WO2019194360A1 - Method of testing transfusion compatibility in canine blood, and kit, monoclonal antibodies, and hybridoma cell lines for same - Google Patents

Method of testing transfusion compatibility in canine blood, and kit, monoclonal antibodies, and hybridoma cell lines for same Download PDF

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WO2019194360A1
WO2019194360A1 PCT/KR2018/007224 KR2018007224W WO2019194360A1 WO 2019194360 A1 WO2019194360 A1 WO 2019194360A1 KR 2018007224 W KR2018007224 W KR 2018007224W WO 2019194360 A1 WO2019194360 A1 WO 2019194360A1
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kai
blood
transfusion
antibody
canine
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Korean (ko)
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WO2019194360A8 (en
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김희영
이재호
이장춘
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김희영
이재호
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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C07K16/28—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
    • C12N5/10—Cells modified by introduction of foreign genetic material
    • C12N5/12—Fused cells, e.g. hybridomas
    • C12N5/16—Animal cells
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
    • C12N5/10—Cells modified by introduction of foreign genetic material
    • C12N5/12—Fused cells, e.g. hybridomas
    • C12N5/16—Animal cells
    • C12N5/163—Animal cells one of the fusion partners being a B or a T lymphocyte
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/564—Immunoassay; Biospecific binding assay; Materials therefor for pre-existing immune complex or autoimmune disease, i.e. systemic lupus erythematosus, rheumatoid arthritis, multiple sclerosis, rheumatoid factors or complement components C1-C9
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/577—Immunoassay; Biospecific binding assay; Materials therefor involving monoclonal antibodies binding reaction mechanisms characterised by the use of monoclonal antibodies; monoclonal antibodies per se are classified with their corresponding antigens
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/80—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving blood groups or blood types or red blood cells
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2510/00—Genetically modified cells
    • C12N2510/02—Cells for production

Definitions

  • the present invention relates to a method for determining the suitability of blood transfusion in dogs, and to kits, monoclonal antibodies and hybridoma cell lines therefor, specifically using antibodies to verify whether the blood of dogs for transfusion causes an immune response in the dog to be transfused.
  • An antigen-antibody reaction is performed on a blood sample of a dog for transfusion and a blood sample of a dog to be transfused, and the monoclonal antibody produced by hybridoma cells of Accession No. KCTC 13495BP and a hybridoma of Accession No.
  • the present invention relates to a method for determining transfusion suitability in dogs, and to kits, monoclonal antibodies and hybridoma cell lines for contacting monoclonal antibodies produced by the cells with each sample and detecting antigen-antibody binding.
  • DEAs dog Erythrocyte Antigens
  • the present inventors have attempted to develop a technology that can supplement or replace the problems of the conventional dog transfusion suitability determination technology.
  • studies have shown that dogs can be distinguished by antigens named Kai 1 and Kai 2 ( J Vet Intern Med 2016; 30: 1642-1647), but no transfusion suitability was known.
  • Subsequent studies further confirmed that the transfusion suitability could be determined by monoclonal antibodies to these antigens, thus completing the present invention.
  • the main object of the present invention is to provide a new method that can complement or replace the existing dog transfusion suitability determination method, such as DEA blood group system.
  • Another object of the present invention is to provide an antibody and a hybridoma cell line for producing the antibody, which are required for the new dog transfusion suitability determination method.
  • the present invention provides an antigen-antibody for a blood sample for transfusion and a blood sample for transfusion using an antibody so as to verify whether the blood for dog transfusion causes an immune response in the dog to be transfused.
  • the antibody comprises contacting each sample with a monoclonal antibody produced by a hybridoma cell of Accession No. KCTC 13495BP and a monoclonal antibody produced by a hybridoma cell of Accession No. KCTC 13496BP. It provides a method for determining the suitability of transfusion of dogs characterized by detecting the antibody binding.
  • the present invention is a kit for performing the above-described determination method, produced by a monoclonal antibody produced by a hybridoma cell of Accession No. KCTC 13495BP and a hybridoma cell of Accession No. KCTC 13496BP. It provides a dog transfusion suitability determination kit comprising a monoclonal antibody.
  • the present invention provides a monoclonal antibody produced by a hybridoma cell of Accession No. KCTC 13495BP and a hybridoma of Accession No. KCTC 13495BP which produces the monoclonal antibody.
  • a hybridoma cell of Accession No. KCTC 13495BP and a hybridoma of Accession No. KCTC 13495BP which produces the monoclonal antibody.
  • the present invention provides a monoclonal antibody produced by a hybridoma cell of Accession No. KCTC 13496BP and a hybridoma of Accession No. KCTC 13496BP producing the monoclonal antibody.
  • a hybridoma cell of Accession No. KCTC 13496BP and a hybridoma of Accession No. KCTC 13496BP producing the monoclonal antibody.
  • the method of the present invention uses the antigen of the dog blood independent from the Dal and Shigeta system as well as the DEA blood type system, which is a typical representative dog transfusion suitability method, and can effectively solve the problem of the conventional dog transfusion suitability determination.
  • the monoclonal antibodies of the present invention have high specificity for each antigen, thereby enabling very accurate and easy identification.
  • A Monoclonal antibody produced by hybridoma cells of Accession No. KCTC 13495BP (hereinafter referred to as 'anti-Kai 1 antibody')
  • B Single monoclonal antibody produced by hybridoma cells of Accession No. KCTC 13496BP Clone antibodies (hereinafter referred to as 'anti-Kai 2 antibodies')
  • M marker (kDa)
  • Lane 1 mouse IgG
  • Lane 2 and 3 purified antibody.
  • Figure 2 shows the ELISA results for identifying the homotype of the monoclonal antibody of the present invention.
  • A anti-Kai 1 antibody
  • B anti-Kai 2 antibody.
  • Figure 3 shows the immunoblot results for identifying the antigen of the monoclonal antibody of the present invention.
  • A anti-Kai 1 antibody
  • B anti-Kai 2 antibody
  • M marker (kDa)
  • Lane 1 Kai 1 + / Kai 2- erythrocytes
  • Lane 2 Kai 1- / Kai 2+ erythrocytes
  • Lane 3 Kai 1- / Kai 2- erythrocytes.
  • Figure 4 shows the affinity chromatography results for identifying the antigen of the monoclonal antibody of the present invention.
  • A Schematic diagram showing affinity chromatography procedure
  • B Protein binding to anti-Kai 1 antibody in membrane proteins of Kai 1+ erythrocytes by affinity chromatography-M: marker (kDa)
  • Lane 1 Kai Membrane protein purified from 1+ erythrocytes (10 ⁇ g)
  • C Kai 2+ erythrocyte membrane proteins were identified by affinity chromatography for binding to anti-Kai 2 antibodies-M: marker (kDa)
  • Lane 1 Bovine serum albumin (2 ⁇ g)
  • Lane 2 membrane protein purified from Kai 2+ erythrocytes (10 ⁇ g).
  • Figure 5 shows the aggregation results after 21 days of Kai mismatched transfusion model.
  • the method for determining the suitability of transfusion of a dog of the present invention uses an antibody to perform an antigen-antibody reaction on a blood sample of a transfusion dog and a blood sample of a dog to be transfused so as to verify whether the blood for dog transfusion causes an immune response in a dog to be transfused.
  • a monoclonal antibody produced by a hybridoma cell of Accession No. KCTC 13495BP (hereinafter referred to as an “anti-Kai 1 antibody”) and a single monoclonal antibody produced by a hybridoma cell of Accession No. KCTC 13496BP.
  • a cloned antibody (hereinafter referred to as an "anti-Kai 2 antibody”) is contacted with each of the above samples, and the antigen-antibody binding is detected.
  • Monoclonal antibodies of the present invention can be obtained according to the conventional monoclonal antibody production method using each of the hybridoma cell lines.
  • the hybridoma cells of the present invention can be obtained by inoculating mice intraperitoneally to obtain ascites and purify it.
  • Antibodies of the present invention are antibodies that specifically bind to antigens named Kai 1 and Kai 2, respectively, wherein the anti-Kai 1 antibody is an IgM kappa type antibody, and the anti-Kai 2 antibody is IgG3 lambda. Type of antibody.
  • the antigens Kai 1 and Kai 2 are membrane proteins of red blood cells (RBCs), of which Kai 1 represents a molecular weight of about 200 kDa and about 50 kDa, and Kai 2 of about 80 kDa.
  • the detection of antigen-antibody binding may be carried out according to conventional antigen-antibody reaction detection methods, for example, sedimentation, aggregation, complement binding, immunoadhesion.
  • a method of visually confirming that aggregation occurs by reacting a reagent containing an antibody with red blood cells on a test tube or plate, such as a method commonly used for human ABO blood group test, can be used.
  • dog blood is Kai 1 + / Kai 2- (hereinafter referred to as 'Kai 1 blood type'), Kai 1- / Kai 2+ (hereinafter referred to as 'Kai 2 blood type') or Kai 1- / Kai 2- (hereinafter referred to as 'Kai-blood type') can be classified into three types.
