Immunomyosin immunoaffinity column, preparation method and application thereof

Abstract

The invention belongs to the technical field of food detection, and discloses an immunoaffinity column of tropomyosin, a preparation method and an application thereof. The immunoaffinity column of tropomyosin comprises a carrier and a tropomyosin antibody, wherein the antibody is an antibody produced by immunization of tropomyosin of white shrimp, and tropomyosin is a protein obtained by extraction and purification of white shrimp; the tropomyosin antibody is coupled to the carrier by a chemical bond, and the tropomyosin antibody is cross-linked with a cross-linking agent after being combined with the carrier. The affinity column of the invention utilizes a chemically modified solid phase carrier, and then tropomyosin is covalently coupled to the carrier to load the affinity column. The immunoaffinity column is mainly used for the purification of tropomyosin, an allergen in crustacean-containing foods, so as to facilitate the detection of tropomyosin in samples at a later stage. The immunoaffinity column of the invention has the advantages of being fast, simple, accurate, having good specificity and high sensitivity for the detection of tropomyosin in food, and provides a powerful means for food inspection and analysis.

Description

Technical Field

[0001] The invention belongs to the technical field of food detection, and relates to detection technology and application of crustacean organisms such as shrimps and crabs, and in particular to an immunoaffinity column of tropomyosin, a preparation method and application thereof. Background Art

[0002] At present, food allergy is the fourth major health problem in the world today, and is listed by the World Health Organization as one of the three major diseases that should be focused on prevention and treatment in the 21st century. According to statistics, 5-8% of children and 2-3% of adults in the world are allergic to food, and more than 90% of food allergies are caused by eight major types of food: milk, peanuts, eggs, soybeans, wheat, nuts, fish, and crustaceans. Survey data show that about 40% of children and 33% of adults in Asia are allergic to aquatic products. Tropomyosin exists in the muscle and non-muscle cells of invertebrates. It is a cross-reactive allergen in crustaceans and the main allergen for species such as shrimp, lobster, crab and Antarctic krill. Allergies to crustacean aquatic products such as shrimp and crab can cause patients to experience allergic symptoms such as skin redness, asthma, and rhinitis. In severe cases, it is accompanied by collapse, shock, and even life-threatening, which seriously affects the physical health and quality of life of allergic people.

[0003] The main methods for allergen detection are enzyme-linked immunosorbent assay (ELISA), nucleic acid amplification (PCR) and liquid chromatography-mass spectrometry (LC-MS). The ELISA method detects the target allergenic protein through specific antibodies. It is a relatively mature method and has commercial kits. However, various processing methods denature the protein, resulting in the inability to accurately bind to the antibody, and there are problems such as cross-reaction and low detection throughput. The PCR method amplifies specific DNA fragments in allergenic foods, but this type of method does not directly measure allergenic proteins and is prone to false positives. LC-MS overcomes the disadvantages of low throughput and cross-reaction of the ELISA method, as well as the disadvantage that PCR technology cannot directly detect allergenic proteins. It detects based on the peptide level, which solves the problem of quantitative detection of denatured proteins and directly detects allergenic proteins with high throughput, high sensitivity, specificity and accuracy.

[0004] Sample pretreatment is also an important part of sample determination. Conventional sample pretreatment processes include extraction and purification steps. Immunoaffinity technology is a widely used purification method in sample pretreatment. This technology uses the principle of antigen-antibody specific adsorption, has good purification effect, and has an enrichment effect. Since tropomyosin in different types of shrimp and crabs is highly homologous, the usual tropomyosin detection method is to detect the total amount of tropomyosin in crustacean aquatic products.

[0005] Through the above analysis, the problems and defects of the existing technology are: the existing method for detecting the allergen tropomyosin in food has low accuracy, complex detection process and high detection cost. Summary of the invention

[0006] In view of the problems existing in the prior art, the present invention provides an immunoaffinity column, a preparation method and an application of tropomyosin, and in particular relates to an immunoaffinity column for purifying tropomyosin in crustacean products such as shrimps and crabs and a preparation method thereof.

