CN109696461B

CN109696461B - Release detection method of therapeutic antibody

Info

Publication number: CN109696461B
Application number: CN201711004653.7A
Authority: CN
Inventors: 贾晓青; 陈敏; 黄岗; 周伟昌; 陈智胜
Original assignee: Wuxi Biologics Shanghai Co Ltd
Current assignee: Wuxi Biologics Shanghai Co Ltd
Priority date: 2017-10-24
Filing date: 2017-10-24
Publication date: 2023-05-26
Anticipated expiration: 2037-10-24
Also published as: CN109696461A

Abstract

The invention discloses a biological activity method based on electrochemiluminescence technology, which can be used for releasing antibody medicines, and is characterized in that the method can detect the biological activity (cell binding method) of a sample antibody by using an electrochemiluminescence method, and the biological activity is calculated by performing four-parameter fitting on the concentration of the sample antibody and the electrochemiluminescence intensity. The method can be used for release detection of antibody medicines through methodology confirmation. Antibodies and test antibodies include anti-PD-1 antibodies or anti-PD-L1 antibodies.

Description

Release detection method of therapeutic antibody

Technical Field

The present invention relates generally to the field of life sciences, and in particular to quality control and establishment of release standard methods in antibody production processes.

Background

PD-1 (programmed death 1) receptor 1 is an important immunosuppressive molecule. The immunoregulation with PD-1 as a target spot has important significance for resisting tumors, infections, autoimmune diseases, organ transplantation survival and the like. The ligand PD-L1 can also serve as a target point, and the corresponding antibody can also play the same role. Therapeutic antibodies can therefore be used clinically to inhibit PD-1 or PD-L1 immunomodulation and thus inhibit disease.

One feature of the binding of PD-1 and PD-L1 is that they bind both rapidly and dissociate rapidly upon elution, so that the binding of both occurs in a "homogenous" state without elution, which more accurately mimics in vivo conditions (biological). None of the previous methods, such as flow cytometry (FACS) or Surface Plasmon Resonance (SPR) cell binding assays, are homogenization experiments and do not meet this requirement. And because of the lack of sensitivity and specificity of these methods, they are not suitable for use in therapeutic antibody production processes for developing therapeutic antibody release standards, there is a need for further optimization of the detection methods in order to obtain high sensitivity readings.

Electrochemiluminescence (ECL) is a specific chemiluminescent reaction initiated electrochemically at the electrode surface. The new generation of labeled immunoassay technology has both high sensitivity of luminescence analysis and high specificity of antigen-antibody reaction, so that the technology has wide application value. The invention establishes a release method and release standard in the antibody production process by using the electrochemiluminescence technology, and the method is different from the conventional FACS-based cell binding measurement, has more concise steps and more stable and accurate data, and is beneficial to quality control in the therapeutic antibody production process.

Disclosure of Invention

In one aspect of the invention, an antibody release method is provided, which utilizes electrochemiluminescence to detect the cell binding activity of a test sample antibody; and calculating biological activity by performing four-parameter fitting on the antibody concentration and the electrochemiluminescence intensity of the test sample to obtain a release standard. The method can be used for the biological activity release detection of the antibody through methodology confirmation.

In one embodiment of the invention, the antibody comprises an antibody drug, preferably a therapeutic antibody drug.

A test antibody as described above includes an anti-PD-1 antibody or an anti-PD-L1 antibody, preferably an anti-PD-1 therapeutic antibody or an anti-PD-L1 therapeutic antibody.

In one embodiment of the invention, the steps of the release method of the anti-PD-1 antibody comprise:

a) Plating cells expressing PD-1 on the cell surface into a cell plate, after which no washing is required;

b) Adding a PD-L1 recombinant protein, a detection antibody and the PD-1 antibody, and incubating without washing; wherein the PD-1 antibody is preferably a PD-1 therapeutic antibody;

c) Adding a reading buffer without surfactant; the read buffer is preferably an MSD read buffer;

d) And reading the plate by using equipment to obtain the electrochemiluminescence intensity.

