Application of culture with transplantation-related antigen in preparation of related kit

Abstract

The invention relates to a culture related to a transplantation-related antigen sensitization state of capillary endothelial cells, application of the culture, and a detection kit containing the culture.

Description

[0001] This application is a divisional application. The original application has the application number 202210216930.5 and the application date is March 7, 2022. The invention is entitled "Cultures related to the sensitization status of transplantation-associated antigens of capillary endothelial cells, their uses and detection kits containing them". Technical Field

[0002] This invention relates to cultures related to the sensitization status of transplantation-associated antigens in capillary endothelial cells in the field of biotechnology, to the uses of said cultures, and to detection kits containing said cultures. Background Technology

[0003] China is the world's second-largest organ transplant country after the United States. Except for identical twins, all allogeneic organ transplants result in rejection, with chronic rejection being the most significant pathway leading to post-transplant organ dysfunction. While high-dose immunosuppressants can prevent acute rejection after organ transplantation, there is still no solution for chronic rejection. A study involving eight European countries revealed the impact of immunosuppressant use on post-transplant organ dysfunction rates between 1989 and 2014. They indicated that the use of highly effective immunosuppressants only reduced the post-transplant dysfunction rate from 16.8% to 4.6% within 0-1 years; while the changes in dysfunction within 1-3 years, 3-5 years, and 5-10 years remained within the range of 3.7%-4.2% (Legendre C. et al., Transplantation, 102(9S1):S1-S4, 2018 DOI:10.1097 / TP.00000002316). Therefore, the use of immunosuppressants to improve long-term survival has reached or is nearing its limit.

[0004] For the past decade or so, how to use biotechnology for early laboratory diagnosis of rejection has been one of the core issues in contemporary transplant science. Since the 1990s, complement-dependent cytotoxicity / autolysis has been the core of transplant immunopathological diagnosis and the primary means of diagnosing organ rejection before and after transplant surgery in hospitals. However, with increasing understanding of the mechanisms of organ transplant rejection, we recognize that the above tests only target short-term acute rejection; chronic rejection is a non-complement-dependent cytotoxic / cellular activation process. Specifically, peripheral blood lymphocytes are used to detect complement-dependent cytotoxicity / autolysis, while immune rejection occurs within capillary tissue. More importantly, newly synthesized specific anti-donor antibodies (DSA) after organ transplantation tend to indicate non-complement-dependent cytotoxicity / cellular activation. Therefore, current hospital-based diagnostic methods cannot comprehensively and accurately assess whether and when pathogenic rejection occurs in organ transplant patients. A significant reason for this is inappropriate diagnostic methods. We have not yet established a suitable method for measuring chronic organ rejection because its mechanisms are fundamentally different from acute rejection.

[0005] 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, katubulin, protein kinase Cζ, and major histocompatibility complex class I-associated A chain are all recognized as capable of stimulating the formation of anti-endothelial cell antibodies. However, many more transplant-associated antigens remain undisclosed or undiscovered (Delville M et al., J Am Soc Nephrol 30:692 2019 DOI:10.1681 / ASN.2018080868). Thirdly, it is particularly noteworthy that when vascular endothelial cells are activated by anti-graft antibodies, they actively participate in the rejection process. Its early pathological basis is capillary vasculitis; in later stages, it involves arterial intimal fibrosis and interstitial fibrosis. Pathogenic antibodies must bind tightly to HLA and non-HLA antigens on the surface of vascular endothelial cells to sensitize the cell surface, thereby triggering intracellular signal transduction pathways and causing endothelial cell activation (Bian H and Reed EF, J Immunol 163(2):1010-10181999). Continuously activated endothelial cells accelerate the release of inflammatory factors on the one hand, and increase adhesion proteins on the cell surface on the other.The combined result of these responses is the infiltration of large numbers of inflammatory cells and cell proliferation around the capillaries, which further develops into vascular obstruction, atrophy, and eventually tissue necrosis (Haas M, Am J Transplant 18(12):2849-2856 2018 doi:10.1111 / ajt.15088).

[0006] Currently, the biotechnology company CareDx (www.caredxinc.com) has developed molecular diagnostic technologies directly related to organ transplantation, based on a nucleic acid molecular hybridization technology platform. The core technology of CareDx products uses gene expression chips to measure the expression levels of rejection-related genes in peripheral blood lymphocytes to determine the patient's immune rejection status.

