CN112505026A - Visual gel detection device and method for soybean allergen - Google Patents
Visual gel detection device and method for soybean allergen Download PDFInfo
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- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
- G01N33/531—Production of immunochemical test materials
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- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
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- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
- G01N33/6854—Immunoglobulins
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- G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
- G01N2333/415—Assays involving biological materials from specific organisms or of a specific nature from plants
Abstract
The invention provides a soybean allergen visual gel detection device and method, wherein the device comprises: the solid-phase extraction column is arranged in the solid-phase extraction column, and the detection layer, the air layer and the quality control layer are sequentially arranged along the direction from the liquid inlet to the liquid outlet; the detection layer consists of a first upper-layer sieve plate, a coupling adhesive containing an anti-soybean protein antibody and a first lower-layer sieve plate, and the quality control layer consists of a second upper-layer sieve plate, a coupling adhesive containing an anti-horseradish peroxidase antibody and a second lower-layer sieve plate. The visual gel detection device provided by the invention can be used for detecting the soybean or the allergen in the soybean-containing sample by utilizing the reaction between the allergen and the antibody, can be used for qualitatively and quantitatively detecting the soybean allergen, has the characteristics of rapidness, simplicity, convenience, high sensitivity and strong specificity, and can be used for detecting the soybean allergen on a large scale without any expensive instrument.
Description
Technical Field
The invention relates to the technical field of food safety detection, in particular to a soybean allergen visual gel detection device and method.
Background
The soybean is rich in protein and is an important vegetable protein and vegetable oil source for human and animal bodies. The planting area is wide, the yield is high, and the planting area is widely applied to food and feed raw materials for a long time. However, soybean is also one of the 8 most important food allergens identified by the food and agriculture organization of the united nations. Soybean is a main food raw material, and meanwhile, soybean protein is also commonly used as a food ingredient to be added into various foods, so that the establishment of a rapid, specific and sensitive detection technology for soybean allergens has very important significance.
At present, various soybean allergen detection methods are most commonly Enzyme-linked immunosorbent assay (ELISA) methods, have the characteristics of high sensitivity and strong specificity, and can be used for qualitatively and quantitatively detecting soybean allergens. However, the conventional ELISA method is not suitable for rapid screening of a large number of samples in practical use. In the face of practical application, an immunological method which is rapid, simple, convenient, high in sensitivity, free of any expensive instrument and suitable for large-scale detection of soybean allergens is urgently needed to be established.
Accordingly, the prior art is yet to be improved and developed.
Disclosure of Invention
In view of the defects of the prior art, the invention aims to provide a soybean allergen visual gel detection device and method, and aims to solve the problem that the existing detection method is not suitable for large-scale detection of soybean allergens.
The technical scheme adopted by the invention for solving the technical problems is as follows:
a visual gel detection device of soybean allergen, which comprises: the solid-phase extraction column is arranged in the solid-phase extraction column, and the detection layer, the air layer and the quality control layer are sequentially arranged along the direction from the liquid inlet to the liquid outlet; the detection layer consists of a first upper-layer sieve plate, a coupling adhesive containing an anti-soybean protein antibody and a first lower-layer sieve plate, and the quality control layer consists of a second upper-layer sieve plate, a coupling adhesive containing an anti-horseradish peroxidase antibody and a second lower-layer sieve plate.
The soybean allergen visual gel detection device comprises a gel detection device body, wherein the preparation of the coupling gel containing the anti-soybean protein antibody comprises the following steps:
the cyanogen bromide activated Sepharose 4B is swollen with HCl, washed thoroughly with HCl solution and then with NaHCO3Fully balancing the solution to prepare activated glucose gel;
dissolving an anti-soybean protein antibody in a gel coupling solution, and mixing with the activated glucose gel to obtain a first mixed solution;
with NaHCO3Washing the first mixed solution with solution, adding glycine solution, and adding acetate buffer solution and NaHCO3And washing the solution alternately to prepare the coupling gel containing the anti-soybean protein antibody.
