CN101122603A - Use of soluble co-signaling molecules in detecting rheumatoid arthritis - Google Patents

Abstract

The invention discloses a method for measuring soluble signal molecules in a liquid sample, and discloses a reagent and a kit for preparing in vitro auxiliary detection of rheumatoid arthritis by using an antibody of PD-1 or its ligand. Using the detection method provided by the present invention, the existence of soluble PD-1 and soluble PD-L1 in human body is confirmed for the first time. The kit provided by the invention is beneficial to clinical testing and laboratory research.

Description

Use of soluble co-signaling molecules in detecting rheumatoid arthritis

technical field

The present invention relates to a method for detecting samples in vitro, more particularly to a method for detecting soluble co-signaling molecules using immunological techniques.

Background technique

Autoimmune diseases seriously endanger human health. Taking rheumatoid arthritis (RA) as an example, it is distributed worldwide, rare in tropical regions, and more common in temperate and frigid regions. In the Chinese population, the incidence rate is 0.3%-0.5%, and there are about 4 to 5 million patients. With the increase of age, the prevalence rate tends to increase. The ratio of female to male incidence is 3:1.

The disorder of autoimmune response is an important reason leading to the continuous progression of these diseases. Taking RA as an example, autoreactive T cells infiltrating in the synovial tissue play a key pathogenic role in cellular immunity and humoral immunity. Autoreactive T cells are activated by exogenous antigens or altered self-antigens, enter the joints through the action of adhesion molecules, mediate cellular immunity and humoral immunity, and cause synovial tissue damage. This group of T cells expressed activation/memory markers such as CD45R, CD44, HLA-DR, and VLA-1, indicating that they were in a state of continuous high activation.

For the reasons for the continuous activation of self-reactive T cells, the classic dual-signal theory holds that the activation of T cells requires the first signal mediated by the complex of TCR and antigenic peptide:MHC molecules and the second signal provided by the co-signaling molecule B7-CD28 pathway. Two signal mediation. Co-signaling molecules are glycoproteins expressed on the cell surface, they participate in the regulation of TCR signaling to promote or inhibit the sensitization, activation, growth and differentiation of T cells. According to their positive or negative regulatory effect on T cell function, they can be divided into co-stimulatory molecules or co-inhibitory molecules.

PD-1 (programmed death-1, Programmed death-1) protein is a co-inhibitory receptor expressed on the surface of activated T, B cells and myeloid cells. PD-1 is a synergistic inhibitory molecule with immunomodulatory function, binding to its ligand can inhibit downstream signaling events and inhibit T and B cell proliferation and cytokine production. The inhibitory signaling pathway is mediated by the ITSM in the cytoplasm, and the phosphorylated tyrosine residues recruit SHP-2 phosphatase to dephosphorylate the TCR and BCR signaling pathway components to exert an inhibitory effect. The effect of PD-1 is closely related to the dose of antigen, and it has a strong inhibitory effect on the proliferative response induced by low-concentration antigen stimulation.

Co-inhibitory molecules such as CTLA-4 and PD-1 are up-regulated after T cell activation to inhibit T cell immune response and play an important role in maintaining peripheral immune tolerance. Once the balance of co-stimulatory signals and inhibitory signals is broken, it will lead to the occurrence of immunopathological phenomena such as autoimmune diseases.

However, the expression of CD28 and ICOS in the synovial fluid infiltrating cells of RA patients has only been partially studied, and there is still a lack of in-depth mechanism research. The exact relationship between co-signaling molecules such as PD-1 and RA is still unknown, and systematic research on co-signaling molecules and their soluble forms in RA is urgently needed.

It can be seen that soluble PD-1 and its ligands are one of the key factors to further understand and study the disordered state of autoimmune response in autoimmune diseases such as RA. Therefore, it is urgent to provide a method in this field, which can detect human The natural soluble PD-1 and its ligand PD-L1 in the body, so as to carry out research related to such diseases more effectively.

Contents of the invention

The present invention aims to provide a method for detecting soluble PD-1 (sPD-1) and its ligands in liquid samples in vitro.

Another object of the present invention is to provide the use of this method.

In a first aspect of the present invention, a method for in vitro detection of soluble PD-1 protein or its ligand in a liquid sample is provided, comprising the steps of:

(a) contacting the sample with an anti-PD-1 antibody or an anti-PD-1 ligand antibody, thereby forming a soluble PD-1 protein-antibody complex or a soluble PD-1 ligand-antibody complex;

(b) detecting the soluble PD-1 protein-antibody complex or the soluble PD-1 ligand-antibody complex, thereby determining the presence or absence of the soluble PD-1 protein or its ligand.

In another preferred embodiment, in step (b), the soluble PD-1 protein-antibody complex or the soluble PD-1 ligand-antibody complex is detected by a sandwich method.

In another preferred embodiment, it includes the steps of:

(1) Coating the antibody of PD-1 or its ligand on a solid phase carrier to obtain a solid phase carrier coated with the antibody of PD-1 or its ligand;

(2) adding a liquid sample on the solid phase carrier coated with the antibody of PD-1 or its ligand;

(3) adding enzyme-labeled anti-PD-1 or its ligand antibodies of different species;

(4) Add substrate to carry out enzyme-catalyzed reaction.

In another preferred example, the antibody against PD-1 or its ligand is a polyclonal antibody.

In another preferred example, the ligand is PD-L1.

In another preferred example, the PD-1 or its ligand is human PD-1 or its ligand.

In another preferred example, the liquid sample includes serum, plasma, synovial fluid, urine or milk, etc.

In the second aspect of the present invention, the use of an antibody against soluble PD-1 protein or its ligand is provided, which can be used to prepare a reagent or kit for auxiliary detection of rheumatoid arthritis.

In another preferred embodiment, the reagent is a solid-phase carrier, and antibodies against PD-1 or its ligands are immobilized on the solid-phase carrier.

In another preferred example, the kit includes:

solid phase carrier,

Antibodies to PD-1 or its ligands,

Enzyme-labeled different species of anti-PD-1 or its ligand antibodies, substrates.

In another preferred example, the solid phase carrier is coated with an antibody to PD-1 or its ligand.

In another preferred example, the ligand is PD-L1.

In another preferred example, the PD-1 or its ligand is human PD-1 or its ligand.

In another preferred example, the antibody against PD-1 or its ligand is a polyclonal antibody.

In another preferred example, the anti-human PD-1 or its ligand antibodies of different species are monoclonal antibodies.

In a third aspect of the present invention, a kit for in vitro detection of soluble PD-1 protein or its ligand in a liquid sample is provided, comprising:

solid phase carrier,

Antibodies to PD-1 or its ligands,

Enzyme-labeled different species of anti-PD-1 or its ligand antibodies, substrates.

In another preferred example, the solid phase carrier described in the above kit is coated with an antibody to PD-1 or its ligand.

In another preferred example, the antibody against PD-1 or its ligand described in the above kit is a polyclonal antibody.

In another preferred example, the different species of anti-human PD-1 or its ligand antibodies described in the above kit are monoclonal antibodies.

In another preferred example, the PD-1 or its ligand described in the above kit is human PD-1 or its ligand.

In another preferred example, the kit further includes a buffer for coating the antibody and a substrate buffer.

In another preferred example, the kit further includes washing solution, blocking solution and stop solution.

In another preferred example, the kit further includes a negative control and a positive control.

In another preferred example, the kit further includes instructions.

Accordingly, the present invention provides a method by which the natural soluble PD-1 and its ligand PD-L1 in the human body can be detected, so as to assist in the detection of rheumatoid arthritis more effectively.

Description of drawings

Figures 1A and B show the mRNA expression levels of mononuclear cell co-signaling molecules in peripheral blood and synovial fluid of RA patients; ^* indicates p<0.05 compared with other groups.

HC denotes healthy controls, RA denotes rheumatoid arthritis patients, OA denotes osteoarthritis controls, PB denotes peripheral blood, and SF denotes synovial fluid.

Figure 1C shows the mRNA expression levels of co-signaling molecules in different cell subsets of RA patients.

^* indicates p<0.05 compared with the peripheral blood (PB) group of RA patients,

+ indicates p<0.05 compared with the synovial fluid-derived CD8+ T cell group of RA patients.

Figure 2 shows the expression levels of co-signaling molecules in different cell subsets of RA patients analyzed by flow cytometry (FACS).

Figure 3 shows the mRNA expression levels of Fas in mononuclear cells in peripheral blood and synovial fluid.

Figure 4 shows the induction of co-signaling molecules by cytokines.

Figure 5 shows the detection results of soluble co-signaling molecules in the synovial fluid or serum of RA patients and controls.

Figure 6 shows the expression of PD-1 full-length mRNA and spliced bodies of different lengths.

Figure 7 shows the effects of recombinant human PD-1-Ig and PD-L1-Ig on the activation and proliferation of T cells.

Detailed ways

After in-depth research on the pathological mechanism of RA, the inventors found that soluble PD-1 and its soluble ligand PD-L1 were significantly increased in the serum and synovial fluid of RA patients, and the increased soluble PD-1 and There is a significant correlation between soluble PD-L1 (sPD-1 and sPD-L1) and rheumatoid factor (RF), revealing that soluble PD-1 and other molecules have certain pathological regulatory effects on abnormal activation of T cells.

