CN110836966A

CN110836966A - Detection nanoparticles, detection method and kit for detecting content of antigen-specific T cells and the like

Info

Publication number: CN110836966A
Application number: CN201810929510.5A
Authority: CN
Inventors: 刘密; 王镕
Original assignee: Individual
Current assignee: Suzhou Ersheng Biopharmaceutical Co Ltd
Priority date: 2018-08-15
Filing date: 2018-08-15
Publication date: 2020-02-25
Also published as: CN118033116A

Abstract

The invention provides a detection particle which can be effectively used for detecting the content of antigen-specific T cells, a corresponding preparation method thereof, a kit comprising the detection particle and a detection method for detecting the content of antigen-specific T cells by using the detection particle, wherein the detection particle is characterized in that: the method is used for activating antigen-specific T cells in detection of the content of the antigen-specific T cells based on any one or more of cell secretion secreted by the activated antigen-specific T cells, the proliferation state of the activated antigen-specific T cells or cell surface markers of the activated antigen-specific T cells in a sample to be tested, and the antigen-specific T cells can be phagocytized by antigen-presenting cells in the sample to be tested to release antigen or generate antigen based on genetic material when the sample to be tested is processed so that the antigen-presenting cells can present the epitope of the released or generated antigen to the surface of the antigen-presenting cells so as to allow the corresponding antigen-specific T cells to recognize and activate the antigen-specific T cells.

Description

Detection nanoparticles, detection method and kit for detecting content of antigen-specific T cells and the like

Technical Field

The invention belongs to the field of immunodetection, and particularly relates to a detection nanoparticle capable of being effectively used for detecting the content of antigen-specific T cells, a corresponding preparation method thereof, a kit comprising the detection nanoparticle and a detection method for detecting the content of antigen-specific T cells by using the detection nanoparticle.

Background

The development of immunological techniques has been extremely rapid in recent years, and particularly, breakthrough progress has been made in the field of immunotherapy of cancer. With the continuous improvement of understanding of various diseases, people find that immune cells play an increasingly important role in the occurrence and treatment of various diseases.

The immune cells include B cells, T cells and the like. T cells play a tremendous role in both the autoimmune and adaptive immune response processes. Antigen-specific T cells are one of the major components of the adaptive immune response. Antigen-specific T cells play an important role in the development and progression of certain diseases, such as cancer and autoimmune diseases.

Therefore, it is important how to effectively detect antigen-specific T cells, especially in peripheral blood, but because of the scarcity of the number of certain antigen-specific T cells in peripheral blood of patients with certain diseases such as type 1 diabetes, there is no very effective technical method for detecting the content of antigen-specific T cells associated with certain diseases.

Disclosure of Invention

The invention provides a detection nanoparticle which can be effectively used for detecting the content of antigen-specific T cells, a corresponding preparation method thereof, a kit comprising the detection nanoparticle and a detection method for detecting the content of antigen-specific T cells by using the detection nanoparticle.

In order to achieve the purpose, the invention adopts the following technical scheme:

one object of the present invention is to provide a detection nanoparticle for antigen-specific T cell content detection, characterized in that: the method is used for activating the antigen-specific T cells in the detection of the content of the activated antigen-specific T cells based on any one or more of cell secretion secreted by the activated antigen-specific T cells, the proliferation state of the activated antigen-specific T cells or cell surface markers of the activated antigen-specific T cells in a sample to be detected, and detection nanoparticles are coated with antigens relevant to diseases or genetic materials required for synthesis of the antigens relevant to the diseases, wherein the particle size of the detection nanoparticles is nano-scale, and the detection nanoparticles can be phagocytized by antigen-presenting cells in the sample to be detected to release the antigens or generate the antigens based on the genetic materials when the sample to be detected is treated so that the antigen-presenting cells can present the antigen epitopes of the released or generated antigens to the surfaces of the antigen-presenting cells to allow the corresponding antigen-specific T cells to recognize and activate the antigen-specific T cells, antigen-specific T cells are antigen-specific T cells associated with a disease.

The detection nano-particles provided by the invention are also characterized in that the particle size of the detection nano-particles is 10-2000 nm.

The detection nano-particles provided by the invention are also characterized in that the particle size of the detection nano-particles is 30-1000 nm.

The detection nano-particle provided by the invention also has the characteristics that the particle size of the detection nano-particle is 100-500 nm.

The detection nanoparticle provided by the invention is also characterized in that the antigen comprises one or more of an antigen associated with a cancer disease and an antigen associated with an autoimmune disease.

The detection nanoparticle provided by the invention also has the characteristic that the autoimmune disease can be type 1 diabetes.

The detection nanoparticle provided by the present invention is also characterized in that the detection nanoparticle comprises an antigen associated with a disease, and the antigen is any one or more of a cell lysate, a protein, a plasmid, DNA, RNA, a polypeptide, a tissue lysate, an exosome lysate, a cell secretory particle lysate, a carbohydrate, and a lipid.

The detection nanoparticle provided by the present invention is also characterized in that the detection nanoparticle contains genetic material required for antigen synthesis associated with a disease, the genetic material being derived from any one or more of cell lysate, plasmid, DNA, RNA, tissue lysate, exosome lysate, and cell secretory particle lysate.

The detection nanoparticle provided by the invention also has the characteristics that the detection nanoparticle is prepared from any one or more of medical high polymer materials, materials required for preparing liposome, metal materials required for preparing the nanoparticle and non-metal materials required for preparing the nanoparticle.

The detection nanoparticle provided by the invention is also characterized in that the detection nanoparticle is any one or more of nanoparticles, liposomes, nanoemulsions, exosomes, exosome analogs, viruses, cell secretory particles and cell secretory particle analogs.

The detection nanoparticle provided by the present invention is also characterized in that the antigen is distributed at either one or both of the inside of the detection nanoparticle and the surface of the detection nanoparticle.

The detection nanoparticle provided by the present invention is also characterized in that the sample to be detected is one or more of blood, peripheral blood mononuclear cells, a lesion site tissue, and a lesion site cell.

The detection nanoparticle provided by the invention is also characterized in that the cell secretion comprises interferon-gamma, interleukin 1, interleukin 2, transforming growth factor- β, tumor necrosis factor- α, interleukin 6, interleukin 7, interleukin 15, interleukin 21, IP-10, interleukin 17, perforin and one or more of granzyme and interleukin 10.

The detection nanoparticle provided by the invention is also characterized in that any one or more of interleukin endotoxin, cytokine endotoxin, chemical factor endotoxin, chemotactic factor, metformin, berberine, artemisinin and artemisinin analogue is added into the detection nanoparticle when a sample to be detected is processed.

The detection nanoparticle provided by the present invention is also characterized in that the cellular secretion is detected by one or more of a cellular secretion detection method, an immunological assay method, a biological activity assay method, and a molecular biological detection method.

The detection nanoparticle provided by the invention is also characterized in that the cell secretion detection method is any one or more of an enzyme-linked immunospot assay, an enzyme-linked immunosorbent assay, an intracellular staining flow cytometry assay, a Cytometric Bead Array, a polymer technology, a multi-cytokine detection method, a high performance liquid chromatography and a liquid chromatography-mass spectrometry.

The detection nanoparticle provided by the invention is also characterized in that the type of the cell secretion is one or more.

The detection nanoparticle provided by the present invention is also characterized in that the detection of the proliferation state after activation of antigen-specific T cells is performed by any one or more of a decrease detection method based on an exogenous marker, an expression detection method based on a molecular marker, an increase detection method based on a metabolic marker, a change detection method based on the biomass of a cell population, and a change detection method based on the number of cells.

The detection nanoparticle provided by the invention is also characterized in that the reduction detection method based on the exogenous markers is carried out by adopting a flow cytometer after the protein binding dyes CFDA-SE and CellTrace and lipophilic dyes PKH67 and PKH26 are labeled.

The detection nanoparticle provided by the invention also has the characteristic that the molecular marker based in the expression detection method based on the molecular marker is Ki-67 and PCNA.

The detection nanoparticle provided by the present invention is also characterized in that the metabolic marker based on the metabolic marker in the metabolic marker-based increase detection method is a redox metabolic marker and a DNA metabolic marker.

