Culture related to the antigen sensitization status of the transplantation of capillary endothelial cells, uses thereof and test kits comprising the same

Abstract

The present invention relates to a culture related to the antigen sensitization status of the transplantation of capillary endothelial cells, to the use of said culture, and also to a test kit comprising said culture.

Description

TECHNICAL FIELD

[0001] The present invention relates to a culture related to the antigen sensitization status of capillary endothelial cells in connection with transplantation, to the use of said culture, and to a test kit comprising said culture. BACKGROUND

[0002] Apart from identical twins, all organ transplants are subject to rejection, with chronic rejection being the most important pathway leading to loss of function after transplantation. While acute rejection can be avoided after organ transplantation using high doses of immunosuppressants, there is no method to address chronic rejection. A study involving eight European countries revealed the impact of immunosuppressants on loss of function after organ transplantation between 1989 and 2014. They showed that the use of high-performance immunosuppressants only reduced the loss of function after transplantation within 0-1 years from 16.8% to 4.6%; while the loss of function within 1-3 years, 3-5 years and 5-10 years remained in the range of 3.7% - 4.2% (Legendre C. et al. Transplantation, 102(9S1): S1-S4, 2018 DOI: 10.1097 / TP.00000002316). Thus, the expectation of improving long-term survival using immunosuppressants has reached or is close to a bottleneck.

[0003] For the last decade, one of the core problems in contemporary transplantation science has been how to use biotechnology to diagnose rejection in the early laboratory. Since the 1990s, complement-dependent cytotoxicity / cell lysis has been the core of the immunopathological diagnosis of transplantation and is currently the main means of diagnosing whether organ rejection is triggered and exists before / after transplantation surgery in hospitals. However, as our understanding of the mechanism of organ transplant rejection has improved, we have realized that the above-mentioned detection is only for short-term acute rejection, and chronic rejection is a process of complement-independent cytotoxicity / cell activation. In particular, the detection of complement-dependent cytotoxicity / cell lysis uses peripheral blood lymphocytes, while immune rejection occurs in capillary tissue. More importantly, the newly synthesized specific anti-donor antibodies, abbreviated as DSA, in patients after organ transplantation tend to be complement-independent cytotoxicity / cell activation. Therefore, the diagnostic methods currently used in hospitals cannot comprehensively and accurately assess whether and when pathogenic rejection occurs in organ transplant patients. One important reason is that the diagnostic method is not appropriate. We have not yet established a method for measuring chronic organ rejection because the mechanism of chronic rejection is completely different from that of acute rejection.

[0004] Surprisingly, basic research in transplantation has provided valuable theories and data for the development of our laboratory techniques. Firstly, many researchers have recognized that our graft antigens over the past few decades have focused on HLA-associated rejection antibodies and their activation of complement. However, substantial clinical evidence shows that immune rejection can occur even between HLA-matched relatives, and non-HLA transplantation-associated antigens are also closely related to immune rejection (Jackson AM et al., J AmSoc Nephrol, 26:1161, 2015, DOI: 10.1681 / ASN.2013121277). A large number of transplant patients still reject their transplanted organs even in the absence of HLA antibodies (Butler CL et al., Am J Transplantation 2020, 20:2768, DOI:10.1111 / ajt.15863; Filippone EJ et al., Transplantation, 105:181, 2021). Secondly, transplant-associated antigens (TAAs) on the surface of capillary endothelial cells play a crucial role in mediating chronic antibody rejection and have attracted considerable attention, as they represent the first point of contact between the organ transplant recipient and the donor tissue. To date, G-protein-coupled receptors (GPCRs), angiotensin I receptor (AT1R), endothelin A receptor (ETAR), vimentin, perlecan, and tubulin have been identified as key antigens. - tubulin), protein kinase C zeta, major histocompatibility complex class I-related A chain, have been recognized to elicit the formation of antibodies against endothelial cells, but more transplant-related antigens remain undiscovered or discovered (Delville M et al. J Am Soc Nephrol 30:692 2019 DOI: 10.1681 / ASN.2018080868). Thirdly, it is particularly worth mentioning that when the vascular endothelial cells are activated by anti-graft antibodies, they are more actively involved in the rejection process. Its early pathological basis is capillaritis; the late stage is arterial intimal fibrosis and interstitial fibrosis. The pathogenic antibodies not only bind tightly to the HLA and non-HLA antigens on the surface of vascular endothelial cells, sensitizing the cell surface, but also trigger intracellular signaling pathways, causing endothelial cell activation (Bian H and Reed EF, J Immunol 163(2): 1010-1018 1999). Continuously activated endothelial cells not only accelerate the release of inflammatory factors, but also increase adhesion proteins on the cell surface. The combined results of these reactions are a large number of inflammatory cells and cell proliferation around the capillaries, further developing into vascular obstruction, atrophy, and even tissue necrosis (Haas M, Am J Transplant 18(12): 2849-2856 2018 doi: 10.1111 / ajt.15088).

