WO2009113742A1 - Method of producing genetically engineered antibody - Google Patents

Abstract

A genetically engineered antibody is produced by isolating antibody-producing cells from a rabbit having been immunized with an antigen, proliferating cells carrying a target antibody gene and then screening, isolating heavy chain and light chain antibody genes from the screened cells and amplifying the same, transferring the same into an expression vector and further transferring into host cells to thereby express a genetically engineered rabbit monoclonal antibody.

Description

 Description Method for producing recombinant antibody Technical field

 The present invention relates to a method for producing a recombinant rabbit monoclonal antibody. Background art

 Monoclonal antibodies are widely used not only as experimental reagents for detecting specific substances, but also as core materials for cancer therapeutics and immunodiagnostics because of their high specificity and affinity.

 Speaking of monoclonal antibodies, mouse-derived monoclonal antibodies are generally recognized. For antibodies or immunoassay reagents used in normal experiments, the degree of affinity of mouse-derived monoclonal antibodies is often sufficient. However, mouse-derived monoclonal antibodies may have insufficient affinity in areas where measurement sensitivity has become increasingly demanded in recent years, such as immunodiagnostic drugs. For example, in the competition method, it can be theoretically proved that the higher the affinity of the antibody, the higher the detection sensitivity. In order to construct a highly sensitive competitive method, an antibody with high affinity is required. Become. In addition, even with the sandwich method, it becomes possible to detect a small amount of antigen by using an antibody with high affinity, and a highly sensitive measurement system can be constructed.

 For example, steroids are small molecules and are measured by competition methods. In particular, among the steroids, estradiol (E 2) requires a highly sensitive measurement system, and there is a strong demand for antibodies with high affinity.

Various monoclonal antibodies other than mice have also been reported. As introduced in bridoma, 1 9, 2 0 1: 2 0 0 0 (Non-patent Document 1), antibodies with high affinity tend to be isolated from antibodies of large animal species . However, even if the same animal species is immunized with the same antigen, the effect of individual differences is great, and the antisera obtained are also different.Therefore, at least several animals must be isolated in order to isolate antibodies with high affinity. It is necessary to immunize an individual with an antigen and isolate a monoclonal antibody from an individual with a high immune response. Therefore, in reality, Usagi is chosen as an immunized animal. Rabbits are also considered to be suitable as immunized animal species, because Rabbit's serum is often used as an antiserum for immunodiagnostics.

 A method for producing a mouse monoclonal antibody has been established and is produced by the method shown below. First, spleen cells (antibody-producing cells) from mice immunized with antigen are fused with mouse myeloma cells (myeloma cells) to produce a hybridoma that has infinite proliferative capacity and produces the desired antibody. . Thereafter, the antibody produced by culturing the cells is purified to produce a mouse monoclonal antibody.

Usagi monoclonal antibodies can also be produced theoretically in the same manner as mouse monoclonal antibodies. However, Usagimyeloma cells used for cell fusion with antibody-producing cells (Proc. Nat. A cad. Sci. USA, 9 2, 9 3 4 8; 1 9 9 5: Non-patent document 2 However, unlike mouse myeloma cells, they are not generally distributed, so there are only a few facilities that can be produced in the same way as mouse monoclonal antibody production methods. Heron monoclonal antibodies cannot be produced. For this reason, several methods have been investigated for producing rabbit antibody monoclonal antibodies without using rabbit cells. First, there is a method of fusing rabbit antibody-producing cells with mouse myeloma cells. However, this method is fusion between different animals, and the fusion efficiency is low. Therefore, the number of hybridomas obtained is small, and the obtained hybridomas lose their antibody-producing ability within a short period of time (

Proc. Natl. A cad. S c in USA, 5 8, 1 1 0 4; 1 9 6 7: Non-patent literature 3, Genetics, 5 4, 1 0 9 5; 1 9 6 6: Non-patent Reference 4). Furthermore, although it has been reported that the ability to produce antibodies can be maintained by culturing the fused cells in the presence of rabbit sera, it is difficult to purify the monoclonal antibodies produced, and long-term antibody productivity has been reported. There is also no report (Science 2 40, 1 7 8 8; 1 9 8 8: Non-patent document 5).

Next, after isolating and amplifying the heavy chain (H chain) and light chain (L chain) antibody genes from the rabbit spleen that had been immunized with the antigen, a rabbit antibody library was prepared, and the rabbit antibody antibody was obtained by the phage display method. (J. Biol. Chem., 2 75, 1 3 6 6 8; 2 0 0: Non-patent document 6; Gene, 1 7 2, 2 9 5; 1 996: Non-patent document 7; J. Immunol. Method., 2 13, 20.1; 1 998: Non-patent document 8). However, according to this method, an operation called “banning” is essential in the process of selecting antibodies from the library. Briefly describing the procedure, a water-insoluble carrier on which an antigen is immobilized is brought into contact with a library, and an antibody bound to the antigen is recovered. Then, the antibody bound to the antigen is amplified by some method to create a new library, and the reaction of the antigen with the water-insoluble carrier is repeated. By this operation, the antibody that binds to the antigen is concentrated, and the antibody that finally binds to the antigen can be isolated. However, as long as the antigen-antibody reaction is an equilibrium reaction, the binding between the antigen and the antibody is affected by the concentration. For example, in the library In principle, it is impossible to selectively concentrate high-affinity antibodies that are present in only amounts. In order to obtain antibodies with high affinity in general,

The antibody needs to be a combination of the H and L chains originally produced by the Usagi. In the antibody obtained by this operation, the combination of the H and L chains originally produced by the Usagi may be reproduced. Since it is extremely low, it is extremely difficult to recover an antibody with improved affinity by this operation.