The detection nanoparticle provided by the invention is also characterized in that the cell population biomass based detection method comprises one or more of DNA content and protein content.

The detection nanoparticle provided by the invention is also characterized in that the cell surface marker for antigen-specific T cell activation is one or more of CD137, CD69, CD38, CD44, CD62L or CD 25.

Another object of the present invention is to provide a method for preparing nanoparticles for detecting antigen-specific T cell content, wherein the method comprises the following steps: step 1, adding a first preset volume of antigen or genetic material required by antigen synthesis with a first preset concentration into a second preset volume of organic phase containing a medical high molecular material with a second preset concentration; step 2, carrying out ultrasonic treatment on the anti-mixture obtained in the step 1 for more than 3 seconds; step 3, adding the mixture obtained after the treatment in the step 2 into a third preset volume of emulsifier aqueous solution with a third preset concentration, and carrying out ultrasonic treatment for more than 5 seconds; step 4, adding the mixture obtained after the treatment in the step 3 into a fourth predetermined volume of emulsifier aqueous solution with a fourth predetermined concentration, and stirring until a predetermined stirring condition is met; step 5, centrifuging the suspension which is processed in the step 4 and meets the preset stirring condition for more than 7 minutes at the rotating speed of more than 8000RPM, removing the supernatant, re-suspending the remaining precipitate in water, repeating the steps for many times, and finally obtaining the final precipitate from which the supernatant is removed; step 6, suspending the final precipitate obtained in the step 5 in a fifth predetermined volume of a lyoprotectant with a fifth predetermined concentration to obtain a suspension; and 7, performing freeze drying treatment on the suspension obtained in the step 6 to obtain freeze-dried powder, and using the freeze-dried powder containing the detection nanoparticles to detect the content of the antigen-specific T cells.

The preparation method provided by the invention also has the following characteristics: the ratio of the first predetermined volume to the second predetermined volume ranges from 1:3 to 1:50, the ratio of the second predetermined volume to the third predetermined volume ranges from 1:1.5 to 1:100, and the ratio of the third predetermined volume to the fourth predetermined volume ranges from 1:3 to 1: 50.

The preparation method provided by the invention also has the following characteristics: wherein the organic solvent in the organic phase is dichloromethane.

The preparation method provided by the invention also has the following characteristics: wherein the medical high polymer material is polylactic acid-glycolic acid copolymer.

The preparation method provided by the invention also has the following characteristics: wherein the emulsifier aqueous solution is polyvinyl alcohol aqueous solution.

The preparation method provided by the invention is also characterized in that the first preset concentration is more than 0.01ng/mL, the second preset concentration range is 0.5mg/mL-500mg/mL, the third preset concentration range is 0.5mg/mL-500mg/mL, and the fourth preset concentration range is 0.5mg/mL-500 mg/mL.

The preparation method provided by the invention also has the following characteristics: wherein the predetermined stirring condition is until the organic solvent is completely volatilized.

The preparation method provided by the invention also has the following characteristics: wherein the lyoprotectant is saccharide such as trehalose, and the content percentage of the lyoprotectant is more than 1%.

The preparation method provided by the invention also has the following characteristics: wherein the freeze drying treatment specifically comprises the following steps: freezing the suspension obtained in the step 5 at-80 deg.C for more than 8 hr, and freeze-drying with a freeze-drying machine for more than 12 hr.

It is still another object of the present invention to provide a kit for detecting antigen-specific T cells, comprising: and detecting nanoparticles, wherein the detecting nanoparticles are the detecting nanoparticles.

Still another object of the present invention is to provide a method for detecting the content of antigen-specific T cells, comprising the steps of: treating a sample to be detected containing antigen-presenting cells and T cells with detection nanoparticles containing an antigen associated with a disease or containing genetic material required for synthesis of the antigen associated with a disease to present an epitope of the antigen released or synthesized using the genetic material to the surface of the antigen-presenting cells to allow the corresponding antigen-specific T cells to recognize and activate the antigen-specific T cells; and detecting the antigen-specific T cell content of the sample to be detected based on cell secretion secreted by the activated antigen-specific T cells, the proliferation state of the activated antigen-specific T cells or cell surface markers of the activated antigen-specific T cells, wherein the detection nanoparticles are the detection nanoparticles.

Action and Effect of the invention

According to the detection nanoparticle for detecting the content of antigen-specific T cells, the kit for detecting antigen-specific T cells, and the method for detecting the content of antigen-specific T cells provided by the present invention, the detection nanoparticle containing an antigen associated with a disease or a genetic material required for synthesizing the antigen, and the size of the detection nanoparticle being nano-sized, can be phagocytized by antigen-presenting cells in a sample to be detected, and release the antigen or the genetic material required for antigen synthesis to synthesize the corresponding antigen, and further, the released antigen or newly synthesized antigen can be presented, so that the corresponding antigen-specific T cells associated with a disease can be activated, and by detecting any one or more of a cell secretion secreted by the activated antigen-specific T cells, a proliferation state of the activated antigen-specific T cells, or a cell surface marker of the activated antigen-specific T cells, based on the relationship between cell secretion, state or cell surface marker and corresponding antigen specific T cell, the content of corresponding antigen specific T cell can be determined, so that the method can be used for monitoring the progress of diseases, predicting and monitoring the treatment effect of disease treatment schemes or evaluating the treatment effect of treatment schemes in the treatment process, thereby providing conditions for implementing accurate treatment and avoiding the waste of medical resources, and providing conditions for selecting proper experimental objects for relevant research to obtain good experimental results and avoid unnecessary waste.

Drawings

FIG. 1 is a schematic diagram of a detection mechanism of a detection method for detecting the content of antigen-specific T cells when detecting antigen-entrapped nanoparticles. In fig. 1:1, antigen presenting cells; 2, antigen-specific T cells; 3, detecting nanoparticles coated with antigens; 4, an antigenic molecule; 5: an antigenic epitope; 6, major histocompatibility complex; 7, T cell receptor; cell secretions, such as interferon-gamma.

FIG. 2 is a schematic diagram of the detection mechanism of the detection method for detecting the content of antigen-specific T cells when nanoparticles are used to encapsulate genetic material capable of generating antigen. In fig. 2: 1, antigen presenting cells; 2, antigen-specific T cells; 3, detection nanoparticles loaded with genetic material capable of generating an antigen; 4, genetic material capable of producing an antigen; 5: an antigen produced by genetic material; 6: an antigenic epitope; 7, major histocompatibility complex; 8, T cell receptor; 9, cell secretions, e.g. interferon-gamma

Detailed Description

The principle of detection of cellular secretions secreted by activated antigen-specific T cells:

FIG. 1 is a schematic diagram of the detection mechanism of the method for detecting the content of antigen-specific T cells.

In FIG. 1, antigen presenting cells; 2, antigen-specific T cells; 3, detecting nanoparticles coated with antigens; 4, an antigenic molecule; 5: an antigenic epitope; 6, major histocompatibility complex; 7, T cell receptor; cell secretions, such as interferon-gamma.

Referring to fig. 1, the principle of the present invention is to treat a sample to be tested containing antigen-presenting cells (APCs) and T cells with detection nanoparticles having a nanoparticle size of the antigen-or genetic material required for synthesis of the antigen. By utilizing the property of phagocytosis of nano-scale particles by antigen-presenting cells, antigens or genetic materials required for antigen synthesis can enter the antigen-presenting cells through nano-sized detection nanoparticles, and after being treated by the antigen-presenting cells, the antigen or genetic materials can be combined with Major Histocompatibility Complex (MHC), so that the antigens entrapped in the detection nanoparticles can be released in the antigen-presenting cells, or the genetic materials can be released to generate the antigens in the antigen-presenting cells. The released or produced antigen is processed by and presented to the surface of the antigen-presenting cell. The epitopes presented on the surface of antigen presenting cells are recognized by the corresponding antigen-specific T cells and activate the antigen-specific T cells. After being activated, the antigen-specific T cells secrete a plurality of cell secretions, and the content of the antigen-specific T cells can be known by detecting the content of the relevant cell secretions by a certain detection means.

