Preparation method and application of vomitoxin immunomagnetic beads

Abstract

The invention discloses a preparation method and application of vomitoxin immunomagnetic beads, and the preparation method of the vomitoxin immunomagnetic beads comprises the following steps: S11) taking magnetic beads with surface active groups, washing the magnetic beads with a coupling buffer solution for 1-5 times, adding a vomitoxin antibody, fully and uniformly mixing at 4-40 DEG C, and reacting for 1-24 hours, so as to obtain the vomitoxin immunomagnetic beads. After the reaction is finished, washing the magnetic beads by adopting a coupling buffer solution, and washing off the unreacted vomitoxin antibody; and S12) adding a blocking reagent, fully and uniformly mixing under the condition of 4-40 DEG C, reacting for 1-5 hours, and cleaning the magnetic beads by adopting a cleaning buffer solution after the reaction is finished, so as to prepare the vomitoxin immunomagnetic beads. The magnetic beads with the surface active groups are adopted as raw materials to be coupled with the vomitoxin antibody, the prepared vomitoxin immunomagnetic beads have excellent stability and uniformity, vomitoxin can be more effectively and evenly fixed, and the preparation method has the advantages of being convenient, rapid, stable and the like.

Description

Technical Field

[0001] The invention relates to the technical field of magnetic microspheres, and in particular to a preparation method and application of vomitoxin immune magnetic beads. Background Art

[0002] Deoxynivalenol (DON) belongs to the trichothecenes family of compounds. It is named because it can cause vomiting in animals. It has certain harmful effects on the human body and is classified as a Class 3 carcinogen by the World Health Organization's Institute for Research on Cancer. Since DON is highly stable and has high resistance to acid, alkali, pressure and heat, it is difficult to remove it during the processing of grain or feed.

[0003] At present, the detection methods of vomitoxin (DON) are mainly colloidal gold method and high performance liquid chromatography. Although the colloidal gold method is convenient, rapid and suitable for on-site detection, its detection results are not accurate and cannot be quantitatively detected; although the high performance liquid chromatography method can provide accurate detection results, it requires the use of solid phase extraction products in the sample pretreatment process, and cooperates with special equipment and uses a large amount of organic reagents, which is costly and a large amount of organic reagents will cause harm to the human body. Summary of the invention

[0004] The purpose of the present invention is to provide a method for preparing and applying vomitoxin immune magnetic beads, using magnetic beads with active groups on the surface ( The vomitoxin immunomagnetic beads are prepared by using a magnetic resonance imaging (MRI) technology (Analog Devices, Inc., San Diego, CA). The preparation method has the characteristics of convenience, speed, and stability. The prepared vomitoxin immunomagnetic beads are used to extract vomitoxin from the sample.

[0005] In order to achieve the above object, the technical solution adopted by the present invention is: a method for preparing vomitoxin immune magnetic beads, comprising the following steps:

[0006] S11) taking magnetic beads with surface active groups, washing them with coupling buffer for 1-5 times, adding vomitoxin antibodies, mixing them thoroughly at 4-40° C., reacting for 1-24 hours, and washing the magnetic beads with coupling buffer after the reaction to wash away unreacted vomitoxin antibodies;

[0007] S12) adding a blocking reagent, mixing thoroughly at 4-40° C., reacting for 1-5 hours, and washing the magnetic beads with a washing buffer after the reaction to obtain vomitoxin immune magnetic beads.

[0008] As a further optimization, the mass ratio of the DON antibody to the magnetic beads is 1-10%.

[0009] As a further optimization, the magnetic beads are polystyrene magnetic microspheres or silica magnetic microspheres or agarose magnetic microspheres.

[0010] As a further optimization, the surface active group is one or more of epoxy group, p-toluenesulfonyl group, active ester group, chloromethyl group and Protein A.

[0011] As a further optimization, the DON monoclonal antibody or DON polyclonal antibody.

[0012] As a further optimization, the coupling buffer is one or more of a citrate buffer, a phosphate buffer, a Tris-HCL buffer, a carbonate buffer, and a borax buffer.

[0013] As a further optimization, the blocking reagent is one or more of bovine serum albumin, casein, Tris, ethylamine, and ethanolamine.

