Monoclonal antibody and detection application thereof

Abstract

The invention discloses a monoclonal antibody and a detection application thereof. The invention also discloses an antibody derivative, a nucleic acid sequence for coding the antibody, and detection application of the antibody, especially application in ELISA and FACS detection. The invention also discloses a method for preparing the antibody. The monoclonal antibody provided by the invention has wide application prospect and practical value.

Description

Technical Field

[0001] The present invention relates to the field of biomedicine technology, and in particular to a monoclonal antibody and a detection application thereof. Background Art

[0002] Killer cell immunoglobulin-like receptor (KIR3DL2) is a member of the killer cell immunoglobulin-like receptor (KIR) family. It is mainly expressed on memory T cells and natural killer (NK) cells and is a key regulator of NK cell function. The interaction between KIR3DL2 and its ligand plays an important role in the incidence, progression and outcome of a variety of diseases, including pregnancy complications, viral infections, autoimmune diseases and hematological malignancies. Antibodies or antigen-binding fragments of KIR3DL2 have broad application potential in the fields of diagnosis, treatment and research.

[0003] Monoclonal antibodies play an important role in medical diagnosis, treatment and research. They are used to treat various diseases, including cancer, autoimmune diseases and infectious diseases. However, traditional methods for producing monoclonal antibodies are often costly and complex. Therefore, it is of great significance to develop an efficient nucleic acid molecule encoding monoclonal antibodies. Currently, there are many technologies for producing monoclonal antibodies, and these antibodies have shown their unique value in applications such as enzyme-linked immunosorbent assay (ELISA) and flow cytometry (FACS).

[0004] Although the existing monoclonal antibody technology has made significant progress in many aspects, there is still a need for new monoclonal antibodies in terms of improving specificity and expanding the scope of application. At present, the development of highly specific monoclonal antibodies against KIR3DL2 protein remains a challenge. In addition, there is also a need for further research and development of derivatives of these antibodies, nucleic acid molecules encoding their amino acid sequences, and their application in experimental techniques such as ELISA and FACS. Summary of the invention

[0005] To make up for the deficiencies of the prior art, the present invention aims to provide a novel monoclonal antibody which has high specificity for KIR3DL2 protein and is innovative in its amino acid sequence, derivatives and applications.

[0006] In order to achieve the above object, the present invention provides the following technical solutions:

[0007] The first aspect of the present invention provides an antibody or antigen-binding fragment against KIR3DL2 protein, the antibody or antigen-binding fragment comprising a heavy chain variable region and a light chain variable region, the heavy chain variable region comprising CDR1, CDR2, and CDR3 in the heavy chain variable region as shown in SEQ ID NO:2; the light chain variable region comprising CDR1, CDR2, and CDR3 in the light chain variable region as shown in SEQ ID NO:6.

[0008] In the context of the present invention, the term "antibody" is used in the broadest sense in the present invention, and explicitly covers monoclonal antibodies, polyclonal antibodies, humanized antibodies, chimeric antibodies, and multispecific antibodies (e.g., bivalent antibodies) formed from at least two complete antibodies, as long as they exhibit the desired biological activity. In the present invention, the antibody sequences obtained by modification also belong to the protection scope of the present invention. The term "modification" refers to any form of modification of the amino acid sequence, such as substitution, deletion, insertion and / or addition of amino acids. The term "substitution" refers to the replacement of one or more amino acids in the original amino acid sequence with different amino acids. The term "deletion" refers to the reduction of one or more amino acids in the original amino acid sequence. The term "insertion" or "addition" refers to the change in the amino acid sequence resulting in the addition of one or more amino acids compared to the original amino acid sequence. In the present invention, the modification preferably occurs in an area outside the variable region, such as the constant region or framework region of the antibody, and the modified antibody still retains the desired functional properties of the antibody or antigen-binding fragment thereof of the present invention, or has improved antigen-binding properties.

[0009] Furthermore, the antibody includes a monoclonal antibody, a polyclonal antibody, a chimeric antibody, a humanized antibody or a murine antibody.

[0010] Furthermore, the antibody of the present invention is a monoclonal antibody.

[0011] The amino acid sequences of CDR1, CDR2 and CDR3 in the heavy chain variable region are shown as SFPMA, TIISSGGSSYYRDSVKG and GGSFAY, respectively; the amino acid sequences of CDR1, CDR2 and CDR3 in the light chain variable region are shown as KASQNINNYLN, NTNNLQM and FQHNNWPLT, respectively.