  • 'Kai 1+' indicates an antigen-antibody response to the anti-Kai 1 antibody
  • 'Kai 1-' indicates no antigen-antibody response to the anti-Kai 1 antibody
  • 'Kai 2+' indicates anti- Showing an antigen-antibody response to Kai 2 antibody
  • "Kai 2-" means not showing an antigen-antibody response to anti-Kai 2 antibody.
  • Kai 1 or Kai 2 acts as an antigen during blood transfusion of the dog, causing an immune response. That is, the blood of Kai 1+ is inappropriate for transfusion to Kai 1- dog, and the blood of Kai 2+ is inappropriate for transfusion to Kai 2- dog.
  • the method of the present invention is a biomarker of a new dog blood antigen different from the existing system. It can be said that it is a method for determining the suitability of blood transfusion.
  • the kit of the present invention is a kit capable of efficiently performing the method for determining the transfusion suitability of the dog of the present invention as described above, and includes the anti-Kai 1 antibody and the anti-Kai 2 antibody.
  • Reagents, instruments, etc. required for the method may be further included.
  • a reagent for stabilizing an antibody such as a buffer, an apparatus required for performing an antigen-antibody reaction such as a slide glass, and the like may be further included.
  • mice Female mice (BALB / c, 6 weeks old) were used. All mice were given free food and water intake and maintained a 12-hour light and dark cycle. EDTA blood samples from untransfused and transfused dogs were provided by Animal Hospital and Korea Animal Blood Bank (KABB). Written consent was obtained from dog owners for use of blood samples collected during treatment. All animal experiments were performed with the approval of the Experimental Animal Steering Committee.
  • Hybridomas producing monoclonal antibodies were prepared by obtaining RBCs from two Mastiff dogs in Korea and sensitizing them to six mice.
  • Rapid Vet-H DEA 1.1 (DMS Laboratories, Inc, Flemington, NJ, USA), classified as DEA 1.1+, Shigeta 1.1B + (Shigeta Animal Pharmaceutical Inc, Oyabe, Japan), polyclonal antiserum (KABB, Sokchosi, South Korea) was applied to the production of monoclonal anti-Kai 1 antibodies in dogs classified as DEA 1.1+.
  • the ascites were filtered through a 0.45 ⁇ m syringe filter, diluted 5 times with PBS, and passed through a protein L or protein G column (GenScript, Piscataway, NJ, USA). Column resin was washed three times with PBS, immunoglobulins were eluted with 0.1 M glycine (pH 3.0) buffer and neutralized by addition of 1 M Tris-HCl (pH 8.0). Purified monoclonal antibody was further diluted with PBS and analyzed by comparison with mouse IgG (Abcam, Cambridge, UK) by bicinchoninic acid (BCA) and sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE).
  • BCA bicinchoninic acid
  • SDS-PAGE sodium dodecyl sulfate polyacrylamide gel electrophoresis
  • Isoforms were determined by adding purified antibodies to two blood samples to each well and measuring absorbance at 450 nm at an optical density of at least 0.2.
  • the membranes were treated with Tween 20 containing Tris-buffered saline (TBST) containing 5% skim milk powder for 90 minutes at room temperature (about 22 ° C.). The membranes were reacted overnight at 4 ° C. with TBST containing 3% skim milk powder containing Kai 1 (1: 250 titer) or Kai 2 (1: 100) antibodies. Membranes were washed three times with TBST for 30 minutes and reacted for 90 minutes at room temperature with TBST containing 3% skim milk powder containing goat anti-mouse IgM or IgG HRP conjugated secondary antibody (1: 3000). After reacting with the secondary antibody, the membrane was washed three times for 30 minutes at room temperature with TBST. Proteins were detected with a chemiluminescent detection system.
  • Affinity purification was performed using Affi-Gel affinity chromatography according to the manufacturer's manual (Bio-Rad, CA, USA). More specifically, 1 mg of anti-Kai 1 antibody or anti-Kai 2 antibody was added to a column filled with Affi-Gel slurry, reacted overnight at 4 ° C, and the column resin was blocked with 100 mM ethanolamine and 3 times with PBS. Washed. Affinity proteins for IgM or IgG were purified using the same columns used for antibody purification.
  • a blood sample is obtained from a dog supplying blood and a dog to be transfused with Kai 1+ or Kai 2+ blood, washed with physiological saline and resuspended in physiological saline at 2-3%, and the same in a 3 ml tube.
  • a volume of plasma and RBC suspension were mixed and reacted at 37 ° C. for 15 minutes.
  • the tube was centrifuged at 1000 g for 1 minute and then gently mixed to remove pelletized RBC. The degree of aggregation was visually observed and graded from negative (0) to positive (4+).
  • Hybridoma cells specific for dog blood used for sensitization of mice were screened in a total of 4800 wells. Cell lines that were nonspecifically aggregated or cross-reacted to all RBCs were removed. Two hybridoma cell lines that produced different antibodies to two RBCs classified as DEA 1.1 and DEA 1.2, but did not cross-react, were isolated and named Kai 1 (KCTC13495BP) and Kai 2 (KCTC13496BP). Kai means 'dog'. The anti-Kai 1 and anti-Kai 2 antibodies contained in the ascites responded to the RBC aggregation test up to 1: 2056 and 1:32 dilution, respectively, and showed no hemolytic activity.
  • Protein L and G columns and SDS-PAGE were used to identify anti-Kai 1 immunoglobulins (Ig) with bands of ⁇ 80 kDa and ⁇ 25 kDa, respectively, on Protein L columns.
  • IgM immunoglobulins
  • anti-Kai 2 Ig was shown in the Protein G column with bands of ⁇ 50 kDa and ⁇ 20 kDa, corresponding to the heavy and light chains of mouse IgG (see FIG.
  • anti-Kai1 antibody to IgM with kappa light chain by ELISA coated with goat anti-mouse heavy chain antibody (IgG1, IgG2a, IgG2b, IgG3, IgA and IgM) or goat anti-mouse light chain antibody (kappa or lambda).
  • IgG1, IgG2a, IgG2b, IgG3, IgA and IgM goat anti-mouse light chain antibody
  • kappa or lambda goat anti-mouse light chain antibody
  • anti-Kai 1 monoclonal antibodies detected major bands of> 170 kDa and minor bands of ⁇ 55 and ⁇ 70 kDa in Kai 1+ samples, but not in Kai 1-blood (FIG. 3A).
  • anti-Kai 2 monoclonal antibodies were used, only single bands between 70 and 130 kDa appeared in the Kai 2+ samples (see FIG. 3B).
  • Affinity chromatography was performed to more accurately identify protein bands specific for the Kai 1 or Kai 2 antibodies.
  • anti-Kai 1 monoclonal antibody showed a major band of ⁇ 200 kDa and a split band of ⁇ 50 kDa.
  • anti-Kai 2 monoclonal antibody bound to prominent bands at ⁇ 80 kDa (see FIG. 4).
  • the DEA 1 blood group test indicated that both the blood type of the transfused dog and the transfused blood group were classified as DEA 1+ and that the transfusion was appropriate, but if the Kai types were different (experimental groups 1 and 2 in Table 2), DEA 1 Regardless of the type, tests with different types of Kai (experimental group 3 and 4 in Table 2) showed that the initial cross-linking test did not show any coagulation reactions, but it appeared appropriate after 21 days of coagulation test. Responses were observed, especially when the Kai type was different from the DEA 1 blood group.
  • the blood type of the transfused dog is Kai 2- / DEA 1+ and the blood type of the transfused blood is Kai 2 + / DEA 1+, and the blood type of the transfused dog is Kai 1- / DEA 1+ and the blood type of the transfused blood is Kai 1 In the case of + / DEA 1+, many thrombi were observed 21 days after the aggregation test (see FIG. 5).

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Abstract

The present invention relates to a method of testing transfusion compatibility in canine blood, and a kit, monoclonal antibodies, and hybridoma cell lines for same. More specifically, to verify whether a canine donor blood would cause an immune response in a patient dog, the present invention carries out an antigen-antibody reaction on a canine donor blood sample and a canine patient blood sample by using antibodies, wherein as the antibodies, monoclonal antibodies produced by hybridoma cells of the accession number KCTC 13495BP and monoclonal antibodies produced by the hybridoma cells of the accession number KCTC 13496BP are each brought in contact with each sample before detecting antigen-antibody bonds therefrom. Independent of the DEA blood-grouping system, which is the conventional representative method for testing transfusion compatibility in canine blood, as well as the Dal and Shigeta systems, the method of the present invention makes use of antigens in canine blood, thereby effectively addressing the shortcoming of existing methods for testing transfusion compatibility in canine blood. In particular, the monoclonal antibodies of the present invention each have high specificity to the antigen, and thus can enable extremely accurate and easy assessment.

