JP7216980B2 - Monoclonal antibody that recognizes porcine CD4 - Google Patents

Description

NPMD NITE P-02666 NPMD NITE P-02667

The present invention relates to a monoclonal antibody that recognizes porcine CD4.

Inbred pigs are “the only medium-sized experimental animals in which transplantation-related genes including MHC are fixed” to replace rodents such as mice, which are evolutionarily distant from humans. In addition, pigs are known to be carriers of human infectious diseases such as influenza. It is important for the elucidation of the virus transmission mechanism to humans, healthy livestock breeding and food supply. Pigs are also considered to be the best xenotransplantation donors for humans due to their high reproductive efficiency, organ size, and physiological similarity to humans. .

Multiple subtypes have been reported for porcine CD4. The present inventors also reported the presence of porcine CD4 subtypes in microminipigs that are not recognized by currently commercially available CD4 antibodies (Non-Patent Documents 1 and 2).

Identification and characterization of two CD4 alleles in Microminipigs. , Asako Ando, Hitoshi Kitagawa BMC Veterinary Research 2016 Oct 7;12(1):222. Tatsuya Matsubara, Naohito Nishii, Satoshi Takashima, Masaki Takasu, Noriaki Imaeda, Kayo Aiki-Oshimo, Kazuaki Yamazoe, Yoshie Kametani, Asako Ando, Hitoshi Kitagawa 2015 Veterinary immunology and Immunopathology 168(3-4):176-183 (IF 1.535) doi:10.1016/j.vetimm.2015.09.008

The microminipigs developed in Japan are small, and their usefulness as experimental animals has been pointed out. is occupying. Therefore, production of a monoclonal antibody that recognizes CD4B is strongly desired for analysis of the immune system of these pigs. Similar CD4 subtypes have also been reported in NIH (National Institutes of Health) minipigs, and the existence of CD4B subtypes in pigs worldwide is presumed. In addition, pigs are being widely used not only in transplantation immunity but also in regenerative medicine research, and identification of species-specific immunocompetent cells is an urgent need. However, the commercially available porcine CD4 antibodies are specific only to CD4A, and no antibodies recognizing other porcine CD4 have been produced so far.

An object of the present invention is to provide a monoclonal antibody that recognizes porcine CD4 in a species-specific manner. Another object of the present invention is to provide a monoclonal antibody that subtype-specifically recognizes porcine CD4B.

As a result of intensive studies to solve the above problems, the present inventors succeeded in obtaining a monoclonal antibody that recognizes porcine CD4 but does not recognize the peripheral blood of humans, mice and common marmosets, and completed the present invention. Arrived.

That is, according to the present invention, the following inventions are provided.
(1) A monoclonal antibody that recognizes porcine CD4B and does not recognize human peripheral blood, mouse peripheral blood and common marmoset peripheral blood.
(2) The monoclonal antibody according to (1), which recognizes porcine CD4A.
(3) The monoclonal antibody according to (1), which does not recognize porcine CD4A.
(4) The monoclonal according to any one of (1) to (3), which is an antibody produced by antibody-producing cells obtained from an immunized animal obtained by immunizing an immunized animal with porcine CD4B-expressing cells as an immunogen. antibody.
(5) Any one of (1) to (4), wherein the heavy chain amino acid sequence comprises the amino acid sequence set forth in SEQ ID NO: 2, and the light chain amino acid sequence comprises the amino acid sequence set forth in SEQ ID NO: 4. monoclonal antibody.
(6) The heavy chain amino acid sequence comprises the amino acid sequence set forth in SEQ ID NO: 6, and the light chain amino acid sequence comprises the amino acid sequence set forth in SEQ ID NO: 8 and/or the amino acid sequence set forth in SEQ ID NO: 10, (1 ) to the monoclonal antibody according to any one of (4).
(7) A monoclonal antibody produced by a hybridoma having accession number NITE AP-02666 or accession number NITE AP-02667.
(8) A hybridoma having accession number NITE AP-02666 or accession number NITE AP-02667.
(9) A method for detecting porcine CD4, comprising the step of contacting the monoclonal antibody according to any one of (1) to (7) with a sample containing porcine CD4 or a cell expressing porcine CD4.

The monoclonal antibody of the present invention can species-specifically recognize the CD4 gene product of most pigs, including not only the minipigs developed in Japan but also the pig strains owned by the NIH. CD4 is one of the most important molecules involved in the immune system, and the monoclonal antibody of the present invention is useful in immunological research, such as regenerative medicine, transplant immunity, and infection immunity, which are related to the porcine immune system.

FIG. 1 shows the immunization schedule and plasma cross-reactivity antibody titers of immunized mice. FIG. 2 shows the results of measuring the cross-reactivity of immunized mouse plasma with CD4A and CD4B transfectants. FIG. 3 shows the result of analyzing the specificity of clone x1E10, which recognizes porcine CD4AB, by FCM. FIG. 4 shows the results of analyzing the specificity of clone b1D7, which specifically recognizes porcine CD4B, by FCM. FIG. 5 shows the results of analyzing the species specificity of clone b1D7 that specifically recognizes porcine CD4B.

