CN115201472A - Method for detecting interaction between cells - Google Patents

Abstract

The invention relates to a method for detecting interaction between cells. The method for detecting the interaction between the cells can reflect the strength of the antibody-dependent cell-mediated cytotoxicity (ADCC) between the effector cells and the target cells by detecting the affinity between the antibody capable of being specifically bound with the target cells and the receptor of the effector cells. The method can simplify the detection process, greatly shorten the detection period, reduce the cost, and deduce the strength of the ADCC effect without actually detecting the death of the target cells. The method of non-cell rapid detection replaces cell detection, and can accelerate the release of the medicine which depends on the biological activity of the antibody and can promote ADCC effect.

Description

Method for detecting cell-cell interaction

Technical Field

The invention relates to the field of biotechnology, in particular to a method for detecting interaction between cells.

Background

The mode of intercellular interaction can be roughly divided into two types, one is independent of intercellular contact, and the cells interact by secreting soluble proteins such as cytokines or chemokines; the other relies on cell-cell contact, which is mediated by the binding of cell-surface ligands to receptors, which in turn transduce signals and ultimately function.

Antibody-dependent cell-mediated cytotoxicity (ADCC) is one of intercellular interactions that depend on intercellular contact, and refers to binding of a Fab fragment of an antibody to an epitope of a virus-infected cell or a tumor cell, binding of an Fc fragment thereof to an Fc receptor (FcR) on the surface of a killer cell (NK cell, eosinophilic granulocyte, neutrophil, or the like), and direct killing of the target cell by the killer cell, which is also an important mechanism for the development of an antitumor therapeutic antibody drug.

At present, the ADCC effect is mainly reflected by detecting the death rate or the survival rate of target cells, and the main detection methods comprise a Lactate Dehydrogenase (LDH) release method, an EuTDA cytotoxicity method, a Calcein-AM release assay method and an ADCC Reporter gene bioactivity detection (ADCC Reporter Bioassay). However, these methods are all based on cell detection, and require a target cell line and an effector cell that bind to a specific antibody, which is costly and requires a long period of cell processing.

Disclosure of Invention

Accordingly, there is a need for a method for detecting cell-cell interactions that is cost effective.

A method of detecting cell-cell interactions, the method comprising the steps of:

respectively obtaining an antibody capable of specifically binding with target cells to be detected and a target receptor on effector cells to be detected, wherein the target cells to be detected comprise virus-infected cells or tumor cells, and the effector cells to be detected comprise at least one of NK cells, eosinophilic granulocytes and neutrophils;

detecting and calculating an affinity constant between the antibody and the target receptor by adopting a biological membrane interference technology; and

and analyzing the interaction strength between the effector cell to be detected and the target cell mediated by the antibody according to the affinity constant.

The method for detecting the interaction between cells can reflect the strength of antibody-dependent cell-mediated cytotoxicity (ADCC) between effector cells and target cells by detecting the affinity between an antibody capable of specifically binding to the target cells and a receptor of the effector cells. The method can simplify the detection process, greatly shorten the detection period, reduce the cost, and deduce the strength of the ADCC effect without actually detecting the death of the target cells. The release of the drug which depends on the biological activity of the antibody and can promote the ADCC effect can be accelerated by a non-cell rapid detection method instead of cell detection.

In one embodiment, the target receptor comprises fcyriii or fceri.

In one embodiment, the antibody comprises an IgG antibody or an IgE antibody.

In one embodiment, the biofilm interference technique is performed on an Octet system.

In one embodiment, the step of detecting and calculating the affinity constant between the antibody and the target receptor using a biofilm interference technique comprises:

preparing a detection buffer solution, wherein the detection buffer solution comprises a phosphate buffer solution containing 0.02% (v/v) to 0.5% (v/v) Tween-20;

wetting a sensor with the detection buffer solution, and fixing the target receptor on the sensor; and

after binding the antibodies of multiple concentration gradients to the target receptor, the data collected by the Octet system are globally fitted to determine the affinity constant.