Description

개의 수혈 적합성 판별 방법, 및 이를 위한 키트, 단일클론항체 및 하이브리도마 세포주Methods of Determining Transfusion Suitability in Dogs, and Kits, Monoclonal Antibodies, and Hybridoma Cell Lines
본 발명은 개의 수혈 적합성 판별 방법, 및 이를 위한 키트, 단일클론항체 및 하이브리도마 세포주에 관한 것으로, 구체적으로 수혈용 개 혈액이 수혈받을 개에서 면역반응을 일으키는지를 검증할 수 있도록 항체를 사용하여 수혈용 개 혈액 시료와 수혈받을 개의 혈액 시료를 대상으로 항원-항체 반응을 수행하되, 상기 항체로 기탁번호 KCTC 13495BP의 하이브리도마 세포에 의해 생산되는 단일클론항체 및 기탁번호 KCTC 13496BP의 하이브리도마 세포에 의해 생산되는 단일클론항체를 상기 각 시료와 접촉시키고, 항원-항체 결합을 검출하는 것을 특징으로 하는 개의 수혈 적합성 판별 방법 및, 이를 위한 키트, 단일클론항체 및 하이브리도마 세포주에 관한 것이다.The present invention relates to a method for determining the suitability of blood transfusion in dogs, and to kits, monoclonal antibodies and hybridoma cell lines therefor, specifically using antibodies to verify whether the blood of dogs for transfusion causes an immune response in the dog to be transfused. An antigen-antibody reaction is performed on a blood sample of a dog for transfusion and a blood sample of a dog to be transfused, and the monoclonal antibody produced by hybridoma cells of Accession No. KCTC 13495BP and a hybridoma of Accession No. KCTC 13496BP The present invention relates to a method for determining transfusion suitability in dogs, and to kits, monoclonal antibodies and hybridoma cell lines for contacting monoclonal antibodies produced by the cells with each sample and detecting antigen-antibody binding.
사람이 수혈을 받기 위해서는 수혈부작용을 최소화하기 위하여 면역거부반응을 일으키지 않는지 등을 사전에 확인하는 과정이 필수적이며, 적합하지 않은 혈액을 수혈할 경우 즉각 사망에 이를 수 있을 정도로 위험하다. 사람 이외의 다른 동물 또한 면역거부반응을 일으키는 혈액을 수혈할 경우 사망에 이를 수 있으므로, 수혈하기 전 적합성을 판별하는 것이 필요하다.In order to receive a blood transfusion, it is essential to check whether or not to cause an immune rejection reaction in order to minimize the side effect of blood transfusion, and if the blood is not properly transfused, it is dangerous enough to cause death immediately. Animals other than humans can also cause death if blood transfused to cause an immune rejection reaction, so it is necessary to determine suitability before transfusion.
사람에 대해서는 많은 연구가 이루어져 혈액형 검사, 항체 선별 검사, 교차 적합 시험 등 수혈 적합성을 판별하기 위한 효과적인 방법들이 개발되어 사용되고 있다. 반면, 개의 경우 몇몇 적합성 판별 방법이 개발되어 있기는 하지만 이에 대한 문제가 발생하고 있다.Many studies have been conducted on humans, and effective methods for determining transfusion suitability, such as blood typing, antibody screening, and cross-fit tests, have been developed and used. On the other hand, although some suitability determination methods have been developed for dogs, problems have arisen.
대표적으로 개 수혈에 따른 민감성 실험을 기초로 1974년 국제위원회에 의해 8개의 DEA(Dog Erythrocyte Antigen)가 다클론 동종항체로 분류되었고, 이들 항원을 이용하여 개의 혈액형을 판별하기 위한 기술들이 개발된 바 있다. 그러나 이들 항원 중 DEA 1에 대한 시약 만이 상업적으로 이용 가능하며, DEA 1의 경우 DEA 1.1과 1.2의 구분이 필요한데 아직까지 이것이 명확하지 않다는 문제와 민감도의 개선이 필요하다는 문제가 있다. 또한 Dal(Dalmatian) 및 Shigeta 개 혈액형을 포함하여 공식적으로 분류되지 않은 적혈구 항원(RBC)이 개에서 확인되었고, 2차 수혈로 급성 용혈성 수혈 반응을 보이는 경우 뿐만 아니라 1차 수혈 후 주요 교차적합 불화합성을 보이는 경우도 보고되어, DEA 혈액형 시스템을 보완하거나 대체하기 위한 새로운 수혈 적합성 판별 방법의 개발이 필요하다.Representatively, eight dog Erythrocyte Antigens (DEAs) were classified as polyclonal allogeneic antibodies by the International Committee in 1974 based on the sensitivity test of dog blood transfusions. Techniques for identifying dog blood types using these antigens were developed. have. But among these antigens, DEA Only reagents for 1 are commercially available, and DEA 1 requires a distinction between DEA 1.1 and 1.2, but this is not clear and there is a need for improved sensitivity. In addition, officially unclassified erythrocyte antigens (RBCs), including Dal (Dalmatian) and Shigeta dog blood types, have been identified in dogs and have major crossfit incompatibility after primary transfusion as well as those with acute hemolytic transfusion in secondary transfusions. In some cases, new transfusion suitability determination methods are needed to supplement or replace the DEA blood group system.
이에, 본 발명자들은 기존 개 수혈 적합성 판별 기술의 문제점들을 보완 또는 대체할 수 있는 기술을 개발하고자 하였다. 이와 관련하여 연구를 통해 Kai 1 및 Kai 2라고 명명한 항원으로 개의 혈액형을 구분할 수 있음을 증명한 바 있으나(J Vet Intern Med 2016;30:1642-1647), 수혈 적합성에 대해서는 알 수 없었다. 이후 추가 연구를 통해 이들 항원에 대한 단일클론항체로 수혈 적합성을 판별할 수 있음을 확인하고 본 발명을 완성하게 되었다.Accordingly, the present inventors have attempted to develop a technology that can supplement or replace the problems of the conventional dog transfusion suitability determination technology. In this regard, studies have shown that dogs can be distinguished by antigens named Kai 1 and Kai 2 ( J Vet Intern Med 2016; 30: 1642-1647), but no transfusion suitability was known. Subsequent studies further confirmed that the transfusion suitability could be determined by monoclonal antibodies to these antigens, thus completing the present invention.
따라서 본 발명의 주된 목적은 DEA 혈액형 시스템과 같은 기존 개의 수혈 적합성 판별 방법을 보완 또는 대체할 수 있는 새로운 방법을 제공하는데 있다.Therefore, the main object of the present invention is to provide a new method that can complement or replace the existing dog transfusion suitability determination method, such as DEA blood group system.
본 발명의 다른 목적은 상기 새로운 개의 수혈 적합성 판별 방법에 필요한 항체 및 이 항체를 생산하는 하이브리도마 세포주를 제공하는데 있다.Another object of the present invention is to provide an antibody and a hybridoma cell line for producing the antibody, which are required for the new dog transfusion suitability determination method.
본 발명의 한 양태에 따르면, 본 발명은 수혈용 개 혈액이 수혈받을 개에서 면역반응을 일으키는지를 검증할 수 있도록 항체를 사용하여 수혈용 개 혈액 시료와 수혈받을 개의 혈액 시료를 대상으로 항원-항체 반응을 수행하되, 상기 항체로 기탁번호 KCTC 13495BP의 하이브리도마 세포에 의해 생산되는 단일클론항체 및 기탁번호 KCTC 13496BP의 하이브리도마 세포에 의해 생산되는 단일클론항체를 상기 각 시료와 접촉시키고, 항원-항체 결합을 검출하는 것을 특징으로 하는 개의 수혈 적합성 판별 방법을 제공한다.According to one aspect of the present invention, the present invention provides an antigen-antibody for a blood sample for transfusion and a blood sample for transfusion using an antibody so as to verify whether the blood for dog transfusion causes an immune response in the dog to be transfused. Performing a reaction, wherein the antibody comprises contacting each sample with a monoclonal antibody produced by a hybridoma cell of Accession No. KCTC 13495BP and a monoclonal antibody produced by a hybridoma cell of Accession No. KCTC 13496BP. It provides a method for determining the suitability of transfusion of dogs characterized by detecting the antibody binding.
본 발명의 다른 양태에 따르면, 본 발명은 상기 판별 방법을 수행하기 위한 키트로, 기탁번호 KCTC 13495BP의 하이브리도마 세포에 의해 생산되는 단일클론항체 및 기탁번호 KCTC 13496BP의 하이브리도마 세포에 의해 생산되는 단일클론항체를 포함하는 개의 수혈 적합성 판별용 키트를 제공한다.According to another aspect of the present invention, the present invention is a kit for performing the above-described determination method, produced by a monoclonal antibody produced by a hybridoma cell of Accession No. KCTC 13495BP and a hybridoma cell of Accession No. KCTC 13496BP. It provides a dog transfusion suitability determination kit comprising a monoclonal antibody.
본 발명의 또 다른 양태에 따르면, 본 발명은 상기 판별 방법에 필요한 기탁번호 KCTC 13495BP의 하이브리도마 세포에 의해 생산되는 단일클론항체, 및 이 단일클론항체를 생산하는 기탁번호 KCTC 13495BP의 하이브리도마 세포주를 제공한다.According to another aspect of the present invention, the present invention provides a monoclonal antibody produced by a hybridoma cell of Accession No. KCTC 13495BP and a hybridoma of Accession No. KCTC 13495BP which produces the monoclonal antibody. Provide a cell line.