The soybean allergen visual gel detection device comprises a soybean allergen visual gel detection device body, wherein the preparation of the coupling gel containing the anti-horseradish peroxidase antibody comprises the following steps:
the cyanogen bromide activated Sepharose 4B is swollen with HCl, washed thoroughly with HCl solution and then with NaHCO3Fully balancing the solution to prepare activated glucose gel;
dissolving an anti-horseradish peroxide antibody diluted by PBS buffer solution into a gel coupling solution, and mixing with the activated glucose gel to obtain a second mixed solution;
with NaHCO3Washing the second mixed solution with a solution, adding a glycine solution, and then adding an acetate buffer solution and NaHCO3And washing the solution for 3 times alternately to prepare the coupling gel containing the anti-horseradish peroxidase antibody.
Visual gel detection device of soybean allergen, wherein, the thickness of air bed is 0.5 ~ 1.0 cm.
A soybean allergen visual gel detection method based on the soybean allergen visual gel detection device comprises the following steps:
carrying out degreasing treatment on soybeans or soybean-containing samples to prepare sample liquid to be detected;
injecting a sample solution to be detected from a liquid inlet of the solid phase extraction column, and then injecting an HRP-labeled anti-soybean protein antibody from the liquid inlet of the solid phase extraction column;
after incubation for a preset time, injecting a PBS buffer solution into the liquid inlet of the solid-phase extraction column, and finally injecting a TMB color developing solution into the liquid inlet of the solid-phase extraction column;
and judging whether the sample solution to be detected contains soybean allergen or not by observing whether the detection layer shows blue or not.
The soybean allergen visual gel detection method comprises the following steps of degreasing soybean or a soybean-containing sample, and preparing a sample solution to be detected, wherein the sample solution comprises:
adding acetone into semen glycines or semen glycines-containing sample for defatting, leaching at 4 deg.C overnight, centrifuging to remove supernatant to obtain precipitate;
and repeatedly leaching the precipitate for several times, ventilating, adding PBS, leaching overnight at 4 ℃, centrifuging, taking supernatant, removing precipitate, and preparing the sample solution to be detected.
The soybean allergen visual gel detection method is characterized in that the detection limit of the soybean allergen is 0.3 ng/mL.
The soybean allergen visual gel detection method further comprises the following steps:
taking a soybean allergen standard substance, respectively diluting with PBS solution to obtain allergen solutions with different concentrations, and respectively adding the allergen solutions with different concentrations to liquid inlets of different solid phase extraction columns;
sequentially injecting an HRP (horse radish peroxidase) marked soybean protein antibody diluted by adding a PBS (phosphate buffer solution) and a TMB (tetramethylbenzidine) color developing solution into a liquid inlet of the solid-phase extraction column, reading a color gray value of a detection layer, and establishing an allergen solution concentration-gray value standard curve;
and acquiring a color gray value of the detection layer when the sample solution to be detected is introduced, and calculating to obtain the soybean allergen content of the sample solution to be detected according to the allergen solution concentration-gray value standard curve and the color gray value of the detection layer of the sample solution to be detected.
Has the advantages that: the invention provides a soybean allergen visual gel detection device and method, wherein the device comprises: the solid-phase extraction column is arranged in the solid-phase extraction column, and the detection layer, the air layer and the quality control layer are sequentially arranged along the direction from the liquid inlet to the liquid outlet; the detection layer consists of a first upper-layer sieve plate, a coupling adhesive containing an anti-soybean protein antibody and a first lower-layer sieve plate, and the quality control layer consists of a second upper-layer sieve plate, a coupling adhesive containing an anti-horseradish peroxidase antibody and a second lower-layer sieve plate. The visual gel detection device provided by the invention can be used for detecting the soybean or the allergen in the soybean-containing sample by utilizing the reaction between the allergen and the antibody, can be used for qualitatively and quantitatively detecting the soybean allergen, has the characteristics of rapidness, simplicity, convenience, high sensitivity and strong specificity, and can be used for detecting the soybean allergen on a large scale without any expensive instrument.