[0007] Biotechnology company One Lambda has developed a technology that uses Luminex single antigen microspheres (SABs) to coat a certain number of HLA molecular fragments—synthetic peptides with varying numbers of amino acids—on the surface of microspheres as targets, thereby binding to anti-graft antibodies in the patient's serum. However, current artificial peptide synthesis technologies, labeling techniques, and the complexity of HLA molecular groups cannot cover all antigens used in clinical xenotransplantation.

[0008] As Albrecht et al. pointed out, the antibodies produced by organ transplant recipients in response to donor antibodies are influenced by many factors, including subclass, titer, epitope, and valence. These factors are essential for maintaining the three-dimensional spatial structure of antigen-antibody binding molecules. Therefore, whether artificially synthesized peptide fragments can represent antigen molecules remains a highly controversial topic. In 2017, the FDA in the United States stated that the use of artificially coated antigens is a medically uncertain aspect in understanding antibody-mediated rejection reactions, their diagnosis, and prognosis, and should be approached with caution, especially in clinical applications (Albrecht R et al., Transplantation 102: e257, 2018). Although the XM-ONE method, marketed as a commercial product, utilizes endothelial progenitor cells isolated from peripheral blood, its clinical efficacy has not been universally accepted (Breimer ME et al., Transplantation 87:549 2009, doi:10.1097 / TP.0b013e3181949d4e.). Therefore, accurately assessing the pathogenicity of transplant antibodies, which depends on the interaction between anti-graft antibodies and vascular endothelial cells, is a crucial indicator for evaluating long-term survival after organ transplantation (Dragun D et al., Kidney International 90:2802016 DOI1:10.1016 / jkint.2016.03.019). Consequently, current diagnostic techniques suffer from high false-positive and false-negative rates in clinical use, hindering clinicians' accurate assessment of recipient status post-transplantation and reducing transplant success rates and survival rates.

[0009] Therefore, given the current scarcity of transplant organs, there is an urgent need for testing kits that can accurately monitor transplant recipients after surgery, guide postoperative treatment plans, and thereby improve transplant success rates and survival rates. Summary of the Invention

[0010] In view of the current technological background, this invention enhances the unique advantages of detecting HLA and non-HLA molecules on the surface of endothelial cells, and, combined with the immunohistochemical pathway, opens up new applications for it in measuring anti-donor antibodies in recipient serum after organ transplantation. A multifunctional kit has been developed, aiming to provide new data from a laboratory diagnostic perspective for the early detection of chronic rejection to guide clinical practice.

[0011] This invention relates to a kit for detecting the sensitization status of transplantation-associated antigens in capillary endothelial cell cultures, comprising capillary endothelial cells carrying the transplantation-associated antigens, wherein sensitization of the transplantation-associated antigens is caused by the binding reaction of the transplantation-associated antigens with anti-graft antibodies; the transplantation-associated antigens include anti-donor HLA molecules and anti-donor non-HLA molecules on the surface of the capillary endothelial cells; sensitization of the transplantation-associated antigens can be detected when the anti-graft antibodies are present in the serum of transplant recipients.

[0012] In this kit, the capillary endothelial cell culture is derived from organ transplant donor or non-donor tissue, the latter being a fully combined mixed culture based on donor HLA antigen typing requirements, wherein the HLA antigen typing is based on serum typing standards.

[0013] In this kit, sensitization to the transplant-associated antigen is detected by a signal displayed on the endothelial cell membrane, the signal being a signal from indirect immunohistochemical staining or indirect immunofluorescence staining.

[0014] This kit contains a labeled anti-human IgG antibody against the graft antibody, the labeling molecule being selected from any one of the following groups: horseradish peroxidase, alkaline phosphatase, colloidal gold-silver solution, fluorescein isothiocyanate (FITC), tetraethylrhodamine (Rhodamine), and tetramethylisothiocyanate (TRIC).

[0015] In this kit, the endothelial cell markers in the capillary endothelial cell culture have been identified by intracytoplasmic VWF labeling.

[0016] 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.

[0017] Another aspect of the present invention relates to a kit for predicting / monitoring 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.

[0018] 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.

[0019] Another aspect of the present invention relates to the use of the capillary endothelial cell culture containing the aforementioned transplant-associated antigen contained in the kit in the preparation of kits for detecting the sensitization status of the transplant-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 transplant-associated antigen in organ transplant recipients, and kits for monitoring / predicting endothelial arteritis in organ transplant recipients.

