WO2023133895A1 - 获取特异性识别抗原的纳米抗体信息的方法 - Google Patents

获取特异性识别抗原的纳米抗体信息的方法 Download PDF

Info

Publication number
WO2023133895A1
WO2023133895A1 PCT/CN2022/072393 CN2022072393W WO2023133895A1 WO 2023133895 A1 WO2023133895 A1 WO 2023133895A1 CN 2022072393 W CN2022072393 W CN 2022072393W WO 2023133895 A1 WO2023133895 A1 WO 2023133895A1
Authority
WO
WIPO (PCT)
Prior art keywords
cells
droplet
cell
antigen
nanobody
Prior art date
Application number
PCT/CN2022/072393
Other languages
English (en)
French (fr)
Inventor
吕孟华
孙娜
刘杨
刘亚
刘龙奇
王媚娘
王诗雨
高开
刘小盼
任丙昭
郑越
梁元庆
沈梦哲
Original Assignee
深圳华大生命科学研究院
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by 深圳华大生命科学研究院 filed Critical 深圳华大生命科学研究院
Priority to PCT/CN2022/072393 priority Critical patent/WO2023133895A1/zh
Publication of WO2023133895A1 publication Critical patent/WO2023133895A1/zh

Links

Images

Classifications

    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
    • G01N21/62—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light
    • G01N21/63—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light optically excited
    • G01N21/65—Raman scattering

