KR101555030B1 - A kit for examining organtransplant rejection - Google Patents

Abstract

The present invention relates to a test kit and a test method capable of effectively confirming a transplant rejection reaction in organ transplantation of different organs, specifically, a buffer solution containing human-derived albumin and a buffer solution containing 1,3-alpha galactose transporter gal galactosyltransferase, α-1,3-gal), wherein said α-galactosyltransferase is a glycoprotein produced by α-1,3-gal -Gal to an antigen-antibody reaction by an antibody (Anti-Gal), that is, a test kit for effectively confirming a transplant rejection reaction by organ transplantation of a heterologous organs measuring an anti-alpha-gal antibody reaction.

Description

{KIT FOR EXAMINING ORGANTRANSPLANT REJECTION}

The present invention relates to a kit for examining rejection of organ transplantation, specifically, a transplant rejection reaction by a heterologous antibody induced by organ transplantation of a heterologous organs by 1,3-alpha galactosyltransferase (α-1,3 (anti-Gal) antibody against the gal-α-1,3-gal (Gal α-1,3 Gal), a glycoprotein produced by the anti- The present invention relates to a test kit capable of measuring whether or not a transplant rejection reaction has occurred.

In various diseases, organ transplantation has been accepted as a clinically effective treatment, but a limited supply of organs has been the most serious limiting factor in clinical treatment. In this respect, heterologous organs are being accepted as new new technologies to replace donor organs that are lacking in the future. In particular, organs of pigs are accepted as substitutes that are readily available and conceptually almost unlimited. Among these, islet pancreas transplantation using pancreatic islets has been confirmed to be the most closely related field of clinical application. However, there are still many barriers in the clinical application of organ transplantation using heterologous organs, especially immunological barriers.

In this connection, gal-α-1,3-gal (Gal α-1-galactosyltransferase, α-1,3-gal) , 3-Gal, and -Agal), that is, anti-alpha-Gal antibody, are responsible for hyperacute rejection in non-human primate laboratory animals after heterologous transplantation. It has been reported that this can be overcome by using a porcine organs lacking genetically engineered α-1,3-galactosyltransferase.

The above-mentioned α-Gal is a protein identified in lower animals of primates or below. When transplanting a long-term animal or a heterologous organ of a lower animal, it causes a rejection reaction in the primate. Therefore, the measurement of the α- It can be an important criterion in monitoring long-term damage and monitoring other immune responses induced by? -Gal. Therefore, accurate measurement of the rejection response to the [alpha] -Gal is very important in providing basic data relating to the judgment of the high-risk patient group and the use of the immunosuppressive agent in xenogeneic organ transplantation.

Therefore, the development of an experimental model or measurement method capable of accurately measuring the rejection response to? -Gal has been increasingly emphasized in relation to treatment with xenotransplantation.

KR10-0313841B KR10-2002-0090087A KR10-0987547B

It is an object of the present invention to solve the problems of the prior art as described above, and an object of the present invention is to provide an examination method and an examination kit which can effectively confirm a transplant rejection reaction by organ transplantation of a dissimilar organs.

In order to accomplish the above object, the present invention provides an inspection kit and an inspection method which can effectively confirm a transplant rejection reaction in organ transplantation. The test kit and the test method may be an assay kit and an assay method for the heterologous antibody.

The test kit can effectively identify a rejection reaction by a heterologous antibody due to the presence or absence of an antibody against a heterogeneous antigen comprising a buffer solution containing no heterologous antigen and an antigen, or the content of an antibody against a heterologous antigen Which may be an inspection kit.

Specifically, the present invention relates to a buffer solution containing human serum albumin (HA) and a non-primate animal-derived protein, specifically α-Gal, for example, a 1,3-galactosyltransferase gal-α-1,3-gal (Gal α-1,3 Gal, α-Gal) which is a glycoprotein produced by α-1,3-gal, or artificially synthesized gal- (Gal-1, Gal, -Gal), and the antigen-antibody reaction (anti-Gal) against the -Gal, that is, the anti- The present invention relates to a test kit for effectively confirming a graft rejection reaction caused by organ transplantation. The anti-stock solution may be an anti-stock solution containing a protein derived from a primate, preferably an HA-conjugated? -Gal.

