WO2015101163A1 - 抗hla-g的单克隆抗体偶联免疫磁珠在肿瘤细胞分选中的应用 - Google Patents

抗hla-g的单克隆抗体偶联免疫磁珠在肿瘤细胞分选中的应用 Download PDF

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WO2015101163A1
WO2015101163A1 PCT/CN2014/093816 CN2014093816W WO2015101163A1 WO 2015101163 A1 WO2015101163 A1 WO 2015101163A1 CN 2014093816 W CN2014093816 W CN 2014093816W WO 2015101163 A1 WO2015101163 A1 WO 2015101163A1
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hla
beads
tumor cells
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卢英
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卢英
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    • C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
    • C12N5/06—Animal cells or tissues; Human cells or tissues
    • C12N5/0602—Vertebrate cells
    • C12N5/0693—Tumour cells; Cancer cells

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  • the invention relates to a method for sorting tumor cells, and a method for sorting tumor cells by using magnetic beads coated with anti-human leukocyte antigen-G (HLA-G) antibody.
  • HLA-G anti-human leukocyte antigen-G
  • the number of circulating tumor cells in the peripheral blood is extremely small, usually only a few tumor cells in about 100 million white blood cells and 50 billion red blood cells, and only one circulating tumor cells per 10 5 to 10 7 monocytes. Therefore, in order to increase the detection rate of circulating tumor cells, it is usually necessary to perform enrichment of circulating tumor cells before detection.
  • Cell enrichment can be achieved by specific markers (immunoisolation) of tumor cells or cell morphological features such as cell volume and density.
  • Commonly used cell enrichment techniques include immunomagnetic sorting, density gradient centrifugation, and cell filtration.
  • Density Gradient Centrifugation It is a technique based on physical properties to separate cells by different sedimentation rates between cells under a certain centrifugal force. This is a commonly used technique in the laboratory for the separation of monocytes and the like.
  • the Onclquick (Greiner Bio-One, Frickenhausen, Germany) and Ficoll-Hypaque separation methods established by this technique were used to separate tumor cells under a certain centrifugal force using a dedicated porous barrier and density gradient separation solution.
  • the device is simple and easy to operate, and can obtain the complete morphology of the cells, which is convenient for further identification. It shows certain clinical significance in some clinical studies, but the sensitivity and specificity are not high, and the recovery rate of the fat tumor cells is low, which limits Its clinical application.
  • Membrane filtration method the difference between the diameter of the tumor cells and the blood cells is utilized, and the cells are filtered through a membrane having a certain pore size to achieve the purpose of separating the cells.
  • this technique is a technique that uses the morphological features of cells to enrich tumor cells.
  • the blood cell diameter is between 8-llum, and the tumor cells are relatively large in diameter (for example, breast cancer cells are about 30 um in diameter), thereby distinguishing between tumor cells and normal blood cells.
  • the difference between tumor cells and some blood cells is not so obvious. It is the aspect of the technology being humanized, but its advantages are equally obvious: more types of tumors are suitable for using size to distinguish normal blood cells without missing surface markers.
  • the first method to use this method is ISET (Isolation by Size of Epithilial Tumor cells), which is simple in equipment, easy to operate, and the cell surface markers can be completely retained; the aggregated tumor cells are more easily separated, which is beneficial for further follow-up. Detection.
  • ISET Isolation by Size of Epithilial Tumor cells
  • the pore size of the tumor cells in the circulation is not uniform, and deformation is often caused by the attack of the immune system and the shearing force of the blood, so the size setting of the membrane pore size is a problem.
  • the microfiltration technology for separating tumor cells according to the cell size has been successfully developed, and the initial experimental results show that it is more sensitive than the antigen-antibody binding separation method, and is more convenient for molecular detection.
  • the following is a specific description.
  • Cell size-based microchip filtration The enrichment platform based on tumor cell size has been reported more and more, and the tumor cells obtained by this method are beneficial for the next step of gene and molecular analysis.
  • One of the high-density micropore techniques greatly improved the enrichment rate of circulating tumor cells in this method.
  • the cultured cells were added to peripheral blood and found to recover more than 90% of tumor cells, and 7.5 mL of blood samples. Separation takes less than 3 minutes to complete.
  • the chip found tumor cells in 51 patients, while the CellSearch system was only positive in blood samples from 26 patients. And the number of circulating tumor cells recovered by the chip is 5.5 times that of the CellSearch system.
  • Antigen-antibody binding separation method Circulating tumor cell separation method based on antigen-antibody binding is currently the most commonly used method. Using different surface antigen markers expressed by tumor cells and normal blood cells, different antibodies are designed to bind to them, and the high specificity and sensitivity of antigen-antibody binding can be used to distinguish and enrich circulating tumor cells (mostly antibodies and The magnetic beads combine to achieve the purpose of separation in a magnetic field. The effectiveness of the immunomagnetic bead enrichment method is much higher than the traditional method. Compared with many enrichment techniques, this technology is relatively mature and has high cell recovery rate. The reported tumor cell recovery rate is about 85%.
  • the magnetic bead-based enrichment method is divided into two principles: immunopositive enrichment and negative enrichment.
  • EpCAM epithelial cell adhesion factor
  • EpCAM epithelial cell adhesion factor
  • EMT epithelial-mesenchymal transition
  • the newly developed biochip technology using EpCAP-coated chip microgrooves, can filter blood-enriched tumor cells and increase enrichment efficiency by about 100 times.
  • EMT epithelial to mesenchymal transition
  • EpCAM epithelial marker antigen
  • one of the characteristics of tumor cells is heterogeneity.
  • Some CTCs do not express EpCAM, and positive selection may lead to false negative results.
  • the quality and quantity of the magnetic beads will affect their identification.
  • the volume of the magnetic beads has been reduced by several tens of times, and this effect is not obvious.