[0012] In the present invention, the term "antigen-binding fragment" includes a part of a complete antibody, and generally refers to one or more fragments of an antibody that play a specific antigen-binding function. The antigen-binding function of an antibody can be achieved by a full-length fragment of the antibody. The "antigen-binding fragment" can be an antibody fragment in the form of Fab, Fv, ScFv, etc., which retains the ability to bind to the KIR3DL2 protein and has a smaller molecular size, which is convenient for penetrating tissues or for specific therapeutic applications.

[0013] The "heavy chain variable region and light chain variable region" of the antibody respectively comprise amino acid sequences, which can specifically bind to a specific epitope of the KIR3DL2 protein.

[0014] The second aspect of the present invention provides a nucleic acid molecule capable of encoding the above-mentioned antibody or antigen-binding fragment thereof.

[0015] In the present invention, the term "nucleic acid molecule" refers to any polymeric form of any length and composed of ribonucleotides or deoxyribonucleotides. Typically, the nucleic acid molecule is a coding sequence, which may include, but is not limited to, prokaryotic sequences, cDNA from eukaryotic mRNA, genomic DNA sequences from eukaryotic (e.g., mammalian) DNA, and even recombinant DNA sequences. The transcription termination sequence will usually be located 3' of the coding sequence.

[0016] Furthermore, the nucleic acid molecule encoding the above antibody includes a heavy chain variable region (VH) coding sequence, a light chain variable region (VL) coding sequence, a heavy chain constant region (CH) coding sequence, and a light chain constant region (CL) coding sequence.

[0017] The present invention provides one or more source species for preparing monoclonal antibodies, including but not limited to mice, rats, rabbits, hamsters, humans, non-human primates, goats, transgenic animals, in vitro cultured cell lines, etc.

[0018] Furthermore, the source of the monoclonal antibody prepared in the present invention is rat.

[0019] The third aspect of the present invention provides a vector, which comprises the nucleic acid molecule described above.

[0020] In the present invention, a vector comprising the nucleic acid molecule of the present invention is provided. The term "vector" refers to an artificial construct that can deliver and preferably express one or more target genes or sequences in a host cell. The vector of the present invention can be a plasmid vector, a viral vector, etc. In some embodiments, the vector refers to a linear or circular nucleic acid molecule that comprises a nucleic acid of the present invention that is operably connected to other segments of autonomous replication provided in a recombinant host cell, or an expression cassette according to a nucleic acid molecule. "Operably connected" means that the nucleic acid sequence of interest is connected to a regulatory sequence (e.g., in an in vitro transcription / translation system or when the vector is introduced into a host cell) in a manner that allows the expression of the nucleotide sequence.

[0021] Furthermore, the vector includes plasmid, bacteriophage, mammalian cell expression vector, baculovirus, yeast virus, plant expression vector and the like.

[0022] The vector of the present invention is a pcDNA series vector used for expressing proteins in mammalian cells.

[0023] Furthermore, the vector of the present invention is a pcDNA3.1 vector.

[0024] A fourth aspect of the present invention provides a recombinant cell, wherein the recombinant cell comprises the aforementioned monoclonal antibody, the aforementioned nucleic acid molecule, and / or the aforementioned vector.

[0025] Furthermore, the cells include B lymphocytes, myeloma cells, lymphoma cells, hybridoma cells, CHO cells, HEK293 cells, insect cell lines, and the like.

[0026] The fifth aspect of the present invention provides an antibody derivative, which comprises a detectable label directly or indirectly coupled to the monoclonal antibody according to the first aspect of the present invention.

[0027] The detectable markers include enzyme markers, fluorescent markers, radioactive markers, chemiluminescent markers, magnetic markers, and the like.

[0028] Furthermore, the detectable marker is an enzyme marker.

[0029] Furthermore, the enzyme marker is horseradish peroxidase.

[0030] The sixth aspect of the present invention provides a product for detecting KIR3DL2 protein, wherein the product comprises the aforementioned antibody or antigen-binding fragment thereof, the aforementioned nucleic acid molecule, the aforementioned vector, and / or the aforementioned recombinant cell.

[0031] Furthermore, the product was detected by ELISA and FACS methods.