The present invention also relates to a buffer solution containing human serum albumin (HA) and a protein derived from a non-primate animal, specifically α-Gal, for example, a 1,3-galactosyltransferase, gal-α-1,3-gal (Gal α-1,3 Gal, α-Gal) or an artificially synthesized gal-α-1,3-gal (Gal-1, 3-Gal, -Gal), and the content of the anti-Gal against? -Gal is measured. The present invention relates to a test kit which can be verified.

The present invention also relates to a method for the treatment of transplant rejection by a heterologous antibody according to the organ transplantation which measures the content of the anti-Gal or the antigen-antibody reaction level including the buffer solution containing no antigen and the anti- The present invention relates to an inspection method that can be effectively confirmed.

Specifically, the test method is a method of measuring the content of the anti-Gal comprising the buffer solution containing no different antigens, specifically the buffer solution containing HA, and the anti-Gal solution containing the HA junction? -Gal, Which can effectively confirm the transplantation rejection reaction by the heterologous antibody.

The inventors of the present invention have found that organ transplantation of a heterologous antibody, specifically, a porcine-derived organs-derived porcine pancreatic islet is transplanted into a primate, specifically, a rhesus monkey, During the study of the antigen-antibody reaction, the immune response related to the rejection reaction was as follows: Anti-Gal antibody against α-Gal, which is a cause of hyperacidity rejection, and anti-PA antibody against pig albumin (Anti-HA) against human plasma albumin, that is, human serum albumin (HA), under various experimental conditions. , It was confirmed that the antibody reaction caused by the human albumin (BSA) was hardly caused, and the bovine albumin was removed from the buffer containing the existing bovine albumin (BSA) When HA conjugated α-Gal conjugated with human-derived albumin is used instead of BSA-conjugated α-Gal conjugated with bovine albumin as an antigen, interference with antigen antibody reaction is eliminated by the existing bovine albumin Gal can not accurately confirm the anti-Gal antibody reaction against? -Gal. Thus, the present invention has been completed.

Hereinafter, the present invention will be described in more detail.

In the present invention, a subject or an experimental animal is not particularly limited to any animal capable of carrying out an immune reaction by transplantation of a heterologous tissue or organ, and preferably is a human, a monkey, a cattle A vertebrate such as a horse, a sheep, a pig, a goat, a camel, a nutrition, a dog, a rabbit, and a mouse, more preferably a primate, and still more preferably a primate excluding a human.

In the present invention, an enzyme-linked immunosorbent assay (ELISA) is one of enzyme immunoassay methods. The enzyme immunoassay refers to a quantitative analysis technique for an antigen and an antibody, and refers to a method of measuring the result of an antigen and an antibody reaction using the degree of discoloration of the substance as a result of the reaction of the enzyme and the substrate. An enzyme such as peroxidase, alkaline-phosphatase, or beta-galactosidase is chemically bound to an antigen or an antibody to perform enzyme-staining after a specific antigen-antibody reaction, and the degree of discoloration is measured. The enzyme immunoassay is a method of measuring an amount of an antigen or an antibody using an antigen-antibody reaction using an enzyme as a marker.

The present invention relates to an examination kit which can effectively confirm a transplant rejection reaction by a heterologous antibody due to organ transplantation of a dissimilar organs.

Specifically, the present invention relates to an assay kit for assaying a transplant rejection reaction by a heterologous antibody according to transplantation of a heterologous organs by measuring an anti-αGal antibody reaction, wherein the assay kit comprises albumin A buffer solution not containing a heterologous protein, which is a primate-derived albumin, preferably HA, and an anti-stock solution containing α-Gal, specifically a primate-derived protein, preferably a primate-derived albumin, The present invention relates to a kit for organ transplant rejection test, which measures transplant rejection according to organ transplantation.

The kit for testing organ rejection reaction may be a kit for assaying an antigen-antibody binding reaction, preferably an assay kit for performing an enzyme-linked immunosorbent assay (ELISA) In this respect, a buffer used for enzyme immunoassay, a dilution buffer used for diluting the reaction solution, a blocking buffer for inhibiting non-specific reactions, and an anti- . The buffer solution is prepared by replacing a commonly used bovine albumin, that is, BSA with an albumin derived from primate, preferably HA derived from human albumin. In this respect, the buffer is preferably a buffer solution containing no heterologous protein, Primate-derived albumin, and even more preferably HA. In addition, the anti-stock solution contains α-Gal as an antigen and α-Gal not bound to a heterologous protein, and preferably an anti-stock solution containing albumin derived from primate, more preferably HA conjugated α-Gal .