The present invention will be described in more detail below.
Prior to the present invention, the only way to confirm the expression of porcine CD4B was to use the mRNA RT-PCR method, as described in Non-Patent Document 1 and Non-Patent Document 2. We confirmed the predominant presence of the CD4 subtype in the microminipig colonies. However, the expression of mRNA and the expression of protein, especially the expression on the cell surface, did not necessarily match, and the behavior of the protein could not be clarified. In addition, we were unable to clarify functional differences in signal transduction of these molecules.

Antibodies that react in common with the CD4A and CD4B antigens and antibodies that specifically react with the CD4B antigen are used in porcine allogeneic or xenogeneic cells, organ transplantation experiments, analysis of antibody-producing ability after various vaccinations, peptides, etc. It is useful in a wide range of fields such as medical laboratory animal science, veterinary medicine, and animal husbandry as an essential antibody for analysis of lymphocyte subset dynamics in immune response experiments against foreign antigens.

The monoclonal antibody of the present invention is characterized by recognizing porcine CD4B and not recognizing human peripheral blood, mouse peripheral blood and common marmoset peripheral blood.
A first example of the monoclonal antibody of the present invention is a monoclonal antibody that recognizes porcine CD4A and porcine CD4B but does not recognize human peripheral blood, mouse peripheral blood and common marmoset peripheral blood.
A second example of the monoclonal antibody of the present invention is a monoclonal antibody that does not recognize porcine CD4A, porcine CD4B, human peripheral blood, mouse peripheral blood and common marmoset peripheral blood.

As used herein, the term “not recognizing” means not substantially recognizing, that is, not substantially cross-reacting with human peripheral blood, mouse peripheral blood, and common marmoset peripheral blood, and not necessarily reacting at all. does not mean only Substantially no cross-reaction means that the binding affinity of the monoclonal antibody of the present invention to human peripheral blood, mouse peripheral blood and common marmoset peripheral blood is lower than the binding affinity to pig peripheral blood mononuclear cells under the same conditions. It means at least 1/10, more preferably 1/100, even more preferably 1/1000.

Antibodies of the present invention are not particularly limited, and mouse antibodies, rat antibodies, rabbit antibodies, sheep antibodies, camel antibodies, chimeric antibodies, humanized antibodies, human antibodies, and the like can be used as appropriate.

An example of the monoclonal antibody of the present invention is an antibody produced by antibody-producing cells obtained from an immunized animal by immunizing an immunized animal with porcine CD4B-expressing cells as an immunogen.
Monoclonal antibodies can be produced by methods known to those skilled in the art.
A desired antigen or a cell expressing the desired antigen is used as a sensitizing antigen to immunize an immunized animal according to a conventional immunization method. Examples of animals to be immunized include mammals such as mice, rats, rabbits, sheep and monkeys. Administration of the antigen to the immunized animal is carried out according to a conventional method. For example, a suspension or emulsion of an adjuvant such as complete Freund's adjuvant or incomplete Freund's adjuvant and an antigen is prepared, and this is administered intravenously, subcutaneously, intracutaneously, intraperitoneally, etc. several times to immunize animals. can be

In the present invention, preferably, BALB/c mice are immunized twice with CD4B homotypic swine peripheral blood mononuclear cells and further immunized with CD4B transgenic A20 cells. It was shown that the BALB/c mouse plasma immunized in this manner produced antibodies capable of binding not only CD4B gene-transfected HEK293 cells but also CD4A gene-transfected HEK293 cells (see FIG. 1).

Next, antibody-producing cells are obtained from the immunized animal. As antibody-producing cells, splenocytes, lymph node cells, B lymphocytes, etc. from immunized animals can be used.

Cells that produce the monoclonal antibodies of the present invention are not particularly limited, but can be obtained as hybridomas, for example, by cell fusion between antibody-producing cells and myeloma cell lines. A hybridoma that produces the monoclonal antibody of the present invention can be obtained by the following cell fusion method. That is, hybridomas can be produced by obtaining, for example, splenocytes as antibody-producing cells from an immunized animal and fusing them with myeloma cells by a known method.

Myeloma cell lines used for cell fusion include, for example, mouse P3X63Ag8, P3U1, and Sp2/0 strains. Fusion promoters such as polyethylene glycol and Sendai virus can be used for cell fusion, and hypoxanthine-aminopterin-thymidine (HAT) medium can be used in a conventional manner for selection of hybridomas after cell fusion. Hybridomas obtained by cell fusion can be cloned by limiting dilution or the like.

Then, by screening the resulting hybridomas, it is possible to obtain hybridomas that produce a monoclonal antibody that recognizes porcine CD4B but does not recognize human peripheral blood, mouse peripheral blood, and common marmoset peripheral blood.

