WO2017206713A1 - 一种磁微粒偶联抗体分子的方法 - Google Patents
一种磁微粒偶联抗体分子的方法 Download PDFInfo
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- C—CHEMISTRY; METALLURGY
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- 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
一种磁微粒偶联抗体分子的方法，所述方法顺序包括如下步骤：磁微粒的平衡；磁微粒的醛基修饰与还原；活化磁微粒与抗体的偶联。该方法大幅度提高了检测效率，缩短检测时间。该方法操作简单，只需两步即得到稳定的磁微粒偶联的抗体，反应条件温和，对原料无浪费，易于大规模制备；节约生产成本；具有极大的市场前景和经济价值。
Description
本发明属于生物技术领域，具体涉及一种磁微粒偶联抗体分子的方法。
磁性微粒具有超顺磁性、较高的比表面积、可修饰功能基团等特性。在磁微粒表面通过化学修饰与抗体分子进行偶联，在外磁场的控制下，通过免疫吸附、洗涤、解吸等步骤，可以从血液、尿液、白带等复杂的生物样本中分离出目标分子，再利用酶联免疫吸附、化学发光等技术手段可快速完成目标分子的浓度或含量测定。磁微粒偶联抗体具有分离简单、亲和吸附高特异性及高敏感性以及物理和化学性能稳定等众多优点，与抗体偶联后磁微粒主要用于细胞分选、生物分子检测、微生物分离等领域。
目前磁微粒与抗体分子的偶联主要通过表面的活性基团如氨基、羧基、巯基、羟基等与蛋白质分子进行共价结合，公开号为CN 101348517A的中国专利将表面氨基修饰的磁微粒与蛋白质分子表面的氨基分别活化成马来酰亚胺与巯基，然后将二者偶联，此法偶联效率较高，但操作复杂，对试剂尤其是需偶联蛋白质的前处理要求尤其高，而且蛋白质表面活化的巯基容易被氧化成二硫键使其团聚。不利于大规模生产应用。公开号为CN103357359A的中国专利采用二醛试剂作为偶联剂，通过醛胺缩合生成Schiff碱将抗体偶联到磁微粒上，此方法反应条件温和、操作简单、对试剂的要求低。但是由于偶联物的亚胺单元在中性条件下容易发生逆反应，使得抗体分子与磁微粒解离，会造成抗体的自偶联以及磁微粒的自偶联，因此造成偶联效率降低，浪费原物料，同时使得检测的准确性、重复性大大降低。
因此，对这一方法进行改进，保证其偶联产物的稳定性以及偶联后抗体的高活性，确保试剂的灵敏度，是该平台发展的核心技术之一。
发明内容
为此，本发明所要解决的技术问题在于克服现有技术的技术瓶颈，从而提出一种偶联效率高、实际灵敏度高、制得的磁微粒活性高的磁微粒偶联抗体分子的方法。
为解决上述技术问题，本发明公开了一种磁微粒偶联抗体分子的方法，所述方法顺序包括如下步骤：
a.磁微粒的平衡：取含有磁微粒的分散液，磁分离，去上清，用活化缓冲液洗涤；
b.磁微粒的醛基修饰：将平衡后的磁微粒加至二醛溶液中，同时加入选择性还原剂固体，反应；磁分离，得到活化的表面醛基修饰的磁微粒；
c.活化磁微粒与抗体的偶联：溶解在偶联缓冲液中的抗体其表面游离氨基与醛基活化的磁微粒通过醛胺缩合被共价偶联在磁微粒表面，同时加入选择性还原剂将亚胺还原得到稳定的偶联物。
优选的，所述的选择性还原剂为氰基硼氢化钠、三乙酰氧基硼氢化钠中的一种。
优选的，所述的二醛溶液中的二醛为丁二醛、戊二醛、己二醛中的一种。
优选的，所述的二醛溶液浓度为0.5％～10％。
优选的，所述的活化缓冲液的pH为7.0～8.0，为MES缓冲液、Tris-HCl缓冲液、PBS缓冲液、三乙醇胺缓冲液中的一种。
优选的，所述的偶联缓冲液的pH为7.0～7.5，为Tris-HCl缓冲液或PBS缓冲液。
优选的，所述的贮存缓冲液为pH值为7.2，为10mM Tris-HCl、150mM NaCl、1mM EDTA、0.5％BSA、0.05％叠氮化钠的缓冲液中的一中。
优选的，所述的磁微粒表面应经过氨基修饰，并可稳定分散于水溶液中。
优选的，所述的磁微粒偶联效果检测方法为酶联免疫化学发光法。