[0007] The present invention is achieved by providing a tropomyosin immunoaffinity column, which comprises a carrier and a tropomyosin antibody. The carrier is commercial agarose gel.

[0008] Furthermore, the antibody is an antibody produced by immunization with tropomyosin of Penaeus vannamei.

[0009] Furthermore, the tropomyosin is a protein obtained by extracting and purifying white shrimp.

[0010] Furthermore, the tropomyosin antibody is coupled to the carrier via chemical bonds, and the tropomyosin antibody is cross-linked with a cross-linking agent after being combined with the carrier.

[0011] Furthermore, the carrier is a Sepharose 4B or a chemically synthesized solid phase carrier.

[0012] Wherein, the carrier is agarose gel 4B including agarose gel 4B activated by cyanogen bromide;

[0013] The chemically synthesized solid phase carrier includes polystyrene, porous polystyrene and cross-linked porous polystyrene filler.

[0014] Another object of the present invention is to provide a method for preparing an immunoaffinity column of tropomyosin using the immunoaffinity column of tropomyosin, the method for preparing the immunoaffinity column of tropomyosin comprising the following steps:

[0015] Step 1, activating the cyanogen bromide activated Sepharose 4B carrier;

[0016] Step 2, coupling the tropomyosin antibody to the activated carrier;

[0017] Step 3, washing the tropomyosin-carrier and loading the column.

[0018] Further, the activation of the cyanogen bromide-activated Sepharose 4B carrier in step 1 comprises:

[0019] A cyanogen bromide-activated Sepharose 4B carrier was selected, and the carrier dry powder was activated by soaking in 1 mmol / L HCl overnight, and then rapidly pumped in a sand core funnel for 15 minutes.

[0020] Furthermore, the step 2 of coupling the tropomyosin antibody with the activated carrier comprises:

[0021] Add 30-200 nmol / mL tropomyosin antibody per gram of activated carrier in 0.1 mol / L NaHCO3, 0.5 mol / L NaCl, pH 8.3 buffer and couple at room temperature for 2 h or at 4°C overnight;

[0022] The coupling product was reacted with 1 mol / L Tris-HCl pH 8.0 at room temperature for 2 h.

[0023] Further, the step 3 of washing the tropomyosin-carrier and loading the column comprises:

[0024] Wash away unconjugated antibody with conjugation buffer;

[0025] The tropomyosin antibody-support was washed alternately with 0.1 mol / L acetate buffer containing 0.5 mol / L NaCl, pH 4.0, and 0.1 mol / L Tris-HCl containing 0.5 mol / L NaCl, pH 8.0;

[0026] Take a 1mL column tube, pad the sieve plate, load the coupling gel suspended in 0.01mol / L pH 7.4 PBS buffer onto the column until the gel height is 0.5mL, balance with PBS, seal the bottom, and store in a refrigerator at 4℃.

[0027] Another object of the present invention is to provide an application of the tropomyosin immunoaffinity column in the enrichment and purification of tropomyosin in a test object, wherein the test object includes seafood and products such as shrimp and crab.

[0028] In combination with the above technical solutions and the technical problems solved, please analyze the advantages and positive effects of the technical solutions to be protected by the present invention from the following aspects:

[0029] First, in view of the technical problems existing in the above-mentioned prior art and the difficulty of solving the problems, the technical solutions to be protected by the present invention and the results and data during the research and development process are closely combined to analyze in detail and deeply how the technical solutions of the present invention solve the technical problems, and some creative technical effects brought about after solving the problems. The specific description is as follows:

[0030] The affinity column provided by the present invention utilizes a chemically modified solid phase carrier, and then covalently couples tropomyosin with the carrier, and then fills the affinity column. The immunoaffinity column is mainly used for the purification of allergen tropomyosin in crustacean-containing foods, so as to facilitate the detection of tropomyosin in samples later. The immunoaffinity column provided by the present invention has the advantages of rapid, simple, accurate, good specificity, high sensitivity, etc. for the detection of tropomyosin in food, and provides a powerful means for food inspection and analysis.