In one embodiment, the concentration of the target receptor after being immobilized is controlled to be 1 to 2 mug/mL.

In one embodiment, the concentration of the antibody is controlled between 12.5nM and 800nM.

In one embodiment, the concentration of the antibody is controlled to be 12.5nM to 500nM.

In one embodiment, the antibody binds to the target receptor for a period of time ranging from 60s to 120s.

In one embodiment, the data acquisition rate of the Octet system is between 2Hz and 10Hz.

Drawings

FIG. 1 is a graph showing the results of measurement of the affinity of IgG antibodies to Fc γ RIIa (F176) by adding different proportions of Tween-20 as a detection buffer to phosphate buffer when the concentration of IgG antibodies is 500nM and the concentration of Fc γ RIIIIa (F176) is 2 μ g/mL;

FIG. 2 is a graph showing the results of measurement of the affinity of IgG antibodies to Fc γ RIIa (F176) by adding different proportions of Tween-20 to phosphate buffer as a detection buffer when the concentration of IgG antibodies is 250nM and the concentration of Fc γ RIIIIa (F176) is 2 μ g/mL;

FIG. 3 is a graph showing the results of measuring the affinity of an IgG antibody for FcyRIIa (F176) under the conditions of an IgG antibody concentration of 250nM and an FcyRIIIIa (F176) concentration of 2. Mu.g/mL, an IgG antibody concentration of 250nM and an FcyRIIIIa (F176) concentration of 0, and an IgG antibody concentration of 0 and an FcyRIIIIa (F176) concentration of 2. Mu.g/mL, respectively, when the Tween-20 concentration in the detection buffer is 0.02% (v/v);

FIG. 4 is a graph showing the results of measuring the affinity of an IgG antibody for various concentrations of Fc γ RIIa (F176) when the concentration of Tween-20 in the detection buffer was 0.02% (v/v) and the concentration of the IgG antibody was 250 nM;

figure 5 is a graph of the results of the binding model 1 for Fc γ riiia (F176) with varying concentrations of IgG, measured and fully dissociated;

FIG. 6 is a graph of the results of the binding model of 1;

fig. 7 to 10 are graphs showing the results of measuring the affinity of the IgG antibody for fcyrila (F176) in examples 1 to 4, respectively.

Detailed Description

The present invention will be described in detail with reference to the following embodiments in order to make the aforementioned objects, features and advantages of the invention more comprehensible. In the following description, numerous specific details are set forth in order to provide a thorough understanding of the present invention. This invention may, however, be embodied in many different forms and should not be construed as limited to the embodiments set forth herein.

Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. The terminology used in the description of the invention herein is for the purpose of describing particular embodiments only and is not intended to be limiting of the invention. As used herein, the term "and/or" includes any and all combinations of one or more of the associated listed items.

"biofilm interference technique" (BLI) described herein is a technique for detecting a sensor surface reaction by detecting a shift change in interference spectrum; when a visible light beam is emitted from the spectrometer, two reflection spectra are formed on two interfaces of the optical film layer at the tail end of the sensor, and an interference spectrum is formed. Any change in the thickness and density of the film layer due to molecular association or dissociation can be reflected by the shift value of the interference spectrum, and a real-time response monitoring map can be made through the shift value.

One embodiment of the present application provides a method for detecting interaction between cells, the method comprising steps S11, S12 and S13, specifically:

step S11: respectively obtaining an antibody capable of being specifically combined with a target cell to be detected and a target receptor on an effector cell to be detected, wherein the target cell to be detected comprises a virus-infected cell or a tumor cell, and the effector cell to be detected comprises at least one of NK cells, eosinophilic granulocytes and neutrophils.

In one embodiment, the effector cell to be tested is an NK cell.

In one embodiment, the target receptor comprises fcyriii or fceri.

In one embodiment, the receptor of interest is Fc γ RIIIa (F176).

In one embodiment, the antibody comprises an IgG antibody or an IgE antibody.