更为优选的，所述步骤c.后还包括如下步骤：
d.偶联磁微粒的保存与效果检测：将偶联后的磁微粒分装于贮存缓冲液
中保存。
本发明的上述技术方案相比现有技术具有以下优点：
1、本发明偶联的抗体磁微粒复合体可以替代抗体的包被用于酶联免疫吸附、化学发光、荧光等的检测，合成的复合微粒能稳定的悬浮于缓冲液中与抗原进行类均相反应，大幅度提高了检测效率，缩短检测时间；本发明操作简单，只需两步即得到稳定的磁微粒偶联的抗体，反应条件温和，对原物料的无浪费，易于大规模制备。
2、本发明将氨基修饰的磁微粒加入至较高浓度二醛试剂中，可较大程度降低磁微粒之间的自偶联，同时形成亚胺中间体可被反应体系中的特异性还原剂还原为仲胺，而引入的醛基则未被还原，使得后续磁微粒与抗体偶联过程中不会由于亚胺的逆反应使得磁微粒脱落，从而大大减少了磁微粒或者抗体的自偶联，大幅度提高偶联的效率以及目标分子检测的灵敏度与准确性。
3、本发明得到的中间品-表面醛基修饰的磁微粒作为“原材料”可长期稳定保存，并且可用一步法与抗体、抗原、亲和素等进行偶联，是一种通用磁性载体，因此其在试剂盒生产中可大幅度提高效率，节约生产成本。
4、本发明不会影响偶联在磁微粒上的抗体的生物学活性：a)原物料本身不会破坏生物分子的活性，偶联反应的温度、离子强度、pH值等条件，不会引起抗体的变性；b)抗体与磁微粒通过多个碳分子的长臂与抗体分子桥联，有效的避免了固相载体的空间位阻效应对抗体活性的影响。
实施例1本实施例公开了一种磁微粒偶联抗体分子(兔抗雌三醇多克隆抗体)的方法，包括如下步骤：
1.磁微粒的平衡：取100mg表面氨基修饰的磁微粒溶胶在磁场作用下沉降(磁分离)10分钟，去上清液，沉降的磁微粒用20mM pH为7.4的磷酸盐活化缓冲液清洗2～3次，每次用量10ml。
2.磁微粒表面醛基活化：
2.1.将25％～50％戊二醛溶液用磷酸盐活化缓冲液稀释为5％，取10mL并加
入平衡后的磁微粒，室温下振荡混悬反应1小时后
2.2.加入5.6mg三乙酰氧基硼氢化钠，继续反应2小时。
2.3.磁分离10分钟，去上清，磁微粒用偶联缓冲液洗涤3次，每次用量10mL。
2.4.活化后的磁微粒用1mL偶联缓冲液重分散后4℃保存备用，可长期保存。
3.磁微粒与抗体的偶联：
3.1.抗体预处理：取3mg兔抗雌三醇多克隆抗体装入透析袋，4℃条件下用1000mL抗体活化缓冲液透析超过12小时。透析后抗体用紫外分光光度法测量浓度，浓度应大于1mg/ml，否则通过浓缩调整浓度。
3.2.取2mg透析后抗体，加入保存的醛基活化的磁微粒，室温下振荡反应1小时。
3.3.加入5.6mg三乙酰氧基硼氢化钠，继续反应3～4小时。
3.4.磁分离10分钟，将上清液小心移出后保存于2～8℃，用于偶联效率检测。
3.5.分离后的磁微粒偶联抗体用贮存缓冲液洗涤3次，每次用10ml。最后重分散于贮存缓冲液中，终浓度为10mg/mL，2～8℃保存。
4.磁微粒与抗体偶联效率测定：用Bradford法测定磁微粒偶联后上清液内抗体浓度，计算未结合抗体质量。用参与偶联的抗体总质量减去未结合抗体质量再除以抗体总量即为抗体偶联率。
5.抗体偶联后磁微粒活性检测：
5.1.将酶标抗原(碱性磷酸酶标记的雌三醇抗原)稀释至浓度为0.02μg/ml。
5.2.用贮存缓冲液将偶联后磁微粒(浓度10mg/ml)倍比稀释，相应浓度为10mg/ml、5mg/ml、2.5mg/ml、1.25mg/ml等。
5.3.将稀释后的磁微粒30μl与酶标抗原溶液30μl混合，混匀后37℃温育5分钟。
5.4.磁分离去上清，磁微粒洗涤3次，每次用200ul。
5.5.加入碱性磷酸酶催化化学发光底物3-(2-螺旋金刚烷)-4-甲氧基-4-(3-磷氧酰)-苯基-1，2-二氧环乙烷二钠盐(AMPPD)30ul，37℃温育5min后测量其发光值，根据发光值可确定偶联物的活性。
实验例
1、将实施例1所述方法的磁微粒偶联效率与现有技术未加特异性还原剂的偶联效率比较，得到的结果如表1所示
表1磁微粒偶联效率的比较(磁微粒质量为500mg)
2、将实施例1所述方法所得到的磁微粒与现有技术未加特异性还原剂制得的磁微粒进行活性检测比较，得到的结果如表2所示。
表2抗体偶联后磁微粒活性检测