In one embodiment of the invention, the steps of the release method of the anti-PD-L1 antibody comprise:

a) Plating cells expressing PD-L1 on the cell surface into a cell plate, after which no washing is required;

b) Adding PD-1 recombinant protein, detection antibody and the anti-PD-L1 antibody, and incubating without washing; wherein the PD-L1 antibody is preferably a PD-L1 therapeutic antibody;

In another aspect of the invention, there is also provided a method of detecting the activity of a PD-1 antibody by electrochemiluminescence, the method comprising the steps of:

a) Plating cells expressing PD-1 on the surface of the cells in a cell plate, and then washing the cells without buffer solution;

b) Adding PD-L1 recombinant protein, a detection antibody and the PD-1 antibody, incubating, and then washing without buffer solution; wherein the PD-1 antibody is preferably a PD-1 therapeutic antibody;

c) Adding a reading buffer without a surfactant, and then washing without the buffer; the reading buffer is preferably MSD read buffer;

In another aspect of the present invention, there is also provided a method for detecting the activity of a PD-L1 antibody by electrochemiluminescence, the method comprising the steps of:

a) Spreading cells expressing PD-L1 on the surface of the cells in a cell plate, and then washing the cells without buffer solution;

b) Adding a PD-1 recombinant protein, a detection antibody and the PD-L1 therapeutic antibody, incubating, and then washing without a buffer solution; wherein the PD-L1 antibody is preferably a PD-L1 therapeutic antibody.

In one embodiment of the present invention, the step of the aforementioned method for releasing PD-1 antibody or method for detecting activity or the step of releasing PD-L1 antibody or method for detecting activity may further comprise:

a') before step a), a blocking solution is added to the cell plate, incubated for about 1 to 3 hours, after which the cell plate is washed with a washing buffer.

Any of the methods described above, wherein the sample antibody concentration and the electrochemiluminescence intensity are subjected to four-parameter fitting by using the formula (1), and the C value, i.e. IC, is calculated according to the four-parameter fitting of the reference sample, the quality control sample and the sample ₅₀ The relative activity of the test sample is calculated by using the formula (2):

wherein x represents an independent variable, A represents a left platform, B represents a curvature parameter, C represents a half-maximal inhibition amount, the unit is ng/mL, and D represents a right platform.

In one embodiment of the invention, the test article release acceptable standard is one or more of the following:

1) The A parameter/D parameter is more than or equal to 2;

2) Parallelism analysis: the slope ratio of the curve is between 0.7 and 1.4; the high plateau ratio is between 0.8 and 1.25;

4) CV of all the linear section compound holes is less than or equal to 30 percent;

5) The biological activity of the quality control product is between 75% and 125%

6) R of four-parameter fitting S-shaped curve ² ≥0.95。

In one embodiment of the invention, the blocking solution is a buffer containing 1 to 5% by volume of bovine serum albumin or a buffer containing 10 to 30% of fetal bovine serum, preferably 15 to 30% of fetal bovine serum or a buffer containing 1 to 3% by volume of bovine serum albumin.

In one embodiment of the invention, the wash buffer is a PBS buffer.

In one embodiment of the invention, the cell plate is an MSD 96-well plate, more preferably an MSD highly binding 96-well plate.

In one embodiment of the invention, the cell is a CHO cell expressing PD-1 or PDL 1.

In one embodiment of the invention, the cell density in the cell plate is 2.5E4/well to 20E 4/well, preferably 3.5E4/well to 7.5E4/well, more preferably 3.5E4/well to 5E 4/well.

In one embodiment of the invention, the PD-1 recombinant protein or PD-L1 recombinant protein is present at a concentration of 50ng/mL to 200ng/mL, preferably 80ng/mL to 160ng/mL.

In one embodiment of the invention, the concentration of the detection secondary antibody is 500ng/mL to 1000ng/mL, preferably 1000ng/mL.

