WO2017206713A1

WO2017206713A1 - Method for coupling magnetic particles with antibody molecules

Info

Publication number: WO2017206713A1
Application number: PCT/CN2017/084741
Authority: WO
Inventors: 郑招荣; 刘金超
Original assignee: 深圳市瀚德标检生物工程有限公司
Priority date: 2016-06-01
Filing date: 2017-05-17
Publication date: 2017-12-07
Also published as: CN106046169A

Abstract

A method for coupling magnetic particle with antibody molecules, comprising the following steps in sequence: balancing of magnetic particles, aldehyde modification and reduction of the magnetic particles, coupling of activated magnetic particles with an antibody. The method greatly improves the detection efficiency and shortens the detection time. The method is simple to operate, can obtain a stable antibody coupled with magnetic particles by two steps only, has mild reaction conditions, wastes no raw material, facilitates large-scale preparation, saves production costs, and has great market prospect and economic value.

Description

Method for coupling magnetic particle to antibody molecule

Technical field

The invention belongs to the field of biotechnology, and in particular relates to a method for conjugated antibody molecules with magnetic particles.

Background technique

The magnetic particles have characteristics such as superparamagnetism, high specific surface area, and functional groups. The surface of the magnetic particle is coupled with the antibody molecule by chemical modification. Under the control of the external magnetic field, the target molecule can be separated from complex biological samples such as blood, urine, leucorrhea, etc. by immunoadsorption, washing, desorption and the like. The concentration or content of the target molecule can be quickly determined by means of enzyme-linked immunosorbent or chemiluminescence. Magnetic particle-conjugated antibodies have many advantages such as simple separation, high specificity and sensitivity of affinity adsorption, and stable physical and chemical properties. After coupling with antibodies, magnetic particles are mainly used for cell sorting, biomolecule detection, microbial separation, etc. field.

At present, the coupling of magnetic particles and antibody molecules is mainly covalently bonded to protein molecules through active groups on the surface such as amino groups, carboxyl groups, sulfhydryl groups, hydroxyl groups, etc. The Chinese patents disclosed in CN 101348517A have surface-amino-modified magnetic particles and proteins. The amino groups on the surface of the molecule are respectively activated into maleimide and sulfhydryl groups, and then the two are coupled. This method has high coupling efficiency, but the operation is complicated, and the pretreatment of reagents, especially the proteins to be coupled, is particularly demanding, and The sulfhydryl groups activated on the surface of the protein are easily oxidized to disulfide bonds to agglomerate them. Not conducive to large-scale production applications. The Chinese patent publication CN103357359A uses a dialdehyde reagent as a coupling agent to couple the antibody to magnetic particles by aldehyde amine condensation to form a Schiff base. This method has mild reaction conditions, simple operation, and low reagent requirements. However, since the imine unit of the conjugate is susceptible to a reverse reaction under neutral conditions, dissociation of the antibody molecule from the magnetic particles causes self-coupling of the antibody and self-coupling of the magnetic particles, thereby causing a decrease in coupling efficiency and waste. The raw materials also greatly reduce the accuracy and repeatability of the test.

Therefore, the improvement of this method, the stability of the coupled product and the high activity of the coupled antibody, and the sensitivity of the reagent are one of the core technologies for the development of the platform.

Summary of the invention

Therefore, the technical problem to be solved by the present invention is to overcome the technical bottleneck of the prior art, and to propose a method of coupling magnetic antibody particles with magnetic particles having high coupling efficiency, high practical sensitivity, and high magnetic particle activity.

In order to solve the above technical problems, the present invention discloses a method for coupling magnetic particle-coupled antibody molecules, and the method sequence includes the following steps:

a. Balance of magnetic particles: take a dispersion containing magnetic particles, magnetically separate, remove the supernatant, and wash with an activation buffer;

b. aldehyde-based modification of magnetic particles: adding the balanced magnetic particles to a dialdehyde solution, simultaneously adding a selective reducing agent solid, reacting; magnetic separation to obtain activated surface aldehyde-modified magnetic particles;

c. Coupling of activated magnetic particles with antibodies: the surface free amino groups and the aldehyde-activated magnetic particles of the antibody dissolved in the coupling buffer are covalently coupled to the surface of the magnetic particles by aldehyde condensation, and selective reduction is added. The agent reduces the imine to give a stable conjugate.

Preferably, the selective reducing agent is one of sodium cyanoborohydride and sodium triacetoxyborohydride.

Preferably, the dialdehyde in the dialdehyde solution is one of succinaldehyde, glutaraldehyde and adipaldehyde.

Preferably, the dialdehyde solution has a concentration of 0.5% to 10%.

Preferably, the activation buffer has a pH of 7.0 to 8.0 and is one of MES buffer, Tris-HCl buffer, PBS buffer, and triethanolamine buffer.

Preferably, the coupling buffer has a pH of 7.0 to 7.5 and is a Tris-HCl buffer or a PBS buffer.

