PD-1 protein expression detection quality control microsphere as well as preparation method and application thereof

Abstract

The invention provides a PD-1 protein expression detection quality control microsphere as well as a preparation method and application thereof. The microsphere product for PD-1 protein expression detection quality control comprises a positive microsphere coupled with the PD-1 protein and a negative microsphere not coupled with the PD-1 protein. According to the method, the microspheres containing ferroferric oxide magnetic cores and with the surfaces modified with streptavidin are obtained through screening, the PD-1 quality control microspheres with the fixed positive percentage are prepared by coupling different contents of PD-1 proteins on the surfaces of the microspheres through the determined preparation process, and the preparation process is stable and low in cost. By using the quality control product, the quality control of the PD-1 protein detection operation process in a biological sample can be enhanced, and early warning can be given to the detection fluctuation exceeding the 95% confidence interval of the quality control microsphere positive percentage.

Description

Technical Field

[0001] The present invention belongs to the field of biological sample immunoassay quality control products, and specifically relates to a PD-1 protein expression detection quality control microsphere, a preparation method and application thereof. Background Art

[0002] Programmed cell death protein 1 (PD-1), also known as CD279, is a type I transmembrane glycoprotein belonging to a class of immunoglobulin superaccelerator members. Its structure consists of 288 amino acids. It is an important immunosuppressive molecule on the surface of T cells, mainly expressed on the surface of T lymphocytes. It transmits inhibitory signals by binding to receptors on target cells, thereby inhibiting the proliferation of T lymphocytes. Under normal circumstances, the immune system will respond to foreign antigens that accumulate in lymph nodes or spleens, promoting the proliferation of antigen-specific T cells. The binding of programmed cell death protein-1 (PD-1) to programmed cell death protein-ligand 1 (PD-L1) can transmit inhibitory signals, thereby inhibiting T cell proliferation, inhibiting T cell inflammatory activity, regulating the immune system and promoting self-tolerance. In clinical applications, detecting the expression level of PD-1 on the surface of total T cells (CD3+), helper T cells (CD4+), and cytotoxic T cells (CD8+) in biological samples has important clinical significance in diseases such as tumors, infectious diseases, autoimmune diseases, and blood diseases.

[0003] At present, flow cytometry is often used in clinical practice to detect the expression of PD-1 protein on the surface of lymphocytes in biological samples. Flow cytometry uses scattered light signals and fluorescent signals generated by cells in the liquid flow through laser irradiation to reflect the physical and chemical characteristics of cells, such as cell size, granularity, and surface antigen expression. By using different antibodies to label lymphocyte surface antigens in biological samples, the proportion of PD-1 protein-positive cells in different types of lymphocytes can be obtained, thereby assisting clinicians in making judgments on the patient's condition.

[0004] To obtain accurate and stable analysis results by analyzing biological samples through flow cytometry, quality control should be carried out throughout. There are two types of existing flow cytometry quality control products. One type is the quality control microspheres used for quality control of flow cytometer equipment, which are used for daily maintenance and status monitoring of instruments and equipment; the other type comes from real clinical samples, including whole blood quality control products and blood cell products preserved by freeze-drying. Since they come from clinical samples, the detection process is the same as that of real samples, and can be used for operation process quality control, especially as positive controls, used to calibrate analytical methods when analyzing results, or used for absolute cell counting. Although the above two types of flow cytometry quality control products have been widely used, they have the following problems:

[0005] 1) Quality control microspheres: Quality control microspheres prepared by existing technologies are mostly equipment calibration microspheres. Although they have the advantages of mature preparation process, controllable quality and stable performance, due to the optical differences between ordinary microspheres and lymphocytes, they cannot be detected simultaneously with lymphocytes using the same detection procedure on the flow platform; and microspheres and cells have significant biological differences, which makes it impossible for microspheres to be directly used for quality control of specific target staining processes on flow platforms. Therefore, they can only be used for standardized settings and compensation adjustments between photoelectric detectors, fluid parts and detection channels of flow cytometer equipment. In principle, the quality control microspheres of the instrument should be run after the daily startup process. The ideal state is to calibrate the optical path before using each different detection item to ensure that the instrument is in the best performance state and the coefficient of variation is less than the acceptable range in the instrument software, but it is more difficult to achieve.

[0006] 2) Whole blood quality control products prepared from real samples: Most of them are freeze-dried products of blood products or cell products. On the one hand, the expression of PD-1 protein in biological samples is continuous expression, that is, there is no obvious grouping boundary between positive and negative cells, and it is difficult to clearly divide the positive and negative boundaries of PD-1 protein expression by analyzing the flow detection result graph. At the same time, the half-life of PD-1 protein in the blood is short and its stability is poor. There is currently no gold standard for accurate quantification of PD-1 protein on the surface of lymphocytes, making the quality control product assignment process difficult. These all make it difficult to use clinical samples to prepare PD-1 positive quality control products, that is, it is difficult to accurately quantify or preserve them for a long time, which is also the biggest risk of instability of such quality control products. Therefore, there are currently no commercialized PD-1 positive quality control products. On the other hand, the preparation process of quality control products using real samples is complicated. For example, the freeze-drying process is highly dependent on equipment and process conditions, which can easily cause batch-to-batch differences in products. At the same time, freeze-dried quality control products need to be reconstituted when used, which not only increases the complexity of the operation, but also introduces unstable factors. Non-freeze-dried quality control products have harsh transportation and storage conditions and a short shelf life. The shelf life of other types of existing whole blood quality control products is only 3 months. The above problems not only make it difficult to prepare PD-1 quality control products using real samples, but also introduce higher risks to the PD-1 detection process in biological samples using such quality control products.

[0007] Therefore, there is an urgent need in this field to develop a quality control product with stable quality control performance, simple and reliable preparation process, and relatively simple use and storage conditions, which can meet the needs of quality control of the stability of the whole process of PD-1 protein detection in clinical samples and provide a reminder of related risks. The present invention overcomes the shortcomings of the prior art and develops a quality control microsphere for the whole process of PD-1 detection in blood samples and its preparation process and quality control method. Summary of the invention

[0008] Therefore, the purpose of the present invention is to overcome the defects in the prior art and provide a PD-1 protein expression detection quality control microsphere, a preparation method and application thereof.

[0009] Before describing the present invention, the terms used in this article are defined as follows:

[0010] The term "PD-1" refers to programmed cell death protein 1, also known as PD-1 or CD279, which is an important immunosuppressive molecule that mainly exists on the surface of immune cells, such as T cells and B cells. The term "PD-1" herein includes human PD-1 protein and its recombinant protein. The full length of human PD-1 protein contains 288 amino acids, which can be further divided into the following regions: signal peptide: located at amino acids 1-20; extracellular region: located at amino acids 21-170; transmembrane region: located at amino acids 171-191; intracellular region: located at amino acids 192-288.

[0011] The term "total T cells" refers to the sum of various T lymphocytes.

[0012] The term "helper T cells" refers to: CD4 + T cells, also known as helper T cells, abbreviated as Th, express CD4.

[0013] The term "cytotoxic T cells" refers to: CD8+ T cells, also known as cytotoxic T lymphocytes, abbreviated as CTL, expressing CD8.

[0014] The term "Biotin" means: Biotin.

[0015] The term "SA" refers to: streptavidin, also known as streptavidin in English.

[0016] The term "NHS carboxyl" refers to: NHS-activated carboxyl, NHS refers to N-hydroxysuccinimide, the English name is N-hydroxy succinimide.

