Method and kit for detecting anti-stem cell antibody

Abstract

The invention provides an anti-stem cell antibody detection method and a kit. According to the method provided by the invention, the anti-stem cell antibody can be detected by adopting an MSD platform, and magnetic beads can be used for replacing stem cells to serve as an immunosuppression competition confirmation reagent to carry out a confirmation experiment. The detection method based on the MSD platform provided by the invention can improve the sensitivity of the method for detecting the stem cell antibody and / or improve the detection flux.

Description

Technical Field

[0001] This application generally relates to the field of stem cells, and more specifically, to methods and kits for detecting anti-stem cell antibodies. Background Technology

[0002] Currently, most assessments of stem cell immunogenicity rely on indirect methods to evaluate the overall immune system response. These methods include detecting the ratio of T cells / B cells / NK cells through immunophenotyping, or reflecting the activation status of the immune system by detecting the levels of biomarkers such as cytokines, chemokines, and adhesion factors.

[0003] Therefore, there is an urgent need for new methods and kits for detecting anti-stem cell antibodies. Summary of the Invention

[0004] In a first aspect, this application provides a method for detecting anti-stem cell antibodies in a biological sample of an individual, comprising the following steps: (a) treating a first biological sample and a second biological sample from the individual with an acid-dissolving reagent to dissociate the anti-stem cell antibodies in the first biological sample and the second biological sample; (b) treating the first biological sample with a neutralizing reagent, while simultaneously or subsequently contacting the first biological sample with a carrier bound to the stem cells, thereby causing the anti-stem cell antibodies in the first biological sample to bind to the stem cells bound to the carrier; (c) treating the second biological sample with a neutralizing reagent, while simultaneously or subsequently removing at least a portion of the antibodies in the second biological sample by magnetic bead treatment, and then contacting the second biological sample with a carrier bound to the stem cells, thereby causing the anti-stem cell antibodies in the second biological sample to bind to the stem cells bound to the carrier; (d) adding an anti-anti-stem cell antibody with a detection label to the carrier from steps (b) and (c), causing the anti-anti-stem cell antibody to bind to the anti-stem cell antibody; and (e) detecting the detection label to determine the anti-stem cell antibody.

[0005] Secondly, this application provides a kit for detecting anti-stem cell antibodies in biological samples, comprising magnetic beads, the stem cells, a carrier, and an anti-anti-stem cell antibody with a detection label, wherein the carrier is capable of binding the stem cells.

[0006] The advantage of the detection method in this application is that it can improve the sensitivity of detecting stem cell antibodies and / or increase the detection throughput. Attached Figure Description

[0007] Figure 1 The screening sensitivity of the method established in this application is shown to be approximately 38.2 ng / mL; where “Run” indicates an analysis plate.

[0008] Figure 2The confirmatory sensitivity of the method established in this application is shown to be approximately 23.7 ng / mL; where “Run” indicates an analytical plate. Detailed Implementation

[0009] Unless otherwise specified, this application employs conventional molecular biology, microbiology, cell biology, biochemistry, and immunology techniques in the art.

[0010] Although the broad scope of this application indicates numerical ranges and parameter approximations, the numerical values ​​are described as accurately as possible in the specific embodiments. However, any numerical value inherently contains a certain degree of error, which is due to the standard deviation present in their respective measurements. Furthermore, all ranges disclosed herein should be understood to encompass any and all subranges contained therein. For example, a range described as "1 to 10" should be considered to include any and all subranges between the minimum value of 1 and the maximum value of 10 (inclusive); that is, it should be considered to include all subranges starting with a minimum value of 1 or greater, such as 1 to 6.1, and all subranges ending with a maximum value of 10 or less, such as 5.5 to 10. Additionally, any references marked "incorporated herein" should be understood to be incorporated herein in their entirety.

[0011] As used in this article, the term "anti-stem cell antibody" can refer to an antibody that can bind to stem cells, such as an antibody that can bind to stem cell surface proteins (e.g., CD90, CD105, and CD73). "Anti-anti-stem cell antibody" can refer to an antibody that can bind to the aforementioned antibodies that can bind to stem cells (e.g., anti-CD90, anti-CD105, and anti-CD73).