In one embodiment of the invention, the incubation time in step b) is 60 minutes to 90 minutes, preferably 90 minutes.

In one embodiment of the invention, the recombinant protein is a protein fused to a tag, wherein the tag is preferably a murine Fc.

In one embodiment of the invention, the detection antibody is ruthenium-conjugated anti-mouse IgG.

Drawings

Figure 1 shows the specificity of the biological activity-releasing method of anti-PD-1 therapeutic antibodies. Wherein the abscissa is the concentration of the product and the reference substance, the ordinate is ECL signal value, the blue circle is marked as the reference substance, the red square is marked as the quality control substance, the orange diamond is marked as the reference substance Rituximab, and the green triangle is marked as the reference substance Xolair.

Figure 2 shows a linear fit of the biological activity release method for anti-PD-1 therapeutic antibodies.

Figure 3 shows a bar graph between cell plating concentration and ECL signal values.

Fig. 4 shows a dose-dependent histogram between PD-L1 concentration and ECL signal values.

Figure 5 shows graphs of the activity results of stress samples under different condition treatments.

Detailed Description

The following detailed description of the invention is provided in connection with the accompanying drawings that are presented to illustrate the invention and not to limit the scope thereof. The experimental methods in the following examples are conventional methods unless otherwise specified. Materials, reagents and the like used in the examples described below are commercially available unless otherwise specified.

Before describing the present invention in detail, it is to be understood that this invention is not limited to particular biological systems or cell types. It is also to be understood that the terminology used herein is for the purpose of describing particular embodiments only, and is not intended to be limiting. As used in this specification and the appended claims, the singular forms "a", "an", and "the" include plural referents unless the content clearly dictates otherwise. Thus, for example, reference to "a cell" includes a combination of two or more cells or an entire culture of cells; reference to a "polynucleotide" actually includes many copies of the polynucleotide. Unless defined otherwise in the reminders of this specification, 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 term "test sample" as used herein refers to a test sample to be identified, to be tested. In the present invention, the test antibody includes an antibody sample to be detected for biological activity, which is produced in industrial production, specifically including a PD-1 antibody or a PD-L1 antibody.

The term "antibody" as used herein includes whole antibodies and any antigen-binding fragment (i.e., an "antigen-binding portion") or single chain thereof. An "antibody" comprises a protein having at least two heavy (H) chains and two light (L) chains or antigen binding portions thereof linked to each other by disulfide bonds. Each heavy chain consists of a heavy chain variable region (abbreviated herein as VH) and a heavy chain constant region. The heavy chain constant region consists of three domains, CH1, CH2 and CH 3. Each light chain consists of a light chain variable region (abbreviated herein as VL) and a light chain constant region. The light chain constant region consists of one domain CL. The VH and VL regions can be further subdivided into regions of higher variability, termed Complementarity Determining Regions (CDRs), interspersed with regions that are more conserved, termed Framework Regions (FR). Each VH and VL consists of three CDRs and four FRs arranged from amino-terminus to carboxy-terminus in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4. The variable regions of the heavy and light chains contain binding domains that interact with antigens. Wherein the term "antibody" refers to an immunoglobulin or fragment or derivative thereof, and includes any polypeptide comprising an antigen binding site, whether or not it is produced in vitro or in vivo. The term includes, but is not limited to, polyclonal, monoclonal, monospecific, multispecific, nonspecific, humanized, single-chain, chimeric, synthetic, recombinant, hybrid, mutant, and grafted antibodies. The term "antibody" also includes antibody fragments that retain antigen binding function, such as Fab, F (ab') 2, fv, scFv, fd, dAb, and other antibody fragments, such as the ability to specifically bind to PD-1 or PD-L1. Typically, such fragments will comprise antigen-binding fragments.

As used herein, the terms "therapeutic antibody," "therapeutic antibody" are used interchangeably and include, but are not limited to, antibodies that have been used in clinical or clinical studies, but also include potential antibodies of clinical use that are being developed that can be administered to a subject to treat a disease; the term "therapeutic antibody" or "therapeutic antibody" also includes detectable antibodies useful for in vitro diagnostics, in vitro research, in vitro development, which antibodies can be used for cytological level or immunological detection.