[0017] The term "TrisHCl buffer" refers to a buffer containing tris (hydroxymethyl)aminomethane (Tris).

[0018] The term "EDTA buffer" refers to a buffer containing ethylenediaminetetraacetic acid (EDTA).

[0019] The term "MES buffer" refers to a buffer containing 2-morpholineethanesulfonic acid (MES).

[0020] The term "CD45" refers to a membrane protein on white blood cells that is expressed in all white blood cells. T lymphocytes belong to white blood cells. Clinically, CD45 expression is used to distinguish white blood cells on flow cytometry, and then T lymphocytes are screened out from white blood cells.

[0021] To achieve the above-mentioned object, the first aspect of the present invention provides a positive microsphere for quality control of PD-1 protein expression detection, wherein the positive microsphere is prepared by coupling a microsphere having a modified group on the surface with a PD-1 protein; wherein:

[0022] The microspheres are magnetic or non-magnetic microspheres, preferably selected from one or more of the following: microspheres containing a ferroferric oxide magnetic core, microspheres containing a ferrous oxide magnetic core, and microspheres without a magnetic core, more preferably microspheres containing a ferroferric oxide magnetic core or microspheres containing a ferrous oxide magnetic core, and most preferably microspheres containing a ferroferric oxide magnetic core.

[0023] According to the first aspect of the present invention, the positive microspheres, wherein:

[0024] The modifying group is selected from one or more of the following: streptavidin, hydroxyl, carboxyl, NHS carboxyl, preferably streptavidin or NHS carboxyl, most preferably streptavidin; and / or

[0025] The PD-1 protein is bound to biotin;

[0026] Preferably, the mass ratio of the positive microspheres to the PD-1 protein is 800 to 6667:1, more preferably 1000 to 6250:1, and further preferably 1587 to 3226:1.

[0027] The second aspect of the present invention provides a microsphere product for quality control of PD-1 protein expression detection, the microsphere product comprising: the positive microspheres described in the first aspect and negative microspheres not coupled to the PD-1 protein;

[0028] Preferably, the volume ratio of the negative microspheres to the positive microspheres is 1-4:1-4, more preferably 1-2:1-2, and most preferably 2:1.

[0029] According to the positive microspheres described in the first aspect of the present invention and the microsphere product described in the second aspect, wherein:

[0030] The particle size of the negative microspheres and / or the positive microspheres is 1 to 6 μm, preferably 1.5 to 5 μm, and more preferably 2 to 4 μm.

[0031] The specific gravity of the positive microspheres and / or microsphere products is 1-5 g / cm 3 , preferably 2 to 4 g / cm 3 , more preferably 2 to 3 g / cm3 : and / or

[0032] The background fluorescence of the negative microspheres and / or the positive microspheres is different from that of lymphocytes.

[0033] The third aspect of the present invention provides a method for preparing the microsphere product according to the second aspect, the method comprising the following steps:

[0034] 1) suspending magnetic or non-magnetic material microspheres in a buffer for dispersion, and separating the dispersed microsphere suspension buffer into negative microspheres and positive microsphere raw materials;

[0035] 2) coupling the dispersed positive microsphere raw material in step 1) with the PD-1 protein bound to biotin;

[0036] 3) purifying the microspheres obtained by coupling in step 2) to obtain the positive microspheres;

[0037] 4) Mixing the negative microspheres and the positive microspheres to a fixed volume to obtain the microsphere product.

[0038] According to the method of the third aspect of the present invention,

[0039] In the step 1), the final concentration of the magnetic or non-magnetic material microsphere suspension is 0.1 mg / mL to 0.5 mg / mL, preferably 0.1 mg / mL to 0.3 mg / mL, and most preferably 0.1 mg / mL; and / or

[0040] The step 3) further comprises: washing the microspheres coupled in step 2) to remove the free PD-1 protein, centrifuging and discarding the supernatant, adding a buffer, and vortexing to mix, so as to obtain the positive microspheres;

[0041] Preferably, in step 3), the centrifugation time is 2 to 10 min, more preferably 2 to 6 min, and even more preferably 2 to 4 min.

[0042] According to the method of the third aspect of the present invention, the step 2) further comprises: adding the positive microsphere material dispersed in step 1) to the PD-1 protein bound to biotin, vortexing to obtain a dispersion, and then vertically mixing the dispersion to obtain the coupled microspheres;

[0043] Preferably, the concentration of the biotin-bound PD-1 protein added is 0.15 ng / μg to 1.25 ng / μg, more preferably 0.16 ng / μg to 1.00 ng / μg, and further preferably 0.31 ng / μg to 0.63 ng / μg; and / or

[0044] Preferably, the coupling time is 30 min to 1.5 h, more preferably 40 min to 1.0 h, most preferably 1.0 h.

[0045] According to the method of the third aspect of the present invention, the step 4) further comprises: vortexing the negative microspheres in step 1) and the positive microspheres in step 3) and resuspending them in a buffer to obtain the microsphere product;

[0046] Preferably, the buffer is selected from one or more of the following: phosphate buffer, carbonate buffer, TrisHCl buffer, EDTA buffer, borate buffer, acetate buffer, MES buffer, more preferably selected from one or more of the following: phosphate buffer, TrisHCl buffer, carbonate buffer, most preferably phosphate buffer; and / or

[0047] Preferably, the pH value of the buffer solution is 7 to 8.5, more preferably 7.0 to 8.0, and even more preferably 7.2 to 7.5.

[0048] The fourth aspect of the present invention provides the use of the positive microspheres described in the first aspect or the microsphere products described in the second aspect in the preparation of products for detecting cell PD-1 protein expression;

[0049] Preferably, the product is a quality control product.

[0050] According to the application of the fourth aspect of the present invention, the cells are T lymphocytes, and the T lymphocytes are preferably selected from one or more of the following: total T cells, helper T cells, and cytotoxic T cells.

[0051] According to a specific embodiment of the present invention, the present invention provides a method for preparing a quality control product for detecting PD-1 expression in lymphocytes of a blood sample, comprising the following steps:

[0052] Step 1. Dispersion of microsphere material.

[0053] Step 2. Coupling of microspheres with PD-1 protein.

[0054] Step 3. Purification of quality control microspheres.

[0055] Step 4. Mix and fix the volume of quality control microspheres.

[0056] Microsphere materials specifically refer to: particle size 2.5-3.5μm, specific gravity 2-3g / cm 3 , microspheres with surface modified with streptavidin.

[0057] The components of the microsphere dispersion liquid are: phosphate buffer (pH 7-8), and the microspheres are in the dispersion liquid at a suitable concentration.

[0058] The microspheres are coupled to the PD-1 protein, and the amount of coupled protein ranges from 0.15ng / μg to 1.25ng / μg.

[0059] Quality control microspheres are divided into negative quality control microspheres and positive quality control microspheres. The mixing ratio of the two microspheres is related to clinical needs. Quality control microspheres with different positive percentages are prepared, and the mixing ratio of the two microspheres is 20%-80%.

[0060] A quality control product prepared according to the method for detecting PD-1 expression in lymphocytes.

[0061] The quality control method of the quality control product comprises the steps of:

[0062] 1). Binding of quality control microspheres and PD-1 antibodies.

[0063] 2). Use cleaning solution to clean the combined product.

[0064] 3). Detection of reaction products.

[0065] 4). Analysis of test results.

