Method for efficiently detecting antibody endocytosis activity through combined application of nano antibody and living cell imaging system and application

Abstract

The invention relates to a method for efficiently detecting the endocytosis activity of an antibody through combined application of a nano antibody and a living cell imaging system and application, and the method comprises the following steps: incubating the nano antibody coupled with a pH-sensitive luminous group and a sample to be detected, and then detecting by adopting the living cell imaging system. On the basis of an Inucyte living cell imaging system, a high-affinity nano-antibody for recognizing an Fc region of a detected antibody is utilized, a pH-dependent fluorescent probe coupled to the nano-antibody can emit a fluorescent signal under acidic environment conditions such as lysosome and the like, and through high-flux continuous detection, the detection sensitivity is high, and the detection sensitivity is high. And detecting the endocytosis fluorescence signals of the same tumor cell sample at different time points, and evaluating the endocytosis activity of the monoclonal antibody for constructing the ADC drug.

Description

Technical Field

[0001] This invention relates to the field of antibody detection technology, and in particular to a method and application for the efficient detection of antibody endocytosis activity by combining nanobodies with a live-cell imaging system. Background Technology

[0002] Antibody-adjuvant (ADC) drugs are a novel class of large-molecule targeted therapies, composed of monoclonal antibodies (mAbs) that specifically bind to one or more antigens, coupled with a small-molecule drug payload possessing cytotoxic activity via a specific linker. The linker reduces the hydrophobicity of the ADC drug, decreases plasma clearance, and prolongs its half-life to 5-7 times that of traditional chemotherapy drugs. After binding to tumor cells, the ADC drug triggers endocytosis of the antigen-antibody complex, internalizing the ADC drug into the tumor cells. In the development and screening of ADC drugs, the endocytosis activity of the ADC drug by tumor cells is a crucial factor. Insufficient endocytosis may result in the drug remaining on the cell membrane, leading to payload release failure.

[0003] Several methods and strategies exist for assessing antibody endocytic activity. One approach is based on cytotoxic substances such as ribosome-inactivating proteins like saponins (ZAP), diphtheria toxins (DT3C), and microtubule inhibitors (VC-MMAE), using indirect conjugation techniques like immune recognition to label antibodies for cytotoxicity detection. This method indirectly reflects antibody endocytic activity by detecting cytotoxic activity. While this approach mimics the mechanism of action of ADC drugs, once antibody endocytic activity reaches a certain level, the conjugated toxins become highly toxic, and the method exhibits strong non-specific cytotoxicity, reducing sensitivity and discriminative power. Indirectly reflecting endocytic activity through cytotoxic activity can mask differences in antibody endocytic strength to some extent. Another approach is cytocytosis detection based on pH-sensitive fluorescent probes. This method conjugates a pH-sensitive fluorescent probe (pHrodo) to a Fab secondary antibody fragment that recognizes the Fc region of the antibody being tested, forming a fluorescent probe complex. After binding to the antibody being tested, high-content imaging (HCS) is used to detect the cytocytic activity signal. While high-content automated microscopy systems can simultaneously assess antibody endocytic activity in multiple samples, they suffer from drawbacks such as expensive equipment, complex operation, and the inability to continuously analyze the same live cell sample for extended periods. The Fab antibody fragments used in this method to identify the antibody being tested have relatively large molecular weights (approximately 50 kDa), which may interfere with the internalization of the antibody. Furthermore, Fab fragments are typically polyclonal antibodies, and the pHrodo probe is randomly conjugated to the Fab label, leading to significant batch-to-batch variability.