Preferably, the storage buffer is one of a buffer having a pH of 7.2 of 10 mM Tris-HCl, 150 mM NaCl, 1 mM EDTA, 0.5% BSA, 0.05% sodium azide.

Preferably, the surface of the magnetic particles should be modified with an amino group and stably dispersed in an aqueous solution.

Preferably, the magnetic particle coupling effect detecting method is an enzyme-linked immunochemiluminescence method.

More preferably, after the step c., the method further comprises the following steps:

d. Preservation and effect detection of coupled magnetic particles: the coupled magnetic particles are dispensed into storage buffer Saved in.

The above technical solution of the present invention has the following advantages over the prior art:

1. The coupled antibody magnetic particle complex of the present invention can be used for the detection of enzyme-linked immunosorbent, chemiluminescence, fluorescence, etc. in place of antibody coating, and the synthesized composite particles can be stably suspended in a buffer and antigen-like. The phase reaction greatly improves the detection efficiency and shortens the detection time; the invention is simple in operation, and the stable magnetic particle-coupled antibody is obtained in two steps, the reaction condition is mild, the raw material is not wasted, and the large-scale preparation is easy.

2. The invention adds amino-modified magnetic particles to a higher concentration of dialdehyde reagent, which can reduce the self-coupling between the magnetic particles to a large extent, and at the same time form an imine intermediate which can be a specific reducing agent in the reaction system. The reduction is a secondary amine, and the introduced aldehyde group is not reduced, so that the magnetic particles are not detached due to the reverse reaction of the imine during the coupling process of the subsequent magnetic particles and the antibody, thereby greatly reducing the self-coupling of the magnetic particles or the antibody. The efficiency of coupling and the sensitivity and accuracy of target molecule detection are greatly improved.

3. The intermediate-surface aldehyde-modified magnetic particle obtained by the present invention can be stably stored as a "raw material" for a long period of time, and can be coupled with an antibody, an antigen, an avidin or the like in a one-step method, and is a general-purpose magnetic carrier. It can greatly improve efficiency and save production costs in the production of kits.

4. The present invention does not affect the biological activity of the antibody coupled to the magnetic particles: a) the raw material itself does not destroy the activity of the biomolecule, and the conditions of the coupling reaction temperature, ionic strength, pH value, etc., do not cause Denaturation of the antibody; b) The antibody and the magnetic particles are bridged with the antibody molecule through the long arm of the plurality of carbon molecules, effectively avoiding the influence of the steric hindrance effect of the solid phase carrier on the activity of the antibody.

detailed description

EXAMPLE 1 This example discloses a method of magnetic particle-conjugated antibody molecule (rabbit anti-estriol polyclonal antibody) comprising the following steps:

1. Balance of magnetic particles: 100 mg of surface-amino-modified magnetic particle sol was sedimented (magnetic separation) under a magnetic field for 10 minutes, the supernatant was removed, and the precipitated magnetic particles were washed with 20 mM phosphate activation buffer of pH 7.4. ~ 3 times, each dose 10ml.

2. Surface aldehyde activation of magnetic particles:

2.1. Dilute 25% to 50% glutaraldehyde solution to 5% with phosphate activation buffer, take 10mL and add The magnetic particles after the equilibrium are shaken and shaken at room temperature for 1 hour.

2.2. Add 5.6 mg of sodium triacetoxyborohydride and continue the reaction for 2 hours.

2.3. Magnetic separation for 10 minutes, the supernatant was removed, and the magnetic particles were washed 3 times with a coupling buffer for 10 mL each time.

2.4. The activated magnetic particles are redispersed in 1 mL of coupling buffer and stored at 4 ° C for later storage.

3. Coupling of magnetic particles with antibodies:

3.1. Antibody pretreatment: 3 mg rabbit anti-estrogen polyclonal antibody was placed in a dialysis bag and dialyzed against 1000 mL of antibody activation buffer for more than 12 hours at 4 °C. After dialysis, the antibody is measured by ultraviolet spectrophotometry, and the concentration should be greater than 1 mg/ml, otherwise the concentration is adjusted by concentration.

3.2. Take 2 mg of the dialyzed antibody, add the preserved aldehyde-activated magnetic particles, and shake the reaction for 1 hour at room temperature.

3.3. Add 5.6 mg of sodium triacetoxyborohydride and continue the reaction for 3-4 hours.

3.4. Magnetic separation for 10 minutes, the supernatant was carefully removed and stored at 2-8 ° C for coupling efficiency detection.

3.5. The separated magnetic particle-conjugated antibody was washed 3 times with storage buffer, 10 ml each time. Finally, it was redispersed in storage buffer at a final concentration of 10 mg/mL and stored at 2-8 °C.