The following examples, which are based on the secretion of cells by activated antigen-specific T cells, illustrate embodiments of the present invention in conjunction with the drawings. For the specific methods or materials used in the embodiments, those skilled in the art can make routine alternatives based on the existing technologies based on the technical idea of the present invention, and not limited to the specific descriptions of the embodiments of the present invention.

The methods used in the examples are conventional methods unless otherwise specified; the materials, reagents and the like used are commercially available unless otherwise specified.

Example 1 detection of cancer cell antigen-specific T cells in cancer patients

This example will be described by taking as an example the detection of antigen-specific T cells in cancer cells.

Immunotherapy for cancer has been greatly advanced in recent years, such as anti-PD-1 antibody, anti-PD-L1 antibody and CAR-T cell therapy, and many related anti-cancer drugs have been approved by the Food and Drug Administration (FDA) for use in the market. The therapeutic mechanism of cancer immunotherapy is the de-recognition and killing of cancer cells by cancer-associated antigen-specific T cells of the autoimmune system. Because of the specificity and memory characteristics of cancer antigen-specific T cells, there are few side effects and long-lasting effects once cancer antigen-specific T cells are able to recognize and attack cancer cells. However, the overall response rate of the current cancer immunotherapy is low, and is only about 20% -40%. One reason this is important is because there are not enough cancer cell antigen-specific T cells in some patients. Generally, cancer patients with high content of cancer antigen-specific T cells in vivo have better cancer immunotherapy effects, while patients with low content of cancer antigen-specific T cells in vivo have poorer cancer immunotherapy effects. The amount of cancer cell antigen-specific T cells in a cancer patient is determined prior to the cancer patient beginning cancer immunotherapy. If the number of cancer cell antigen-specific T cells in a patient is large, the therapeutic effect of immunotherapy is good; if the number of cancer cell antigen-specific T cells in the patient is small, this indicates that the prognosis for immunotherapy may be poor.

To this end, this example examined the antigen-specific T cell content in cancer cells.

In this embodiment, the detection nanoparticles are prepared first, and then the prepared detection nanoparticles are used to detect the content of antigen-specific T cells in cancer cells.

1. Preparation of detection nanoparticles

The detection nanoparticles used in the embodiment are nanoparticles, and the preparation method specifically comprises the following steps:

step 1, adding a first predetermined volume of an aqueous phase solution containing an antigen with a first predetermined concentration into a second predetermined volume of an organic phase containing a medical polymer material with a second predetermined concentration;

in this example, the antigen is present in a cancer cell lysate.

In this embodiment, the cancer cell lysate (biological sample) containing the antigen is dissolved in water or any aqueous solution containing water and having no influence on the subsequent detection, so as to prepare an antigen solution in an aqueous phase, and the concentration of the obtained antigen solution, i.e. the first predetermined concentration, requires that the antigen concentration content is greater than 0.1mg/mL, so that the concentration is selected, it is found by a lot of experiments that the larger the antigen concentration is, the more the antigen is loaded in the prepared nanoparticles, and when the prepared antigen concentration is greater than 0.1mg/mL, i.e. the first predetermined concentration is greater than 0.1mg/mL, the enough antigen is available for the subsequent detection. In addition, the first predetermined volume is 200 μ L in this embodiment.

Dissolving the medical polymer material in an organic solvent to obtain a second predetermined volume of an organic phase containing the medical polymer material with a second predetermined concentration, in this embodiment, the medical polymer material is poly (lactic-co-glycolic acid) (PLGA), the organic solvent is dichloromethane, and the volume of the obtained organic phase, that is, the second predetermined volume is 2 mL. In the present embodiment, the second predetermined concentration of the medical polymer material is in the range of 0.5mg/mL to 500mg/mL, preferably 100 mg/mL.

PLGA was chosen in this example because the inventors found that this material is a pH sensitive material that does not release antigen at physiologically neutral pH, but once phagocytosed into antigenic cells, enters the endosome, which is acidic, and under acidic conditions PLGA is rapidly degraded and thus antigen can be released rapidly.

In practice, the second predetermined volume of organic phase is set according to the ratio between it and the first predetermined volume of aqueous phase, in this embodiment the ratio between the first predetermined volume of aqueous phase and the second predetermined volume of organic phase ranges from 1:2 to 1:50, preferably 1: 10. The first predetermined volume, the second predetermined volume, and the ratio of the first predetermined volume to the second predetermined volume can be adjusted as needed to adjust the size of the nanoparticles produced during the implementation.

And 2, carrying out ultrasonic treatment on the antigen polymer mixed liquid obtained in the step 1 for more than 2 seconds.

The step is to perform nanocrystallization, the size of the prepared nanoparticles can be controlled by the length of the ultrasonic time, and the change of the particle size can be caused by too long or too short ultrasonic time, so that the proper ultrasonic time needs to be selected, and the ultrasonic time in the embodiment is more than 2 seconds.

And 3, adding the mixture obtained after the treatment in the step 2 into a third preset volume of aqueous solution containing a third preset concentration of emulsifier, and carrying out ultrasonic treatment for more than 5 seconds.

Adding the mixture obtained in the step 2 into an emulsifier aqueous solution to continue ultrasonic nano-crystallization.

In this example, the aqueous emulsifier solution is an aqueous polyvinyl alcohol (PVA) solution, the third predetermined volume is 5mL, and the third predetermined concentration is 20 mg/mL. The third predetermined volume is adjusted according to its ratio to the second predetermined volume, which in this embodiment is set in the range of 1:1.5-1:100, preferably 2: 5. The ratio of the second predetermined volume to the third predetermined volume may be adjusted during the implementation in order to control the size of the nanoparticles.

Similarly, the ultrasonic time, the volume of the emulsifier aqueous solution and the concentration of the emulsifier aqueous solution are all taken according to the steps to obtain the nanoparticles with proper size.

And 4, adding the liquid obtained after the treatment in the step 3 into a fourth preset volume of emulsifier aqueous solution with a fourth preset concentration, and stirring until preset stirring conditions are met.

In this step, the emulsifier aqueous solution is still PVA, the fourth predetermined volume is greater than 50mL, the fourth predetermined concentration is 5mg/mL, and the fourth predetermined concentration is selected based on obtaining nanoparticles with a suitable size. The fourth predetermined volume is selected based on a ratio of the third predetermined volume to the fourth predetermined volume. In this embodiment, the ratio of the third predetermined volume to the third predetermined volume is in the range of 1:3 to 1:50, preferably 1: 10. The ratio of the third predetermined volume to the fourth predetermined volume may be adjusted during the implementation to control the size of the nanoparticles.

In this embodiment, the predetermined stirring condition in this step is until the volatilization of the organic solvent is completed, that is, the volatilization of dichloromethane in step 1 is completed.

And 5, centrifuging the mixed solution which is processed in the step 4 and meets the preset stirring condition for more than 7 minutes at the rotating speed of more than 8000RPM, removing the supernatant, re-suspending the rest precipitate in water, repeating the steps for multiple times, and finally obtaining the final precipitate with the supernatant removed.

The purpose of this step is to remove some residual polymer and unencapsulated antigen, and in this example, this step is specifically:

step 5.1, centrifuging the mixed solution which is processed in the step 4 and meets the preset stirring condition on an ultra-high speed centrifuge at more than 8000RPM for more than 7 minutes, and discarding the supernatant;

step 5.2, resuspending the precipitate left in step 5.1 in water;

step 5.3, repeating the step 5.1;

step 5.4, repeating the step 5.2;

and 5.5, repeating the step 5.1 to obtain the final precipitate.

In practice, depending on the actual situation, the cycle of step 5.1 to step 5.5 can be repeated to obtain the desired final precipitate.

And 6, suspending the final precipitate obtained in the step 5 in a third predetermined volume of the lyoprotectant with a third predetermined concentration to obtain a suspension.

Trehalose (trehalase) is used as the lyoprotectant in this example.

In this embodiment, the fifth predetermined volume of the lyoprotectant in this step is 20mL, and the fifth predetermined concentration is 4% by mass, which are set so as not to affect the lyophilization effect in the subsequent lyophilization.

And 7, performing freeze drying treatment on the suspension obtained in the step 6, and using the freeze-dried substance to detect the nano-particles for detecting the content of the antigen specific T cells.