Drawings
Fig. 1 is a structural diagram of a visual gel detection device for soybean allergens according to the present invention.
Fig. 2 is a flowchart of a method for visually detecting soybean allergen in gel according to a preferred embodiment of the present invention.
FIG. 3 is a diagram showing the method for determining the result of soybean allergen detection.
Fig. 4 is a graph showing the experimental results of different concentrations of soy allergen solution in a visual gel detection device of the present invention.
Detailed Description
In order to make the objects, technical solutions and advantages of the present invention clearer and clearer, the present invention is further described in detail below with reference to the accompanying drawings and examples. It should be understood that the specific embodiments described herein are merely illustrative of the invention and are not intended to limit the invention.
The invention provides a visual gel detection device which has good specificity and high sensitivity, is rapid, simple and convenient and is suitable for detecting soybean allergens on a large scale, as shown in figure 1, the visual gel detection device comprises: the detection layer 10 is arranged in the solid-phase extraction column and consists of an upper-layer sieve plate, a coupling adhesive containing an anti-soybean protein antibody and a lower-layer sieve plate; a quality control layer 20 composed of an upper sieve plate, a coupling adhesive containing an anti-horseradish peroxidase antibody and a lower sieve plate, and an air layer 30 positioned between the quality control layer 10 and the detection layer 20.
Specifically, in this embodiment, the sample solution to be detected is injected from the liquid inlet of the solid phase extraction column, generating antigen-antibody immunoreaction in the detection layer, after the antigen and antibody are fully reacted to form antigen-antibody compound, adding an HRP-labeled anti-soybean protein antibody into the visual gel detection device, wherein the HRP-labeled anti-soybean protein antibody is combined with the antigen-antibody complex to form an antibody-antigen-HRP-labeled antibody complex, incubating for a preset time, injecting PBS buffer solution into the liquid inlet of the solid phase extraction column to wash and remove unbound antibody, adding TMB color development solution at the moment, observing whether the detection layer is blue to judge whether the sample solution to be detected contains soybean allergen, and the quantitative detection can be carried out on the sample liquid to be detected according to the color development gray value of the detection layer, and the content of the allergen protein in the sample to be detected is calculated.
Specifically, the preparation method of the coupling gel of the anti-soybean protein antibody comprises the following steps:
a10, activation of glucose gel: cyanogen bromide-activated Sepharose 4B (Sepharose 4B) after swelling with 1mM HCl, washing well with 200mL HCl solution, and adding sufficient NaHCO 0.1mol/L to the swollen gel3Balancing the solution for later use;
a11, coupling: dissolving 0.2mL of 2.5g/L anti-soybean protein antibody in 5mL of gel coupling solution, mixing with the activated gel, stirring on a shaking table, and reacting for 20 h;
a12, washing: with 50mL of 0.1mol/L NaHCO3Washing the coupled gel by the solution to remove the unbound anti-soybean protein antibody;
a13, sealing: adding 20mL of glycine solution into the gel solution to block the activated sites on the gel;
a14, washing: 100mL of pH4.0 acetate buffer solution and pH8.4 NaHCO were used3The solution was washed 3 times alternately with the blocked gel;
a15, storage: 1.0mL of PBS solution was added to the prepared gel, and the gel was transferred to a sterile centrifuge tube, and 0.01mL of Procline 300 was added to the solution and stored in a refrigerator at 4 ℃ for further use.
Wherein the PBS solution in the step A15 is a buffer solution and plays a role of a dissolution protection reagent, and the proper pH buffer can be adjusted to prevent the structure and the biological characteristics of the biological protein from being damaged; the Procline 300 is a diagnostic reagent preservative and can help the prepared gel to be well preserved as it is.