[0031] Second, considering the technical solution as a whole or from the perspective of the product, the technical effects and advantages of the technical solution to be protected by the present invention are described in detail as follows:

[0032] The present invention provides a tropomyosin immunoaffinity column with high specificity, which can specifically identify tropomyosin in a sample, effectively solve the problem of unsatisfactory sample purification in the pretreatment process, reduce sample matrix interference, increase method recovery rate, and fill the gap of immunoaffinity technology in the field of tropomyosin detection.

[0033] Third, as auxiliary evidence of the inventiveness of the claims of the present invention, it is also reflected in the following important aspects:

[0034] (1) The expected benefits and commercial value of the technical solution of the present invention after transformation are:

[0035] The present invention provides a method for preparing an immunoaffinity column for purifying and enriching tropomyosin. By optimizing the method for preparing tropomyosin antibodies, the prepared high-performance antibodies are characterized, and the preparation process of the immunoaffinity column is further optimized, which can be used for the preparation of commercial immunoaffinity columns. Crustacean aquatic products are the largest allergens in my country, and the supervision and inspection of aquatic products is an important measure to ensure food safety. Therefore, the present invention provides a new idea for the accurate quantification of tropomyosin in crustacean aquatic products, and has broad application prospects.

[0036] (2) The technical solution of the present invention fills the technical gap in the industry at home and abroad:

[0037] The present invention provides a new idea for the detection of allergen proteins in food. To achieve the purpose of accurate quantification, we can improve the pertinence and effectiveness of sample pretreatment by preparing a specific allergen protein immunoaffinity column, and then capture the target protein through immunoaffinity column purification for subsequent treatment. This technical solution can be used for accurate quantitative analysis of allergen proteins in food, and by improving the effectiveness of the pretreatment purification process, the specificity of the detection method can be improved. BRIEF DESCRIPTION OF THE DRAWINGS

[0038] In order to more clearly illustrate the technical solutions of the embodiments of the present invention, the following briefly introduces the drawings required for use in the embodiments of the present invention. Obviously, the drawings described below are only some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without creative work.

[0039] Figure 1 It is a flow chart of the method for preparing the immunoaffinity column of tropomyosin provided in an embodiment of the present invention;

[0040] Figure 2 is a structural diagram of the appearance of a tropomyosin immunoaffinity column provided in an embodiment of the present invention;

[0041] Figure 3 is an extracted ion chromatogram of tropomyosin in shrimp provided by an embodiment of the present invention;

[0042] In the figure: A, injection port plug; B, upper sieve plate; C, packing; D, column; E, lower sieve plate; F, outlet plug. DETAILED DESCRIPTION

[0043] In order to make the purpose, technical solution and advantages of the present invention more clearly understood, the present invention is further described in detail below in conjunction with the embodiments. It should be understood that the specific embodiments described herein are only used to explain the present invention and are not used to limit the present invention.

[0044] In view of the problems existing in the prior art, the present invention provides an immunoaffinity column of tropomyosin, a preparation method and an application thereof. The present invention is described in detail below in conjunction with the accompanying drawings.

[0045] 1. Explanatory Examples In order to enable those skilled in the art to fully understand how to implement the present invention, this section provides an illustrative example that expands and describes the technical solution of the claims.

[0046] The tropomyosin immunoaffinity column provided in the embodiment of the present invention comprises a carrier and a tropomyosin antibody.

[0047] The antibody provided in the embodiment of the present invention is an antibody produced by immunization with tropomyosin of Penaeus vannamei. Tropomyosin is a protein obtained by extraction and purification of Penaeus vannamei.

[0048] The tropomyosin antibody provided in the embodiment of the present invention is coupled to the carrier through a chemical bond, and the tropomyosin antibody is cross-linked with a cross-linking agent after being combined with the carrier.