Hybridoma screening is performed using methods known in the art such as ELISA assay, Western blot analysis, radioimmunoassay, FACS using cells expressing porcine CD4B, and hybridomas that produce antibodies that recognize porcine CD4B. Cells can be selected. Cloning a hybridoma that secretes the desired antibody, culturing it under appropriate conditions, collecting the secreted antibody, and purifying it using methods known in the art, such as ion exchange columns, affinity chromatography, etc. can be done.

An example of the antibody of the present invention is the monoclonal antibody produced by the hybridoma having the accession number NITE AP-02666 obtained in the examples below. This monoclonal antibody contains the amino acid sequence of SEQ ID NO: 2 as the heavy chain amino acid sequence and the amino acid sequence of SEQ ID NO: 4 as the light chain amino acid sequence. Hybridomas with accession number: NITE AP-02666 are also within the scope of the present invention.

Another example of the antibody of the present invention is the monoclonal antibody produced by the hybridoma having accession number NITE AP-02667 obtained in the Examples below. This monoclonal antibody comprises the amino acid sequence set forth in SEQ ID NO: 6 as the heavy chain amino acid sequence, and the amino acid sequence set forth in SEQ ID NO: 8 and/or the amino acid sequence set forth in SEQ ID NO: 10 as the light chain amino acid sequence. is. Hybridomas with accession number: NITE AP-02667 are also within the scope of the present invention.

DNA encoding the monoclonal antibody is readily isolated and sequenced by conventional methods (e.g., using oligonucleotide probes capable of specifically binding to the genes encoding the heavy and light chains of the monoclonal antibody). can.

Recombinant antibodies produced by cloning antibody genes from hybridomas, inserting them into appropriate vectors, introducing them into hosts, and producing them using genetic recombination techniques can be used. Specifically, cDNA of an antibody variable region (V region) can be synthesized from hybridoma mRNA using reverse transcriptase. Once the DNA encoding the V region of the antibody of interest is obtained, it is ligated with the DNA encoding the desired antibody constant region (C region) and incorporated into an expression vector. Alternatively, the DNA encoding the antibody V region may be incorporated into an expression vector containing the antibody C region DNA. It is incorporated into an expression vector so that it is expressed under the control of an expression control region such as an enhancer or promoter. This expression vector can then be used to transform a host cell to express the antibody.

As described above, once the antibody gene is isolated and introduced into a suitable host to produce the antibody, a combination of a suitable host and an expression vector can be used. When using eukaryotic cells as hosts, animal cells, plant cells, and fungal cells can be used. Animal cells include (1) mammalian cells such as CHO, COS, myeloma, BHK (baby hamster kidney), HeLa, Vero, (2) amphibian cells such as Xenopus oocytes, or (3) insect cells. , for example, sf9, sf21, Tn5, etc. are known. As plant cells, cells derived from the genus Nicotiana, for example, Nicotiana tabacum, are known, and these cells may be cultured as callus. As fungal cells, yeast such as the genus Saccharomyces such as Saccharomyces cerevisiae, and filamentous fungi such as the genus Aspergillus such as Aspergillus niger are known. When using prokaryotic cells, there are production systems using bacterial cells. As bacterial cells, E. coli and Bacillus subtilis are known. An antibody can be obtained by introducing the desired antibody gene into these cells by transformation and culturing the transformed cells in vitro.

In addition, the monoclonal antibody of the present invention may be a low-molecular-weight antibody such as an antibody fragment, or a modified antibody, as long as the property of recognizing porcine CD4B is not lost. Specific examples of antibody fragments include Fab, Fab', F(ab')2, Fv, Diabody and the like. Such antibody fragments can be obtained by constructing genes encoding these antibody fragments, introducing them into expression vectors, and expressing them in suitable host cells. Antibodies conjugated with various molecules such as polyethylene glycol (PEG) can also be used as modified antibodies.

Antibodies expressed or produced as described above can be purified by known methods commonly used for purification of proteins. For example, antibodies can be separated and purified by appropriately selecting and combining affinity columns such as protein A columns, chromatography columns, filters, ultrafiltration, salting out, dialysis, and the like.

According to the present invention, porcine CD4 can be detected by contacting the monoclonal antibody of the present invention with a sample containing porcine CD4 or cells expressing porcine CD4. Preferably, the monoclonal antibody of the present invention is contacted with cells expressing porcine CD4, and porcine CD4 can be detected by a flow cytometric assay.

The present invention will be described in more detail with reference to the following examples, but the present invention is not limited by the examples.

<Material>
Eight-week-old female BALB/c mice were purchased from CLEA Japan.
Pig blood was obtained from domestic universities. Anti-mouse IgG APC antibody used as a secondary antibody in screening was from BioLegend. PEG solution and P3X63-U1 myeloma cell line were used for cell fusion. HAT supplement from SIGMA and HT supplement from Gibco (registered trademark) were used as selective media for hybridoma culture. Monoclonal antibody isotypes were determined using Roche's IsoStrip ^™ Mouse Monoclonal Antibody Isotyping Kit.