[0032] The seventh aspect of the present invention provides a method for preparing the above-mentioned antibody or antigen-binding fragment thereof, the method comprising culturing the recombinant cell described in the fourth aspect of the present invention.

[0033] Furthermore, the specific steps include: 1) using the aforementioned vector to transform into host cells; 2) culturing the host cells in step 1) under suitable conditions; 3) isolating and purifying the aforementioned monoclonal antibody from the culture medium of the host cells.

[0034] The eighth aspect of the present invention provides a method for detecting KIR3DL2 protein or its functional domain, the method comprising incubating the KIR3DL2 protein or its functional domain with the antibody or its antigenic fragment as described above.

[0035] Preferably, the functional domain of the KIR3DL2 protein is the extracellular domain where amino acids 22-337 of the KIR3DL2 protein are located.

[0036] Preferably, the KIR3DL2 protein is of murine origin.

[0037] Furthermore, the rat anti-mouse KIR3DL2 antibody is 12C5E6.

[0038] The ninth aspect of the present invention provides the use of the aforementioned monoclonal antibody or antigen-binding fragment thereof in preparing a product, wherein the use is the use in preparing a product for detecting KIR3DL2 protein.

[0039] Preferably, the KIR3DL2 protein is a free protein or a protein expressed on the cell membrane.

[0040] Preferably, the product is detected by ELISA and FACS methods.

[0041] Preferably, the functional domain of the KIR3DL2 protein is the extracellular domain where amino acids 22-337 of the KIR3DL2 protein are located.

[0042] Furthermore, the antibody can bind to the mKIR3DL2-His recombinant protein in a gradient-dependent manner.

[0043] Furthermore, the antibody can bind well to cells overexpressing mKIR3DL2 protein.

[0044] The term "CDR sequence" used in the present invention refers to the amino acid sequence in the complementary determining region of an antibody molecule. These sequences are part of the variable region of an antibody and play a decisive role in the binding of the antibody to an antigen. The CDR sequence is the key region in which an antibody can recognize and bind to a specific antigen. In an antibody molecule, the CDR sequence is divided into three regions, namely: CDR1, CDR2, and CDR3. The term "CDR" may refer to a CDR determined by any method recognized in the art.

[0045] Advantages and beneficial effects of the present invention: The present invention provides a 12C5E6 antibody, which has good binding activity with cells overexpressing mKIR3DL2 protein and has broad application prospects. BRIEF DESCRIPTION OF THE DRAWINGS

[0046] Figure 1 This is a graph of Elisa testing the binding activity of 12C5E6 antibody to antigen.

[0047] Figure 2 This is a graph of cell apoptosis analyzed by flow cytometry (Ctrl represents untreated cells, Flag antibody is used as a positive control, the horizontal axis PE represents the fluorescence intensity of the specific PE-labeled antibody on the cells, and the vertical axis SSC represents the internal granularity and complexity of the cells). DETAILED DESCRIPTION

[0048] The invention will be further described below in conjunction with specific examples. It should be understood that the specific embodiments described herein are presented by way of example and are not intended to limit the invention. The main features of the invention may be used in a variety of embodiments without departing from the scope of the invention.

[0049] Example Preparation and Physical and Chemical Property Testing of mKIR3DL2-His Recombinant Protein

[0050] 1. Experimental Materials

[0051] Reagents: HAT (Sigma, H0262); complete culture medium (Beijing Zhongke Maichen Technology Co., Ltd., CM10040); 96-well transparent flat-bottom polystyrene microplate (Corning, CLS 9018); anti-mouse secondary antibody (Jackson ImmunoResearch, 115-035-003); HT (Sigma, H0137); human embryonic kidney HEK293T cells (System Biosciences); Chinese hamster ovary carcinoma CHO cells (ECACC, 85051005); Protein A affinity chromatography (Nanomicron Technology, NMabTM ProProtein A, 17010-150100); high affinity 96-well plate (Corning, 3590); PBST (PBS, Xi'an Het Biotechnology, BF001; Tween-20, Sangon Biotechnology, A100777); BSA (Sangon Biotechnology, A500023-0100); TMB (Beijing Meikowande, 1001).