[0005] Currently, the biotechnology company CareDx (www.caredxinc.com) has developed a molecular diagnostic technology directly related to organ transplantation based on the nucleic acid molecule hybridization technology platform. The core technology of CareDx products is to use gene expression chips to measure the expression level of rejection-related genes in peripheral blood lymphocytes to determine the immune rejection status of patients.

[0006] The biotechnology company One Lambda has developed a technology that coats a certain number of HLA molecular fragments, i.e. synthetic peptides of unequal amino acid numbers, on the surface of microspheres as targets, i.e. Luminex single antigen microspheres (SAB), to bind to anti-graft antibodies in patient serum. Because of the current artificial peptide synthesis technology, labeling technology, and the complexity of HLA molecular groups, it cannot cover all antigens in clinical xenotransplantation.

[0007] As Albrecht et al. pointed out: organ transplant recipients produce antibodies to the donor are affected by sub-class, titer, epitope and valence, and many other factors necessary to maintain the three-dimensional spatial structure of antigen-antibody binding reaction molecules, so whether synthetic peptide fragments can represent antigen molecules is still a controversial issue. For this reason, the FDA agency in the United States in 2017 considered that the use of artificial coated antigens to understand antibody-mediated rejection and its diagnosis, prognosis is a part of medical uncertainty, especially in clinical application should be cautious (Albrecht R et al., Transplantation 102: e257, 2018). Although the commercial name is XM-ONE method uses the separation of peripheral blood from the endothelial progenitor cells, but its clinical effect has not been consistently recognized (Breimer ME et al., Transplantation 87:549 2009, doi:10.1097 / TP.0b013e3181949d4e.). Therefore, the precise assessment of the pathogenicity of transplantation antibodies depends on the interaction of anti-graft antibodies with vascular endothelial cells is an important indicator for evaluating long-term survival after organ transplantation (Dragun D et al., Kidney International 90:280 2016 DOI1: 10.1016 / jkint.2016.03.019). Therefore, the current diagnostic technology has a high problem of false positive and false negative rate in clinical use, which is difficult to guide clinicians to accurately determine the status of recipients after transplantation, and reduces the success rate of transplantation, survival rate and other indicators of transplant recipients.

[0008] Therefore, in the current situation of the shortage of transplanted organs, there is an urgent need for a detection kit that can truly monitor recipients after transplantation and guide postoperative treatment programs, thereby improving the success rate and survival rate of transplantation. SUMMARY

[0009] In view of the above current technical background, the present application deepens the unique advantages of detecting endothelial cell surface molecules HLA and non-HLA, combined with immunohistochemical approach, opens up its new use in determining anti-donor antibodies in serum of recipients after organ transplantation, and develops a number of functional kits, aiming to provide new data for early detection of chronic rejection from the perspective of laboratory diagnosis to guide clinical practice.

[0010] The present application relates to a kit for detecting the sensitization state of a transplant-related antigen of a culture of capillary endothelial cells, comprising capillary endothelial cells bearing said transplant-related antigen, the sensitization of said transplant-related antigen resulting from a binding reaction of said transplant-related antigen with an anti-graft antibody; said transplant-related antigen including anti-donor HLA molecules and anti-donor non-HLA molecules on the surface of said capillary endothelial cells; the sensitization of said transplant-related antigen being detectable in the presence of said anti-graft antibody in the serum of a transplant recipient.

[0011] In such a kit, the culture of capillary endothelial cells is from an organ transplant donor or from non-donor tissue, the latter being a mixed culture completely assembled according to the donor HLA antigen typing criteria, said HLA antigen typing being based on a serum typing standard.

[0012] In such a kit, the detection of the sensitization of said transplant-related antigen is by means of a signal displayed on the membrane of the endothelial cells, said signal being from an indirect immunohistochemical staining or an indirect immunofluorescent staining.