In addition, the kit for organ transplant rejection test for measuring transplant rejection reaction according to the organ transplantation of the present invention comprises a primer-derived albumin, preferably a buffer containing HA and a primate-derived albumin, preferably a HA junction? -Gal An anti-proliferative agent; Measuring the antigen-antibody reaction by Anti-Gal or measuring the content of Anti-Gal.

The test kit can be prepared by measuring the content of Anti-Gal, which is an antibody contained in a sample separated from an animal such as blood of an experimental animal, using an albumin derived from a primate, preferably HA-conjugated? -Gal, By measuring the antibody reaction, it is possible to confirm a hyperacute rejection reaction caused by an anti-Gal antibody reaction that may occur in an experimental animal or an individual. It is also possible that the albumin contained in the buffer used in the test kit is albumin derived from primate, Is HA, and the antibody is a primate-derived albumin other than BSA, PSA or non-primate animal albumin conjugated alpha -Gal, preferably HA conjugated alpha -Gal, It is possible to minimize the antigen antibody reaction by primate animal-derived albumin, for example, BSA, and to minimize the interference reaction to the anti-Gal antibody reaction in the final confirmation result There are features that can improve the accuracy in the face.

Quantitative confirmation of the anti-Gal antibody reaction may be performed by a method of measuring an antigen-antibody binding reaction, preferably an enzyme immunoassay, more preferably an enzyme-linked immunosorbent assay (ELISA) . In this regard, the kits of the present invention may be used in conjunction with additional constituent means required to carry out enzyme immunoassays, in particular with respect to the performance of enzyme immunoassays or enzyme immunoassays, to those skilled in the art to which the present invention pertains For example, a secondary antibody against Anti-Gal, and the like.

The method for measuring the antigen-antibody binding reaction may be a radioimmunoassay (RIA), a sandwich assay, an immunoblot assay or the like in addition to the enzyme immunoassay, but is not limited thereto.

In another aspect, the present invention relates to a method for detecting an albumin-bound albumin-bound albumin-bound albumin-bound albumin-bound albumin-bound albumin from the serum of an individual transplanted with an enzyme- Immunospecific assay, Enzyme-linked immunosorbent assay (ELISA)) to determine the degree of anti-Gal immune response of an individual.

The primate-derived albumin may be human-derived albumin (HA), and the primate-derived albumin-bonded α-Gal may be an HA-bonded α-Gal.

Specifically, the test method is a method of detecting the presence or absence of albumin in a buffer solution, wherein the albumin contained in the buffer is albumin derived from primate, preferably HA; And an antigen-linked immunosorbent assay (ELISA) is carried out using an anti-Gal-immunosorbent assay (ELISA) using an anti-Gal-immune serum-containing albumin, preferably an HA- Or a transplant rejection test that measures transplant rejection according to organ transplantation.

The subject may be an animal other than a human, preferably a primate except for a human.

More specifically, the present invention relates to a method for screening an ELISA comprising: applying an ELISA well to an anti-parental solution comprising a non-primate animal-derived albumin non-primate albumin, preferably an HA junction? -Gal; Blocking with a blocking buffer containing a heterologous protein, that is, a non-primate animal-derived protein, and a primate-derived albumin, preferably HA; Diluting the serum of the recipient with the heterologous organ using a dilution buffer containing primer-derived albumin, preferably HA, which is not a non-primate-derived protein; Adding the serum of the individual transplanted with the diluted heterologous organ to an ELISA well to perform an anti-Gal antibody reaction; And stopping the reaction, and then reacting the secondary antibody against Anti-Gal.

The kit for examining transplant rejection according to the present invention and the test method for the transplant rejection reaction according to the present invention can be applied to a conventional method using antioxidants such as bovine serum albumin, Gal has been developed to eliminate the interference effect of the antigen-antibody reaction by Gal, thereby enabling to more accurately confirm the antigen-antibody reaction by Anti-Gal, and to study the hyperacute rejection which is the main failure cause of organ transplantation in the organ transplantation And in future organ transplantation, can be usefully applied to the selection of appropriate immunosuppressive agents.