In one embodiment, the antibody is an IgG antibody.

Step S12: and detecting and calculating the affinity constant between the antibody and the target receptor by adopting a biological membrane interference technology.

In particular, kinetic analysis on biosensors used in biofilm interference technology can provide binding data in real time, yielding more information about the activity and behavior of molecular interactions, while end-point analysis such as ELISA can only yield one reading per sample.

In an alternative specific example, the biosensor may be selected from a Streptavidin (SA) biosensor or an anti-histidine (HIS 1K) biosensor.

In one embodiment, the biofilm interference technique is performed on an Octet system.

In one embodiment, step S12 further includes step S121, step S122, and step S123, specifically:

step S121: preparing a detection buffer solution, wherein the detection buffer solution comprises a phosphate buffer solution containing 0.02% (v/v) to 0.5% (v/v) Tween-20.

In one embodiment, the detection buffer is a phosphate buffer solution of 0.02% (v/v) to 0.5% (v/v) tween-20. Preferably, non-specific binding during the assay is minimized by using a phosphate buffer containing 0.02% (v/v) Tween-20 as the assay buffer.

Step S122: the sensor is wetted with a detection buffer and the target receptor is immobilized on the sensor.

In one embodiment, the concentration of the target receptor after immobilization is controlled to be 1. Mu.g/mL-2. Mu.g/mL. Further, the concentration of the target receptor after immobilization is controlled to 1. Mu.g/mL-1.5. Mu.g/mL.

In particular, the effective surface density of the receptor must be low enough to allow sufficient spacing between the receptor molecules, which helps to ensure that each antibody molecule binds to a single receptor molecule. Reducing the receptor density can be achieved by reducing the concentration of receptor used in the curing step or by shortening the time of the curing step or a combination of both. However, fewer receptor molecules on the surface means fewer sites available for analyte binding, which reduces the analytical signal. Thus, maintaining sufficient signal limits the extent to which the receptor cure density decreases.

Step S123: after the antibodies with multiple concentration gradients are combined with the target receptor, the data collected by the Octet system are globally fitted to determine the affinity constant.

In one embodiment, 4 to 6 concentration gradients of antibody are bound to the target receptor. Further, 5 concentration gradients of antibody were bound to the target receptor.

In one embodiment, the concentration of antibody is controlled between 12.5nM and 800nM. Further, the concentration of the antibody was controlled to 12.5nM to 500nM. Further, the concentration of the antibody is controlled to be 12.5nM to 250nM.

In one embodiment, the time for binding of the antibody to the target receptor is 60s to 120s. Further, the time for binding of the antibody to the receptor is 60 to 90 seconds.

In particular, as long as there is curvature in the binding step data traces and equilibrium is reached for higher analyte concentrations, a shorter binding step can improve the kinetic fit of the 1.

In one embodiment, the data acquisition rate of the Octet system is between 2Hz and 10Hz.

Preferably, the data acquisition rate of the Octet system is 10Hz.

Specifically, the data acquisition rate refers to the number of bound signal data points in the report, produced by the octet system per second, expressed in hertz. Higher acquisition rates produce more data points per second. Fc γ riiia (F176) typically has a fast binding rate with IgG interaction, and therefore requires a higher collection rate.

Step S13: and analyzing the strength of the interaction between the effector cell to be detected and the target cell mediated by the antibody according to the affinity constant.

Specifically, the strength of the affinity between the antibody and the target receptor can be determined according to the magnitude of the affinity constant. In an alternative embodiment, the affinity constant can be compared to a reference affinity constant to derive a particular strength of the antibody-mediated interaction between the test effector cell and the target cell. In other embodiments, the specific strength of the antibody-mediated interaction between the test effector cell and the target cell can also be determined based on the affinity constant between the reference antibody and the target receptor.