  • Immunomagnetic beads negative sorting similar to positive capture, negative sorting also uses the principle of magnetic bead sorting. However, antibodies that bind negatively to magnetic beads are antigens that bind to the surface of normal blood cells, and tumor cells do not express such antigens on the surface, such as CD45. The principle of negative enrichment is to remove all other cells in the blood, and then the remaining cells are all rare cells including CTCs, and then identified. The red blood cells in the blood are lysed, and the white blood cells bind to the antibody and the magnetic beads and remain in the magnetic field. The rest are tumor cells and a small amount of red and white blood cells, and then the tumor cells are identified and counted by immunostaining.
  • the disadvantage of negative enrichment is that the recovery rate is less than positive enrichment.
  • the advantage is that the identified CTCs are not labeled by magnetic beads and are relatively less affected during sample processing and enrichment. Compared with positive capture, negative sorting is not affected by changes in tumor cell surface antigens, so it can also be applied to tumors with inconsistent and specific specificity and sensitivity of circulating tumor cells, such as melanoma. The sensitivity is relatively high, but it interferes with more cell residues. This technology has not developed an automatic system at present, and the operation is relatively complicated. The influence of human factors is relatively large, but it is still a possible future development direction.
  • the CellSearch system is currently the only FDA-approved circulating tumor cell enrichment technology that has been approved for large-scale clinical validation for prognostic evaluation and efficacy testing of metastatic adenocarcinoma, colorectal cancer, and prostate cancer. A number of studies have confirmed that the system can effectively predict the survival of patients with metastatic breast, colorectal and prostate cancer, and more importantly, can predict the efficacy early after routine treatment in a cycle, so it has been approved by the FDA for the above several advanced stages. Tumor patients.
  • the CellSearch system is an automated circulatory spleen cell detection system whose detection stability has been validated in multiple centers. The system uses the principle of immunomagnetic beads positive capture.
  • the workflow is: using immunomagnetic beads combined with EpCAM antibody to bind tumor cells in a magnetic field, and then using keratin (CK) antibody to detect circulating tumor cells, and also can determine CD45, with CrCD45_ as the tumor cell standard Quasi, finally count the circulating tumor cells.
  • the system can also give partial cell morphology data such as nuclear cytoplasmic ratio, and can collect the obtained tumor cells for artificial identification.
  • the CellSearch system typically requires 7.5 ml of peripheral blood to perform the above operations, and can additionally detect HER2 expression if needed, but with a general ability.
  • Immunomagnetic beads have been widely used in the detection of circulating tumor cells due to their high sensitivity, specificity and rapid separation.
  • EMT epithelial-mesenchymal transition
  • some epithelial-specific antigen expression is weakened when tumor cells enter the circulatory system. Or disappear; while the tumor cells entering the circulatory system under the influence of human autoimmunity and hemorheology, their own apoptosis and decreased antigen expression may lead to a weakening of the ability of positive capture technology to detect circulating tumor cells. Therefore, this technique has certain limitations for certain tumors.
  • Antigen-antibody-binding microstrip technology Several microchip technologies have been reported for spleen tumor cell detection. Recently, the microchips have been studied by surface-arranged coated EpCAM antibodies. The recycling efficiency is over 60%. The chip can be applied to the detection of a variety of tumor circulating tumor cells, including lung cancer, prostate cancer, pancreatic cancer, breast cancer and colorectal cancer, and has been verified in the prediction experiment of lung cancer. The tumor cells obtained by this technology can be used. The next step in the detection of EGFR. The chip technology requires blood flow at a very slow rate to allow antigenic antibodies to bind well, and 7.5 ml of blood takes approximately 10 h.
  • New technologies based on microfluidic technology and cell size separation are also being developed.
  • blood samples are passed through a certain space of the pores, and then arranged through the microcolumns to form a space of a certain size and spacing.
  • Cells above a certain size are retained, and others are retained. Is separated. Separation of different cells and plasma can be achieved using pores of different spacing.
  • microelectrode array technology has facilitated the development of two-dimensional electrophoretic separation technology to enrich circulating tumor cells.
  • Cells with different characteristics will find their own position at different distances of the microelectrodes and accumulate cells with different characteristics because the difference between hydrodynamics and conductivity is separated, and the characteristics of the cells themselves are completely protected.
  • the cultured tumor cells were used to test the enrichment rate of the technique, and the recovery rate was found to exceed 90%.
  • the shortcoming of these techniques is that the amount of blood used is too small, and must be diluted with the corresponding isotonic working solution. If the experiment is carried out according to the blood volume commonly used in clinical practice, the working fluid amount can reach 70 ml, and the passing speed is only 0.5. Ml / h, the dilution process of the liquid will also cause the loss of circulating tumor cells.
  • HLA-G leukocyte antigen G
  • the immune system has the function of repelling and eliminating these cancerous cells, which is called host immune surveillance.
  • cancer cells have clearly escaped the host's immune surveillance. This phenomenon is called the immune escape of tumors, and the mechanism is that tumor cells have an anti-ancestor phenomenon in the early stage, which can secrete and express. HLA-G, which allows it to escape the host's immune surveillance and killing.
  • HLA-G is abnormally expressed only in malignant tumors, and is not expressed in normal tissues other than the placenta. It is a new tumor marker with high malignant tumor specificity.
  • the conference summarized the progress of the HLA-G study since the first academic seminar in 1999, clearly stating that the abnormal expression of HLA-G in tumor cells is Indisputable facts.
  • the technical problem to be solved by the present invention provides a method for sorting breast cancer tumor cells.
  • the tumor cells include breast cancer tumor cells, esophageal cancer, non-small cell lung cancer, uterine cancer, gastric cancer, colon cancer and the like.
  • a method for sorting tumor cells comprising the following steps:
  • the anti-HLA-G monoclonal antibody to which the magnetic beads are coupled is MEM-G/2, MEM-G/9 or 4H84.
  • the anti-HLA-G monoclonal antibody and the immunomagnetic beads are subjected to antibody saturation coupling, preferably using a mass ratio of 200 ug antibody: 1000 ug to activate the magnetic beads.