[0066] The results analysis method is as follows:

[0067] The positive percentage of PD-1 quality control microspheres should be within its 95% confidence interval. If the positive percentage fluctuation range exceeds the 95% confidence interval, it indicates that the test is abnormal and the test process needs to be checked and calibrated.

[0068] According to another specific embodiment, based on step 1 to step 4: the microspheres have a particle size of 1-6 μm and a specific gravity of 1-4 g / cm 3 , magnetic or non-magnetic microspheres (materials) with streptavidin modified on the surface.

[0069] The dispersion of the microsphere material refers to suspending the microsphere material in a phosphate buffer at a certain final concentration, and the final concentration of the suspension can be 0.1 mg / mL to 0.5 mg / mL, and 0.1 mg / mL is preferred in this embodiment.

[0070] The present invention first screens the microspheres, and the screening conditions include the material, particle size, specific gravity, background fluorescence and properties of the surface modification groups of the microspheres.

[0071] Currently commonly used microspheres include microspheres containing ferroferric oxide magnetic cores, ferric oxide magnetic cores, and microspheres without magnetic cores, and surface modifications with hydroxyl, carboxyl, NHS carboxyl or streptavidin. Considering that the selected microsphere raw materials need to take into account the stability of the production process and the convenience of use. The present embodiment gives priority to microspheres containing ferroferric oxide magnetic cores and surface modifications with streptavidin. Because the microspheres contain ferroferric oxide magnetic cores, their specific gravity can be maintained within a suitable range, so that they can be unified with the operating conditions of clinical samples and are easy to use. The streptavidin modification on the surface can ensure the stability of the production process.

[0072] At the same time, the material and particle size of the microspheres determine the voltage range of the flow detection. Considering that the quality control microspheres of the present invention will be tested together with clinical samples in the future, microspheres whose detection conditions are compatible with clinical samples should be selected. In this embodiment, microspheres with a particle size of 2-5 μm and containing ferroferric oxide magnetic cores are preferred;

[0073] In addition, the specific gravity of the microspheres affects the centrifugal rate and time during the operation. The present invention screened microspheres with different specific gravity and determined that the specific gravity was greater than 2g / cm 3 The centrifugation conditions of the microspheres are consistent with those of the corresponding cells in clinical samples;

[0074] The background fluorescence of the microspheres and the properties of the surface modification groups will also affect the performance of the quality control microspheres. Combined with the subsequent protein labeling and detection requirements, the background fluorescence of the microsphere raw materials can be controlled to be distinguished from lymphocytes.

[0075] The type of modification group on the surface of the microsphere determines the subsequent coupling method between the microsphere and the target protein. Currently, the more common modification groups include chemical groups such as hydroxyl, carboxyl, and amino groups, and biological protein groups such as IgG and streptavidin. Microsphere surface protein coupling methods include chemical cross-linking methods such as the glutaraldehyde method and the carbodiimide method, as well as biological coupling methods represented by the streptavidin-biotin system. The present invention selects the streptavidin-biotin coupling method. Compared with chemical cross-linking, the streptavidin-biotin coupling process is simple, the process is controllable, the coupling efficiency is high, and the product stability is good. The specific principles are as follows:

[0076] Streptavidin (SA) is a protein secreted by Streptomyces, with a molecular weight of about 65 kD. It is composed of four identical peptide chains, each of which can bind to one biotin, and the affinity constant (K) of the two is 10 15 mol / L, it has a stronger binding force than that between antigen and antibody and is less affected by temperature, pH and reagents, so it is widely used in immune response.

[0077] Biotin is a common small molecule conjugate in immune response, and is often used to label proteins and antibodies. Antigens labeled with biotin can be quickly and stably coupled to a solid phase carrier (microsphere) modified with streptavidin with the help of the streptavidin-biotin system.

[0078] Furthermore, the present invention optimizes the protein coupling conditions.

[0079] In order to meet the needs of clinical use, the present invention studies the concentration of coupled protein, and studies the coupling concentrations of 1.25 ng / μg, 0.63 ng / μg, 0.31 ng / μg and 0.16 ng / μg, respectively, and judges the suitability of the coupling concentration by the fluorescence value of the labeled quality control microspheres in the detection channel. In this embodiment, a coupling concentration of 0.31 ng / μg is preferred.

[0080] Furthermore, the present invention optimizes the coupling reaction time, coupling for different times such as 10 min, 20 min, and 40 min, respectively, and uses a vertical mixer to rotate and mix for 20 minutes (20 rpm / min) at room temperature (25° C.) to assist the coupling reaction process.

[0081] The coupled microspheres are washed to remove the free PD-1 protein. The steps include:

[0082] Centrifuge at 10000 rpm for 3 min and carefully aspirate the supernatant;

[0083] Add 50 μL 1× PBS (pH 7.4) and vortex to mix;

[0084] Repeat the above steps 2 times.

[0085] Furthermore, the present invention optimizes the mixing ratio of negative quality control microspheres and positive quality control microspheres.

[0086] In this embodiment, the mixing ratio of positive quality control microspheres and negative quality control microspheres is optimized. The microspheres are mixed at conditions of 4:1, 2:1, 1:1, 1:2 and 1:4, respectively, to obtain quality control microspheres with positive percentages of approximately 80%, 66%, 50%, 33%, and 20%. Preferably, the positive quality control microspheres and the negative quality control microspheres are mixed at a ratio of 1:2 to prepare quality control microspheres with a positive percentage of approximately 34%.

[0087] The mixed microspheres were fixed to a fixed volume, and the quality control microspheres were resuspended in 1× PBS to a final concentration of 1 mg / mL.

[0088] The quality control microspheres prepared by the above method are easy to use and have stable performance. Specifically, the quality control microspheres are involved in the entire process of lymphocyte PD-1 detection, including the PD-1 antibody staining process, the flow detection process and the data analysis process. The conditions applicable to the overall process are consistent with those of lymphocytes. The quality control microspheres themselves have stable performance, and the fluorescence is stable on the calibrated flow device. They can give warnings for fluctuations that exceed the 95% confidence interval of the quality control microsphere positive percentage, and prompt the PD-1 staining process and flow device fluctuations.

[0089] On the other hand, the present invention provides a quality control microsphere for PD-1 detection prepared based on the above process.

[0090] In another aspect, the present invention provides a method for quality control using the quality control microspheres, comprising the steps of:

[0091] (1) Quality control microsphere staining

[0092] (2) Flow cytometry detection of reaction products

[0093] (3) Result analysis: Based on the staining conditions of the positive and negative quality control microspheres, quality control of the operation process and the testing equipment was performed.

[0094] The present invention optimizes the preparation process of quality control microspheres to make the properties of quality control microspheres close to those of lymphocytes in clinical samples. The staining operation process and flow platform detection parameters can be consistent with clinical samples, without the need for separate operations, thus simplifying the quality control process. It is only necessary to set the data collection method of the quality control microspheres during data collection, and the process is as follows:

[0095] Before using PD-1 quality control microspheres for the first test, you need to ensure that the flow cytometer has undergone daily quality control and is operating normally, so that you can set up the test template on the flow cytometer. The specific method for setting up the test template is as follows:

[0096] A) Open the flow cytometry software and use the original lymphocyte PD-1 detection template to collect samples of quality control microspheres. The detection conditions and data collection conditions are consistent with the original lymphocyte PD-1 detection conditions;

[0097] B) After completing the data collection of PD-1 quality control microspheres, select the gate tool, select the scatter group of PD-1 quality control microspheres in the CD45 fluorescence and SSC scatter plot, and set the gate name to "PD-1Beads";

[0098] C) Select the scatter plot tool, create a new scatter plot of PD-1 fluorescence and SSC, select the gate tool, and circle the positive gate based on the ratio of PD-1 negative and positive quality control microspheres;

[0099] D) After completing the above operations, save the template as a lymphocyte PD-1 expression detection template containing PD-1 quality control microspheres.