[0012] As used herein, the term "individual" refers to a mammal, including but not limited to primates, cattle, horses, pigs, sheep, goats, dogs, cats, and rodents such as rats and mice. Preferably, the mammal is a non-human primate or a human. A particularly preferred mammal is a human. The terms "individual," "patient," and "subject" are used interchangeably herein.

[0013] As used in this article, the term "biological sample" refers to any sample taken from an individual (e.g., a human or other animal), such as a person receiving stem cell therapy, and that may contain anti-stem cell antibodies. Biological samples can be bodily fluids, such as blood, plasma, serum, urine, vaginal fluid, fluid from the scrotum (e.g., ascites from the testes), vaginal douches, pleural fluid, ascites, cerebrospinal fluid, saliva, sweat, tears, sputum, bronchoalveolar lavage fluid, fluid expelled from the nipple, fluid aspirated from different parts of the body (e.g., the thyroid gland, the breast), intraocular fluid (e.g., aqueous humor), etc.

[0014] In this specification and claims, the words “comprising,” “including,” and “containing” mean “including but not limited to” and are not intended to exclude other parts, additives, components, or steps.

[0015] It should be understood that the features, characteristics, components or steps described in a particular aspect, embodiment or example of this application may be applied to any other aspect, embodiment or example described herein, unless there is any contradiction.

[0016] Current systems for assessing stem cell immunogenicity mainly rely on two types of indirect methods: one is to analyze immune cell subsets (e.g., CD4+) using techniques such as flow cytometry. + T cells, CD8 + Changes in the proportion or activation status of T cells, B cells, NK cells, etc. (e.g., increased levels of regulatory T cells, Tregs) can indirectly infer the degree of stem cell stimulation of the immune system. Secondly, techniques such as qPCR can be used to detect fluctuations in the concentrations of inflammation-related cytokines (e.g., IL-6, TNF-α), chemokines (e.g., CXCR4 ligand SDF-1), and immunomodulatory molecules (e.g., TGF-β) in serum or tissue samples, serving as surrogate indicators of immune response strength. Although these assessment methods are widely used in scientific research and preclinical studies, novel methods for assessing stem cell immunogenicity remain needed.

[0017] Based on the exploration of the above problems, this application provides the following implementation plan: In a first aspect, this application provides a method for detecting anti-stem cell antibodies in a biological sample of an individual, comprising the following steps: (a) treating a first biological sample and a second biological sample from the individual with an acid-dissolving reagent to dissociate the anti-stem cell antibodies in the first biological sample and the second biological sample; (b) treating the first biological sample with a neutralizing reagent, while simultaneously or subsequently contacting the first biological sample with a carrier bound to the stem cells, thereby causing the anti-stem cell antibodies in the first biological sample to bind to the stem cells bound to the carrier; (c) treating the second biological sample with a neutralizing reagent, while simultaneously or subsequently removing at least a portion of the antibodies in the second biological sample by magnetic bead treatment, and then contacting the second biological sample with a carrier bound to the stem cells, thereby causing the anti-stem cell antibodies in the second biological sample to bind to the stem cells bound to the carrier; (d) adding an anti-anti-stem cell antibody with a detection label to the carrier from steps (b) and (c), causing the anti-anti-stem cell antibody to bind to the anti-stem cell antibody; and (e) detecting the detection label to determine the anti-stem cell antibody.

[0018] In some embodiments, the acid hydrolysis agent may be selected from one or more of acetic acid, glycine, and hydrochloric acid. In a preferred embodiment, the acid hydrolysis agent contains about 50-150 mM of glycine and has a pH of about 1-3. In a more preferred embodiment, the acid hydrolysis agent contains about 100 mM of glycine and has a pH of about 2.0.

[0019] In some embodiments, the neutralizing agent is selected from one or more of Trizma (prepared using Tris Base) and Tris (prepared using Tris-HCl). In a preferred embodiment, the neutralizing agent contains about 0.25-1 M Tris and has a pH of about 8.5-10.5. In a more preferred embodiment, the neutralizing agent contains about 0.5 M Tris and has a pH of about 9.5.

[0020] In some implementations, the magnetic beads are selected from Protein G beads, Protein A beads, and Protein A / G beads.