Definitions

  • the present invention relates to the field of biology.
  • the present invention relates to methods for obtaining information on Nanobodies that specifically recognize antigens.
  • Antibodies have the characteristics of high affinity with cognate antigens, high specificity, and can be obtained in monoclonal form. They are the preferred tools for many applications including scientific research, in vivo and in vitro medical diagnosis, and immunotherapy. However, due to the large molecular weight (150kDa) of traditional antibodies and their relatively complex protein folding characteristics, the sequence amplification and expression of antibodies are limited, resulting in high production costs and limited applications. In camelids, in addition to the above-mentioned conventional antibodies produced by B cells, there is also an antibody lacking light chains. The structure of its VHH fragment cloned and expressed separately is the smallest unit known to bind the target antigen, so it is also Known as nanobody (nanobody).
  • nanobodies are also expected to have a significant role in the market and clinic because of their low molecular weight (15kDa), high affinity, strong thermal stability, strong tissue permeability, good solubility, and easy modification. Wider application, and greatly reduce the production cost of antibody drugs.
  • the screening methods for antibodies mainly include phage display technology, yeast surface display technology, ribosome display technology, etc., but there are some shortcomings.
  • phage display technology belongs to prokaryotic expression system and lacks proper post-translational processing. Folding of the antibody sometimes occurs incorrectly, resulting in the problem that the antibodies screened by this method sometimes have low affinity; the library capacity (10 7 ) of the yeast display technology is much smaller than that of the phage display technology (10 9 ), and the throughput is small.
  • the yeast The growth and reproduction speed is not as fast as that of phage; although there are corresponding commercial reagents available for ribosome display technology, this method is technically difficult and requires high technical requirements for operators.
  • Microfluidics is a multi-interdisciplinary subject including engineering, physics, chemistry, microfabrication and bioengineering.
  • Microfluidic technology has the ability to shrink the basic functions of biological and chemical laboratories onto a chip of a few square centimeters. It can precisely control and manipulate micro-scale fluids to complete different biological or chemical reaction processes, and to carry out their products. analyze.
  • the basic characteristics and greatest advantages of microfluidic technology are the flexible combination and scale integration of various unit technologies on an overall controllable micro platform, which has the advantages of controllable liquid flow, minimal consumption of samples and reagents, high throughput, and fast analysis speed. and other advantages. Then, how to use microfluidic technology to realize the study of nanobody and immune response mechanism is an urgent problem to be solved.
  • the present invention aims to solve at least one of the technical problems existing in the prior art at least to a certain extent. For this reason, the present invention proposes a method for obtaining information on nanobodies that specifically recognize antigens.
  • This method uses microfluidic technology to screen out B cells that can secrete nanobodies at high throughput, and has the advantages of short time-consuming, high efficiency, and complete retention. Cell information and other advantages.
  • single-cell sequencing is used to sequence and analyze the screened B cells, so that the sequence information of the nanobody can be accurately obtained, the nanobody can be synthesized, and the role of the nanobody and the B cells that produce the nanobody in the immune system can also be studied The mechanism has a good application prospect.
  • the invention proposes a method for obtaining the information of the nanobody that specifically recognizes the antigen.
  • the method includes: Step 1: Enriching mature B cells from the subject's peripheral blood lymphocytes; Step 2: Using microfluidic droplets to generate a chip, which will contain antigens and signal detection reagents The solution and the single B cell are wrapped in a single droplet, and the signal detection reagent can generate a detectable signal when the antigen is combined with the target nanobody; step 3: incubating the generated single droplet, So that the B cells secrete the nanobody and the antigen specifically binds to the nanobody; step 4: sort the incubated droplet using a droplet sorting chip, and analyze the collected detectable signal , so as to sort out the target cells that can secrete the target nanobody, and collect the positive droplets containing the target cell; step 5: perform single-cell sequencing on the target cell, so as to obtain the target nanobody information.
  • a microfluidic droplet generation chip is used to obtain a single B cell wrapped with an antigen and a signal detection reagent.
  • the resulting signal changes, thereby screening out the B cells that secrete the target nanobody.
  • the B cell is sequenced to obtain the antigen receptor (namely nanobody) and its mRNA sequence information on the surface of the B cell, which can be used to accurately synthesize the nanobody and make it functional.
  • the method according to the embodiment of the present invention uses microfluidic technology to screen out B cells that can secrete nanobodies in high throughput, which has the advantages of short time, high efficiency, and the ability to retain complete cell information. Further, single-cell sequencing is used to sequence and analyze the screened B cells, so that the sequence information of the nanobody can be accurately obtained, the nanobody can be synthesized and used, and the nanobody and the B cells that produce the nanobody can also be studied in the immune system. The mechanism of action is played, and the application prospect is good.
  • the above-mentioned method for obtaining information on Nanobodies that specifically recognize antigens may also have the following additional technical features:
  • the subject is selected from the family Camelidae, preferably an alpaca.
  • camelids plasma cells produce antibodies whose Fab-terminal VHH portions are nanobodies.
  • the subject has previously been immunized with the antigen in vivo. This facilitates the acquisition of a large number of B cells.
  • step 1 includes: step 1-1: incubating the peripheral blood lymphocytes with a biotin-labeled antibody that can bind to mature B cells; step 1-2: incubating the Wash, filter and resuspend the incubated cells to obtain a single cell suspension; Step 1-3: incubate the single cell suspension with anti-biotin magnetic beads, and the anti-biotin-labeled magnetic beads The beads can be combined with the biotin-labeled antibody, and the magnetic beads in the incubated cell fluid can be collected to obtain enriched mature B cells.
  • B cells Based on the characteristics of B cells, antibodies that can bind to them are selected for subsequent enrichment. After the B cells are combined with the antibody, they are washed and filtered. The purpose of the filtration is to remove the united cells and minimize the situation where multiple cells are wrapped into the same droplet. Finally, they are resuspended to obtain a single cell suspension. Since the antibody is labeled with biotin, incubating the single cell suspension with anti-biotin magnetic beads can make the anti-biotin magnetic beads capture the antibody and form anti-biotin magnetic beads-biotin-labeled antibody-B cells , to achieve the enrichment of B cells.
  • the antibody is selected from mouse anti-alpaca BCMA monoclonal antibody.
  • the antibody can specifically bind B cells.
  • step 1-1 0.01-1 ⁇ L of antibody is incubated for every 1 ⁇ 10 5 cells; the incubation is performed at room temperature for 0.5-1 hour in the dark.
  • antibodies can be sufficiently bound to B cells.
  • the cell density in the single cell suspension is not more than 10 7 cells/mL; the incubation is carried out at 2-6°C for 10-20 minutes. If the cell density is too large, the number of cells wrapped in a single droplet is greater than 1 when the droplet is generated.
  • the signal detection reagent is selected from fluorescent secondary antibodies and streptomycin affinity magnetic beads.
  • the antigen is provided in the form of streptavidin-affinity magnetic beads-biotin-antigen.