Similarly, the preparation method of the coupling gel of the anti-horseradish peroxidase antibody comprises the following steps:
b10, activation of glucose gel: after swelling cyanogen bromide-activated Sepharose 4B with 1mM HCl, the gel was washed thoroughly with 200mL HCl solution, and sufficient 0.1mol/L NaHCO was added to the swollen gel3Balancing the solution for later use;
b11, coupling: dissolving an anti-horseradish peroxidase (HRP) antibody diluted by 2000 times in 0.2mL of 0.01M PBS buffer solution into 5mL of gel coupling solution, mixing with the activated gel, stirring on a shaking table, and reacting for 20 h;
b12, washing: with 50mL of 0.1mol/L NaHCO3Washing the coupled gel by using the solution to remove the unbound anti-HRP antibody;
b13, sealing: adding 20mL of glycine solution into the gel solution to block the activated sites on the gel;
b14, washing: 100mL of pH4.0 acetate buffer solution and pH8.4 NaHCO were used3The solution was washed 3 times alternately with the blocked gel;
b15, storage: 1.0mL of PBS solution was added to the prepared gel, and the gel was transferred to a sterile centrifuge tube, and 0.01mL of Procline 300 was added to the solution and stored in a refrigerator at 4 ℃ for further use.
The specific assembling method of the visual gel detection device is as follows:
adding a solid phase extraction column with the specification of 1mL into a second lower-layer sieve plate, then adding 200 mu L of the coupling adhesive containing the anti-horseradish peroxidase antibody, and adding a second upper-layer sieve plate after natural deposition to form a column bed, thus forming a quality control layer;
and arranging an air layer on the second upper-layer sieve plate of the quality control layer, adding the first lower-layer sieve plate, adding 200 mu L of the coupling adhesive containing the anti-soybean protein antibody, and adding the first upper-layer sieve plate after natural deposition to form a column bed, namely forming a detection layer, so that the visual gel detection device is assembled.
Preferably, the thickness of the air layer may be set to 0.5 to 1.0 cm.
The present invention also provides a visualized gel detection method for detecting soybean allergen, please refer to fig. 2, fig. 2 is a flowchart of a preferred embodiment of the visualized gel detection method for soybean allergen provided by the present invention, as shown in the figure, it includes the following steps:
s10, carrying out degreasing treatment on the soybeans or the samples containing the soybeans to prepare sample liquid to be detected;
s20, injecting a sample solution to be detected from a liquid inlet of the solid phase extraction column, and then injecting an HRP-labeled anti-soybean protein antibody from the liquid inlet of the solid phase extraction column;
s30, after incubation for a preset time, injecting a PBS buffer solution into the liquid inlet of the solid phase extraction column, and finally injecting a TMB color development solution into the liquid inlet of the solid phase extraction column;
and S40, judging whether the sample liquid to be detected contains soybean allergen or not by observing whether the detection layer shows blue or not.
Specifically, the preparation method of the detection solution of the sample to be detected in step S10 includes: weighing 10g of soybean or a sample containing the soybean, adding 100mL of acetone for degreasing, leaching at a constant temperature of 4 ℃ overnight, centrifuging for 10min at 5000 Xg, removing supernatant, repeatedly leaching the precipitate for 3 times, ventilating in a fume hood to make the acetone completely volatilize, adding PBS (phosphate buffer solution) with the mass volume ratio of 1:10 and the pH value of 7.4, leaching overnight, centrifuging for 10min at 5000 Xg again, removing the precipitate, and taking supernatant to obtain the required sample liquid to be detected.
In order to ensure the accuracy of sample detection, in this embodiment, the step S20 is specifically: adding the sample solution to be detected into the visual gel detection column at the flow rate of 1 drop/second to ensure that the antigen and the antibody can fully react, adding 200 mu L of HRP-labeled anti-soybean protein antibody with pH7.4 and 0.01M PBS buffer solution for diluting 4000 times into the visual gel detection column, and closing a liquid outlet at the lower end.
In this embodiment, the step S30 specifically includes: after incubation at room temperature for 3min, the cells were washed 3 times with 0.01M PBS buffer at pH7.4 containing Tween-20 to remove unbound antibody, and then TMB developing solution was added and allowed to stand for 3 min.