[0049] The carrier provided by the embodiment of the present invention is agarose gel 4B or a chemically synthesized solid phase carrier; wherein the carrier is agarose gel 4B including agarose gel 4B activated by cyanogen bromide, and the chemically synthesized solid phase carrier includes polystyrene, porous polystyrene and cross-linked porous polystyrene filler.

[0050] like Figure 1 As shown, the method for preparing the immunoaffinity column of tropomyosin provided in the embodiment of the present invention comprises the following steps:

[0051] S101, activation of cyanogen bromide-activated Sepharose 4B carrier;

[0052] S102, coupling tropomyosin antibody to the activated carrier;

[0053] S103, washing the tropomyosin-carrier and loading into the column.

[0054] As a preferred embodiment, the method for preparing an immunoaffinity column for tropomyosin in shrimp and crab provided in the embodiment of the present invention specifically comprises the following steps:

[0055] 1. Select the cyanogen bromide activated agarose gel carrier Sepharose 4B for activation

[0056] Take 1g of cyanogen bromide activated agarose gel, add 50mL 1mmol / L HCl, soak overnight for activation, wash the gel with 100mL 1mmol / L HCl 6 times, then wash with 100mL ultrapure water, and quickly pump and wash in a sand core funnel for 15min;

[0057] 2. Add 30-200 nmol / mL tropomyosin antibody to the activated agarose gel Sepharose 4B, and couple at room temperature for 2 hours or at 4°C overnight in a buffer solution of 0.1 mol / L NaHCO3, 0.5 mol / L NaCl, pH 8.3;

[0058] 3. The coupling product was reacted with 1 mol / L Tris-HCl pH 8.0 at room temperature for 2 h;

[0059] 4. Wash away uncoupled antibodies with coupling buffer;

[0060] 5. Wash the tropomyosin antibody-carrier alternately with 0.1mol / L acetate buffer (pH 4.0, containing 0.5mol / L NaCl) and 0.1mol / L Tris-HCl (pH 8.0, containing 0.5mol / L NaCl);

[0061] 6. Take a 1mL column tube, pad the sieve plate, and load the coupling gel suspended in PBS buffer (0.01mol / L pH 7.4) onto the column until the gel height is 0.5mL. Balance with PBS, seal the bottom, and store in a refrigerator at 4℃.

[0062] Preferably, in step 2, the tropomyosin antibody is prepared by the following method:

[0063] (a) Extraction and purification of tropomyosin

[0064] Remove the head, tail and shrimp thread of Penaeus vannamei; add Buffer A (50mmol / L KCl and 2mmol / L NaHCO3) at a ratio of 1:10 (g / mL), fully homogenize and extract at 4℃ for 20min; centrifuge at 4℃ and 10000r / min for 20min and collect the precipitate; resuspend the precipitate in 10 times the volume of Buffer A, centrifuge at 4℃ and 10000r / min for 20min and collect the precipitate, repeat steps 2 to 4 5 times; wash the precipitate with pre-cooled acetone until it is colorless, filter it with 6 layers of gauze, dry it at room temperature, remove impurities such as fat and fat-soluble pigments to obtain shrimp acetone powder; dissolve the acetone powder in Buffer B (0.02mol / L Tris-HCl, 1mol / L KCl and 0.1mmol / L DTT, pH 7.5), extract for 72h; filter the extract with 6 layers of gauze to obtain the supernatant, heat it in water for 20min; centrifuge at 4℃, 10000r / min for 20min, take the supernatant, slowly add 30% ammonium sulfate, and let it stand at 4℃ for 1h; centrifuge at 4℃, 10000r / min for 20min, take the precipitate, and re-dissolve it with 1MPBS; adjust the pH value to 4.6 with 1mol / L HCl solution, centrifuge at 4℃, 8000r / min for 10min, take the precipitate, and re-dissolve it with PBS.

[0065] The extracted ion chromatogram of shrimp tropomyosin provided in the embodiment of the present invention is as follows: Figure 3 shown.