<Method>
(1) Transfection CD4A and CD4B genes are mRNA sequences of CD4 genes of microminipigs analyzed by the inventors' group (Non-Patent Document 1) (CD4A: GeneBank accession number LC064059.1; CD4B: GeneBank accession number LC064060 1) Primers were designed, cDNA was synthesized using total RNA of microminipigs, and constructs were prepared. A modified S/MAR (scaffold/matrix attachment region) episomal vector was used as a vector (Modified S/MAR episomal vectors for stably expressing fluorescent-protein tagged transgenes with small cell-to-cell fluctuations. Akiko Mizutani, Eri Kikkawa, 2013; 443(1):113-6. PMID: 23969013 doi: 10.1016/j.ab.2013.08.009). The gene sequences of the constructs are shown in SEQ ID NOS: 11-15.

SEQ ID NO: 11: ori2 pEPI Km CAG(K499) Age SLA1-BamHI IRES/EM7 917/921/919/920 mVenus-Km fusion noBam (vector)
SEQ ID NO: 12: CD4A (cDNA)
SEQ ID NO: 13: CD4B (cDNA)
SEQ ID NO: 14: Lig 13-117 (expression vector incorporating CD4A)
SEQ ID NO: 15: Lig 13-118 (expression vector incorporating CD4B)

HEK293 cells were cultured in D-MEM-10% FCS medium, and gene transfer was performed using the Invitrogen Neon transfect system under the conditions of 1100V 10ms 3pulses and placed in a 37°C incubator with a ₅ % CO concentration for 42 to 48 hours. After culturing, G418 was added to the medium and only transfected cells were extracted. A20 cells were cultured in RPMI1640-10% FCS medium, transfected with either CD4A or CD4B under the conditions of 1500 V 10 ms 3 pulses, and cultured in a 37° C. incubator with 5% CO ₂ concentration for 18 to 24 hours. The cultured gene-introduced A20 cells were collected, and the level of mVenus gene expression was confirmed by flow cytometry.

(2) Immunization/confirmation of mouse antibody titer Mononuclear cells were collected from swine peripheral blood using LYMPHOPREP. After lysing erythrocytes with RBC Lysis Buffer and treating with mitomycin C (MMC) at 37° C. for 40 minutes, 3×10 ⁶ cells were intraperitoneally administered to BALB/c mice at intervals of 2 weeks. Two weeks later, the CD4B gene-introduced A20 cells were treated with MMC by the method described above, and intraperitoneally administered. After this booster immunization was performed twice, MMC-treated porcine peripheral blood mononuclear cells were administered twice with an interval of 2 weeks. Mouse antibody titers were obtained by adding mouse plasma to HEK293 cells and CD4A/B-introduced cells using HEK293 cells as the parent strain, staining with anti-mouse IgG APC as a secondary antibody, and producing polyclonal antibodies in mouse plasma. It was confirmed.

(3) Cell fusion The applicants used the conventional method for producing hybridomas (Production of a Locus and Allele-Specific Monoclonal Antibody for the Characterization of SLA-1*0401 mRNA and Protein Expression Levels in MHC-Defined Microminipigs. Yoshie Kametani*, Shino Ohshima, Asuka Miyamoto, Atsuko Shigenari, Masaki Takasu, Noriaki Imaeda, Tatsuya Matsubara, Masafumi Tanaka, Takashi Shiina, Hiroshi Kamiguchi, Ryuji Suzuki, Hitoshi Kitagawa, Jerzy K. Kulski, Noriaki Hirayama, Hidetoshi Inoko, Asako Ando PLOS ONE 2016 11(10):e0164995. doi: 10.1371/journal.pone.0164995.).

Immunized BALB/c mice were anesthetized with isoflurane, heart blood was collected, euthanized by cervical dislocation, and spleens were removed. The spleen was ground with a glass slide in RPMI 1640-10% FCS medium, passed through a mesh (77 µm), and centrifuged at 300 xg for 5 minutes at 4°C. The supernatant was removed, the precipitate was suspended in RBC Lysis Buffer, and immediately after suspension, centrifugation was performed at 300 xg for 5 minutes at 4°C. After centrifugation, the supernatant was removed, the cells were suspended in 10 ml of RPMI1640-10% FCS medium, and the cell count was determined. P3X63-U1 cells were prepared so that the ratio of P3X63-U1 cells and spleen cells was 1:10, mixed with spleen cells, and centrifuged at 300 xg for 5 minutes at 4°C. The supernatant was removed, 1 ml of a PEG solution warmed to 37°C was added over 1 minute, and the mixture was allowed to stand in a constant temperature water bath at 37°C for 1 minute. Then 2 ml of RPMI1640-10% FCS medium was added over 1 minute, followed by 10 ml of RPMI1640-10% FCS medium over 1 minute. Centrifugation was performed at 90 xg for 5 minutes at 4°C, and the cells were suspended in a medium adjusted to 5 x ¹⁰⁶ /ml of RPMI1640-10% FCS medium and HAT supplement at a ratio of 50:1 after removing the supernatant. After turbidity, 100 μL/well was dispensed into a Corning 96-well plate and cultured in a 37° C. incubator with a 5% CO ₂ concentration. Ten days later, the number of hybridoma colonies was counted, and the medium was replaced.