  • Fluorescent secondary antibodies can be nanobody-conjugated. After the fluorescent secondary antibody needs to be combined with the antibody first, when the antibody can bind to the antigen (the antibody is antigen-specific), the fluorescent secondary antibody is indirectly linked to the antigen. Therefore, in the solution containing the antigen and the signal detection reagent, the fluorescent secondary antibody The anti-antibody exists free and produces a weak fluorescent signal. Streptavidin magnetic beads bind to the antigen through biotin.
  • the fluorescent secondary antibody When the antigen binds to the nanobody on the B cell, the fluorescent secondary antibody will bind to the antigen and the nanobody respectively, so that the fluorescent secondary antibody can be indirectly connected to the magnetic bead , due to the magnetism between the magnetic beads, multiple magnetic beads gather together, and then drive multiple fluorescent secondary antibodies to gather together, which increases the intensity of the fluorescent signal. In this way, it can be judged based on the strength of the fluorescent signal whether it is a positive droplet containing the target nanobody.
  • the present invention does not strictly limit the type of the fluorescent secondary antibody, which can be selected based on the characteristics of the antigen and the nanobody, as long as it can bind to the antigen and the nanobody.
  • the fluorescent secondary antibody is selected from 488-conjugated AffiniPure Goat Anti-Alpaca IgG, subclasses 2+3 specific.
  • step 3 the incubation is carried out at 30-40° C. for 1-4 hours. In this way, the cells can fully secrete the antibody and the antigen can be fully combined with the antibody. In a preferred embodiment, the incubation is carried out at 37°C for 1-4 hours.
  • the parameters used in the sorting are as follows: laser power: 1-15mW; signal gain: 0.37-0.56; waveform generator setting: square wave, voltage 2-3VPP, 40cycles/ s, frequency is 20K HZ, external trigger, divided voltage output load; voltage magnification: 200 times.
  • the inventor obtained the above sorting conditions through a large number of experiments, thereby effectively improving the sorting efficiency and accuracy. Other parameter conditions are not effective. For example, if the laser power is too high, the background noise will increase, and if the laser power is too low, the signal strength will increase. Not enough; if the voltage is too high, the droplets will fuse, and if the voltage is too small, the droplets will not be deflected.
  • the flow rate at which the incubated droplets are injected into the droplet phase inlet of the droplet sorting chip is 10-100 ⁇ L/h, and the oil phase is injected into the droplet sorting chip
  • the flow rate of the oil phase inlet of the chip is 800-1200 ⁇ L/h.
  • the inventor obtained the above-mentioned optimal flow rate through a large number of experiments. If the flow rate of the oil phase is too high, the liquid droplets will not be easily deflected, and if the flow rate of the oil phase is too small, the distance between the droplets will be too small, which will reduce the accuracy of sorting.
  • the droplet flow rate is related to the sorting flux. If the droplet velocity is too small, the throughput will decrease, if the droplet velocity is too high, the sorting accuracy will decrease.
  • the analysis includes: separately sorting the incubated droplets and negative droplets on the same droplet sorting chip to obtain a sample signal data set and a negative signal Data set, wherein, the negative droplets contain antibodies and signal detection reagents, but do not contain cells; respectively calculate the ratio of the maximum signal value to the sum of all signal values in the sample signal data set and the negative signal data set, and record it as the sample signal ratio and negative signal ratio; the sample signal ratio being greater than the negative signal ratio is an indication that the post-incubation droplet is a positive droplet containing the target cell.
  • the method of the present invention for obtaining information on nanobodies that specifically recognize antigens enables high-throughput screening of B cells that secrete target nanobodies, and the analysis of millions of cells can be completed within 4 hours, thereby greatly increasing the throughput of screening , shortening the time required for screening (traditional methods need more than 2 weeks, this method shortens the time to a few hours). It can directly detect primary cells without cell cloning, and can obtain natural nanobody secreted cell RNA and mRNA information, making the synthesized antibody more functional, and can realize the analysis of B cell secreted receptors, that is, antibodies, without the need Restricted to cell surface molecules.
  • Figure 1 shows a schematic diagram of the operation process according to an embodiment of the present invention
  • Fig. 2 has shown the electron micrograph according to one embodiment of the present invention, and wherein, A is before sorting (fluorescent field); B is before sorting (bright field); C is after sorting (fluorescent field); D is sorting after (brightfield).
  • Figure 3 shows the electrophoresis of alpaca BCR amplification, in which the BCR fragment is mainly enriched at about 500bp.
  • the working buffer working solution, used for droplet generation and cell culture in the droplet
  • the main reagent dosage is from the reference (High-throughput single-cell activity-based screening and sequencing of antibodies using droplet microfluidics.DOI :10.1038/s41587-020-0466-7).
  • the fluorescent secondary antibody Alexa 488-conjugated AffiniPure Goat Anti-Alpaca IgG, subclasses 2+3 specific, Jackson Company
  • Affinity streptomycin-coated magnetic beads were co-incubated with biotin-antigen.
  • Use a pipette gun to draw 10 ⁇ l magnetic beads (Strep Plus, 300nm, ADEMTECH, 03233, 5mg/ml) into a 1.5ml low adsorption centrifuge tube, add 50 ⁇ l 1.5 ⁇ mol biotin-S1 (for bitoin-Goat IgG anti-Alpaca IgG (VHH ), using a concentration of 100nmol), and incubated at room temperature for 30min to fully bind the biotin-antigen to the SA magnetic beads.
  • the target cells i.e. cryopreserved alpaca peripheral blood lymphocytes
  • the target cells i.e. cryopreserved alpaca peripheral blood lymphocytes
  • Alpaca B cell single cell suspension preparation Alpaca B cells collected in 4.2.1 were centrifuged at 500 ⁇ g for 5 minutes at room temperature, and the supernatant was removed;
  • 4.2.2 Use the configured working buffer to resuspend the enriched alpaca B cells, and pipette to mix well.
  • the incubated droplets were placed in the ring magnet for 2 minutes, and then the droplets were loaded onto the droplet sorting chip to complete signal detection and droplet sorting.
  • the specific operation method is as follows:
  • Sorting platform hardware parameter setting Set the parameters of the laser, comparator, waveform generator, and signal amplifier. The parameters are as follows:
  • Waveform generator settings square wave, voltage 3VPP, 40cycles/s, frequency 20K HZ, external trigger, voltage divider output load;
  • the percentage of positive droplets was observed and estimated under a fluorescent microscope in advance (assumed to be 10% positive rate). Collect the signal values of 50,000 droplets (experience value, the droplet signal is between 1-2), and set the signal value in the 10% percentile as the threshold according to the collected signal values.
  • the threshold setting can be adjusted according to the actual situation: in order to ensure the positive rate, a more rigorous threshold can be set, such as the signal value of 5%. In order to ensure the yield, the threshold can be relaxed, for example, the signal value of 15% is set as the threshold. Then start sorting, and record the droplet signal throughout.
  • the ratio of the concentrated maximum signal value to the sum of all signal values is recorded as the sample signal ratio and the negative signal ratio; collect positive droplets whose sample signal ratio is greater than the negative signal ratio.
  • the recovered cells were divided into two parts, and the following operations were performed respectively:
  • the B cells are lysed, reverse transcribed, and amplified with the primer sequences for classic alpaca BCR amplification.
  • Primer 2 GATGTGCAGCTGCAGGAGTCTGGRGGAGG, R stands for A or G
  • sequence information obtained by sequencing was compared on NCBI, and it was found that it could be compared to the IGH chain of Camelidae, and the comparison rate was distributed from >80% to >90%. This shows that the present invention can obtain the nanobody sequence information with high accuracy.