[0020] The core technology of this invention uses donor tissue or capillary cultures identified by HLA, whose cell surfaces contain all natural antigens. Therefore, it can accurately and specifically identify antibodies that sensitize cells, thereby enhancing their importance in the diagnosis of chronic rejection.

[0021] This invention utilizes donor or donor-identical HLA-typed tissue cells in its experimental design, while simultaneously providing anti-graft antibodies and sensitizing capillary endothelial cells. Therefore, it offers clinicians a window to monitor the long-term survival of transplant recipients. Clinicians can obtain the most direct and robust pathological diagnostic results without the need for biopsies, predicting whether capillary endarteritis will occur within the graft. It is well known that early rejection is reversible and treatable. Typically, doctors take biopsies within one week, one month, three months, six months, and one year after surgery to observe changes in the condition. This is not only a matter of cost, but biopsies are also invasive, and multiple long-term biopsies are not the first choice in clinical practice. More importantly, abnormal pathological biopsies usually indicate irreversible lesions. Therefore, there is an urgent need to develop non-invasive molecular diagnostic techniques to detect rejection at an earlier stage.

[0022] This invention achieves the following effects, including but not limited to:

[0023] One Lambda's single-antigen microsphere technology detects population-response antibodies, thus relying solely on the manufacturer-specified antigen to detect antibodies reacting with synthetic peptide fragments; this is a purely chemical antibody detection method. The kit in this application differs in mechanism, rapidly detecting whether an individual receiving a xenograft has produced any anti-graft antibodies against the xenograft based on the presence of cellular sensitization; this is a biological antibody detection method.

[0024] Because endothelial cell cultures using donor tissue or formulated based on individualized tissue typing provide all recognition regions of donor antigens in their natural tertiary structure or HLA isotype antigens obtained from a typing library according to HLA type on their cell membrane surface, they cover a more comprehensive range of HLA-related antigen recognition regions compared to synthetic peptide fragments artificially cross-linked on microspheres. Therefore, antibody formation can be detected more closely to the actual in vivo situation by detecting the sensitization status of capillary endothelial cells. This assay is a sensitive, specific, and efficient method. This invention has clinical guiding significance.

[0025] This invention uses serum testing, and the serum collection technology is mature and can be carried out in different locations. It is convenient for patients through fast mail delivery, thus enabling a wider range of patients to undergo regular testing in different locations.

[0026] This invention uses donor or capillary endothelial cells with the same HLA type as the donor as the detection object, which is different from the cross-matching test using lymphocytes. It is mainly used for long-term monitoring of chronic rejection after organ transplantation. Attached Figure Description

[0027] The accompanying drawings illustrate only some embodiments and should not be considered as a limitation on the scope.

[0028] Figure 1 Characterization refers to microscopic images of the morphological features of in vitro capillary cultures sensitized with cell antibodies.

[0029] Figure 2A The results of the positive reaction test sensitized by the antibody using indirect fluorescence (FITC-labeled secondary antibody) showed that anti-endothelial cell sensitizing antibodies were present on the cell membrane surface (appearing green); and endothelial cell-specific VWF molecules were present in the cytoplasm (appearing red fluorescence).

[0030] Figure 2B The results of the negative reaction test using indirect fluorescence (FITC-labeled secondary antibody) on cells not sensitized by the antibody showed that only endothelial cell-specific VWF molecules were present in the cytoplasm (exhibiting red fluorescence).

[0031] Figure 3A Positive results of antibody sensitization testing using the horseradish peroxidase / DBA staining system: Sensitizing antibodies against microvascular cultures were present on the cell surface (dark brown, with dark blue in the cell nucleus).

[0032] Figure 3B Negative results of antibody sensitization test using the horseradish peroxidase / DBA staining system: no anti-microvascular culture was observed on the cell surface, only the cell nucleus was stained (dark blue). Detailed Implementation

[0033] The following detailed explanation of the invention, using specific operating steps of the kit, along with data and charts, aims to provide a comprehensive overview of the invention for those skilled in the art. Its scope of protection includes, but is not limited to, the content claimed in the claims, and the reagents and steps used can be modified and adjusted accordingly in a manner understandable to those skilled in the art.

[0034] definition

[0035] The “chronic antibody-mediated rejection” mentioned in this article refers to the pathological process in which organ function is gradually lost one year after transplantation, in the absence of clear factors such as acute rejection and drug poisoning.