In the method for detecting an interaction between cells, the strength of antibody-dependent cell-mediated cytotoxicity (ADCC) between an effector cell and a target cell can be reflected by detecting the affinity between an antibody capable of specifically binding to the target cell and a receptor of the effector cell. The method can simplify the detection process, greatly shorten the detection period, reduce the cost, and deduce the strength of the ADCC effect without actually detecting the death of the target cells. The method of non-cell rapid detection replaces cell detection, and can accelerate the release of the medicine which depends on the biological activity of the antibody and can promote ADCC effect.

The following detailed description is made with reference to specific embodiments and parameter optimization processes. The following examples are not specifically described, and other components except inevitable impurities are not included. Reagents and instruments used in the examples are all conventional in the art and are not specifically described. The experimental procedures, in which specific conditions are not indicated in the examples, were carried out according to conventional conditions, such as those described in the literature, in books, or as recommended by the manufacturer.

The specific detection steps are as follows:

1. buffer solution preparation

Phosphate Buffer (PBS, national drug, cat # 72013561) with pH 7.4 was prepared, and Tween-20 (Tween-20) was added as a detection Buffer (Q Buffer).

2. Wetting sensor

Streptavidin (SA) biosensors (Octet, cat # 18-5020) were wetted in 200. Mu.l of Q Buffer for 10 minutes.

3. Solidification receptor Fc gamma RIIa (F176)

The receptor Fc γ RIIa (F176) (ACRO Biosystems, cat # CDA-H82E 8) was diluted with Q Buffer, 200. Mu.l were added to the latter 96-well plate and a reaction time of 300s (curing height 0.25 nm) was set.

4. Analysis of antibodies

The antibody was diluted with Q Buffer and 200 μ l was added to a post 96 well plate, one analyte concentration per SA sensor, and the antibody was set to 90s binding time to Fc γ rliiia (F176), 90s dissociation time, and 1000RPM. Determination of the affinity constant K _D The value is obtained.

And (3) parameter optimization process:

as with almost any kinetic assay, proper assay development is critical in assaying binding interactions of antibodies with Fc γ riiia (F176) in order to obtain accurate, reliable and reproducible affinity and kinetic constants. The quality of the kinetic data depends on the format of the biosensor used and the conditions of the binding pair, and the detection conditions, including reagent quality, buffer conditions, ligand immobilization density, analyte concentration, binding step time, data acquisition rate, and data analysis process, need to be considered.

1. Optimizing reagent quality

Reagent quality is a critical factor for kinetic analysis. Since aggregation of antibodies or receptors leads to increased affinity and non-specific binding, thereby affecting kinetics, analytical techniques should be used to fully evaluate the purity, activity and quality of antibody samples prior to use in kinetic experiments. Reagents should not be used which are stored at 4 ℃ for a long period of time, especially at very high or very low concentrations. Purification, storage conditions and handling of the receptor protein should be carefully considered and multiple freeze-thaw cycles are avoided. Commercial Fc γ riiia (F176) is of varying quality, so determining a reliable source of high quality Fc γ riiiia (F176) is crucial to exhibiting good detection performance.

2. Optimized detection buffer

For kinetic detection of antibodies and the receptor Fc γ rliiia (F176) using biofilm interference techniques, the analyte sample was diluted with phosphate detection buffer pH 7.4 and the equilibration and dissociation detection steps. The experiment was carried out using a phosphate buffer containing 0% (v/v), 0.02% (v/v), 0.1% (v/v) and 0.5% (v/v) Tween-20 at an IgG concentration of 500nM and 250nM, respectively, by setting the concentration of Fc γ RIIa (F176) to 2 μ g/mL (see FIGS. 1 and 2). As can be seen from FIGS. 1 and 2, when the IgG concentration of the analyte was 250nM and 500nM, the superior detection effect was obtained using the phosphate buffer containing 0.02% (v/v) Tween-20.

