence signal intensity with the standard curve.
[0054] The competitive inhibition curves and standard curves were plotted based on the results obtained from measuring different concentrations of OVA-D antigen, as shown below. Figure 6 As shown.
[0055] The above description is merely a preferred embodiment of the present invention, and therefore should not be construed as limiting the scope of the present invention. All equivalent changes and modifications made in accordance with the scope of the patent and the contents of the specification should still fall within the scope of the present invention.
Claims
1. An OVA-D fluorescent immunochromatographic test strip in a surimi product, characterized by: The fish surimi product comprises a substrate and a sample pad, a binding pad, a reaction membrane and an absorbent pad arranged on the substrate, the reaction membrane is arranged at the middle part of the substrate, one end of the sample pad contacts the other end of the binding pad, one end of the binding pad contacts the other end of the reaction membrane, and one end of the reaction membrane contacts the other end of the absorbent pad. The binding pad contains fluorescent quantum dot labeled rabbit anti-OVA-D polyclonal antibody and fluorescent quantum dot labeled goat IgG, and the reaction membrane is provided with a detection zone, the detection zone is provided with a detection line and a control line from the other end to one end of the reaction membrane, the detection line is coated with OVA-D antigen, and the control line is coated with rabbit anti-goat IgG antibody.
2. The OVA-D fluorescent immunochromatographic test strip for surimi products according to claim 1, characterized in that: The reaction membrane is a nitrocellulose membrane.
3. The OVA-D fluorescent immunochromatographic test strip for surimi products according to claim 1, characterized in that: The substrate is a PVC plate.
4. The OVA-D fluorescent immunochromatographic test strip in a surimi product according to claim 1, characterized by: The reaction membrane is a nitrocellulose membrane, and the substrate is a PVC plate.
5. A method for detecting OVA-D in a surimi product, characterized by: The fish surimi sample is dissolved with a sample treatment buffer, and the supernatant is obtained by standing, then the supernatant is added dropwise to the sample pad of the OVA fluorescent immunochromatographic detection test strip in the fish surimi product to react, and the fluorescence signal is detected by a dry-type fluorescence detector after the reaction is completed, so as to detect.
6. The method for detecting OVA-D in a surimi product according to claim 5, wherein: The sample treatment buffer is PBS with pH of 7.4 and containing Tween-20.
7. The method for detecting OVA-D in a surimi product according to claim 6, wherein: The content of Tween-20 in the PBS is 0.5%.
8. The method for detecting OVA-D in a surimi product according to claim 7, wherein: The concentration of the PBS is 10 mmol / L.
9. The detection method according to any one of claims 5 to 8, characterized in that: The detection is qualitative detection.
10. The assay method according to any one of claims 5 to 8, wherein: The detection is quantitative detection.