In this embodiment, the steps specifically include:

step 7.1: freezing the suspension obtained in the step 6 at-80 ℃ for more than 8 hours to obtain a frozen product;

step 7.2: freeze-drying the frozen product obtained in the step 7.1 by using a freeze dryer to obtain freeze-dried detection nanoparticles;

step 7.3: the freeze-dried particles obtained in step 7.2 were analysed for particle size, surface charge and antigen loading. Based on these analysis results, appropriate detection nanoparticles are selected for detecting the content of antigen-specific T cells.

The particle size of the detection nanoparticle is on the order of nanometers so as to be phagocytosed by antigen-presenting cells when used for detection of antigen-specific T cells, and is within a suitable range for enhancing phagocytosis efficiency, and in this embodiment, the particle size of the detection nanoparticle is 10-2000nm, more preferably 30-1000nm, and most preferably 100-500 nm.

In addition, in the preparation process of the detection nanoparticle in this embodiment, an antigen is directly used for encapsulation, and finally the nanoparticle containing the antigen is obtained, and the antigen is a cell lysate, in practice, when the antigen is directly encapsulated, the antigen may also be any one or more of a cell lysate, a protein, a plasmid, DNA, RNA, a polypeptide, a tissue lysate, an exosome lysate, a cell secretory particle lysate, a saccharide, and a lipid.

In addition, during the preparation process of the detection nanoparticles, genetic material required for synthesis of antigen associated with disease can be entrapped, thus finally obtaining detection nanoparticles entrapping the genetic material from any one or more of cell lysate, plasmid, DNA, RNA, tissue lysate, exosome lysate, cell secretory particle lysate

In addition, the prepared detection nano-particles can be distributed with antigens on the inner part or the surface of the detection nano-particles, or both the inner part and the surface.

In addition, the detection nanoparticles used in the embodiments are nanoparticles, and in practice, any one or more of liposomes, nanoemulsions, exosomes, exosome analogs, viruses, cell secretory particles, and cell secretory particle analogs may also be used.

2. Detection of antigen-specific T cell content in cancer cells

The detection process indirectly obtains the content of the T cells by detecting the content of cell secretion secreted by the activated T cells, and comprises the following steps: treating a sample to be detected containing antigen presenting cells and T cells by using the detection nanoparticles obtained by the preparation method so as to present the antigen epitope of the antigen released or synthesized by using genetic materials to the surface of the antigen presenting cells to allow the antigen specific T cells to recognize and activate the antigen specific T cells; and detecting the content of the antigen-specific T cells in the sample to be detected based on the cell secretion secreted by the activated antigen-specific T cells.

In this example, as a method for detecting the number of antigen-specific T cells, a method for detecting a cell secretion by an ELISPOT detection technique is used, and the cell secretion may be selected from one or more of Interferon- γ, interleukin 1(IL-1), interleukin 2(IL-2), Transforming Growth Factor (TGF) - β, interleukin 6(IL-6), interleukin 7(IL-7), interleukin 15(IL-15), interleukin 21(IL-21), IP-10(Interferon gamma-induced protein 10, CXCL10), interleukin 17(IL-17), and interleukin 10 (IL-10).

The cellular secretion in this example is interferon-gamma. In specific practice, the detection process is as follows:

step 1, sampling

Several milliliters of peripheral blood of a patient is extracted, and heparin anticoagulation is added in the blood extraction process.

Step 2, preparation of sample to be tested-extraction separation of PBMCs (peripheral blood mononuclear cells)

The samples to be tested in the implementation capacity are PBMCs.

The PBMCs are extracted and separated by extraction and separation methods which are conventionally adopted in laboratories, such as a Ficoll density gradient centrifugation method and a percoll layering liquid method. The isolated PBMCs containing antigen presenting cells and T cells are added to AIM V cell culture medium.

And 3, suspending the prepared detection nanoparticles in an AIM V cell culture medium, namely redissolving the detection nanoparticles in a simple AIM V culture medium.

Step 4, mixing the PBMCs in the AIM V medium and the detection nanoparticles in the AIM V medium into a 96-well PVDF plate previously coated with interferon-gamma antibody a (capture antibody) and subjected to the AIM V medium blocking treatment for 1 hour or more.

Unbound interferon-gamma antibody a in the 96-well plate was washed away before the mixed addition of PBMCs and detection nanoparticles, and AIM V medium was added to the 96-well plate at 37 deg.C (5% CO)₂) The incubator was left to stand for more than 1 hour and closed to avoid nonspecific adsorption.

Step 5, obtaining the PBMCs obtained in the step 4 and the redissolution detection nanoparticles obtained in the step 3The products of (A) were mixed to obtain a mixture at 37 deg.C (5% CO)₂) The culture is carried out in an incubator for 24 to 96 hours.

Step 6: the mixture of PBMCs and detection nanoparticles was discarded and the 96-well plate was washed.

And 7: interferon-gamma antibody b (detection antibody) was added and incubated at 37 deg.C (5% CO)₂) Culturing in an incubator for more than 2 hours.

And 8: the solution containing interferon-gamma antibody b was discarded, and the 96-well plate was washed and developed using a corresponding method to form spots on the surface of the 96-well plate.

And step 9: and reading data by adopting an ELISPOT analyzer and analyzing an experimental result.

In the ELISPOT detection method, a cell secretion such as interferon-gamma secreted after an antigen-specific T cell is activated is adsorbed by an antibody a coated on a 96-well plate, and a double antibody sandwich structure is formed after an antibody b is added. When the double-antibody sandwich structure is treated by a certain color development method, spots are formed at the positions of the cells. The formation of one spot represents one activated antigen-specific T cell, so by measuring the number of spots formed by color development in each well of a 96-well plate, we can know the number of antigen-specific T cells in the sample to be tested. Thus, depending on the amount of the cancer cell antigen-specific T cells in the subject, the immunotherapy effect is evaluated from the following points:

1. can be used as a reference index for judging whether a patient selects the immunotherapy or not so as to avoid unnecessary financial burden, waste of precious treatment time and unnecessary side effects of the patient caused by the unsuitability of the immunotherapy and effectively prevent the waste of unnecessary medical resources;

2. the method is used as a reference index for selecting a research object in tumor prevention and treatment research, so that the tumor prevention and treatment research can effectively select the research object to perform immunotherapy research, thereby obtaining scientific and reasonable research results, better promoting the scientific progress of tumor prevention and treatment, avoiding unnecessary waste of research expenses, manpower and material resources, and effectively preventing and treating waste of public resources.

Example 2

The following is a description of example 2. In this example, the same principle of detection and selection of substances and parameters involved in the respective production processes as those in example 1 are based on the same parts, and the same explanation is omitted.

This example illustrates the detection of antigen-specific T cells associated with type 1 diabetes.

Once β cells are completely killed, the human body cannot synthesize and secrete insulin capable of reducing blood sugar, so that the blood sugar is increased, and only insulin can be supplemented by an external way to maintain the balance of blood sugar, so that β cell-specific T cells play a key role in the occurrence and development processes of type 1 diabetes.

To this end, the present example examined β cell-specific T cell levels associated with type 1 diabetes.

1. Preparation of detection nanoparticles

The detection nanoparticles used in the embodiment are nanoparticles which entrap type 1 diabetes associated antigens GAD65 and preproinsulin, and the preparation method specifically comprises the following steps:

step 1, adding a predetermined first volume of antigen solution containing a first predetermined concentration of antigen protein into a predetermined second volume of organic phase of a predetermined second predetermined concentration of medical polymer material.

In this example, the antigens are derived from type 1 diabetes associated antigen proteins GAD65 and preproinsulin. The first predetermined volume is 200 μ L. The first predetermined concentration is a concentration of GAD65 and preproinsulin greater than 0.01mg/mL, respectively.

In this embodiment, the type 1 diabetes-associated antigen proteins GAD65 and preproinsulin are dissolved in water or any aqueous solution containing water that does not affect the subsequent detection, to prepare an antigen solution.