Furthermore, for biosensor kinetic analysis, non-specific binding (NSB) of analyte to the biosensor during the binding step should be minimized, as it will alter the analyte binding pattern and interfere with accurate calculation of kinetic rates. A positive signal in the binding step indicates non-specific binding of the analyte to the biosensor. If the NSB signal is minimal, it can be subtracted by reference during data analysis. However, the NSB nm shift signal above 20% of the maximum detection signal should be reduced by optimizing the detection conditions. Steps that can be used to mitigate NSB include optimizing the detection buffer and/or adding a blocking step after ligand immobilization. Increasing the amount of tween-20 in the buffer or increasing the salt concentration can improve binding and eliminate non-specific signal. To test for nonspecific binding of analytes, a preliminary experiment was performed by setting three groups of 250nM IgG concentration and zero Fc γ RIIa (F176) concentration, 2 μ g/mL IgG zero concentration and zero Fc γ RIIa (F176) concentration, and 250nM IgG concentration and 2 μ g/mL Fc γ RIIIIa (F176) concentration, using a phosphate buffer containing 0.02% (v/v) Tween-20 as a detection buffer, and the results are shown in FIG. 3. As can be seen from FIG. 3, the use of a phosphate buffer containing 0.02% (v/v) Tween-20 as a detection buffer minimizes non-specific binding.

When changing the buffer between detection steps, i.e. from 1 × kinetic buffer in the immobilization step to detection buffer in the equilibration and binding step, it is necessary to ensure that the baseline runs are run long enough for the biosensor to reach equilibrium in the new buffer and for any signal drift to stabilize. When switching buffers, an equilibration step of 1 to 5 minutes is usually required.

3. Optimizing ligand cure density

The amount of ligand immobilized (loaded) onto the biosensor can have a significant impact on the detection results in terms of signal intensity, apparent kinetic behavior, and non-specific binding. Although curing as much ligand as possible in the curing step will certainly maximize the detected signal, this approach may also produce undesirable artifacts. Effects such as molecular crowding, avidity, non-specific binding and/or mass transfer all influence the observed binding kinetics. Conversely, if too little ligand is immobilized, the detection signal can be very low, resulting in poor data trace separation and insufficient signal-to-noise ratio. Therefore, ligand immobilization levels should be optimized for each assay and biosensor format. For the immobilization optimization experiments, several dilutions of ligand molecules were immobilized in parallel onto the biosensor using phosphate buffer containing 0.02% (v/v) Tween-20 as detection buffer, at an IgG antibody concentration set at 250nM, with ligand concentrations of 1. Mu.g/mL, 2. Mu.g/mL, 4. Mu.g/mL and 8. Mu.g/mL, respectively. FIG. 4 is a graph of the results of a cure optimization experiment. The preferred ligand concentration is selected to be a lower concentration of immobilized ligand that produces an acceptable signal response during the analyte binding step. Therefore, according to the results of FIG. 4, the preferred ligand immobilization concentration is 1. Mu.g/mL-2. Mu.g/mL.

4. Optimizing analyte concentration

In the binding step, the rate of binding of the analyte to the immobilized ligand is measured. Measuring the concentration of a single analyte is sufficient for a simple binding assay or a qualitative assay. However, when precise kinetics and affinity constants are required, a dilution series measuring 4 to 6 analyte concentrations in the binding step is better. The multiple analyte concentrations support global curve fitting, i.e., fitting all curves in the dataset simultaneously to produce a set of results. The range of analyte concentrations to be used will depend on the sensitivity of the detection and the affinity of the interaction.

5. Optimizing binding step time

Fc γ RIIa (F176) -IgG interactions generally have higher binding rates (R) ((R))>1E5 M ^-1 s ^-1 ) Wherein the primary binding interaction is able to rapidly reach equilibrium. When the interaction is fast, a shorter run time of the binding step is sufficient to bring the interaction to equilibrium (indicated when the binding track is flattened). This time can be as short as 60 seconds. As long as there is curvature in the binding step data traces and equilibrium is reached for higher analyte concentrations, a shorter binding step can improve the kinetic fit of the 1.