[0013] Such a kit comprises a labeled anti-human IgG antibody against said anti-graft antibody, the label molecule being selected from any one of the following group: horseradish peroxidase, alkaline phosphatase, colloidal gold-silver solution, fluorescein isothiocyanate FITC, tetraethyl rhodamine and tetramethyl isothiocyanate TRIC.

[0014] In such a kit, the endothelial cell marker in the culture of capillary endothelial cells has been labeled with intracytoplasmic VWF.

[0015] The present application relates to a kit for detecting the sensitization state of a transplant-related antigen of a culture of capillary endothelial cells, comprising capillary endothelial cells bearing said transplant-related antigen, the sensitization of said transplant-related antigen resulting from a binding reaction of said transplant-related antigen with an anti-graft antibody; said transplant-related antigen including anti-donor HLA molecules and anti-donor non-HLA molecules on the surface of said capillary endothelial cells; the sensitization of said transplant-related antigen being detectable in the presence of said anti-graft antibody in the serum of a transplant recipient.

[0016] The present application relates to a kit for detecting the sensitization state of a transplant-related antigen of a culture of capillary endothelial cells, comprising capillary endothelial cells bearing said transplant-related antigen, the sensitization of said transplant-related antigen resulting from a binding reaction of said transplant-related antigen with an anti-graft antibody; said transplant-related antigen including anti-donor HLA molecules and anti-donor non-HLA molecules on the surface of said capillary endothelial cells; the sensitization of said transplant-related antigen being detectable in the presence of said anti-graft antibody in the serum of a transplant recipient.

[0017] The two aforementioned kits further comprise: a membrane intra- / co-staining medium, a washing medium, a cell plate fixing medium, a cell growth promoting incubation medium, an incubation blocking medium, and a substrate required for the development of said label, optionally comprising a positive serum, a negative serum, a washing medium and an incubation dilution medium.

[0018] The present application also relates to the use of the culture of capillary endothelial cells bearing said transplantation-associated antigens contained in the aforementioned kit for the preparation of a kit for detecting the sensitization status of the transplantation-associated antigens of a culture of capillary endothelial cells, for detecting / monitoring the presence and / or the titer of antibodies sensitizing the transplantation-associated antigens of the surface of capillary endothelial cells in a recipient of an organ transplant, and for monitoring / predicting endarteritis of the intima in a recipient of an organ transplant.

[0019] The core technology of the present application uses donor tissue or HLA-defined capillary cultures, whose cell surface contains all the natural antigens, thus allowing the accurate and specific identification of those antibodies sensitizing the cells, thus deepening their importance in the diagnosis of chronic rejection.

[0020] The present application uses cells of the donor or of the same HLA typing as the donor in the design of the test, while providing the sensitization of the anti-graft antibodies with the capillary endothelial cells, thus providing a window for the clinician to monitor the long-term survival of the graft in a patient receiving an organ transplant. The clinician can obtain the most direct and powerful pathological diagnosis without the need for biopsy material, predicting whether capillary endarteritis has occurred in the graft. It is known that early rejection is a reversible reaction, which can be treated. In the usual case, the doctor will take a biopsy from the patient over time, at one week, one month, three months, six months, one year after surgery, to observe the changes in the patient's condition. This is not only a matter of cost, but also a matter of trauma, and multiple long-term biopsies are not the first choice in clinical practice. More importantly, when the patient's pathological biopsy is abnormal, it usually indicates irreversible damage. Therefore, we urgently need to establish a non-traumatic molecular diagnostic technique that can detect rejection earlier.

[0021] The present application achieves the following effects, including but not limited to:

[0022] One Lambda's monoclonal antigen microsphere technology detects panel reactive antibodies, so only according to the manufacturer's specified antigens, it detects antibodies that react with synthetic peptide fragments, which is a pure chemical method of antibody detection. The mechanism of the kit of the present application is different, and it can quickly detect whether the recipient of a xenogeneic organ has produced any anti-graft antibodies against the allograft according to the presence or absence of cell sensitization, which is a biological method of antibody detection.