[0014] The present invention also provides an application of vomitoxin immunomagnetic beads, wherein the vomitoxin immunomagnetic beads are prepared by the above-mentioned method for preparing vomitoxin immunomagnetic beads, and the vomitoxin immunomagnetic beads are used to extract vomitoxin, comprising the following steps:

[0015] S21) adding 0.01-0.1M PBS buffer to the sample and mixing well, adding vomitoxin immune magnetic beads thereto, mixing well at room temperature, and then placing the mixture on a magnetic stand for magnetic attraction, and after all the vomitoxin immune magnetic beads are attracted to one side, pouring out the clear liquid on the other side;

[0016] S22) adding 1-10% methanol solution, magnetically aspirating and washing the vomitoxin immune magnetic beads, and discarding the washing solution;

[0017] S23) adding methanol to elute the vomitoxin adsorbed on the surface of the vomitoxin immunomagnetic beads, collecting the eluate, and performing detection.

[0018] As a further optimization, the sample in S21 is feed or soy sauce.

[0019] As a further optimization, the detection in S23 is high performance liquid chromatography detection or colloidal gold detection.

[0020] Compared with the prior art, the present invention has the following beneficial effects:

[0021] 1. The present invention uses magnetic beads with surface active groups as raw materials to couple vomitoxin antibodies. The prepared vomitoxin immune magnetic beads have excellent stability and uniformity, and can more effectively and evenly fix vomitoxin;

[0022] 2. The preparation method of the present invention is convenient, fast and stable. BRIEF DESCRIPTION OF THE DRAWINGS

[0023] Figure 1This is a scanning electron micrograph of the vomitoxin immunomagnetic beads prepared in Example 1 of the present invention.

[0024] Figure 2 This is a graph showing the HLPC test results of Application Example 1 of the present invention. DETAILED DESCRIPTION

[0025] The following are specific embodiments of the present invention, which further describe the technical solutions of the present invention, but the present invention is not limited to these embodiments.

[0026] Embodiment 1:

[0027] A method for preparing vomitoxin immunomagnetic beads comprises the following steps:

[0028] S11) taking magnetic beads having surface active groups, wherein the surface active groups are epoxy groups, washing them with phosphate buffer for 5 times, adding vomitoxin monoclonal antibodies at a ratio of 2% antibody per mg of magnetic beads, mixing them thoroughly at 20° C., reacting for 12 hours, and after the reaction, washing the magnetic beads with phosphate buffer to wash away unreacted vomitoxin antibodies;

[0029] S12) adding bovine serum albumin, mixing thoroughly at 20° C., reacting for 5 hours, and after the reaction, washing the magnetic beads with a washing buffer to obtain vomitoxin immune magnetic beads.

[0030] Tested and combined Figure 1 As shown, the particle size of the prepared vomitoxin immunomagnetic beads is relatively uniform, 1-2 μm, and the CV value is 2.6%.

[0031] Embodiment 2:

[0032] A method for preparing vomitoxin immunomagnetic beads comprises the following steps:

[0033] S11) taking magnetic beads with surface active groups, wherein the surface active groups are epoxy groups, washing them with Tris-HCL buffer for 5 times, adding vomitoxin monoclonal antibodies at a ratio of 5% antibody per mg of magnetic beads, mixing them thoroughly at 20° C., reacting for 12 hours, and after the reaction, washing the magnetic beads with Tris-HCL buffer to wash away unreacted vomitoxin antibodies;

[0034] S12) adding casein, mixing thoroughly at 20° C., reacting for 5 hours, and after the reaction, washing the magnetic beads with a washing buffer to obtain vomitoxin immune magnetic beads.

[0035] After testing, the prepared vomitoxin immunomagnetic beads had a relatively uniform particle size of 1-2 μm, and a CV value of 2.8%.

[0036] Embodiment 3:

[0037] A method for preparing vomitoxin immunomagnetic beads comprises the following steps:

[0038] S11) taking magnetic beads having surface active groups, wherein the surface active groups are epoxy groups, washing them with phosphate buffer for 5 times, adding vomitoxin polyclonal antibodies at a ratio of 8% antibody per mg of magnetic beads, mixing them thoroughly at 20° C., reacting for 12 hours, and after the reaction, washing the magnetic beads with phosphate buffer to wash away unreacted vomitoxin antibodies;

[0039] S12) adding casein, mixing thoroughly at 20° C., reacting for 5 hours, and after the reaction, washing the magnetic beads with a washing buffer to obtain vomitoxin immune magnetic beads.