본 발명의 또 다른 양태에 따르면, 본 발명은 상기 판별 방법에 필요한 기탁번호 KCTC 13496BP의 하이브리도마 세포에 의해 생산되는 단일클론항체, 및 이 단일클론항체를 생산하는 기탁번호 KCTC 13496BP의 하이브리도마 세포주를 제공한다.According to another aspect of the present invention, the present invention provides a monoclonal antibody produced by a hybridoma cell of Accession No. KCTC 13496BP and a hybridoma of Accession No. KCTC 13496BP producing the monoclonal antibody. Provide a cell line.
본 발명의 방법은 기존의 대표적인 개 수혈 적합성 판별 방법인 DEA 혈액형 시스템 뿐만 아니라 Dal 및 Shigeta 시스템과는 별개의 독립적인 개 혈액의 항원을 이용하는 방법으로, 기존 개 수혈 적합성 판별의 문제점을 효과적으로 보완할 수 있다. 특히 본 발명의 단일클론항체는 각 항원에 대해 특이성이 높아 매우 정확하고 용이한 판별을 가능하게 한다.The method of the present invention uses the antigen of the dog blood independent from the Dal and Shigeta system as well as the DEA blood type system, which is a typical representative dog transfusion suitability method, and can effectively solve the problem of the conventional dog transfusion suitability determination. have. In particular, the monoclonal antibodies of the present invention have high specificity for each antigen, thereby enabling very accurate and easy identification.
도 1은 본 발명의 단일클론항체를 SDS-PAGE로 분리한 결과이다. (A) 기탁번호 KCTC 13495BP의 하이브리도마 세포에 의해 생산되는 단일클론항체(이하, '항-Kai 1 항체'라 한다), (B) 기탁번호 KCTC 13496BP의 하이브리도마 세포에 의해 생산되는 단일클론항체(이하, '항-Kai 2 항체'라 한다), M: 마커(kDa), Lane 1: 생쥐의 IgG, Lane 2 및 3: 정제된 항체.1 is a result of separation of the monoclonal antibody of the present invention by SDS-PAGE. (A) Monoclonal antibody produced by hybridoma cells of Accession No. KCTC 13495BP (hereinafter referred to as 'anti-Kai 1 antibody'), (B) Single monoclonal antibody produced by hybridoma cells of Accession No. KCTC 13496BP Clone antibodies (hereinafter referred to as 'anti-Kai 2 antibodies'), M: marker (kDa), Lane 1: mouse IgG, Lane 2 and 3: purified antibody.
도 2는 본 발명의 단일클론항체의 동종형을 확인하기 위한 ELISA 결과를 나타낸 것이다. (A) 항-Kai 1 항체, (B) 항-Kai 2 항체.Figure 2 shows the ELISA results for identifying the homotype of the monoclonal antibody of the present invention. (A) anti-Kai 1 antibody, (B) anti-Kai 2 antibody.
도 3은 본 발명의 단일클론항체의 항원을 확인하기 위한 면역블롯 결과를 나타낸 것이다. (A) 항-Kai 1 항체, (B) 항-Kai 2 항체, M: 마커(kDa), Lane 1: Kai 1+/Kai 2- 적혈구, Lane 2: Kai 1-/Kai 2+ 적혈구, Lane 3: Kai 1-/Kai 2- 적혈구.Figure 3 shows the immunoblot results for identifying the antigen of the monoclonal antibody of the present invention. (A) anti-Kai 1 antibody, (B) anti-Kai 2 antibody, M: marker (kDa), Lane 1: Kai 1 + / Kai 2- erythrocytes, Lane 2: Kai 1- / Kai 2+ erythrocytes, Lane 3: Kai 1- / Kai 2- erythrocytes.
도 4는 본 발명의 단일클론항체의 항원을 확인하기 위한 친화 크로마토그래피 결과를 나타낸 것이다. (A) 친화 크로마토그래피 과정을 나타낸 모식도, (B) Kai 1+ 적혈구의 막단백질 중 항-Kai 1 항체에 결합하는 단백질을 친화 크로마토그래피로 확인한 결과 - M: 마커(kDa), Lane 1: Kai 1+ 적혈구에서 정제된 막단백질(10㎍), (C) Kai 2+ 적혈구의 막단백질 중 항-Kai 2 항체에 결합하는 단백질을 친화 크로마토그래피로 확인한 결과 - M: 마커(kDa), Lane 1: 소혈청알부민(2㎍), Lane 2: Kai 2+ 적혈구에서 정제된 막단백질(10㎍).Figure 4 shows the affinity chromatography results for identifying the antigen of the monoclonal antibody of the present invention. (A) Schematic diagram showing affinity chromatography procedure, (B) Protein binding to anti-Kai 1 antibody in membrane proteins of Kai 1+ erythrocytes by affinity chromatography-M: marker (kDa), Lane 1: Kai Membrane protein purified from 1+ erythrocytes (10 μg), (C) Kai 2+ erythrocyte membrane proteins were identified by affinity chromatography for binding to anti-Kai 2 antibodies-M: marker (kDa), Lane 1 : Bovine serum albumin (2 μg), Lane 2: membrane protein purified from Kai 2+ erythrocytes (10 μg).
도 5는 Kai 불일치 수혈 모델의 21일 후 응집 결과를 나타낸 것이다. (A) Kai 2+/DEA 1+ 혈액을 수혈받은 Kai 2-/DEA 1+ 개의 응집 결과, (B) Kai 1+/DEA 1+ 혈액을 수혈받은 Kai 1-/DEA 1+ 개의 응집 결과. 기준막대 : 200㎛.Figure 5 shows the aggregation results after 21 days of Kai mismatched transfusion model. (A) Kai 2 + / DEA 1+ blood transfusion results in Kai 2- / DEA 1+ blood; (B) Kai 1 + / DEA 1+ blood transfusion results in Kai 1- / DEA 1+ blood transfusions. Reference bar: 200 μm.
본 발명의 개의 수혈 적합성 판별 방법은 수혈용 개 혈액이 수혈받을 개에서 면역반응을 일으키는지를 검증할 수 있도록 항체를 사용하여 수혈용 개 혈액 시료와 수혈받을 개의 혈액 시료를 대상으로 항원-항체 반응을 수행하되, 상기 항체로 기탁번호 KCTC 13495BP의 하이브리도마 세포에 의해 생산되는 단일클론항체(이하, '항-Kai 1 항체'라 한다) 및 기탁번호 KCTC 13496BP의 하이브리도마 세포에 의해 생산되는 단일클론항체(이하, '항-Kai 2 항체'라 한다)를 상기 각 시료와 접촉시키고, 항원-항체 결합을 검출하는 것을 특징으로 한다.The method for determining the suitability of transfusion of a dog of the present invention uses an antibody to perform an antigen-antibody reaction on a blood sample of a transfusion dog and a blood sample of a dog to be transfused so as to verify whether the blood for dog transfusion causes an immune response in a dog to be transfused. A monoclonal antibody produced by a hybridoma cell of Accession No. KCTC 13495BP (hereinafter referred to as an “anti-Kai 1 antibody”) and a single monoclonal antibody produced by a hybridoma cell of Accession No. KCTC 13496BP. A cloned antibody (hereinafter referred to as an "anti-Kai 2 antibody") is contacted with each of the above samples, and the antigen-antibody binding is detected.
본 발명의 단일클론항체는 상기 각 하이브리도마 세포주를 사용하여 통상적인 단일클론항체 생산방법에 따라 수득할 수 있다. 예를 들어, 본 발명의 하이브리도마 세포를 쥐에 복강 내 접종하여 복수를 얻고 정제하는 방법으로 수득할 수 있다.Monoclonal antibodies of the present invention can be obtained according to the conventional monoclonal antibody production method using each of the hybridoma cell lines. For example, the hybridoma cells of the present invention can be obtained by inoculating mice intraperitoneally to obtain ascites and purify it.
본 발명의 항체는 각각 Kai 1, Kai 2라 명명된 항원에 특이적으로 결합하는 항체로 항-Kai 1 항체는 IgM 카파(kappa) 타입의 항체이며, 항-Kai 2 항체는 IgG3 람다(lamda) 타입의 항체이다. 항원인 Kai 1 및 Kai 2는 개의 적혈구(red blood cell, RBC)의 막단백질(membrane protein)로 이중 Kai 1은 약 200kDa 및 약 50kDa의 분자량 크기, Kai 2는 약 80kDa의 분자량 크기를 나타낸다.Antibodies of the present invention are antibodies that specifically bind to antigens named Kai 1 and Kai 2, respectively, wherein the anti-Kai 1 antibody is an IgM kappa type antibody, and the anti-Kai 2 antibody is IgG3 lambda. Type of antibody. The antigens Kai 1 and Kai 2 are membrane proteins of red blood cells (RBCs), of which Kai 1 represents a molecular weight of about 200 kDa and about 50 kDa, and Kai 2 of about 80 kDa.
본 발명에서 항원-항체 결합의 검출은 통상적인 항원-항체 반응 검출 방법, 예를 들어 침강반응, 응집반응, 보체결합반응, 면역부착반응 등의 방법에 따라 수행될 수 있다. 사람의 ABO 혈액형 검사를 위해 통상적으로 사용되는 방법과 같이 항체가 함유된 시약을 시험관 또는 플레이트 상에서 적혈구와 반응시켜 응집이 일어나는지를 육안으로 확인하는 방법을 사용할 수 있다.In the present invention, the detection of antigen-antibody binding may be carried out according to conventional antigen-antibody reaction detection methods, for example, sedimentation, aggregation, complement binding, immunoadhesion. A method of visually confirming that aggregation occurs by reacting a reagent containing an antibody with red blood cells on a test tube or plate, such as a method commonly used for human ABO blood group test, can be used.