[0023] The use of donor tissue or endothelial cell cultures prepared according to individualized tissue typing provides all the recognition regions of donor antigens in their natural tertiary structure or HLA homologous antigens obtained from a matching library according to HLA type on the cell membrane surface, thus covering a more complete recognition region of HLA-related antigens than synthetic peptide fragments artificially cross-linked on microspheres, and thus the formation of antibodies can be detected more closely to the actual in vivo situation by the sensitization state of capillary endothelial cells. The test is a sensitive, specific and efficient method. The present application has clinical guiding significance.

[0024] The serum test of the present application is mature in serum taking technology, and can be operated in different places. The patient can be conveniently delivered by express mail, so that a larger range of patients can be regularly tested in different places.

[0025] The present application uses donor or capillary endothelial cells with the same HLA typing as the donor as the detection object, which is different from the cross matching test using lymphocytes, and is mainly used for long-term monitoring of chronic rejection after organ transplantation. BRIEF DESCRIPTION OF DRAWINGS

[0026] The accompanying drawings only illustrate some embodiments, and therefore should not be considered as limiting the scope.

[0027] Figure 1 Characterization is a microscopic photograph of the morphological characteristics of the in vitro capillary culture for testing cell antibody sensitization.

[0028] Figure 2A . Positive reaction test results using indirect fluorescence (FITC-labeled secondary antibody) for antibody sensitization: presence of sensitized antibodies against endothelial cells on the cell membrane surface (presenting green); VWF molecules specific to endothelial cells are contained in the cytoplasm (presenting red fluorescence).

[0029] Figure 2B . Negative reaction test results using indirect fluorescence (FITC-labeled secondary antibody) for antibody sensitization: only VWF molecules specific to endothelial cells are contained in the cytoplasm (presenting red fluorescence).

[0030] Figure 3A . Positive reaction results using horseradish peroxidase / DBA staining system for antibody sensitization: presence of sensitized antibodies against microvascular cultures on the cell surface (presenting dark brown, cell nucleus is dark blue).

[0031] Figure 3B . Negative reaction results using horseradish peroxidase / DBA staining system for antibody sensitization: no anti-microvascular culture on the cell surface only presents cell nucleus staining (presenting dark blue). DETAILED DESCRIPTION

[0032] The following detailed description of the application is presented in the context of the specific procedures of the kit, combined with data and figures, to provide a detailed understanding of the application to those skilled in the art. The scope of protection includes but is not limited to the content claimed by the claims, and the reagents and steps used can also be modified and adjusted accordingly as understood by those skilled in the art.

[0033] Definitions

[0034] "Chronic antibody-mediated rejection" as used herein refers to a pathological process of gradual loss of function of an organ after one year of transplantation, in the absence of acute rejection and drug toxicity, among other clear factors.

[0035] "Anti-HLA antibody" as used herein refers to an antibody synthesized by the body against an allogeneic HLA molecule due to exposure to non-self HLA profile by blood transfusion, pregnancy, or organ transplantation. This includes, among others, de novo antibodies associated with chronic transplantation, which are basically of the IgG type.

[0036] "Anti-non-HLA antibody" as used herein refers to an antibody synthesized by the body against molecules other than allogeneic HLA molecules due to exposure to non-self non-HLA antigens by blood transfusion, pregnancy, or organ transplantation.

[0037] "Capillary endothelial cell crossmatch" as used herein refers to a test in which in vitro cultured capillary endothelial cells isolated and expanded from donor tissue are incubated with the recipient's serum antibodies to detect whether an immunological reaction occurs.

[0038] "Immunohistochemistry and immunofluorescence" as used herein refers to the indication of the presence or absence of the expected antigen-antibody immune reaction by labeling the reactive enzyme molecules or fluorescent molecules on the antibody molecules, selected from any of the following groups: horseradish peroxidase, alkaline phosphatase, colloidal gold-silver solution, fluorescein isothiocyanate (FITC), tetraethyl rhodamine (Rhodamine), and tetramethyl isothiocyanate (TRIC).

[0039] "Capillary cell sensitization" as used herein refers to the binding of antigen molecules on the surface of capillary cells by the corresponding antibody molecules.

[0040] "Transplantation-associated antigen" as used herein refers to a biological molecule involved in the rejection reaction related to self-recognition between allogeneic individuals.