[0100] E) When using PD-1 quality control microspheres for subsequent flow cytometry analysis, the saved lymphocyte PD-1 expression detection template containing PD-1 quality control microspheres can be used.

[0101] After flow cytometry detection, first analyze the test results of the quality control microspheres, and then perform quality control on the detection process. The method is as follows:

[0102] a) Analysis of quality control microsphere results: If the positions of the PD-1 negative and positive quality control microspheres on the PD-1 fluorescence-SSC scatter plot are normal and the positive ratio is normal, then the flow cytometry PD-1 channel detection light path is normal and the staining process is normal;

[0103] b) The position of the PD-1 negative quality control microspheres on the PD-1 fluorescence-SSC scatter plot is normal, the positive percentage is low or there are no positive particles, which indicates that the flow cytometry PD-1 channel detection light path is normal and the staining process is abnormal;

[0104] c) The positions of the PD-1 negative and positive quality control microspheres on the PD-1 fluorescence-SSC scatter plot were shifted overall, and the positive percentage detection value of the quality control microspheres exceeded the 95% confidence interval, indicating that there was an abnormality in the detection optical path of the flow PD-1 channel and that instrument calibration was required.

[0105] Based on the above problems, the present invention mainly solves the following technical problems:

[0106] An innovative quality control microsphere for PD-1 detection has been developed. By screening parameters such as microsphere material, particle size, sedimentation coefficient, and optimizing the microsphere surface labeling process, the preparation of quality control microspheres with two different PD-1 protein contents was achieved with a stable process, providing sensitive system error quality control products for the entire process of PD-1 clinical sample detection. The quality control effect of system errors such as the PD-1 protein immunoassay operation process and the status of the detection equipment can be achieved: a warning is given for fluctuations that exceed the 95% confidence interval of the quality control microsphere positive percentage. The user is guided to judge the reliability of the sample test results. At the same time, the preparation process of the quality control microspheres is stable and the cost is controllable. The present invention provides a preparation process and a method for using the quality control microspheres.

[0107] The present invention provides a quality control microsphere for detecting PD-1 protein in a biological sample and a method for preparing and using the same. Microspheres containing a ferroferric oxide magnetic core and a 2.5-3.5 μm surface modified with streptavidin are obtained by screening, and different amounts of PD-1 protein are coupled to the surface of the microspheres using the preparation process determined by the present invention to prepare PD-1 quality control microspheres with a fixed positive percentage. The preparation process is stable and low-cost; at the same time, the present invention provides a method for using the above-mentioned quality control microspheres in the process of biological sample detection. The use of the above-mentioned quality control products can strengthen the quality control of the PD-1 protein detection operation process in biological samples, and give an early warning for detection fluctuations that exceed the 95% confidence interval of the positive percentage of the quality control microspheres.

[0108] The positive microspheres, microsphere products and preparation methods thereof for PD-1 protein expression detection quality control of the present invention may have but are not limited to the following beneficial effects:

[0109] 1. For the first time, protein-labeled microspheres were used to prepare quality control products for PD-1 detection. This quality control product is an external quality control product that can perform quality control on the entire process of PD-1 protein detection in samples and provide prompts for fluctuations that exceed the 95% confidence interval of the positive percentage of the quality control microspheres.

[0110] 2. Through screening and surface modification, the quality control microspheres are consistent with clinical samples in terms of operation and detection process. The operation is simple and can achieve quality control of system errors in the entire process of PD-1 protein detection.

[0111] 3. The quality control microsphere preparation process based on microsphere surface modification is simple, reliable and stable. The CV of the fluorescence mean value of the three batches of quality control microspheres PD-1 detection is <3%. There is no need for freeze-drying and other processes that have high requirements for equipment and process.

[0112] 4. The quality control microspheres include two quality control microspheres labeled with PD-1 protein at different concentrations. After the two microspheres are mixed, they are used as an independent sample to participate in the entire process of staining and detection. The systematic error of the staining and detection process is judged by the proportion of PD-1 positive quality control microspheres in the flow detection results. The quality control microspheres are sensitive to operational errors or systematic errors in the process, and can give warnings for fluctuations that exceed the 95% confidence interval of the positive percentage of the quality control microspheres, effectively indicating the reliability of the clinical sample test results. The operation process is the same as that of clinical samples, and no additional operation is required.

[0113] 5. The quality control microspheres have good stability and can be stored at 4°C for more than 12 months. Compared with quality control products prepared by existing technologies, they are easier to store and transport. BRIEF DESCRIPTION OF THE DRAWINGS

[0114] The embodiments of the present invention are described in detail below with reference to the accompanying drawings, wherein:

[0115] Figure 1-Figure 7 In the lymphocyte flow cytometry results shown in , the horizontal and vertical axes "SSC-H" are the side light intensity, "FITC-H" is the PD-1 fluorescence intensity, and "CD45 Percp-H" is the CD45 fluorescence intensity.

[0116] Figure 1 The lymphocyte flow cytometry results of the screening of the microsphere product material for PD-1 protein expression detection quality control of the present invention in Examples 1-4 are shown; wherein, Figure 1 A shows the lymphocyte flow cytometry results of the microsphere product material in Example 1; Figure 1 B shows the lymphocyte flow cytometry results of the microsphere product material in Example 2; Figure 1 C shows the lymphocyte flow cytometry results of the microsphere product material in Example 3; Figure 1 D shows the lymphocyte flow cytometry results of the microsphere product material in Example 4.

[0117] Figure 2 The screening of microsphere product materials for PD-1 protein expression detection quality control of the present invention in Comparative Examples 1-3 is shown; wherein, Figure 2 A shows the lymphocyte flow cytometry results of the microsphere product material in Comparative Example 1; Figure 2 The lymphocyte flow cytometry results of the microsphere product material in B; Figure 2 C shows the lymphocyte flow cytometry results of the microsphere product material.

[0118] Figure 3 The fluorescence detection spectrum of the positive microsphere PD-1 protein coupling concentration in the microsphere product for PD-1 protein expression detection quality control prepared in Example 1 is shown; wherein, Figure 3 A shows the fluorescence detection spectrum of the positive microsphere PD-1 protein coupling concentration of 1.25 ng / μg; Figure 3 B shows the fluorescence detection spectrum of the positive microsphere PD-1 protein coupling concentration of 0.63 ng / μg; Figure 3 C shows the fluorescence detection spectrum of the positive microsphere PD-1 protein coupling concentration of 0.31 ng / μg; Figure 3 D shows the fluorescence detection spectrum of the positive microsphere PD-1 protein coupling concentration of 0.16 ng / μg.

