CN115343468A

CN115343468A - Coating method of Tosyl magnetic bead and application of coating method in kit

Info

Publication number: CN115343468A
Application number: CN202210731642.3A
Authority: CN
Inventors: 张馨予; 赵存洋; 汪云峰; 柳建敏
Original assignee: Ningbo Haiershi Intelligent Manufacturing Co ltd
Current assignee: Ningbo Haiershi Intelligent Manufacturing Co ltd
Priority date: 2022-06-24
Filing date: 2022-06-24
Publication date: 2022-11-15

Abstract

The invention relates to a method for coating a Tosyl magnetic bead and application of the Tosyl magnetic bead in a kit, and belongs to the technical field of kits. The invention discloses a method for coating a Tosyl magnetic bead, which is characterized by comprising the following steps: uniformly mixing and coating the magnetic beads and the antibodies pretreated by the pretreatment solution in a coating solution, then sealing by adopting a sealing solution, and then washing and resuspending by using a PBS (phosphate buffer solution) to obtain a working solution of the magnetic bead coated antibodies; the pretreatment solution comprises 3-6mM Tris, 10-20mM serine and 0.5-2M ammonium sulfate, and the pH value is 9.5.

Description

Coating method of Tosyl magnetic bead and application of coating method in kit

Technical Field

The invention belongs to the technical field of kits, and relates to a method for coating a Tosyl magnetic bead and application of the Tosyl magnetic bead in a kit.

Background

Immunomagnetic beads (IMB), also known as immunomagnetic microspheres, are small, uniform, superparamagnetic, protective shell spherical particles, and are essentially formed by the combination of carrier microspheres and an immunopigand. The carrier microsphere mainly comprises magnetic substances (metal small particles such as ferric oxide and ferroferric oxide) and a high molecular layer (such as polyethyleneimine, polyvinyl alcohol, polyacrylic acid and the like). Common functional groups mainly comprise tosyl magnetic beads, carboxyl magnetic beads, amino magnetic beads, epoxy magnetic beads, sulfydryl magnetic beads, silicon-based magnetic beads, physical adsorption magnetic beads and the like, and the surfaces of the functional groups have different physical properties of hydrophobicity, hydrophilicity, nonpolar polarity, positive charge, negative charge and the like. When the surface of the immunomagnetic bead is tosyl, the immunomagnetic bead is hydrophobic, is easy to agglomerate, mainly points to an antibody or antigen containing amino or sulfhydryl, and the coating process needs to be carried out at 37 ℃ and under the condition of pH7.5-9.5. However, in the current coating method, the coating solution of the Tosyl magnetic beads generally uses a boric acid system, the reaction promoter is ammonium sulfate, and the magnetic beads before and after coating are often agglomerated, which is not favorable for the subsequent application in a kit.

Disclosure of Invention

The present invention is directed to solving the above problems of the prior art, and provides a method for coating Tosyl magnetic beads by using a pretreatment solution containing serine to perform pretreatment, so as to improve the dispersibility of the magnetic bead working solution.

The purpose of the invention can be realized by the following technical scheme: a method for coating a Tosyl magnetic bead, the method comprising: uniformly mixing and coating the magnetic beads pretreated by the pretreatment solution and the antibody in a coating solution, sealing by adopting a sealing solution, washing by using a PBS (phosphate buffer solution), and carrying out resuspension to obtain a working solution of the magnetic bead coated antibody;

the pretreatment solution comprises 3-6mM Tris, 10-20mM serine and 0.5-2M ammonium sulfate, and the pH value is 9.5.

Preferably, the pretreatment solution comprises 5-6mM Tris, 14-16mM serine, 0.8-1.2M ammonium sulfate.

The amino acid selected in the pretreatment solution can only be serine, because the steric hindrance of the polar side chain amino acid-serine is smaller than that of glycine and threonine, the polar side chain amino acid-serine has a good hydrophilic effect, a side chain is easy to form a hydrogen bond with tosyl on the surface of a magnetic bead, and the clustered magnetic bead becomes dispersed due to the action of the serine under a hydrophilic environment due to the hydrophobic effect.

Preferably, the coating solution comprises 10-20mM Tris, 0.4-1M ammonium sulfate, 0.05-0.2% Tween 20, and has a pH of 9.5.

More preferably, the concentration ratio of Tris in the pretreatment solution and Tris in the coating solution is 1: 2-4.

More preferably, the concentration ratio of ammonium sulfate in the pretreatment solution and the coating solution is 1: 0.2-0.5.

Preferably, the blocking solution is 50mM Tris buffer pH7.4 containing 0.1-1% fish skin gelatin, 0.05-0.2% mannose.

