WO2017206713A1 - Procédé de couplage de particules magnétiques à des molécules d'anticorps - Google Patents

Procédé de couplage de particules magnétiques à des molécules d'anticorps Download PDF

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Publication number
WO2017206713A1
WO2017206713A1 PCT/CN2017/084741 CN2017084741W WO2017206713A1 WO 2017206713 A1 WO2017206713 A1 WO 2017206713A1 CN 2017084741 W CN2017084741 W CN 2017084741W WO 2017206713 A1 WO2017206713 A1 WO 2017206713A1
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WIPO (PCT)
Prior art keywords
magnetic particles
magnetic
coupling
buffer
coupled
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Application number
PCT/CN2017/084741
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English (en)
Chinese (zh)
Inventor
郑招荣
刘金超
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深圳市瀚德标检生物工程有限公司
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Application filed by 深圳市瀚德标检生物工程有限公司 filed Critical 深圳市瀚德标检生物工程有限公司
Publication of WO2017206713A1 publication Critical patent/WO2017206713A1/fr

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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K17/00—Carrier-bound or immobilised peptides; Preparation thereof
    • C07K17/14—Peptides being immobilised on, or in, an inorganic carrier

Definitions

  • the invention belongs to the field of biotechnology, and in particular relates to a method for conjugated antibody molecules with magnetic particles.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • the improvement of this method is one of the core technologies for the development of the platform.
  • 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.
  • the present invention discloses a method for coupling magnetic particle-coupled antibody molecules, and the method sequence includes the following steps:
  • Balance of magnetic particles take a dispersion containing magnetic particles, magnetically separate, remove the supernatant, and wash with an activation buffer;
  • 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;
  • 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 selective reducing agent is one of sodium cyanoborohydride and sodium triacetoxyborohydride.
  • the dialdehyde in the dialdehyde solution is one of succinaldehyde, glutaraldehyde and adipaldehyde.
  • the dialdehyde solution has a concentration of 0.5% to 10%.
  • 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.
  • the coupling buffer has a pH of 7.0 to 7.5 and is a Tris-HCl buffer or a PBS buffer.
  • 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.
  • the surface of the magnetic particles should be modified with an amino group and stably dispersed in an aqueous solution.
  • the magnetic particle coupling effect detecting method is an enzyme-linked immunochemiluminescence method.
  • the method further comprises the following steps:
  • 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.
  • 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.
  • 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.
  • 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.
  • EXAMPLE 1 This example discloses a method of magnetic particle-conjugated antibody molecule (rabbit anti-estriol polyclonal antibody) comprising the following steps:
  • the activated magnetic particles are redispersed in 1 mL of coupling buffer and stored at 4 ° C for later storage.
  • 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.
  • 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.
  • the enzyme-labeled antigen (alkaline phosphatase-labeled estriol antigen) was diluted to a concentration of 0.02 ⁇ g/ml.
  • 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.
  • 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.

Abstract

L'invention concerne un procédé de couplage de particules magnétiques à des molécules d'anticorps, comprenant les étapes suivantes, en séquence : équilibrage des particules magnétiques, modification d'aldéhyde et réduction des particules magnétiques, couplage de particules magnétiques activées avec un anticorps. Le procédé améliore considérablement l'efficacité de détection et raccourcit le temps de détection. Le procédé est simple à utiliser, permet d'obtenir un anticorps stable couplé à des particules magnétiques en seulement deux étapes, présente des conditions de réaction douces, ne gaspille pas de matière première, facilite la préparation à grande échelle, économise des coûts de production et a une grande perspective de marché et une grande valeur économique.
PCT/CN2017/084741 2016-06-01 2017-05-17 Procédé de couplage de particules magnétiques à des molécules d'anticorps WO2017206713A1 (fr)

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
CN201610388140.XA CN106046169A (zh) 2016-06-01 2016-06-01 一种磁微粒偶联抗体分子的方法
CN201610388140.X 2016-06-01

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WO2017206713A1 true WO2017206713A1 (fr) 2017-12-07

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CN (1) CN106046169A (fr)
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CN110632290A (zh) * 2019-09-27 2019-12-31 昆山迪安医学检验实验室有限公司 一种抗双链dna抗体检测试剂
CN114264810A (zh) * 2021-12-08 2022-04-01 南京诺唯赞医疗科技有限公司 一种抗体偶联胶乳微球的监控方法及其应用

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CN106046169A (zh) * 2016-06-01 2016-10-26 深圳市瀚德标检生物工程有限公司 一种磁微粒偶联抗体分子的方法
CN110133271B (zh) * 2018-02-09 2022-12-13 北京豪迈生物工程股份有限公司 一种将抗体或其抗原结合片段共价结合至颗粒表面的方法
CN109061186A (zh) * 2018-08-14 2018-12-21 深圳天辰医疗科技有限公司 一种降钙素原检测试剂盒和降钙素原检测方法
CN109725148B (zh) * 2018-12-30 2022-05-03 广东环凯微生物科技有限公司 一种免疫磁珠保存液

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CN110632290A (zh) * 2019-09-27 2019-12-31 昆山迪安医学检验实验室有限公司 一种抗双链dna抗体检测试剂
CN114264810A (zh) * 2021-12-08 2022-04-01 南京诺唯赞医疗科技有限公司 一种抗体偶联胶乳微球的监控方法及其应用
CN114264810B (zh) * 2021-12-08 2024-03-15 南京诺唯赞医疗科技有限公司 一种抗体偶联胶乳微球的监控方法及其应用

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