由上表可以看出，实施例1所述的方法，大幅度提高了检测效率，缩短检测时间；用实施例所述方法制得的磁微粒，活性高；较现有技术有着显著的进步和实用性价值。
显然，上述实施例仅仅是为清楚地说明所作的举例，而并非对实施方式的限定。对于所属领域的普通技术人员来说，在上述说明的基础上还可以做出其它不同形式的变化或变动。这里无需也无法对所有的实施方式予以穷举。而由此所引伸出的显而易见的变化或变动仍处于本发明创造的保护范围之中。
Claims (10)
- 一种磁微粒偶联抗体分子的方法，其特征在于，所述方法顺序包括如下步骤：a.磁微粒的平衡：取含有磁微粒的分散液，磁分离，去上清，用活化缓冲液洗涤；b.磁微粒的醛基修饰：将平衡后的磁微粒加至二醛溶液中，同时加入选择性还原剂固体，反应；磁分离，得到活化的表面醛基修饰的磁微粒；c.活化磁微粒与抗体的偶联：溶解在偶联缓冲液中的抗体其表面游离氨基与醛基活化的磁微粒通过醛胺缩合被共价偶联在磁微粒表面，同时加入选择性还原剂将亚胺还原得到稳定的偶联物。
- 如权利要求1所述的磁微粒偶联抗体分子的方法，其特征在于，所述的选择性还原剂为氰基硼氢化钠、三乙酰氧基硼氢化钠中的一种。
- 如权利要求2所述的磁微粒偶联抗体分子的方法，其特征在于，所述的二醛溶液中的二醛为丁二醛、戊二醛、己二醛中的一种。
- 如权利要求3所述的磁微粒偶联抗体分子的方法，其特征在于，所述的二醛溶液浓度为0.5％～10％。
- 如权利要求4所述的磁微粒偶联抗体分子的方法，其特征在于，所述的活化缓冲液的pH为7.0～8.0，为MES缓冲液、Tris-HCl缓冲液、PBS缓冲液、三乙醇胺缓冲液中的一种。
- 如权利要求5所述的磁微粒偶联抗体分子的方法，其特征在于，所述的偶联缓冲液的pH为7.0～7.5，为Tris-HCl缓冲液或PBS缓冲液。
- 如权利要求6所述的磁微粒偶联抗体分子的方法，其特征在于，所述的贮存缓冲液为pH值为7.2，为10mM Tris-HCl、150mM NaCl、1mM EDTA、0.5％BSA、0.05％叠氮化钠的缓冲液中的一中。
- 如权利要求7所述的磁微粒偶联抗体分子的方法，其特征在于，所述的磁微粒表面应经过氨基修饰，并可稳定分散于水溶液中。
- 如权利要求8所述的磁微粒偶联抗体分子的方法，其特征在于，所述的磁微粒偶联效果检测方法为酶联免疫化学发光法。
- 如权利要求9所述的磁微粒偶联抗体分子的方法，其特征在于，所述步骤c.后还包括如下步骤：d.偶联磁微粒的保存与效果检测：将偶联后的磁微粒分装于贮存缓冲液中保存。
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| Application Number | Priority Date | Filing Date | Title |
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| CN201610388140.X | 2016-06-01 | ||
| CN201610388140.XA CN106046169A (zh) | 2016-06-01 | 2016-06-01 | 一种磁微粒偶联抗体分子的方法 |
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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 |
| CN115639361A (zh) * | 2018-02-09 | 2023-01-24 | 北京豪迈生物工程股份有限公司 | 表面结合有抗体的颗粒 |
| 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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| 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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