[0036] The "anti-HLA antibodies" discussed in this article refer to antibodies against allogeneic HLA molecules synthesized by the body due to exposure to non-autologous HLA profiles caused by blood transfusions, pregnancy, or organ transplantation. This includes, in particular, de novo antibodies synthesized post-transplant and associated with chronic transplantation, primarily IgG.

[0037] The “anti-non-HLA antibody” mentioned in this article refers to an antibody synthesized by the body against molecules other than allogeneic HLA molecules due to exposure to non-autologous non-HLA antigens caused by blood transfusion, pregnancy, or organ transplantation.

[0038] The “capillary endothelial cell cross-matching” described in this article refers to a test that uses in vitro cultured capillary endothelial cells isolated and expanded from donor tissue and recipient serum antibodies to detect whether an immunological response occurs.

[0039] The “immunohistochemistry and immunofluorescence” described herein refer to the indication of the presence of a desired antigen-antibody immune reaction by labeling antibody molecules with reactive enzyme molecules or fluorescent molecules, wherein the reactive enzyme molecules or fluorescent molecules are selected from any one of the following groups: horseradish peroxidase, alkaline phosphatase, colloidal gold-silver solution, fluorescein isothiocyanate (FITC), tetraethylrhodamine, and tetramethylisothiocyanate (TRIC).

[0040] The “capillary cell sensitization” mentioned in this article refers to the binding of antigen molecules on the surface of capillary cells to corresponding antibody molecules.

[0041] The “transplantation-associated antigens” mentioned in this article refer to biomolecules that participate in rejection reactions between allogeneic individuals through self-recognition.

[0042] The "anti-graft antibodies or DSA" mentioned in this article include, but are not limited to, "anti-human HLA antibodies." Historically, HLA molecules have been considered a core component of major histocompatibility molecules; they are protein molecules that cross the leukocyte membrane and are the primary components of the immune system's recognition of foreign tissues. However, with the continuous development of scientific research, antigens that can mediate secondary rejection reactions also exist on the surface of non-leukocyte cells, thereby stimulating the body to produce corresponding antibodies. Unlike HLA, many non-HLA antigens lack specific and well-defined molecular characteristics. Therefore, anti-graft antibodies encompass antibodies produced against both HLA and non-HLA molecules.

[0043] The terms "detection" and "monitoring" used in this article are interchangeable. "Prediction" refers to both the current situation and future conditions.

[0044] Specifically, one aspect of the present invention relates to a kit for detecting the sensitization status of transplantation-related antigens in capillary endothelial cell cultures, comprising capillary endothelial cells carrying the transplantation-related antigens, wherein sensitization of the transplantation-related antigens is caused by the binding reaction of the transplantation-related antigens with anti-graft antibodies; the transplantation-related antigens include anti-donor HLA molecules and anti-donor non-HLA molecules on the surface of the capillary endothelial cells; sensitization of the transplantation-related antigens can be detected when the anti-graft antibodies are present in the serum of the transplant recipient.

[0045] In this kit, the capillary endothelial cell culture is derived from organ transplant donor or non-donor tissue, the latter being a fully combined mixed culture based on donor HLA antigen typing requirements, wherein the HLA antigen typing is based on serum typing standards.

[0046] In this kit, sensitization to the transplant-associated antigen is detected by a signal displayed on the endothelial cell membrane, the signal being a signal from indirect immunohistochemical staining or indirect immunofluorescence staining.

[0047] This kit contains a labeled anti-human IgG antibody against the graft antibody, the labeling molecule being selected from any one of the following groups: horseradish peroxidase, alkaline phosphatase, colloidal gold-silver solution, fluorescein isothiocyanate (FITC), tetraethylrhodamine, and tetramethylisothiocyanate (TRIC).

[0048] In this kit, the endothelial cells in the capillary endothelial cell culture have been labeled with intracytoplasmic VWF.

[0049] 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.

[0050] 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.

[0051] 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.

[0052] 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.

[0053] 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).