Dissolving a medical polymer material in a predetermined second volume of organic solvent to obtain an organic solution, in this embodiment, the medical polymer material is poly (lactic-co-glycolic acid) (PLGA), the organic solvent is dichloromethane, and the predetermined organic volume is 2 mL.

Likewise, the ratio of the first predetermined volume of the aqueous phase to the second predetermined volume of the organic phase is 1: 10. The first predetermined volume, the second predetermined volume, and the ratio of the first predetermined volume to the second predetermined volume may be adjusted as desired during implementation.

And 2, carrying out ultrasonic treatment on the mixed liquor obtained in the step 1 for more than 2 seconds.

And 3, adding the liquid obtained after the treatment in the step 2 into a third preset volume of emulsifier aqueous solution with a third preset concentration, and carrying out ultrasonic treatment for more than 5 seconds.

In this example, the aqueous emulsifier solution is an aqueous polyvinyl alcohol (PVA) solution, the third predetermined volume is 5mL, and the third predetermined concentration is 20 mg/mL. In this embodiment, the ratio of the second predetermined volume to the third predetermined volume is 2: 5. The ratio of the second predetermined volume to the third predetermined volume may be adjusted during the implementation to control the size of the nanoparticles.

In this step, the emulsifier aqueous solution is still PVA, the fourth predetermined volume is greater than 50mL, the fourth predetermined concentration is 5mg/mL, and the selection of the fourth predetermined volume is determined according to the ratio of the third predetermined volume to the fourth predetermined volume. In this embodiment, the ratio of the third predetermined volume to the fourth predetermined volume is 1: 10. The ratio of the third predetermined volume to the fourth predetermined volume may be adjusted during the implementation to control the size of the nanoparticles.

In this embodiment, the predetermined stirring condition in this step is until the organic solvent is completely volatilized, that is, the dichloromethane in step 1 is completely volatilized.

And 5, centrifuging the liquid which is processed in the step 4 and meets the preset stirring condition for more than 7 minutes at the rotating speed of more than 8000RPM, removing the supernatant, resuspending the rest precipitate in water, repeating the steps for multiple times, and finally obtaining the final precipitate with the supernatant removed.

step 5.1, centrifuging the liquid meeting the preset stirring condition in the step 4 on an ultra-high speed centrifuge at more than 8000RPM for more than 7 minutes, and discarding the supernatant;

step 5.2, resuspending the precipitate left in step 5.1 in water;

step 5.3, repeating the step 5.1;

step 5.4, repeating the step 5.2;

and 5.5, repeating the step 5.1 to obtain the final precipitate.

And 6, suspending the final precipitate obtained in the step 5 in a fifth predetermined volume of the lyoprotectant with a fifth predetermined concentration to obtain a suspension.

Trehalose (trehalase) is used as the lyoprotectant in this example.

In this embodiment, the fifth predetermined volume of the lyoprotectant in this step is 20mL, and the fifth predetermined concentration is 4%, which are set so as not to affect the lyophilization effect in the subsequent lyophilization.

And 7, performing freeze drying treatment on the suspension obtained in the step 6 to obtain freeze-dried powder, wherein the freeze-dried powder containing the detection nanoparticles can be used for detecting the content of the antigen-specific T cells.

In this embodiment, the steps specifically include:

step 7.2: freeze-drying the frozen product obtained in the step 7.1 by using a freeze dryer for more than 12 hours to obtain detection nanoparticles;

step 7.3: and (3) analyzing the particle size, the surface charge and the antigen loading amount of the detected nano particles obtained in the step 7.2.

In this embodiment, the particle size of the detection nanoparticles is 10-2000nm, more preferably 30-1000nm, and most preferably 100-500 nm.

2.1 content detection of antigen-specific T cells in diabetic patients

In this example, a method of detecting cell secretion by enzyme-linked immunospot (ELISPOT) detection is still used as a method of detecting the number of antigen-specific T cells. In practice, any one or more of enzyme-linked immunosorbent assay (ELISA), flow cytometry detection of intracellular cytokines, polymer technology, Luminex's multi-cytokine detection, high performance liquid chromatography, and liquid chromatography may be used.

In practice, the secretion of the cell may be one or more of Interferon- γ, interleukin 1(IL-1), interleukin 2(IL-2), Transforming Growth Factor (TGF) - β, interleukin 6(IL-6), interleukin 7(IL-7), interleukin 15(IL-15), interleukin 21(IL-21), IP-10(Interferon gamma-induced protein 10, CXCL10), interleukin 17(IL-17), and interleukin 10(IL-10) as required, and the sample to be tested is still peripheral blood.

The sample to be tested and the specific detection process in this embodiment are the same as those in embodiment 1.

In the ELISPOT assay, the formation of a spot represents an activated antigen-specific T cell, so by measuring the number of spots formed by color development in each well of a 96-well plate, we can know the number of antigen-specific T cells in the sample to be tested. Thus, according to the content of the antigen-specific T cells related to the type 1 diabetes in the body of the detected object, the progress of the type 1 diabetes can be monitored or the treatment effect of different treatment methods for the type 1 diabetes can be evaluated:

1. the antigen-specific T cells are already present in the patient before the onset of the type 1 diabetes mellitus patient, so that the disease progress of the type 1 diabetes mellitus patient can be known by detecting the number of the antigen-specific T cells in peripheral blood, and the more the number of the antigen-specific T cells in the patient is, the larger the killing and destruction to β cells of the patient is, so that the patient can know the own condition in advance, and certain preventive measures can be taken in advance;

2. in the treatment of type 1 diabetes patients, detection of antigen-specific T cells can be used to assess the efficacy of the treatment: if a certain treatment means can obviously reduce the number of antigen-specific T cells, the treatment effect of the treatment means is good, so that the treatment means can be used as an evaluation basis for whether a patient adopts the treatment means or not in the follow-up process, or can be used as a basis for evaluating the treatment effects of different treatment means or treatment methods by a medicine enterprise or a medicine development institution, the precision of medical treatment can be improved, and the treatment resources or experimental resources can be saved.

Example 3

The following is a description of example 3. In this example, the same principle of detection and selection of substances and parameters involved in the respective production processes as those in example 1 are based on the same parts, and the same explanation is omitted.

The present example is also described with reference to the detection of antigen-specific T cells associated with type 1 diabetes, as well as the detection of antigen-specific T cells associated with type 1 diabetes, and also with reference to the detection of the content of β cell antigen-specific T cells in peripheral blood of patients with insulin-dependent diabetes (type 1 diabetes).

1. Preparation of detection nanoparticles

The detection nanoparticles used in the present embodiment are nanoparticles that entrap genetic material DNA required for synthesis of type 1 diabetes associated antigen GAD65, and the preparation method specifically includes the following steps:

step 1, adding a first predetermined volume of antigen solution containing DNA required for antigen synthesis at a first predetermined concentration into a second predetermined volume of organic solution containing medical polymer material at a second predetermined concentration.

In this example, the DNA required for antigen synthesis is the DNA required for antigen GAD synthesis.

In this example, the DNA required for synthesizing the antigen is dissolved in water or any aqueous solution containing water that does not affect the subsequent detection to prepare an aqueous solution, the first predetermined volume is 200. mu.L, and the first predetermined concentration is greater than 0.01 ng/mL.

The medical polymer material is dissolved in a predetermined organic solvent to obtain a polymer organic solution, in this embodiment, the medical polymer material is poly (lactic-co-glycolic acid) (PLGA), the organic solvent is dichloromethane, and the predetermined organic volume is 2 mL.

The ratio of the first predetermined volume of the aqueous phase to the second predetermined volume of the organic phase is 1: 10. The first predetermined volume, the second predetermined volume, and the ratio of the first predetermined volume to the second predetermined volume may be adjusted as desired during implementation.

And 2, carrying out ultrasonic treatment on the mixture obtained in the step 1 for more than 3 seconds.

In this example, the aqueous emulsifier solution is an aqueous polyvinyl alcohol (PVA) solution, the third predetermined volume is 5mL, and the third predetermined concentration is 20 mg/mL.