[0040] After testing, the prepared vomitoxin immunomagnetic beads had a relatively uniform particle size of 1-2 μm, and a CV value of 2.6%.

[0041] Embodiment 4:

[0042] A method for preparing vomitoxin immunomagnetic beads comprises the following steps:

[0043] S11) taking magnetic beads with surface active groups, wherein the surface active groups are p-toluenesulfonyl groups, washing them with carbonate buffer for 5 times, adding vomitoxin polyclonal antibodies at a ratio of 8% antibody per mg of magnetic beads, mixing them thoroughly at 20° C., reacting for 12 hours, and after the reaction, washing the magnetic beads with carbonate buffer to wash away unreacted vomitoxin antibodies;

[0044] S12) adding casein, mixing thoroughly at 20° C., reacting for 5 hours, and after the reaction, washing the magnetic beads with a washing buffer to obtain vomitoxin immune magnetic beads.

[0045] After testing, the prepared vomitoxin immunomagnetic beads had a relatively uniform particle size of 1-2 μm, and a CV value of 2.7%.

[0046] Application Example 1:

[0047] The vomitoxin immune magnetic beads prepared in Example 2 were used to extract and purify the vomitoxin in the feed:

[0048] S21) The feed is crushed with a grinder, 5 g of the crushed and sieved sample is taken, 20 mL of 0.1 M pH 7.4 PBS buffer and 1 mg of vomitoxin standard are added and mixed, and after standing, 10 mL of the supernatant is taken, and 2 mg of vomitoxin immune magnetic beads are added thereto, and mixed at room temperature for 5 min, and then the sample-vomitoxin immune magnetic beads mixture is placed on a magnetic rack and magnetically attracted for 1 min. After all the magnetic beads are attracted to one side, the clear liquid on the other side is poured out;

[0049] S22) adding 10% methanol solution, washing the magnetic beads by magnetic suction and discarding the washing solution;

[0050] S23) Add 200 μL methanol to elute the vomitoxin adsorbed on the surface of the magnetic beads, collect the eluate in a centrifuge tube, wait for HPLC detection, and compare with the blank test. The HPLC detection result is: the spike recovery rate is 92.8%

[0051] It should be noted that, after adding 500 μL of 0.1M pH7.4 PBS buffer to the eluate in S23 and mixing, it was used for colloidal gold detection, and the detection result was: positive.

[0052] Application Example 2:

[0053] This application example is different from application example 1 in that the vomitoxin immune magnetic beads prepared in example 3 are used to extract and purify the vomitoxin in the feed, and the HPLC test result thereof is: the spike recovery rate is 95.1%.

[0054] Application Example 3:

[0055] This application example is different from application example 1 in that the vomitoxin immune magnetic beads prepared in example 4 are used to extract and purify the vomitoxin in the feed, and the HPLC detection result thereof is: the spiked recovery rate is 95.3%.

[0056] Application Example 4:

[0057] The vomitoxin immunomagnetic beads prepared in Example 2 were used to extract and purify the vomitoxin in soy sauce:

[0058] S21) Take 5 mL of soy sauce, add 15 mL of 0.1 M pH 7.4 PBS buffer and mix well. Then add 2 mg of vomitoxin immune magnetic beads and mix well at room temperature for 5 min. Then put the sample-vomitoxin immune magnetic beads mixture on a magnetic rack and magnetically absorb for 1 min. After all the magnetic beads are attracted to one side, pour out the clear liquid on the other side.

[0059] S22) adding 10% methanol solution, washing the magnetic beads by magnetic suction and discarding the washing solution;

[0060] S23) 200 μL methanol was added to elute the vomitoxin adsorbed on the surface of the magnetic beads, and the eluate was collected in a centrifuge tube for HPLC detection and compared with the blank test. The HPLC detection result showed that the recovery rate of the spiked product was 90.4%.