[0004] In addition, flow cytometry based on temperature changes is also a commonly used method for detecting endocytosis. This method involves staining cells with the detection antibody and fluorescent secondary antibody at 4°C and incubating for endocytosis at 37°C. Afterward, the fluorescence signal on the cell surface is removed by acid washing, and the remaining fluorescence signal after endocytosis is detected by high-content imaging or flow cytometry to assess antibody endocytosis activity. Alternatively, cells can be bound to the detection antibody at 4°C and 37°C for endocytosis, followed by staining with fluorescent secondary antibody. The change in the average fluorescence intensity of the samples under the two conditions can be used to detect antibody endocytosis activity. Although flow cytometry is highly sensitive and quantitatively accurate, its disadvantages include the need for complex equipment and operating techniques, and the inability to perform high-throughput screening at multiple time points continuously. This method also suffers from the relatively large molecular weight of the secondary antibody (around 145 kDa), the fact that flow cytometry fluorescent secondary antibodies are usually polyclonal antibodies, and significant batch-to-batch variability, which affects the endocytosis activity of the detected antibody. Furthermore, the acid washing method for removing the fluorescence signal from the cell surface is unstable and prone to false positives related to endocytosis. Therefore, developing a method that can accurately and efficiently detect antibody endocytic activity can greatly improve the screening efficiency of antibodies with high endocytic activity and promote the progress of ADC drug development. Summary of the Invention

[0005] To address the aforementioned technical problems, this invention provides a method and application for the efficient detection of antibody endocytosis activity using a combination of nanobodies and a live-cell imaging system. Based on the Incucyte live-cell imaging system, this invention utilizes high-affinity nanobodies that recognize the Fc region of the antibody being detected. A pH-dependent fluorescent probe coupled to the nanobodies emits a fluorescent signal under acidic conditions such as lysosomes. Through high-throughput continuous detection, the endocytosis fluorescence signal of the same tumor cell sample at different time points is detected, evaluating the endocytosis activity of monoclonal antibodies used to construct ADC drugs.

[0006] To achieve this objective, the present invention adopts the following technical solution:

[0007] In a first aspect, the present invention provides a method for efficiently detecting antibody endocytosis activity by combining nanobodies with a live-cell imaging system. The method includes incubating a sample to be tested with a nanobodies coupled with a pH-sensitive luminescent group, and then performing detection using a live-cell imaging system.

[0008] The method of this invention involves seeding target cells into 96-well flat-bottomed plates with black walls and transparent bottoms. After the cells adhere, the antibody sample to be tested is pre-incubated with a nanobody conjugated with a pH-sensitive fluorescent group, thus labeling the antibody with the pH-sensitive fluorescent group. The labeled antibody sample is then added to the culture wells seeded with target cells and placed directly into an Incucyte live-cell imaging system. Images are acquired cyclically at fixed time intervals to detect changes in intracellular signals.

[0009] This invention, based on the Incucyte live-cell imaging system, utilizes high-affinity nanobodies that recognize the Fc region of the antibody being tested. A pH-dependent fluorescent probe coupled to the nanobodies emits a fluorescent signal under acidic conditions such as lysosomes. Through high-throughput continuous detection, the internalization fluorescence signals of the same tumor cell sample at different time points are measured to evaluate the internalization activity of monoclonal antibodies used to construct ADC drugs. This method uses recombinantly expressed high-affinity nanobodies with a small molecular weight. The nanobodies bind to the Fc region of the antibody being tested. The pH-dependent fluorescent probes, site-labeled on the nanobodies, only emit fluorescence under lysosome-related acidic conditions, directly reflecting the level of antibodies internalized into the lysosomes of tumor cells.

[0010] Preferably, the pH-sensitive luminescent group includes any one or a combination of at least two of pHrodo iFL Red, pH-sensitive Red 600, or pHAb Amine Reactive Dye.

[0011] In this invention, a pH-sensitive luminescent group is linked to nanobodies, wherein the fluorescence intensity of the luminescent group increases with increasing acidity. The intensity change is particularly significant within the pH range of 4.5-9. In the extracellular environment (neutral, pH approximately 7.2-7.4), there is virtually no fluorescence; however, after internalization, a bright fluorescent signal is emitted in the acidic environment of integrons and lysosomes.

[0012] Preferably, the nanobody includes any one or a combination of at least two of the following: monoclonal nanobody, a mixture of multiple clonal nanobody, and multispecific nanobody.