[0119] Figure 4 The PD-1 protein fluorescence detection spectrum of the microsphere product prepared in Example 1 for quality control of PD-1 protein expression detection with different mixing ratios of positive microspheres and negative microspheres is shown; wherein, Figure 4A shows the PD-1 protein fluorescence detection results with a mixing ratio of positive microspheres to negative microspheres of 4:1; Figure 4 B shows the PD-1 protein fluorescence detection results with a mixing ratio of positive microspheres to negative microspheres of 2:1; Figure 4 C shows the PD-1 protein fluorescence detection result when the mixing ratio of positive microspheres to negative microspheres is 1:1; Figure 4 D shows the PD-1 protein fluorescence detection result when the mixing ratio of positive microspheres to negative microspheres is 1:2; Figure 4 E shows the PD-1 protein fluorescence detection results when the mixing ratio of positive microspheres to negative microspheres is 1:4.

[0120] Figure 5 The quality control analysis results in Example 6 are shown; wherein, Figure 5 A shows that when all conditions are normal, the positive percentage of flow cytometry after quality control microsphere staining is within the 95% confidence interval; Figure 5 B and Figure 5 C shows that when the staining process is abnormal or the staining antibody fails, the positive percentage of the flow cytometry test after the quality control microsphere staining is lower than the lower limit of the 95% confidence interval; Figure 5 D shows that when the device is in an abnormal state, the positive percentage of flow cytometry after quality control microsphere staining exceeds the 95% confidence interval.

[0121] Figure 6 The quality control microsphere products for quality control of PD-1 protein expression detection prepared in Examples 8-13 are shown; wherein, Figure 6 A shows the flow cytometry effect diagram of the quality control microsphere product for quality control of PD-1 protein expression detection prepared in Example 8; Figure 6 B shows the flow cytometry effect diagram of the quality control microsphere product for quality control of PD-1 protein expression detection prepared in Example 9; Figure 6 C shows the flow cytometry effect diagram of the quality control microsphere product for quality control of PD-1 protein expression detection prepared in Example 10; Figure 6 D shows the flow cytometry effect diagram of the quality control microsphere product for quality control of PD-1 protein expression detection prepared in Example 11; Figure 6 E shows a flow cytometry effect diagram of the quality control microsphere product for quality control of PD-1 protein expression detection prepared in Example 12; Figure 6 F shows the flow cytometry effect diagram of the quality control microsphere product prepared in Example 13 for quality control of PD-1 protein expression detection.

[0122] Figure 7 The quality control microsphere products for quality control of PD-1 protein expression detection prepared in Examples 14-17 are shown; wherein, Figure 7A shows the flow cytometry effect diagram of the quality control microsphere product for quality control of PD-1 protein expression detection prepared in Example 14; Figure 7 B shows the flow cytometry effect diagram of the quality control microsphere product for quality control of PD-1 protein expression detection prepared in Example 15; Figure 7 C shows the flow cytometry effect diagram of the quality control microsphere product for quality control of PD-1 protein expression detection prepared in Example 16; Figure 7 D shows the flow cytometry effect diagram of the quality control microsphere product prepared in Example 17 for quality control of PD-1 protein expression detection. DETAILED DESCRIPTION

[0123] The present invention is further described below by means of specific examples. However, it should be understood that these examples are only used for more detailed and specific description and should not be understood as limiting the present invention in any form.

[0124] This section generally describes the materials and test methods used in the experiments of the present invention. Although many materials and operating methods used to achieve the purpose of the present invention are well known in the art, the present invention is still described as detailed as possible. It is clear to those skilled in the art that in the context, if not specifically stated, the materials and operating methods used in the present invention are well known in the art.

[0125] The reagents and instruments used in the following examples are as follows:

[0126] Reagents:

[0127] Streptavidin microspheres containing ferroferric oxide magnetic core were purchased from Suzhou Beaver Biomedical Engineering Co., Ltd.; phosphate buffer was purchased from Beijing Solebow Technology Co., Ltd.; Biotin-coupled PD-1 protein was purchased from Beijing Biopsies Biotechnology Co., Ltd.

[0128] instrument:

[0129] Flow cytometry instrument, purchased from Beijing Layer Biotechnology Co., Ltd., model LongCyte TM .

[0130] The PD-1 protein in the PD-1 protein coupled with Biotin used in the following Examples 1-17 is recombinant human PD-1, Avi His HEK293 (HEK293 expression), also known as PD1, CD279, PDCD1, SLEB2, and its specific amino acid sequence is as follows:

[0131] LDSPDRPWNPPTFSPALLVVTEGDNATFTCSFSNTSESFVLNWYRMSPSNQTDKLAAFPEDRSQPGQDCRFRVTQLPNGRDFHMSVVRARRNDSGTYLCGAISLAPKAQIKESLRAELRVTERRAEVPTAHPSPSPRPAGQFQ.

[0132] Example 1

[0133] This example is used to illustrate a positive microsphere for quality control of PD-1 protein expression detection of the present invention comprising a ferroferric oxide magnetic core, a quality control microsphere product thereof and a preparation method thereof.

[0134] Step 1. Dispersion of microsphere material

[0135] Microspheres containing ferroferric oxide magnetic cores were selected as raw materials, with a particle size of 2.8 μm and a specific gravity of 2.3 g / cm 3 , the surface is modified with streptavidin.

[0136] The microsphere material was suspended in phosphate buffer at a final concentration of 0.1 mg / mL and divided into two parts with a volume ratio of 2:1. The part accounting for 2 / 3 of the total volume was used as negative microspheres for standby use, and the part accounting for 1 / 3 of the total volume was used as positive microsphere raw material.

[0137] Step 2. Coupling of microspheres with PD-1 protein

[0138] In order to meet the clinical quality control needs, quality control microspheres include negative microspheres and positive microspheres. The method of coupling the positive microsphere raw material with PD-1 protein is as follows:

[0139] Add the recombinant PD-1 protein coupled with Biotin to the dispersed positive microsphere raw material. The concentration of the recombinant PD-1 protein coupled with Biotin is controlled at 0.31 ng / μg microsphere. After the recombinant protein is added, it is thoroughly vortexed and mixed. Place the positive microsphere raw material dispersion containing the recombinant PD-1 protein coupled with Biotin on a vertical mixer to ensure that the microspheres can be fully dispersed in the system during the coupling process while maintaining the uniformity of the coupling. The coupling time is 1 hour.

[0140] Step 3. Purification of quality control microspheres

[0141] The coupled positive microspheres are washed to remove the free PD-1 protein. The following steps are included:

[0142] Centrifuge at 10000 rpm for 3 min and carefully aspirate the supernatant;

[0143] Add 10 mM phosphate buffer, the pH value of the buffer needs to be stable between 7-8, the pH value of the buffer used in this example is 7.3, and vortex to mix;

[0144] Repeat the above steps twice to obtain positive microspheres. The ratio of the obtained positive microspheres to the coupled PD-1 protein is 3226:1.

[0145] Step 4. Mixing and fixing the quality control microspheres

[0146] The negative and positive microspheres prepared in steps 1-3 were thoroughly vortexed and resuspended in 10 mM phosphate buffer solution to a final concentration of 1 mg / mL.

[0147] The mixed quality control microspheres can be stored at 2-8°C for more than one year. Figure 1 A shows the lymphocyte flow cytometry results of the microsphere product material in Example 1 (alternative microsphere 4).

[0148] Examples 2-4, Comparative Examples 1-3

[0149] Examples 2 to 4 and Comparative Examples 1 to 3 are used to illustrate the screening of microsphere product materials for quality control of PD-1 protein expression detection of the present invention.

[0150] The preparation methods of Examples 2 to 4 and Comparative Examples 1-3 are exactly the same as Steps 1 to 3 in Example 1, except that the materials shown in Table 1 below are different.