Specifically, the inventors originally speculated that the local expression level of co-stimulatory molecules in the joints of RA patients increased, while the expression of co-inhibitory molecules on the surface of synovial infiltrating T cells and macrophages may be down-regulated, and the lack of negative regulatory mechanisms leads to the persistence of T cells. Activation mediates cellular and humoral immune responses, and joint damage continues to progress.

However, the inventors found that the expressions of synergistic inhibitory molecules PD-1 and CTLA-4 were significantly increased in synovial fluid-infiltrated T cells of RA patients, and their corresponding ligands were also up-regulated on the surface of synovial fluid-infiltrated macrophages, and then invented It was found that soluble co-signaling molecules antagonized the regulation of co-inhibitory molecules and played a pathological role in the continuous activation of autoreactive T cells.

On the basis of the above findings, the inventors prepared a kit, using the ELISA method, for the first time confirmed the existence of soluble PD-1 and soluble PD-L1 in the human body, and also established the detection of soluble PD-1 in liquid samples or soluble PD-L1 approach.

Auxiliary method for detecting rheumatoid arthritis

The method for detecting rheumatoid arthritis provided by the present invention is to detect soluble co-signaling molecules in a liquid sample, preferably soluble PD-1 and/or its ligand, and the ligand is preferably PD-L1.

The liquid sample is serum, plasma, synovial fluid, urine, or breast milk, among which serum or synovial fluid is preferably used.

The soluble co-signaling molecules in the liquid sample can be detected by conventional methods in the art, preferably using the immunological method of antigen-antibody combination, that is, the soluble co-signaling molecules in the sample are combined with the known antibody of the signal molecule to judge the sample Whether the soluble co-signaling molecule exists in More preferably, enzyme-linked immunosorbent assay (ELISA) is used.

In a preferred example of the present invention, detect through the following steps:

(a) Coating the ELISA plate with human PD-1 or human PD-L1 antibody;

(b) adding a liquid sample to form soluble human PD-1 and human PD-1 antibody complex or soluble human PD-L1 and human PD-L1 antibody complex I;

(c) adding enzyme-labeled sheep/or mouse anti-human PD-1 antibody or anti-human PD-L1 antibody to generate enzyme-labeled double antibody sandwich complex II;

(d) adding substrate to carry out enzyme-catalyzed reaction, judging and detecting according to OD value.

In another preferred example of the present invention, detect through the following steps:

(a) Coating the ELISA plate with human PD-1 or human PD-L1 antibody;

(b) adding a liquid sample to form soluble human PD-1 and human PD-1 antibody complex or soluble human PD-L1 and human PD-L1 antibody complex I;

(c) adding goat/or mouse anti-human PD-1 antibody or anti-human PD-L1 antibody to generate double antibody sandwich complex II;

(d) adding enzyme-labeled mouse anti-goat or goat anti-mouse anti-antibodies to produce enzyme-labeled complex III;

(e) adding substrate to carry out enzyme-catalyzed reaction, judging and detecting according to OD value.

In another preferred embodiment of the present invention, goat/or mouse anti-human PD-1 antibody or anti-human PD-L1 antibody is labeled with biotin, and then combined with enzyme-labeled streptavidin for detection.

The human PD-1 or human PD-L1 antibody is commonly used in the field, preferably a polyclonal antibody. The polyclonal antibody can be prepared by methods well known to those skilled in the art.

The goat/or mouse anti-human PD-1 antibody or anti-human PD-L1 antibody is commonly used in the art, preferably a monoclonal antibody. Monoclonal antibodies can be prepared using hybridoma technology (see Kohler et al., Nature 256; 495, 1975; Kohler et al., Eur. J. Immunol. 6:511, 1976; Kohler et al., Eur. J. Immunol. 6: 292, 1976; Hammerling et al., In Monoclonal Antibodies and TCell Hybridomas, Elsevier, N.Y., 1981).

The detection method provided by the invention can be qualitative or quantitative.

Auxiliary detection reagents for rheumatoid arthritis

The invention provides a reagent for detecting soluble PD-1 or its ligand in a liquid sample in vitro. The reagent is a solid phase carrier immobilized with an antibody against PD-1 or its ligand.

The solid-phase carrier is used as an adsorbent and a container in the ELISA determination process, and does not participate in chemical reactions, which is commonly called an ELISA plate. Its material can be familiar to those skilled in the art, including (but not limited to) polyvinyl chloride, polystyrene, polyacrylamide, cellulose and the like. Shapes are familiar to those skilled in the art and include, but are not limited to, microtiter plates, beads, and small test tubes, among others. Among them, polystyrene microtiter plates are preferred. The microtiter plate can be a single well or a multi-well strip.

The PD-1 or PD-L1 antibody is used to coat the solid phase carrier, preferably goat anti-human PD-1 or goat anti-human PD-L1 antibody, which can be polyclonal antibody or monoclonal antibody, preferably polyclonal antibody Cloned antibodies, such as those from R&D Systems.

Antibody coating can be carried out by methods well known in the art. The preferred method is to prepare a solution with a concentration of 1-10 μg/ml of human PD-1 or human PD-L1 antibody with coating buffer, and add it to the surface of the solid phase carrier , 4°C for 18-24 hours or 37°C for 2 hours, wash off the antibody not adsorbed on the surface of the solid-phase carrier with washing buffer; then add blocking solution. The coating buffer is selected from (but not limited to): carbonate buffer at pH 9.6, phosphate buffer at pH 7.2, and Tris-HCL buffer at pH 7-8. The washing buffer is selected from (but not limited to): phosphate buffer containing 0.05% Tween 20. The blocking solution is selected from (but not limited to): fetal bovine serum, goat serum or rabbit serum.

A kit for auxiliary detection of rheumatoid arthritis

The invention provides a kit for detecting soluble PD-1 or its ligand in liquid samples in vitro. The kit includes a solid phase carrier, an antibody to PD-1 or an antibody to PD-L1, enzyme-labeled anti-PD-1 or anti-PD-L1 antibodies of different species, and a substrate.

The solid-phase carrier is used as an adsorbent and a container in the ELISA determination process, and does not participate in chemical reactions, which is commonly called an ELISA plate. Its material can be familiar to those skilled in the art, including (but not limited to) polyvinyl chloride, polystyrene, polyacrylamide, cellulose and the like. Shapes are familiar to those skilled in the art and include, but are not limited to, microtiter plates, beads, and small test tubes, among others. Among them, polystyrene microtiter plates are preferred. The microtiter plate can be a single well or a multi-well strip.

The PD-1 or PD-L1 antibody is used to coat the solid phase carrier, preferably goat human PD-1 or goat human PD-L1 antibody, which can be polyclonal antibody or monoclonal antibody, preferably polyclonal antibody , such as products from R&D Systems.

Antibody coating can be carried out by methods well known in the art. The preferred method is to prepare a solution with a concentration of 1-10 μg/ml of human PD-1 or human PD-L1 antibody with coating buffer, and add it to the surface of the solid phase carrier , 4°C for 18-24 hours or 37°C for 2 hours, wash off the antibody not adsorbed on the surface of the solid-phase carrier with washing buffer; then add blocking solution. The coating buffer is selected (but not limited to): pH9.6 carbonate buffer, pH7.2 phosphate buffer, pH7-8 Tris-HCL buffer. The washing buffer is selected from (but not limited to): phosphate buffer containing 0.05% Tween 20. The blocking solution is selected from (but not limited to): fetal bovine serum, goat serum or rabbit serum.

The kit of the present invention may include a mouse/goat anti-human PD-1 antibody or a human PD-L1 antibody as an antibody reactive with a liquid sample. The antibody used for the reaction may have an enzyme-labeled non-human anti-human PD-1 or anti-human PD-L1 antibody that constitutes the enzyme label. The non-human antibody is a monoclonal or polyclonal antibody, preferably a monoclonal antibody.

The present invention can mark the enzyme on the goat anti-mouse or mouse anti-goat antibody. The present invention can also label biotin on mouse/goat anti-human PD-1 antibody or human PD-L1 antibody, and then combine with enzyme-labeled streptavidin for detection.

Enzymes well known in the art can be used, such as (but not limited to): horseradish peroxidase (horseradishperoxidase, HRP), alkaline phosphatase (alkaline phosohatase, AP), glucose oxidase and β-D-galactoside Enzymes, urease, etc., among which HRP is preferred.

The kit of the present invention comprises substrates well known in the art, including (but not limited to): o-phenylenediamine (O-phenylenediamine, OPD), tetramethylbenzidine (3,3',5,5'-tetramethylbenzidine, TMB ), 2,2'-azino-bis-(3-ethylbenzodihydrothiazoline-6-sulfonic acid) diammonium salt [2,2'-azino-di-(3-ethylbenz iazobine sulfonate- 6), ABTS], 3-(4-hydroxy)phenylpropionic acid [3-(4-hydroxy)phenly propionic acid, HPPA], p-nitrophenylphosphate (p-nitrophenylphosphate, p-NPP), etc., among which preferred TMB.

The substrate buffer used is well known in the art, for example (but not limited to): PH5.0 phosphate citric acid, in a preferred example of the present invention, prepare TMB use liquid with this substrate buffer, in use liquid Contains: TMB (10mg/5ml absolute ethanol) 0.5ml, substrate buffer (PH5.5) 10ml, 0.75% H ₂ O ₂ 32μl.

In a preferred embodiment of the present invention, the kit further includes a stop solution for the enzyme-catalyzed color reaction, the stop solution has a pH of 1-6, preferably 2NH ₂ SO ₄ , or 1% sodium dodecyl sulfate.