Preferably, 0.3 to 0.6mg of magnetic beads is added to 100. Mu.l of the pretreatment solution.

Preferably, the mass ratio of the magnetic beads to the antibodies is 100 to (0.3-2).

Preferably, the concentration of the magnetic beads in the magnetic bead-coated antibody working solution is 3 to 5mg/ml.

The invention also discloses an application of the coating method of the Tosyl magnetic bead in a kit.

The prepared magnetic bead working solution and the alkaline phosphatase labeled antibody working solution are combined according to a conventional method to prepare a kit with high sensitivity.

Compared with the prior art, the invention has the following beneficial effects:

1. the pretreatment solution can uniformly disperse the Tosyl magnetic beads, and is beneficial to the subsequent reaction.

2. Through the comprehensive action of the pretreatment solution, the coating solution and the confining solution, the magnetic bead working solution prepared by the method has good reactivity.

3. The magnetic bead working solution prepared by the invention can be combined with an alkaline phosphatase labeled antibody working solution, so that the detection sensitivity of the reagent is improved.

Drawings

FIG. 1 is a microscopic view of the working fluids obtained in examples 1, 4 to 6 and comparative examples 3 to 10 using the present invention.

FIG. 2 is a microscopic view of the working solutions of magnetic beads prepared in comparative examples 11 and 12, in examples 11, 12, 14, 16, 18, 19, 21, 22, 34, 35, 36, 38, 40, and 42, according to the present invention.

Detailed Description

The following are specific examples of the present invention and further describe the technical solutions of the present invention, but the present invention is not limited to these examples. If not specifically stated, the raw materials adopted by the invention are all common materials sold in the market; and tested according to conventional testing methods.

Example 1

Resuspending 2.5mg of Tosyl magnetic beads in 500ul of pretreatment solution (pH 9.5) containing 5mM Tris, 15mM serine and 1M ammonium sulfate, mixing well, placing the centrifuge tube on a magnetic separation frame until the supernatant is completely clear, carefully removing the supernatant by using a pipette, repeating the operation for three times, and mixing well for 20min on a blood mixer.

Examples 2 to 10

The difference is that the pretreatment solution has different components compared with example 1, and specifically, as shown in table 1 below, the dispersibility is better in examples 1 to 10, and the optimum is in example 1.

Example 11

Immediately re-suspending the solution pretreated by the pretreatment solution in example 1 to the target volume with a coating solution containing 15mM Tris, 1M ammonium sulfate and 0.1% Tween 20, adding the antibody according to the antibody-magnetic bead feeding ratio of 20ug/mg, mixing uniformly, and coating for 20h at 37 ℃ by using a blood mixer; after the reaction is finished, placing the centrifugal tube on a magnetic separation frame until the supernatant is completely thorough, carefully removing the supernatant by using a pipette gun, adding 500ul of Tris confining liquid containing 5% fish skin gelatin and having pH of 7.4, fully mixing, and then uniformly mixing and sealing for 20 hours at 37 ℃ by using a blood mixer; after the sealing is finished, placing the centrifugal tube on a magnetic separation rack until the supernatant is completely thorough, carefully removing and discarding the supernatant by using a pipette, cleaning magnetic beads by using 0.1M PBS (phosphate buffer solution) containing 0.1% BSA (bovine serum albumin) and having the pH of 7.4, wherein the concentration of the magnetic beads is 5mg/ml, placing the centrifugal tube on the magnetic separation rack until the supernatant is completely thorough, carefully removing and discarding the supernatant by using the pipette, repeatedly cleaning for 2 times, then resuspending the magnetic beads by using the buffer, and storing to 2-8 ℃. The prepared magnetic bead working solution was observed under an electron microscope for aggregation, and the result is shown in fig. 1.

Examples 12 to 24

Compared with example 11, the difference is the coating liquid composition, which is shown in Table 2 below. The combination of reactivity and dispersibility is best in example 19, and slightly poor in examples 14 and 15 due to too high concentration of Tris; in examples 22-23, the reactivity values were lower with lower pH, and lower with higher pH, so the optimum was 9.5.

Examples 25 to 33

The difference from example 19 is that the solutions pretreated with the pretreatment solution were examples 2 to 10. From the dispersion and reactivity data, it is seen that the overall performance of examples 25-33 remains superior.

Examples 34 to 50

Compared with the example 11, the difference is that the confining liquid component and the feeding ratio are different. In the selection of the feed ratios, the ratio results obtained for the different feed ratios of examples 37, 40-42 were similar, but the background of example 40 was lower and therefore better.

Examples 51 to 59

The difference from example 40 is that the solutions pretreated with the pretreatment liquid were examples 2 to 10. From the dispersion and reactivity data, it is also clear that the overall performance in example 40 remains superior.