[0151] Currently commonly used microspheres include those containing ferroferric oxide magnetic cores, ferrous oxide magnetic cores, and those without magnetic cores and modified with hydroxyl groups, carboxyl groups, NHS carboxyl groups or streptavidin on the surface. Considering that the selected raw materials of microspheres need to take into account the stability of the production process and the convenience of use. This patent gives priority to microspheres containing ferroferric oxide magnetic cores and modified with streptavidin on the surface. At the same time, in order to make it consistent with the operation and detection conditions of clinical samples, parameters such as particle size, specific gravity and background fluorescence of the microspheres should also be considered.

[0152] The alternative microspheres include one containing ferroferric oxide magnetic core and one without magnetic core, and all of them are modified with streptavidin on the surface, for a total of 7 types.

[0153] The first consideration is that the quality control microspheres should be able to be detected using the same flow cytometry procedure as lymphocytes. Therefore, the microspheres are screened using flow cytometry lymphocyte detection conditions. Microspheres that can be detected using the flow cytometry conditions for lymphocytes and do not overlap with the lymphocyte position in the lymphocyte detection scatter plot are preferred.

[0154] Attached Figure 1 and 2The following are the figures and results of flow cytometry tests of 7 microspheres. Among them, Examples 1-4 (corresponding to candidate microspheres 4, 2, 5, and 7, respectively) can meet the screening conditions and can be used as candidate microsphere raw materials. Among them, Example 1 (candidate microsphere 4) is the most preferred in this screening, please refer to Example 1 for details.

[0155] At the same time, for ease of operation, the centrifugation conditions of the quality control microspheres should be the same as those of the clinical samples, that is, the same as the collection conditions of lymphocytes. The clinical lymphocyte collection conditions are 300-500g centrifugation and the centrifugation time is 5-10min. This screening uses the lower limit of the centrifugation conditions for collecting lymphocytes clinically, that is, 300g and collection can be completed within 5min as the conditions for screening alternative microspheres, and the cell recovery efficiency under the same conditions is used as a control. The above four alternative microspheres can meet the requirements.

[0156] Combining the flow detection requirements and the collection requirements, Examples 1-4 (corresponding to candidate microspheres 4, 2, 5, and 7, respectively) can meet the screening conditions and can be used as candidate microsphere raw materials. Among them, Example 1 (candidate microsphere 4) is the most preferred in this screening.

[0157] Table 1 Screening of quality controlled microsphere product materials in Examples 2 to 4 and Comparative Examples 1 to 3

[0158]

[0159]

[0160] Table 2 Recovery of microsphere product materials of quality control in Examples 1 to 4

[0161]

[0162] Example 5

[0163] This example is used to illustrate the effect of the microsphere product for quality control of PD-1 protein expression detection in Example 1 at different PD-1 protein coupling concentrations.

[0164] 5.1 Comparison of the effects of different PD-1 protein coupling concentrations

[0165] In this example, the microsphere product of Example 1 was used as an example to study the concentration of the recombinant PD-1 protein coupled with Biotin, and the concentrations of 1.25 ng / μg, 0.63 ng / μg, 0.31 ng / μg, and 0.16 ng / μg were added to study the coupling effects of different added concentrations. Figure 3 .

[0166] At different PD-1 protein coupling concentrations, as the coupling concentration decreases, the fluorescence value of the positive quality control microspheres after staining gradually decreases, and gradually approaches the fluorescence value of the negative quality control microspheres. In order to facilitate the equipment and process quality control of PD-1 staining, the scatter point groups of positive quality control microspheres and negative quality control microspheres should be as close as possible while being clearly distinguishable. Therefore, the two coupling concentrations of 0.63ng / ug and 0.31ng / ug can be used as the PD-1 protein coupling concentrations of the positive quality control microspheres, of which 0.31ng / ug is more preferred.

[0167] 5.2 Comparative study of different proportions of positive quality control microspheres and negative quality control microspheres

[0168] In this embodiment, the microsphere product of Example 1 is taken as an example, and the mixing ratio of the positive quality control microsphere and the negative quality control microsphere is optimized. The microspheres are mixed at conditions of 4:1, 2:1, 1:1, 1:2 and 1:4 respectively. Figure 4 Different mixing ratios of PD-1 positive quality control microspheres and negative quality control microspheres do not affect the actual application. It is easier to interpret when the ratio is 1:2, so 1:2 is the preferred mixing ratio.

[0169] Example 6

[0170] This example is used to illustrate the quality control operation process, analysis method and results of the microsphere product prepared in Example 1 for quality control of PD-1 protein expression detection.

[0171] Part 1: Quality Control Operation Procedure

[0172] The quality control microspheres prepared in Example 1 have stable performance. Specifically, the positive percentage on the flow detection platform is stable. Therefore, the quality control microspheres can be used in daily testing to perform quality control on the detection, machine operation, and flow device status. The specific process is as follows:

[0173] Step 1. Staining of quality control microspheres

[0174] Based on the optimization of the quality control microsphere preparation process in the present invention, the staining process of the quality control microsphere is consistent with that of the clinical sample. The staining process includes:

[0175] Take one person's 5 μL quality control microspheres, add 200 ng of fluorescently labeled PD-1 antibody, and incubate for 20 min in the dark;

[0176] After incubation, centrifuge at 300 g for 5 min and discard the supernatant directly;

[0177] Add 300 μL of phosphate buffer to resuspend the microspheres, centrifuge at 300 g for 5 min, and discard the supernatant directly;

[0178] Add 300 μL of phosphate buffer to resuspend the microspheres and perform flow cytometry detection.

[0179] Step 2. Flow cytometry detection of reaction products

[0180] Flow analysis of reaction products refers to the detection of dye products based on a flow fluorescence platform. The flow detection parameter settings are consistent with those of clinical samples. The specific methods are as follows:

[0181] Template setting: Before using PD-1 quality control microspheres for the first test, you need to ensure that the flow cytometer has undergone daily quality control and is operating normally, so that you can set the test template on the flow cytometer. The specific method for setting the test template is as follows:

[0182] A) Open the flow cytometry software and use the original lymphocyte PD-1 detection template to collect samples of quality control microspheres. The detection conditions and data collection conditions are consistent with the original lymphocyte PD-1 detection conditions;

[0183] B) After completing the data collection of PD-1 quality control microspheres, select the gate tool, select the scatter group of PD-1 quality control microspheres in the CD45 fluorescence and SSC scatter plot, and set the gate name to "PD-1Beads";

[0184] C) Select the scatter plot tool, create a new scatter plot of PD-1 fluorescence and SSC, select the gate tool, and circle the positive gate based on the ratio of PD-1 negative and positive microspheres;

[0185] D) After completing the above operations, save the template as a lymphocyte PD-1 expression detection template containing PD-1 quality control microspheres.

[0186] E) When using PD-1 quality control microspheres for subsequent flow cytometry analysis, the saved lymphocyte PD-1 expression detection template containing PD-1 quality control microspheres can be used.

[0187] Daily testing: Place the stained quality control microsphere sample on the sample rack of the flow cytometer, open the flow cytometry software, and select the lymphocyte PD-1 expression detection template containing PD-1 quality control microspheres;

[0188] Enter the sample name and click the data collection icon to collect samples. When the collected data meets the conditions required by the template, the collection will automatically end and subsequent data analysis will be carried out.