6. Optimizing data acquisition rates

Because Fc γ rliiia (F176) -IgG interactions generally have higher binding rates, a standard data acquisition rate (5.0 Hz) in octet system data acquisition software may not be an ideal setting. When data is acquired by the Octet system, there is a delay from the time the biosensor dips into the sample to the time the first data point is reported to allow the software to average the data collected. This delay can cause the reporter signal of the binding step to start above the baseline in the rapid binding interaction, resulting in inaccurate data fitting results. Thus at higher binding rates, the data acquisition rate can be increased to more quickly report binding data. The data acquisition rate refers to the number of binding signal data points in the report, produced by the octet system per second, expressed in hertz. Higher acquisition rates produce more data points per second and better monitor for rapid binding events. Preferably, in the data acquisition software, a high concentration kinetic acquisition rate (10.0 Hz) is selected. The acquisition rate setting should always be determined based on the binding rate, the amount of signal generated and the experimental setting.

7. Optimizing data analysis

In a suitably optimized biosensor assay, the kinetic curve will follow a single stoichiometric binding curve representing a single analyte molecule per binding site, so the 1. When only the first part of the dissociation step is included in the analysis, a 1. Shortening the dissociation step fit to 5-10 seconds captures the initial dissociation rate in the biphasic curve, improving the fit and allowing calculation of reproducible off-rates and K _D The value is obtained. This method is preferably curve fitted when comparing the binding capacity to Fc γ riiia (F176) between IgG samples or to reference samples to determine loss or increase in activity.

Example 1

1. Buffer solution preparation

Phosphate Buffer (PBS) with pH of 7.4 is prepared, and then Tween-20 (Tween-20) is added to prepare PBS containing 0.02% (v/v) Tween-20 as detection Buffer (Q Buffer).

2. Wetting sensor

Streptavidin (SA) biosensors (Octet, cat # 18-5020) were wetted in 200. Mu.l of Q Buffer for 10 minutes.

3. Cure acceptors Fc γ RIIa (F176)

The receptor Fc γ RIIa (F176) (ACRO Biosystems, cat # CDA-H82E 8) was diluted to 1.5. Mu.g/ml with Q Buffer, 200. Mu.l were added to a post 96-well plate and a reaction time of 300s (0.25 nm curing height) was set.

4. Analysis of antibodies

IgG1 antibody (antibody concentration: 0.06 mg/mL) produced using fucosyltransferase 8 (FUT 8) -deficient CHO cell line A was diluted with Q Buffer to 200nM, 100nM, 50nM, 25nM, 12.5nM and 0nM, respectively, 200. Mu.l of the IgG1 antibody was added to the post-96-well plate, one analyte concentration for each SA sensor, and the binding time of the IgG1 antibody to Fc γ RIIa (F176) was set to 90s, the dissociation time to 90s, and the rotation speed to 1000RPM. The results of the affinity assay for Fc γ riiia (F176) for IgG1 antibody produced by cell line a are shown in fig. 7, and the specific data are shown in table 1.

Example 2

1. Buffer solution preparation

Phosphate Buffer Solution (PBS) with the pH value of 7.4 is prepared, and then Tween-20 (Tween-20) is added to prepare PBS containing 0.02% (v/v) Tween-20 to serve as detection Buffer solution (Q Buffer).

2. Wetting sensor

Streptavidin (SA) biosensors (Octet, cat # 18-5020) were wetted in 200. Mu.l of Q Buffer for 10 minutes.

3. Cure acceptors Fc γ RIIa (F176)

The receptor Fc γ RIIa (F176) (ACRO Biosystems, cat # CDA-H82E 8) was diluted to 1.5. Mu.g/ml with Q Buffer, 200. Mu.l were added to a post 96-well plate and a reaction time of 300s (0.25 nm curing height) was set.