[0021] In some embodiments, the detection label may be an electrochemiluminescent label, preferably a ruthenium-based label, and step (e) may be based on electrochemiluminescent immunoassay, wherein the carrier may be selected to mediate the reaction of the electrochemiluminescent label.

[0022] In some implementations, the biological sample can be a serum sample, a plasma sample, or a whole blood sample.

[0023] In some implementation schemes, stem cells may be selected from hematopoietic stem cells, human umbilical cord mesenchymal stem cells, spinal cord mesenchymal stem cells, adipose mesenchymal stem cells, skin mesenchymal stem cells, neural stem cells, adipose stem cells, and induced pluripotent stem cells, etc.

[0024] For ease of description and understanding, each step has been assigned a letter code. However, it should be noted that these codes do not strictly limit the order in which these steps are performed. For example, the processing and testing of the first biological sample can be performed simultaneously with the processing and testing of the second biological sample, or they can be performed separately.

[0025] In an implementation scheme in which both steps are performed simultaneously, for example, but not limited to, steps (a) through (e) can be performed in sequence (e.g., where steps (b) and (c) are performed substantially simultaneously).

[0026] In separate implementation schemes, for example but not limited to, the first biological sample can be acid-digested, neutralized, contacted with a carrier, contacted with anti-anti-stem cell antibodies with detection markers, and then detected. Then, the second biological sample can be acid-digested, neutralized, treated with magnetic beads, contacted with a carrier, contacted with anti-anti-stem cell antibodies with detection markers, and then detected. That is, in such an implementation scheme, a large-scale screening experiment can be conducted first, and then confirmatory experiments can be performed on the screened positive samples to eliminate false positive results.

[0027] For example, after the first biological sample is screened as positive, a confirmatory test can be performed on the second biological sample. In the confirmatory test, for example, the second biological sample can be divided into two portions and then processed according to the method provided in the first aspect as follows: (a) treating the first and second samples with an acid-dissolving reagent to dissociate the anti-stem cell antibodies in the first and second samples; (b) treating the first sample with a neutralizing reagent, while simultaneously or subsequently contacting the first sample with a carrier bound to the stem cells, thereby allowing the anti-stem cell antibodies in the first sample to bind to the stem cells bound to the carrier; (c) treating the second sample with a neutralizing reagent, while simultaneously or subsequently removing at least a portion of the antibodies in the second sample by magnetic bead treatment, and then contacting the second sample with a carrier bound to the stem cells, thereby allowing the anti-stem cell antibodies in the second sample to bind to the stem cells bound to the carrier; (d) adding an anti-anti-stem cell antibody with a detection label to the carrier from steps (b) and (c), such that the anti-anti-stem cell antibody binds to the anti-stem cell antibody; and (e) detecting the detection label to determine the anti-stem cell antibody. For ease of description, the terms "first sample" and "second sample" have been used above. However, those skilled in the art will understand that "first sample" and "second sample" here can actually correspond to "first biological sample" and "second biological sample." The above exemplary embodiments (i.e., embodiments in which screening and confirmation are carried out separately) also fall within the protection scope of this application.

[0028] In some implementations, the first and second biological samples may originate from the same biological sample collected from an individual, for example, by dividing a serum sample into two.

[0029] Secondly, this application provides a kit for detecting anti-stem cell antibodies in biological samples, comprising magnetic beads, the stem cells, a carrier, and an anti-anti-stem cell antibody with a detection label, wherein the carrier is capable of binding the stem cells.

[0030] In some embodiments, the detection tag is an electrochemiluminescent tag, preferably a ruthenium-based tag, and the carrier is capable of mediating the reaction of the electrochemiluminescent tag.

[0031] Where there is no conflict, the technical features of the first aspect also apply to the second aspect.

[0032] Example

[0033] The examples are provided by way of illustration only and not by way of limitation. Those skilled in the art will readily recognize that various non-critical parameters can be changed or modified to produce substantially the same or similar results. Where specific techniques or conditions are not specified in the experimental examples, they were performed according to the techniques or conditions described in the literature in the art or according to the product instructions. Reagents or instruments used, unless otherwise specified, are all commercially available conventional products.