In the case of the test kit of the present invention, in relation to the immune response normally caused by antibody transplantation of a heterologous organ, measurement of the heterologous antigen, i.e.,? -Gal, which is a protein expressed in lower animals, , It is possible to minimize the interference phenomenon by using primer-derived albumin, specifically human-derived albumin, that is, HA, without using BSA which is non-primate animal-derived albumin capable of showing an interference phenomenon, The antibody reaction of Gal can be accurately measured.

FIG. 1 is a graph showing the production of anti-Gal and anti-non-Gal antibodies induced by heterologous transplantation in a primate animal after transplantation of pork pancreas according to an embodiment of the present invention. FIG. 1B is a graph showing the results of anti-Gal-IgG before and after post-transplantation, and FIG. 1B is a graph showing the results of anti- (Porcine endothelial cells, PECs) of 1,3-αgalactosyltransferase gene knockout (GTKO).
FIG. 2 is a graph showing the results of Western blotting (2-DE gel) using 2-DE gel to identify specific anti-non-Gal antibodies induced by heterologous transplantation in primate animals after pig pancreas transplantation according to an embodiment of the present invention Western blot). The results of the antigen-antibody reaction were indicated by arrows, 1 means BA (bovine serum albumin, gi 1351907), 2 means PA (albumin, partial (Sus scrofa), gi 164318), 3 means calreticulin (Sus scrofa, gi 291622246), 4 means poly 4-hydroxylase beta polypeptide (Sus scrofa, gi 358009193), and 5 means cytoskeletal beta actin (partial scintillation sus scrofa, gi 45269029).
FIG. 3 is a graph showing the results of immunohistochemical analysis of human albumin (HA), pig albumin (PA) and the like in the plasma of non-human primates before and after the transplant to confirm the induction of porcine albumin by the heterologous transplantation in the primate animal after pancreas transplantation according to an embodiment of the present invention. ), Small albumin (BA), and HA conjugated? -Gal with an ELISA.
FIG. 4 is a graph showing the increase of IgG antibody against human albumin (HA), porcine albumin (PA), small albumin (BA) and HA conjugated? -Gal by heterologous transplantation according to an embodiment of the present invention 4A is an ELISA result for each antibody before and after heterologous organ transplantation for group 1, and FIG. 4B is an ELISA result for each antibody before and after heterogeneous organ transplantation for group 2-A. In each graph, the white bar graph means before the organ transplantation and the black bar graph means after the organ transplantation.
FIG. 5 is a graph showing an IgG antibody response to human albumin (HA), porcine albumin (PA), small albumin (BA) and HA-conjugated? -Gal according to an embodiment of the present invention, The results of the ELISA for each of the antibodies confirmed before and after the test. In each graph, the white bar graph means before the organ transplantation and the black bar graph means after the organ transplantation.

Hereinafter, embodiments of the present invention will be described in detail so that those skilled in the art can easily carry out the present invention. The present invention may, however, be embodied in many different forms and should not be construed as limited to the embodiments set forth herein.

< Example > Induced by pig pancreas transplantation Antigen-antibody reaction  Confirm

In order to solve the problem of the system for diagnosing the rejection reaction by the transplantation of the existing heterologous organ, that is, the transplantation of the pancreas in the present invention, in order to confirm the type of the antibody in the primate animal caused by the transplantation of the pancreas first, A rhesus monkey, a medical laboratory monkey with Rh factor with non human primates (NHPS), was subjected to intraportal porcine islet transplantation (PITX) (Kang HJ et al ., Transplantation 97: 999-1008 (2014)), and the occurrence of antibodies was confirmed according to the type of immunosuppressant administered.

Briefly, the porcine islets of the pigs were separated by the modified Ricordi method from a miniature adult pig (Seoul National University, Korea) (Kim HI, et. al ., Xenotransplantation 16: 11-18 (2009), Jin SM et al, Xenotransplantation 18:. 369 - 379 (2011)), pig pancreas transplantation (porcine islet transplantation ((PITX) ) with 10% swine serum (porcine serum) for 24 hours before addition of Medium 199 (Gibco BRL, Life Technologies Ltd , UK) at 37 &lt; 0 &gt; C using the free-floating method.