As used herein, the terms "programmed cell death 1", "protein PD-1", "PD1", "PDCD1", "hPD-1" and "hPD-F" are used interchangeably and include variants, isoforms, species homologs of human PD-1 and analogs having at least one common epitope of PD-1.

As used herein, the terms "programmed death ligand 1", "PD-L1", "PD L1", "B7 homolog 1", "B7-H1", "CD274" are used interchangeably and include variants, isoforms, species homologs of human PD-L1 and analogs having at least one common epitope of PD-L1.

As used herein, the term "CV" refers to a coefficient of variation, which is an absolute value reflecting the degree of data dispersion. The data size is influenced not only by the degree of variance of the variable values, but also by the average level of the variable values. The ratio of standard deviation to average is called coefficient of variation. The coefficient of variation may eliminate the effect of unit and/or average differences on the comparison of the degree of variation of two or more data. The calculation formula of the variation coefficient is as follows: coefficient of variation cv= (standard deviation SD/Mean) ×100%.

As used herein, the term "recombinant protein" refers to a recombinant vector to which a gene fragment that can be translated into a protein of interest is linked using gene recombination techniques, and then transferred into a host cell that can express the protein of interest to express a specific recombinant protein molecule.

As used herein, the term "read buffer" refers to a buffer for reading, including but not limited to MSD read buffer.

As used herein, the terms "let-off," real-time let-off, "" let-off detection, "" carry out let-off detection, "RTR," "RTRT" are used interchangeably to refer to the ability to evaluate and ensure acceptable quality of intermediate and/or finished products based on process data; it generally effectively combines data of measured material properties and process control, etc., thereby evaluating and guaranteeing the quality of intermediate products or final finished products.

Examples

The invention is further illustrated by the following examples. The examples are provided for illustrative purposes only and should not be construed as limiting the scope or content of the invention in any way.

Example 1: reference, quality control and test preparation (for anti-PD-1 antibodies)

According to the protein labeling amount, the reference, quality control and test substances are diluted into 10 or 11 concentration points by gradient dilution or targeted dilution with an analysis culture medium, and the 10 or 11 points are used as a standard curve. Wherein the quality control is the same as the reference but is diluted independently of the reference.

Example 2: preparation of PD-1/CHO-S cell lines

Either continuously cultured cells or freshly thawed cryopreserved cells are suitable for use in this method.

1. Cells in continuous culture: the cells were centrifuged and the supernatant discarded.

2. Cryopreserved cells: PD-1/CHO-S cells were removed from the liquid nitrogen tank and quickly placed into a 37℃water bath for thawing. The cell suspension in the freezing tube was added to a CHO medium containing about n×9mL (N represents the cell count) and gently swirled to mix. The supernatant was discarded by centrifugation.

3. Cells were washed once with cold DPBS. The supernatant was discarded after centrifugation. The cells are resuspended by adding assay medium according to the desired cell suspension volume. Cell suspension is taken for cell counting, and cell density and cell activity are determined. The cell density was adjusted to 16.7E5/mL with the assay medium and the cell suspension at the adjusted concentration was homogenized.

Example 3: test method

1. Closing: mu.L of blocking solution (10% FBS/PBS or 3% BSA/PBS) was added to the MSD high binding 96 wells. The MSD plate is placed on a shaker and shaken at a speed of 150-220 rpm and incubated for 2.+ -.1 hours at room temperature (this step may be omitted if no blocking is required).

2. Reference, quality control and test solutions were prepared as described in example 1. PD-L1-mFc stock was diluted to a final concentration of 800ng/mL and vortexed. The secondary Antibody (MSD SULFO-TAG coat Antibody-mouse Antibody) stock solution was diluted to a final concentration of 2.857. Mu.g/mL and vortexed. Preparation of PD-1/CHO-S cell lines is described in example 2.