(4) Screening Screening was performed using HEK293 cells and CD4A- or CD4B-introduced cells using the HEK293 cells as parent strains.
On the day before the screening, mixed cells of HEK293 cells and HEK293 CD4A/B-introduced cells were dispensed at 1×10 ⁴ /well into Thermo 96-well plates, and cultured in a 37° C. incubator with 5% CO ₂ concentration. On the day of screening, 160 μL of hybridoma culture supernatant was removed.

The medium of HEK293 cells and HEK293 CD4A/B-introduced cells was removed, and the removed hybridoma culture supernatant was slowly added at 70 μL/well so as not to detach the cells, and incubated at room temperature for 15 minutes. 100 μL/well of 1% BSA/1×PBS was added, and 120 μL of the supernatant was removed so that the tip of the tip did not touch the bottom of the plate. After adding 150 μL/well of 1% BSA/1×PBS, 180 μL of supernatant was removed. 50 μL/well of BioRegen's anti-mouse IgG APC diluted 500-fold with 1% BSA/1×PBS was slowly added and incubated at room temperature for 15 minutes. 1% BSA/1×PBS was added at 100 μL/well, centrifugation was performed at 300×g for 5 minutes at 4° C., and 120 μL of the supernatant was removed so that the tip of the tip did not touch the bottom of the plate. After adding 150 μL/well of 1% BSA/1×PBS, centrifugation was performed at 300×g for 5 minutes at 4° C., and 150 μL of the supernatant was removed so that the tip of the chip did not touch the bottom of the plate. 100 μL/well of Hoechst 33342 diluted twice was added and incubated at room temperature for 1 hour.

The reactivity of hybridoma supernatants, HEK293 cells, and CD4A/B-introduced cells was analyzed using Array Scan VTI from Thermo Scientific. After analysis by Array Scan, similar analysis was performed using flow cytometry. HEK293 cells and HEK293 CD4A and CD4B-introduced cells were placed in Fisher tubes, respectively, and hybridoma culture supernatants were added at 50 μL/tube and incubated at 4° C. for 15 minutes. After incubation, 1 ml of 1×PBS was added and centrifuged at 1000 g for 5 minutes (KUBOTA2420), leaving 50 μL of the supernatant and removing the other supernatant. 10 μL/tube of 100-fold diluted anti-mouse IgG APC from BioLegend was added and incubated at 4° C. for 15 minutes. After incubation, 1 ml of 1×PBS was added, centrifugation was performed at 1000 g for 5 minutes, the supernatant was removed, 200 μL/well of 1% BSA/1×PBS was added, and FALCON (registered trademark) rounds were passed through a mesh. It was transferred to a bottom tube and analyzed by flow cytometry (BD FACS VERSE). With a fluorescence intensity of 3 x 10 ¹ as a cutoff value, there are 20% or more positive cells that show fluorescence above the cutoff value by staining CD4 transfectants, and the parent strain has a condition that 90% or more is below the cutoff value. Clones producing antibodies were designated as positive clones.

(5) Analysis of species specificity and allotype specificity Human lymphocytes, microminipig lymphocytes, and mouse lymphocytes were isolated from peripheral blood by gravity separation using Ficoll (human) or LYMPHOPREP ^™ (microminipig/mouse) to obtain mononuclear cells. A spherical fraction was collected. Using the culture supernatant of the clone obtained by screening as the primary antibody, staining was performed as follows, and analysis was performed by flow cytometry in the same manner as above. A porcine CD4 antibody (SouthenBiotech 74-12-4) was used as a positive control.

(6) Antibody primary structure analysis PCR was performed using cDNA derived from total RNA extracted from the hybridoma, and after confirming that a specific band was amplified, the gene sequence was analyzed with a next-generation sequencer. The read IDs identified in the cDNA representative sequences were sorted, only long ones were extracted, and the full-length sequences were determined using software such as Genetyx (registered trademark).

<Results>
(1) Immunization for production of antibodies that specifically recognize both porcine CD4A/B BALB/c mice were immunized twice with CD4B homotype porcine peripheral blood mononuclear cells, and further immunized with CD4B gene-introduced A20 cells. BALB/c mouse plasma was shown to produce antibodies capable of binding not only to CD4B gene-transfected HEK293 cells but also to CD4A gene-transfected HEK293 cells (FIGS. 1A and 1B).