The kit of the present invention may also include a negative control and a positive control. The negative control substance is a liquid that does not contain PD-1 or PD-L1, and can be (but not limited to) recalcified human plasma (recalcified human plasma) as a raw material, that is, calcium ions are added to the plasma to make the Fibrin coagulation, the fluid obtained after removal of the clot, similar in composition to serum; or normal human serum. The positive control substance is based on a buffer solution containing a protein protective agent, and a certain amount of the substance to be tested is added to it. In a preferred example of the present invention, the positive control substance is recombinant human PD-1 or PD- L1 chimera.

The kit provided by the invention can be used for both qualitative and quantitative detection.

When detecting with the kit of the present invention, methods well known in the art can be used, and a preferred method includes steps:

(1) Coat the polyclonal antibody of human PD-1 or human PD-L1 on the ELISA plate, overnight at 37°C, wash the plate with washing buffer, add blocking solution and then wash the plate;

(2) Add normal human serum, sample serum or synovial fluid and human PD-1 or human PD-L1 fusion protein standard into different wells, incubate at 37°C for 2 hours, and wash the plate;

(3) Add reactive antibody, i.e. mouse anti-human PD-1 monoclonal antibody or mouse anti-human PD-L1 monoclonal antibody, incubate at 37°C for 2 hours, and wash the plate;

(4) Add HRP-labeled goat anti-mouse IgG (the antibody against human Ig has been absorbed), incubate at 37°C for 2 hours, and wash the plate;

(5) After adding TMB substrate for color development, add stop solution to terminate the reaction;

(6) Read the OD value on a microplate reader.

For quantitative determination, plot the concentration of the human PD-1 or human PD-L1 fusion protein standard dilution gradient and the corresponding OD value to obtain a standard curve; measure the amount of the sample from the standard curve according to the OD value of the sample.

In another preferred example, the above steps (3) and (4) become:

(3') Add biotin-labeled reactive antibody, i.e. mouse anti-human PD-1 monoclonal antibody or mouse anti-human PD-L1 monoclonal antibody, incubate at 37°C for 2 hours, and wash the plate;

(4') Add HRP-labeled streptavidin, incubate at 37°C for 2 hours, and wash the plate.

The main advantages of the present invention are:

1. Established the method for detecting soluble PD-1 and its ligands in liquid samples for the first time, and confirmed the existence of soluble PD-1 and soluble PD-L1 in the human body for the first time;

2. The method provided by the present invention can be used in clinical testing and laboratory research as an effective means for auxiliary detection of rheumatoid arthritis in vitro;

3. The detection method provided by the present invention can utilize existing equipment (such as a microplate reader), has low cost, and is suitable for popularization.

Below in conjunction with specific embodiment, further illustrate the present invention. It should be understood that these examples are only used to illustrate the present invention and are not intended to limit the scope of the present invention. For the experimental methods without specific conditions indicated in the following examples, usually follow the conventional conditions or the conditions suggested by the manufacturer. All percentages and parts are by weight unless otherwise indicated.

Unless otherwise defined, all professional and scientific terms used herein have the same meanings as commonly understood by those skilled in the art. In addition, any methods and materials similar or equivalent to those described can be applied to the method of the present invention. The preferred implementation methods and materials described herein are for demonstration purposes only.

Example 1

Detection of soluble co-signaling molecules

American College of Rheumatology 1987 diagnostic criteria for rheumatoid arthritis

1. Morning stiffness for at least 1 hour (≥ 6 weeks).

2. Three or more joints are swollen (≥6 weeks).

3. Symmetrical joint swelling (≥6 weeks).

4. Swelling of wrist, metacarpophalangeal and proximal phalangeal joints (≥6 weeks).

5. Subcutaneous nodules.

6. Hand X-ray changes (at least osteoporosis and narrow joint space).

7. Rheumatoid factor positive (titer> 1:20).

A diagnosis can be made if four or more of the above seven items are met.

Materials and methods

1 material

Lymphocyte Separation Liquid LymphoprepTM (AXIS-SHIELD);

Mouse anti-human monoclonal antibody anti-CD4-PercP, anti-CD4-FITC, anti-CD8-FITC, anti-CD14-FITC, anti-CTLA-4-APC, anti-PD-1-FITC, anti-CD80- PE, anti-PD-L1-PE-CY7 and corresponding isotype control antibody (BD Bioscience);

RNeasy Mini Kit (Qiagen); Sensiscript RT Kit (Qiagen); Human Soluble CTLA-4 ELISA Kit (Bender MedSystems); Human Soluble CD80 ELISA Kit (Bender MedSystems);

Recombinant human cytokines IFN-γ, IL-10, TNF-α (R&D Systems);

CD4+T cell, CD8+T cell, CD14+ monocyte sorting kit (Dynal Biotech);

Anti-human CD3 and CD28 monoclonal antibody (eBioscience); [3H]-TdR (Shanghai Nuclear Research Institute); human CTLA-4, PD-1, BTLA, CD28, ICOS, CD80, CD86, PD-L1, PD-L2, ICOSL, fulllength PD-1(flPD-1), PD-1 delete exon 3(PD-1Δex3), PD-1 delete exon 2(PD-1Δex2), PD-1 delete exon 2,3(PD-1Δex2,3 ) fluorescent quantitative PCR primers (Invitrogen); ELISA detection antibodies and related reagents; SYBR Green (ABI), etc.

2 experimental equipment

Flow cytometer FACS Aria ^TM (Becton Dickinson); low temperature high-speed centrifuge (BECKMAN); _CO2 incubator (Heraus, Germany); real-time quantitative PCR instrument (ABI); spectrophotometer (BECKMAN); instrument; gel scanning imaging system (UVP-GDS8000); microplate reader (Spectra Max250); multi-head collector (TOMTEC Harvester96); β liquid scintillation instrument (Perkin Elmer); ultraviolet nucleic acid analyzer (BECKMAN); (Eppendorf); inverted microscope (Nikon); clean bench, etc.

3 Cases and samples

The present invention includes 95 cases of patients with rheumatoid arthritis (RA), who are outpatients or inpatients in the Department of Orthopedics and Internal Medicine of Shanghai Guanghua Hospital of Integrated Traditional Chinese and Western Medicine. All cases met the ACR criteria established by the American College of Rheumatology in 1987.

Patient's age: 54±15 years old; gender: male 24/female 71; disease duration: 15±12 years. Synovial fluid and whole blood samples were obtained from 37 patients, and synovial fluid and serum samples were obtained from the other 58 patients for ELISA detection. Synovial fluid and whole blood samples from 30 OA patients (age: 64±17 years old; sex: male 12/female 18; course of disease: 9±4 years) were used as controls (Shanghai Guanghua Hospital of Integrated Traditional Chinese and Western Medicine, Shanghai No. People's Hospital). All specimens were collected from patients who did not take hormones and immunosuppressants within 2 months. The peripheral blood samples of 50 healthy volunteers were used to separate mononuclear cells and serum as normal control group.

Synovial fluid specimens were aseptically obtained through synovial cavity puncture or joint surgery, anticoagulated with heparin, centrifuged at 350g for 3min, and the supernatant was collected and stored in a -80°C refrigerator. Mononuclear cells from peripheral blood or synovial fluid were separated by LymphoprepTM.

4 Preparation of mononuclear cells from peripheral blood and joint synovial fluid

Mononuclear cells (peripheral blood mononuclear cell, PBMC; synovial fluid mononuclear cell, SFMC) were separated from anticoagulated whole blood and joint synovial fluid by LymphoprepTM density gradient centrifugation, washed twice with 2% FCS/PBS, and set aside.

5 Isolation and identification of T cells and monocyte subsets

5.1 Use Dynabeads positive selection to isolate CD4+T cells, CD8+T cells or CD14+ monocytes.

According to the product manual, the process is briefly described as follows:

(1) Resuspend and mix the Dynabeads solution;

(2) Take 72 μl of Dynabeads that have been inoculated with CD4 or CD8 monoclonal antibody (for sorting CD14+ monocytes, add 25 μl of Dynabeads coated with CD14 monoclonal antibody) and wash once with 1 ml of 0.1% BSA/PBS;

(3) Add 1ml of mononuclear cell suspension (1×10 ⁷ /ml), and incubate on a rotary shaker at 4°C for 20min;

(4) Place the Eppendorf tube into the MPC-S magnetic field (Dynal Biotech) and incubate for 3 min;

(5) Discard the supernatant, wash the magnetic bead-bound cells with 0.1% BSA/PBS for 3 times, and the target cells are bound to the magnetic beads.

5.2 Use Dynabeads (Dynal Biotech) to isolate CD4+ T cells by negative selection

Proceed as follows:

(1) 1×10 ⁷ mononuclear cells were resuspended in 100 μl BufferI (0.1% BSA/PBS containing 2mM EDTA);

(2) Add 20 μl FCS, 20 μl anti-CD14, anti-CD16, anti-CD56, anti-CDw123, anti-CD36, anti-CD8, anti-CD19, anti-Glycophorin antibody mixture, and incubate at 4°C for 20 minutes;

(3) Add 2ml Buffer I, mix well, centrifuge at 300g at 4°C for 8min, discard the supernatant;

(4) Cells were resuspended in 900 μl Buffer I, plus 100 μl pre-washed Dynabeads, and incubated at room temperature for 15 minutes;

(5) Add 1ml Buffer I, mix well, divide into two equal parts, and transfer to new Eppendorf tubes respectively;

(6) Place the Eppendorf tube into the MPC-S magnetic field (Dynal Biotech) and incubate for 3 min;

(7) Aspirate the supernatant and move to a new sterile Eppendorf tube;

(8) Repeat steps (5)-(8) once, and collect the supernatant, which contains the isolated CD4+T cells.