[0054] Specifically, the vascular endothelial cell cultures are highly selective and have a defined HLA antigen profile. The high selectivity of the vascular endothelial cells means that they are derived from (1) donor tissue; (2) the HLA typing of the implanted cells is designed and specially formulated by laboratory personnel for the examinee based on the donor's HLA antigen type. Specifically, when the physician applies for this diagnostic reagent, they also submit the typing of human leukocyte tissue compatibility antigens (HLA-A, HLA-B, HLA-C, HLA-DR, HLA-DQ, HLA-DP) of the donor organ that has been implanted into the patient. Based on this antigen typing profile, cells expressing these antigens are extracted, revived, and implanted at the bottom of a 96-well cell culture microplate, with a planting quantity of 3000-4000 live cells / well. Therefore, capillary endothelial cell cultures inherently express non-HLA and / or endothelial cell-related transplantation antigens, containing antigenic determinants for detecting anti-HLA and anti-non-HLA antibodies.

[0055] The specific operating procedure of the kit includes: adding patient serum containing anti-HLA and anti-non-HLA antibodies to capillary culture, incubating under specified conditions, allowing the antigens to bind to the corresponding antibodies, followed by an elution step, and then adding labeled anti-human antibodies (secondary antibodies). If color development or fluorescence occurs (positive), it indicates the presence of anti-HLA and anti-non-HLA antibodies in the patient serum; if no anti-HLA and anti-non-HLA antibodies are present in the patient serum, there will be no color development or a fluorescence reaction (negative).

[0056] In specific implementations, 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, or anti-human IgG antibody labeled with colloidal gold-silver solution, fluorescein isothiocyanate (FITC)-labeled anti-human IgG antibody, rhodamine-labeled anti-human IgG antibody, and tetramethylisothiocyanate (TRIC)-labeled anti-human IgG antibody.

[0057] In a specific implementation plan, cells that show color development or exhibit a fluorescent reaction (positive) are located at the cell membrane.

[0058] In specific implementation schemes, the colorimetric reaction substrates are, but are not limited to: 3,3'-diaminobenzidine (DAB) and its derivatives, nitrotetrazole blue chloride (NBT) and a solution of 5-bromo-4-chloro-3-indolyl phosphate toluidine salt (BCIP).

[0059] In a specific implementation scheme, the kit includes positive control serum, negative control serum, blank reagent, co-staining medium, blocking buffer, incubation medium 1, and incubation medium 2.

[0060] The components and exemplified concentrations of the blank reagent, blocking buffer, incubation medium 1, incubation medium 2, and incubation medium 3 solutions are as follows:

[0061] 1. Blank reagent: Phosphate buffer containing 1% albumin (weight / volume)

[0062] 2. Co-staining medium: Contains anti-human VWF molecular antibody diluted 1:1000.

[0063] 3. Blocking buffer: 10% (volume / volume) sheep serum phosphate buffer

[0064] 4. Incubation medium 1: 100 U / mL tumor necrosis factor Phosphate buffer

[0065] 5. Incubation medium 2: 0.1% Tween-20 phosphate buffer

[0066] 6. Incubation medium 3: Nitrotetrazole blue chloride (NBT) and 5-bromo-4-chloro-3-indolyl phosphate toluidine salt (BCIP) solution.

[0067] Example

[0068] Preparation example: Preparation of capillary endothelial cell culture:

[0069] 1. Separation: Capillary endothelial cells were separated from adipose tissue using 0.1% collagenase;

[0070] 2. Pretreatment of culture: Before using cell culture, remove the cell growth medium, add incubation medium 1, and incubate at 37°C and 5% CO2 for 18-24 hours.

[0071] 3. After incubation, rinse the cells with washing medium 1, repeating three times;

[0072] 4. Add fixation medium to the cell wells and incubate at room temperature for 30 minutes, or at 4°C for 18-20 hours;

[0073] 5. After fixation, rinse the cells with washing medium 1, repeating three times;

[0074] 6. Add incubation medium 3 at room temperature to block and fix the cells, and let stand for 1-2 hours;

[0075] 7. Add co-staining medium at room temperature, let stand for 1-2 hours, and then aspirate all the liquid.

[0076] 8. Rinse the cells with washing medium 1, repeat three times.