In order to ensure the accuracy of the experimental result, negative and positive controls are independently made in the experiment and are used as the judgment basis of the experimental result, the specific result judgment is shown in figure 3, and the experimental result shows that when the detection layer displays blue, the detection layer is positive, and the sample contains the allergen; when the detection layer appears colorless, it is negative, indicating that the sample does not contain the allergen.
Specifically, in this embodiment, the color of the detection layer is changed by a double antibody sandwich method, when the sample solution to be detected contains soybean allergen, the soybean allergen is combined with an anti-soybean protein antibody to form an antigen-antibody complex, and then HRP is added to label the anti-soybean protein antibody, so that the soybean allergen is combined to form an antibody-antigen-HRP labeled antibody complex, and at this time, TMB color developing solution is added to generate a soluble blue product under the catalysis of HRP, i.e. the detection layer is made to be blue. And the quality control layer is used for judging the effectiveness of the detection result, specifically, after the anti-soybean protein antibody marked by the HRP is fully combined with the antigen-antibody complex, the rest of the HRP continuously flows to the quality control layer, is combined with the coupling glue containing the anti-HRP antibody in the quality control layer, and is blue under the action of the TMB color development liquid, so that the detection result is proved to be effective.
In this embodiment, when the concentration of the standard substance for detecting soybean allergen is greater than 0.3ng/mL, the color of the detection layer is significantly different from that of the blank sample, i.e. the detection limit of the sample is 0.3 ng/mL.
Similarly, in order to analyze the content of the allergen protein in the sample, the quantitative detection analysis can be performed on the sample liquid to be detected, and the quantitative detection analysis method comprises the following specific steps:
taking a soybean allergen standard substance, respectively diluting with PBS solution to obtain allergen solutions with different concentrations, and respectively adding the allergen solutions with different concentrations to liquid inlets of different solid phase extraction columns;
sequentially injecting an HRP (horse radish peroxidase) marked soybean protein antibody diluted by adding a PBS (phosphate buffer solution) and a TMB (tetramethylbenzidine) color developing solution into a liquid inlet of the solid-phase extraction column, reading a color gray value of a detection layer, and establishing an allergen solution concentration-gray value standard curve;
and acquiring a color gray value of the detection layer when the sample solution to be detected is introduced, and calculating to obtain the soybean allergen content of the sample solution to be detected according to the allergen solution concentration-gray value standard curve and the color gray value of the detection layer of the sample solution to be detected.
In this embodiment, the specific detection process is as follows: taking a soybean allergen standard substance, diluting the standard substance with a PBS solution respectively to obtain an allergen solution 1 with the concentration of 1.2ng/L, an allergen solution 2 with the concentration of 0.6ng/L, an allergen solution 3 with the concentration of 0.3ng/L, an allergen solution 4 with the concentration of 0.2ng/L, an allergen solution 5 with the concentration of 0.1ng/L and an allergen solution 6 with the concentration of 0ng/L, respectively taking 1mL of the allergen solutions with different concentrations, adding the visual gel detection column at the flow rate of 1 drop/second to enable the antigen and the antibody to be fully reacted, adding 200 muL of HRP labeled anti-soybean protein antibody diluted by 4000 times with the PBS buffer solution with the pH of 7.4 and the PBS buffer solution with the concentration of Tween-20, washing the column for 3 times with the PBS buffer solution with the pH of 7.4 and the PBS buffer solution with the concentration of 0.01M, washing and removing the unbound antibody, adding 150 mu L of TMB chromogenic substrate solution, drying, observing the colors of the detection layer and the quality control layer after 5min, reading the gray value of the color of the detection layer by adopting software, taking the read gray value as a Y axis and the concentration of the allergen solution as an X axis, establishing a standard curve, taking the sample solution to be detected, detecting according to the steps, and obtaining the content of the allergen protein in the sample to be detected according to the standard curve.
The experimental result is shown in fig. 4, and it can be seen that the quality control layer shows blue, which proves that the experimental result is effective, and the experimental result shows that the degree of blue display of the detection layer gradually increases with the increase of the concentration of the allergen solution.