본 발명에 따르면, 개 혈액은 Kai 1+/Kai 2-(이하, 'Kai 1 혈액형'이라 한다), Kai 1-/Kai 2+(이하, 'Kai 2 혈액형'이라 한다) 또는 Kai 1-/Kai 2-(이하, 'Kai - 혈액형'이라 한다)의 3가지로 분류될 수 있다. 이때 'Kai 1+'는 항-Kai 1 항체에 항원-항체 반응을 나타내는 것, 'Kai 1-'는 항-Kai 1 항체에 항원-항체 반응을 나타내지 않는 것, 'Kai 2+'는 항-Kai 2 항체에 항원-항체 반응을 나타내는 것, 'Kai 2-'는 항-Kai 2 항체에 항원-항체 반응을 나타내지 않는 것을 의미한다.According to the present invention, dog blood is Kai 1 + / Kai 2- (hereinafter referred to as 'Kai 1 blood type'), Kai 1- / Kai 2+ (hereinafter referred to as 'Kai 2 blood type') or Kai 1- / Kai 2- (hereinafter referred to as 'Kai-blood type') can be classified into three types. In this case, 'Kai 1+' indicates an antigen-antibody response to the anti-Kai 1 antibody, 'Kai 1-' indicates no antigen-antibody response to the anti-Kai 1 antibody, and 'Kai 2+' indicates anti- Showing an antigen-antibody response to Kai 2 antibody, "Kai 2-" means not showing an antigen-antibody response to anti-Kai 2 antibody.
본 발명에 따르면, Kai 1 또는 Kai 2가 개의 수혈 시 항원으로 작용하여 면역반응을 일으킨다. 즉, Kai 1+인 개 혈액은 Kai 1-인 개에게 수혈하는 것이 부적합하고, Kai 2+인 개 혈액은 Kai 2-인 개에게 수혈하는 것이 부적합하다.According to the present invention, Kai 1 or Kai 2 acts as an antigen during blood transfusion of the dog, causing an immune response. That is, the blood of Kai 1+ is inappropriate for transfusion to Kai 1- dog, and the blood of Kai 2+ is inappropriate for transfusion to Kai 2- dog.
따라서 다음과 같이 수혈 적합성을 판별할 수 있다.Therefore, the suitability of blood transfusion can be determined as follows.
1) Kai 1 혈액형(Kai 1+/Kai 2-)의 개에게는 Kai 1 혈액형(Kai 1+/Kai 2-)의 혈액 또는 Kai - 혈액형(Kai 1-/Kai 2-)의 혈액을 수혈하는 것이 적합1) For dogs with Kai 1 blood type (Kai 1 + / Kai 2-), transfusion of Kai 1 blood type (Kai 1 + / Kai 2-) or Kai-blood type (Kai 1- / Kai 2-) fitness
2) Kai 2 혈액형(Kai 1-/Kai 2+)의 개에게는 Kai 2 혈액형(Kai 1-/Kai 2+)의 혈액 또는 Kai - 혈액형(Kai 1-/Kai 2-)의 혈액을 수혈하는 것이 적합2) For dogs with Kai 2 blood type (Kai 1- / Kai 2+), transfusion of Kai 2 blood type (Kai 1- / Kai 2+) or Kai-blood type (Kai 1- / Kai 2-) fitness
3) Kai - 혈액형(Kai 1-/Kai 2-)의 개에게는 Kai - 혈액형(Kai 1-/Kai 2-)의 혈액을 수혈하는 것이 적합3) Kai-blood type (Kai 1- / Kai 2-) dogs are best suited for blood transfusions of Kai- blood type (Kai 1- / Kai 2-)
상기와 같이 본 발명의 항체는 기존 DEA 혈액형 시스템, Dal 및 Shigeta 시스템과 전혀 다른 양상의 항원-항체 반응을 나타내므로, 본 발명의 방법은 기존의 시스템과는 다른 새로운 개 혈액의 항원을 바이오마커로 이용하는 개의 수혈 적합성 판별 방법이라고 할 수 있다.As described above, since the antibody of the present invention exhibits an antigen-antibody reaction in a completely different form from the existing DEA blood group system, the Dal and Shigeta system, the method of the present invention is a biomarker of a new dog blood antigen different from the existing system. It can be said that it is a method for determining the suitability of blood transfusion.
본 발명의 키트는 상기와 같은 본 발명의 개의 수혈 적합성 판별 방법을 효율적으로 수행할 수 있도록 할 수 있는 키트로, 상기 항-Kai 1 항체 및 항-Kai 2 항체를 포함하며, 이 밖에 본 발명의 방법에 필요한 시약, 기구 등이 더 포함될 수 있다. 예를 들어, 완충제 등의 항체의 안정화를 위한 시약, 슬라이드 글라스 등의 항원-항체 반응을 수행하는데 필요한 기구 등이 더 포함될 수 있다.The kit of the present invention is a kit capable of efficiently performing the method for determining the transfusion suitability of the dog of the present invention as described above, and includes the anti-Kai 1 antibody and the anti-Kai 2 antibody. Reagents, instruments, etc. required for the method may be further included. For example, a reagent for stabilizing an antibody such as a buffer, an apparatus required for performing an antigen-antibody reaction such as a slide glass, and the like may be further included.
이하, 실시예를 통하여 본 발명을 더욱 상세히 설명하기로 한다. 이들 실시예는 단지 본 발명을 예시하기 위한 것이므로, 본 발명의 범위가 이들 실시예에 의해 제한되는 것으로 해석되지는 않는다.Hereinafter, the present invention will be described in more detail with reference to Examples. Since these examples are only for illustrating the present invention, the scope of the present invention is not to be construed as being limited by these examples.
[[ 실시예Example ]]
1-1. 실험방법1-1. Experiment method
1-1-1. 동물 및 샘플1-1-1. Animals and samples
암컷 생쥐(BALB/c, 6주령)를 사용하였다. 모든 생쥐는 음식과 물을 자유롭게 섭취할 수 있도록 하였으며, 12시간의 명암주기를 유지하였다. 수혈받지 않은 개와 수혈받은 개의 EDTA 혈액 샘플은 동물병원과 한국동물혈액은행(KABB)에서 제공받았다. 치료 중에 수집된 혈액샘플을 실험에 사용할 수 있도록 개 소유자로부터 서면 동의를 얻었다. 모든 동물실험은 대구한의대의 실험동물운영위원회의 승인을 받아 수행되었다.Female mice (BALB / c, 6 weeks old) were used. All mice were given free food and water intake and maintained a 12-hour light and dark cycle. EDTA blood samples from untransfused and transfused dogs were provided by Animal Hospital and Korea Animal Blood Bank (KABB). Written consent was obtained from dog owners for use of blood samples collected during treatment. All animal experiments were performed with the approval of the Experimental Animal Steering Committee.
1-1-2. 개 혈액에 대한 1-1-2. For dog blood 단일클론항체의Monoclonal antibody 스크리닝 및 생산 : Kai 1 및 Kai 2 Screening and Production: Kai 1 and Kai 2
한국의 마스티프(Mastiff)견 두 마리로부터 RBC를 얻어 6마리의 쥐에 감작하는 방법으로 단일클론항체를 생산하는 하이브리도마를 제작하였다.Hybridomas producing monoclonal antibodies were prepared by obtaining RBCs from two Mastiff dogs in Korea and sensitizing them to six mice.
1. Rapid Vet-H DEA 1.1(DMS Laboratories, Inc, Flemington, NJ, USA)을 통해 DEA 1.1+로 분류되고, Shigeta 1.1B+이며(Shigeta Animal Pharmaceutical Inc, Oyabe, Japan), 다클론 항혈청(KABB, Sokchosi, South Korea)을 통해 DEA 1.1+로 분류된 개를 단클론 항-Kai 1 항체의 생산에 적용1. Rapid Vet-H DEA 1.1 (DMS Laboratories, Inc, Flemington, NJ, USA), classified as DEA 1.1+, Shigeta 1.1B + (Shigeta Animal Pharmaceutical Inc, Oyabe, Japan), polyclonal antiserum (KABB, Sokchosi, South Korea) was applied to the production of monoclonal anti-Kai 1 antibodies in dogs classified as DEA 1.1+.