5.3 Identification of isolated cells

Take part of negatively selected CD4+ T cells and positively selected CD4+ T cells, CD8+ T cells or CD14+ monocytes, remove Dynabeads, count, and take 2×105 cells to mix with FITC-labeled CD4 monoclonal antibody and CD8 mononuclear cells respectively. Anti- or CD14 monoclonal antibody (BD Bioscience) was incubated on ice for 20 min, washed twice with 2% FCS/PBS, fixed with 300 μl 1% paraformaldehyde, and detected by FACS. The results showed that the purity of each isolated cell subpopulation was greater than 97%. CD4+T cells, CD8+T cells or CD14+ monocytes isolated by positive selection were lysed with 350 μl RLT respectively, and used for downstream RNA extraction, cDNA synthesis and real-time quantitative PCR analysis. The CD4+ T cells isolated by negative selection were used for cell culture.

6 Induction of co-signaling molecules by cytokines

PBMC from healthy people were inoculated in 24-well plates at 1×106/ml, cultured in RPMI 1640 medium containing 100 U/ml penicillin, 100 μg/ml streptomycin, and 10% FCS, and different concentrations of recombinant human IFN-γ, IL-10 or TNF-α (R&D Systems), set up medium control group, culture at 37°C, 5% CO2 for 12 hours, collect cells, sort CD4+ T cells or CD14+ monocytes, extract RNA, Fluorescent quantitative PCR was used to detect the mRNA levels of various co-signaling molecules, and the mononuclear cells of each cytokine stimulation group and control group with a concentration of 25ng/ml were selected for FACS analysis.

Detection of expression abundance of PD-1 mRNA spliced bodies of different lengths after activation of 7T cells

The isolated RA, OA patient SFMC, PBMC and normal PBMC were inoculated in 96-well plates at a density of 2×10 ⁵ /well, in the presence of 1 μg/ml anti-CD3 monoclonal antibody (clone OKT-3, eBioscience) and 0.05 μg/ml ml anti-CD28 monoclonal antibody (clone CD28.2, eBioscience), 100 U/ml penicillin, 100 μg/ml streptomycin, and 10% FCS were cultured in 1640 culture medium for 48 hours, and no antibody control group was set. Collect cells, extract RNA, and use fluorescent quantitative PCR to detect full-length PD-1 (flPD-1), PD-1 delete exon 3 (PD-1Δex3), PD-1 delete exon2 (PD-1Δex2), PD-1 Delete exon 2, 3 (PD-1Δex2, 3) and other PD-1 molecule mRNA expression abundance of different length spliced bodies.

8 RNA extraction

Use Qiagen's Rneasy Mini Kit to extract PBMC and SFMC total RNA, the steps are as follows:

Cells were lysed by adding 350 μl lysis solution (RLT) repeatedly by pipetting

↓

Add an equal volume of 350μl 70% ethanol, mix well

↓

Aspirate 700μl sample to the Rneasy mini column, put it on a 2ml collection tube ≥8000g (≥10000rpm), centrifuge for 15s, pour off the liquid in the collection tube (if the sample exceeds 700μl, repeat it again)

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Add 350μl RW1≥8000g (≥10000rpm), centrifuge for 15s, pour off the liquid in the collection tube

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Add 80μl DNase, room temperature for 15min

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Add 350μl RW1≥8000g (≥10000rpm), centrifuge for 15s, discard the collection tube

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Put the Rneasy mini column on a new 2ml collection tube, add 500μl RPE (working concentration) ≥ 8000g (≥ 10000rpm), centrifuge for 15s, pour off the liquid in the collection tube

↓

Add 500μl RPE (working concentration) ≥ 8000g (≥ 10000rpm), centrifuge for 2min, discard the collection tube

↓

Put the RNeasy mini column on a new 1.5ml collection tube

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Add 30-50μl RNase-free water ≥ 8000g (≥ 10000rpm), centrifuge for 1min, collect the liquid in the tube, measure the OD value

↓

Detection of RNA concentration and purity by UV spectrophotometer

9 cDNA synthesis

Use Qiagen’s Sensiscript RT Kit and follow the instructions, as follows:

React component Volume (μl) 10× reverse transcription buffer 2.0 dNTP (5.0mM) 2.0 Random primer (0.15μg/ml) 1.0 RNase inhibitor 40U/μl 0.25

reverse transcriptase 1.0 RNase-free water variable RNA template (<50ng) variable total capacity 20

Incubate at 37°C for 60 minutes, incubate at 93°C for 5 minutes, centrifuge, and store cDNA at -200°C.

10 Fluorescent quantitative PCR detection

Primers for CTLA-4, PD-1, BTLA, CD28, ICOS, CD80, CD86, PD-L1, PD-L2 and ICOSL genes were designed using Applied Biosystems Prime Express 2.0 software, flPD-1, PD-1Δex3, PD- 1Δex2, PD-1Δex2, 3 primer design sequences are as follows (Seq.ID.No.1-30):

Specific primer sequence for gene fluorescence quantitative PCR

Gene Seq.ID.No. Sequence (5'-3') GAPDH 12 Upstream primer: GTGAAGGTCGGAGTCAACG Downstream primer: TGAGGTCAATGAAGGGGTC CTLA-4 34 Upstream primer: TGCTTGATTGCGTGGAATTG Downstream primer: CCACCAGCTGTGGCTTCCT PD-1 56 Upstream primer: CAGTGGCATCCCGAAACG Downstream primer: CACAGCCAGGAGCTCTTCTGA CD28 78 Upstream primer: CCTCCTCCCTTACCTAGACAATGAGA Downstream primer: GGCTTAGAAGGTCCGGGAAA ICOS 910 Upstream primer: GCACGACCCTAACGGTGAATAC Downstream primer: TGGGTGCCAGAGTTCCATATTA CD80 1112 Upstream primer: GTGTTATCCACGTGACCAAGGA Downstream primer: TTGCCAGTAGATGCGAGTTTGT CD86 1314 Upstream primer: TGCCATGCCAATTTGCAA Downstream primer: GCCTAAGTATACCTCATTCAGAACCAA PD-L1 1516 Upstream primer: CTTCAAGCAGGGATTCTCAACCT Downstream primer: TAAGTCCCACATTGCCTGCAT PD-L2 1718 Upstream primer: GAATTGCAGCTTCACCAGATAGC Downstream primer: AAGTTGCATTCCAGGGTCACA ICOSL 1920 Upstream primer: CTGGGCCTTTAGGTGAATGTG Downstream primer: AATGCCCGGTGATGACAACT

flPD-1 2122 Upstream primer: CTCAGGGTGACAGAGAGAAG Downstream primer: GACACCAACCACCAGGGTTT PD-1Δex3 2324 Upstream primer: AGG GT GACAG GGACAATAGG Downstream primer: CCATAGTCCACAGAGAACAC PD-1Δex2 2526 Upstream primer: GGTTCTTAGAGAGAAGGGCA Downstream primer: GACACCAACCACCAGGGTTT PD-1Δex2.3 2728 Upstream primer: TGGTTCTTAGGGACAATAGG Downstream primer: TCTTCTCTCGCCACTGGAAA BTLA 2930 Upstream primer: AGCCACTCAACAACTCTTTATGTGA Downstream primer: TTGCCACTTCGTCCTTGGA

Gene expression was performed by fluorescent quantitative PCR method, GAPDH gene was used as an internal reference, and H ₂ O was used as a negative control. reaction system:

1:5 diluted cDNA template 4.85μl Fluorescent quantitative PCR primers (upstream primers, downstream primers 5 μM each) 0.15μl 2×SYBR Green PCR buffer system (Applied Biosystems) 5μl total capacity 10μl

Add a 384-well PCR reaction plate, and the reaction is carried out in the ABI 7900 Sequence Detection System. The reaction process is as follows:

^* Determination of flPD-1, PD-1Δex3, PD-1Δex2, PD-1Δex2, 3 mRNAs with different lengths cut body circulation 50 times.

Relative expression level of the target gene = 2 ^{- (target gene CT-GAPDHCT)} (CT: cycle threshold).

11 Flow Cytometry (FACS)

Resuspend 5×10 ⁵ -1×10 ⁶ cells in 50 μl containing 2% FCS/PBS, add appropriate amount of fluorescently labeled antibody of the target molecule or isotype control, mix well, incubate at room temperature in the dark for 30 minutes, add 2% FCS/PBS 2ml , after mixing, centrifuge at 300g for 8min, discard the supernatant, and repeat washing once. Cells were resuspended in 300 μl 2% FCS/PBS, mixed evenly and detected by flow cytometry. Because CTLA-4 molecules are easy to be endocytized, transmembrane staining is required. Cells were fixed in 4% paraformaldehyde at room temperature for 20 minutes, centrifuged at 300g for 8 minutes, supernatant was discarded, and cells were resuspended in 500 μl 2% FCS/0.5% saponin/PBS , incubate at room temperature for 10 minutes, centrifuge, remove the supernatant, add 50 μl 2% FCS/0.5% saponin/PBS and 10 μl anti-human CTLA-4 monoclonal antibody, incubate at room temperature for 1 hour, wash once, and resuspend the cells with 300 μl 1% paraformaldehyde. Use FACS-Aria ^TM to analyze on the machine.