  • the preparation of the immunomagnetic beads taking activated magnetic beads, adding a saturated coupling amount of anti-HLA-G monoclonal antibody and PBS, mixing at room temperature for 3 hours, washing three times with PBS, adding glycine to mix, The remaining aldehyde groups were blocked; mixed with BSA-containing PBS solution for 30 min, the non-specific adsorption sites were blocked, washed three times with PBS, and vortexed with PBS buffer to obtain a uniform modified immunomagnetic beads.
  • the volume ratio of the cell fluid to be sorted to the HLA-G immunomagnetic beads is 100: (20-100).
  • the present invention also provides an anti-HLA-G immunomagnetic bead for isolating tumor cells. And a sorting kit containing anti-HLA-G immunomagnetic beads.
  • HLA-G was used to sort tumor cell markers.
  • HLA-G was a tumor-specific broad-spectrum marker. After coupling with immunomagnetic beads, the specificity was good and the tumor efficiency was high.
  • HLA-G coupled with immunomagnetic beads has good sensitivity and reproducibility, and can detect relevant tumor cells.
  • HLA-G monoclonal antibodies MEM-G/2, MEM-G/9 and 4H84, source company: Santa Cruz Biotechnology.
  • Nanoimmune magnetic bead preparation involves the coupling of activated magnetic beads to the monoclonal antibody. By optimizing the particle size of the nanoparticles and the attachment efficiency of the antibody, the adsorption efficiency of the nano-immunized magnetic beads on the antigen cells is improved.
  • the magnetic beads are selected to activate the magnetic beads at 50 nm.
  • Antibody coupling 1) Before antibody coupling, anti-HLA-G antibody (using MEM-G/2, MEM-G/9 and 4H84, respectively) to PBS at 4 ° C, after three times of dialysis, overnight, Store in PBS and adjust the concentration to 5mg/ml.
  • reaction medium 500 ⁇ L of nano magnetic beads were placed in 7 centrifuge tubes, numbered, and the medium in tubes 1 and 2 was replaced with phosphate buffer solution under the action of external magnetic field.
  • 0.1M PB 50 ⁇ L of nano magnetic beads were placed in 7 centrifuge tubes, numbered, and the medium in tubes 1 and 2 was replaced with phosphate buffer solution under the action of external magnetic field.
  • 0.1M PB 50 ⁇ L of nano magnetic beads were placed in 7 centrifuge tubes, numbered, and the medium in tubes 1 and 2 was replaced with phosphate buffer solution under the action of external magnetic field.
  • 40 ⁇ L (5mg/ml) antibody was added
  • the content of the antibody in the supernatant after the coupling reaction was detected by using a (BCA) kit (Shanghai Shenggong Bioengineering Technology Service Co., Ltd.) in combination with a microplate reader (PerKin EImer, USA).
  • the anti-HLA-G monoclonal antibody solution was diluted to a standard solution of 0.5 mg/ml with a pH of 7.4, 0.01 mol of phosphate tween (0.05% Tween-20) solution, and taken as 0, 1, 2, 4, 8, respectively.
  • a standard curve is prepared according to the concentration of the standard solution and the absorbance value, and the amount of the antibody in the supernatant is obtained according to the absorbance value of the supernatant to be detected, thereby obtaining the antibody content coupled to the surface of the magnetic bead per unit mass.
  • reaction medium The suitable ion concentration and pH of the reaction medium play an important role in the efficient coupling of magnetic beads with proteins to reduce non-specific adsorption between them.
  • the experimental results show that all three antibodies are suitable.
  • Optimal coating time It can be seen from Table 1 that the three antibodies basically reached saturation after being adsorbed by the magnetic beads for 3 hours, and the OD280 value tends to be stable, so the coating time was determined to be 3 hours.
  • Table 1 The best time for antibody coated magnetic beads (take 4H84 as an example)
  • antibody magnetic bead coupling ratio the results finally showed anti-HLA-G 4H84 monoclonal antibody, the antibody concentration was set to 5mg / ml, the magnetic bead concentration was 2mg / ml, the antibody and magnetic beads mass ratio was 200ug: 1000ug, Magnetic bead coupling is performed at this time, and antibody coupling can be substantially saturated.
  • Magnetic Bead Surface Antibody Quantification The amount of anti-HLA-G antibody attached to the surface of the magnetic beads was measured using a BCA protein quantification kit. The absorbance value corresponding to the protein standard solution having a known concentration is obtained. According to the amount of protein contained in different standard solutions and their absorbance values, a standard curve is made.
  • the absorbance value of the supernatant sample is substituted into the formula, and the protein content in the supernatant sample is 71.2, respectively, because the reaction antibody is 200 ⁇ g, that is, the amount of antibody attached per 1000 ug of magnetic beads is 128.8; It was calculated that the amount of anti-HLA-G antibody attached to the surface of the magnetic beads was 128.8 ⁇ g/mg on average.
  • the three anti-HLA-G immunomagnetic beads prepared in step 2.2 of the present embodiment were used for artificially cultured tumor cell line cells for tumor cell collection; the artificially cultured tumor cell line cells were mixed with healthy human blood to prepare artificial samples, and used. Anti-HLA-G immunomagnetic beads were used to enrich tumor cells in artificial samples, and the enriched samples were counted and the enrichment efficiency was calculated.
  • the anti-HLA-G immunomagnetic beads can achieve a recovery rate of 85%-93% for the corresponding tumor cell enrichment rate.
  • the enrichment rate of tumor cells in blood can reach 77%-84%, which indicates that the prepared anti-HLA-G immunomagnetic beads have higher enrichment rate and better reproducibility, and can recover related tumor cells.
  • the amount of nano-immunized magnetic beads see Table 3 tested, the ratio of tumor sample to nano-magnetic beads is 100ul (10 7 /ml): 40ul (2mg / ml), the enrichment effect is better and will not cause magnetic bead waste .
  • the optimal reaction time of nano-immunized magnetic beads and tumor cells see Table 4.
  • the optimal time for the reaction of the nano-immunized magnetic beads with the sample is 30 min.