2. Rapid Vet-H DEA 1.1을 통해 DEA 1.1-로 분류되지만, Shigeta 1.2B+이고, 다클론 항혈청(KABB, Sokchosi, South Korea)을 통해 DEA 1.2+로 분류된 개를 단클론 항-Kai 2 항체의 생산에 적용2. Dogs classified as DEA 1.1- through Rapid Vet-H DEA 1.1 but with Shigeta 1.2B + and DEA 1.2+ via polyclonal antiserum (KABB, Sokchosi, South Korea) were tested for monoclonal anti-Kai 2 antibodies. Applied to production
항응고 처리한 개의 혈액을 인산염 완충 식염수 (PBS, pH 7.2)로 세척한 후, 샘플을 1 x 108 cells/㎖의 RBC로 조절하였다. 수집하기 17, 10 및 3일 전에 RBC 현탁액 1㎖를 생쥐에 복강 내 주사하였다. 이소플루란 마취 하에 생쥐를 희생시키고, 비장세포를 얻었다. 50% 폴리에틸렌 글리콜을 사용하여 5 : 1의 비율로 세포를 생쥐 골수종 세포(P3X63Ag8.653; ATCC, Manassas, VA, USA)와 융합시켰다. 100μM 히포크산틴, 0.4μM 아미노프테린 및 16μM 티미딘이 함유된 히포크산틴 - 아미노프테린 - 티미딘 배지로 하이브리도마 세포를 분리한 후, 감작에 사용된 혈액에 특이적인 응집을 나타내는 하이브리도마 세포를 직접 적혈구응집반응 분석으로 선별하였다. 양성 하이브리도마 세포를 새롭게 준비된 암컷 BALB/c 생쥐(n = 6)에 복강 내 접종하여 단클론항체를 함유하는 복수를 얻었다.Blood from anticoagulated dogs was washed with phosphate buffered saline (PBS, pH 7.2) and the samples were then adjusted to 1 × 10 8 cells / ml RBC. Mice were injected intraperitoneally with 1 ml of RBC suspension 17, 10 and 3 days prior to collection. Mice were sacrificed under isoflurane anesthesia and splenocytes were obtained. Cells were fused with mouse myeloma cells (P3X63Ag8.653; ATCC, Manassas, VA, USA) at a ratio of 5: 1 using 50% polyethylene glycol. After isolation of hybridoma cells with hypoxanthine-aminopterin-thymidine medium containing 100 μM hypoxanthine, 0.4 μM aminopterin and 16 μM thymidine, the cells exhibited specific aggregation to blood used for sensitization. Bridoma cells were selected by direct hemagglutination assay. Positive hybridoma cells were inoculated intraperitoneally into freshly prepared female BALB / c mice (n = 6) to obtain ascites containing monoclonal antibodies.
1-1-3. 항체 정제1-1-3. Antibody purification
복수를 0.45㎛ 시린지 필터로 여과하고 PBS로 5배 희석한 다음, protein L 또는 protein G 컬럼(GenScript, Piscataway, NJ, USA)에 통과시켰다. 컬럼 레진은 PBS로 3회 세척하였고, 면역글로불린은 0.1M 글리신(pH 3.0) 완충액으로 용출하였으며, 1M Tris-HCl(pH 8.0)을 첨가하여 중화시켰다. 정제된 단클론항체를 PBS로 추가 희석하고 BCA(bicinchoninic acid) 및 SDS-PAGE(sodium dodecyl sulfate polyacrylamide gel electrophoresis)로 생쥐 IgG(Abcam, Cambridge, UK)와 비교하여 분석하였다.The ascites were filtered through a 0.45 μm syringe filter, diluted 5 times with PBS, and passed through a protein L or protein G column (GenScript, Piscataway, NJ, USA). Column resin was washed three times with PBS, immunoglobulins were eluted with 0.1 M glycine (pH 3.0) buffer and neutralized by addition of 1 M Tris-HCl (pH 8.0). Purified monoclonal antibody was further diluted with PBS and analyzed by comparison with mouse IgG (Abcam, Cambridge, UK) by bicinchoninic acid (BCA) and sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE).
1-1-4. Kai 항체의 동종형 결정1-1-4. Homotype Determination of Kai Antibodies
염소 항-생쥐 중쇄 항체(IgG1, IgG2a, IgG2b, IgG3, IgA 및 IgM) 또는 염소 항-생쥐 경쇄 항체(카파 또는 람다)가 코팅된 ELISA 플레이트(Thermo Scientific, Rockford, IL, USA)를 사용하였다. 두 개 혈액 샘플에 대한 정제된 항체를 각 웰에 첨가하고 광학밀도 0.2 이상에서 450nm에서 흡광도를 측정하여 동종형을 결정하였다.ELISA plates (Thermo Scientific, Rockford, IL, USA) coated with goat anti-mouse heavy chain antibodies (IgG1, IgG2a, IgG2b, IgG3, IgA and IgM) or goat anti-mouse light chain antibodies (kappa or lambda) were used. Isoforms were determined by adding purified antibodies to two blood samples to each well and measuring absorbance at 450 nm at an optical density of at least 0.2.
1-1-5. 개 1-1-5. dog RBCRBC 막 단백질을 사용한  Using membrane proteins 웨스턴블롯Western Blot
당단백질 분리 키트(Thermo Scientific)를 사용하여 개 RBC 막 단백질을 침전시키고, 단백질 농도를 결정하였다(Bio-Rad DC protein assay kit II, Bio-Rad, CA, USA). 50±100㎍의 단백질을 함유하는 세포용해물을 4x NuPAGETM-LDS 샘플 완충액에 혼합하고, 단백질을 1x NuPAGE MES SDS 전개 완충액을 사용한 NuPAGF™ Bis-Tris 겔로 분리하였다(Thermo Scientific, Rockford, IL, USA). 전이 완충액(25mM Tris, 250mM 글리신, 20% 메탄올)을 사용하여 300mA로 90분간 겔을 Hybond ECL 전이 막으로 전기-전이시켰다. 비특이적 결합부위를 차단하기 위해, 막을 5% 탈지분유가 함유된 Tween 20 함유 Tris-완충 식염수(TBST)로 상온(약 22℃)에서 90분간 처리하였다. 막을 Kai 1(1:250 titer) 또는 Kai 2(1:100) 항체가 함유된 3% 탈지분유 함유 TBST로 4℃에서 하룻밤동안 반응시켰다. 막을 TBST로 30분간 3회 세척하고 염소 항-생쥐 IgM 또는 IgG HRP 접합 2차 항체(1:3000)가 함유된 3% 탈지분유 함유 TBST로 상온에서 90분간 반응시켰다. 2차 항체로 반응시킨 후, 막을 TBST로 상온에서 30분간 3회 세척하였다. 단백질을 화학발광 검출 시스템으로 검출하였다.Glycoprotein separation kit (Thermo Scientific) was used to precipitate dog RBC membrane proteins and determine protein concentration (Bio-Rad DC protein assay kit II, Bio-Rad, CA, USA). Cytolysates containing 50 ± 100 μg of protein were mixed in 4 × NuPAGE ™ -LDS sample buffer and proteins were separated on NuPAGF ™ Bis-Tris gel using 1 × NuPAGE MES SDS development buffer (Thermo Scientific, Rockford, IL, USA). Gels were electro-transferred into Hybond ECL transition membranes at 300 mA for 90 minutes using transition buffer (25 mM Tris, 250 mM glycine, 20% methanol). To block nonspecific binding sites, the membranes were treated with Tween 20 containing Tris-buffered saline (TBST) containing 5% skim milk powder for 90 minutes at room temperature (about 22 ° C.). The membranes were reacted overnight at 4 ° C. with TBST containing 3% skim milk powder containing Kai 1 (1: 250 titer) or Kai 2 (1: 100) antibodies. Membranes were washed three times with TBST for 30 minutes and reacted for 90 minutes at room temperature with TBST containing 3% skim milk powder containing goat anti-mouse IgM or IgG HRP conjugated secondary antibody (1: 3000). After reacting with the secondary antibody, the membrane was washed three times for 30 minutes at room temperature with TBST. Proteins were detected with a chemiluminescent detection system.
1-1-6. Kai 항체의 친화 크로마토그래피1-1-6. Affinity Chromatography of Kai Antibodies
친화 정제를 Affi-Gel affinity chromatography를 사용하여 제조사의 매뉴얼에 따라 수행하였다(Bio-Rad, CA, USA). 보다 구체적으로, 1㎎의 항-Kai 1 항체 또는 항-Kai 2 항체를 Affi-Gel 슬러리가 채워진 컬럼에 첨가하고, 4℃에서 하룻밤 반응시킨 다음 컬럼 레진을 100mM 에탄올아민으로 블록시키고 PBS로 3회 세척하였다. IgM 또는 IgG에 대한 친화 단백질을 항체 정제에 사용된 것과 같은 컬럼을 사용하여 정제하였다.Affinity purification was performed using Affi-Gel affinity chromatography according to the manufacturer's manual (Bio-Rad, CA, USA). More specifically, 1 mg of anti-Kai 1 antibody or anti-Kai 2 antibody was added to a column filled with Affi-Gel slurry, reacted overnight at 4 ° C, and the column resin was blocked with 100 mM ethanolamine and 3 times with PBS. Washed. Affinity proteins for IgM or IgG were purified using the same columns used for antibody purification.