[0052] 2. Experimental plan

[0053] 1. Antibody Preparation

[0054] (1) Preparation of mKIR3DL2-His recombinant protein: The mouse KIR3DL2 extracellular domain (amino acids 22-337) was constructed into the pCDNA3.1 vector, and a His tag was added to the C-terminus. The constructed vector was transfected into HEK293 transient transfection cells, and the mKIR3DL2-His recombinant protein was obtained through cell sample treatment, affinity purification and multi-step purification.

[0055] SEQ ID of mKIR3DL2-His recombinant protein NO:1:HVGSHDKPFLYAWPSYVVPLGQNVTLTCDSHRGSNIFKLYKEEGSPIPQLHETTFQKSQVFGPVTTEHAGTYRCFHPQYANVLSAHSEPLKIIISGIYLKPFLLILQSPLVNSGGNVTLECHSENMFDTYILISHRMGIIKNSVQVSAEHHESGSHVTYS IGPMTPDLVGTYTCYGANSYYPYEWSDPSDPIDIKITGVYKKPSLSVLMGPVLMMSGETMTLSCISDHQFDMFHMSREGVPQGQGMPGVQIHSGKFEAKFLLSSMIQKGNYRCYGSFRNSSHVWSSPSDPLYLPAKGNCPACTEEDPKIHNCKNLRHHHHHHHH.

[0056] (2) Preparation of mouse KIR3DL2 monoclonal antibody: ① Immunization: emulsify the mKIR3DL2-His recombinant protein with adjuvant and immunize 6-8 week old female rats at 2 points on the back and 1 point in the abdominal cavity, injecting 1 / 3 of the volume each time, avoiding injection into the left spleen of the mouse. The first immunization dose is 100 μg, and the second to fourth immunization doses are 50 μg, and immunization is performed every 14 days. ELISA is used to detect the polyclonal antibody titer against the immunogen in rat serum, and the rat with the highest titer is immunized with 100 μg protein without adjuvant by intraperitoneal injection.

[0057] ②Cell fusion: Aseptically prepare the immune-qualified rat spleen cell suspension, fuse it with mouse myeloma cells sp2 / 0 (ATCC) at a ratio of 5:1, then place it in a 96-well cell culture plate, and screen it with complete medium containing HAT. Replace half of the medium once a week, and clone formation can be seen in 2 weeks.

[0058] ③ Hybridoma supernatant screening: Enzyme-linked immunosorbent assay (ELISA) was used to screen the culture supernatant of hybridoma cells: the 96-well transparent flat-bottom polystyrene microplate was coated with recombinant mKIR3DL2-His protein, and the hybridoma cell culture supernatant obtained in the previous step was added after blocking, and then the anti-mouse secondary antibody labeled with horseradish peroxidase (HRP) was added. The clone wells whose supernatant reacted positively with mKIR3DL2-His protein and negatively with the control His protein were selected, that is, the hybridoma cells secreting rat anti-mouse KIR3DL2 antibodies, and cultured with complete culture medium containing HT, and subcloned.

[0059] ④ Subcloning: For each selected hybridoma cell line, count the cells and dilute them to 1 cell / 200 μL in complete culture medium containing HT, plate them in a 96-well cell culture plate, and culture them at 37°C. After the formation of a single clone, screen the hybridoma subclones whose supernatants react positively with the mKIR3DL2-His protein by ELISA again.

[0060] ⑤ Flow cytometry to detect the ability of antibodies to bind to cell surface N1: Human embryonic kidney HEK293T cells expressing exogenous mKIR3DL2-Flag were used to screen positive hybridoma strains by FACS using BD Accuri™ C6. The cells were stained with hybridoma supernatant samples, followed by secondary antibody binding with goat anti-rat IgG linked to FITC. The corresponding cell lines transfected with empty vector plasmids were used as negative controls. After flow cytometry detection, FlowJo software was used for analysis, and hybridoma cell lines that were positive in both FACS and ELISA were selected for cloning.

[0061] ⑥ Hybridoma cell sequencing and antibody purification: The cDNA coding sequence of the antibody secreted by the hybridoma cell line that is positive in both FACS and Elisa is determined and translated into an amino acid sequence. Finally, the hybridoma with clone number 12C5E6 is obtained for antibody purification. The codons preferred by Chinese hamster ovarian cancer CHO cells are selected for reverse translation, thereby determining the coding cDNA sequence of 12C5E6, inserting it into the pcDNA3.1 vector after synthesis, and transfecting it into CHO cells by electroporation for expression. After expression, the CHO cell culture supernatant is harvested, and the antibody protein in the cell culture supernatant is separated and purified by Protein A affinity chromatography, and the purity and other quality control tests are performed and quantified.