[0041] The "anti-graft antibodies or DSAs" described herein, including but not limited to "anti-human HLA antibodies", are antibodies against HLA molecules, which are considered to be the core part of the major histocompatibility molecules, and are protein molecules across the white blood cell membrane, and are the primary component of the immune system to recognize allogeneic tissues. However, with the continuous development of scientific research, non-white cell surface tissue cells also have antigens that can mediate secondary rejection reactions, thereby stimulating the body to produce corresponding antibodies. Unlike HLA, many non-HLA antigens lack specific and clear molecular characteristics. Therefore, anti-graft antibodies include antibodies against both HLA and non-HLA molecules.

[0042] The "detection" and "monitoring" described herein are used interchangeably, and "prediction" is for the present and future conditions.

[0043] Specifically, the present application relates to a kit for detecting the sensitization of a capillary endothelial cell culture to a transplantation-related antigen, which comprises capillary endothelial cells with said transplantation-related antigen, the sensitization of said transplantation-related antigen being caused by the binding reaction of said transplantation-related antigen with anti-graft antibodies; said transplantation-related antigen includes anti-donor HLA molecules and anti-donor non-HLA molecules on the surface of said capillary endothelial cells; and the sensitization of said transplantation-related antigen can be detected in the presence of said anti-graft antibodies in the serum of a transplant recipient.

[0044] In such a kit, the capillary endothelial cell culture is from an organ transplant donor or non-donor tissue, which is a mixed culture completely assembled according to the requirements of donor HLA antigen typing, which is based on serum typing as a standard.

[0045] In such a kit, the detection of the sensitization of said transplantation-related antigen is through a signal displayed on the endothelial cell membrane, which is a signal from indirect immunohistochemical staining or indirect immunofluorescent staining.

[0046] Such a kit contains a labeled anti-human IgG antibody against said graft antibodies, and the label molecule is selected from any one of the following group: horseradish peroxidase, alkaline phosphatase, colloidal gold-silver solution, fluorescein isothiocyanate (FITC), tetraethyl rhodamine (Rhodamine) and tetramethyl isothiocyanate (TRIC).

[0047] In such a kit, the endothelial cells in the capillary endothelial cell culture have been labeled with intracytoplasmic VWF.

[0048] Specifically, another aspect of the present invention relates to a kit for detecting / monitoring the presence and / or titer of anti-graft antibodies in organ transplant recipients, comprising a capillary endothelial cell culture containing the transplant-associated antigen contained in the aforementioned kit, and a labeled anti-human IgG antibody against the transplant antibodies contained in the aforementioned kit.

[0049] Specifically, another aspect of the present invention relates to a kit for monitoring / predicting endarteritis in organ transplant recipients, comprising a capillary endothelial cell culture containing the transplant-associated antigen contained in the aforementioned kit, and a labeled anti-human IgG antibody against the transplant antibody contained in the aforementioned kit.

[0050] The two kits mentioned above also include: intramembrane / membrane co-staining medium, washing medium, cell plate fixation medium, cell growth incubation medium, incubation blocking medium, and substrates required for the labeling and color development, optionally including positive serum, negative serum, washing medium, and incubation dilution medium.

[0051] Specifically, another aspect of the present invention relates to the use of the capillary endothelial cell culture containing the aforementioned transplantation-associated antigen contained in the kit in the preparation of kits for detecting the sensitization status of the transplantation-associated antigen in the capillary endothelial cell culture, kits for detecting / monitoring the presence and / or titer of antibodies sensitizing the surface of capillary endothelial cells to transplantation-associated antigen in organ transplant recipients, and kits for monitoring / detecting endothelial arteritis in organ transplant recipients.

[0052] The kits of the present invention for detecting the sensitization status of transplantation-related antigens in capillary endothelial cell cultures, and for detecting / monitoring the presence and / or titer of antibodies sensitizing the surface of capillary endothelial cells to transplantation-related antigens in organ transplant recipients, employ cellular methods, specifically characterized by in vitro culture of capillary cultures using organ transplant donors or those with known histocompatibility antigen typing (see attached). Figure 1 As a target of transplant-related antigens, the presence of antibodies against transplant-related antigens in the serum of patients who have received organ transplants is measured (Figure 2). The detection kit described in this invention can simultaneously measure the sensitization status of human leukocyte-associated antigens (HLA) and / or non-HLA on the surface of capillary endothelial cells (Figure 3).