1-1-7. Kai 타입과 1-1-7. Kai type DEADEA 1 테스트  1 test 키트의Of kit 개 혈액형 비교 Dog Blood Group Comparison
Kai 타입과 DEA 1 사이의 관계를 결정하기 위해, Rapid Vet-H DEA 1 카드 테스트(DMS Laboratories, Flemington, NJ, USA)로 분류된 DEA 1+ 또는 DEA 1- 샘플(총 n = 50)을 Kai 항체로 분류된 결과와 비교하였다.To determine the relationship between Kai type and DEA 1, a sample of DEA 1+ or DEA 1- (total n = 50) categorized as Rapid Vet-H DEA 1 card test (DMS Laboratories, Flemington, NJ, USA) Comparison was made with the results classified as antibodies.
1-1-8. 동종항체의 검출1-1-8. Detection of homologous antibodies
항-Kai 1 항체 및 항-Kai 2 항체를 적용하는 것 뿐만 아니라 항-Kai 1 항체 및/또는 항-Kai 2 항체의 존재를 확인하기 위해 개(n = 4)의 혈액을 튜브 응집 분석으로 테스트하였다. 보다 구체적으로, 혈액을 공급하는 개와 Kai 1+ 또는 Kai 2+ 혈액을 수혈받을 개에게서 혈액 샘플을 얻고, 생리식염수로 세척한 다음 생리식염수에 2 ~ 3%로 재현탁하고, 3㎖ 튜브에서 같은 부피의 플라스마와 RBC 현탁액을 혼합하고 37℃에서 15분간 반응시켰다. 튜브를 1000g에서 1분간 원심분리한 다음 부드럽게 혼합하여 펠렛화한 RBC를 제거하였다. 응집 정도는 육안으로 관찰하여 음성(0) ~ 양성(4+)으로 등급화하였다.In addition to applying anti-Kai 1 antibody and anti-Kai 2 antibody, blood from dogs (n = 4) was tested by tube aggregation assay to confirm the presence of anti-Kai 1 antibody and / or anti-Kai 2 antibody. It was. More specifically, a blood sample is obtained from a dog supplying blood and a dog to be transfused with Kai 1+ or Kai 2+ blood, washed with physiological saline and resuspended in physiological saline at 2-3%, and the same in a 3 ml tube. A volume of plasma and RBC suspension were mixed and reacted at 37 ° C. for 15 minutes. The tube was centrifuged at 1000 g for 1 minute and then gently mixed to remove pelletized RBC. The degree of aggregation was visually observed and graded from negative (0) to positive (4+).
1-2. 실험결과1-2. Experiment result
1-2-1. 1-2-1. 단클론항체Monoclonal antibody 생산 production
생쥐의 감작에 사용된 개 혈액에 특이적인 하이브리도마 세포를 총 4800 웰에서 스크리닝하였다. 비특이적으로 응집되거나 모든 RBC에 교차반응하는 세포주는 제거하였다. 각각 DEA 1.1과 DEA 1.2로 분류된 두 개의 RBC에 대한 서로 다른 항체를 생산하지만 교차반응하지 않는 2가지의 하이브리도마 세포주를 분리하고, Kai 1(KCTC13495BP) 및 Kai 2(KCTC13496BP)라 명명하였다. Kai는 '개'를 의미한다. 복수에 함유된 항-Kai 1 항체 및 항-Kai 2 항체는 각각 1:2056 및 1:32 희석까지 RBC 응집 테스트에 반응하였으며, 어떠한 용혈활성도 나타내지 않았다.Hybridoma cells specific for dog blood used for sensitization of mice were screened in a total of 4800 wells. Cell lines that were nonspecifically aggregated or cross-reacted to all RBCs were removed. Two hybridoma cell lines that produced different antibodies to two RBCs classified as DEA 1.1 and DEA 1.2, but did not cross-react, were isolated and named Kai 1 (KCTC13495BP) and Kai 2 (KCTC13496BP). Kai means 'dog'. The anti-Kai 1 and anti-Kai 2 antibodies contained in the ascites responded to the RBC aggregation test up to 1: 2056 and 1:32 dilution, respectively, and showed no hemolytic activity.
1-2-2. 정제된 항-Kai 항체의 동종형1-2-2. Homotypes of Purified Anti-Kai Antibodies
Protein L 및 G 컬럼, 그리고 SDS-PAGE를 사용하여, Protein L 컬럼에서 각각 ~80kDa 및 ~25kDa의 밴드로 항-Kai 1 면역글로불린(Ig)을 확인하였다. 이것은 생쥐의 IgG와 다른 중쇄 및 경쇄의 Ig를 나타내는 것이므로 IgM이라는 것을 의미한다(도 1A 참조). 반면, 항-Kai 2 Ig는 Protein G 컬럼에서 쥐의 IgG의 중쇄 및 경쇄에 해당하는 ~50kDa 및 ~20kDa의 밴드로 나타났다(도 1B 참조).Protein L and G columns and SDS-PAGE were used to identify anti-Kai 1 immunoglobulins (Ig) with bands of ˜80 kDa and ˜25 kDa, respectively, on Protein L columns. This means IgM because it represents Ig of heavy and light chains different from IgG of mice (see FIG. 1A). On the other hand, anti-Kai 2 Ig was shown in the Protein G column with bands of ˜50 kDa and ˜20 kDa, corresponding to the heavy and light chains of mouse IgG (see FIG.
또한, 염소 항-생쥐 중쇄 항체(IgG1, IgG2a, IgG2b, IgG3, IgA 및 IgM) 또는 염소 항-생쥐 경쇄 항체(카파 또는 람다)로 코팅된 ELISA에 의해 항-Kai1 항체가 카파 경쇄를 갖는 IgM으로 분류되었고, 항-Kai 2 항체는 IgG3 중쇄 및 람다 경쇄에 대한 항체와 반응하였다. 이러한 결과는 항-Kai 1 항체 및 항-Kai 2 항체가 각각 IgM 카파와 IgG 람다의 서브클래스에 속한다는 것을 나타낸다(도 2 참조).In addition, the anti-Kai1 antibody to IgM with kappa light chain by ELISA coated with goat anti-mouse heavy chain antibody (IgG1, IgG2a, IgG2b, IgG3, IgA and IgM) or goat anti-mouse light chain antibody (kappa or lambda). Classified and anti-Kai 2 antibodies reacted with antibodies against IgG3 heavy and lambda light chains. These results indicate that anti-Kai 1 antibodies and anti-Kai 2 antibodies belong to subclasses of IgM kappa and IgG lambda, respectively (see FIG. 2).
1-2-3. 1-2-3. DEA DEA 1+ 및  1+ and DEADEA 1- 개에서 Kai 타입의 분포 1- distribution of Kai type
기존 튜브 응집 분류 분석에 이들 두 단일클론항체를 이용하면 개 혈액과 강한 양성(3+/4+) 또는 음성(0) 응집 반응을 나타내었다. 한국의 203마리의 개(대부분 한국 마스티프 종)를 조사한 결과, Kai 1+/Kai 2-가 42%, Kai 1-/Kai 2+가 37%, Kai 1-/Kai 2-가 20%로 나타났다. 그러나 항-Kai 1 및 항-Kai 2 단일클론항체 모두에 응집 반응을 나타내는 혈액 샘플은 단 하나도 없었다.The use of these two monoclonal antibodies in conventional tube aggregation classification assays showed strong positive (3 + / 4 +) or negative (0) aggregation reactions with dog blood. A survey of 203 dogs in Korea (mostly Korean mastiff) found 42% in Kai 1 + / Kai 2-, 37% in Kai 1- / Kai 2+, and 20% in Kai 1- / Kai 2- . However, none of the blood samples showed agglutination with both anti-Kai 1 and anti-Kai 2 monoclonal antibodies.
추가로, 상업적으로 이용할 수 있는 DEA 1 테스트 키트(Rapid Vet-H DEA 1)로 50마리 이상을 확인한 결과와 이들의 Kai 타입의 결과를 비교하였을 때, DEA 1+ 개는 Kai 1+/Kai 2- 또는 Kai 1-/Kai 2+인 반면, DEA 1- 개는 Kai 1+/Kai 2- 또는 Kai 1-/Kai 2+, 또는 Kai 1-/Kai 2-가 될 수 있었다. 이러한 결과는 DEA 1+ 혈액이 Kai 1+, Kai 2+ 또는 완전히 Kai -가 될 수 있다는 것을 의미한다(표 1 참조).In addition, when comparing 50 or more of the Kai type results with the commercially available DEA 1 test kit (Rapid Vet-H DEA 1), the DEA 1+ dogs were Kai 1 + / Kai 2 -Or Kai 1- / Kai 2+, while DEA 1- dogs could be Kai 1 + / Kai 2- or Kai 1- / Kai 2+, or Kai 1- / Kai 2-. These results indicate that DEA 1+ blood can be Kai 1+, Kai 2+ or completely Kai − (see Table 1).