[0077] Example 1: Sensitization state reagent kit

[0078] (I) Operating Procedures

[0079] 1. While sealing the cell plate, dilute the patient's serum with incubation medium 2 at ratios of 1:2 and 1:4;

[0080] 2. Add the diluted serum to be tested to the designated cell wells of the capillary endothelial cell culture;

[0081] 3. Incubation: 1 hour at 37℃ and humid conditions, or 18-20 hours at 4℃ and humid conditions;

[0082] 4. After completing the incubation of patient serum antibodies, rinse the cells with washing medium 2, repeating three times, each time for at least 5 minutes;

[0083] 5. Add the labeled secondary antibody diluted 1:10 to 1:100 and incubate at 37°C for 1 hour under humid conditions;

[0084] 6. Rinse the cells with washing medium 2, repeating three times, each time for at least 5 minutes;

[0085] 7. Add the appropriate substrate (only when using oxidase-labeled anti-human IgG antibody or alkaline phosphatase-labeled anti-human IgG antibody), and monitor the staining intensity under a microscope;

[0086] 8. Terminate the reaction with purified water, and counterstain the cell nuclei with hematoxylin or DAPI;

[0087] 9. Seal the slide using resin or fluorescent protective solution.

[0088] Positive control and negative control

[0089] Perform the same procedures on the positive and negative controls as described in steps 2-9 of the kit.

[0090] (II) Data Collection and Results Analysis

[0091] Under a light microscope or fluorescence microscope, the main observations are of the substrate or fluorescence reaction intensity on the cell membrane. Substrate reaction intensity is determined by optical density, while fluorescence reaction intensity is determined by the percentage of positive cells in the field of view, graded from 0 to 4, as follows:

[0092] 0 degrees - Compared to blank and negative serum controls, there is no perceptible change in substrate color, or the percentage of fluorescently positive cells in the field of view is 0-2%.

[0093] Grade 1 - Compared to blank and negative serum controls, weak, discontinuous changes in substrate color are visible, or the percentage of fluorescently positive cells in the field of view is >2% and ≤10%.

[0094] Grade 2 - Visible weak, continuous change in substrate color, or >10% and ≤25% of fluorescently positive cells in the field of view.

[0095] Grade 3 - Visible strong and continuous substrate color change, or >25% and ≤50% of fluorescently positive cells in the field of view.

[0096] Grade 4 - Visible strong continuous substrate color change, or the percentage of fluorescently positive cells in the field of view >50% and ≤100% (consistent with the reaction intensity of the positive control).

[0097] The sensitization result is calculated by recording the substrate intensity or fluorescence reaction intensity, and expressed as a positive rate (%).

[0098] The above statistical analysis data are as follows:

[0099] 1) Within a field of view with at least ten random magnifications of 400×, photograph and retain digital records of the staining results of 50 cells.

[0100] 2) Within a field of view with at least ten random magnifications of 1000×, photograph and retain digital records of the staining results of 10 cells.

[0101] <Conclusion>

[0102] We used this method to examine 13 post-kidney transplant patients and measured the positive status of cell plate sensitization results, expressed as a percentage.

[0103] Biochemical tests were performed on transplant recipients to determine their disease stage. Glomerular filtration rate (GFR) and creatinine levels are commonly used clinical indicators. The criteria for mild rejection were a GFR of 121-160 ml / min and a serum creatinine level of 100-130 μmol / L. The criteria for severe rejection were a GFR above 160 ml / min and a serum creatinine level above 130 μmol / L.

[0104] Based on the above criteria, these patients were divided into an early rejection group (7 patients) and a severe rejection group (6 patients).

[0105] The test results are shown in Table 1.

[0106]

[0107] The results showed that 6 out of 7 patients in the early rejection group were positive, and 5 out of 6 patients in the severe rejection group were positive.

[0108] The detection rate of positive results in the early rejection group is of great clinical significance because at this stage, immunosuppressants can be adjusted in time to achieve reversible organ transplant rejection.

[0109] Although the results in the severe rejection group did not reach 100%, the immune mechanism in patients with severe rejection after transplantation 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 alternative immune rejection mechanisms. Therefore, this study can provide further auxiliary guidance for the research on clinical immunopathological mechanisms.

[0110] Example 2: Kit for detecting anti-graft antibody titers

[0111] The preparation of this kit follows the same procedure as described above.

[0112] To determine the titer of anti-graft antibodies, the patient's serum was first serially diluted to five different concentrations: 1:2, 1:4, 1:16, 1:64, and 1:256, and then added to the designated wells of the capillary endothelial cell culture. For the remaining kit operation steps, please refer to Example 1.

[0113] The lowest titer at which a positive result was obtained was taken as the final measured titer of anti-graft antibody.

[0114] The antibody positive titer results of the above 13 patients are shown in Table 2 below.