[0053] In particular, the said cultures of vascular endothelial cells are highly selective and have a defined HLA antigen profile. The said cultures of vascular endothelial cells are highly selective in that they are derived from (1) donor tissue; (2) the HLA typing of the cells is specifically designed for the recipient by the physician based on the type of HLA antigens of the donor of the organ to be implanted in the patient. In particular, when the physician requests the use of the diagnostic reagent, he also submits the typing of the human leukocyte histocompatibility antigens, i.e. HLA-A, HLA-B, HLA-C, HLA-DR, HLA-DQ, HLA-DP, of the donor of the organ to be implanted in the patient. Based on this antigen profile, cells expressing these antigens are extracted, recovered and seeded in the bottom of 96-well cell culture microplates in the cell bank. The number of cells seeded is 3000-4000 viable cells / well. Thus, the cultures of capillary endothelial cells inherently express non-HLA and / or graft-related antigens associated with endothelial cells, containing antigenic determinants for the detection of anti-HLA and anti-non-HLA antibodies.

[0054] The specific operating procedure of the kit comprises: the addition of the patient's serum containing anti-HLA and anti-non-HLA antibodies to the capillary culture, after incubation under defined conditions, the antigen binds to the corresponding antibody, after the elution step, the addition of the labeled anti-human antibody (secondary antibody). If there is a color development or a fluorescent reaction (positive), it indicates the presence of anti-HLA and anti-non-HLA antibodies in the patient's serum; if there is no color development or fluorescent reaction (negative), it indicates the absence of anti-HLA and anti-non-HLA antibodies in the patient's serum.

[0055] In a particular embodiment, the labeled anti-human antibody (secondary antibody) includes, but is not limited to: horseradish peroxidase-labeled anti-human IgG antibody, alkaline phosphatase-labeled anti-human IgG antibody, colloidal gold-silver solution-labeled anti-human IgG antibody, fluorescein isothiocyanate (FITC)-labeled anti-human IgG antibody, tetraethyl rhodamine-labeled anti-human IgG antibody, and tetramethyl isothiocyanate (TRIC)-labeled anti-human IgG antibody.

[0056] In a particular embodiment, the cells with color development or fluorescent reaction (positive) are localized to the cell membrane.

[0057] In a particular embodiment, the color development substrate includes, but is not limited to: 3,3'-diaminobenzidine (DAB) and its derivatives, nitro blue tetrazolium chloride (NBT) and 5-bromo-4-chloro-3-indolyl phosphate toluene amine salt (BCIP) solution.

[0058] In a particular embodiment, the kit includes positive control serum, negative control serum, blank reagent, co-staining medium, blocking buffer, incubation medium 1, incubation medium 2.

[0059] The components and concentrations of the blank reagent, blocking buffer, incubation medium 1, incubation medium 2, incubation medium 3 solutions are as follows:

[0060] 1. Blank reagent: contains 1% albumin (w / v) in phosphate buffered saline

[0061] 2. Co-staining medium: contains 1:1000 dilution of anti-human VWF molecule antibody

[0062] 3. Blocking buffer: 10% (v / v) goat serum in phosphate buffered saline

[0063] 4. Incubation medium 1: 100 U / mL tumor necrosis factor (TNF-α) in phosphate buffered saline

[0064] 5. Incubation medium 2: 0.1% Tween-20 in phosphate buffered saline

[0065] 6. Incubation medium 3: nitro blue tetrazolium chloride (NBT) and 5-bromo-4-chloro-3-indolyl phosphate toluidine salt (BCIP) solution.

[0066] Examples

[0067] Preparation Example: Preparation of capillary endothelial cell culture:

[0068] 1. Isolation: capillary endothelial cells are isolated from adipose tissue using 0.1% collagenase;

[0069] 2. Pre-treatment of culture: before use of the cell culture, the cell growth medium is removed and incubation medium 1 is added and incubated at 37°C in 5% CO2 humidified conditions for 18-24 hours;

[0070] 3. After completion of incubation, the cells are washed with washing medium 1, repeated three times;

[0071] 4. Fixing medium is added to the cell wells, left for 30 minutes at room temperature or 18-20 hours at 4°C;

[0072] 5. After completion of fixation, the cells are washed with washing medium 1, repeated three times;

[0073] 6. Incubation medium 3 is added at room temperature to block the fixed cells, left for 1-2 hours;

[0074] 7. Co-staining medium is added at room temperature, left for 1-2 hours, after which all the liquid is aspirated;

[0075] 8. The cells are washed with washing medium 1, repeated three times. ​

[0076] Example 1: Sensitization status kit

[0077] (I) Operation steps

[0078] 1. Dilute patient serum with incubation medium 2 at a ratio of 1:2 and 1:4 while closing the cell plate;