[0013] Preferably, the nanobody is sourced from any one or a combination of at least two of alpacas, llamas, and camels.

[0014] Preferably, the nanobody includes an anti-species immunoglobulin nanobody.

[0015] Preferably, the anti-species immunoglobulin nanobody includes any one or a combination of at least two of anti-human IgG, anti-mouse IgG, anti-rabbit IgG, anti-rat IgG, anti-sheep IgG, or anti-monkey IgG.

[0016] Preferably, the sample to be tested includes an antibody that recognizes a target cell surface protein.

[0017] Preferably, the antibody against the target cell surface protein includes any one or a combination of at least two of mouse-derived antibodies, rat-derived antibodies, human-derived antibodies, engineered humanized antibodies, or rabbit-derived antibodies.

[0018] Preferably, the target cells include human tumor cells and / or mouse cells.

[0019] Preferably, the human tumor cells include any one or a combination of at least two of NCI-H2073, MDA-MB-468, HPAF-II, T84, SKBR-3, HT29, HepG2, ASPC-1, or PC-3.

[0020] Preferably, the mouse-derived tumor cells include any one or a combination of at least two of MC38, 4T1, or CT26.

[0021] Preferably, the molar ratio of the sample to be tested to the nanobody coupled with a pH-sensitive luminescent group is 1:(2-10).

[0022] The molar ratio of the sample to be tested to the nanobody coupled with the pH-sensitive luminescent group is 1:(2-10). The (2-10) can be, for example, 2, 3, 4, 5, 6, 7, 8, 9, or 10. Preferably, the incubation temperature is 35-40°C, and the time is 10-40 min. The 35-40°C can be, for example, 35°C, 36°C, 37°C, 38°C, 39°C, or 40°C. The 10-40 min can be, for example, 10 min, 15 min, 20 min, 25 min, 30 min, 35 min, or 40 min.

[0023] Preferably, the live-cell imaging system includes the Incucyte live-cell analysis system.

[0024] Preferably, the time interval for image acquisition using the live cell imaging system is 0.25-4 h. For example, it can be 0.25 h, 0.5 h, 1 h, 1.5 h, 2 h, 2.5 h, 3 h, 3.5 h, or 4 h, etc.

[0025] Preferably, the method further includes pretreatment of the sample to be tested.

[0026] Preferably, the pretreatment includes preparing a single-cell suspension of target cells, coating it with a coating solution in a microplate, seeding and culturing the cells until the cell density reaches 40%-50%, and then adding the sample to be tested. For example, the concentration could be 40%, 42%, 44%, 46%, 48%, or 50%.

[0027] In this invention, if the target cells are poorly adherent or suspended cells, they need to be coated with corresponding substances before inoculation. If the target cells are well adherent, they do not need to be coated with substances such as L-ornithine that promote cell adhesion.

[0028] Preferably, the coating solution comprises a 0.001-0.05% poly-L-ornithine solution and / or a 0.05%-0.3% bovine serum albumin solution containing 1-10 μg / mL fibronectin. The 0.001-0.05% can be, for example, 0.001%, 0.005%, 0.01%, 0.02%, 0.03%, 0.04%, or 0.05%, etc.

[0029] Secondly, the present invention provides an application of the method for efficiently detecting antibody endocytosis activity by combining nanobodies with a live-cell imaging system according to the first aspect in the detection of antibody endocytosis activity.

[0030] Compared with the prior art, the present invention has at least the following beneficial effects:

[0031] 1. This invention provides a pH-dependent fluorescent probe for site-specific labeling on nanobodies. The labeling amount is predetermined, eliminating the need for washing or quenching steps, reducing background interference signals, and facilitating quantitative comparisons. The pH-dependent fluorescent probe emits fluorescence only under lysosome-related acidic conditions, directly and accurately reflecting the antibody level endocytosed into tumor cell lysosomes, thus better assessing the endocytic activity of antibodies playing a role in ADC drugs.