Landscapes

  • Health & Medical Sciences (AREA)
  • Chemical & Material Sciences (AREA)
  • Immunology (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Biochemistry (AREA)
  • Organic Chemistry (AREA)
  • General Health & Medical Sciences (AREA)
  • Medicinal Chemistry (AREA)
  • Genetics & Genomics (AREA)
  • Molecular Biology (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • Biophysics (AREA)
  • Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
  • Physics & Mathematics (AREA)
  • Analytical Chemistry (AREA)
  • General Physics & Mathematics (AREA)
  • Pathology (AREA)
  • Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)

Abstract

提供了获取特异性识别抗原的纳米抗体信息的方法，包括：从受试者的外周血淋巴细胞中富集B细胞；利用微流控液滴生成芯片，将含有抗原和信号检测试剂的溶液与单个B细胞包裹在单个液滴内；将生成的单个液滴进行孵育，以使B细胞分泌纳米抗体以及抗原与纳米抗体发生特异性结合；利用液滴分选芯片对孵育后的液滴进行分选，对收集的可检测信号进行分析，以便分选出可分泌目标纳米抗体的目标细胞，收集包含目标细胞的阳性液滴；对目标细胞进行单细胞测序，以便获取目标纳米抗体信息。

Description

获取特异性识别抗原的纳米抗体信息的方法 技术领域
本发明涉及生物领域。具体地，本发明涉及获取特异性识别抗原的纳米抗体信息的方法。
背景技术
抗体有与同源抗原具有高度亲和力、高度特异性、能够以单克隆形式获得等特点，是包括科学研究、体内外医学诊断和免疫治疗在内的许多应用的首选工具。然而由于传统抗体的分子量大(150kDa)以及其蛋白折叠相对复杂的特点，使其在抗体的序列扩增及表达受到限制，因而生产成本高，在应用中受到限制。在骆驼科动物中，B细胞除了产生上述常规的抗体，还存在一种缺失轻链的抗体，其VHH片段单独克隆并表达出来的结构是目前已知的可结合目标抗原的最小单位，故也被称作纳米抗体(nanobody)。纳米抗体除了具有上述传统抗体的优点外，还因其低分子量(15kDa)、高亲和力、热稳定性强、组织渗透性强、溶解性好、易改造等优点使其有望在市场和临床中有着更广泛的应用，并大大降低抗体药物的生产成本。
目前，针对抗体的筛选方法主要有噬菌体展示技术、酵母菌表面展示技术、核糖体展示技术等，但均存在着一些不足之处，如噬菌体展示技术属于原核表达系统，缺乏适当的翻译后加工，其抗体折叠有时会发生错误，导致该方法筛选出的抗体有时存在亲和力不高的问题；酵母展示技术的库容量(10 7)远小于噬菌体展示技术(10 9)，通量小，此外酵母的生长及繁殖速度也不及较噬菌体快；核糖体展示技术虽然有相应的商业化试剂可用，但是该方法的技术难度较大，对操作人员的技术要求较高，此外由于其产物为蛋白一级结构，因此不适用于高级结构的蛋白筛选。以上几种方法，有一个共同特点，一是筛选耗时长，二是筛选过程中丢失了抗原特异性B细胞的基因组信息。
微流控是一个包括了工程学、物理学、化学、微加工和生物工程等领域的多交叉学科。微流控技术具有将生物、化学等实验室的基本功能缩微到一个几平方厘米芯片上的能力，可以精确控制和操控微尺度流体，以完成不同的生物或化学反应过程，并对其产物进行分析。微流控技术的基本特征和最大优势是多种单元技术在整体可控的微小平台上灵活组合、规模集成，具有液体流动可控、消耗试样和试剂极少、高通量、分析速度快等优势。那么，如何利用微流控技术实现研究纳米抗体及免疫反应机制是亟待解决的问题。
发明内容
本发明旨在至少在一定程度上解决现有技术中存在的技术问题至少之一。为此，本发明提出了获取特异性识别抗原的纳米抗体信息的方法，该方法采用微流控技术高通量筛选出可分泌纳米抗体的B细胞，具有用时短、效率高、可以保留完整的细胞信息等优点。进一步地，采用单细胞测序对筛选的B细胞进行测序分析，从而可以准确获知纳米抗体的序列信息，合成纳米抗体，也可以研究纳米抗体及产生该纳米抗体的B细胞在免疫系统中发挥的作用机制，应用前景好。
本发明提出了一种获取特异性识别抗原的纳米抗体信息的方法。根据本发明的实施例，所述方法包括：步骤1：从受试者的外周血淋巴细胞中富集成熟B细胞；步骤2：利用微流控液滴生成芯片，将含有抗原和信号检测试剂的溶液与单个所述B细胞包裹在单个液滴内，所述信号检测试剂可在所述抗原与目标纳米抗体结合时产生可检测信号；步骤3：将生成的所述单个液滴进行孵育，以使所述B细胞分泌纳米抗体以及所述抗原与纳米抗体发生特异性结合；步骤4：利用液滴分选芯片对所述孵育后的液滴进行分选，对收集的可检测信号进行分析，以便分选出可分泌目标纳米抗体的目标细胞，收集包含所述目标细胞的阳性液滴；步骤5：对所述目标细胞进行单细胞测序，以便获取目标纳米抗体信息。
根据本发明实施例的方法中，利用微流控液滴生成芯片，获得包裹抗原和信号检测试剂的单个B细胞，对该单个B细胞共培养，再利用液滴分选芯片，基于信号检测试剂产生的信号变化，从而筛选出分泌目标纳米抗体的B细胞。最后，对该B细胞进行测序，获得B细胞表面的抗原受体(即纳米抗体)及其mRNA序列信息，利用这些信息可以准确地合成纳米抗体，并使其具有功能性。
由此，根据本发明实施例的方法采用微流控技术高通量筛选出可分泌纳米抗体的B细胞，具有用时短、效率高、可以保留完整的细胞信息等优点。进一步地，采用单细胞测序对筛选的B细胞进行测序分析，从而可以准确获知纳米抗体的序列信息，合成并利用该纳米抗体，也可以研究纳米抗体及产生该纳米抗体的B细胞在免疫系统中发挥的作用机制，应用前景好。
根据本发明的实施例，上述获取特异性识别抗原的纳米抗体信息的方法还可以具有下列附加技术特征：
根据本发明的实施例，所述受试者选自骆驼科，优选羊驼。在骆驼科动物中，浆细胞会产生抗体，该抗体的Fab端的VHH部分即为纳米抗体。
根据本发明的实施例，所述受试者预先经过所述抗原的体内免疫。由此，以便于获取大量的B细胞。
根据本发明的实施例，步骤1包括：步骤1-1：将所述外周血淋巴细胞与生物素标记的抗体进行孵育，所述抗体可与成熟的B细胞结合；步骤1-2：将所述孵育后的细胞进行洗涤、过滤和重悬，以便得到单细胞悬液；步骤1-3：将所述单细胞悬液与抗生物素的磁珠进行孵育，所述抗生物素标记的磁珠可与所述生物素标记的抗体结合，收集孵育后的细胞液中的磁珠，以便得到富集的成熟B细胞。
基于B细胞的特性，选择可以与其结合的抗体，以便于后续进行富集。B细胞与抗体结合后，进行洗涤和过滤，过滤的目的是为了除去团结的细胞，尽量减少多个细胞包裹到同一个液滴中的情况，最后经重悬，以便得到单细胞悬液。由于抗体携带有生物素标记，所以再将单细胞悬液与抗生物素的磁珠进行孵育，可以使得抗生物素磁珠捕获抗体，形成抗生物素磁珠-生物素标记的抗体-B细胞，实现B细胞的富集。
根据本发明的实施例，所述抗体选自鼠抗羊驼BCMA单克隆抗体。该抗体可以特异性地结合B细胞。