[0061] It should be noted that after adding 500 μL 0.1M pH7.4 PBS buffer to the S23 eluate and mixing evenly, the mixture was used for colloidal gold detection, and the detection result was positive.

[0062] Application Example 5:

[0063] The difference between this application example and application example 4 is that the vomitoxin immune magnetic beads prepared in example 3 are used to extract and purify the vomitoxin in soy sauce, and the test result of HPLC test is: the spiked recovery rate is 93.2%.

[0064] Application Example 6:

[0065] The difference between this application example and application example 4 is that the vomitoxin immune magnetic beads prepared in example 4 are used to extract and purify the vomitoxin in soy sauce, and the test result of HPLC test is: the spiked recovery rate is 94.1%.

[0066] It can be seen from the above application examples that the vomitoxin immune magnetic beads prepared by the present invention have excellent extraction effects on vomitoxin in feed and soy sauce.

[0067] The specific embodiments described herein are merely examples of the spirit of the present invention. Those skilled in the art may make various modifications or additions to the specific embodiments described or replace them in similar ways, but they will not deviate from the spirit of the present invention or exceed the scope defined by the appended claims.

Claims

1. A method for preparing vomitoxin immunomagnetic beads, characterized in that: The steps include: S11) taking magnetic beads with surface active groups, washing them with coupling buffer for 1-5 times, adding vomitoxin antibodies, mixing them thoroughly at 4-40° C., reacting for 1-24 hours, and washing the magnetic beads with coupling buffer after the reaction to wash away unreacted vomitoxin antibodies; S12) adding a blocking reagent, mixing thoroughly at 4-40° C., reacting for 1-5 hours, and washing the magnetic beads with a washing buffer after the reaction to obtain vomitoxin immune magnetic beads.

2. The method for preparing vomitoxin immunomagnetic beads according to claim 1, characterized in that: The mass ratio of the DON antibody to the magnetic beads is 1-10%.

3. The method for preparing vomitoxin immunomagnetic beads according to claim 1, characterized in that: The magnetic beads are polystyrene magnetic microspheres, silica magnetic microspheres or agarose magnetic microspheres.

4. The method for preparing vomitoxin immunomagnetic beads according to claim 1, characterized in that: The surface active groups are one or more of epoxy groups, p-toluenesulfonyl groups, active ester groups, chloromethyl groups, and Protein A.

5. The method for preparing vomitoxin immunomagnetic beads according to claim 1, characterized in that: The deoxynivalenol monoclonal antibody or deoxynivalenol polyclonal antibody.

6. The method for preparing vomitoxin immunomagnetic beads according to claim 1, characterized in that: The coupling buffer is one or more of a citrate buffer, a phosphate buffer, a Tris-HCL buffer, a carbonate buffer, and a borax buffer.

7. The method for preparing vomitoxin immunomagnetic beads according to claim 1, characterized in that: The blocking reagent is one or more of bovine serum albumin, casein, Tris, ethylamine and ethanolamine.

8. An application of vomitoxin immune magnetic beads, characterized in that: The vomitoxin immunomagnetic beads are prepared by the method for preparing the vomitoxin immunomagnetic beads according to any one of claims 1 to 7, and the vomitoxin immunomagnetic beads are used to extract vomitoxin, comprising the following steps: S21) adding 0.01-0.1M PBS buffer to the sample and mixing well, adding vomitoxin immune magnetic beads thereto, mixing well at room temperature, and then placing the mixture on a magnetic stand for magnetic attraction, and after all the vomitoxin immune magnetic beads are attracted to one side, pouring out the clear liquid on the other side; S22) adding 1-10% methanol solution, magnetically aspirating and washing the vomitoxin immune magnetic beads, and discarding the washing solution; S23) adding methanol to elute the vomitoxin adsorbed on the surface of the vomitoxin immunomagnetic beads, collecting the eluate, and performing detection.

9. The use of vomitoxin immunomagnetic beads according to claim 8, characterized in that: S21 The sample is feed or soy sauce.

10. The use of vomitoxin immunomagnetic beads according to claim 8, characterized in that: The detection described in S23 is high performance liquid chromatography detection or colloidal gold detection.

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