[0032] 2. This invention, based on the Incucyte live-cell imaging system, enables high-throughput detection of endocytosis at different time points in the same sample within a single experiment, facilitating the identification of the optimal endocytosis time point within the experimental window. It eliminates the need for repeated instrument-based detection, reducing the risk of experimental errors caused by operational procedures. Experimental results can include multi-dimensional evaluations such as fluorescence intensity in the field of view, percentage of positive fluorescence area, and visualized cell images, providing a more intuitive and clear reflection of the endocytic activity of the detected antibody.

[0033] 3. The nanobodies used in this invention are recombinantly expressed nanobodies, exhibiting minimal batch-to-batch variation and a small molecular weight of approximately 15 kDa, which substantially does not affect the endocytic activity of the antibody being tested. These high-affinity nanobodies can rapidly and stably bind to the Fc region of the antibody being tested, requiring short incubation times and offering convenient operation without the need for additional conjugation or purification steps. Attached Figure Description

[0034] Figure 1 This is a schematic diagram illustrating a method for efficiently detecting antibody endocytosis activity using a live-cell imaging system.

[0035] Figure 2 The image shows the data analysis results based on cell fluorescence intensity after detecting antibodies for tumor cell endocytosis.

[0036] Figure 3 The graph shows the results of data analysis based on the proportion of positive cells after detecting antibodies for tumor cell endocytosis.

[0037] Figure 4 This figure shows the analysis results after tumor endocytosis under different antibody concentrations.

[0038] Figure 5 Image of cells after incubation with a test antibody labeled with a pH-sensitive fluorescent group.

[0039] Figure 6 This image shows the endocytosis results of conventional PE fluorescently labeled test antibodies detected using a high-content cell imaging system.

[0040] Figure 7 This image shows the results of endocytosis of pH-sensitive luminescent group-labeled antibodies detected using the Incucyte imaging system.

[0041] Figure 8 This is a graph showing the in vivo efficacy data of ADC drugs in animals. Detailed Implementation

[0042] The technical solution of the present invention will be further described below with reference to the accompanying drawings and specific embodiments. However, the following examples are merely simplified examples of the present invention and do not represent or limit the scope of protection of the present invention. The scope of protection of the present invention is determined by the claims.

[0043] The sources of reagents used in the following examples are:

[0044] Nanobodies coupled with pH-sensitive luminescent groups: Anti-Human IgG, AlpSdAbs® VHH, pH-Red600, Critical Point Bio, 023-101-012.

[0045] Example 1

[0046] This embodiment detects antibody endocytosis activity.

[0047] The experiment was conducted according to Figure 1The following is a schematic diagram illustrating the operation of a method for efficiently detecting antibody endocytosis activity using a live-cell imaging system. Cultured NCI-H2073 adherent tumor cells (preserved by the storage center of the Biopharmaceutical R&D Service Department, Shanghai WuXi Biologics Co., Ltd.) were digested with 0.25% Trypsin-EDTA (Thermo Fisher, 25200-072). The NCI-H2073 tumor cells were resuspended in RPMI-1640 complete medium containing 10% fetal bovine serum (ExCell Bio, FND500) to collect the cells, preparing a single-cell suspension and determining the cell density. The adjusted cell concentration was seeded into 96-well culture plates at a rate of 10,000 cells / 50 µL / well. The side of the culture plate was gently tapped to ensure even distribution of the liquid in the wells. The culture plate was placed in a clean bench on an undisturbed horizontal surface at room temperature for 10 min. After the cells settled to the bottom, the culture plate was placed in a 37°C, 5% CO2 incubator for further culture. After overnight incubation, once the cells have adhered normally and their morphology and growth have returned to normal, the antibodies to be tested are prepared.