[0115]

[0116] Example 3: Kit for detecting endarteritis in organ transplant recipients

[0117] Patients suspected of having post-transplant endoarteritis were diagnosed via pathological biopsy (positive or negative), and capillary cell sensitization was also performed. The criteria for assessing transplant rejection in kidney transplant recipients include testing for endoarteritis, as well as glomerulonephritis, leiomyomyitis, and CD4+. + T-cell infiltration, etc. If three out of the five indicators are positive, transplant immune rejection can be diagnosed.

[0118] Table 3. Sensitivity and specificity of detecting endarteritis in organ transplant recipients

[0119]

[0120]

[0121] The test results from the above kit show that it can sensitively and specifically reflect whether transplant recipients have endarteritis. Moreover, since it only requires a body fluid sample for testing, it has unforeseen clinical significance in the future compared to pathological biopsy, which is painful and has poor compliance as a regular follow-up test for transplant rejection.

[0122] While this disclosure has been provided according to the illustrated implementation, those skilled in the art will readily recognize that variations of the embodiments may exist and that such variations will be within the scope of this disclosure. Therefore, many modifications can be made by those skilled in the art without departing from the scope of the appended claims.

Claims

1. Use of capillary endothelial cell cultures carrying transplantation-associated antigens in the preparation of a kit for detecting the sensitization status of transplantation-associated antigens in capillary endothelial cell cultures, the kit comprising capillary endothelial cells carrying said transplantation-associated antigens, said sensitization being caused by the binding reaction of said transplantation-associated antigens with anti-graft antibodies; The transplant-related antigens include anti-donor HLA molecules and anti-donor non-HLA molecules on the surface of the capillary endothelial cells; When the anti-graft antibody is present in the serum of the transplant recipient, sensitization to the transplant-related antigen can be detected. The capillary endothelial cell culture mentioned therein comes from organ transplant donor or non-donor tissues, the latter being a mixed culture that is completely combined according to the donor HLA antigen typing requirements, and the HLA antigen typing is based on serum typing as the standard.

2. The use according to claim 1, wherein the detection of sensitization to the transplant-associated antigen is by means of a signal displayed on the endothelial cell membrane, said signal being a signal from indirect immunohistochemical staining or indirect immunofluorescence staining.

3. The use according to claim 2, wherein the kit comprises a labeled anti-human IgG antibody against the graft antibody, the labeling molecule being selected from any one of the following groups: horseradish peroxidase, alkaline phosphatase, colloidal gold-silver solution, fluorescein isothiocyanate (FITC), tetraethylrhodamine (Rhodamine), and tetramethylisothiocyanate (TRIC).

4. The use according to claim 3, wherein the endothelial cells in the capillary endothelial cell culture have been labeled with intracytoplasmic VWF.

5. The use according to any one of claims 1-4, wherein the kit further comprises: intramembrane / membrane co-staining medium, washing medium, cell plate fixation medium, cell growth incubation medium, incubation blocking medium, and substrate required for the labeling and staining, optionally including positive serum, negative serum, washing medium and incubation dilution medium.

6. Use of capillary endothelial cell cultures carrying transplant-associated antigens in the preparation of a kit for detecting / monitoring the presence and / or titer of anti-graft antibodies in organ transplant recipients, the kit comprising capillary endothelial cell cultures carrying said transplant-associated antigens and labeled anti-human IgG antibodies against said graft antibodies, wherein said capillary endothelial cell cultures are derived from organ transplant donor or non-donor tissues, the latter being a fully combined mixed culture according to donor HLA antigen typing requirements, said HLA antigen typing being based on serum typing standards.

7. Use of capillary endothelial cell cultures carrying transplant-associated antigens in the preparation of a kit for monitoring / predicting endarteritis of transplant recipients, the kit comprising capillary endothelial cell cultures carrying said transplant-associated antigens and labeled anti-human IgG antibodies against said transplant antibodies, wherein said capillary endothelial cell cultures are derived from organ transplant donor or non-donor tissues, the latter being a fully combined mixed culture according to donor HLA antigen typing requirements, said HLA antigen typing being based on serological typing.

8. The use according to claim 6 or 7, wherein the kit further comprises: intramembrane / membrane co-staining medium, washing medium, cell plate fixation medium, cell growth incubation medium, incubation blocking medium, and substrate required for the labeling and staining, optionally including positive serum, negative serum, washing medium and incubation dilution medium.

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