In summary, the present invention provides a soybean allergen visual gel detection device and method, wherein the device comprises: the solid-phase extraction column is arranged in the solid-phase extraction column, and the detection layer, the air layer and the quality control layer are sequentially arranged along the direction from the liquid inlet to the liquid outlet; the detection layer consists of a first upper-layer sieve plate, a coupling adhesive containing an anti-soybean protein antibody and a first lower-layer sieve plate, and the quality control layer consists of a second upper-layer sieve plate, a coupling adhesive containing an anti-horseradish peroxidase antibody and a second lower-layer sieve plate. The visual gel detection device provided by the invention can be used for detecting the soybean or the allergen in the soybean-containing sample by utilizing the reaction between the allergen and the antibody, can be used for qualitatively and quantitatively detecting the soybean allergen, has the characteristics of rapidness, simplicity, convenience, high sensitivity and strong specificity, and can be used for detecting the soybean allergen on a large scale without any expensive instrument.
It is to be understood that the invention is not limited to the examples described above, but that modifications and variations may be effected thereto by those of ordinary skill in the art in light of the foregoing description, and that all such modifications and variations are intended to be within the scope of the invention as defined by the appended claims.
Claims (8)
1. A visual gel detection device of soybean allergen, characterized by comprising: the solid-phase extraction column is arranged in the solid-phase extraction column, and the detection layer, the air layer and the quality control layer are sequentially arranged along the direction from the liquid inlet to the liquid outlet; the detection layer consists of a first upper-layer sieve plate, a coupling adhesive containing an anti-soybean protein antibody and a first lower-layer sieve plate, and the quality control layer consists of a second upper-layer sieve plate, a coupling adhesive containing an anti-horseradish peroxidase antibody and a second lower-layer sieve plate.
2. The visual soybean allergen gel detection device according to claim 1, wherein the preparation of the coupling gel containing the anti-soybean protein antibody comprises the steps of:
the cyanogen bromide activated Sepharose 4B is swollen with HCl, washed thoroughly with HCl solution and then with NaHCO3Fully balancing the solution to prepare activated glucose gel;
dissolving an anti-soybean protein antibody in a gel coupling solution, and mixing with the activated glucose gel to obtain a first mixed solution;
with NaHCO3Washing the first mixed solution with solution, adding glycine solution, and adding acetate buffer solution and NaHCO3And washing the solution alternately to prepare the coupling gel containing the anti-soybean protein antibody.
3. The visual soybean allergen gel detection device according to claim 1, wherein the preparation of the conjugate gel containing the anti-horseradish peroxidase antibody comprises the steps of:
the cyanogen bromide activated Sepharose 4B is swollen with HCl, washed thoroughly with HCl solution and then with NaHCO3Fully balancing the solution to prepare activated glucose gel;
dissolving an anti-horseradish peroxide antibody diluted by PBS buffer solution into a gel coupling solution, and mixing with the activated glucose gel to obtain a second mixed solution;
with NaHCO3Washing the second mixed solution with a solution, adding a glycine solution, and then adding an acetate buffer solution and NaHCO3And washing the solution for 3 times alternately to prepare the coupling gel containing the anti-horseradish peroxidase antibody.
4. The visual soybean allergen gel detection device according to claim 1, wherein the thickness of the air layer is 0.5-1.0 cm.
5. A soybean allergen visual gel detection method based on the soybean allergen visual gel detection device of any one of claims 1-4, comprising the steps of:
carrying out degreasing treatment on soybeans or soybean-containing samples to prepare sample liquid to be detected;
injecting a sample solution to be detected from a liquid inlet of the solid phase extraction column, and then injecting an HRP-labeled anti-soybean protein antibody from the liquid inlet of the solid phase extraction column;
after incubation for a preset time, injecting a PBS buffer solution into the liquid inlet of the solid-phase extraction column, and finally injecting a TMB color developing solution into the liquid inlet of the solid-phase extraction column;
and judging whether the sample solution to be detected contains soybean allergen or not by observing whether the detection layer shows blue or not.