12 Detection of soluble co-signaling molecules in serum and synovial fluid by enzyme-linked immunosorbent assay (ELISA)

The main reagents for soluble CTLA-4, PD-1 and PD-L1 (sCTLA-4, sPD-1 and sPD-L1) ELISA are shown in Table 1.

Other reagents include:

PBS: NaCl 8.00g, KCl 0.20g, Na ₂ HPO ₄ ×12H ₂ O, KH ₂ PO ₄ 0.20g;

Wash Buffer: 0.05% Tween20/PBS;

Blocking Buffer: 0.5%BSA/0.05%Tween/PBS;

TMB substrate;

Stop solution: 2N H ₂ SO ₄ .

Table 1 Main reagents for soluble CTLA-4, PD-1 and PD-L1 ELISA detection

Soluble protein (lower limit of detection) Coating antibody standard protein Detection antibody Color rendering system Soluble CTLA-4 (0.16ng/ml) Anti-human CTLA-4 antibody (2μg/m1) (BenderMedSystemskit) Soluble CTLA-4 standard protein Biotin-labeled anti-human CTLA-4 antibody, diluted 1:1000 Horseradish peroxidase-labeled streptavidin, diluted 1:5000 Soluble PD-1 (0.3ng/ml) Goat anti-human PD-1 (1.5μg/ml) (R&D Systems) Recombinant human PD-1/Fc fusion protein (R&D Systems) Mouse anti-human PD-1 monoclonal antibody (J116, 4μg/ml) (eBioscience) Horseradish peroxidase-labeled goat anti-mouse immunoglobulin (Southern Biotechnology) (1:500 dilution)

Soluble PD-L1 (0.3ng/ml) Goat anti-human PD-L1 (1.5μg/ml) (R&D Systems) Recombinant human PD-L1/Fc fusion protein (R&DSystems) Mouse anti-human PD-L1 monoclonal antibody (MIH1, 1μg/ml) (eBioscience) Antibody same as above (1:2000 dilution)

Specific steps:

(1) Coating plate: add coating antibody 100 μl/well, 37°C, overnight;

(2) Plate washing: add 300 μl washing solution/well, wash the plate, x 2 times;

(3) Blocking: add 250 μl blocking solution/well, incubate at room temperature for 2 h;

(4) Plate washing: add 300 μl washing solution/well, wash the plate, x 3 times;

(5) Add samples (1:2 dilution) and 100 μl/well of serially diluted standards, set up duplicate wells, and incubate at 37°C for 2 hours;

(6) Plate washing: add 300 μl washing solution/well, wash the plate, x 3 times;

(7) Add 100 μl/well of reaction antibody, incubate at 37°C for 2 hours;

(8) Plate washing: add 300 μl washing solution/well, wash the plate, x 3 times;

(9) Add streptomycin-HRP or antibody-HRP, 370C, avoid direct light, incubate for 1h;

(10) Plate washing: add 300 μl washing solution/well, wash the plate, x 6 times;

(11) Add 100 μl/well of TMB substrate, and develop color in the dark;

(12) Add 100 μl/well of stop solution to stop the reaction;

(13) Reading: Read the OD value on a microplate reader (Bio-Rad Laboratories) (measurement wavelength 450nm, reference wavelength 540nm or 620nm).

Human soluble CD80 (sCD80) was detected by human sC80Instant ELISA Kit (Bender Medsystems), and the steps are briefly described as follows:

(1) Add 150 μl double-distilled water to the standard well (the gradient diluted standard has been coated in the well);

(2) Add 150 μl double distilled water to the blank well;

(3) Add 100 μl double-distilled water to the sample well (coated with ami-human sCD80mAb, HRP-Conjugate-anti-sCD80mAb and sample dilution components, all are dry-freeze powder);

(4) Add 50 μl of synovial fluid or serum to the sample wells, duplicate the wells, seal the membrane, and incubate at room temperature for 3 hours;

(5) Add 400 μl washing solution/well, wash the plate, ×3 times;

(6) Add 100 μl/well of TMB substrate, and develop color for 10 minutes in the dark;

(7) Add 100 μl/well of stop solution to stop the reaction;

(8) Reading: read the OD value on a microplate reader (Bio-Rad Laboratories) (measurement wavelength 450nm, reference wavelength 620nm), the detection limit of the kit is 0.32ng/ml.

13 Functional test of soluble PD-1 and PD-L1

Peripheral blood CD4+ T cells (4×104/well) and 4000Gry irradiated PBMCs (2×10 ⁵ /well, as APC) from healthy people were inoculated into 96-well plates pre-coated with anti-CD3 monoclonal antibody (1μg/ml) , cultured in RPMI 1640 medium containing 10% FCS, with or without gradient concentrations of human PD-1-Ig, PD-L1-Ig, control Ig (0 μg/ml, 0.2 μg/ml, 1 μg/ml, 5 μg/ml, 25 μg/ml), cultured in 37°C, 5% CO2 incubator for 4 days, 1 μCi [3H] was added 18 hours before collecting the cells, the cells were collected, and the incorporation of [3H] was detected by a β liquid scintillation instrument. Analyze the effects of PD-1-Ig and PD-L1-Ig on the activation and proliferation of CD4+ T cells.

14 Statistical Analysis

Mann-Whitney U test was used for the gene expression level of each experimental group and control group, and Student’s st test was used for the difference between the two groups, and single-factor ANOVA analysis was used before U test or t test.

p<0.05 was considered statistically significant.

result

1 Synergistic inhibitory molecules are highly expressed in synovial fluid infiltrating T cells and macrophages in patients with rheumatoid arthritis

Mononuclear cells were isolated from the peripheral blood and synovial fluid of 23 RA patients, 10 OA patients, and 22 healthy people, and analyzed by fluorescent quantitative PCR method, including CTLA-4, PD-1, BTLA, CD28, ICOS, CD80 , CD86, PD-L1, PD-L2 and ICOSL and other molecules including CD28 family synergistic molecules mRNA expression level. see picture 1.

The results showed that, compared with healthy people and OA controls, synergistic inhibitory molecules including CTLA-4, PD-1, PD-L1, PD-L2, and the ligand CD80 of CTLA-4 were significantly increased in the synovial fluid of RA patients. The selective expression was increased in infiltrating mononuclear cells, among which the levels of CTLA-4 and PD-1 mRNA were increased by 11.9 times and 5.1 times compared with healthy controls, respectively; they were increased by 3.3 times and 1.5 times compared with OA SFMC. Their corresponding ligands CD80 and PD-L1 mRNA were also significantly increased compared with the control group (Figure 1A), while the expressions of CD28, ICOS, CD86 and ICOSL had no obvious trend of change (Figure 1B).

CTLA-4 and PD-1 signaling pathways play an important role in regulating T cell immune response and maintaining peripheral immune tolerance, so the inventors further studied the expression of these synergistic signaling molecules in immune cell subsets. The inventors sorted out CD4+, CD8+ T cell subsets and CD14+ macrophages in the peripheral blood and synovial fluid of 9 patients with RA. The expressions of CD4+ and CD8+ T cells infiltrated in synovial fluid were both increased, and CTLA-4 mRNA was highly expressed in CD4+ T cells infiltrated in synovial fluid, while the expression of CD80 and PD-L1 mRNA in CD14+ macrophages infiltrated in synovial fluid was significantly increased (Fig. 1C).

The inventors further used FACS research to analyze the expression of co-inhibitory molecules in the synovial fluid infiltrating immune cells of RA patients (because CTLA-4 molecules were quickly endocytosed after being expressed on the cell membrane surface, transmembrane staining was also performed at the same time, marked as iCTLA-4, CTLA-4 expressed on the membrane surface is identified as sCTLA-4), consistent with the results of fluorescent quantitative PCR.

The results showed that the expressions of CTLA-4 and PD-1 molecules were significantly increased in synovial fluid infiltrating CD4+ T cells, and the expressions of the corresponding ligands CD80 and PD-L1 were also increased on the surface of synovial fluid infiltrating CD14+ macrophages (Fig. 2A , B).

The inventor also used fluorescent quantitative PCR to study the expression of Fas, another negative regulatory molecule, and found that the expression of Fas molecule mRNA in the peripheral blood and synovial fluid infiltrated mononuclear cells of RA patients increased (Fig. 3A), and Fas in The expression level of synovial fluid infiltrating CD4+ T cell subsets in RA patients was significantly higher than that of CD8+ T cells and peripheral blood T cells (Fig. 3B).

The inventors used fluorescent quantitative PCR and FACS analysis, and the results showed that negative regulatory molecules such as cooperative inhibitory molecules CTLA-4, PD-1, and PD-L1, as well as CD80, were significantly expressed in the synovial fluid infiltrating T cells and macrophages of RA patients. Increase. This result is unexpected, indicating that after the body suffers from RA, the immune regulation mechanism in the body still plays a regulatory role on the excessive immune response.

2 Cytokines IFN-γ, TNF-α can induce the expression of CD80 and PD-L1 in monocytes

The inventors used the cytokines IFN-γ, TNF-α, and IL-10, which were significantly increased in the synovial fluid of RA patients, to stimulate and culture healthy PBMCs for 12 hours, and analyzed molecules such as CTLA-4, PD-1, CD80, and PD-L1 expression. See Figure 4.