[0066] (2) Preparation and purification of tropomyosin monoclonal antibodies

[0067] Three female Balb / c mice aged 6 to 8 weeks were selected, tropomyosin was diluted with physiological saline, and an equal volume of Freund's complete adjuvant was added and injected into the back of the neck. Immunization was boosted every 2 weeks. An equal volume of Freund's incomplete adjuvant was added for the 2nd and 3rd immunizations. Blood was collected by tail cutting 7 to 10 days after the 3rd immunization, and the serum antibody titer was detected by indirect ELISA.

[0068] The mice with the best immune effect were selected for preparing hybridoma cells, and the spleen cells of Balb / c mice with the highest titer were fused with myeloma cells Sp2 / 0 to obtain hybridoma cells through screening.

[0069] Using the method of inducing ascites in mice, take healthy Balb / c female mice and inject 0.5 ml of paraffin oil into each mouse for later use. Blow off the positive clone hybridoma cells from the expanded culture, centrifuge at 1000 rpm for 5 minutes, suspend the cells with incomplete culture medium, and adjust the cell number to 10 6 / ml, 1ml of positive clone hybridoma cells was injected intraperitoneally into each mouse, and ascites was collected 7-9 days later, centrifuged, and the supernatant was collected. The supernatant was purified by saturated ammonium sulfate method, and further purified by Protein G affinity chromatography to obtain tropomyosin monoclonal antibody.

[0070] Preferably, in step 2, the coupling buffer has a formula of: 0.1 mol / L NaHCO3, 0.5 mol / L NaCl, pH 8.3.

[0071] Preferably, in step 3, the formula of the blocking solution is: 1 mol / L Tris-HCl pH 8.0.

[0072] The technical solution of the present invention is further described below in conjunction with specific embodiments.

[0073] Example 1: Preparation of Tropomyosin Immunoaffinity Column

[0074] 1. Agarose gel activation

[0075] Take 1g of cyanogen bromide activated agarose gel, add 50mL 1mmol / L HCl, soak overnight for activation, wash the gel with 100mL 1mmol / L HCl for 6 times, then wash with 100mL ultrapure water, and quickly pump and wash in a sand core funnel for 15min.

[0076] 2. Coupling of Tropomyosin

[0077] Add 30-200 nmol / mL tropomyosin antibody to the activated agarose gel Sepharose 4B, and couple in 0.1 mol / L NaHCO3, 0.5 mol / L NaCl, pH 8.3 buffer at room temperature for 2 h, or overnight at 4°C; react the coupling product with 1 mol / L Tris-HCl pH 8.0 at room temperature for 2 h; wash away the uncoupled antibody with coupling buffer.

[0078] The tropomyosin antibody-carrier was washed alternately with 0.1 mol / L acetate buffer (pH 4.0, containing 0.5 mol / L NaCl) and 0.1 mol / L Tris-HCl (pH 8.0, containing 0.5 mol / L NaCl).

[0079] 3. Column Packing

[0080] Take a 1mL column tube, pad the sieve plate, load the coupling gel suspended in PBS buffer (0.01mol / L pH 7.4) onto the column until the gel height is 0.5mL, balance with PBS, seal the bottom, and store in a refrigerator at 4℃.

[0081] Example 2: Determination of the capacity of the tropomyosin immunoaffinity column

[0082] according to Figure 2 After filling the immunoaffinity column, 1 mL of a certain concentration of target solution was passed through the column. After the liquid surface entered the affinity column, 10 mL of phosphate buffer (pH 7.4) was added to wash the affinity column; the phosphate buffer in the column was drained with an ear-washing bulb or syringe, 300 μL of neutralization buffer (1M Tris, pH 9.0) was added to the test tube for collecting the eluted liquid, and 2.7 mL of elution buffer (0.1M glycine buffer, pH 2.5) was added to the affinity column, and dripped out by gravity flow rate, with a flow rate of ≤1 drop / second. After all the liquid entered the sieve plate, the eluent in the column was drained with an ear-washing bulb or syringe. In order to test the column capacity of the homemade immunoaffinity column, we tested the recovery rate of different sample loadings, and the results are shown in Table 1.