3. Diluted reference and test samples, diluted 800ng/mL PD-L1-mFc and diluted 2.857. Mu.g/mL detection antibody were transferred to the second column to the twelfth column of the U-bottom 96-well plate (transfer plate) in a ratio of 1:1:2 (v: v) (e.g., 45. Mu.L: 90. Mu.L), and mixed well. In the first column, assay medium and detection antibody mixed at 1:1 (v: v) (e.g., 90. Mu.L: 90. Mu.L) are added as background. And storing in a refrigerator at the temperature of 2 to 8 ℃ in a dark place.

4. MSD was washed 3 times with DPBS or 1 XPBS in high binding 96-well plates (this step was omitted if no blocking of the MSD plates was required).

5. PD-1/CHO-S cell plating: add to the MSD high binding plate at 30 μl/well. The mixture of reference and test antibodies, PD-L1-mFc and detection antibody was added at 70. Mu.L/well to the MSD high binding plates containing PD-1/CHO-S cells in a total volume of 100. Mu.L per well.

6. MSD plates were incubated at room temperature or 22-28℃in a constant temperature incubator for 60 to 90 minutes in the absence of light.

7. To each well of the MSD plate was added 100. Mu.L of 2 Xsurfactant-free Read Buffer.

8. The plate is read with MSD MESOTM SECTORS or equivalent device.

9. Data analysis:

although multiple plates were operated simultaneously for each experiment, each plate was calculated independently.

Fitting of antibody concentration and electrochemiluminescence intensity with four parameters

Wherein x=argument

A=left platform

B = curvature parameter

C=half inhibition (ng/mL)

D=right platform

Parallelism analysis: b value is obtained according to four-parameter fitting calculation of the reference and the test sample, namely the Slope of a four-parameter fitting curve, and the Ratio (Slope Ratio) of the Slope of the test sample or the quality control sample is obtained by calculating the following formula:

the relative activities of the quality control product and the sample are calculated as follows: c value is obtained by fitting and calculating four parameters of reference, quality control and test sample, namely IC ₅₀ The biological activity of the test sample was calculated using the following formula.

10. In one particular embodiment, system applicability

Only when the quality control product meets all the following criteria, the experiment is effective,

a) The A parameter/D parameter is more than or equal to 2;

b) Parallelism analysis: the slope ratio of the curve is between 0.7 and 1.4; a high plateau ratio between 0.8 and 1.25;

c) The double complex holes comprise complex holes CV less than or equal to 30% in the linear section;

d) The biological activity of the quality control product is between 75% and 125%;

e) R of four-parameter fitting S-shaped curve ² ≥0.95；

Experiments were considered ineffective if the results of the quality control product did not meet the above criteria.

Reference acceptable standard

The A parameter/D parameter is more than or equal to 2

All linear section compound holes CV are less than or equal to 30 percent.

R of four-parameter fitting S-shaped curve ² ≥0.95

Acceptable standard of test sample

The A parameter/D parameter is more than or equal to 2

Parallelism: the slope ratio of the curve is between 0.7 and 1.4; high plateau ratio of between 0.8 and 1.25

CV of all linear section compound holes of the test sample is less than or equal to 30 percent.

R of four-parameter fitting S-shaped curve ² ≥0.95

Example 4: method validation

1. Specialization of

Irrelevant Isotype-matched antibodies did not inhibit the binding of PD-L1 to the PD-1 expressing CHO cell line, and the control samples had no specific biological activity (FIG. 1). The method has good specificity for investigating the biological activity of the PD-1 resistant product.

2. Accuracy of

Average recovery of samples at 50%, 71%, 100%, 141% and 200% target concentrations ranged from 80% to 120% (table 1).

3. Precision of

Samples of 50%, 71%, 100%, 141%, and 200% target concentration, each independently 8 times; the intermediate precision assay results were less than 25% RSD per sample (table 1).