As shown in Figure 4A, the results showed that IFN-γ and TNF-α could selectively induce CD80 and PD-L1 mRNA expression in CD14+ monocytes in a dose-dependent manner, while CTLA-4 and PD-1 expression were not significantly Influence. IL-10 had no effect on the mRNA expression of co-inhibitory molecule ligands and receptors.

The inventors further selected 25ng/ml concentrations of IFN-γ, TNF-α and IL-10 to stimulate PBMC cells from healthy people for 12 hours for FACS analysis, and further confirmed that IFN-γ and TNF-α can induce CD80 and PD-L1 in CD14+ Expression on the surface of monocytes (Fig. 4B). The above cytokines also failed to induce the expression of CTLA-4 and PD-1 in the activated state of the cells.

3 Soluble PD-1 was significantly increased in serum and synovial fluid of RA patients and was associated with rheumatoid factor

The CTLA-4 and PD-1 signaling pathways play a negative regulatory role in the immune response, but the expression of CTLA-4 and PD-1 in the synovial fluid infiltrating T cells of RA patients is increased, and the corresponding ligands CD80 and PD-L1 are in The surface of CD14+ macrophages was also increased, while T cells were in a paradoxical state of constant activation. The inventors believe that there are two main possibilities: first, there are autoreactive antibodies against these molecules, which block the inhibitory pathway; second, there are soluble co-signaling molecules such as soluble CTLA-4 (soluble CTLA-4, sCTLA-4), Soluble PD-1 (soluble PD-1, sPD-1), soluble CD80 (soluble CD80, sCD80) or PD-L1 (solublePD-L1, sPD-L1) blocked the inhibitory pathway.

The inventors investigated the possibility of the existence of soluble co-signaling molecules. The inventor measured the serum of 95 RA patients, the synovial fluid of 37 RA patients, and the serum of 14 patients with osteoarthritis (OA) in the control group, the synovial fluid of 30 OA patients, and sCTLA-4, sCTLA-4, Expression levels of sPD-1, sCD80 and sPD-L1. See Figure 5 and Table 2.

The results in Figure 5A and Table 2 show that, compared with the control group, sPD-1 and sPD-L1 were significantly increased in the serum and synovial fluid of RA patients, and sCD80 was elevated to a certain extent in the synovial fluid and serum of RA patients , no increase in sCTLA-4 was detected. Elevated sPD-1 and sPD-L1 in serum were significantly correlated with rheumatoid factor (RF), but not with C-reactive protein (C Reactive Protein, CRP) and erythrocyte sedimentation rate (ESR). Significant correlation. sCD80 was also correlated with RF (Fig. 5B-D).

Table 2 Concentration of soluble co-signaling molecules in serum and synovial fluid (ng/ml, mean ± standard error)

HC sera RA sera RA SF OA sera OA SF sPD-1 0.30±0.04 3.40±0.45 ^* 2.65±0.70 ^* 0.08±0.01 0.12±0.03 sCTLA-4 0.08±0.01 0.10±0.01 0.09±0.01 0.03±0.004 0.04±0.003 sPD-L1 0.58±0.07 3.52±0.61 ^*# 1.82±0.47 ^* 0.47±0.03 0.38±0.02 sCD80 0.18±0.01 0.28±0.02 ^* 0.52±0.07 ^* 0.14±0.01 0.13±0.01

*p<0.05 compared with HC and OA control group, #p<0.05 compared with RA SF, +p<0.05 compared with RA sera

Alternative splicing of PD-1 mRNA in 4RA patients: PD-1Δex3 mRNA splicing body is selectively increased

The full-length mRNA of PD-1 molecule contains exon1 (encoding signal peptide), exon2 (encoding extracellular Ig-like region), exon3 (encoding transmembrane region), exon4 and 5 (encoding intracellular region) (Genebank accession number: NM_005018) . In addition to the full-length mRNA (flPD-1), there are several different splice forms, including PD-1Δex2, PD-1Δex3, PD-1Δex2,3, PD-1Δex2,3,4.

PD-1Δex2 removes exon2 (extramembrane Ig-like region); PD-1Δex3 cuts out exon3 (transmembrane region), but the extracellular Ig-like region remains intact; PD-1Δex2, 3 removes the encoding cell membrane Sequences of the outer Ig-like region and the transmembrane region. It can therefore be inferred that PD-1Δex3 encodes a soluble PD-1 molecule. As shown in Figure 6A, among the three PD-1 mRNA splicing forms whose reading frame was not affected, PD-1Δex3 was selectively increased in the peripheral blood and synovial fluid mononuclear cells of RA patients, while the other two mRNA splicing Excitosomes could not be detected due to their low expression abundance.

The inventors further tested whether the increased expression of PD-1Δex3 mRNA in peripheral blood and synovial fluid mononuclear cells of RA patients was related to the activation state of T cells. To this end, the inventors used anti-CD3 monoclonal antibody and anti-CD28 monoclonal antibody to stimulate PBMCs and SFMCs of RA patients, PBMCs and SFMCs of OA patients and PBMCs of healthy people for 48 hours, and collected cells to measure the level of PD-1Δex3 mRNA.

The results showed that the expression level of PD-1Δex3mRNA did not increase after T cells were stimulated by monoclonal antibodies, suggesting that the increase in the expression level of PD-1Δex3mRNA was specific to RA patients, and there was an alternative splicing phenomenon of PD-1Δex3mRNA in RA patients (Figure 6B) .

5 Soluble PD-1 blocks the inhibitory signal mediated by the PD-1 pathway and promotes the activation and proliferation of T cells

The inventors used human PD-1-Ig and PD-L1-Ig to carry out functional experiments (PD-1-Ig and PD-L1-Ig contain the complete extracellular region of PD-1 and PD-L1 respectively, so they can be well Biological functions of simulated soluble PD-1 and soluble PD-L1:

Amino acid sequence of the extracellular domain of PD-1 molecule contained in human PD-1-Ig (Extracellular domain: Met1-Gln 167) (Seq.ID.No.31)

1 mqipqapwpv vwavlqlgwr pgwfldspdr pwnpptffpa llvvtegdna tftcsfsnts

61 esfvlnwyrm spsnqtdkla afpedrsqpg qdcrfrvtql pngrdfhmsv vrarrndsgt

121 ylcgaislap kaqikeslra elrvterrae vptahpsspsp rpagqfqtlv vgvvggllgs

181 lvllvwvlav icsraargti garrtgqplk edpsavpvfs vdygeldfqw rektpeppvp

241 cvpeqteyat ivfpsgmgts sparrgsadg prsaqplrpe dghcswpl

Amino acid sequence of the extracellular domain of PD-L1 molecule contained in human PD-L1-Ig (Extracellular domain: Met1-Thr239) (Seq.ID.No.32)

1 mrifagiift acchllraft itapkdlyvv eygsnvtmec rfpvereldl lalvvyweke

61 deqviqfvag eedlkpqhsn frgraslpkd qllkgnaalq itdvklqdag vycciisygg

121 adykritlkv napyrkinqr isvdpatseh elicqaegyp eaeviwtnsd hqpvsgkrsv

181 ttsrtegmll nvtsslrvna tandvfyctf wrsqpgqnht aeliipelpa thppqnrthw

241 vllgsilllfl ivvstvllfl rkqvrmldve kcgvedtssk nrndtqfeet

The results showed that the proliferation of CD4+ T cells in the PD-1-Ig and PD-L1-Ig treatment groups was significantly higher than that in the control group (Figure 7), suggesting that soluble PD-1 and soluble PD-L1 can effectively block PD-1 Inhibitory signals mediated by signaling pathways promote T cell activation and proliferation.

Example 2-4

Preparation of ELISA kits

Example 2 Example 3 Example 4

ELISA plate (12×8 wells) + + + Goat anti-human PD-1 polyclonal antibody (1.5μg/ml) (R&D Systems) + + + HRP-mouse anti-human PD-1 monoclonal antibody (4μg/ml) (eBioscience) + Mouse anti-human PD-1 monoclonal antibody (J116, 4μg/ml) (eBioscience) + Biotin-mouse anti-human PD-1 monoclonal antibody (1μg/ml) (eBioscience) + HRP-goat anti-mouse IgG (Southern Biotechnology) (1:500 dilution) + HRP-streptavidin (appropriate dilution) (Sigma company) +

Note: 1. + indicates that the item is included in the kit

2. The mouse anti-human PD-1 monoclonal antibody was purchased from eBioscience, and then labeled with horseradish peroxidase or biotin by methods well known in the art

Also included in the kit:

Phosphate buffer solution (PBS): NaCl 8.00g, KCl 0.20g, Na ₂ HPO ₄ ×12H ₂ O, KH ₂ PO ₄ , 0.20g;

Wash buffer: the above PBS containing 0.05% Tween 20;

Blocking solution: the above PBS containing 0.5% fetal bovine serum (BSA) and 0.05% Tween;

TMB substrate;

Enzyme-catalyzed reaction termination solution: 2N H ₂ SO ₄ ;

Negative control substance: normal human serum;

Positive control substance: recombinant human PD-1/Fc chimera (R&D Systems).