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Abstract

提供了一种抗HLA-G的单克隆抗体偶联免疫磁珠在肿瘤细胞分选中的应用。还提供了一种肿瘤细胞的分选方法，包括以下步骤：（1）抗HLA-G的单克隆抗体偶联免疫磁珠得HLA-G免疫磁珠；（2）将HLA-G免疫磁珠混匀后加入到待分选的细胞液中，用免疫磁珠分离方法进行分离。

Description

抗HLA-G的单克隆抗体偶联免疫磁珠在肿瘤细胞分选中的应用 技术领域
本发明涉及一种分选肿瘤细胞的方法，一种以应用抗人白细胞抗原G(human leukocyte antigen-G， HLA-G)抗体偶联磁珠分选肿瘤细胞的方法。
背景技术
循环肿瘤细胞在外周血中的数量极少，通常在约1亿个白细胞和500亿个红细胞中仅有数个肿瘤细胞，大约每105～107个单核细胞中才有一个循环肿瘤细胞。因此，为了提高循环肿瘤细胞的检出率，通常需在检测前行循环肿瘤细胞的富集。细胞富集可通过肿瘤细胞的特异性标志物(免疫分离)或细胞形态特征如细胞体积和密度等来实现。
常用的细胞富集技术主要有免疫磁分选、密度梯度离心和细胞过滤等。
1.1密度梯度离心法：是一种基于物理学特性、利用在一定离心力下细胞之间沉降率不同分离细胞的技术。这是实验室一项常用的技术，用于分离单核细胞等。利用这项技术建立的Onclquick(Greiner Bio-One，Frickenhausen，Germany)和Ficoll-Hypaque分离法，利用专用的多孔屏障和密度梯度分离液，在一定离心力下分离出肿瘤细胞。这项技术设备简单操作简便，能得到细胞的完整形态，便于进一步鉴别，在部分临床研究中显示出一定的临床意义，但是敏感性和特异性均不高，胖瘤细胞回收率低，限制了其临床应用。
1.2膜过滤法：是利用肿瘤细胞直径与血液细胞的区别，通过一定孔径的膜来过滤细胞而达到分离细胞的目的。同密度梯度离心法不一样，这项技术是利用细胞的形态学特点来富集肿瘤细胞的技术。一般血细胞直径在8-llum之间，而肿瘤细胞相对直径较大(例如乳腺癌细胞直径约30um)，借此区别以分离肿瘤细胞和正常血液细胞。肿瘤细胞与部分血液细胞的差别并没有那么明显，是该技术被人话病的方面，但是它的优势同样明显：更多类型的肿瘤都适合利用大小来区分正常血液细胞而不因表面标志缺失或者肿瘤异质性导致的漏检。第一个采用此法的是ISET(Isolation by Size of Epithilial Tumor cells)，该方法设备简单，操作方便，而且细胞表面标记能完整保留；聚集的在一起的肿瘤细胞更容易分离，利于后续的进一步检测。但是，循环中的肿瘤细胞孔径不是均一的，在免疫***攻击及血液剪切力的作用下往往发生变形，因此膜孔径的大小设定是个难题。有研究认为Sum的孔径分离效果好，但是敏感性受到质疑。在此基础上新发展的ScreenCell分离法，得到的胖瘤细胞除可常规检测外还可用于细胞培养，且有较好的敏感性。
鉴于依照肿瘤细胞大小的分离方法的优势，目前依据细胞大小分离肿瘤细胞的微过滤技术研发成功，初期的实验结果显示其比抗原抗体结合分离法有更高的灵敏性，更方便进行分子检测，下面为相关的专门描述。
以细胞大小为基础的微芯片过滤：以肿瘤细胞大小为基础建立的富集平台逐渐有了更多的报道，这种方法得到的肿瘤细胞有利于进行下一步的基因和分子的分析。其中一种采用高密度微孔工艺技术大大提高了该方法对循环肿瘤细胞的富集率，以培养细胞加入外周血进行实验发现其对肿瘤细胞的回收率超过90％，并且7.5mL血液标本的分离只需要不到3分钟的时间就能完成。与CellSearch***比较，在57例转移性***癌、大肠癌、乳腺癌和膀胱癌患者中，该芯片在51例患者中发现肿瘤细胞，而CellSearch***只在其中26例患者血液标本中为阳性结果，并且芯片回收的循环肿瘤细胞数量是CellSearch***的5.5倍。有一篇报道比较了依照大小富集和CellSearch***富集循环肿瘤细胞效率的实验，结果也是类似的，这些都显示出CellSearch***的局限性。以大小为基础的微芯片分离技术的富集效率取决于微孔的密度和规律，尽管显示出一定的优势，目前仍没有应该该方法的临床大宗报道。
1.3抗原抗体结合分离法：以抗原抗体结合为基础的循环肿瘤细胞分离方法是目前最常用的方法。利用肿瘤细胞和正常血液细胞表达的不同表面抗原标记，设计不同的抗体与之结合，利用抗原抗体结合的高度特异性和灵敏性，达到区别和富集循环肿瘤细胞的目的(多是将抗体和磁珠结合，在磁场中达到分离的目的)。免疫磁珠法富集法的有效性要比传统方法高很多。和众多富集技术相比，此技术较为成熟，细胞回收率高，文献报道肿瘤细胞回收率约为85％左右。基于磁珠的富集方法从原理上分为两种为免疫阳性富集和阴性富集两种方式。
1.3.1免疫磁珠阳性扑获：