[0048] The test antibody, conjugated to the MUC13 antigen, was obtained through immune screening by the Biopharmaceutical R&D Service Department of Shanghai WuXi Biologics Co., Ltd., which was also responsible for recombinant expression and purification. The sequence of the positive control antibody was synthesized based on the clone number C14 disclosed in the University of Tennessee Research Foundation patent WO2016 / 168607A1. The test antibody was diluted to a 4x working concentration (40 nM) using RPMI-1640 growth medium, and Anti-Human IgG, AlpSdAbs® VHH (pH-Red 600, Critical Point Bio, 023-101-012) was diluted to a 4x working concentration (80 nM) using cell growth medium. An equal volume of the diluted test antibody and the diluted labeled nanobody was mixed to prepare a 2x working solution. The prepared 2x working solution was incubated at 37°C for 20 min to allow the nanobody to fully bind to the test antibody, resulting in the antibody-labeled complex. To avoid experimental errors, replicates were used. Using a pipette, add 50 µL of 2x antibody-labeled complex working solution to each corresponding cell culture well to achieve the final working concentration of 10 nM. After adding the working solution and control solution to the designated wells, remove all air bubbles and immediately place the culture plate in the Incucyte live cell analysis system and begin scanning. Set the Incucyte integrated software to perform image acquisition every 2 hours, continuing image acquisition for 68 hours.

[0049] The acquired images were analyzed using the data analysis module in the Incucyte integrated software. Based on the cell fluorescence intensity data, the results were... Figure 2 The results presented show the detection of antibodies endocytosis in tumor cells. As antibody incubation time increased, the cell fluorescence intensity gradually increased, and the signal differences between different test antibodies gradually became larger, showing significant distinction. Simultaneously, data based on the proportion of positive cells... Figure 3 The results showed that as the antibody incubation time increased, the proportion of fluorescently positive cells gradually increased and then stabilized. The differences in the positive proportions between different test antibodies gradually increased, showing significant distinctions. Figure 3 The analytical methods described can, to some extent, mitigate the impact of excessively high abnormal signal values ​​on the analysis results.

[0050] Example 2

[0051] This embodiment optimizes the optimal time window and antibody concentration for evaluating antibody endocytosis activity.

[0052] The method described in Example 1 was used for detection. HepG2 adherent tumor cells (preserved by the storage center of the Biopharmaceutical R&D Service Department of Shanghai WuXi Biologics Co., Ltd.) were seeded at a rate of 20,000 cells / well in 96-well plates. After the cells had adhered normally and their morphology and growth had reached normal, the antibody to be tested was prepared. The anti-GPC3 protein antibody sequence used in the experiment was synthesized based on the hGC33 sequence disclosed in Chugai's patent WO2006006693.

[0053] Antibody-labeled complex working solutions were prepared at final antibody concentrations of 40 nM, 8 nM, and 1.6 nM, respectively. These solutions were added to cell culture wells, and images were acquired every 6 hours using the Incucyte live-cell imaging system for a total of 96 hours. Based on the cell fluorescence intensity data from the acquired images, the changes in the detection window under different antibody concentrations were analyzed. Results were presented in... Figure 4 As shown, with increasing antibody concentration and prolonged incubation time, the cell fluorescence intensity gradually increased, and the detection window expanded, exhibiting significant differences. The cell field of view for the group with a final antibody concentration of 40 nM is shown below. Figure 5 Cell images after incubation with the test antibody labeled with a pH-sensitive fluorescent group show red fluorescence in the cytoplasm. Cells treated with the labeled isotype control did not show obvious cellular fluorescence, and the difference from the isotype control group was particularly significant, indicating a good detection window.

[0054] Example 3

[0055] This embodiment compares methods for antibody endocytosis activity.