Comparative example 1

The difference from example 40 was that the pretreatment solution was a 0.1M sodium borate solution at pH9.5, the coating solution was 0.3-3M ammonium sulfate (0.1M boric acid) at pH7.5-9.5, and the blocking solution was a PBS solution at pH7.4 containing 0.5% BSA. The properties are shown in Table 3.

Comparative example 2

The difference compared to example 40 is that the pretreatment solution was a 0.1M sodium borate solution at pH 9.5. The properties are shown in Table 3.

Comparative example 3

The difference compared to example 1 is that the amino acid is glycine.

Comparative example 4

The difference compared to example 1 is that the amino acid is threonine.

Comparative example 5

The difference compared with example 1 is that the ammonium sulfate concentration in the pretreatment solution was 0.3mM.

Comparative example 6

The difference compared to example 1 is that the ammonium sulfate concentration in the pretreatment solution was 3mM.

Comparative example 7

The difference compared to example 1 is that the concentration of Tris in the pretreatment solution was 1mM.

Comparative example 8

The difference from example 1 is that the concentration of Tris in the pretreatment solution was 10mM.

Comparative example 9

The difference compared with example 1 is that the buffer in the pretreatment solution was Hepes.

Comparative example 10

The difference from example 1 is that the buffer in the pretreatment solution was Mops.

Comparative example 11

The difference compared to example 40 is that span 80 is the surfactant in the coating solution.

Comparative example 12

The difference compared to example 40 is that the surfactant in the coating liquid is polyethylene glycol 400.

TABLE 1 pretreatment solution composition and Performance Table

TABLE 2 composition and Property of coating and confining liquids

TABLE 3 reactivity test chart for different pretreatment processes

As can be seen from Table 1, the working solution for magnetic beads has better dispersibility in the pretreatment solution with pH of 9.5, which comprises 3-6mM Tris, 10-20mM serine and 0.5-2M ammonium sulfate; in a coating solution with pH of 9.5 and comprising 10-20mM Tris, 0.4-1M ammonium sulfate and 0.05-0.2% Tween 20, the magnetic bead working solution has better reactivity; the magnetic bead working solution has better reactivity in 50mM Tris confining liquid with pH7.4 containing 0.1-1% of fish skin gelatin and 0.05-0.2% of mannose. Comparative example 1 the conventional coating method is adopted, and both dispersibility and reactivity are poor; compared with the comparative example 1, the dispersibility and the reactivity of the pretreatment solution are improved to a certain extent by adopting the conventional pretreatment solution, and the pretreatment solution only adopts the coating solution and the sealing solution of the invention but not adopts the pretreatment solution of the invention, but are still lower than those of the pretreatment solution in the example 1 of the invention. The reactivity middle ratio of the working solution prepared by the method is more than 12.5, the background is kept at a slightly low level, and the agglomeration condition is good.

In conclusion, the coating method of the invention can uniformly disperse the Tosyl magnetic beads in the working solution and has better reactivity.

The specific embodiments described herein are merely illustrative of the spirit of the invention. Various modifications or additions may be made to the described embodiments or alternatives may be employed by those skilled in the art without departing from the spirit or ambit of the invention as defined in the appended claims.

Claims

1. A method for coating Tosyl magnetic beads, the method comprising: uniformly mixing and coating the magnetic beads pretreated by the pretreatment solution and the antibody in a coating solution, sealing by adopting a sealing solution, washing by using a PBS (phosphate buffer solution), and carrying out resuspension to obtain a working solution of the magnetic bead coated antibody;

2. The coating method according to claim 1, wherein the coating solution comprises 10-20mM Tris, 0.4-1M ammonium sulfate, 0.05-0.2% Tween 20, and has a pH of 9.5.

3. The coating method according to claim 1, wherein the blocking solution is 50mM Tris buffer pH7.4 containing 0.1-1% fish skin gelatin, 0.05-0.2% mannose.

4. The coating method according to claim 1, wherein the PBS buffer comprises 0.1% BSA, the PBS buffer has a concentration of 0.01-0.2M, and the pH is 7.4.

5. The coating method according to claim 1, wherein 0.3 to 0.6mg of magnetic beads is added to 100. Mu.l of the pretreatment solution.

6. The coating method according to claim 1, wherein the mass ratio of the magnetic beads to the antibodies is 100: (0.3-2).

7. The coating method according to claim 1, wherein the concentration of the magnetic beads in the magnetic bead-coated antibody working solution is 3 to 5mg/ml.

8. Use of the method for coating Tosyl magnetic beads according to claim 1 in a kit.