Similarly, the ultrasonic time, the volume of the emulsifier aqueous solution and the concentration of the emulsifier aqueous solution are all taken according to the steps to obtain the nanoparticles with proper size. The third predetermined volume is selected based on a ratio of the second predetermined volume to the third predetermined volume. In this embodiment, the ratio of the second predetermined volume to the third predetermined volume is 2: 5. The ratio of the second predetermined volume to the third predetermined volume may be adjusted during the implementation to control the size of the nanoparticles.

In this step, the emulsifier aqueous solution is still PVA, the fourth predetermined volume is more than 50mL,

the fourth predetermined concentration is 5mg/mL, and the selection of the fourth predetermined volume and the fourth predetermined concentration is still based on obtaining nanoparticles of a suitable size. The fourth predetermined volume is selected based on a ratio of the third predetermined volume to the fourth predetermined volume. In this embodiment, the ratio of the third predetermined volume to the fourth predetermined volume is 1: 10. The ratio of the third predetermined volume to the fourth predetermined volume may be adjusted during the implementation to control the size of the nanoparticles.

The purpose of this step is to remove some residual polymers and unencapsulated DNA, and in this example, this step is specifically:

step 5.2, resuspending the precipitate left in step 5.1 in water;

step 5.3, repeating the step 5.1;

step 5.4, repeating the step 5.2;

and 5.5, repeating the step 5.1 to obtain the final precipitate.

Trehalose (trehalase) is used as the lyoprotectant in this example.

And 7, performing freeze drying treatment on the suspension obtained in the step 6 to obtain freeze-dried powder. The lyophilized powder containing the detection nanoparticles can be used to detect the content of antigen-specific T cells.

In this embodiment, the steps specifically include:

step 7.2: freeze-drying the frozen product obtained in the step 7.1 by using a freeze dryer for more than 12 hours to obtain freeze-dried detection nanoparticles;

step 7.3: and (3) analyzing the particle size, the surface charge and the DNA encapsulation amount of the detected nanoparticles obtained in the step 7.2.

Content detection of antigen-specific T cells of type 21 diabetes patients

In this example, a method of detecting cell secretion by a conventional enzyme-linked immunosorbent assay (ELISA) detection technique was used as a method of detecting the number of antigen-specific T cells. The specific application can be adjusted according to the actual conditions of different ELISA reagents and detection kits.

Step 1, sampling

Several milliliters of peripheral blood of a patient is extracted, and heparin anticoagulation is added in the blood extraction process. This experiment was performed using freshly collected peripheral blood.

And 2, suspending the prepared detection nanoparticles in an AIM V cell culture medium.

Step 3, mixing the detection nanoparticles in peripheral blood and AIM V media at 37 deg.C (5% CO)₂) The culture is carried out in an incubator for 24 to 96 hours.

Step 4, removing blood cells by centrifugation and collecting supernatant.

And 5, directly or after diluting the supernatant, detecting the content of the interferon-gamma in the supernatant by adopting an ELISA detection method of the interferon-gamma. The specific detection steps are the same as the ELISA routine detection. Briefly described as follows:

the supernatant was added to a 96-well plate coated with anti-interferon-gamma antibody a and blocked with 0.5% BSA either directly or after dilution with PBS. After at least 2 hours of action, the cells are washed and the anti-interferon-gamma antibody b is added. The antibody b is linked to a substance capable of color reaction or fluorescence detection. And detecting the content of the interferon-gamma in the supernatant by a color development method, a chemiluminescence method or a fluorescence method after reacting with the antibody b for a period of time.

In this embodiment, the method of detecting the secretion of the T cell by enzyme-linked immunosorbent assay (ELISA) is used as a method for detecting the number of the antigen-specific T cell, and in practice, any one or more of enzyme-linked immunospot (ELISPOT), enzyme-linked immunosorbent assay (ELISA), flow cytometry detection of intracellular cytokines, multimer technology, Luminex multi-cytokine detection method, high performance liquid chromatography, and LC-MS may be used.

The sample to be detected and the specific detection process in this embodiment are the same as those in

embodiments

1 and 2, and differently, in this detection process, in this embodiment, after the genetic material DNA required for detecting the synthetic antigen contained in the nanoparticle enters the antigen-presenting cell and is released, the corresponding antigen is synthesized in the antigen-presenting cell, and then subsequent presentation and activation are performed.

FIG. 2 is a schematic diagram of the detection mechanism of the detection method for detecting the content of antigen-specific T cells when nanoparticles are used to encapsulate genetic material capable of generating antigen.

In FIG. 2, 1, antigen presenting cells; 2, antigen-specific T cells; 3, detection nanoparticles loaded with genetic material capable of generating an antigen; 4, genetic material capable of producing an antigen; 5: an antigen produced by genetic material; 6: an antigenic epitope; 7, major histocompatibility complex; 8, T cell receptor; 9, cell secretion, such as interferon-gamma.

As can be seen from fig. 2, the detection mechanism of this embodiment is also based on the secretion of cells, and different from that of example 1, because the entrapped material is genetic material, after the entrapped material is released into the cells, the antigen is first generated in the cells, and then the antigen is processed and presented.

In the ELISA detection method, the content of the cell secretion is different, and the corresponding chromogenic or fluorescent readings are different, so that the content of the cell secretion can be judged according to the final reading, and further the content of the antigen-specific T cells can be determined. Therefore, according to the content of the antigen-specific T cells related to the type 1 diabetes in the body of the detected object, the disease progression of the type 1 diabetes can be monitored and the treatment effect of different treatment methods of the type 1 diabetes can be evaluated.

Examples effects and effects

According to the detection nanoparticle for antigen-specific T cell content detection and the method for detecting antigen-specific T cell content provided in examples 1 to 3, by preparing a detection nanoparticle containing an antigen associated with a disease or a genetic material required for synthesizing the antigen, and the size of the detection nanoparticle being nano-sized, it is possible to phagocytize the detection nanoparticle by antigen-presenting cells in a sample to be detected and release the antigen or the genetic material required for antigen synthesis to synthesize the corresponding antigen, and further, by presenting the released antigen or newly synthesized antigen, it is possible to activate the corresponding antigen-specific T cell associated with a disease, and by detecting a cell secretion secreted from the activated antigen-specific T cell and based on the relationship between the cell secretion and the corresponding antigen-specific T cell, it is possible to determine the content of the corresponding antigen-specific T cell, therefore, the method can be used for monitoring the progress of diseases or evaluating treatment schemes for comparing diseases, provides conditions for implementing accurate treatment, provides conditions for selecting proper experimental objects for relevant research to obtain good experimental results and avoid unnecessary waste, and has the advantages of low cost, relatively simple operation and convenient use.

In addition, in examples 1 to 3, the detection of the content of antigen-specific T cells is based on the secretion of cells secreted by activated antigen-specific T cells, and as the present invention, the detection of the content of antigen-specific T cells may also be based on the proliferation status of activated antigen-specific T cells or cell surface markers of activated antigen-specific T cells:

when the proliferation state of the activated antigen-specific T cells is detected, the detection principle is that the antigen-specific T cells can proliferate after being activated, and the proliferation speed of the antigen-specific T cells is higher than that of the antigen-specific T cells which are not activated, so that whether the antigen-specific T cells are activated or not can be determined by detecting the proliferation speed and the proliferation state of the antigen-specific T cells, and the content of the antigen-specific T cells can be determined.

When the cell surface marker based on the activated antigen specific T cell is used for detection, the detection principle is that after the antigen specific T cell is activated, the cell surface can express a plurality of specific molecules, which indicates that the antigen specific T cell is the activated antigen specific T cell, and whether the antigen specific T cell is activated or not and the content of the antigen specific T cell can be determined by detecting the existence and the content of the specific molecules on the cell surface.

The detection of the proliferation state of the antigen-specific T cells after activation is carried out by any one of a decrease detection method based on an exogenous marker, an expression detection method based on a molecular marker, an increase detection method based on a metabolic marker, a change detection method based on the biomass of a cell population, and a change detection method based on the number of cells; wherein, the reduction detection method based on the exogenous markers is that protein binding dyes CFDA-SE, CellTrace and lipophilic dyes PKH67 and PKH26 are labeled and then detected by a flow cytometer, and the molecular markers can be Ki-67 and PCNA; the metabolic marker based on in the metabolic marker based increase detection method may be a redox metabolic marker and a DNA metabolic marker; in the method for detecting a change in biomass based on a cell population, the biomass based on the cell population may include one or more of a DNA content and a protein content.