[0079] 2. Add the diluted serum to be tested to the designated cell well position of the capillary endothelial cell culture;

[0080] 3. Incubate: 1 hour at 37°C in a humidified condition, or 18-20 hours at 4°C in a humidified condition;

[0081] 4. After completing the incubation of patient serum antibodies, rinse the cells with washing medium 2, repeat three times, each for at least 5 minutes;

[0082] 5. Add labeled secondary antibody diluted at 1:10 to 1:100, incubate for 1 hour at 37°C in a humidified condition;

[0083] 6. Rinse the cells with washing medium 2, repeat three times, each for at least 5 minutes;

[0084] 7. Add the corresponding substrate (only when using oxidase-labeled anti-human IgG antibody, alkaline phosphatase-labeled anti-human IgG antibody), observe the intensity of staining under a microscope;

[0085] 8. Terminate the reaction with pure water, and restain the cell nucleus with hematoxylin or DAPI;

[0086] 9. Mount the slide with resin or fluorescence protection fluid.

[0087] Positive and negative controls

[0088] The same operation is performed on the positive and negative controls according to the operation steps 2-9 of the reference kit.

[0089] (II) Data collection and result analysis

[0090] Under a general light microscope or a fluorescence microscope, the intensity of the substrate or fluorescence reaction of the cell membrane is mainly observed. The intensity of the substrate reaction is determined by the degree of optical density, and the intensity of the fluorescence reaction is determined by the proportion of positive cells in the field of view, which is divided into 0-4 degrees, as follows:

[0091] 0 degrees - no detectable change in substrate color compared with blank and negative serum controls, or the proportion of positive cells in the field of view is 0-2%

[0092] 1 degree - weak, discontinuous change in substrate color is visible, or > 2% and < 10% of the field is fluorescently positive

[0093] 2 degree - weak, continuous change in substrate color is visible, or > 10% and < 25% of the field is fluorescently positive

[0094] 3 degree - stronger, continuous change in substrate color is visible, or > 25% and < 50% of the field is fluorescently positive

[0095] 4 degree - strong, continuous change in substrate color is visible, or > 50% and < 100% of the field is fluorescently positive (consistent with positive control intensity)

[0096] The intensity of the substrate or the intensity of the fluorescent reaction is recorded and the sensitization result is calculated as a positive rate (%).

[0097] The statistical analysis data is as follows:

[0098] 1) In at least ten random fields at 400x magnification, take and retain digital records of the staining results of 50 cells.

[0099] 2) In at least ten random fields at 1000x magnification, take and retain digital records of the staining results of 10 cells.

[0100] <CONCLUSION>

[0101] We used this method to detect 13 patients after kidney transplantation, and detected the positive state of the cell plate sensitization result, expressed as a percentage.

[0102] Biochemical tests were performed on patients after transplantation to determine their disease course. Clinically, glomerular filtration rate and creatinine level are commonly used indicators. The judgment standard for the mild rejection group is glomerular filtration rate of 121-160 ml / min and serum creatinine level of 100-130 μmol / L. The judgment standard for the severe rejection group is glomerular filtration rate of more than 160 ml / min and serum creatinine level of more than 130 μmol / L.

[0103] According to the above standard, these patients are divided into early rejection group (7 patients) and severe rejection group (6 patients).

[0104] The test results are shown in Table 1

[0105] Positive rate (%) of capillary endothelial cell sensitization results Early rejection group (n=7) 6 / 7(86％) Severe rejection group (n=6) 5 / 6(83％)

[0106] The results are that 6 of the 7 patients in the early rejection group are positive, and 5 of the 6 patients in the severe rejection group are positive.

[0107] The positive rate detected in the early rejection group has important clinical significance because at this stage the immunosuppressants can be adjusted in time to reverse the treatment of organ transplant rejection.

[0108] Although the results of the severe rejection group did not reach 100%, because when the patient develops severe rejection after transplantation, the immune mechanism is not entirely antibody-mediated chronic transplant immune rejection, the antibodies involved include not only immunoglobulins such as IgG2 and IgG4, but also cell-mediated or other bypass immune rejection, so the study of the clinical immunopathological mechanism can provide further auxiliary guidance.