Therefore, after immunization with CD4B gene-introduced A20 cells, mice exhibiting increased plasma cross-reactivity antibody titers against two types of HEK293 cells into which each gene of CD4A and CD4B was introduced were selected, and mouse splenocytes and P3X myeloma cells were selected. performed cell fusion with Culture supernatants of the produced hybridomas were screened using similar two types of CD4 gene-introduced cells. Clones recognizing CD4AB were picked up as those staining both CD4AB, and clones recognizing only CD4B were picked as those recognizing only CD4B.

(2) Measurement and screening of mouse plasma cross-reactivity after immunization Figure 2 shows CD4A in mouse plasma obtained by immunizing each of 12 immunized mice twice with PBMC and then immunizing with CD4A gene-introduced A20 cells. and cross-reactivity with CD4B transfectants. FIG. 2 shows that many mice immunized with CD4A still produce antibodies that cross-react with CD4B.

In particular, the plasma of 28-week-old R cut mice that had been immunized four times was strongly stained with both HEK293 cells transfected with the CD4A gene and HEK293 cells transfected with the CD4B gene. increased. Therefore, this mouse splenocyte cell fusion (Fig. 2: Fusion x) was performed. In addition, we screened hybridoma supernatants produced from these cell fusions.
Using similar immunization and checking methods, mice with elevated titers of antibodies recognizing both CD4AB were obtained. Splenocyte fusion was performed using these. After that, positive cells were selected by Array scan.

Hybridoma cells that produce monoclonal antibody x1E10 were deposited on March 7, 2018 with the receipt number: NITE AP-02666 at the National Institute of Technology and Evaluation Patent Microorganisms Depositary Center (292-0818, Kisarazu, Chiba Prefecture, Japan). It has been deposited at Ichi Kazusa Kamatari 2-5-8).
The hybridoma cells that produce the monoclonal antibody b1D7 were registered on March 7, 2018 with the receipt number: NITE AP-02667 at the National Institute of Technology and Evaluation Patent Microorganisms Depositary Center (292-0818, Chiba Prefecture, Japan). It is deposited at 2-5-8 Kazusa Kamatari, Kisarazu City.

(3) Monoclonal antibody <1> Clone x1E10 that recognizes CD4AB
(1) Analysis of specificity by FCM Specificity analysis was performed using both HEK293-CD4A and HEK293-CD4B transfectants. As a result, x1E10 recognized both CD4A and CD4B transfectants. As a result, it was revealed that x1E10 is specific for both CD4A and CD4B and recognizes both CD4A and CD4B in common (Fig. 3A). Moreover, the antibody of this clone did not recognize the peripheral blood of humans, mice, and common marmosets (CM), and reacted only with porcine peripheral blood mononuclear cells (Fig. 3B). Therefore, this monoclonal antibody was found to be specific only to pigs.

(2) Structural Analysis of Monoclonal Antibody Gene by Next-Generation Sequencer Gene sequence analysis was performed from the hybridoma mRNA of this antibody by a next-generation sequencer. The nucleotide sequence and amino acid sequence of the heavy chain of clone x1E10 that recognizes CD4AB and the nucleotide sequence and amino acid sequence of the light chain of x1E10 are described below.

Heavy chain:
Nucleotide sequence (SEQ ID NO: 1)
CGCTCTCACTGGAGGCTGATCTCTGAAGATAAGGAGGTGTAGCCTAAAAGATGAGAGTGCTGATTCTTTTGTGGCTGTTCACAGCCTTTCCTGGTATCCTGTCTGATGTGCAGCTTCAGGAGTCGGGACCTGGCCTGGTGAAACCTTCTCAGTCTCTGTCCCTCACCTGCACTGTCACTGGCTACTCAATCATCAGTGATTATGCCTGGAACTGGATCCGGCAGTTTCCAGGAAACAAGATGGAGTGGATGGGCTACATACTCTACAGTGGAACCACTAACTACAACCCCTCTCTCAAAAGTCGAATCTCTATCACTCGAGACACATCCAAGAACCAGTTCTTCCTGCAGTTGAATTCTGTGACTACTGAGGACACAGCCACATATTACTGTGCACGAAGGACCTACTATGATTACGATTACTATGGTATGGACTACTGGGGTCAAGGAACCTCAGTCACCGTCTCCTCAGCCAAAACAACAGCCCCATCGGTCTATCCACTGGCCCCTGTGTGTGGAGATACAACTGGCTCCTCGGTGACTCTAGGATGCCTGGTCAAGGG

Amino acid sequence (SEQ ID NO: 2)
MRVLILLWLFTAFPGILSDVQLQESGPGLVKPSQSLSLTCTVTGYSIISDYAWNWIRQFPGNKMEWMGYILYSGTTNYNPSLKSRISITRDTSKNQFFLQLNSVTTEDTATYYCARRTYYDYDYYGMDYWGQGTSVTVSSAKTTAPSVYPLAPVCGDTTGSSVTLGCLVK