4. Analysis of antibodies

IgG1 antibody (antibody concentration: 0.43 mg/mL) produced using fucosyltransferase 8 (FUT 8) -deficient CHO cell line B was diluted with Q Buffer to 200nM, 100nM, 50nM, 25nM, 12.5nM and 0nM, respectively, 200. Mu.l of the IgG1 antibody was added to the post-96-well plate, one analyte concentration for each SA sensor, and the binding time of the IgG1 antibody to Fc γ RIIa (F176) was set to 90s, the dissociation time to 90s, and the rotation speed to 1000RPM. The affinity assay results for Fc γ riiia (F176) and Fc γ riiiia for IgG1 antibody produced by cell line B are shown in fig. 8, and the specific data are shown in table 1.

Example 3

1. Buffer solution preparation

Phosphate Buffer Solution (PBS) with the pH value of 7.4 is prepared, and then Tween-20 (Tween-20) is added to prepare PBS containing 0.02% (v/v) Tween-20 to serve as detection Buffer solution (Q Buffer).

2. Wetting sensor

Streptavidin (SA) biosensors (Octet, cat # 18-5020) were wetted in 200. Mu.l of Q Buffer for 10 minutes.

3. Cure acceptors Fc γ RIIa (F176)

The receptor Fc γ RIIa (F176) (ACRO Biosystems, cat # CDA-H82E 8) was diluted to 1.5. Mu.g/ml with Q Buffer, 200. Mu.l were added to the latter 96-well plate and a reaction time of 300s was set (curing height 0.25 nm).

4. Analysis of antibodies

IgG1 antibody (antibody concentration: 0.28 mg/mL) produced by wild-type CHO cell line C was diluted to 200nM, 100nM, 50nM, 25nM, 12.5nM and 0nM, respectively, with QBuffer, 200. Mu.l was added to the rear 96-well plate, one analyte concentration per SA sensor, and the binding time of IgG1 antibody to Fc γ RIIa (F176) was set for 90s, dissociation time for 90s, and rotation speed for 1000RPM. The results of the affinity assay of the IgG1 antibody produced by cell line C with Fc γ RIIa (F176) are shown in FIG. 9, and the specific data are shown in Table 1.

Example 4

1. Buffer solution preparation

Phosphate Buffer Solution (PBS) with the pH value of 7.4 is prepared, and then Tween-20 (Tween-20) is added to prepare PBS containing 0.02% (v/v) Tween-20 to serve as detection Buffer solution (Q Buffer).

2. Wetting sensor

Streptavidin (SA) biosensors (Octet, cat # 18-5020) were wetted in 200. Mu.l of Q Buffer for 10 minutes.

3. Solidification receptor Fc gamma RIIa (F176)

The receptor Fc γ RIIa (F176) (ACRO Biosystems, cat # CDA-H82E 8) was diluted to 1.5. Mu.g/ml with Q Buffer, 200. Mu.l were added to the latter 96-well plate and a reaction time of 300s was set (curing height 0.25 nm).

4. Analysis of antibodies

IgG1 antibody (antibody concentration: 0.28 mg/mL) produced by wild-type CHO cell line D was diluted to 800nM, 400nM, 200nM, 100nM, 50nM and 0nM, respectively, with QBuffer, 200. Mu.l was added to the post-96 well plate, one analyte concentration for each SA sensor, and the IgG1 antibody binding time to Fc γ RIIa (F176) was set for 90s, dissociation time for 90s, and rotation speed for 1000RPM. The affinity assay results for Fc γ riiia (F176) for IgG1 antibody produced by cell line D are shown in fig. 10, and the specific data are shown in table 1.