6. The visual gel detection method for soybean allergen according to claim 5, wherein the step of preparing the sample solution to be tested by performing a degreasing treatment on soybean or a sample containing soybean comprises:
adding acetone into semen glycines or semen glycines-containing sample for defatting, leaching at 4 deg.C overnight, centrifuging to remove supernatant to obtain precipitate;
and repeatedly leaching the precipitate for several times, ventilating, adding PBS, leaching overnight at 4 ℃, centrifuging, taking supernatant, removing precipitate, and preparing the sample solution to be detected.
7. The visual gel detection method of soybean allergen according to claim 5, wherein the detection limit of soybean allergen is 0.3 ng/mL.
8. The visual gel detection method of soybean allergen according to claim 5, further comprising the steps of:
taking a soybean allergen standard substance, respectively diluting with PBS solution to obtain allergen solutions with different concentrations, and respectively adding the allergen solutions with different concentrations to liquid inlets of different solid phase extraction columns;
sequentially injecting an HRP (horse radish peroxidase) marked soybean protein antibody diluted by adding a PBS (phosphate buffer solution) and a TMB (tetramethylbenzidine) color developing solution into a liquid inlet of the solid-phase extraction column, reading a color gray value of a detection layer, and establishing an allergen solution concentration-gray value standard curve;
and acquiring a color gray value of the detection layer when the sample solution to be detected is introduced, and calculating to obtain the soybean allergen content of the sample solution to be detected according to the allergen solution concentration-gray value standard curve and the color gray value of the detection layer of the sample solution to be detected.
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Citations (17)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO1987007384A1 (en) * | 1986-05-30 | 1987-12-03 | Quidel | Enzyme immunoassay device |
| WO2005085855A2 (en) * | 2004-02-27 | 2005-09-15 | Board Of Regents, The University Of Texas System | System and method for integrating fluids and reagents in self-contained cartridges containing sensor elements and reagent delivery systems |
| RU2374649C1 (en) * | 2008-05-04 | 2009-11-27 | Государственное образовательное учреждение высшего профессионального образования "Саратовский государственный университет им. Н.Г. Чернышевского" | Test system for immunoenzymometric determination of toxicants |
| CN102680696A (en) * | 2012-06-08 | 2012-09-19 | 江南大学 | Double antibody sandwich enzyme-linked immuno sorbent assay (ELISA) method for detecting peanut allergic component Arah1 |
| CN102707064A (en) * | 2011-07-14 | 2012-10-03 | 集美大学 | Method for sensitive detection of freshwater fish main allergen parvalbumin |
| CN102798721A (en) * | 2012-05-28 | 2012-11-28 | 上虞常春生物技术有限公司 | Food allergen detection kit and food allergen detection method |
| CN202649216U (en) * | 2012-05-28 | 2013-01-02 | 上虞常春生物技术有限公司 | Food allergen detection kit |
| CN102879585A (en) * | 2012-09-28 | 2013-01-16 | 安徽农业大学 | Indirect ELISA (enzyme linked immunosorbent assay) method for detecting soybean antigenic protein serum antibody |
| CN103439502A (en) * | 2013-06-28 | 2013-12-11 | 北京新华联协和药业有限责任公司 | Rapid specific antibody IgE detection kit and preparation method thereof |
| CN103616520A (en) * | 2013-12-05 | 2014-03-05 | 浙江天科高新技术发展有限公司 | High-sensitivity food allergen detecting method and detecting kit |
| CN103675256A (en) * | 2012-09-12 | 2014-03-26 | 天津科技大学 | Chloramphenicol immunoaffinity gel detection column |