[0034] Example 1. Detection of anti-stem cell antibodies based on the MSD platform

[0035] 1. Sample preparation

[0036] 1.1. Sample Pretreatment: Serum samples were pretreated with an acid hydrolysis reagent (100 mM glycine, pH 2.0) at a ratio of 1:10 (7 μL serum + 63 μL acid hydrolysis solution), mixed well, and incubated at room temperature for 30–40 min. Positive serum samples were artificial serum samples obtained by incorporating anti-stem cell antibodies (1.56–200 ng / mL) into the matrix serum. Considering the unavailability of positive control antibodies of the same species in clinical projects, this example used the murine anti-stem cell antibody BD Pharmingen. TM Purified mouse anti-human CD90 (ADA PC1) and human IgG (ADA PC2) were used as positive controls. The matrix serum was derived from a mixture of healthy human serum, which was prepared by purchasing and recruiting normal healthy individuals in China.

[0037] 1.2. Magnetic Bead Treatment and Neutralization: Protein G agarose magnetic beads (manufacturer: BEAVER; catalog number: 70805-100) or cell culture medium (basal medium manufacturer: Corning; product number: 10-022-CV; cell culture medium: basal medium + 10% fetal bovine serum (Gibco; catalog number: A5669701)) were used to treat the acid-digested serum samples, and neutralization was performed simultaneously. The neutralization reagent used was Tris buffer (pH 9.5, 0.5 M; prepared by mixing 1 M Tris buffer with cell culture medium at a 1:1 volume ratio).

[0038] For samples treated with culture medium (screening samples), mix the cell culture medium, acid-hydrolyzed serum sample, and neutralizing reagent thoroughly, and then incubate overnight at room temperature with shaking at 800-1200 rpm. The volume ratio of cell culture medium, acid-hydrolyzed serum sample, and neutralizing reagent is 3:2:1. For samples treated with magnetic beads (confirmatory test samples), mix Protein G agarose magnetic beads, acid-digested serum samples, and neutralizing reagent, and then incubate overnight at room temperature with shaking at 800-1200 rpm. The volume ratio of Protein G agarose magnetic beads, acid-digested serum samples, and neutralizing reagent is 3:2:1.

[0039] 1.3. Magnetic bead separation: Place the sample after shaking incubation in a magnetic separator and allow it to adsorb for 3-5 min. Then, aspirate the supernatant to remove the magnetic beads from the sample. Repeat this process twice.

[0040] 2. Preparation of MSD microplates

[0041] 2.1. Capture reagent coating: The density of resuscitated human umbilical cord mesenchymal stem cells was adjusted to approximately 5 × 10⁻⁶. 4 Cells / mL were added to MSD microplates at 100 μL / well and incubated overnight (22-24 hours) at 37°C with 5% CO2. Human IgG (Jackson ImmunoResearch, catalog number 009-000-003) (positive control) and 1% BSA in PBS (negative control 3 / dilution) were coated similarly. See the table below for sample loading diagram.

[0042]

[0043] 2.2. Blocking: Take out the MSD microplate obtained in step 2.1, discard the supernatant and pat dry; add blocking solution (30% FBS in PBS) to the MSD microplate at 150 μL / well and block at room temperature for at least 1.5 hours.

[0044] 3. Anti-stem cell antibody detection

[0045] 3.1. Plate Transfer and Sample Addition: Remove the MSD microplate obtained in step 2.2, discard the supernatant and blot dry. Add the screening and confirmatory samples (after removing the magnetic beads) to the MSD microplate (50 μL / well), and then incubate overnight (16-18 hours) at 2-8℃ with shaking at 600 rpm. Add 1% BSA in PBS to the negative control and positive control wells, and the remaining operations are similar.

[0046] 3.2. Washing the plate: Take out the MSD microplate obtained in step 3.1, discard the supernatant, wash the plate twice, discard the supernatant again, and pat dry.

[0047] 3.3. Adding test reagents: As shown in the plate diagram, add working solutions of test reagents 1 and 2 (ruthenium-labeled anti-mouse antibody (Ru-AffiniPure Goat Anti-Mouse IgG (H+L), catalog number before ruthenium labeling: 115-005-166) and ruthenium-labeled anti-human antibody (Ru-Goat anti-Human IgA+IgG+IgM, catalog number before ruthenium labeling: 109-005-064), 50 μL / well, and incubate with shaking at 2-8℃ for 2.5-3 hours.