[0189] Part II: Quality Control Results Analysis Method

[0190] Step 1. Result analysis

[0191] After the test, the flow test results are analyzed to perform quality control on the test process. The analysis method is as follows:

[0192] a) Analysis of quality control microsphere results: If the positions of PD-1 negative and positive microspheres on the PD-1 fluorescence-SSC scatter plot are normal and the positive ratio is normal, then the flow cytometry PD-1 channel detection light path is normal and the staining process is normal;

[0193] b) The position of PD-1 negative microspheres on the PD-1 fluorescence-SSC scatter plot is normal, the positive percentage is low or there are no positive particles, which indicates that the flow cytometry PD-1 channel detection light path is normal and the staining process is abnormal;

[0194] c) The positions of PD-1 negative and positive microspheres on the PD-1 fluorescence-SSC scatter plot are generally offset. When the positive percentage of the quality control microsphere detection exceeds the 95% confidence interval, it indicates that the detection is abnormal and the detection process needs to be checked and calibrated.

[0195] Part III: Quality Control Analysis Results

[0196] like Figure 5 As shown in A, when all conditions are normal (including equipment, staining process, and staining antibodies), the positive percentage of flow cytometry after quality control microsphere staining is within the 95% confidence interval;

[0197] like Figure 5 B and Figure 5 As shown in C, when the staining process is abnormal or the staining antibody fails, the positive percentage of the flow cytometry test after quality control microsphere staining is lower than the lower limit of the 95% confidence interval;

[0198] like Figure 5 As shown in D, when the equipment is in an abnormal state, the positive percentage of flow cytometry after quality control microsphere staining exceeds the 95% confidence interval.

[0199] Example 7

[0200] This example is used to illustrate the precision and stability of the microsphere product prepared in Example 1 for quality control of PD-1 protein expression detection.

[0201] According to the method of Example 1, microsphere products for quality control of PD-1 protein expression detection were prepared and divided into three batches (Batch 1 to 3).

[0202] The quality control microspheres prepared by the process of the present invention are simple, reliable and stable. The precision of quality control microspheres prepared in different batches was studied. The specific methods and results are as follows:

[0203] Each quality control microsphere prepared in the same batch was tested 20 times, and the results were stable. The results of batch 1 are recorded in Table 3. Similarly, as shown in Table 4, the 95% confidence interval of the positive percentage of the microsphere product prepared in batch 1 ranged from 32.14% to 35.80%.

[0204] The quality control microspheres prepared in the above batches 1 to 3 were tested repeatedly 20 times, and the average positive percentages were all within the range of 34% ± 2%. The production process between different batches was stable, as shown in Table 5.

[0205] Table 3 Repeatability of PD-1 quality control microspheres prepared in batch 1

[0206]

[0207]

[0208] Table 4 95% confidence interval range of the positive percentage of PD-1 quality control microspheres prepared in batch 1

[0209] Mean Standard Deviation (SD) Coefficient of variation (CV) SSC 363464.80 4848.46 1.33％ Negative microsphere fluorescence 29057.55 325.40 1.12％ Positive microsphere fluorescence 63461.45 1539.54 2.43％ Positive percentage 33.97％ 0.93％ 2.75％

[0210] Table 5 Repeatability of PD-1 quality control microspheres prepared in batches 1 to 3

[0211]

[0212] At the same time, the detection performance of the quality control microspheres prepared in the above three batches at different storage times was tested, see Table 6.

[0213] After the PD-1 quality control microspheres were produced, they were stored at 2-8°C and tested at 1 month, 3 months, 6 months, 9 months, 10 months, 11 months and 12 months of storage. The mean positive percentages of the PD-1 quality control microspheres were all within the 95% confidence interval. It is proved that the PD-1 quality control microspheres prepared according to the preparation method of Example 1 can be stably stored for 12 months at 2-8°C.

[0214] Table 6 Stability of PD-1 quality control microspheres prepared in batches 1 to 3

[0215]

[0216]

[0217] Embodiments 8 to 17

[0218] This example is used to illustrate other positive microspheres and quality control microsphere products suitable for quality control of PD-1 protein expression detection according to the present invention.

[0219] The preparation methods of Examples 8 to 17 are the same as those of Example 1 in terms of preparation processes and steps, except for the differences in materials, PD-1 protein coupling amount, and mixing ratio of cationic and cationic microspheres shown in Table 7 below.

[0220] Table 7 Quality control microsphere products prepared in Examples 8 to 17

[0221]

[0222]

[0223] Figure 6 Other quality control microsphere products suitable for the quality control of PD-1 protein expression detection of the present invention prepared in Examples 8-13 are shown; wherein, Figure 6 A shows the flow cytometry effect diagram of the quality control microsphere product for PD-1 protein expression detection quality control, which is prepared using ferroferric oxide magnetic core microspheres as raw materials, with a coupling concentration of 0.63 ng / μg and a negative microsphere to positive microsphere mixing ratio of 2:1; Figure 6 B shows the flow cytometry effect diagram of the quality control microsphere product for PD-1 protein expression detection quality control, which is prepared using ferroferric oxide magnetic core microspheres as raw materials, with a coupling concentration of 0.63 ng / μg and a negative microsphere to positive microsphere mixing ratio of 1:1; Figure 6 C shows the flow cytometry effect diagram of the quality control microsphere product for PD-1 protein expression detection quality control, which is prepared using ferroferric oxide magnetic core microspheres as raw materials, with a coupling concentration of 0.31 ng / μg and a negative microsphere to positive microsphere mixing ratio of 1:1; Figure 6 D shows the flow cytometry effect diagram of the quality control microsphere product for PD-1 protein expression detection quality control, which is prepared using ferric oxide magnetic core microspheres as raw materials, with a coupling concentration of 0.63 ng / μg and a negative microsphere to positive microsphere mixing ratio of 2:1; Figure 6 E shows the flow cytometry effect diagram of the quality control microsphere product for PD-1 protein expression detection quality control, which is prepared using iron oxide magnetic core microspheres as raw materials, with a coupling concentration of 0.31 ng / μg and a negative microsphere to positive microsphere mixing ratio of 1:1; Figure 6 F shows the flow cytometry effect diagram of a quality control microsphere product for quality control of PD-1 protein expression detection, which is prepared using ferroferric oxide magnetic core microspheres as raw materials, with a coupling concentration of 0.63 ng / μg and a mixing ratio of negative microspheres to positive microspheres of 1:1.

[0224] Figure 7 The quality control microsphere products for quality control of PD-1 protein expression detection prepared in Examples 14-17 are shown; wherein, Figure 7 A shows the flow cytometry effect diagram of the quality control microsphere product for PD-1 protein expression detection quality control, which is prepared using ferroferric oxide magnetic core microspheres as raw materials, with a coupling concentration of 0.31 ng / μg and a negative microsphere to positive microsphere mixing ratio of 2:1; Figure 7B shows the flow cytometry effect diagram of the quality control microsphere product for PD-1 protein expression detection quality control, which is prepared using ferroferric oxide magnetic core microspheres as raw materials, with a coupling concentration of 1.25 ng / μg and a negative microsphere to positive microsphere mixing ratio of 4:1; Figure 7 C shows the flow cytometry effect diagram of the quality control microsphere product for PD-1 protein expression detection quality control, which is prepared using ferroferric oxide magnetic core microspheres as raw materials, with a coupling concentration of 1.25 ng / μg and a negative microsphere to positive microsphere mixing ratio of 1:1; Figure 7 D shows the flow cytometry effect diagram of a quality control microsphere product for quality control of PD-1 protein expression detection, which is prepared using iron oxide magnetic core microspheres as raw materials, with a coupling concentration of 1.25 ng / μg and a mixing ratio of negative microspheres to positive microspheres of 1:4.