The cell surface marker activated by the antigen-specific T cells may be one or more of CD137, CD69, CD38, CD44, CD62L, or CD25 when detected based on the cell surface marker of activated antigen-specific T cells.

In addition, in examples 1 and 2, the antigen selected is a cancer cell antigen and a type 1 diabetes-associated antigen, and as the present invention, the antigen may be used as needed in association with any disease, for example, an antigen associated with autoimmune disease, regardless of whether the detection is performed based on cell secretion secreted by activated antigen-specific T cells, proliferation status of activated antigen-specific T cells, or cell surface markers of activated antigen-specific T cells.

In addition, in example 1, the antigen is a cancer cell lysate, and in example 2, the antigen is an antigen protein associated with type 1 diabetes, and as the present invention, the antigen may be any one or more of a cancer cell lysate, an antigen protein, a polypeptide, a cell lysate, a tissue lysate, DNA, RNA, an exosome (exosome) lysate, a cell secretory particle lysate, a carbohydrate, and a lipid, regardless of whether detection is performed based on a cell secretion secreted from an activated antigen-specific T cell, a proliferation state of an activated antigen-specific T cell, or a cell surface marker of an activated antigen-specific T cell.

In addition, in examples 1 to 3, the material for preparing nanoparticles selected was PLGA, and as the present invention, the material for preparing nanoparticles may be any other material for preparing nanoparticles, other than PLGA, according to the use requirements, regardless of whether the detection is performed based on the cell secretion secreted by the activated antigen-specific T cells, the proliferation state of the activated antigen-specific T cells, or the cell surface marker of the activated antigen-specific T cells.

In addition, in the preparation process of the detection nanoparticles of example 1 and example 2, the antigen is directly used for entrapment, and finally the nanoparticle containing the antigen is obtained, and in example 1, the antigen is a cell lysate, and in example 2, the antigen is a protein, and in practice, no matter which one of the cell secretion secreted by the activated antigen-specific T cell, the proliferation state of the activated antigen-specific T cell, or the cell surface marker of the activated antigen-specific T cell is used for detection, when the antigen is directly entrapped, the antigen can be any one or more of a cell lysate, a protein, a plasmid, a polypeptide, DNA, RNA, a tissue lysate, an exosome lysate, a cell secretory particle lysate, a carbohydrate, and a lipid.

In addition, in the preparation of the detection nanoparticle as in example 3, whether the detection is based on any one of the cell secretion secreted from the activated antigen-specific T cell, the proliferation state of the activated antigen-specific T cell, or the cell surface marker of the activated antigen-specific T cell, genetic material required for antigen synthesis associated with a disease is entrapped, thus finally obtaining the detection nanoparticle entrapping such genetic material, which may be derived from any one or more of cell lysate, plasmid, DNA, RNA, tissue lysate, exosome lysate, cell secretion particle lysate.

In addition, in examples 1 to 3, no other substance is added to the mixture of the nanoparticles and the sample to be tested during the detection of the antigen-specific T cells to enhance the detection, and in practical use, no matter which one of the cell secretion secreted by the activated antigen-specific T cells, the proliferation status of the activated antigen-specific T cells, or the cell surface marker of the activated antigen-specific T cells is used for detection, any one or more of interleukin, endotoxin, cytokine, chemical factor, chemokine, metformin, berberine, artemisinin, and the like can be added as required during the treatment of the sample to be tested by the nanoparticles to enhance the sensitivity of the detection technique.

In addition, in the embodiment 1-3, the detected cell secretion is one, in practice, a plurality of cell secretions can be selected for detection according to needs, the cell secretions in the embodiment 1-3 are interferon-gamma, and in practice, the cell secretions can be any one or more of interleukin 1, interleukin 2, transforming growth factor- β, tumor necrosis factor- α, interleukin 6, interleukin 7, interleukin 15, interleukin 21, IP-10, interleukin 17, perforin and granzyme and interleukin 10;

in addition, in examples 1 to 3, the method for detecting a cell secretion by detecting a T cell secretion is an immunological detection method, and in practice, any of an immunological detection method, a biological activity measurement method, and a molecular biological detection method may be used regardless of whether the detection is performed based on a cell secretion secreted from an activated antigen-specific T cell, a proliferation state of an activated antigen-specific T cell, or a cell surface marker of an activated antigen-specific T cell; in examples 1 to 3, the immunological detection method is enzyme-linked immunospot (ELISPOT), and practically any one or more of enzyme-linked immunosorbent assay, flow cytometry assay for intracellular staining, Cytometric Bead Array, multimer technique, multi-cytokine assay, high performance liquid chromatography, and LC-MS may be selected.

In examples 1 to 3, the sample to be tested is Peripheral Blood Mononuclear Cells (PBMCs), and in practice, the sample to be tested may be any one or more of peripheral blood mononuclear cells, blood, plasma, serum, lesion site tissue, and lesion site cells, regardless of whether the detection is performed based on cell secretion secreted by activated antigen-specific T cells, proliferation state of activated antigen-specific T cells, or cell surface markers of activated antigen-specific T cells.

In examples 2 and 3, the disease-related antigens include Glutamic Acid Decarboxylase (GAD) and preproinsulin (preproinsulin), and practically, the disease-related antigens include type 1 diabetes mellitus-related antigens such as proinsulin (preproinsulin), insulin (insulin), Islet Cell Antibody (ICA), β cell lysate, ZnT8, chroman A, islet-specific glucose-6-phosphatealytical subunit-related protein (IGRP), CD38, and tyrosine phosphatase (IA-2), as antigens for detecting type 1 diabetes mellitus.

In addition, the detection nanoparticles of examples 1-3 are nanoparticles, and as the present invention, peripheral blood mononuclear cells, the detection nanoparticles can be any substance having a size of nanometer level containing a disease-associated antigen or genetic material required for synthesizing the antigen, such as any one or more of liposomes, nanoemulsions, exosomes, exosome analogs, viruses, cell secretory particles, cell secretory particle analogs, and the like.

In addition, in examples 1 to 3, the material for preparing the detection nanoparticles includes a medical polymer material, and in practice, peripheral blood mononuclear cells, and the material for preparing the detection nanoparticles includes any one or more of a medical polymer material, a material required for preparing liposomes, a metal material required for preparing nanoparticles, and a non-metal material required for preparing nanoparticles.

In addition, the present invention also provides a kit for detecting antigen-specific T cells, which comprises the above-mentioned detection nanoparticles, and therefore, the antigen-specific T cell content can be detected by the antigen-specific T cell detection method of the present invention.

Claims

1. A detection nanoparticle for antigen-specific T cell content detection, comprising:

for activating the antigen-specific T cells in the detection of the amount of the antigen-specific T cells based on any one or more of a cell secretion secreted by the activated antigen-specific T cells, a proliferation status of the activated antigen-specific T cells, or a cell surface marker of the activated antigen-specific T cells in a sample to be tested,

the detection nanoparticles are loaded with an antigen associated with a disease or with genetic material required for synthesis of an antigen associated with a disease,

wherein the detection nanoparticles have a nanometer size, and can be phagocytized by antigen-presenting cells in the sample to be tested to release the antigen or generate the antigen based on the genetic material when the sample to be tested is processed, so that the antigen-presenting cells present the released or generated epitope of the antigen to the surface of the antigen-presenting cells to allow the antigen-specific T cells to recognize and activate the antigen-specific T cells,

the antigen-specific T cell is an antigen-specific T cell associated with the disease.

2. The detection nanoparticle according to claim 1, wherein:

wherein the particle size of the detection nano-particles is 10-2000 nm.

3. The detection nanoparticle of claim 2, wherein:

wherein the particle size of the detection nano-particles is 30-1000 nm.

4. The detection nanoparticle of claim 2, wherein:

wherein the particle size of the detection nanoparticles is 100-500 nm.

5. The detection nanoparticle according to claim 1, wherein:

wherein the antigen comprises one or more of an antigen associated with a cancer disease and an antigen associated with an autoimmune disease.