根据本发明的实施例，步骤1-1中，每1×10 5个细胞与0.01～1μL抗体进行孵育；所述孵育是在室温避光下进行0.5～1小时。由此，可以从使得抗体充分结合B细胞。
根据本发明的实施例，步骤1-3中，所述单细胞悬液中细胞密度不大于10 7个/毫升；所述孵育是在2～6℃下进行10～20分钟。若细胞密度过大会造成在生成液滴时候，单个液滴里包裹的细胞数大于1。
根据本发明的实施例，步骤2中，所述信号检测试剂选自荧光二抗和链霉素亲和磁珠。
根据本发明的实施例，所述抗原是以链霉素亲和磁珠-生物素-抗原的形式提供的。
荧光二抗可以纳米抗体结合。荧光二抗需要先与抗体结合后，在抗体可以结合抗原时(抗体为抗原特异性的)，荧光二抗间接地与抗原相连接，因此，在含有抗原和信号检测试剂的溶液中，荧光二抗是游离存在的，产生的荧光信号较弱。链霉亲和素磁珠通过生物素与抗原结合，当抗原与B细胞上的纳米抗体结合时，荧光二抗将分别与抗原和纳米抗体结合，从而可以使荧光二抗间接连接在磁珠上，由于磁珠之间具有磁性，使得多个磁珠聚集一起，进而带动着多个荧光二抗聚集一起，使得荧光信号强度增强。如此，可以基于荧光信号强弱判断是否为含有目标纳米抗体的阳性液滴。
需要说明的是，本发明对于荧光二抗的种类不做严格限定，可以基于抗原及纳米抗体的特性而选择，只要可以与抗原和纳米抗体结合即可。根据本发明的实施例，所述荧光二抗选自
Figure PCTCN2022072393-appb-000001
488-conjugated AffiniPure Goat Anti-Alpaca IgG,subclasses 2+3 specific。
根据本发明的实施例，步骤3中，所述孵育是在30～40℃下进行1～4小时。由此，以便于使细胞充分分泌抗体以及抗原和抗体充分结合。在一优选实施例中，所述孵育是在37℃ 下进行1～4小时。
根据本发明的实施例，步骤4中，所述分选采用的参数如下：激光功率：1-15mW；信号增益：0.37-0.56；波形发生器设置：方形波，电压为2-3VPP，40cycles/s，频率为20K HZ，外部触发，分压output load；电压放大倍数：200倍。发明人经过大量实验得到上述分选条件，由此，可以有效地提高分选效率和准确性，其他参数条件效果不佳，例如激光功率过高会导致背景噪音变大，过低会导致信号强度不够；电压过大会导致液滴融合，电压过小会导致液滴无法偏转。
根据本发明的实施例，步骤4中，所述孵育后的液滴注入所述液滴分选芯片的液滴相入口的流速为10～100μL/h，油相注入所述液滴分选芯片的油相入口的流速为800～1200μL/h。发明人经过大量实验得到上述较优流速，若油相流速过高会导致液滴不易偏转，油相流速过小，会导致液滴间距过小，降低分选的准确率。另外，液滴流速与分选通量有关。液滴速度过小，通量降低，液滴速度过大，分选准确率降低。
根据本发明的实施例，步骤4中，所述分析包括：在同一液滴分选芯片上，分别将所述孵育后的液滴与阴性液滴进行分选，获得样品信号数据集和阴性信号数据集，其中，所述阴性液滴中含有抗体和信号检测试剂，不含有细胞；分别计算所述样品信号数据集和阴性信号数据集中最大信号值与所有信号值总和的比值，记作样品信号比值和阴性信号比值；所述样品信号比值大于阴性信号比值，是所述孵育后的液滴为含有所述目标细胞的阳性液滴的指示。
有益效果：
本发明的获取特异性识别抗原的纳米抗体信息的方法实现了高通量筛选分泌目标纳米抗体的B细胞，可以在4小时内完成对百万个细胞的分析，从而大大增加了筛选的通量，缩短了筛选所需要的时间(传统方法需要2周以上，本方法将时间缩短至几个小时内)。能够针对原代细胞直接检测，无需进行细胞克隆，可以获取天然的纳米抗体分泌细胞RNA及mRNA信息，使合成的抗体更具功能性，可以实现B细胞分泌型受体，即抗体的分析，不必局限于细胞表面分子。
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。
附图说明
本发明的上述和/或附加的方面和优点从结合下面附图对实施例的描述中将变得明显和容易理解，其中：
图1显示了根据本发明一个实施例的操作流程示意图；
图2显示了根据本发明一个实施例的电镜图，其中，A为分选前(荧光场)；B为分选前(明场)；C为分选后(荧光场)；D为分选后(明场)。
图3显示了对羊驼BCR扩增后的电泳图，其中BCR片段主要富集在500bp左右。
具体实施方式
下面将结合实施例对本发明的方案进行解释。本领域技术人员将会理解，下面的实施例仅用于说明本发明，而不应视为限定本发明的范围。实施例中未注明具体技术或条件的，按照本领域内的文献所描述的技术或条件或者按照产品说明书进行。所用试剂或仪器未注明生产厂商者，均为可以通过市购获得的常规产品。
实施例1
在该实施例中，参考图1，按照下列方法获取特异性识别抗原的纳米抗体信息：
1.配置working buffer(工作液，用于的液滴生成及液滴内的细胞培养)，主要试剂用量来自参考文献(High-throughput single-cell activity-based screening and sequencing of antibodies using droplet microfluidics.DOI:10.1038/s41587-020-0466-7)。
2.配置荧光二抗
使用第一步配置好的working buffer将荧光二抗(Alexa
Figure PCTCN2022072393-appb-000002
488-conjugated AffiniPure Goat Anti-Alpaca IgG,subclasses 2+3 specific，Jackson公司)稀释至100nM。
3.准备磁珠
3.1亲和链霉素包裹的磁珠与biotin-抗原共孵育。用移液枪吸取10μl磁珠(Strep Plus,300nm,ADEMTECH，03233，5mg/ml)到1.5ml低吸附离心管中，加入50μl 1.5μmol biotin-S1(对于bitoin-Goat IgG anti-Alpaca IgG(VHH)，使用浓度为100nmol)，室温孵育30min使biotin-抗原充分结合SA磁珠。
3.2洗涤磁珠以除去游离的biotin-抗原。将离心管放在磁力架上静置3-5min，待上清变澄清后，用移液器移除上清。
3.3重复洗涤磁珠。从磁力架取下离心管，加入500μl含0.1％PF127的1X PBS悬浮磁珠，将离心管放在磁力架上静置3-5min，待上清变澄清后，用移液器移除上清。重复本步骤3次。
3.4取50μl第二步配好的浓度为100nmol的荧光二抗重悬磁珠，避光，置于冰上待 用。此时磁珠浓度为1mg/ml。
4.准备细胞(纳米抗体)
羊驼B细胞富集
4.1.1取目标抗原(SARS-CoV-2(COVID-19)S1protein,His Tag，货号：S1N-C52H2-1mg品牌：百普赛斯)对羊驼进行体内免疫，一般在为2个月完成4-5次免疫，每次免疫间隔两周左右。最后一次免疫完成后，静脉采血50-100ml，利用梯度密度离心法分离羊驼的外周血单核细胞，于液氮中保存备用。
4.1.2液氮中复苏目的细胞(即冻存的羊驼外周血淋巴细胞)于6孔板或合适的细胞培养皿中，置于37℃、5％CO 2培养箱，过夜培养；
4.1.3第二天从孔板中回收细胞至15ml离心管中，500μl PBS洗孔板板底，重复3次，一并回收至15ml离心管中。对细胞进行计数；
4.1.4 500×g室温离心5分钟，去除培养基上清；
4.4.5使用PBS重悬细胞沉淀，并调整细胞密度至5×10 6个/mL；