[0225] As shown in Table 7, Figure 6 and Figure 7 As shown, the quality control microsphere products prepared in Examples 8 to 17 all have good quality control effects, among which Examples 9, 11, 12, and 13 have better quality control effects, and Examples 8, 10, and 14 have better quality control effects.

[0226] Although the above embodiments 1-17 show the effects of some embodiments, those skilled in the art should understand that according to the concept of the present invention, the aforementioned other embodiments that do not specifically show the effects or other technical solutions of the present invention that are not shown in the embodiments or test examples can also achieve the following technical effects stated in the invention content section that are equivalent to embodiments 1-17:

[0227] 1. For the first time, protein-labeled microspheres were used to prepare quality control products for PD-1 detection. This quality control product is an external quality control product that can perform quality control on the entire process of PD-1 protein detection in samples and provide prompts for fluctuations that exceed the 95% confidence interval of the positive percentage of the quality control microspheres.

[0228] 2. Through screening and surface modification, the quality control microspheres are consistent with clinical samples in terms of operation and detection process. The operation is simple and can achieve quality control of system errors in the entire process of PD-1 protein detection.

[0229] 3. The quality control microsphere preparation process based on microsphere surface modification is simple, reliable and stable. The CV of the fluorescence mean value of the three batches of quality control microspheres PD-1 detection is <3%. There is no need for freeze-drying and other processes that have high requirements for equipment and process.

[0230] 4. The quality control microspheres include two quality control microspheres labeled with PD-1 protein at different concentrations. After the two microspheres are mixed, they are used as an independent sample to participate in the entire process of staining and detection. The systematic error of the staining and detection process is judged by the proportion of PD-1 positive microspheres in the flow detection results. The quality control microspheres are sensitive to operational errors or systematic errors in the process, and can give warnings for fluctuations that exceed the 95% confidence interval of the positive percentage of the quality control microspheres, effectively indicating the reliability of the clinical sample test results. The operation process is the same as that of clinical samples, and no additional operation is required.

[0231] 5. The quality control microspheres have good stability and can be stored at 4°C for more than 12 months. Compared with quality control products prepared by existing technologies, they are easier to store and transport.

[0232] Although the present invention has been described to a certain extent, it is obvious that appropriate changes in various conditions can be made without departing from the spirit and scope of the present invention. It is understood that the present invention is not limited to the embodiments described, but belongs to the scope of the claims, which includes equivalent replacements of each factor described.

Claims

1. A positive microsphere for quality control of PD-1 protein expression detection, characterized in that: The positive microspheres are prepared by coupling microspheres with modified groups on the surface with PD-1 proteins; wherein: The microspheres are magnetic or non-magnetic microspheres, preferably selected from one or more of the following: microspheres containing a ferroferric oxide magnetic core, microspheres containing a ferrous oxide magnetic core, and microspheres without a magnetic core, more preferably microspheres containing a ferroferric oxide magnetic core or microspheres containing a ferrous oxide magnetic core, and most preferably microspheres containing a ferroferric oxide magnetic core.

2. The positive microsphere according to claim 1, characterized in that: The modifying group is selected from one or more of the following: streptavidin, hydroxyl, carboxyl, NHS carboxyl, preferably streptavidin or NHS carboxyl, most preferably streptavidin; and / or The PD-1 protein is bound to biotin; Preferably, the mass ratio of the positive microspheres to the PD-1 protein is 800 to 6667:1, more preferably 1000 to 6250:1, and further preferably 1587 to 3226:

1.

3. A microsphere product for quality control of PD-1 protein expression detection, characterized in that: The microsphere product comprises: the positive microspheres according to claim 1 or 2 and the negative microspheres not coupled with the PD-1 protein; Preferably, the volume ratio of the negative microspheres to the positive microspheres is 1-4:1-4, more preferably 1-2:1-2, and most preferably 2:

1.

4. The positive microsphere according to claim 1 or 2 or the microsphere product according to claim 3, characterized in that: The particle size of the negative microspheres and / or the positive microspheres is 1 to 6 μm, preferably 1.5 to 5 μm, and more preferably 2 to 4 μm. The specific gravity of the positive microspheres and / or microsphere products is 1-5 g / cm 3 , preferably 2 to 4 g / cm 3 , more preferably 2 to 3 g / cm 3 : and / or The background fluorescence of the negative microspheres and / or the positive microspheres is different from that of lymphocytes.

5. A method for preparing the microsphere product according to claim 3 or 4, characterized in that: The method comprises the following steps: 1) suspending magnetic or non-magnetic material microspheres in a buffer for dispersion, and separating the dispersed microsphere suspension buffer into negative microspheres and positive microsphere raw materials; 2) coupling the dispersed positive microsphere raw material in step 1) with the PD-1 protein bound to biotin; 3) purifying the microspheres obtained by coupling in step 2) to obtain the positive microspheres; 4) Mixing the negative microspheres and the positive microspheres to a fixed volume to obtain the microsphere product.

6. The method according to claim 5, characterized in that: In the step 1), the final concentration of the magnetic or non-magnetic material microsphere suspension is 0.1 mg / mL to 0.5 mg / mL, preferably 0.1 mg / mL to 0.3 mg / mL, and most preferably 0.1 mg / mL; and / or The step 3) further comprises: washing the microspheres coupled in step 2) to remove the free PD-1 protein, centrifuging and discarding the supernatant, adding a buffer, and vortexing to mix, so as to obtain the positive microspheres; Preferably, in step 3), the centrifugation time is 2 to 10 min, more preferably 2 to 6 min, and even more preferably 2 to 4 min.

7. The method according to claim 5 or 6, characterized in that: The step 2) further comprises: adding the positive microsphere material dispersed in step 1) to the PD-1 protein combined with biotin, vortexing to obtain a dispersion, and then vertically mixing the dispersion to obtain the coupled microspheres; Preferably, the concentration of the biotin-bound PD-1 protein added is 0.15 ng / μg to 1.25 ng / μg, more preferably 0.16 ng / μg to 1.00 ng / μg, and further preferably 0.31 ng / μg to 0.63 ng / μg; and / or Preferably, the coupling time is 30 min to 1.5 h, more preferably 40 min to 1.0 h, most preferably 1.0 h.

8. The method according to any one of claims 5 to 7, characterized in that The step 4) further comprises: vortexing the negative microspheres in step 1) and the positive microspheres in step 3) and resuspending them in a buffer to obtain the microsphere product; Preferably, the buffer is selected from one or more of the following: phosphate buffer, carbonate buffer, TrisHCl buffer, EDTA buffer, borate buffer, acetate buffer, MES buffer, more preferably selected from one or more of the following: phosphate buffer, TrisHCl buffer, carbonate buffer, most preferably phosphate buffer; and / or Preferably, the pH value of the buffer solution is 7 to 8.5, more preferably 7.0 to 8.0, and further preferably 7.2 to 7.

5.

9. Use of the positive microsphere or microsphere product according to any one of claims 1 to 4 in the preparation of a product for detecting cell PD-1 protein expression; Preferably, the product is a quality control product.

10. The use according to claim 9, characterized in that: The cells are T lymphocytes, and the T lymphocytes are preferably selected from one or more of the following: total T cells, helper T cells, and cytotoxic T cells.

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