[0062] 2. Analysis of antibody binding ability

[0063] (1) Elisa detection of 12C5E6 antibody binding ability

[0064] ① Antigen coating: Add 1 μg / mL mouse KIR3DL2-His recombinant protein to a high-affinity 96-well plate, 8 wells in total, 100 μL per well, 16 wells in total, 100 μL per well, seal the plate and incubate at 4°C overnight.

[0065] ②Wash away the antigen: Remove the antigen solution and wash the plate three times with 0.05% PBST buffer, 200 μL / well each time.

[0066] ③ Blocking: Aspirate or invert the plate to remove all the washing solution; block with PBS buffer containing 1% BSA, 300 μL per well, and incubate at room temperature for 1 hour after sealing; remove the blocking solution, wash the plate twice with 0.05% PBST buffer, 200 μL / well each time, and aspirate or invert the plate to remove all the washing solution.

[0067] ④ Incubate with primary antibody: add the experimental group antibody 12C5E6 or the control group antibody Blank, 200 ng / mL in the first well of each group, dilute in 2-fold gradient to 7 wells in sequence, and the last well is blank buffer, a total of 8 wells, 100 μL in each well, seal the plate and incubate at room temperature for 1 hour; remove the antibody solution, wash the plate with 0.05% PBST buffer 5 times, 200 μL / well each time, and aspirate or invert the plate to remove all the washing solution.

[0068] ⑤ Incubate with secondary antibody: Add secondary antibody Goat Anti-Rat IgG-HRP at a dilution of 1:5000, 100 uL per well, seal the plate and incubate at room temperature for 1 hour; remove the secondary antibody solution, wash the plate 5 times with 0.05% PBST buffer, 200 μL / well each time, and aspirate or invert the plate to remove all the washing solution.

[0069] ⑥ Color development: Add TMB substrate solution, 100 uL per well, and develop color at room temperature away from light; after reaching the required intensity, add 1 M H2SO4 solution to terminate the reaction, 50 μL per well; measure the absorbance at 450 nm with an enzyme-linked analyzer; draw a curve using the correlation between the sample readings and the two groups of antibody concentrations.

[0070] (2) FACS detection of the ability of 12C5E6 antibody to bind to cells. ① Human embryonic kidney HEK293T cells were transfected with mKIR3DL2-Flag plasmid, and the cells were digested into single cells 24 h after transfection.

[0071] ② Dilute the 12C5E6 antibody to a concentration of 5 μg / mL, use the Flag antibody as a positive control, and incubate the antibodies at room temperature for 1 h.

[0072] ③ Dilute Goat anti-Rat IgG Alexa Fluor™ 546 secondary antibody at 1:500 and incubate at room temperature for 1 hour.

[0073] ④ The ability of 12C5E6 antibody to bind to cells was verified by FACS using BD Accuri™ C6. Flow cytometry was used for analysis using FlowJo software.

[0074] 3. Experimental Results

[0075] The sequence of a monoclonal antibody 12C5E6 was screened.

[0076] The amino acid sequence encoding antibody 12C5E6 is shown below: the amino acid sequence encoding the heavy chain variable region, sequence number SEQ ID NO: 2: EVQLVESGGVLVQPGRSMKLSCTASGFSFSSFPMAWVRQAPTRGLEWVATIISSGGSSYYRDSVKGRFTISRDNAKSTLYLQMDSLRSEDTATYYCATGGSFAYWGQGTLVTVSS.

[0077] Among them, the amino acid sequences encoding the heavy chain variable regions CDR1, CDR2, and CDR3 are SFPMA, TIISSGGSSYYRDSVKG, and GGSFAY, respectively, and the sequence numbers are SEQ ID NOs: 3, 4, and 5, respectively.

[0078] The amino acid sequence encoding the light chain variable region, sequence number SEQ ID NO:6: DIQMTQSPSLLSAYVGDGVTINCKASQNINNYLNWYQQKLGEAPKLLIYNTNNLQMVTPSRFSGSGSGTDYTLTISSLQPEDFGTYFCFQHNNWPLTFGSGTTLEIK.