4.1.6向细胞悬液中加入合适量的(每5×10 5个细胞加1μL抗体)生物素标记的鼠抗羊驼BCMA单克隆抗体，用移液器轻轻吹打混匀后，室温避光孵育30分钟至1小时；
4.1.7 500×g室温离心5分钟，去除PBS上清,使用PBS洗涤细胞3次；
4.1.8使用PBS重悬细胞沉淀(调整细胞浓度至5×10 6个/ml)，过20μm直径细胞筛，除去细胞团，使成为单细胞悬浮液，备用；
4.1.9将autoMACS rinsing solution(购自MILTENYI，货号130-091-222)与MACS BSA stock solution(购自MILTENYI，货号130-091-376)按照体积比20：1混合，配置成Buffer。使用的试剂应该在4度冰箱预冷，以出去气泡(气泡会堵塞柱子)，下述步骤至4.1.16中均使用此buffer；
4.1.10 1-2ml buffer悬浮羊驼细胞(细胞使用前确保为单细胞悬浮液)，300g 10min，弃上清；
4.1.11 80μl buffer重悬细胞(细胞密度<10 7个/mL)中加入20μl抗生物素磁珠,混匀，4°冰箱孵育15min；
4.1.12 1-2ml buffer洗细胞，300g 10min,弃上清。100μl buffer重悬细胞；
4.1.13 MS管(MiniMACS，美天旎)放在磁力架上，5ml EP管置于MS管下方，500ul buffer预润MS管；
4.1.15细胞悬液加入MS中，收集流出的液体(废液)；
4.1.15 500μl buffer洗MS管，合并收集流出的液体(废液)，重复3次；
4.1.16将MS管从磁力架上移除，放在新的5ml EP管上，加入1ml buffer用塞子推出目的细胞，为保证得率，可加入1ml buffer重复一次；为保证纯度，可以换新的MS管再重复一次磁珠富集步骤。
4.2羊驼B细胞单细胞悬液准备：4.2.1收集到的羊驼B细胞，500×g室温离心5分钟，去除上清；
4.2.2使用配置好的working buffer重悬富集的羊驼B细胞，移液枪吹打混匀。使用20μm直径的滤网过滤，去除成团细胞，然后调整细胞浓度至10 7cells/ml，使用AO/PI染料、countstar芯片和细胞计数仪检测细胞活率。
5.液滴生成
5.1将芯片表面的保护膜撕掉，放置于液滴发生装置的芯片槽区域。
5.2向收集管加入200μl液滴生成油，旋紧收集管盖，并将收集管竖直放置于固定架上。
5.3将收集管盖上的连接管A端(接触收集管底部的连接管)插入芯片的Outlet孔。
5.4将30ml注射器放置在固定架上，并调节推杆到初始位置。用针头连接注射器和收集管盖上的连接管B端(非接触收集管底部的连接管)。
注意：在进行上样操作时，注意细胞和磁珠加样孔的位置，将细胞和磁珠加到对应的孔中。
5.5使用移液器轻轻吹打混匀细胞，向芯片的细胞孔加入50μl单细胞悬液，确保枪头接触孔底部。
5.6轻轻吹打混匀磁珠，向芯片beads孔加入100μl磁珠，确保枪头接触孔底部。
5.7快速添加200μl液滴生成油到芯片的Oil孔。
5.8快速将注射器的推杆拉到液滴发生位置，并固定在液滴发生装置的卡槽中，此时开始生成液滴。在显微镜下观察芯片中的液滴生成情况，防止有堵塞情况出现。
5.9待细胞相将要消耗完毕时，在细胞相中重新加入50μl working buffer，吹打混匀，并在油相中添加100μl Droplet Generation Oil。
5.10约15min后液滴生成结束，从装置中取下液滴收集管。
6液滴内细胞孵育
将液滴在37°细胞培养箱孵育2小时，完成细胞分泌抗体以及抗原抗体结合。
7液滴分选
孵育好的液滴在环形磁铁中放置2min时间，然后将液滴上样到液滴分选芯片中，完成信号检测和液滴分选。具体操作方法如下：
7.1取1ml和5ml的注射器各一只，取掉针头。5ml注射器内吸入5ml油(油相)。 1ml注射器吸取200μl油，润注射器内壁，随后小心吸取孵育后的液滴(液滴相)。随后重新将1ml注射器的针头分别重新盖到两只注射器上。剪取2根长约30cm、内径为1mm的软管，分别将软管的一头链接在1ml和5ml注射器针头上，小心推注射器，排尽注射器及软管中的空气。
7.2装有液滴和油的两个注射器分别安装在压力泵中。对于液滴样本，压力泵设置流速为50μl/h。对于油相，设置压力泵的流速为1000μl/h。将液滴及油相的软管另一头连接分选芯片的对应入口。
7.3分选平台硬件参数设置。设置激光、比较器、设置波形发生器、信号放大器的各项参数。参数如下：
激光功率：10mW；
信号增益：0.56；
波形发生器设置：方形波，电压为3VPP，40cycles/s，频率为20K HZ，外部触发，分压output load；
电压放大倍数：200倍。
7.4打开激光，使用单片机接收液滴信号。事先在荧光显微镜下观察和预估阳性液滴所占的百分比(假设为10％阳性率)。收集5万个液滴的信号值(经验值，液滴信号在1-2之间)，根据收取的信号值，设置处于10％分位的信号值为阈值。该阈值设置可以根据实际情况进行调整：为保证阳性率，可以设置更加严谨的阈值，如5％分为的信号值。为保证得率，可以放宽阈值，如设置15％分为的信号值为阈值。随后开始分选，并全程记录液滴信号。
分别将孵育后的液滴与阴性液滴(含有抗原和荧光二抗，不含细胞)进行分选，获得样品信号数据集和阴性信号数据集；分别计算所述样品信号数据集和阴性信号数据集中最大信号值与所有信号值总和的比值，记作样品信号比值和阴性信号比值；收集样品信号比值大于阴性信号比值的阳性液滴。
结果如图2所示，分选前后可以看出阳性液滴中荧光信号强度明显增强。
8细胞回收
取适量分选后得到的阳性液滴体积(不大于200μl)，按先后顺序加入500μl DMEM培养基和300μl PFO，然后冰上静置10mins，期间可以轻轻摇晃。短暂离心之后，可见明显的相分离，细胞位于最上层的水相。吸取上层水相至新的1.5ml EP管，不要扰动油相，加入100μl DMEM洗涤水油界面，重复洗涤2～3次。回收细胞300g，10mins离心，加入少量培养基重悬，检测总数和活率。
9 B细胞单细胞测序/单孔PCR
回收到的细胞分两部分，分别进行如下操作：
(1)将B细胞进行裂解，反转录，以经典的羊驼BCR扩增的引物序列进行扩增。
引物1：CTAGTGCGGCCGCTGAGGAGACGGTGACCTGGGT
引物2：GATGTGCAGCTGCAGGAGTCTGGRGGAGG，R代表A或G
扩增结果如下图3。可以看出，BCR片段主要富集在500bp左右，与文献报道的纳米抗体长度接近。
(2)使用B细胞单细胞测序平台，进行建库并测序，获取B细胞受体(纳米抗体)信息和序列。
对测序所获得的序列信息在NCBI上进行比对，发现其可以比对到骆驼科的IGH链上，比对率分布在>80％到>90％。由此表明本发明可以获取出纳米抗体序列信息，准确性强。
在本说明书的描述中，参考术语“一个实施例”、“一些实施例”、“示例”、“具体示例”、或“一些示例”等的描述意指结合该实施例或示例描述的具体特征、结构、材料或者特点包含于本发明的至少一个实施例或示例中。在本说明书中，对上述术语的示意性表述不必须针对的是相同的实施例或示例。而且，描述的具体特征、结构、材料或者特点可以在任一个或多个实施例或示例中以合适的方式结合。此外，在不相互矛盾的情况下，本领域的技术人员可以将本说明书中描述的不同实施例或示例以及不同实施例或示例的特征进行结合和组合。
尽管上面已经示出和描述了本发明的实施例，可以理解的是，上述实施例是示例性的，不能理解为对本发明的限制，本领域的普通技术人员在本发明的范围内可以对上述实施例进行变化、修改、替换和变型。