[0083] Table 1 Test results

[0084]

[0085] When the loading amount of tropomyosin is in the range of 40-80μg, the recovery rate is above 95%; when the loading amount is 100μg, the loading recovery rate is 79.1%. This is because the purification of the immunoaffinity column is based on the specific binding of antigen and antibody. Therefore, when the number of antibodies filled in the immunoaffinity column is fixed, there is a limit to the number of antigens that can be adsorbed. When all the specific binding sites of the antibody are occupied, no more specific binding of antigens can be performed. To ensure the purification effect, it is recommended that the injection volume should not exceed 80μg.

[0086] Example 3: Tropomyosin immunoaffinity column recovery test

[0087] Recovery rate = (tropomyosin content in spiked sample - tropomyosin content in blank sample) / spiked amount * 100%. The recovery test results of the two samples are shown in Table 2.

[0088] Table 2 Recovery test results

[0089]

[0090] 2. Application Examples: In order to prove the creativity and technical value of the technical solution of the present invention, this section provides application examples of the technical solution of the claims on specific products or related technologies.

[0091] Example 4: Purification and detection of tropomyosin in Penaeus chinensis using tropomyosin immunoaffinity column

[0092] 1. Sample Extraction

[0093] Weigh 0.1 g of sample, add 10 mL of ultrapure water, and vortex for 1 h. Take 3 mL of extract for subsequent purification.

[0094] 2. Sample Purification

[0095] Balance: Take out the immunoaffinity column, remove the plug at the bottom of the affinity column, and let the liquid flow out by gravity; after the liquid in the column stops dripping out, take 10mL of phosphate buffer to balance the affinity column;

[0096] Loading: When the buffer solution surface is close to the sieve plate, take 3 mL of the extract and load it;

[0097] Washing: After the sample liquid surface enters the affinity column, add 10mL phosphate buffer (pH 7.4) to wash the affinity column;

[0098] Elution: After elution, drain the phosphate buffer in the column with an ear bulb or syringe, add 300 μL of neutralization buffer (1M Tris, pH 9.0) to the test tube for collecting the eluted liquid, and then add 2.7 mL of elution buffer (0.1 M glycine buffer, pH 2.5) into the affinity column. Let it drip out by gravity flow rate, with a flow rate of ≤1 drop / second. After all the liquid enters the sieve plate, drain the eluent in the column with an ear bulb or syringe; quickly mix the liquid collected in the test tube for subsequent enzymatic hydrolysis.

[0099] 3. Detection

[0100] Liquid chromatography-tandem mass spectrometry was used to detect the content of tropomyosin in Chinese shrimp, which was 3730μg / g.

[0101] Example 5: Purification and detection of tropomyosin in shrimp chips using tropomyosin immunoaffinity column

[0102] 1. Sample Extraction

[0103] Weigh 0.1 g of sample, add 10 mL of ultrapure water, and vortex for 1 h. Take 3 mL of extract for subsequent purification.

[0104] 2. Sample Purification

[0105] Balance: Take out the immunoaffinity column, remove the plug at the bottom of the affinity column, and let the liquid flow out by gravity; after the liquid in the column stops dripping out, take 10mL of phosphate buffer to balance the affinity column;

[0106] Loading: When the buffer solution surface is close to the sieve plate, take 3 mL of the extract and load it;

[0107] Washing: After the sample liquid surface enters the affinity column, add 10mL phosphate buffer (pH 7.4) to wash the affinity column;

[0108] Elution: After elution, drain the phosphate buffer in the column with an ear bulb or syringe, add 300 μL of neutralization buffer (1M Tris, pH 9.0) to the test tube for collecting the eluted liquid, and then add 2.7 mL of elution buffer (0.1 M glycine buffer, pH 2.5) into the affinity column. Let it drip out by gravity flow rate, with a flow rate of ≤1 drop / second. After all the liquid enters the sieve plate, drain the eluent in the column with an ear bulb or syringe; quickly mix the liquid collected in the test tube for subsequent enzymatic hydrolysis.