Example 5-7

ELISA kit

Example 5 Example 6 Example 7 ELISA plate (12×8 wells) + + + Goat anti-human PD-L1 polyclonal antibody (1.5μg/ml) (R&D Systems) + + + HRP-mouse anti-human PD-L1 monoclonal antibody (4μg/ml) (eBioscience) + Mouse anti-human PD-L1 monoclonal antibody (J116, 4μg/ml) (eBioscience) + Biotin-mouse anti-human PD-L1 monoclonal antibody (1μg/ml) (eBioscience) + HRP-goat anti-mouse IgG (Southern Biotechnology) (1:500 dilution) + HRP-streptavidin (appropriate dilution) (Sigma company) +

Note: 1. + indicates that the item is included in the kit

2. The mouse anti-human PD-1 monoclonal antibody was purchased from eBioscience, and then labeled with horseradish peroxidase or biotin by methods well known in the art

Also included in the kit:

Phosphate buffer solution (PBS): NaCl 8.00g, KCl 0.20g, Na ₂ HPO ₄ ×12H ₂ O, KH ₂ PO ₄ , 0.20g;

Wash buffer: the above PBS containing 0.05% Tween 20;

Blocking solution: the above PBS containing 0.5% fetal bovine serum (BSA) and 0.05% Tween;

TMB substrate;

Enzyme-catalyzed reaction termination solution: 2N H ₂ SO ₄ ;

Negative control substance: normal human serum;

Positive control: recombinant human PD-L1/Fc chimera (R&D Systems).

Example 8

Use of the kit

specimen:

Without the knowledge of the testers (single-blind), serum samples (RA-sera) from 50 patients with rheumatoid arthritis (RA), synovial fluid samples (RA-SF) from 32 patients with RA, and bone and joint samples from 22 patients were used. Synovial fluid and serum samples (OA-SF and OA-sera) from patients with OA (OA) and serum from 50 healthy volunteers (HC-sera).

Detect with the kit that embodiment 2 makes, concrete steps are as follows:

(1) Packing plate: Add 100 μl of goat anti-human PD-1 polyclonal antibody to each well, and overnight at 37°C;

(2) Plate washing: add 300 μl washing buffer to each well, and wash the plate, ×2 times;

(3) Blocking: Add 250 μl of blocking solution to each well and incubate at room temperature for 2 hours;

(4) Plate washing: add 300 μl washing buffer to each well, and wash the plate, ×3 times;

(5) Add the sample (1:2 dilution) and the gradiently diluted positive control substance into different wells in an amount of 100 μl per well, set up duplicate wells, and incubate at 37°C for 2 hours;

(6) Plate washing: add 300 μl washing buffer to each well, and wash the plate, ×3 times;

(7) Add 100 μl of mouse anti-human PD-1 monoclonal antibody to each well, and incubate at 37°C for 2 hours;

(8) Plate washing: add 300 μl washing buffer to each well, and wash the plate, ×3 times;

(9) Add HRP-goat anti-mouse IgG to each well, and incubate for 1 hour at 37°C, avoiding direct light;

(10) Plate washing: add 300 μl washing buffer to each well, wash the plate, x 6 times;

(11) Add 100 μl of TMB substrate to each well, and develop color in the dark;

(12) Add 100 μl of stop solution to each well to terminate the reaction;

(13) Reading: Read the OD value on a microplate reader (Bio-Rad Laboratories) (measurement wavelength 450nm, reference wavelength 540nm or 620nm).

The results are shown in Table 3.

Table 3 Concentration of soluble PD-1 in serum and synovial fluid (ng/ml, mean ± standard error)

HC sera RA sera RA SF OA sera OA SF sPD-1 0.28±0.03 3.65±0.41 ^* 2.90±0.63 ^* 0.09±0.01 0.15±0.03

*p<0.05 compared with HC and OA control group,

The results show that the kit prepared in Example 2 can be used to detect soluble PD-1 in liquid samples.

Example 9-10

Use of the kit

Example 8 was repeated with the kits prepared in Examples 3 and 4, and similar results were obtained, indicating that the prepared kits could be used to detect soluble PD-1 in liquid samples.

Example 11

Use of the kit

specimen:

Without the knowledge of the testers (single-blind), serum samples (RA-sera) from 50 patients with rheumatoid arthritis (RA), synovial fluid samples (RA-SF) from 32 patients with RA, and bone and joint samples from 22 patients were used. Synovial fluid and serum samples (OA-SF and OA-sera) from patients with OA (OA) and serum from 50 healthy volunteers (HC-sera).

Detect with the kit that embodiment 5 makes, concrete steps are as follows:

(1) Packing plate: Add 100 μl of goat anti-human PD-L1 polyclonal antibody to each well, overnight at 37°C;

(2) Plate washing: add 300 μl washing buffer to each well, and wash the plate, ×2 times;

(3) Blocking: Add 250 μl of blocking solution to each well and incubate at room temperature for 2 hours;

(4) Plate washing: add 300 μl washing buffer to each well, and wash the plate, ×3 times;

(5) Add the sample (1:2 dilution) and the gradiently diluted positive control substance into different wells in an amount of 100 μl per well, set up duplicate wells, and incubate at 37°C for 2 hours;

(6) Plate washing: add 300 μl washing buffer to each well, and wash the plate, ×3 times;

(7) Add 100 μl of mouse anti-human PD-L1 monoclonal antibody to each well, and incubate at 37°C for 2 hours;

(8) Plate washing: add 300 μl washing buffer to each well, and wash the plate, ×3 times;

(9) Add HRP-goat anti-mouse IgG to each well, and incubate for 1 hour at 37°C, avoiding direct light;

(10) Plate washing: add 300 μl washing buffer to each well, wash the plate, x 6 times;

(11) Add 100 μl of TMB substrate to each well, and develop color in the dark;

(12) Add 100 μl of stop solution to each well to terminate the reaction;

(13) Reading: Read the OD value on a microplate reader (Bio-Rad Laboratories) (measurement wavelength 450nm, reference wavelength 540nm or 620nm).

The results are shown in Table 4.

Table 4 Concentration of soluble PD-L1 in serum and synovial fluid (ng/ml, mean ± standard error)

HC sera RA sera RA SF OA sera OA SF sPD-L1 0.60±0.08 3.35±0.54 ^* 2.76±0.49 ^* 0.57±0.06 0.46±0.02

*p<0.05 compared with HC and OA control group.

The results show that the kit prepared in Example 5 can be used to detect soluble PD-L1 in liquid samples.

Example 12-13

Use of the kit

Example 11 was repeated with the kits prepared in Examples 6 and 7, and similar results were obtained, indicating that the prepared kits could be used to detect soluble PD-L1 in liquid samples.

The above description is only a preferred embodiment of the present invention, and is not intended to limit the scope of the essential technical content of the present invention. The essential technical content of the present invention is broadly defined in the scope of the claims of the application, and any technical entity completed by others or method, if it is exactly the same as defined in the scope of the claim, or if it is an equivalent change, it will be deemed to be included in the scope of the claim.