Claims (14)

  1. 一种获取特异性识别抗原的纳米抗体信息的方法，其特征在于，包括：
    步骤1：从受试者的外周血淋巴细胞中富集成熟B细胞；
    步骤2：利用微流控液滴生成芯片，将含有抗原和信号检测试剂的溶液与单个所述B细胞包裹在单个液滴内，所述信号检测试剂可在所述抗原与目标纳米抗体结合时产生可检测信号；
    步骤3：将生成的所述单个液滴进行孵育，以使所述B细胞分泌纳米抗体以及所述抗原与纳米抗体发生特异性结合；
    步骤4：利用液滴分选芯片对所述孵育后的液滴进行分选，对收集的可检测信号进行分析，以便分选出可分泌目标纳米抗体的目标细胞，收集包含所述目标细胞的阳性液滴；
    步骤5：对所述目标细胞进行单细胞测序，以便获取目标纳米抗体信息。
  2. 根据权利要求1所述的方法，其特征在于，所述受试者选自骆驼科；
    所述受试者预先经过所述抗原的体内免疫。
  3. 根据权利要求1所述的方法，其特征在于，步骤1包括：
    步骤1-1：将所述外周血淋巴细胞与生物素标记的抗体进行孵育，所述抗体可与成熟的B细胞结合；
    步骤1-2：将所述孵育后的细胞进行洗涤、过滤和重悬，以便得到单细胞悬液；
    步骤1-3：将所述单细胞悬液与抗生物素标记的磁珠进行孵育，所述抗生物素标记的磁珠可与所述生物素标记的抗体结合，收集孵育后的细胞液中的磁珠，以便得到富集的成熟B细胞。
  4. 根据权利要求3所述的方法，其特征在于，所述抗体选自鼠抗羊驼BCMA单克隆抗体。
  5. 根据权利要求3所述的方法，其特征在于，步骤1-1中，每1×10 5个细胞与0.01～1μL抗体进行孵育；
    所述孵育是在室温避光下进行0.5～1小时。
  6. 根据权利要求3所述的方法，其特征在于，步骤1-3中，所述单细胞悬液中细胞密度不大于10 7个/毫升；
    所述孵育是在2～6℃下进行10～20分钟。
  7. 根据权利要求1所述的方法，其特征在于，步骤2中，所述信号检测试剂选自荧光二抗和链霉素亲和磁珠。
  8. 根据权利要求7所述的方法，其特征在于，所述荧光二抗选自Alexa
    Figure PCTCN2022072393-appb-100001
    488-conjugated AffiniPure Goat Anti-Alpaca IgG,subclasses 2+3 specific。
  9. 根据权利要求1所述的方法，其特征在于，所述抗原是以链霉素亲和磁珠-生物素-抗原的形式提供的。
  10. 根据权利要求1所述的方法，其特征在于，步骤3中，所述孵育是在30～40℃下进行1～4小时。
  11. 根据权利要求1所述的方法，其特征在于，步骤3中，所述孵育是在37℃下进行1～4小时。
  12. 根据权利要求1所述的方法，其特征在于，步骤4中，所述分选采用的参数如下：
    激光功率：1-15mW；
    信号增益：0.37-0.56；
    波形发生器设置：方形波，电压为2-3VPP，40cycles/s，频率为20K HZ，外部触发，分压output load；
    电压放大倍数：200倍。
  13. 根据权利要求1所述的方法，其特征在于，步骤4中，所述孵育后的液滴注入所述液滴分选芯片的液滴相入口的流速为10～100μL/h，油相注入所述液滴分选芯片的油相入口的流速为800～1200μL/h。
  14. 根据权利要求1所述的方法，其特征在于，步骤4中，所述分析包括：
    在同一液滴分选芯片上，分别将所述孵育后的液滴与阴性液滴进行分选，获得样品信号数据集和阴性信号数据集，其中，所述阴性液滴中含有抗体和信号检测试剂，不含有细胞；
    分别计算所述样品信号数据集和阴性信号数据集中最大信号值与所有信号值总和的比值，记作样品信号比值和阴性信号比值；
    所述样品信号比值大于阴性信号比值，是所述孵育后的液滴为含有所述目标细胞的阳性液滴的指示。
PCT/CN2022/072393 2022-01-17 2022-01-17 获取特异性识别抗原的纳米抗体信息的方法 WO2023133895A1 (zh)