[0056] Antibody endocytosis activity was detected by a combination of acid washing and temperature change, and measured using the OperettaCLS (PerkinElmer) high-content imaging and analysis system. On day 1, poly-D-lysine (PDL) was diluted to 10 μg / mL with DPBS and added to 96-well plates (Greiner-655090) at a volume of 100 μL / well. The plates were incubated at 37°C for 2 hours, and the supernatant was discarded. HT29 cells were added to 96-well plates at a volume of 10,000 cells / well and cultured in complete medium containing 10% FBS. The plates were then incubated at 37°C with 5% CO2. On day 3, the cell culture supernatant was discarded. Different concentrations of the test antibody (serially diluted 5-fold in 1% bovine serum albumin, from 150 nM to 0.048 nM, for a total of 6 concentrations) were added to the plates at a volume of 100 μL / well and incubated at 4°C for 1 hour. After incubation, wash the cells once with 100 μL of washing buffer (1% bovine serum albumin), dilute the PE fluorescently labeled goat anti-human immunoglobulin Fc fragment specific secondary antibody (1:150) with 1% bovine serum albumin, and add 100 μL / well to the plate. Incubate at 4°C in the dark for 1 hour, then wash the culture plate as described above.

[0057] Then, 100 μL / well of 1% bovine serum albumin was added, and the cells were incubated at 37°C for 2 hours for endocytosis. After incubation, the cells were washed as described above, and incubated at room temperature in the dark for 15 min with 100 μL / well of Hoechst 33342 (1:5000 diluted in 1 xPBS, Invitrogen-H3570). The cells were then washed as described above. 100 μL / well of wash buffer (0.1 M glycine, 0.15 M NaCl, pH adjusted to 2.5) was added to the culture plate, and the plate was incubated at 4°C for 5 min. After washing once with 100 μL / well of wash buffer, 100 μL of 4% PFA was added to each well. The culture plate was stored at 4°C before Operetta scanning. The amount of antibody endocytosis was assessed using the Operetta CLS high-content analysis system. Background fluorescence was established using wells without antibody or containing only secondary antibody.

[0058] Experimental results are as follows Figure 6 As shown. The sequence of the anti-EGFR / HER3 bispecific antibody BL-B01D1 used in the experiment was synthesized based on the SI-71X14 sequence disclosed in BAILI-BIO's patent WO2022061255A1. The two candidate antibody molecules were obtained through systematic combination screening by the Biopharmaceutical R&D Service Department of Shanghai WuXi Biologics Co., Ltd., which was also responsible for recombinant expression and purification.

[0059] The endocytic activity of the same antibodies used in the above experiments was detected using an experimental method combining pH-sensitive fluorescent nanobodies with a live-cell imaging system. Following the detection method of Example 1, HT29 adherent tumor cells (preserved by the storage center of the Biopharmaceutical R&D Service Department of Shanghai WuXi Biologics Co., Ltd.) were seeded at 10,000 cells / well in 96-well plates. After the cells had adhered normally and their morphology and growth had reached normal, the antibodies to be tested were prepared. Antibody-labeled complex working solution with a final concentration of 10 nM was prepared and added to the cell culture wells. Images were acquired every 4 hours using the Incucyte live-cell imaging system for 112 hours. Based on the cell fluorescence intensity data in the acquired images, the differences in endocytic activity between different antibodies were detected. Results were obtained in… Figure 7 The results showed that, with the extension of antibody incubation time, the endocytic activity of the two candidate molecules was significantly stronger than that of Baili Tianheng's EGFR / HER3 bispecific antibody BL-B01D1.

[0060] Subsequently, the bispecific antibody from this experiment was conjugated with the DXd small molecule drug to prepare an ADC drug with a DAR value of 6 (WuXi AppTec was responsible for conjugation and production), and in vivo animal experiments were conducted. Animal experiment results showed that the tumor-suppressive effect of the two candidate molecules prepared as ADC drugs was significantly stronger than that of the BL-B01D1 antibody. Figure 8 The trend of antibody endocytosis activity obtained using the experimental method in this patent is consistent with the trend of subsequent in vivo pharmacodynamic experiments in animals. This method can more accurately assess the differences in antibody endocytosis activity, and screen suitable antibody molecules for subsequent ADC drug development.