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<400>4

ccaccagctg tggcttcct 19

<210>5

<211>18

<212>DNA

<213> Artificial sequence

<220>

<221>misc_feature

<223> Primer

<400>5

cagtggeatc ccgaaacg 18

<210>6

<211>21

<212>DNA

<213> Artificial sequence

<220>

<221>misc_feature

<223> primer

<400>6

cacagccagg agctcttctg a 21

<210>7

<211>25

<212>DNA

<213> Artificial sequence

<220>

<221>misc_feature

<223> Primer

<400>7

cctcctcctt acctagacaa tgaga 25

<210>8

<211>20

<212>DNA

<213> Artificial sequence

<400>8

ggcttagaag gtccgggaaa 20

<210>9

<211>22

<212>DNA

<213> Artificial sequence

<220>

<221>misc_feature

<223> Primer

<400>9

gcacgaccct aacggtgaat ac 22

<210>10

<211>22

<212>DNA

<213> Artificial sequence

<220>

<221>misc_feature

<223> Primer

<400>10

tgggtgccag agttccatat ta 22

<210>11

<211>22

<212>DNA

<213> Artificial sequence

<220>

<221>misc_feature

<223> primer

<400>11

gtgttatcca cgtgaccaag ga 22

<210>12

<211>22

<212>DNA

<213> Artificial sequence

<220>

<221>misc_feature

<223> Primer

<400>12

ttgccagtag atgcgagttt gt 22

<210>13

<211>18

<212>DNA

<213> Artificial sequence

<220>

<221>misc_feature

<223> Primer

<400>13

tgccatgcca atttgcaa 18

<210>14

<211>27

<212>DNA

<213> Artificial sequence

<220>

<221>misc_feature

<223> Primer

<400>14

gcctaagtat acctcattca gaaccaa 27

<210>15

<211>23

<212>DNA

<213> Artificial sequence

<220>

<221>misc_feature

<223> Primer

<400>15

cttcaagcag ggattctcaa cct 23

<210>16

<211>21

<212>DNA

<213> Artificial sequence

<220>

<221>misc_feature

<223> Primer

<400>16

taagtcccac attgcctgca t 21

<210>17

<211>23

<212>DNA

<213> Artificial sequence

<220>

<221>misc_feature

<223> Primer

<400>17

gaattgcagc ttcaccagat agc 23

<210>18

<211>21

<212>DNA

<213> Artificial sequence

<220>

<221>misc_feature

<223> Primer

<400>18

aagttgcatt ccagggtcac a 21

<210>19

<211>21

<212>DNA

<213> Artificial sequence

<220>

<221>misc_feature

<223> Primer

<400>19

ctgggccttt aggtgaatgt g 21

<210>20

<211>20

<212>DNA

<213> Artificial sequence

<220>

<221>misc_feature

<223> Primer

<400>20

aatgcccggt gatgacaact 20

<210>21

<211>20

<212>DNA

<213> Artificial sequence

<220>

<221>misc_feature

<223> Primer

<400>21

ctcagggtga cagagagaag 20

<210>22

<211>20

<212>DNA

<213> Artificial sequence

<220>

<221>misc_feature

<223> Primer

<400>22

gacaccaacc accagggttt 20

<210>23

<211>20

<212>DNA

<213> Artificial sequence

<220>

<221>misc_feature

<223> Primer

<400>23

agggtgacag ggacaatagg 20

<210>24

<211>20

<212>DNA

<213> Artificial sequence

<220>

<221>misc_feature

<223> Primer

<400>24

ccatagtcca cagagaacac 20

<210>25

<211>20

<212>DNA

<213> Artificial sequence

<220>

<221>misc_feature

<223> Primer

<400>25

ggttcttaga gagaagggca 20

<210>26

<211>20

<212>DNA

<213> Artificial sequence

<220>

<221>misc_feature

<223> Primer

<400>26

gacaccaacc accagggttt 20

<210>27

<211>20

<212>DNA

<213> Artificial sequence

<220>

<221>misc_feature

<223> Primer

<400>27

tggttcttag ggacaatagg 20

<210>28

<211>20

<212>DNA

<213> Artificial sequence

<220>

<221>misc_feature

<223> Primer

<400>28

tcttctctcg ccactggaaa 20

<210>29

<211>25

<212>DNA

<213> Artificial sequence

<220>

<221>misc_feature

<223> Primer

<400>29

agccactcaa caactcttta tgtga 25

<210>30

<211>19

<212>DNA

<213> Artificial sequence

<220>

<221>misc_feature

<223> Primer

<400>30

ttgccacttc gtccttgga 19

<210>31

<211>288

<212>PRT

<213> Homo sapiens

<400>31

Met Gln Ile Pro Gln Ala Pro Trp Pro Val Val Trp Ala Val Leu Gln

1 5 10 15

Leu Gly Trp Arg Pro Gly Trp Phe Leu Asp Ser Pro Asp Arg Pro Trp

20 25 30

Asn Pro Pro Thr Phe Phe Pro Ala Leu Leu Val Val Thr Glu Gly Asp

35 40 45

Asn Ala Thr Phe Thr Cys Ser Phe Ser Asn Thr Ser Glu Ser Phe Val

50 55 60

Leu Asn Trp Tyr Arg Met Ser Pro Ser Asn Gln Thr Asp Lys Leu Ala

65 70 75 80

Ala Phe Pro Glu Asp Arg Ser Gln Pro Gly Gln Asp Cys Arg Phe Arg

85 90 95

Val Thr Gln Leu Pro Asn Gly Arg Asp Phe His Met Ser Val Val Arg

100 105 110

Ala Arg Arg Asn Asp Ser Gly Thr Tyr Leu Cys Gly Ala Ile Ser Leu

115 120 125

Ala Pro Lys Ala Gln Ile Lys Glu Ser Leu Arg Ala Glu Leu Arg Val

130 135 140

Thr Glu Arg Arg Ala Glu Val Pro Thr Ala His Pro Ser Pro Ser Pro

145 150 155 160

Arg Pro Ala Gly Gln Phe Gln Thr Leu Val Val Gly Val Val Gly Gly

165 170 175

Leu Leu Gly Ser Leu Val Leu Leu Val Trp Val Leu Ala Val Ile Cys

180 185 190

Ser Arg Ala Ala Arg Gly Thr Ile Gly Ala Arg Arg Thr Gly Gln Pro

195 200 205

Leu Lys Glu Asp Pro Ser Ala Val Pro Val Phe Ser Val Asp Tyr Gly

210 215 220

Glu Leu Asp Phe Gln Trp Arg Glu Lys Thr Pro Glu Pro Pro Val Pro

225 230 235 240

Cys Val Pro Glu Gln Thr Glu Tyr Ala Thr Ile Val Phe Pro Ser Gly

245 250 255

Met Gly Thr Ser Ser Pro Ala Arg Arg Gly Ser Ala Asp Gly Pro Arg

260 265 270

Ser Ala Gln Pro Leu Arg Pro Glu Asp Gly His Cys Ser Trp Pro Leu

275 280 285

<210>32

<211>290

<212>PRT

<213> Homo sapiens

<400>32

Met Arg Ile Phe Ala Gly Ile Ile Phe Thr Ala Cys Cys His Leu Leu

1 5 10 15

Arg Ala Phe Thr lle Thr Ala Pro Lys Asp Leu Tyr Val Val Glu Tyr

20 25 30

Gly Ser Asn Val Thr Met Glu Cys Arg Phe Pro Val Glu Arg Glu Leu

35 40 45

Asp Leu Leu Ala Leu Val Val Tyr Trp Glu Lys Glu Asp Glu Gln Val

50 55 60

Ile Gln Phe Val Ala Gly Glu Glu Asp Leu Lys Pro Gln His Ser Asn

65 70 75 80

Phe Arg Gly Arg Ala Ser Leu Pro Lys Asp Gln Leu Leu Lys Gly Asn

85 90 95

Ala Ala Leu Gln Ile Thr Asp Val Lys Leu Gln Asp Ala Gly Val Tyr

100 105 110

Cys Cys Ile Ile Ser Tyr Gly Gly Ala Asp Tyr Lys Arg Ile Thr Leu

115 120 125

Lys Val Asn Ala Pro Tyr Arg Lys Ile Asn Gln Arg Ile Ser Val Asp

130 135 140

Pro Ala Thr Ser Glu His Glu Leu Ile Cys Gln Ala Glu Gly Tyr Pro

145 150 155 160

Glu Ala Glu Val Ile Trp Thr Asn Ser Asp His Gln Pro Val Ser Gly

165 170 175

Lys Arg Ser Val Thr Thr Ser Arg Thr Glu Gly Met Leu Leu Asn Val

180 185 190

Thr Ser Ser Leu Arg Val Asn Ala Thr Ala Asn Asp Val Phe Tyr Cys

195 200 205

Thr Phe Trp Arg Ser Gln Pro Gly Gln Asn His Thr Ala Glu Leu Ile

210 215 220

Ile Pro Glu Leu Pro Ala Thr His Pro Pro Gln Asn Arg Thr His Trp

225 230 235 240

Val Leu Leu Gly Ser Ile Leu Leu Phe Leu Ile Val Val Ser Thr Val

245 250 255

Leu Leu Phe Leu Arg Lys Gln Val Arg Met Leu Asp Val Glu Lys Cys

260 265 270

Gly Val Glu Asp Thr Ser Ser Ser Lys Asn Arg Asn Asp Thr Gln Phe Glu

275 280 285

Glu Thr

290

Claims (15)

1. A method for in vitro detection of soluble PD-1 protein or its ligand in a liquid sample, characterized in that it comprises steps: (a) contacting the sample with an anti-PD-1 antibody or an anti-PD-1 ligand antibody, thereby forming a soluble PD-1 protein-antibody complex or a soluble PD-1 ligand-antibody complex; (b) detecting the soluble PD-1 protein-antibody complex or the soluble PD-1 ligand-antibody complex, thereby determining the presence or absence of the soluble PD-1 protein or its ligand. 2. The method of claim 1, wherein in step (b) the soluble PD-1 protein-antibody complex or the soluble PD-1 ligand-antibody complex is detected by a sandwich method. 3. The method of claim 1, comprising the steps of: (1) Coating the antibody of PD-1 or its ligand on a solid phase carrier to obtain a solid phase carrier coated with the antibody of PD-1 or its ligand; (2) adding a liquid sample on the solid phase carrier coated with the antibody of PD-1 or its ligand; (3) adding enzyme-labeled anti-PD-1 or its ligand antibodies of different species; (4) Add substrate to carry out enzyme-catalyzed reaction. 4. The method of claim 1, wherein the ligand is PD-L1. 5. The method of claim 1, wherein the PD-1 or its ligand is human PD-1 or its ligand. 6. The method according to any one of claims 1-5, wherein the liquid sample includes serum, plasma, synovial fluid, urine or milk, etc. 7. The use of an antibody against soluble PD-1 protein or its ligand, characterized in that it is to prepare a reagent or kit for auxiliary detection of rheumatoid arthritis. 8. The use according to claim 7, wherein the reagent is a solid-phase carrier on which an antibody against PD-1 or its ligand is immobilized. 9. purposes as claimed in claim 7, is characterized in that, described test kit comprises: solid phase carrier, Antibodies to PD-1 or its ligands, Enzyme-labeled different species of anti-PD-1 or its ligand antibodies, substrates. 10. The use according to claim 7, wherein the ligand is PD-L1. 11. A kit for in vitro detection of soluble PD-1 protein or its ligand in a liquid sample, characterized in that it comprises: solid phase carrier, Antibodies to PD-1 or its ligands, Enzyme-labeled different species of anti-PD-1 or its ligand antibodies, substrates. 12. The kit according to claim 11, wherein the solid phase carrier is coated with an antibody to PD-1 or its ligand. 13. The kit according to claim 11, wherein the antibody to PD-1 or its ligand is a polyclonal antibody. 14. The kit according to claim 11, wherein the different species of anti-human PD-1 or its ligand antibodies are monoclonal antibodies. 15. The kit according to claim 11, further comprising a negative control and a positive control.

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