TABLE 1 accuracy and precision results list

4. Linearity of

Using the data from the recovery study, the expected relative activity and measured relative activity fit linearly better, with a Pearson correlation coefficient of ≡0.95 (FIG. 2).

5. Test effective range

The test results showed good linearity for recovery in the range of 50% to 200% target concentration, and both accuracy and precision of the results met acceptable standards. Thus, the effective range of the test is 50% to 200% of the target concentration range.

6. Durability of

The parameters of PD-L1 concentration, test incubation time, cell density, detection antibody concentration, cell generation and the like are in the research range, and QC recovery rate is more than or equal to 80% and less than or equal to 120%

7. Stress sample

The activity of heat-treated, acid-treated (about pH 3) and alkali-treated (about pH 11) stress samples was significantly reduced relative to the untreated control (fig. 5).

8. Long term quality control

In the period of at least 6 months, at least two testers detect the quality control product, and in the process, the recovery rate of the quality control product is between 80% and 120%, and the quality control product meets the release requirement, so that the release method is proved to meet the investigation of long-term quality control.

9. Conclusion(s)

The experimental results, including specificity, accuracy, precision, effective range of test and analysis of linear, durable and stress samples, prove that the method is suitable for biological activity detection of anti-PD 1 antibody in various stages of laboratory, including pharmaceutical stock solution, finished product, stable sample and intermediate.

Example 5: optimization of method parameters

1. Cell density

Cell type: CHO cells expressing PD-1 or PD-L1, respectively, explored the cell density range: 6.25E3/Kong E4/well. As a result, cells with the results shown in FIGS. 3,2.5E4/Kong E4/well were all suitable for use in the experiment.

2. PD-L1/PD-1 concentration

Exploring concentration ranges: 4 to 2000ng/mL. As a result, referring to FIG. 4, 100.+ -.50 ng/mL of PD-L1 was suitable for the experiment.

3. Other parameters

3.1 detecting the concentration of the secondary antibody

Exploring concentration ranges: 100ng/mL to 2000ng/mL. The detection secondary antibody concentration of 750+/-250 ng/mL is suitable for experiments.

3.2, test incubation time

Search range: incubation times of 60 to 90 minutes are suitable for the experiment.

3.3 sealing liquid

Search conditions: 1% BSA to 3% BSA, 15% to 30% FBS/medium, no blocking. All three are suitable for the experiment.

3.4, assay buffer

Search conditions: 1% BSA, 10% to 30% FBS/medium, 10% to 30% FBS/PBS. All three are suitable for the experiment.

3.5 comparison of cryopreserved cells and continuously cultured cells

Results: is suitable for experiments.

Incorporated by reference

The entire disclosure of each patent document and scientific document cited herein is incorporated by reference for all purposes.

Equivalent means

The present invention may be embodied in other specific forms without departing from its essential characteristics. Accordingly, the foregoing embodiments are to be considered as illustrative rather than limiting the invention described herein. The scope of the invention is indicated by the appended claims rather than by the foregoing description, and all changes that come within the meaning and range of equivalency of the claims are intended to be embraced therein.

Claims

1. A method for releasing biological activity of an antibody, wherein the method detects cell binding activity of an antibody of a test sample by an electrochemiluminescence method, calculates biological activity by performing four-parameter fitting on the concentration of the antibody of the test sample and the intensity of electrochemiluminescence, and confirms that the method can be used for releasing biological activity of the antibody by a methodology, comprising:

b) Adding PD-L1 recombinant protein, detection antibody and anti-PD-1 antibody, and incubating without washing;

c) Adding a reading buffer without surfactant;

d) Using a device reading plate to obtain electrochemiluminescence intensity;

e) Performing four-parameter fitting on the antibody concentration and the electrochemiluminescence intensity of the sample by using a formula (1), and calculating to obtain a C value, namely IC, according to four-parameter fitting of the reference, the quality control and the sample ₅₀ The relative activity of the test sample is calculated by using the formula (2):