Experiments were carried out on 30 rhesus monkeys, and the separated islet clusters were treated with immunosuppression (IS) treated with 100,000 islet equivalents / kg per kg of experimental animals, (Rhesus monkey) with streptozotocin-induced type 1 diabetes.

Experiments were conducted by varying the type and amount of immunosuppressant treatment for the rhesus monkeys. Depending on the type and content of the immunosuppressant treatment, the animals were divided into groups as shown in Table 1 below. ATG refers to anti-thymocyte globulin (Thymoglobulin ^® , Genzyme, Cambridge, USA), anti-ICAM1 refers to anti-human intercellular adhesion molecule 1 antibody, MMF refers to mycophenolate anti-CD40 (2C10R4, National Institutes of Health, Bethesda, USA) is an antibody against CD154 and anti-CD154 (5C8, National Institutes of Health, Bethesda, USA) , And sirolimus means sirolimus (Rapamune ^® , Wyeth, USA), a commercially available immunosuppressive agent.

Specifically, in group I, five were treated with only conventional immunosuppressive therapy, not the CD40-CD40L blocker. On the other hand, group 2 (group II) was divided into 25 mice receiving immunosuppressants based on CD40-CD40L blockers. Group 2-A was divided into 10 animals treated with anti-ICAM1, sirolimus and anti-CD154. Group 2-B was divided into 7 animals treated with anti-ATG, sirolimus and anti-CD154. Group 2-C Were divided into 7 groups with ATG, sirolimus and anti-CD40, and group 2-C with 1 sirolimus and anti-CD154.

Regarding the administration of the anti-CD154, 20 mg / kg was administered intravenously, 4 days before the organ transplantation, 3 days after the organ transplantation, and 7 days after the organ transplantation. Lt; / RTI &gt; Thereafter, administration of Group 2-A was discontinued, and Group 2-B was administered every other week until the end of the experiment. The anti-CD40 was administered by intravenous injection at a dose of 30 mg / kg after 4 days of organ transplantation, 3 days of organ transplantation, 3 days, 7 days and 12 days after organ transplantation, respectively. , Respectively. Regarding the administration of sirolimus, oral administration was carried out 9 days prior to organs transplantation in such a manner that a stable minimum blood concentration was maintained in the range of 3 μg / ml to 8 μg / ml daily. The anti-ICAM1 was administered intravenously at a dose of 8 mg / kg before 9 days, 6 days and 3 days before organs transplantation, and the administration of ATG was intravenously injected 3 days before and 5 days before transplantation mg / kg &lt; / RTI &gt;

In Table 1 below, Anti-Gal means an antibody to the heterologous gal-α-1,3-gal (Gal α 1,3 Gal, α-gal), Anti-PA means an anti-porcine albumin antibody , Anti-BA means anti-small albumin antibody, and the values in the above table means the number of animals in which the antibody was identified in the test animals.

group Laboratory animal ID
(recipient) Immunosuppressant One month after organ transplantation, the induced antibodies Anti-Gal Anti-PA Anti-BA Group I
(n = 5) M1 campath, tacrolimus, leflunomide, MMF 5 5 3 M2 ATG, bortezomib, leflunomide, MMF M3 anti-ICAM1, rituximab M4 anti-ICAM1 M5 anti-ICAM1, bortezomib, leflunomide Group 2
(n = 5) Total 4 0 0 Group 2-A
(n = 10) M6, M9, M10, M11, M12, M13, M14, M15, M16,
M21 anti-ICAM1, sirolimus, anti-CD154 One 0 0 Group 2-B
(n = 7) M7, M8, M17, M18, M19, M20, M22 ATG, sirolimus, anti-CD154 2 0 0 Group 2-C
(n = 7) M23, M24, M25, M26, M27, M28, M29 ATG, sirolimus, anti-CD40 One 0 0 Group 2-D
(n = 1) M30 sirolimus, anti-CD154 0 0 0

Blood samples of each of the above-mentioned experimental animals were collected once a week until the organ transplantation was confirmed to be unsuccessful after organ transplantation, organ transplantation and organ transplantation, and stored in an EDTA tube (EDTA tube) And stored at -70 [deg.] C until used.

As shown in the above Table 1, in the case of the recipient of Group 1, all of the individuals were identified in terms of Anti-Gal (100%) and Anti-PA (100%), ), Whereas in group 2 recipients, the antibody was detected in 4 individuals (16%) in relation to Anti-Gal, but no antibody was detected in Anti-PA and Anti-BA.