EpCAM(上皮细胞枯附因子)是最常用的免疫磁珠阳性扑获的目标抗原。EpCAM是上皮来源肿瘤最常见的表面标记，EpCAM抗体结合磁珠，与肿瘤细胞表面抗原结合，在磁场中得到分离。但是，基于此原理的分离技术需要肿瘤细胞表面有稳定、丰富的表面标记，否则就容易产生假阴性的结果。据研究，由于转移过程中的上皮间质转化(EMT)、肿瘤细胞的异质性等原因，一些肿瘤细胞表面EpCAM表达减弱或者缺失并且非上皮来源的肿瘤细胞不表达EpCAM，例如黑色素瘤细胞。为了提高该技术的敏感性，可考虑应用联合抗体磁珠来进行扑获。新发展的生物芯片技术，利用EpCAP包被的芯片微槽，可以过滤血液富集肿瘤细胞，提高富集效率约100倍。
正性富集的特点具有较高的富集效率，由于CTCs属于外周血内的稀有细胞，因此能够高效率的回收肿瘤细胞是正性富集的最大优势，其富集的倍数从104到2×105不等。因此正性富集能提高鉴定cTCs的灵敏度。但正性富集仍存在方法学的不足，因其原理是将血液内具有上皮表型的细胞全部回收，富集中使用的主要抗原是EpCAM。但并非只有肿瘤细胞表达EpCAM，血液来源的某些细胞或非恶性上皮细胞表达EpCAM时也会被富集。肿瘤在转移的过程中，部分肿瘤细胞表型发生转化，上皮间皮转化(Epithelial to mesenchymal transition，EMT)现象，上皮标记抗原EpCAM将表达下调，使磁珠的富集效率降低。此外，肿瘤细胞的特点之一就是异质性，有些CTCs不表达EpCAM，阳性选择可能会导致假阴性的结果。另外，由于肿瘤细胞分离后仍然同磁珠相连，磁珠的质量和数量将影响对其鉴定，但目前由于技术进步，磁珠的体积已降低几十倍，此种影响已不明显。
1.3.2免疫磁珠阴性分选：与阳性扑获类似，阴性分选也利用的是磁珠分选原理。但是阴性分选与磁珠结合的抗体是结合正常血液细胞表面的抗原，肿瘤细胞表面不表达此类抗原，比如CD45。负性富集的原理是将血液中其它细胞全部除去，而后剩余细胞即为包含CTCs在内全部稀有细胞，再行鉴定。血液中的红细胞被裂解，白细胞结合抗体和磁珠后在磁场中滞留，剩余的是肿瘤细胞和少量的红、白细胞，然后再利用免疫焚光染色识别和计数肿瘤细胞。阴性富集的缺点是回收率不及阳性富集，优点是鉴定的CTCs没有被磁珠标记，在样本处理和富集的过程相对较少受到影响。与阳性扑获相比，阴性分选不受肿瘤细胞表面抗原变化的影响，因此在循环肿瘤细胞表面抗原表达不一致和特异性、敏感性较低的肿瘤中也可以应用，比如黑色素瘤等，且敏感性相对较高，但干扰细胞残留较多。这项技术目前未发展出自动***，操作较为复杂，人为因素影响相对较大，不过仍然是未来可能的发展方向。
1.3.3CellSearch检测***
CellSearch***是目前FDA唯一批准的循环肿瘤细胞富集技术，该***经过大规模临床验证被批准用于转移性孔腺癌、大肠癌和***癌的预后评价和疗效检测。多项研究证实该***能有效预测转移性乳腺癌、大肠癌和***癌患者的生存期，更重要的是能在一周期常规治疗后早期预测疗效，因此获得了FDA批准应用于以上几种晚期肿瘤患者。CellSearch***是一个自动化的循环脾瘤细胞检测***，其检测稳定性在多个中心得到了验证。该***采用的是免疫磁珠阳性扑获的原理。
其工作流程为：采用免疫磁珠结合EpCAM抗体在磁场中结合肿瘤细胞，然后再用角蛋白(CK)抗体来检测循环肿瘤细胞，同时还可以测定CD45，以CrCD45_为肿瘤细胞标 准，最后给循环肿瘤细胞计数。该***还可以给出部分细胞形态学的数据如胞核胞浆比，并且可以收集所得到的肿瘤细胞进行人工的鉴定。CellSearch***完成上述操作一般需要7.5ml的外周血液，如果需要还可以附加检测HER2的表达，但是能力一般。
免疫磁珠以其高度的灵敏性、特异性和快速分离的特点，在循环肿瘤细胞检测中得到了较多的应用。但是该及预后的相关性研究技术最大的缺点在于，由于很多类型的实体瘤中广泛的存在上皮间质转化(EMT)现象，肿瘤细胞在进入循环***过程中，一些上皮特异性的抗原表达减弱或者消失；同时进入循环***的肿瘤细胞在人自身免疫力和血液流变力学的作用下，自身出现调亡和抗原表达减弱，这些都可能导致阳性扑获技术对循环肿瘤细胞的检测能力减弱。因此，对于某些肿瘤，该技术存在一定的局限性。
1.3.4抗原抗体结合为基袖的微芯片技术：有几种微芯片技术被报道用于循环脾瘤细胞检测，最近报道的微芯片即采用表面阵列性排列地包被的EpCAM抗体研究称其回收效率超过60％。该芯片可以同时应用于多种肿瘤循环肿瘤细胞的检测，包括肺癌、***癌、胰腺癌、乳腺癌和大肠癌等，并在对肺癌的疗效预测实验中得到验证，该技术获得的肿瘤细胞可以进行下一步的EGFR的检测。徵芯片技术需要血流以极慢点速度通过以便使得抗原抗体充分结合，7.5ml血液大约需要10h。
1.4其它富集技术：
基于微流体技术和细胞大小分离法的新技术也在发展，首先让血液样本通过一定空间的孔道，然后再通过微柱规律排列形成大小间距一定的空间，一定大小以上的细胞得以留存，其它则被分离。利用不同间距的孔径，可以实现不同细胞以及血浆的分离。