Priority Applications (1)

Application Number Priority Date Filing Date Title
PCT/CN2022/072393 WO2023133895A1 (zh) 2022-01-17 2022-01-17 获取特异性识别抗原的纳米抗体信息的方法

Applications Claiming Priority (1)

Application Number Priority Date Filing Date Title
PCT/CN2022/072393 WO2023133895A1 (zh) 2022-01-17 2022-01-17 获取特异性识别抗原的纳米抗体信息的方法

Publications (1)

Publication Number Publication Date
WO2023133895A1 true WO2023133895A1 (zh) 2023-07-20

Family

ID=87279864

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/CN2022/072393 WO2023133895A1 (zh) 2022-01-17 2022-01-17 获取特异性识别抗原的纳米抗体信息的方法

Country Status (1)

Country Link
WO (1) WO2023133895A1 (zh)

Citations (7)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2017180738A1 (en) * 2016-04-12 2017-10-19 Medimmune, Llc Immune repertoire mining
CN109627328A (zh) * 2018-12-31 2019-04-16 梁重阳 基于拉曼光谱和微液滴技术的单克隆抗体制备方法
CN109952313A (zh) * 2016-01-15 2019-06-28 伯克利之光生命科技公司 生产患者特异性抗癌治疗剂的方法和其治疗方法
CN110483643A (zh) * 2018-09-19 2019-11-22 程晓东 一种新型的抗体组合物、分离的核酸和制备方法
CN110964101A (zh) * 2019-12-13 2020-04-07 山东民康生物科技有限公司 一种纳米抗体制备方法
CN112165989A (zh) * 2018-04-18 2021-01-01 高保真生物技术有限公司 单细胞分析的微流控方法
WO2021102308A1 (en) * 2019-11-21 2021-05-27 Emulate Inc. Antibody producing microfluidic devices

Patent Citations (7)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN109952313A (zh) * 2016-01-15 2019-06-28 伯克利之光生命科技公司 生产患者特异性抗癌治疗剂的方法和其治疗方法
WO2017180738A1 (en) * 2016-04-12 2017-10-19 Medimmune, Llc Immune repertoire mining
CN112165989A (zh) * 2018-04-18 2021-01-01 高保真生物技术有限公司 单细胞分析的微流控方法
CN110483643A (zh) * 2018-09-19 2019-11-22 程晓东 一种新型的抗体组合物、分离的核酸和制备方法
CN109627328A (zh) * 2018-12-31 2019-04-16 梁重阳 基于拉曼光谱和微液滴技术的单克隆抗体制备方法
WO2021102308A1 (en) * 2019-11-21 2021-05-27 Emulate Inc. Antibody producing microfluidic devices
CN110964101A (zh) * 2019-12-13 2020-04-07 山东民康生物科技有限公司 一种纳米抗体制备方法

Similar Documents

Publication Publication Date Title
CA2146974C (en) Direct selection of cells by secretion product
CN110016462B (zh) 从脾脏细胞中高效分离单个抗原特异性b淋巴细胞的方法
CN113046422A (zh) 基于免疫磁珠和滚环扩增的外泌体膜蛋白的流式检测方法及应用
CN106996976B (zh) 基于微流控技术的ctc蛋白质分型试剂盒
Murray et al. Unsupervised capture and profiling of rare immune cells using multi-directional magnetic ratcheting
Sun et al. Application of microfluidic technology in antibody screening
CN110361442B (zh) 一种用于质谱流式细胞仪检测的外泌体及其制备方法与应用
WO2023133895A1 (zh) 获取特异性识别抗原的纳米抗体信息的方法
CN116515844B (zh) 一种迁移体适配体及其筛选方法和应用
CN108441472A (zh) 一种高效分离单个抗原特异性b细胞的方法
CN115125251B (zh) 一种高效获得特异性全人单克隆抗体基因的方法和应用
CN114990124A (zh) 膜蛋白靶标cd44的核酸适配体、其筛选方法与应用
JP7153493B2 (ja) 生体粒子の測定方法、非特異シグナルの検出方法、生体粒子測定方法及び生体粒子を検出するための試薬キット
CN114181895A (zh) 一种自主构建的SLA-2-HB01-pCDH/sT2细胞系筛选CTL表位方法
CN109439624B (zh) 一种用于识别或富集有核红细胞的方法
CN115819595B (zh) 一种抗lag3纳米抗体及其制备方法与应用
JP7153494B2 (ja) 生体粒子の測定方法、非特異シグナルの検出方法、生体粒子測定方法及び生体粒子を検出するための試薬キット
CN115786350B (zh) 一种特异性识别并结合外周血t淋巴细胞的核酸适体、互补序列及它们的应用
EP3599467B1 (en) Bioparticle measuring method
JP2009106936A (ja) 磁性ナノ粒子の制御された凝集による増大した分離効率
CN115786349B (zh) 一种用于外周血中杀伤性t淋巴细胞无痕分选的核酸适体、互补序列及其应用
US20210096046A1 (en) Methods and Devices for Producing Cellular Suspensions from Tissue Samples
Yang Kinetic Competition Screening of Yeast-Displayed Libraries for Isolating High Affinity Binders
WO2024145974A1 (zh) 一种tigit纳米抗体及其制备方法与应用
KR101616705B1 (ko) 특이적 결합분자―나노섬유 복합체 및 이를 이용한 세포활성화 방법

Legal Events

Date Code Title Description
121 Ep: the epo has been informed by wipo that ep was designated in this application

Ref document number: 22919541

Country of ref document: EP

Kind code of ref document: A1