Specifically, in order to measure anti-non-Gal antibodies, which are not antibodies of gal-α-1,3-gal induced by heterologous transplantation in primate animals after transplantation of pancreas, anti-Gal and anti-non -Gal were measured by ELISA and flow cytometry using a sample of the M4 experimental animal of Table 1, and the results are shown in FIG. As shown in FIG. 1A and FIG. 1B, a rapid increase in blood samples collected from rhesus monkeys, which are recipients of non-human primate, after heterologous transplantation of both Anti-Gal and Anti-non-Gal was confirmed.

From the above results, Western blotting was carried out using a sample of the M4 laboratory animal of Table 1 before and after heterologous transplantation in order to identify anti-non-Gal antibodies that were abruptly increased in the anti-non-Gal antibody Respectively.

This experiment was carried out using 150 μg of protein extracted from GTKO endothelial cells (PEC6) using a DUALXtract total membrane protein extraction kit (Dualsystems biotech AG, Switzerland) in 7 cm immobilized pH gradient strips (Bio-Rad Laboratories, USA) at 20 ° C for 12 hours. Separation was then performed on 2DE gels. Isoelectric focusing was performed using a 2-DE system (PROTEAN ^® IEF Cell, Bio-Rad Laboratories, USA) for 4 hours at 50 V, 20 minutes at 250 V, 40 minutes at 2,000 V, 2 hours horizontally. The IPG strip, which was subjected to the isoelectric focusing, was equilibrated and subjected to a 2-DE process on 10% SDS-PAGE (Bio-Rad Laboratories).

After the 2-DE process was performed, the cells were stained using Coomassie brilliant blue (Coomassie brilliant blue G-250 (Bio-Rad Laboratories, USA) , Western blotting was performed using 1% HA and Amersham (TM) ECL prime western blotting detection reagent (GE Healthcare, USA), followed by confirmation of the protein. As a result of immunohistochemical staining, the protein was identified using trypsin, followed by mass spectrometry using MALDI-TOF / TOF mass spectrometry. The measurement results are shown in Fig.

As shown in FIG. 2, the molecular weight of the first protein of FIG. 2 was 69,248, the second protein 71,348, the third protein 48,429, the fourth protein 56,763, and the fifth protein 40,194. MASCOT analysis MASCOT analysis, MASCOT 2.3.01) showed that the first protein was BA, the second protein was PA, the third protein was calreticulin precursor, the fourth protein was porcine poly 4-hydroxylase beta polypeptide and the fifth protein was porcine cytoskeletal beta actin.

In relation to the above results, in order to clarify which antibody is increased by the organ transplantation itself, blood samples of M4 laboratory animals before and after xenotransplantation were subjected to immunohistochemistry using human albumin (HA), porcine albumin (PA) The above experiment was carried out using an anti-stock solution containing albumin (BA) and HA-conjugated Gal (HA-HA), respectively, and the degree of antibody binding was analyzed by ELISA. The results of the analysis are shown in FIG.

The degree of the reaction of Xenoreactive antibodies by the above-mentioned heterologous transplantation was measured by the following method (Choi HJ, Kim MK, Lee HJ, et. al . Investigative ophthalmology & visual science 52: 6643-6650 (2011)).

Specifically, 100 μl of the stock solution containing HA, PA, BA and HA conjugated α-Gal (Gal-HA, 5 μg / mL; GlycoTech, USA) was added to each well, The antisense solution was discarded, and 100 μl of 5% HA blocking buffer diluted with PBS was added thereto, followed by reaction at 37 ° C for 30 minutes. Thereafter, the reaction solution was discarded, washed with washing buffer containing 0.05% Tween 20 for 5 times, 100 μl of a blood sample of an experimental animal diluted with 1% HA-added PBS was added to each well, Lt; / RTI &gt; for 30 minutes. After the antigen-antibody reaction using peroxidase-conjugated anti-human IgG (Sigma-Aldrich, USA), the color reaction was performed and the absorbance was measured. The result of subtracting the measured absorbance value and the absorbance of PBS sample (negative control) containing 1% HA was shown in FIG.

As shown in FIG. 3, it was confirmed that the reactivity was significantly increased in the cases of BA, PA and Gal-HA, while the antibody reaction was hardly increased in HA after heterologous transplantation.