And (4) analyzing results:

TABLE 1

Fig. 7 to 10 are graphs showing the results of measuring the affinity of the IgG antibody with Fc γ rliiia (F176) in examples 1 to 4, respectively, and table 1 is the statistical data of fig. 7 to 10, in which the column of "same protein concentration" indicates the fold results obtained by comparing the affinity measured in examples 1 and 2 with the affinity measured in example 3, respectively, and the column of "4-fold protein concentration" indicates the fold results obtained by comparing the affinity measured in examples 1 and 2 with the affinity measured in example 4, respectively. As can be seen from Table 1, the affinity constant K between the antibody IgG and the receptor Fc γ RIIa (F176) was determined by an Octet system based on the principle of biofilm interference _D The significantly stronger affinity of the IgG antibodies produced by fucosyltransferase 8 (FUT 8) -deficient CHO cell lines a and B of examples 1 and 2, compared to the IgG antibodies produced from wild-type CHO cell lines C and D of examples 3 and 4, to the receptor Fc γ riiia (F176), demonstrates the stronger antibody-mediated interaction between immune cells and tumor cells and the greater ability of immune cells to kill tumor cells in examples 1 and 2, with example 2 again being superior to example 1. This is in contrast to the conclusions obtained from cell assaysSimilarly, fucosyltransferase 8 (FUT 8) -deficient CHO cell lines produce antibodies that mediate greater ADCC.

Accurate and reliable kinetics of the interaction between Fc γ riiia and monoclonal antibodies are challenging, but are a key application in many stages of biopharmaceutical development. The present invention provides a rapid, flexible and sensitive solution for determining these interactions, whether it be a full kinetic analysis, a steady state analysis or a relative binding capacity analysis.

The technical features of the embodiments described above may be arbitrarily combined, and for the sake of brevity, all possible combinations of the technical features in the embodiments described above are not described, but should be considered as being within the scope of the present specification as long as there is no contradiction between the combinations of the technical features.

The above-mentioned embodiments only express several embodiments of the present invention, and the description thereof is more specific and detailed, but not construed as limiting the scope of the invention. It should be noted that, for a person skilled in the art, several variations and modifications can be made without departing from the inventive concept, which falls within the scope of the present invention. It should be understood that the technical solutions provided by the present invention, which are obtained by logical analysis, reasoning or limited experiments by those skilled in the art, are within the scope of the present invention as set forth in the appended claims. Therefore, the protection scope of the present patent should be subject to the appended claims, and the description and drawings can be used to explain the contents of the claims.

Claims (10)

1. A method of detecting cell-cell interactions, comprising the steps of:

respectively obtaining an antibody capable of specifically binding with target cells to be detected and a target receptor on effector cells to be detected, wherein the target cells to be detected comprise virus-infected cells or tumor cells, and the effector cells to be detected comprise at least one of NK cells, eosinophilic granulocytes and neutrophils;

detecting and calculating an affinity constant between the antibody and the target receptor by adopting a biomembrane interference technology; and

and analyzing the interaction strength between the effector cell to be detected and the target cell mediated by the antibody according to the affinity constant.

2. The method of claim 1, wherein the target receptor comprises fcyriii or fceri.

3. The method of claim 2, wherein the antibody comprises an IgG antibody or an IgE antibody.

4. The method according to any one of claims 1 to 3, wherein the biofilm interference technique is performed on an Octet system.

5. The method of claim 4, wherein the step of detecting and calculating the affinity constant between the antibody and the target receptor using a biofilm interference technique comprises:

preparing a detection buffer solution, wherein the detection buffer solution comprises a phosphate buffer solution containing 0.02% (v/v) to 0.5% (v/v) Tween-20;

wetting a sensor with the detection buffer solution, and fixing the target receptor on the sensor; and

after the antibodies with multiple concentration gradients are combined with the target receptor, the data collected by the Octet system are globally fitted to determine the affinity constant.

6. The method according to claim 5, wherein the concentration of the target receptor after immobilization is controlled to 1 to 2 μ g/mL.

7. The method of claim 5, wherein the concentration of the antibody is controlled to be 12.5nM to 800nM.

8. The method according to claim 7, wherein the concentration of the antibody is controlled to be 12.5nM to 500nM.

9. The method of claim 5, wherein the antibody binds to the target receptor for a period of time ranging from 60s to 120s.

10. The method according to any one of claims 5 to 9, wherein the data acquisition rate of the Octet system is between 2Hz and 10Hz.

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