微电极阵列技术的引入促生了双向电泳分离技术来富集循环肿瘤细胞。不同特征的细胞会在微电极不同的距离找到自己的位置并聚集不同特征的细胞因为流体力学和导电性的区别得到分离，同时细胞本身的特征得以完整保护。用培养的肿瘤细胞来检验该技术的富集率，发现其回收率超过90％。这些技术的不足之处是其用血量太少，而且必须用相应的等张工作液来稀释，如果按照临床常用的血量来进行实验，其工作液体量可达70ml，而通过速度只有0.5ml/h，液体的稀释过程同样会造成循环肿瘤细胞的丢失。
1.5HLA-G：
胎儿基因的一半来自父亲，胎儿对母亲来讲是一个半异体，但胎儿如何能够耐受母亲的免疫***排斥呢？经过十多年来研究已经证明：主要分布在胎盘绒毛细胞表面的白细胞抗原G(HLA-G)起到了主要的功能，HLA-G具有抑制自然杀伤细胞和细胞毒性T淋巴细胞的功能，在胎儿的免疫耐受的机理中起着重要的作用。
肿瘤在发生和发展变化过程中，免疫***对这些发生癌变的细胞有排斥消除的功能，称之为宿主的免疫监视。但在已经患上癌症的病人体内，癌细胞明显地逃过了宿主的免疫监视，这种现象称为肿瘤的免疫逃逸，而其机理在于：肿瘤细胞发生早期具有反祖现象，能够分泌、表达HLA-G，使其逃过宿主的免疫监视和杀伤。
从上世纪九十年代中(1996)起，已有大量的研究结果证明：在大多数常见恶性肿瘤，如乳腺癌，肺癌，食道癌，胃癌，直-结肠癌，卵巢癌，子宫内膜癌，子***的部分或大部分肿瘤细胞出现了HLA-G的异常表达，这是肿瘤生物学中的一个普遍现象。
HLA-G仅在恶性肿瘤中出现异常表达，在除胎盘外的正常组织则无表达，是一种新的、具有高度恶性肿瘤特异性的肿瘤标志物。在2003年巴黎召开的第二届HLA-G学术研讨会上，大会总结了自1999年第一届学术研讨会后HLA-G研究的进展，明确指出：肿瘤细胞出现HLA-G异常表达已是不争之事实。
发明内容
针对上述现有技术中存在的不足，本发明所要解决的技术问题提供一种分选乳腺癌肿瘤细胞的方法。
为了实现上述目的，本发明提出如下技术方案：
抗HLA-G的单克隆抗体偶联免疫磁珠在肿瘤细胞分选中的应用。
所述肿瘤细胞包括乳腺癌肿瘤细胞、食道癌，非小细胞肺癌，子宫癌，胃癌，结肠癌等。
一种肿瘤细胞的分选方法，包括以下步骤：
(1)抗HLA-G的单克隆抗体偶联免疫磁珠得抗HLA-G免疫磁珠；
(2)将抗HLA-G免疫磁珠加入到待分选的细胞液中，用免疫磁珠分离方法进行分离。
优选的，所述偶联磁珠的抗HLA-G的单克隆抗体为MEM-G/2、MEM-G/9或4H84。
优选的，所述抗HLA-G的单克隆抗体和免疫磁珠之间是抗体饱合偶联，优选用料质量比为200ug抗体∶1000ug活化磁珠。
优选的，所述免疫磁珠的制备：取经活化的磁珠，加入饱合偶联用量的抗HLA-G的单克隆抗体和PBS，于室温下圆周混合3h，PBS清洗三次，加入甘氨酸混合，封闭剩余的醛基；加入含有BSA的PBS溶液混合30min，封闭非特异性的吸附位点，PBS清洗3次，加入PBS缓冲液漩涡震荡，即得到均匀的修饰有一抗的免疫磁珠。
优选的，所述待分选的细胞液与HLA-G免疫磁珠的用量体积比为100∶(20-100)。
优选的，所述步骤(2)的具体方法是：取待分选的细胞液100μL(细胞数107/m)，加入HLA-G免疫磁珠40μL，2mg/mL，28℃温箱中孵育30分钟后，在磁力架上利用免疫磁珠磁分离方法，将悬浮液弃之，用适量的0.01M PBS(Ph＝7.4)重复洗涤3-4次，将悬浮液弃之，最后每管用100μL PBS重悬磁珠后，使用细胞计数板对肿瘤细胞数进行计数。
本发明还提供一种用于分离肿瘤细胞的抗HLA-G免疫磁珠。以及含有抗HLA-G免疫磁珠的分选试剂盒。
本发明的有益效果是：
(1)选用HLA-G为分选肿瘤细胞标志物，HLA-G为肿瘤特异的广谱性标志物，偶联免疫磁珠后，特异性好，分选肿瘤效率高；
(2)HLA-G偶联免疫磁珠后敏感性和重复性较好，可以检出相关的肿瘤细胞。
本发明的附加方面和优点将在下面的描述中部分给出，部分将从下面的描述中变得明显，或通过本发明的实践了解到。
具体实施方式
下面通过实施例对本发明的技术方案作进一步说明，但不能理解为对本发明的限制。
1、抗HLA-G单克隆抗体
项目中HLA-G的单克隆抗体MEM-G/2、MEM-G/9和4H84，来源公司：圣克鲁斯生物技术公司。
2、纳米免疫磁珠制备
纳米免疫磁珠制备包括活化磁珠与单抗的偶联关键步骤。通过优化纳米粒子的粒径和抗体的连接效率，提高纳米免疫磁珠对抗原细胞的吸附效率。
2.1纳米磁珠的选择及修饰
试验方法：
1、活化磁珠的购买：本实施例使用的是美天旎公司、Kisker公司生产的磁珠。
2、磁珠选择50nm活化磁珠。
2.2免疫磁珠与单抗偶联制备
试验方法：
1、抗体偶联：1)抗体偶联前，抗HLA-G抗体(分别使用MEM-G/2、MEM-G/9和4H84做实验)对PBS在4度C，经过三次透析，过夜，储存在PBS中，将浓度调整到5mg/ml.