To investigate how the heterologous antigenic antibody response is affected by immunosuppressive agents, the use of a general immunosuppressive agent and a CD40-CD40L blockade of antigen-antibody reaction by CD40 and CD40 ligand was used to inhibit the heterologous antigen antibody response . The experiments were performed on Group 1 and Group 2-A of the experimental animals of Table 1 above. The experimental results are shown in Table 1 and FIGS. 4A and 4B.

As shown in Table 1 and FIG. 4A, antibody responses were induced in all the experimental animals to which no immunosuppressive agent was administered for the CD40-CD40L blockade, whereas in the case of Group 2 , The CD40-CD40L blockade immunosuppressant was administered to the experimental animals. However, the antibody response to PA and BA was not induced, but the antibody against α-Gal was induced. Particularly, in group 2, IgG antibody against anti-BA and IgG antibody against anti-PA did not differ significantly at the end of one month after transplantation, while immunosuppressive agents for CD40-CD40L blockade IgG antibody against anti-BA was detected in 60% of the group 1, whereas IgG antibody against anti-PA was detected in 100% of the antibody reaction, whereas in group 2, antibody reaction Was not confirmed, and the tendency was confirmed to be different. In addition, IgG antibodies against anti-Gal were identified in all experimental animals in group 1, whereas only 4 samples in group 2 showed 16% incidence of heterologous antigen antibody response, and group 1 and group 2 was clearly identified. In addition, in the case of the immune response of the four individuals (0.29 ± 0.15) of the above-identified group 2, it was confirmed that the level was significantly lower than that of the group 1 (1.66 ± 0.49) (P = 0.0275, t test).

From the above results, it was confirmed that the immunosuppressant for the CD40-CD40L blockade can effectively inhibit the antibody reaction to anti-PA and anti-BA, but it was confirmed that it partially inhibited the anti-Gal antibody.

In addition, for group 1 animals in which organ transplantation failed due to the heterologous antigen antibody reaction, porcine pancreas transplantation was performed again for M4 and M5 experimental animals after 6 months. Prior to the second heterogeneous transplantation, anti-ICAM1, anti-CD154 and sirolimus immunosuppressants were administered, as in group 2-A. Blood samples were collected from experimental animals two days before and two days after the second heterologous transplantation, and the result of performing an antigen-antibody reaction to HA, BA, PA and Gal-HA in a blood sample is shown in FIG.

As shown in FIG. 5, in all the experimental results, no antibody reaction to HA was detected. On the other hand, before using the immunosuppressant, the antigen antibody response to BA, PA and Gal-HA was higher than that of rhesus monkey without treatment, but when using immunosuppressive agents, Antibody response was found to be significantly reduced, but BA and PA were found not to be reduced, resulting in failure of organ transplantation. From these results, it was expected that the antibody reaction to Anti-Gal and the antibody reaction to Anti-BA and anti-PA would be regulated by different pathways, and the reaction by Anti-PA and Anti-BA was related .

From the above-identified results, an experiment using an experimental animal was carried out in order to measure the anti-Gal changes in the case of replacing a heterologous protein such as albumin derived from non-primate animal including BA by a primate-derived protein, specifically HA. Specifically, an experiment was conducted on two rhesus monkeys (A and B) that had undergone the transplantation of pancreas after the administration of the immunosuppressant as in the case of the experimental animal M1. ELISA was confirmed by the same method using the sample collected from the same laboratory animal. HA-conjugated? -Gal was used as an antigen for confirming the immune response by Anti-Gal, and the buffers used for ELISA were HA buffer 5% and dilution buffer containing 1% HA, and a buffer solution containing BSA and a dilution buffer containing 1% BSA were used, and samples of each experimental animal Were tested for Anti-Gal IgG, respectively. The experimental results are shown in Table 2 below.