6. The detection nanoparticle of claim 5, wherein:

wherein the autoimmune disease is type 1 diabetes.

7. The detection nanoparticle according to claim 1, wherein:

wherein the detection nanoparticles are loaded with an antigen associated with a disease,

the antigen is any one or more of cell lysate, protein, plasmid, DNA, RNA, polypeptide, tissue lysate, exosome lysate, cell secretory granule lysate, carbohydrate and lipid.

8. The detection nanoparticle according to claim 1, wherein:

wherein the detection nanoparticles contain genetic material required for antigen synthesis associated with a disease,

the genetic material is derived from any one or more of cell lysate, plasmid, DNA, RNA, tissue lysate, exosome lysate, cell secretory granule lysate.

9. The detection nanoparticle according to claim 1, wherein:

the detection nanoparticles are prepared from any one or more of medical high polymer materials, materials required for preparing liposome, metal materials required for preparing nanoparticles and non-metal materials required for preparing nanoparticles.

10. The detection nanoparticle according to claim 1, wherein:

the detection nanoparticles are any one or more of nanoparticles, liposomes, nanoemulsions, exosomes, exosome analogs, viruses, cell secretory granules and cell secretory granule analogs.

11. The detection nanoparticle according to claim 1, wherein:

wherein the antigen is distributed in any one or two positions of the inner part of the detection nano-particle and the surface of the detection nano-particle.

12. The detection nanoparticle according to claim 1, wherein:

wherein the sample to be detected is any one or more of blood, peripheral blood mononuclear cells, focus part tissues and focus part cells.

13. The detection nanoparticle according to claim 1, wherein:

wherein the cell secretion comprises interferon-gamma, interleukin 1, interleukin 2, transforming growth factor- β, tumor necrosis factor- α, interleukin 6, interleukin 7, interleukin 15, interleukin 21, IP-10, interleukin 17, perforin and one or more of granzyme and interleukin 10.

14. The detection nanoparticle according to claim 1, wherein:

wherein, when the detection nano-particles process the sample to be detected, any one or more of interleukin, endotoxin, cytokine, chemical factor, chemotactic factor, metformin, berberine, artemisinin and artemisinin analogue is added.

15. The detection nanoparticle according to claim 1, wherein:

wherein the cellular secretion is detected using any one or more of an immunological assay, a biological activity assay, and a molecular biological detection method.

16. The detection nanoparticle of claim 15, wherein:

the cell secretion detection method is any one or more of an enzyme-linked immunospot assay, an enzyme-linked immunosorbent assay, an intracellular staining flow cytometry assay, a Cytometric Bead Array, a polymer technology, a multi-cytokine detection method, a high performance liquid chromatography and a liquid chromatography-mass spectrometry.

17. The detection nanoparticle according to claim 1, wherein:

wherein the cell secretion is one or more based on the kind of the cell secretion.

18. The detection nanoparticle according to claim 1, wherein:

wherein the detection of the proliferation state of the antigen-specific T cells after activation is performed by one or more of a decrease detection method based on an exogenous marker, an expression detection method based on a molecular marker, an increase detection method based on a metabolic marker, a change detection method based on the biomass of a cell population, and a change detection method based on the number of cells.

19. The detection nanoparticle of claim 18, wherein:

wherein, the reduction detection method based on the exogenous markers is that protein binding dyes CFDA-SE, CellTrace and lipophilic dyes PKH67 and PKH26 are labeled and then detected by a flow cytometer.

20. The detection nanoparticle of claim 18, wherein:

wherein, the molecular marker based in the expression detection method based on the molecular marker is Ki-67 and PCNA.

21. The detection nanoparticle of claim 18, wherein:

wherein the metabolic marker-based increase detection method is based on a redox metabolic marker and a DNA metabolic marker.

22. The detection nanoparticle of claim 18, wherein:

wherein, in the method for detecting the change based on the biomass of the cell population, the biomass based on the cell population comprises one or more of DNA content and protein content.

23. The detection nanoparticle according to claim 1, wherein:

wherein the cell surface marker of antigen-specific T cell activation is one or more of CD137, CD69, CD38, CD44, CD62L or CD 25.

24. A preparation method of detection nanoparticles for detecting the content of antigen-specific T cells is characterized in that the detection nanoparticles are nanoparticles, and the preparation process comprises the following steps:

step 1, adding a first predetermined volume of an aqueous phase solution containing antigens with a first predetermined concentration or genetic materials required for antigen synthesis into a second predetermined volume of an organic phase containing a medical polymer material with a second predetermined concentration;

2, performing ultrasonic treatment on the mixture of the antigen or the genetic material required by antigen synthesis obtained in the step 1 for more than 2 seconds;

step 3, adding the mixture obtained after the treatment in the step 2 into a third preset volume of emulsifier aqueous solution with a third preset concentration, and carrying out ultrasonic treatment for more than 5 seconds;

step 4, adding the mixture obtained after the treatment in the step 3 into a fourth preset volume of the emulsifier aqueous solution with a fourth preset concentration, and stirring until preset stirring conditions are met;

step 5, centrifuging the mixed solution which is processed in the step 4 and meets the preset stirring condition for more than 7 minutes at the rotating speed of more than 8000RPM, removing the supernatant, re-suspending the remaining precipitate in water, repeating the steps for many times, and finally obtaining the final precipitate from which the supernatant is removed;

step 6, suspending the final precipitate obtained in the step 5 in a fifth predetermined volume of a lyoprotectant with a fifth predetermined concentration to obtain a suspension;

and 7, carrying out freeze drying treatment on the suspension obtained in the step 6 to obtain freeze-dried powder. The lyophilized powder containing detection nanoparticles was used to detect the content of the antigen-specific T cells.

25. The method of claim 24, wherein:

wherein the ratio of the first predetermined volume to the second predetermined volume ranges from 1:2 to 1:50, the ratio of the second predetermined volume to the third predetermined volume ranges from 1:1.5 to 1:100, and the ratio of the third predetermined volume to the fourth predetermined volume ranges from 1:3 to 1: 50.

26. The method of claim 24, wherein:

wherein the organic solvent in the organic phase is dichloromethane.

27. The method of any one of claims 24-26, wherein:

wherein the medical high polymer material is polylactic acid-glycolic acid copolymer.

28. The method of claim 24, wherein:

wherein the emulsifier aqueous solution is a polyvinyl alcohol aqueous solution.

29. The method of manufacturing according to claim 24 or 28, wherein:

wherein the first predetermined concentration is in a range greater than 0.01ng/mL, the second predetermined concentration is in a range of 0.5mg/mL-500mg/mL, the third predetermined concentration is in a range of 0.5mg/mL-500mg/mL, and the fourth predetermined concentration is in a range of 0.5mg/mL-500 mg/mL.

30. The method of any one of claims 24-26, wherein:

wherein the preset stirring condition is that the organic solvent is completely volatilized.

31. The method of claim 24, wherein:

wherein the freeze-drying protective agent is sugar, and the content percentage of the freeze-drying protective agent is more than 1%.

32. The method of manufacturing according to claim 24 or 31, wherein:

wherein the freeze drying treatment specifically comprises the following steps:

freezing the suspension obtained in the step 5 at the temperature of-80 ℃ for more than 8 hours, and then using a freeze dryer to freeze and dry for more than 12 hours.

33. A kit for detecting antigen-specific T cells, comprising:

the detection of the nano-particles is carried out,

wherein the detection nanoparticle is the detection particle of any one of claims 1-23.

34. A method for detecting the content of antigen-specific T cells is characterized by comprising the following steps:

treating a test sample containing antigen-presenting cells and T cells with detection nanoparticles containing an antigen associated with a disease or containing genetic material required for synthesis of an antigen associated with a disease to present epitopes of the antigen released or synthesized using the genetic material to the surface of the antigen-presenting cells to allow the antigen-specific T cells to recognize and activate the antigen-specific T cells;

detecting the antigen-specific T cell content of the test sample based on cell secretions secreted by the activated antigen-specific T cells, the proliferation status of the activated antigen-specific T cells, or cell surface markers of the activated antigen-specific T cells,

wherein the detection nanoparticle is the detection nanoparticle of any one of claims 1-23.