Light chain:
Nucleotide sequence (SEQ ID NO: 3)
TCCTCAGGCTGTCTCCTCAGGTTGCCTCCTCAAAATGAAGTTGCCTGTTAGGCTGTTGGTGCTGATGTTCTGGATTCCTGCTTCCAGCAGTGATGTTTTGATGACCCAAATTCCACTCTCCCTGCCTGTCAGTCTTGGAGATCAAGCCTCCATCTCTTGCAGATCTAGTCAGACCATTGTCCATAGTCTTGGTAACACCTATTTAGAATGGTACCTACAGAAACCAGGCCAGTCTCCAAAGCTCCTGATCTACAAAGTTTCCAACCGATTTTCTGGGGTCCCAGACAGGTTCATTGGCAGTGGATCCGGGACAGACTTCACACTCAAGATCACCAGAGTGGAGGCTGAGGATCTGGGAGTTTATTACTGCTTTCAGGGTTCACATGTTCCGTGGACGTTCGGTGGAGGCACCAAGCTGGAAATCAAACGGGCTGATGCTGCACCAACTGTATCCATCTTCCCACCATCCAGTGAGCAGTTAA

Amino acid sequence (SEQ ID NO: 4)
MKLPVRLLVLMFWIPASSSDVLMTQIPLSLPVSLGDQASISCRSSQTIVHSLGNTYLEWYLQKPGQSPKLLIYKVSNRFSGVPDRFIGSGSGTDFTLKITRVEAEDLGVYYCFQGSHVPWTFGGGTKLEIKRADAAPTVSIFPPSSEQL

<2> Clone b1D7 that specifically recognizes porcine CD4B
(1) Specificity analysis by FCM Specificity analysis was performed using both HEK293-CD4A and HEK293-CD4B transfectants. As shown in Figure 4, this clone recognizes the CD4A transfectant. However, only CD4B transfectants were recognized with high avidity. The above results revealed that b1D7 is specific only to CD4B and does not recognize CD4A.

Next, the species specificity of b1D7 was analyzed using porcine, human, and mouse peripheral blood mononuclear cells. As a result, as shown in FIG. 5, b1D7 did not recognize the peripheral blood of humans, mice, and common marmosets (MC), and reacted only with CD4B homozygous swine peripheral blood mononuclear cells. Therefore, this monoclonal antibody was found to be species-specific.

(2) Monoclonal Antibody Gene Structure Analysis Gene sequence analysis was performed from the hybridoma mRNA of this antibody using a next-generation sequencer. The nucleotide sequence and amino acid sequence of the heavy chain of clone b1D7 that specifically recognizes porcine CD4B, and the nucleotide sequence and amino acid sequence of the light chain of b1D7 are described below. It was revealed that this antibody has two types of light chains. Two of these light chains were always expressed by cloning, and it was considered highly likely that the clone was a fusion of two B cells with one myeloma.

Heavy chain:
Nucleotide sequence (SEQ ID NO: 5)
CACTGGAGGCTGATCTCTGAAGATAAGGAGGTGTAGCCTAAAAGATGAGAGTGCTGATTCTTTTGTGGCTGTTCACAGCCTTTCCTGGTATCCTGTCTGATGTGCAGCTTCAGGAGTCGGGACCTGGCCTGGTGAAACCTTCTCAGTCTCTGTCCCTCACCTGCACTGTCACTGGCTACTCAATCACCAGTGATTATGCCTGGAACTGGATCCGGCAGTTTCCAGGAAACAAACTGGAGTGGATGGGCTACATAAGCT T CAGTGGTAGCACTAGCTACAACCCATCTCTCAAAAGTCGAATCTCTATCACTCGAGACACATCCAAGAACCAGTTCTTCCTGCAGTTGAATTCTGTGACTACTGAGGACACAGCCACATATTACTGTGCAAGACAGGGCTATGGAGATTACGACCCCTTTCCTTACTGGGGCCAAGGGACTCTGGTCACTGTCTCTGCAGCCAAAACGACACCCCCATCTGTCTATCCACTGGCCCCTGGATCTGCTGCCCAAACTAACTCCATGGTGACCCTGGGATGCCT[G|T]GTCAA[G|A]GG

Amino acid sequence (SEQ ID NO: 6)
MRVLILLWLFTAFPGILSDVQLQESGPGLVKPSQSLSLTCTVTGYSITSDYAWNWIRQFPGNKLEWMGYIS F SGSTSYNPSLKSRISITRDTSKNQFFLQLNSVTTEDTATYYCARQGYGDYDPFPYWGQGTLVTVSAAKTTPPSVYPLAPGSAAQTNSMVTLGCLVK