2)所述HLA-G免疫磁珠的制备：
取活化磁珠500μL，2mg/mL(以活性磁珠的质量计)，加入40μL 5mg/mL的抗HLA-G的单克隆抗体，在1.5ML微离心管中，于室温下圆周混合3h，1ML PBS清洗三次，加入500μL 0.2mol/L甘氨酸混合30min，封闭剩余的醛基；1MLPBS清洗三次；加入含有质量百分比2.5％BSA的PBS溶液混合30min，封闭非特异性的吸附位点，PBS清洗3次，加入500μL PBS缓冲液漩涡震荡，即得到浓度为2.0mg/ml的均匀的修饰有一抗的免疫磁珠。
2、对单抗标记纳米磁珠的反应条件进行优化，选择最佳的反应条件方法如下：
(1)反应介质的选择：分别取500μL纳米磁珠，置于7个离心管中，编号，在外加磁场的作用下，将1、2管中介质换成磷酸盐缓冲液，浓度以此为0.1M PB(pH＝7.4)和0.01M PB(pH＝7.4)，第3-6管中介质分别为0.1M PBS(pH＝6.0)、0.01M PBS(pH＝6.0)、0.1M PBS(pH＝7.4)和0.01M PBS(pH＝7.4)，第7管中介质为去离子水(pH＝6.0)，分别向管中加入40μL(5mg/ml)抗体，放到恒温振荡器上反应6h后(150r/min)，置于磁场中分离上清液，在紫外分光光度计下测OD280nm，确定最佳的反应介质。
(2)最佳温度的确定：向经过表面修饰后磁珠中加入最适浓度的抗体，分别采用室温(25℃)和37℃两种温度条件下反应4h，然后用0.01M PBS(pH＝7.4)洗涤数次，恢复其原体积并测其上清中OD280nm值。
(3)最佳时间的确定：取6个1.5mL离心管，依次编号1-6，向每管中加入500μL活化处理的纳米磁珠，用0.01M PBS(pH＝7.4)洗涤2-3次，加入40μL(5mg/ml)抗体，加入后立即测上清OD280nm值，然后每隔1h测一次OD280nm值，直至6h。
(4)抗体偶联磁珠的饱和浓度：，500ul 2mg/ml纳米磁珠分别用不同量的抗体5mg/ml，10μL、30μL、40μL、60ul，100μL，补足总体积为600ul，偶联于室温30分钟，加入后立即测上清OD280nm值，偶联完成后，再测一次OD280nm值，确定饱和偶联抗体磁珠比例。
3、抗体偶联量的检测：
采用(BCA)试剂盒(上海生工生物工程技术服务有限公司)结合酶标仪(美国PerKinEImer公司)检测偶联反应后上清液中抗体的含量。抗HLA-G的单克隆抗体溶液用pH7.4，0.01moL磷酸盐吐温(0.05％Tween-20)溶液稀释为0.5mg/ml的标准溶液，分别取0，1，2，4，8，12，16，20μL标准液于酶反应条中，每个标准溶液做1个平行重复，每孔加pH7.4，0.01mol/L磷酸盐吐温(O.05％Tween-20)溶液补足总体积为20μL；将待检测上清液12000r/min，离心10min后，取20μL于酶反应条中，每个待检测上清液做1个平行重复；向所有加了标准液或上清液的酶反应孔中加入200μL BCA反应液，60℃反应30min；用酶标仪检测各孔的吸光度(A) 值。根据标准液的浓度与吸光值做出标准曲线，再根据待检测上清液的吸光度值得到上清液中抗体的量，从而得出单位质量磁珠表面偶联的抗体含量。
试验结果：
1、反应介质的确定：合适的离子浓度和pH的反应介质，对于磁珠与蛋白质高效的偶联减少它们之间的非特异性吸附具有重要作用。实验结果显示，三种抗体均适用，当介质为0.01M PBS(pH＝7.4)时，非特异性吸附不明显，OD280值较高，可以作为包被磁珠的反应介质。
2、最佳温度的确定：在37℃温度偏高，导致部分溶液挥发，且与室温条件相比测得的上清中的OD280数值极其不稳定，故三种抗体的实验选择室温包被。
3、最佳包被时间：从表1中可以看出，三种抗体经磁珠吸附3h后基本上达到了饱和，OD280值趋于稳定，因此确定包被时间为3h。
表1 抗体包被磁珠最佳时间(以4H84为例)
Figure PCTCN2014093816-appb-000001
4、抗体磁珠偶联比例：结果最终显示抗HLA-G的4H84单克隆抗体，将抗体浓度定为5mg/ml，磁珠浓度为2mg/ml，抗体和磁珠质量比为200ug∶1000ug，此时进行磁珠偶联，，抗体偶联基本可达到饱和。
5、磁珠表面抗体定量采用BCA蛋白定量试剂盒检测磁珠表面连接的抗HLA-G抗体的量。得到浓度已知的蛋白标准溶液所对应的吸光度值。根据不同标准溶液所含蛋白量与其吸光度值，做标准曲线。以4H84为例，将上清液样品吸光度值代入公式，得出上清液样品中蛋白含量分别为71.2，因加入反应抗体为200μg，即每1000ug磁珠表面连接的抗体量为128.8；综合以上计算可得，磁珠表面连接的抗HLA-G抗体量平均为128.8μg/mg。
3、抗HLA-G免疫磁珠富集肿瘤细胞试验
使用本实施例步骤2.2制备的三种抗HLA-G免疫磁珠用于人工培养的肿瘤细胞系细胞，进行肿瘤细胞收集；将人工培养的肿瘤细胞系细胞与健康人血液混合制备人工样品，使用抗HLA-G免疫磁珠，对人工样品中肿瘤细胞进行富集，对富集样品进行细胞计数，计算富集效率。
试验方法：
1、细胞样品准备及抗HLA-G免疫磁珠富集肿瘤细胞：
1)实验室培养来自ATCC的6种肿瘤细胞系细胞：乳腺癌症(MCF-7)，食道癌(EC109)，非小 细胞肺癌(A549)，子***(Hela)，胃癌(NCI-N87)，结肠癌(HT29)，制备107/ml(104/ul)培养的各种肿瘤细胞样本；取各种肿瘤细胞，100ul，加入1.5ml离心管中，每种细胞为三管；