DEA 1 DEA 1 Kai 1+ Kai 1+ Kai 2+ Kai 2+ Kai -Kai- 총계%(n)Total% (n)
DEA 1+(n=19) DEA 1+ (n = 19) 1515 44 -- 38%(19)38% (19)
DEA 1-(n=31)DEA 1- (n = 31) 2121 77 33 62%(31)62% (31)
총계%(n)Total% (n) 72%(36)72% (36) 22%(11)22% (11) 6%(3)6% (3) 100%(50)100% (50)
1-2-4. Kai 1-2-4. Kai 단일클론항체에Monoclonal antibody 의한 Kai 1 및 Kai 2 항원의 확인 Identification of Kai 1 and Kai 2 antigens
환원 조건에서의 면역블롯에 의해, 항-Kai 1 단일클론항체는 Kai 1+ 샘플에서 >170kDa의 주요 밴드와 ~55 및 ~70kDa의 마이너 밴드를 검출하였고, Kai 1- 혈액에서는 검출하지 못했다(도 3A 참조). 항-Kai 2 단일클론항체를 사용한 경우에는 Kai 2+ 개의 샘플에서 70 및 130kDa 사이의 단일밴드만이 나타났다(도 3B 참조). Kai 1 또는 Kai 2 항체에 특이적인 단백질 밴드를 보다 정확하게 확인하고자 친화 크로마토그래피를 수행한 결과, 항-Kai 1 단일클론항체로 ~200kDa의 주요 밴드와 ~50kDa의 분할 밴드가 나타났다. 한편, 항-Kai 2 단일클론항체는 ~80kDa에서 두드러진 밴드에 결합하였다(도 4 참조).By immunoblot under reducing conditions, anti-Kai 1 monoclonal antibodies detected major bands of> 170 kDa and minor bands of ˜55 and ˜70 kDa in Kai 1+ samples, but not in Kai 1-blood (FIG. 3A). When anti-Kai 2 monoclonal antibodies were used, only single bands between 70 and 130 kDa appeared in the Kai 2+ samples (see FIG. 3B). Affinity chromatography was performed to more accurately identify protein bands specific for the Kai 1 or Kai 2 antibodies. As a result, anti-Kai 1 monoclonal antibody showed a major band of ~ 200 kDa and a split band of ~ 50 kDa. On the other hand, anti-Kai 2 monoclonal antibody bound to prominent bands at ˜80 kDa (see FIG. 4).
1-2-5. Kai 1 및 Kai 2에 대한 동종항체 검출1-2-5. Homologous Antibody Detection for Kai 1 and Kai 2
Kai 1 및 Kai 2를 이용한 수혈 적합성 판별의 유효성을 검증하기 위하여, 수혈 전 응집 테스트를 통해 동종항체의 검출을 시도하였다.In order to verify the validity of the transfusion suitability determination using Kai 1 and Kai 2, alloantibodies were detected by pre-transfusion aggregation test.
DEA 1 혈액형 테스트를 통해 수혈받는 개의 혈액형과 수혈 혈액의 혈액형이 모두 DEA 1+로 분류되어 수혈이 적합한 것으로 나타났지만, Kai 타입이 서로 다른 경우(표 2의 1번 및 2번 실험군), DEA 1 타입과 상관없이 Kai 타입이 서로 다른 경우(표 2의 3번 및 4번 실험군)를 대상으로 테스트한 결과, 초기 교차검사에서는 모두 응집반응이 나타나지 않아 적합한 것으로 보이지만, 응집 테스트 21일 후에는 모두 응집반응이 나타났으며, 특히 DEA 1 혈액형 테스트를 통해 수혈이 적합한 것으로 판별된 경우에도 Kai 타입이 다르면 응집반응이 나타났다.The DEA 1 blood group test indicated that both the blood type of the transfused dog and the transfused blood group were classified as DEA 1+ and that the transfusion was appropriate, but if the Kai types were different ( experimental groups 1 and 2 in Table 2), DEA 1 Regardless of the type, tests with different types of Kai (experimental group 3 and 4 in Table 2) showed that the initial cross-linking test did not show any coagulation reactions, but it appeared appropriate after 21 days of coagulation test. Responses were observed, especially when the Kai type was different from the DEA 1 blood group.
또한 수혈받는 개의 혈액형이 Kai 2-/DEA 1+이고 수혈 혈액의 혈액형이 Kai 2+/DEA 1+인 경우, 그리고 수혈받는 개의 혈액형이 Kai 1-/DEA 1+이고 수혈 혈액의 혈액형이 Kai 1+/DEA 1+인 경우, 응집 테스트 21일 후에 많은 혈전이 관찰되었다(도 5 참조).The blood type of the transfused dog is Kai 2- / DEA 1+ and the blood type of the transfused blood is Kai 2 + / DEA 1+, and the blood type of the transfused dog is Kai 1- / DEA 1+ and the blood type of the transfused blood is Kai 1 In the case of + / DEA 1+, many thrombi were observed 21 days after the aggregation test (see FIG. 5).
이러한 결과는 Kai 1 및 Kai 2를 이용한 수혈 적합성 판별 방법이 유효하다는 것을 의미하며, 또한 DEA 1 시스템으로 판별할 수 없는 경우도 수혈 적합성을 판별할 수 있다는 것을 의미한다.These results indicate that the transfusion suitability determination method using Kai 1 and Kai 2 is valid, and that the transfusion suitability can be determined even if it cannot be determined by the DEA 1 system.
Day 0Day 0 Day 21Day 21
개dog 수혈받는 개의 혈액형Blood type of transfused dog 수혈 혈액의 혈액형Blood type of transfusion blood 교차검사결과Cross test result 응집 세기Cohesive strength
1One
Kai 1+, DEA 1+Kai 1+, DEA 1+ Kai 2+, DEA 1+ Kai 2+, DEA 1+ 적합 fitness 1+1+
22 Kai 2+, DEA 1+ Kai 2+, DEA 1+ Kai 1+, DEA 1+ Kai 1+, DEA 1+ 적합 fitness 1+1+
33 Kai 1+ Kai 1+ Kai 2+ Kai 2+ 적합 fitness 3+3+
44 Kai 2+ Kai 2+ Kai 1+ Kai 1+ 적합 fitness 3+3+
[수탁번호][Accession number]
기탁기관명 : 한국생명공학연구원Depositary: Korea Research Institute of Bioscience and Biotechnology
수탁번호 : KCTC13495BPAccession number: KCTC13495BP
수탁일자 : 20180315Deposit Date: 20180315
기탁기관명 : 한국생명공학연구원Depositary: Korea Research Institute of Bioscience and Biotechnology
수탁번호 : KCTC13496BPAccession number: KCTC13496BP
수탁일자 : 20180315Deposit Date: 20180315
Figure PCTKR2018007224-appb-I000001
Figure PCTKR2018007224-appb-I000001
Figure PCTKR2018007224-appb-I000002
Figure PCTKR2018007224-appb-I000002
Figure PCTKR2018007224-appb-I000003
Figure PCTKR2018007224-appb-I000003
Figure PCTKR2018007224-appb-I000004
Figure PCTKR2018007224-appb-I000004

Claims (6)

  1. 수혈용 개 혈액이 수혈받을 개에서 면역반응을 일으키는지를 검증할 수 있도록 항체를 사용하여 수혈용 개 혈액 시료와 수혈받을 개의 혈액 시료를 대상으로 항원-항체 반응을 수행하되,Antigen-antibody reactions are performed on transfusion dog blood samples and blood samples of dogs to be transfused using antibodies to verify whether transfusion dog blood causes an immune response in the transfusion dog.
    상기 항체로 기탁번호 KCTC 13495BP의 하이브리도마 세포에 의해 생산되는 단일클론항체 및 기탁번호 KCTC 13496BP의 하이브리도마 세포에 의해 생산되는 단일클론항체를 상기 각 시료와 접촉시키고, 항원-항체 결합을 검출하는 것을 특징으로 하는 개의 수혈 적합성 판별 방법.The monoclonal antibody produced by the hybridoma cells of Accession No. KCTC 13495BP and the monoclonal antibody produced by the hybridoma cells of Accession No. KCTC 13496BP were contacted with each of the samples, and the antigen-antibody binding was detected. Transfusion suitability determination method of a dog.
  2. 제 1항의 방법을 수행하기 위한 키트로,A kit for performing the method of claim 1,
    기탁번호 KCTC 13495BP의 하이브리도마 세포에 의해 생산되는 단일클론항체 및 기탁번호 KCTC 13496BP의 하이브리도마 세포에 의해 생산되는 단일클론항체를 포함하는 개의 수혈 적합성 판별용 키트.A kit for transfusion suitability determination in dogs comprising a monoclonal antibody produced by hybridoma cells of Accession No. KCTC 13495BP and a monoclonal antibody produced by hybridoma cells of Accession No. KCTC 13496BP.
  3. 기탁번호 KCTC 13495BP의 하이브리도마 세포에 의해 생산되는 단일클론항체.Monoclonal antibody produced by hybridoma cells of Accession No. KCTC 13495BP.
  4. 제 3항의 단일클론항체를 생산하는 기탁번호 KCTC 13495BP의 하이브리도마 세포주.A hybridoma cell line of Accession No. KCTC 13495BP, which produces the monoclonal antibody of claim 3.
  5. 기탁번호 KCTC 13496BP의 하이브리도마 세포에 의해 생산되는 단일클론항체.Monoclonal antibody produced by hybridoma cells of Accession No. KCTC 13496BP.
  6. 제 5항의 단일클론항체를 생산하는 기탁번호 KCTC 13496BP의 하이브리도마 세포주.A hybridoma cell line of Accession No. KCTC 13496BP, which produces the monoclonal antibody of claim 5.
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