wherein x represents an independent variable, A represents a left platform, B represents a curvature parameter, C represents a half-number inhibition amount, the unit of the half-number inhibition amount is ng/mL, and D represents a right platform;

or alternatively

b) Adding PD-1 recombinant protein, detection antibody and anti-PD-L1 antibody, incubating, and then eliminating the need for washing;

c) Adding a reading buffer without surfactant;

d) Using a device reading plate to obtain electrochemiluminescence intensity;

wherein x represents an independent variable, A represents a left plateau, B represents a curvature parameter, C represents a half-maximal inhibition amount, the unit of the half-maximal inhibition amount is ng/mL, and D represents a right plateau.

2. A method for detecting the cellular activity of an anti-PD-1 antibody using electrochemiluminescence, said method comprising the steps of:

c) Adding a reading buffer without surfactant;

d) Using a device reading plate to obtain electrochemiluminescence intensity;

3. A method for detecting the cellular activity of an anti-PD-L1 antibody using electrochemiluminescence, said method comprising the steps of:

a) Plating cells expressing PD-L1 on the cell surface into a cell plate, and then eliminating the need for liquid washing;

b) Adding PD-1 recombinant protein, detection antibody and PD-L1 monoclonal antibody, incubating, and then eliminating the need of washing;

c) Adding a reading buffer without surfactant;

d) Using a device reading plate to obtain electrochemiluminescence intensity;

4. A method according to any one of claims 1 to 3, wherein the steps further comprise:

a') adding a blocking solution to the cell plate prior to step a), incubating for 1 to 3 hours, and then

The cell plates were washed with buffer.

5. A method according to any one of claims 1 to 3, wherein the system suitability criteria is one or more of the following criteria:

a) The A parameter/D parameter is more than or equal to 2;

c) The complex aperture CV of all the linear sections is less than or equal to 30 percent;

e) R of four-parameter fitting S-shaped curve ² ≥0.95。

6. The method of claim 4, wherein the blocking solution is a buffer containing bovine serum albumin at a concentration of 1% to 5% by volume or a buffer of 10% to 30% fetal bovine serum.

7. The method of claim 6, wherein the blocking solution is a buffer containing bovine serum albumin at a concentration of 1% to 3% by volume or a buffer of 15% to 30% fetal bovine serum.

8. The method of claim 6 or 7, wherein the buffer is PBS buffer or basal medium.

9. A method according to any one of claims 1 to 3, wherein the cell plate is a 96-well plate.

10. The method of claim 9, wherein the cell plate is a 96-well plate with high MSD binding.

11. The method of any one of claims 1 to 3, wherein the cell is a CHO cell expressing PD-1 or PD-L1.

12. A method according to any one of claims 1 to 3, wherein the cell density in the cell plate is 2.5E4/well to 20E 4/well.

13. The method of claim 12, wherein the cell density in the cell plate is 3.5E4/well to 7.5E4/well.

14. The method of claim 13, wherein the cell density in the cell plate is 3.5E4/well to 5E 4/well.

15. The method of any one of claims 1 to 3, wherein the concentration of the PD-1 recombinant protein or PD-L1 recombinant protein is 50ng/mL to 200ng/mL.

16. The method of claim 15, wherein the concentration of the PD-1 recombinant protein or PD-L1 recombinant protein is 80ng/mL to 160ng/mL.

17. The method of any one of claims 1 to 3, wherein the concentration of the detection antibody is 500ng/mL to 1000ng/mL.

18. The method of claim 17, wherein the concentration of the detection antibody is 1000ng/mL.

19. A method according to any one of claims 1 to 3, wherein the incubation time in step b) is from 60 minutes to 90 minutes.

20. The method of claim 19, wherein the incubation time in step b) is 90 minutes.

21. A method according to any one of claims 1 to 3, wherein the recombinant protein is a tag fused protein.

22. The method of claim 21, wherein the tag is a murine Fc.

23. The method of any one of claims 1 to 3, wherein the detection antibody is ruthenium-conjugated anti-mouse IgG.