2)取5ul上述制备好的各种肿瘤细胞与5mL健康人血液混合，形成肿瘤细胞在外周血中人工样本，肿瘤细胞数：104/ml血液。每种细胞制备三管；
3)将40ul制备的抗HLA-G免疫磁珠，加入100ul肿瘤细胞样品中混匀，孵育，然后使用德国美天泥公司的LS柱和强磁场磁力分离器，进行分离，PBS清洗，然后将磁珠吸附细胞重新悬浮于100ul PBS，进行细胞计数，使用细胞计数板对富集的细胞进行检测，统计肿瘤细胞占富集前细胞总数的比例，计算抗HLA-G磁珠富集肿瘤细胞效率。
4)将40ul 2mg/ml制备的抗HLA-G免疫磁珠，加入1ml人工肿瘤细胞血液样品中，在磁场中进行分离，PBS清洗，然后将磁珠吸附细胞重新悬浮于1ml PBS；取5ul样品，滴加于载玻片上，多聚甲醛固定等IF准备，使用BD公司的抗CK8/18/19-FICT，进行孵育，使用荧光显微镜进行计数，统计回收肿瘤细胞占富集前细胞总数的比例，计算抗HLA-G磁珠富集血液样品中肿瘤细胞效率。
2、纳米免疫磁珠最佳用量确定：取5份MCF-7人工肿瘤样本，每份100μL，分别加入纳米免疫磁珠10、20、40、60、100μL，28℃温箱中孵育1h后，在磁力架上利用免疫磁珠磁分离方法，将悬浮液弃之，用适量的0.01M PBS(Ph＝7.4)重复洗涤2-3次，将悬浮液弃之，最后每管用100μL PBS重悬磁珠后，使用细胞计数板对肿瘤细胞数进行计数。
3、纳米免疫磁珠与肿瘤细胞最佳反应时间的确定：将MCF-7细胞四份体积为1ml的人工肿瘤-血液样本分别加入磁珠40μL，28℃温箱中分别孵育10min、30min、60min后，在磁力架上利用免疫磁珠磁分离方法，将将悬浮液弃之，用适量的0.01M PBS(pH＝7.4)重复洗涤2-3次，将悬浮液弃之，最后每管用100μL PBS重悬磁珠后，使用细胞计数板对肿瘤细胞浓度进行计数。
试验结果：
1、经测试，在制备的各种肿瘤细胞样本中和肿瘤细胞在血液-人工样品中，抗HLA-G免疫磁珠对相对应肿瘤细胞富集率可以达到回收率85％-93％。对血液中肿瘤细胞富集率可达到77％-84％，这说明，制备的抗HLA-G免疫磁珠的富集率较高，可重复性较好，可以回收相关的肿瘤细胞。
表2 抗HLA-G免疫磁珠对不同肿瘤细胞富集敏感性对比
Figure PCTCN2014093816-appb-000002
Figure PCTCN2014093816-appb-000003
2、纳米免疫磁珠用量：见表3经测试，肿瘤样品与纳米磁珠比为100ul(107/ml)∶40ul(2mg/ml)时，富集效果较好且不会造成磁珠浪费。
表3 纳米磁珠用量与肿瘤细胞富集率的关系
Figure PCTCN2014093816-appb-000004
3、纳米免疫磁珠与肿瘤细胞最佳反应时间：见表4，经观察磁珠与肿瘤样本反应30min时，肿瘤细胞数量多，混有的正常细胞少。而磁珠与肿瘤样本反应60min时，正常细胞较多。磁珠与样本结合时间过长或过短，都会增加非特异性反应。因此纳米免疫磁珠与样本反应的最佳时间为30min。
表4 纳米免疫磁珠与肿瘤样本最佳反应时间(以4H84为例)
时间(min) 富集效率
10 62.38％
30 89.12％
60 91.25％
尽管已经示出和描述了本发明的实施例，本领域的普通技术人员可以理解：在不脱离本发明的原理和宗旨的情况下可以对这些实施例进行多种变化、修改、替换和变型，本发明的范围 由权利要求及其等同物限定。

Claims (7)

  1. 抗HLA-G的单克隆抗体偶联免疫磁珠在肿瘤细胞分选中的应用。
  2. 一种肿瘤细胞的分选方法，其特征在于，包括以下步骤：
    (1)抗HLA-G的单克隆抗体偶联免疫磁珠得HLA-G免疫磁珠；
    (2)将HLA-G免疫磁珠混匀后加入到待分选的细胞液中，用免疫磁珠分离方法进行分离。
  3. 根据权利要求2所述的分选方法，其特征在于，所述抗HLA-G的单克隆抗体为MEM-G/2、MEM-G/9或4H84。
  4. 根据权利要求2所述的分选方法，其特征在于，所述抗HLA-G的单克隆抗体和免疫磁珠之间是抗体饱合偶联，优选用料质量比为200ug∶1000ug。
  5. 根据权利要求2所述的分选方法，其特征在于，所述免疫磁珠的制备：
    取经活化的磁珠，加入饱合偶联用量的HLA-G的单克隆抗体和PBS，于室温下混合3h，PBS清洗三次，加入甘氨酸混合，封闭剩余的醛基；加入含有BSA的PBS溶液混合30min，封闭非特异性的吸附位点，PBS清洗3次，加入PBS缓冲液漩涡震荡，即得到均匀的修饰有一抗的免疫磁珠。
  6. 根据权利要求2所述的分选方法，其特征在于，所述待分选的细胞液与HLA-G免疫磁珠的用量体积比为100∶(20-100)。
  7. 根据权利要求2所述的分选方法，其特征在于，所述步骤(2)的具体方法是：取待分选的细胞液100μL(细胞数107/ml)，加入混匀的HLA-G免疫磁珠40μL，2mg/mL，28℃温箱中孵育30分钟后，在磁力架上利用免疫磁珠磁分离方法，将悬浮液弃之，用0.01M PBS(Ph＝7.4)重复洗涤2-3次，将悬浮液弃之，最后每管用100μL PBS重悬磁珠后，使用细胞计数板对肿瘤细胞数进行计数。
PCT/CN2014/093816 2013-12-31 2014-12-15 抗hla-g的单克隆抗体偶联免疫磁珠在肿瘤细胞分选中的应用 WO2015101163A1 (zh)

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