[0079] Among them, the amino acid sequences encoding the light chain variable regions CDR1, CDR2, and CDR3 are KASQNINNYLN, NTNNLQM, and FQHNNWPLT, respectively, and the sequence numbers are SEQ ID NOs: 7, 8, and 9, respectively.

[0080] The binding ability of 12C5E6 antibody was tested by Elisa. The results are as follows Figure 1 As shown, the absorbance value of the antibody is greater than 2, which is much greater than that of the negative control group, indicating that the 12C5E6 antibody has a good affinity for the recombinant protein antigen and that the 12C5E6 antibody can bind to the mKIR3DL2-His recombinant protein in a gradient-dependent manner.

[0081] By FACS analysis, Figure 2 As shown, we observed that the fluorescently labeled 12C5E6 antibody could bind well to cells overexpressing mKIR3DL2 protein.

[0082] The above embodiments are only used to understand the method and core concept of the present invention. It should be noted that, for those skilled in the art, several improvements and modifications can be made to the present invention without departing from the principles of the present invention, and these improvements and modifications will also fall within the scope of protection of the claims of the present invention.

Claims

1. An antibody or antigen-binding fragment thereof against KIR3DL2 protein, characterized in that: The antibody or antigen-binding fragment thereof comprises a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region comprises CDR1, CDR2, and CDR3 in the heavy chain variable region as shown in SEQ ID: 2; and the light chain variable region comprises CDR1, CDR2, and CDR3 in the light chain variable region as shown in SEQ ID NO:

6.

2. The antibody or antigen-binding fragment thereof according to claim 1, characterized in that: The amino acid sequences of CDR1, CDR2 and CDR3 in the heavy chain variable region are shown as SFPMA, TIISSGGSSYYRDSVKG and GGSFAY, respectively; the amino acid sequences of CDR1, CDR2 and CDR3 in the light chain variable region are shown as KASQNINNYLN, NTNNLQM and FQHNNWPLT, respectively; Preferably, the antibody comprises a monoclonal antibody, a polyclonal antibody, a chimeric antibody, a humanized antibody or a murine antibody.

3. A nucleic acid molecule, characterized in that The nucleic acid molecule encodes the antibody or antigen-binding fragment thereof according to any one of claims 1-2.

4. A carrier, characterized in that The vector comprises the nucleic acid molecule of claim 3.

5. A recombinant cell, characterized in that The recombinant cell comprises the antibody or antigen-binding fragment of claim 1 , the nucleic acid molecule of claim 3 , and / or the vector of claim 4 .

6. An antibody derivative, characterized in that: The antibody derivative comprises the antibody or antigen-binding fragment of any one of claims 1 to 2, and a detectable label directly or indirectly coupled to the antibody or antigen-binding fragment; Preferably, the detectable marker includes an enzyme marker, a fluorescent marker, a radioactive marker, a chemiluminescent marker, or a magnetic marker; Preferably, the enzyme marker is horseradish peroxidase.

7. A product for detecting KIR3DL2 protein or its functional fragment, characterized in that: The product comprises the antibody or antigen-binding fragment thereof according to any one of claims 1 to 2, the nucleic acid molecule according to claim 3, the vector according to claim 4, and / or the recombinant cell according to claim 5; Preferably, the product is detected by ELISA or FACS method.

8. A method for preparing the antibody or antigen-binding fragment thereof according to any one of claims 1 to 2, characterized in that: The method refers to culturing the recombinant cell according to claim 5.

9. A method for detecting KIR3DL2 protein or its functional domain, characterized in that: Incubate the KIR3DL2 protein or its functional domain with the antibody or antigenic fragment thereof according to any one of claims 1 to 2; Preferably, the functional domain of the KIR3DL2 protein is the extracellular domain where amino acids 22-337 of the KIR3DL2 protein are located.

10. Use of the antibody or antigen-binding fragment thereof according to any one of claims 1 to 2 in preparing a product, characterized in that: The application is an application in preparing a product for detecting KIR3DL2 protein or its functional domain; Preferably, the KIR3DL2 protein is a free protein or a protein expressed on the cell membrane; Preferably, the product is detected by ELISA and FACS methods; Preferably, the functional domain of the KIR3DL2 protein is the extracellular domain where amino acids 22-337 of the KIR3DL2 protein are located.

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