[0048] 3.4. Washing the plate: Take out the MSD microplate obtained in step 3.3, discard the supernatant, wash the plate twice, discard the supernatant again, and pat dry.

[0049] 3.5. Detection: Add 150 μL of MSD Read Buffer T (2×) working solution to the MSD microplate and place the MSD SA microplate into a MESO QUICKPLEX SQ120 for detection within 15 min.

[0050] 3.6. Analysis: Calculate the results of the sample test.

[0051] 3.6.1 Calculate the signal-to-noise ratio of the sample, which is the average value of the sample instrument response divided by the average value of the negative quality control instrument response.

[0052] 3.6.2 Percentage of immunosuppression inhibited after adding magnetic beads to the sample in the immunosuppression confirmation experiment. .

[0053] The average ECLU value of the screening experiment is as follows Figure 1 As shown, the sensitivity of the screening experiment using this method is approximately 38.2 ng / mL.

[0054] The % inhibition curve of the confirmatory experiment is as follows: Figure 2 As shown, the sensitivity of the confirmatory experiment for this method is approximately 23.7 ng / mL.

Claims

1. A method for detecting anti-stem cell antibodies in an individual's biological sample, comprising the following steps: (a) Treating a first biological sample and a second biological sample from the individual with an acid hydrolysis reagent to dissociate the anti-stem cell antibodies in the first biological sample and the second biological sample; (b) Treat the first biological sample with a neutralizing agent, and simultaneously or subsequently contact the first biological sample with a carrier in which the stem cells are bound, thereby binding the anti-stem cell antibody in the first biological sample to the stem cells bound on the carrier. (c) Treat the second biological sample with a neutralizing agent, and simultaneously or subsequently remove at least a portion of the antibodies in the second biological sample by magnetic bead treatment, and then contact the second biological sample with a carrier in which the stem cells are bound, so that the anti-stem cell antibodies in the second biological sample bind to the stem cells bound on the carrier. (d) Adding the anti-anti-stem cell antibody with a detection label to the vector from steps (b) and (c) such that the anti-anti-stem cell antibody binds to the anti-stem cell antibody; and (e) Detect the detection marker to determine the anti-stem cell antibody.

2. The method according to claim 1, wherein the acid hydrolysis reagent is selected from one or more of acetic acid, glycine, and hydrochloric acid; and / or The neutralizing agent is selected from one or more of Trizma and Tris.

3. The method of claim 2, wherein the acid hydrolysis reagent comprises 50-150 mM glycine and has a pH of 1-3, and / or the neutralization reagent comprises 0.25-1 M Tris and has a pH of 8.5-10.

5.

4. The method of claim 2 or 3, wherein the acid hydrolysis reagent comprises 100 mM glycine and has a pH of 2.0, and / or the neutralizing reagent comprises 0.5 M Tris and has a pH of 9.

5.

5. The method of claim 1, wherein the magnetic bead is selected from Protein G magnetic beads, Protein A magnetic beads, and Protein A / G magnetic beads.

6. The method according to any one of claims 1-5, wherein the detection label is an electrochemiluminescent label, and step (e) is based on electrochemiluminescent immunoassay, wherein the carrier is capable of mediating the reaction of the electrochemiluminescent label.

7. The method of claim 6, wherein the detection marker is a ruthenium-based marker.

8. The method according to any one of claims 1-7, wherein the biological sample is a serum sample, a plasma sample, or a whole blood sample.

9. The method according to any one of claims 1-8, wherein the stem cells are hematopoietic stem cells, human umbilical cord mesenchymal stem cells, spinal cord mesenchymal stem cells, adipose mesenchymal stem cells, skin mesenchymal stem cells, neural stem cells, adipose stem cells, or induced pluripotent stem cells.

10. A kit for detecting anti-stem cell antibodies in a biological sample, comprising magnetic beads, said stem cells, a carrier, and an anti-anti-stem cell antibody with a detection label, wherein said carrier is capable of binding said stem cells.

11. The kit according to claim 10, wherein the detection label is an electrochemiluminescent label, and the carrier is capable of mediating a reaction of the electrochemiluminescent label.

12. The kit of claim 11, wherein the detection marker is a ruthenium-based marker.

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