Light chain:
Nucleotide sequence (SEQ ID NO: 7)
Predicted DNA sequence of IGKV6-20_IGKJ2_CGQGYSYPYTF
TAGAGGCCAGCCCAGCTGTCCATGATTTATAACCTAGGCCTTTGCAGTGAGATCTGAAATGCATCAGACCAGCATGGGCATCAAGATGGAATCACAGACTCTGGTCTTCATATCCATACTGCTCTGGTTATATGG A GCTGATGGGAACATTGTAATGACCCAATCTCCCAAATCCATGTCCATGTCAGTAGGAGAGAGGGTCACCTTGA C CTGCAAGGCCAGTGAGAATGTGG T TACTTATGT T TCCTGGTATCAACAGAAACCAGAGCAGTCTCCTAAACTGCTGATATACGGGGCATCCAACCGGTACACTGGGGTCCCCGATCGCTTCACAGGCAGTGGATCTGCAACAGATTTCACTCTGACCATCAGCAGTGTGCAGGCTGAAGACCTTGCAGATTATCACTGTGGACAGGGTTACAGCTATCCGTACACGTTCGGAGGGGGGACCAAGCTGGAAATAAAACGGGCTGATGCTGCACCAACTGTATCCATCTTCCCACCATCCAGTGAGCAGTTAA

Amino acid sequence (SEQ ID NO: 8)
MESQTLVFISILLWLYGADGNIVMTQSPKSMSMSVGERVTL T CKASENV V TYVSWYQQKPEQSPKLLIYGASNRYTGVPDRFTGSGSATDFTLTISSVQAEDLADYHCGQGYSYPYTFGGGTKLEIKRADAAPTVSIFPPSSEQL

Light chain:
Nucleotide sequence (SEQ ID NO: 9)
AAGAACTGACTAGACTCTATCTTGCTATTTGCATATTACATTTTCAGTAACCACAAATATCTCACAGTTGGTTTATAGCAAAGTACTTATGAGAATAGTAGTAATTAGCTAGGGACCAAAGTTCAAAGACAAAATGGATTTTCAAGTGCAGATTTTCAGCTTCCTGCTAATCAGTGCCTCAGTCATACTGTCCAGAGGACAAATTGTTCTCACCCAGTCTCCA A CAATCATGTCTGCATCTCCAGGGGAGAAGGTCACCATGACCTGCAGTGCCAGCTCAAGTGT T AGTTACATGTACTGGTACCAGCAGAAGCCAGGATCCTCCCCCAGACTCCTGATTTATGACACATCCAACCTGGCTTCTGGAGTCCCTGTTCGCTTCAGTGGCAGTGGGTCTGGGACCTCTTACTCTCTCACAATCAGCCGAATGGAGGCTGAAGATGCTGCCACTTATTACTGCCAGCAGTGGAGTAGTTACCCGTACACGTTCGGAGGGGGGACCAAGCTGGAAATAAAACGGGCTGATGCTGCACCAACTGTATCCATCTTCCCACCATCCAGTGAGCAGTTAA

Amino acid sequence (SEQ ID NO: 10)
MDFQVQIFSFLLISASVILSRGQIVLTQSP T IMSASPGEKVTMTCSASSSVSYMYWYQQKPGSSPRLLIYDTSNLASGVPVRFSGSGSGTSYSLTISRMEAEDAATYYCQQWSSYPYTFGGGTKLEIKRADAAPTVSIFPPSSEQL

From the above results, it can be seen that the present screening method and the present monoclonal antibody are optimal for flow cytometry as a swine-specific CD4 monoclonal antibody preparation method and a monoclonal antibody-producing hybridoma.

Claims (6)

comprising the amino acid sequence set forth in SEQ ID NO: 2 as the heavy chain amino acid sequence, and the amino acid sequence set forth in SEQ ID NO: 4 as the light chain amino acid sequence;
A monoclonal antibody that recognizes porcine CD4A, porcine CD4B, and does not recognize human peripheral blood, mouse peripheral blood, and common marmoset peripheral blood. The heavy chain amino acid sequence comprises the amino acid sequence set forth in SEQ ID NO: 6, and the light chain amino acid sequence comprises the amino acid sequence set forth in SEQ ID NO: 8 and/or the amino acid sequence set forth in SEQ ID NO: 10,
A monoclonal antibody that does not recognize porcine CD4A, porcine CD4B , human peripheral blood, mouse peripheral blood and common marmoset peripheral blood. 3. The monoclonal antibody according to claim 1 or 2, which is an antibody produced by antibody-producing cells obtained from an immunized animal by immunizing an immunized animal with porcine CD4B-expressing cells as an immunogen. A monoclonal antibody produced by a hybridoma having accession number NITE AP-02666 or accession number NITE AP-02667. A hybridoma with accession number NITE AP-02666 or accession number NITE AP-02667. A method for detecting porcine CD4, comprising the step of contacting the monoclonal antibody according to any one of claims 1 to 4 with a sample containing porcine CD4 or a cell expressing porcine CD4.

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