| CN103792363A (en) * | 2012-10-31 | 2014-05-14 | 天津科技大学 | Mung bean allergen antibodies and their preparation method and use |
| CN104931663A (en) * | 2015-06-17 | 2015-09-23 | 深圳大学 | Immune colloidal gold method capable of quickly detecting milk allergen |
| CN105424925A (en) * | 2015-11-20 | 2016-03-23 | 天津科技大学 | Immunoaffinity gel detection column for detecting furaltadone metabolite |
| CN107860927A (en) * | 2017-10-27 | 2018-03-30 | 江苏浩欧博生物医药股份有限公司 | A kind of preparation method and detection kit and its detection method of anaphylactogen Pru p1 albumen |
| WO2018144425A1 (en) * | 2017-01-31 | 2018-08-09 | Vanderbilt University | Generation of human allergen-and helminth-specific ige monoclonal antibodies for diagnostic and therapeutic use |
| CN110672856A (en) * | 2019-09-27 | 2020-01-10 | 广州博厚健康科技有限公司 | Food intolerance specificity IgG detection kit based on multiple microarrays |
-
2020
- 2020-11-23 CN CN202011319847.8A patent/CN112505026A/en active Pending
Patent Citations (17)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO1987007384A1 (en) * | 1986-05-30 | 1987-12-03 | Quidel | Enzyme immunoassay device |
| WO2005085855A2 (en) * | 2004-02-27 | 2005-09-15 | Board Of Regents, The University Of Texas System | System and method for integrating fluids and reagents in self-contained cartridges containing sensor elements and reagent delivery systems |
| RU2374649C1 (en) * | 2008-05-04 | 2009-11-27 | Государственное образовательное учреждение высшего профессионального образования "Саратовский государственный университет им. Н.Г. Чернышевского" | Test system for immunoenzymometric determination of toxicants |
| CN102707064A (en) * | 2011-07-14 | 2012-10-03 | 集美大学 | Method for sensitive detection of freshwater fish main allergen parvalbumin |
| CN102798721A (en) * | 2012-05-28 | 2012-11-28 | 上虞常春生物技术有限公司 | Food allergen detection kit and food allergen detection method |
| CN202649216U (en) * | 2012-05-28 | 2013-01-02 | 上虞常春生物技术有限公司 | Food allergen detection kit |
| CN102680696A (en) * | 2012-06-08 | 2012-09-19 | 江南大学 | Double antibody sandwich enzyme-linked immuno sorbent assay (ELISA) method for detecting peanut allergic component Arah1 |
| CN103675256A (en) * | 2012-09-12 | 2014-03-26 | 天津科技大学 | Chloramphenicol immunoaffinity gel detection column |
| CN102879585A (en) * | 2012-09-28 | 2013-01-16 | 安徽农业大学 | Indirect ELISA (enzyme linked immunosorbent assay) method for detecting soybean antigenic protein serum antibody |
| CN103792363A (en) * | 2012-10-31 | 2014-05-14 | 天津科技大学 | Mung bean allergen antibodies and their preparation method and use |
| CN103439502A (en) * | 2013-06-28 | 2013-12-11 | 北京新华联协和药业有限责任公司 | Rapid specific antibody IgE detection kit and preparation method thereof |
| CN103616520A (en) * | 2013-12-05 | 2014-03-05 | 浙江天科高新技术发展有限公司 | High-sensitivity food allergen detecting method and detecting kit |
| CN104931663A (en) * | 2015-06-17 | 2015-09-23 | 深圳大学 | Immune colloidal gold method capable of quickly detecting milk allergen |
| CN105424925A (en) * | 2015-11-20 | 2016-03-23 | 天津科技大学 | Immunoaffinity gel detection column for detecting furaltadone metabolite |
| WO2018144425A1 (en) * | 2017-01-31 | 2018-08-09 | Vanderbilt University | Generation of human allergen-and helminth-specific ige monoclonal antibodies for diagnostic and therapeutic use |
| CN107860927A (en) * | 2017-10-27 | 2018-03-30 | 江苏浩欧博生物医药股份有限公司 | A kind of preparation method and detection kit and its detection method of anaphylactogen Pru p1 albumen |
| CN110672856A (en) * | 2019-09-27 | 2020-01-10 | 广州博厚健康科技有限公司 | Food intolerance specificity IgG detection kit based on multiple microarrays |
Non-Patent Citations (1)
| Title |
|---|
| 纪卫红: "食品中农药残留免疫亲和凝胶检测柱的研制", 《中国优秀硕士学位论文全文数据库 农业科技辑》 * |
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