Anti-Gal IgG absorbance division HA buffer BSA buffer A (1/6400 dilution) 0.265 ± 0.002 0.357 0.003 A (1/3200 dilution) 0.498 + 0.014 0.685 ± 0.001 A (1/1600 dilution) 0.894 + 0.027 1.300 + 0.012 A (1/800 dilution) 1.460 + 0.012 2.158 + 0.040 B (1/100 dilution) 0.513 + 0.018 0.588 ± 0.002

As shown in Table 2 above, when BSA was used in both of the experimental animals A and B, the absorbance was measured to be about 150% as compared with the case of using HA, and it was estimated that there was a strong antibody reaction . These results were similar in the case of varying degree of dilution. From the above results, it was confirmed that when using buffer solution using BSA or BSA-conjugated? -Gal, anti-Gal It has been confirmed that it is not easy to accurately measure the degree of immunity rejection due to excessive measurement of the reaction. In order to solve this problem, when a buffer solution containing HA or HA-conjugated? -Gal is used, And thus the immune rejection can be precisely measured.

These results suggest that rejection of α-gal, a heterologous causative agent responsible for hyperacute rejection, in xenotransplantation provides important information on the humoral response in xenotransplantation, Considering the anti-BA reaction frequently observed in conjunction with the reaction by the anti-PA, in the enzyme immunoassay for the detection of Anti-Gal, which is an antibody against the -gal, in the ELISA using Anti-Gal, Or BA-conjugated Gal, it is possible to reduce the accuracy of the final result due to the interference effect in the anti-Gal antibody reaction. Therefore, in the ELISA using Anti-Gal, the buffer containing BA or BA -conjugated Gal, etc. It is considered that replacing the components containing BA with HA can contribute to the improvement of the accuracy of this experiment.

While the present invention has been particularly shown and described with reference to exemplary embodiments thereof, it is to be understood that the invention is not limited to the disclosed exemplary embodiments, Of the right.

Claims (8)

A1 The present invention relates to an examination kit for confirming a transplant rejection reaction by heterologous antibody by measuring an anti-alpha-gal antibody reaction according to organ transplantation of a heterologous organs,
Wherein the test kit comprises an anti-coagulant solution containing albumin-bound albumin-derived albumin and an albumin-conjugated alpha -Gal-derived albumin contained in the buffer solution, the organ transplant rejection measuring transplant rejection according to organ transplantation Kits for testing reactions. The method according to claim 1,
The primate-derived albumin is human-derived albumin (HA), and the α-Gal is produced by 1,3-galactosyltransferase (α-1,3-gal) Galactosidase is a galactosidase, gal-α-1,3-gal (Gal α-1,3 Gal) or artificially synthesized gal-α-1,3-gal HA conjugated &lt; RTI ID = 0.0 &gt; a-Gal &lt; / RTI &gt; A buffer solution in which the albumin contained in the buffer solution is human-derived albumin (HA), and an HA-conjugated? -Gal;
Measuring the antigen-antibody reaction by Anti-Gal;
A kit for organ transplant rejection test that measures transplant rejection according to organ transplantation of a dissimilar organs. A buffer solution in which the albumin contained in the buffer solution is human-derived albumin (HA), and an HA-conjugated? -Gal;
Measuring the content of Anti-Gal;
A kit for organ transplant rejection test that measures transplant rejection according to organ transplantation of a dissimilar organs. The organ transplant rejection test kit according to any one of claims 1 to 4, which is an examination kit for performing an enzyme-linked immunosorbent assay (ELISA) A transplant rejection reaction kit according to the present invention. An enzyme-linked immunosorbent assay (ELISA) was performed using a buffer solution in which the albumin contained in the buffer was albumin derived from primate and an anti-stock solution containing albumin-conjugated α-Gal derived from primate, , A method of determining the transplant rejection response by organ transplantation of a heterologous organs confirming the degree of the anti-Gal immune response of an individual. The method according to claim 6,
The primate-derived albumin is human-derived albumin (HA), the primate-derived albumin-conjugated alpha -Gal is HA-conjugated alpha -Gal, and the individual is a primate except human
Transplant rejection test method to measure transplant rejection according to organ transplantation. A step of applying an ELISA well to an anti-parental solution containing human-derived albumin (HA) conjugated? -Gal; Blocking buffer containing HA is added to an ELISA well coated with anti-spinning solution to block the blocking buffer; Diluting the serum of the transplant recipient with dilution buffer containing HA; Performing an anti-Gal antibody reaction by adding the serum of the individual transplanted with the diluted heterologous organ to an ELISA well; And stopping the antibody reaction and then reacting the secondary antibody against Anti-Gal
A transplant rejection test method for measuring transplant rejection according to organ transplantation.

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