[0109] 3. Detection

[0110] Liquid chromatography-tandem mass spectrometry showed that no tropomyosin was detected in the shrimp crackers.

[0111] 3. Evidence of the effects of the embodiments. The embodiments of the present invention have achieved some positive effects during the development or use process, and indeed have great advantages over the prior art. The following content is described in conjunction with the data, charts, etc. of the test process.

[0112] The present invention provides a new way to purify tropomyosin. Based on the immunoaffinity column, the purification effect of sample pretreatment can be greatly improved, the specificity and sensitivity of the method can be improved, and interference removal can be achieved. Compared with the existing pretreatment technology, the immunoaffinity column has a higher purification effect and better specificity, providing method support for the detection of tropomyosin in food.

[0113] The above description is only a specific implementation mode of the present invention, but the protection scope of the present invention is not limited thereto. Any modifications, equivalent substitutions and improvements made by any technician familiar with the technical field within the technical scope disclosed by the present invention and within the spirit and principle of the present invention should be covered by the protection scope of the present invention.

Claims

1. An immunoaffinity column of tropomyosin, characterized in that: The tropomyosin immunoaffinity column comprises a carrier and a tropomyosin antibody; The tropomyosin antibody is coupled to the carrier through a chemical bond, and the tropomyosin antibody is cross-linked with a cross-linking agent after being combined with the carrier; The carrier is agarose gel 4B or a chemically synthesized solid phase carrier; Wherein, the carrier is agarose gel 4B including agarose gel 4B activated by cyanogen bromide; The chemically synthesized solid phase carrier includes polystyrene, porous polystyrene and cross-linked porous polystyrene filler; The method for preparing the immunoaffinity column of tropomyosin using the immunoaffinity column of tropomyosin as described above comprises the following steps: Step 1, activating the cyanogen bromide activated Sepharose 4B carrier; Step 2, coupling the tropomyosin antibody to the activated carrier; Step 3, washing the tropomyosin-carrier and loading into a column; The step 2 of coupling the tropomyosin antibody with the activated carrier comprises: Add 30-200 nmol / mL tropomyosin antibody per gram of activated carrier in 0.1 mol / L NaHCO3, 0.5 mol / L NaCl, pH 8.3 buffer, and couple at room temperature for 2 h or at 4 °C overnight; The coupling product was reacted with 1 mol / L Tris-HCl pH 8.0 at room temperature for 2 h.

2. The immunoaffinity column of tropomyosin according to claim 1, characterized in that The antibody is an antibody produced by immunization with tropomyosin of Penaeus vannamei.

3. The immunoaffinity column of tropomyosin according to claim 1, characterized in that The tropomyosin is a protein obtained by extracting and purifying white shrimp.

4. The method for preparing the immunoaffinity column of tropomyosin according to claim 1, characterized in that: The activation of the cyanogen bromide-activated Sepharose 4B carrier in step 1 comprises: A cyanogen bromide-activated Sepharose 4B carrier was selected, and the carrier dry powder was activated by soaking in 1 mmol / L HCl overnight, and then rapidly pumped in a sand core funnel for 15 minutes.

5. The method for preparing the immunoaffinity column of tropomyosin according to claim 1, characterized in that: The step 3 of washing the tropomyosin-carrier and loading the column comprises: Wash away unconjugated antibody with conjugation buffer; The tropomyosin antibody-support was washed alternately with 0.1 mol / L acetate buffer containing 0.5 mol / L NaCl at pH 4.0 and 0.1 mol / L Tris-HCl containing 0.5 mol / L NaCl at pH 8.0; Take a 1mL column tube, pad the sieve plate, and load the coupling gel suspended in 0.01mol / LpH 7.4 PBS buffer onto the column until the gel height is 0.5mL. Balance with PBS, seal the bottom, and store in a refrigerator at 4℃.

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