4. Measurement of coupling efficiency of magnetic particles and antibodies: The concentration of the antibody in the supernatant after magnetic particle coupling was measured by the Bradford method, and the mass of the unbound antibody was calculated. The antibody coupling rate is obtained by subtracting the mass of the unbound antibody from the total mass of the antibody involved in the coupling and dividing by the total amount of the antibody.

5. Detection of magnetic particle activity after antibody coupling:

5.1. The enzyme-labeled antigen (alkaline phosphatase-labeled estriol antigen) was diluted to a concentration of 0.02 μg/ml.

5.2. Dilute the magnetic particles (concentration 10 mg/ml) after coupling with a storage buffer at a concentration of 10 mg/ml, 5 mg/ml, 2.5 mg/ml, 1.25 mg/ml, and the like.

5.3. Mix 30 μl of the diluted magnetic particles with 30 μl of the enzyme-labeled antigen solution, mix and incubate at 37 ° C for 5 minutes.

5.4. Magnetic separation to remove the supernatant, and the magnetic particles were washed 3 times with 200 ul each time.

5.5. Addition of alkaline phosphatase-catalyzed chemiluminescent substrate 3-(2-helixadamantane)-4-methoxy-4-(3-phosphonooxy)-phenyl-1,2-dioxycyclohexane The disodium salt (AMPPD) was 30 ul, and its luminescence value was measured after incubation at 37 ° C for 5 min, and the activity of the conjugate was determined according to the luminescence value.

Experimental example

1. The magnetic particle coupling efficiency of the method described in Example 1 is compared with the coupling efficiency of the prior art unspecificized reducing agent, and the results obtained are shown in Table 1.

Table 1 Comparison of magnetic particle coupling efficiency (magnetic particle mass is 500mg)

2. The magnetic particles obtained by the method described in the first embodiment are compared with the magnetic particles prepared by the prior art without the specific reducing agent, and the results are shown in Table 2.

Table 2: Detection of magnetic particle activity after antibody coupling

It can be seen from the above table that the method described in Embodiment 1 greatly improves the detection efficiency and shortens the detection time; the magnetic particles prepared by the method described in the embodiment have high activity; and the prior art has significant progress and Practical value.

It is apparent that the above-described embodiments are merely illustrative of the examples, and are not intended to limit the embodiments. Other variations or modifications of the various forms may be made by those skilled in the art in light of the above description. There is no need and no way to exhaust all of the implementations. Obvious changes or variations resulting therefrom are still within the scope of the invention.

Claims

A method of conjugated antibody molecules with magnetic particles, characterized in that the method sequence comprises the following steps:

a. Balance of magnetic particles: take a dispersion containing magnetic particles, magnetically separate, remove the supernatant, and wash with an activation buffer;

b. aldehyde-based modification of magnetic particles: adding the balanced magnetic particles to a dialdehyde solution, simultaneously adding a selective reducing agent solid, reacting; magnetic separation to obtain activated surface aldehyde-modified magnetic particles;

c. Coupling of activated magnetic particles with antibodies: the surface free amino groups and the aldehyde-activated magnetic particles of the antibody dissolved in the coupling buffer are covalently coupled to the surface of the magnetic particles by aldehyde condensation, and selective reduction is added. The agent reduces the imine to give a stable conjugate.
The magnetic particle-coupled antibody molecule according to claim 1, wherein the selective reducing agent is one of sodium cyanoborohydride and sodium triacetoxyborohydride.
The magnetic particle-coupled antibody molecule according to claim 2, wherein the dialdehyde in the dialdehyde solution is one of succinaldehyde, glutaraldehyde, and adipaldehyde.
The method of coupling magnetic particle-coupled antibody molecules according to claim 3, wherein the dialdehyde solution has a concentration of from 0.5% to 10%.
The magnetic particle-coupled antibody molecule according to claim 4, wherein the activation buffer has a pH of 7.0 to 8.0, and is MES buffer, Tris-HCl buffer, PBS buffer, and triethanolamine. One of the buffers.
The magnetic particle-coupled antibody molecule according to claim 5, wherein the coupling buffer has a pH of 7.0 to 7.5 and is a Tris-HCl buffer or a PBS buffer.
The magnetic particle-coupled antibody molecule according to claim 6, wherein the storage buffer has a pH of 7.2 and is 10 mM Tris-HCl, 150 mM NaCl, 1 mM EDTA, 0.5% BSA, 0.05%. One of the sodium azide buffers.
The method of coupling magnetic particle-coupled antibody molecules according to claim 7, wherein the surface of the magnetic particles is modified with an amino group and stably dispersed in an aqueous solution.
The magnetic particle-coupled antibody molecule according to claim 8, wherein the magnetic particle coupling effect detecting method is an enzyme-linked immunochemiluminescence method.
The magnetic particle-coupled antibody molecule according to claim 9, wherein the step c. further comprises the following steps:

d. Preservation and effect detection of coupled magnetic particles: The coupled magnetic particles are stored in a storage buffer.