[0109] Example 2: Kit for detecting anti-graft antibody titers

[0110] The kit is prepared according to the above preparation examples.

[0111] To determine the titer of anti-graft antibodies, the patient's serum needs to be serially diluted 1:2, 1:4, 1:16, 1:64, and 1:256, and added to the designated cell culture well of the capillary endothelial cells. The remaining kit operation steps are described in Example 1.

[0112] The lowest titer that appears positive is the final measured anti-graft antibody titer.

[0113] The antibody positive titer results of the above 13 patients are shown in Table 2 below

[0114]

[0115] Example 3: Kit for detecting intimal arteritis in organ transplant recipients

[0116] Patients suspected of having intimal arteritis after organ transplantation were judged to be positive and negative by pathological biopsy, and capillary cell sensitization reaction determination was performed. The detection criteria for determining whether transplant immune rejection occurs in kidney transplant recipients include detection of intimal arteritis, glomerulitis, smooth muscle inflammation, CD4 + , T cell infiltration, etc. If 3 of the 5 indicators are positive, it is determined that transplant immune rejection has occurred.

[0117] Table 3. Sensitivity and specificity of detecting intimal arteritis in organ transplant recipients

[0118]

[0119] From the above kit detection results, it was found that the kit can compare the sensitivity and specificity of reflecting whether the transplant recipient has intimal arteritis, and because only body fluids need to be taken for detection, compared to pathological biopsy, which causes pain and poor compliance for regular review of transplant rejection detection methods, it has unpredictable replacement significance in the future.

[0120] While the present disclosure has been provided in terms of implementations, those skilled in the art will readily recognize that the embodiments can have applications in other implementations as well. Many modifications and variations of the present disclosure can be made by those of ordinary skill in the art without departing from the spirit and scope of the appended claims. Therefore, it is contemplated that the claims will cover the modifications and variations of this disclosure provided such are within the scope of the appended claims.

Claims

1. A kit for detecting the sensitization status of a transplant associated antigen of a capillary endothelial cell culture, comprising capillary endothelial cells bearing said transplant associated antigen, the sensitization of said transplant associated antigen resulting from a binding reaction of said transplant associated antigen with an anti- graft antibody; said transplant associated antigen comprising anti-donor HLA molecules and anti-donor non-HLA molecules on the surface of said capillary endothelial cells; said sensitization of said transplant associated antigen being detectable in the presence of said anti-graft antibody in the serum of a transplant recipient, wherein said capillary endothelial cell culture is from a tissue of an organ transplant donor.

2. The kit of claim 1, wherein the detection of the sensitization of said transplant associated antigen is by a signal displayed on the endothelial cell membrane, said signal being from an indirect immunohistochemical staining or an indirect immunofluorescent staining.

3. The kit of claim 2, wherein said kit comprises a labeled anti-human IgG antibody against said graft antibody, the label molecule being selected from any one of the following group: horseradish peroxidase, alkaline phosphatase, colloidal gold-silver solution, fluorescein isothiocyanate FITC, tetraethyl rhodamine and tetramethyl isothiocyanate TRIC.

4. The kit of claim 3, wherein the endothelial cells in said capillary endothelial cell culture have been labeled with intracytoplasmic VWF.

5. The kit of any one of claims 3-4, further comprising: intramembrane / membrane co-staining media, washing media, cell plate fixation media, cell growth promoting incubation media, incubation blocking media, and substrates required for the visualization of said label.

6. The kit of claim 5, further comprising positive serum, negative serum and incubation dilution media.

7. A kit for detecting / monitoring the presence and / or titer of an anti-graft antibody in a recipient of an organ transplant, comprising the capillary endothelial cell culture bearing said transplant associated antigen contained in the kit of any one of claims 1-4, and the labeled anti-human IgG antibody against said graft antibody contained in the kit of claim 4.

8. A kit for monitoring / predicting intimal arteriopathy in a graft of a recipient of an organ transplant, comprising the capillary endothelial cell culture bearing said transplant associated antigen contained in the kit of any one of claims 1-4, and the labeled anti-human IgG antibody against said graft antibody contained in the kit of claim 4.

9. The kit of claim 7 or 8, further comprising: intramembrane / membrane co-staining media, washing media, cell plate fixation media, cell growth promoting incubation media, incubation blocking media, and substrates required for the visualization of said label.

10. The kit of claim 9, further comprising positive serum, negative serum and incubation dilution media.

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