[0061] In summary, this invention is based on the Incucyte live-cell imaging system and utilizes high-affinity nanobodies that identify the Fc region of the antibody being tested. The nanobodies are coupled with pH-dependent fluorescent probes that emit fluorescent signals under acidic environments such as lysosomes. Through high-throughput continuous detection, the endocytic fluorescence signals of the same tumor cell samples at different time points are detected, and the endocytic activity of monoclonal antibodies used to construct ADC drugs is evaluated.

[0062] The applicant declares that the above description is only a specific embodiment of the present invention, but the protection scope of the present invention is not limited thereto. Those skilled in the art should understand that any changes or substitutions that can be easily conceived by those skilled in the art within the technical scope disclosed in the present invention fall within the protection and disclosure scope of the present invention.

Claims

1. A method for efficient detection of antibody endocytosis activity by combining nanobodies with a live cell imaging system, characterized in that, The method comprises incubating the nanoantibody coupled with the pH-sensitive luminescent group with the sample to be tested, and then detecting by using a live cell imaging system.

2. The method for detecting antibody endocytosis activity with high efficiency by combining nanobody with live cell imaging system according to claim 1, wherein, The pH-sensitive luminescent group comprises any one or a combination of at least two of pHrodo iFL Red, pH-sensitive Red 600 or pHAb Amine Reactive Dye.

3. The method for high efficient detection of antibody endocytosis activity of nanobody combined with live cell imaging system according to claim 1 or 2, characterized in that, The nanoantibody comprises an anti-species immunoglobulin nanoantibody. Preferably, the anti-species immunoglobulin nanoantibody comprises any one or a combination of at least two of anti-human IgG, anti-mouse IgG, anti-rabbit IgG, anti-rat IgG, anti-goat IgG or anti-monkey IgG.

4. The method for high efficient detection of antibody endocytosis activity of nanobody according to any one of claims 1-3 in combination with live cell imaging system, characterized in that, The sample to be tested comprises an antibody recognizing a target cell surface protein. Preferably, the target cell comprises a human tumor cell and / or a mouse cell.

5. The method for detecting antibody endocytosis activity with high efficiency by combining nanobody with live cell imaging system according to claim 4, wherein, The human tumor cell comprises any one or a combination of at least two of NCI-H2073, MDA-MB-468, HPAF-II, T84, SKBR-3, HT29, HepG2, ASPC-1 or PC-3. Preferably, the mouse tumor cell comprises any one or a combination of at least two of MC38, 4T1 or CT26.

6. The method of using the nanobody of any one of claims 1-5 in combination with a live cell imaging system to efficiently detect antibody endocytosis activity, characterized in that, The molar ratio of the sample to be tested to the nanoantibody coupled with the pH-sensitive luminescent group is 1:(2-10). Preferably, the temperature of the incubation is 35-40℃, and the time is 10-40 min.

7. The method for high efficient detection of antibody endocytosis activity of nanobody combined with live cell imaging system according to claims 1-6, characterized in that, The live cell imaging system comprises an Incucyte live cell analysis system. Preferably, the time interval for image acquisition by using the live cell imaging system is 0.25-4 h.

8. The method of using the nanobody of any one of claims 1-7 in combination with a live cell imaging system to efficiently detect antibody endocytosis activity, characterized in that, The method further comprises pretreatment of the sample to be tested.

9. The method for detecting antibody endocytosis activity with high efficiency by combining nanobody with live cell imaging system according to claim 8, wherein, The pretreatment comprises preparing a single-cell suspension of the target cell, coating a microwell plate with a coating solution, inoculating and culturing the cell, and adding the sample to be tested when the cell growth density is 40%-50%. Preferably, the coating solution comprises 0.001-0.05% poly-L-ornithine solution and / or 0.05%-0.3% bovine serum albumin solution containing 1-10 μg / mL fibronectin.

10. Use of a nanoantibody according to any one of claims 1-9 in combination with a live cell imaging system for efficient detection of antibody endocytosis activity in detection of antibody endocytosis activity.

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