 Other than the above method, after holding the rabbit antibody-producing cells in a soft agar medium, search for antibody-producing cells expressing the target antibody by the erythrocyte hemolysis method, isolate the antibody gene from one antibody-producing cell, There is a method of producing it as a recombinant antibody (Proc. Atl. A cad. Sci. USA, 93, 784 43; 1 996: Non-patent document 9). By using this method, the combination of the H and L chains originally produced by the rabbits can be reproduced, but immunodiagnosis that requires extremely high antibody performance (affinity, specificity, storage stability, productivity, etc.) Antibodies like those used in medicine are not easily obtained. In other words, the method of Non-Patent Document 9 may be used as long as a rabbit monoclonal antibody that binds to an antigen is isolated, but thousands of high-performance monoclonal antibodies that can be used in immunodiagnostics are isolated. Even if a recombinant antibody of tens of thousands of clones is prepared, there is no guarantee that the target antibody can be isolated. Furthermore, since the above work requires a large number of experimental man-hours, it is practically difficult to implement. In addition, since the gene is isolated from a single cell, the efficiency of antibody gene amplification is low even if an antibody-expressing cell is found, and the antibody gene amplification probability reported in Non-Patent Document 9 is H chain. 27%, about 55% for L chain.

From the above, although the performance of the Usagi monoclonal antibody has been reported to be high, the Usagi monoclonal antibody itself is Since it can only be produced in some facilities with mammary cells, it has made it difficult to use the antibody in an industrial manner. Disclosure of the invention

 The problem to be solved by the present invention is that it is possible to produce a Usagi monoclonal antibody that could only be produced by a specific institution having Usagimie mouth cells with knowledge of normal laboratory facilities and molecular biology. It is to provide technology.

 The present invention made in view of the above problems includes the following inventions. The first invention is to isolate antibody-producing cells from rabbits immunized with antigen, to select cells carrying the desired antibody gene, and to isolate and amplify heavy and light chain antibody genes from the selected cells In the method for producing a recombinant antibody, which is introduced into an expression vector and then introduced into a host cell to express the recombinant rabbit monoclonal antibody, a cell having the target antibody gene is selected. A method for producing a recombinant antibody, wherein antibody-producing cells are grown and then selected.

 The second invention is characterized in that the operation of isolating heavy and light chain antibody genes from the selected cells is carried out within a period in which the antibody genes are not dropped from the selected cells. This is a method for producing the genetically engineered antibody described in the invention.

 The third invention is characterized in that the operation of isolating heavy and light chain antibody genes from the selected cells is carried out within 14 days after the start of proliferation of antibody-producing cells. It is a manufacturing method of the gene recombinant antibody of description.

The fourth invention is characterized in that the antibody-producing cells are any of spleen cells, lymph node cells, and peripheral blood cells of a rabbit immunized with an antigen. The method for producing a recombinant antibody according to any one of the first to third inventions is characterized in that the method for growing antibody-producing cells is a method for culturing antibody-producing cells in a medium. The method for producing a recombinant antibody according to any one of the first to fourth inventions.

 The sixth invention is characterized in that the method of growing antibody-producing cells is a method of fusing and culturing cells capable of imparting immortalizing ability, as described in the first to fourth inventions This is a method for producing a recombinant antibody.

 A seventh invention is the method for producing a genetically engineered antibody according to the first to sixth inventions, characterized in that the antigen to immunize the rabbit is a hapten or a peptide.

 The eighth invention is the method for producing a recombinant antibody according to any one of the first to seventh inventions, wherein the host cell is an animal cell. Brief Description of Drawings

 Fig. 1 shows the results of culturing cells derived from the spleen and evaluating the antigen-binding ability of the culture supernatant of 192 clones.

 Figure 2 shows the results of evaluation of antibodies in the culture supernatant after cell fusion using lymphocytes derived from peripheral blood by ELISA.

 FIG. 3 shows the results of screening the culture supernatant of 3840 clones. The vertical axis shows competitive characteristics, the horizontal axis shows specificity, and the size of each plot shows reactivity. In addition, the antibody indicated by the black circle plot in the figure is a clone with high competitive performance, high specificity, and high reactivity, and the antibody gene was isolated from the clone.

FIG. 4 shows the structure of the expression vector p ECE dhfr. SV 40 P in the figure is the SV 40 promoter, dhfr is dihydrophore * Reductorase gene, poly A indicates polyadenylate displacement signal, Amp indicates ampicillin resistance gene, and MCS indicates multiple cloning site.

 FIG. 5 shows the results of comparison of competition characteristics using mouse monoclonal antibody, rat monoclonal antibody, and rabbit monoclonal antibody produced in the present invention. The vertical axis shows the BZB 0 value when measuring E 2 at the concentration of l O O O p gZmL, and the horizontal axis shows the dissociation constant.

 Figure 6 shows the results of evaluating the performance of each antibody by fluorescence depolarization. The vertical axis indicates the degree of polarization, and the horizontal axis indicates the antibody concentration. The degree of polarization of the fluorescent substance label E 2 used as the antigen was set to 20. The black circles in the figure indicate the results when the mouse monoclonal antibody Ma-1 is used, the white circles indicate the rat monoclonal antibody Ra-1 and the black squares indicate the rabbit monoclonal antibody U-1 prepared according to the present invention. It is.

 FIG. 7 shows the result of preparing a calibration curve by preparing a reagent for a fully automatic immunodiagnostic system manufactured by Tosoh Corporation. The white circles in the figure are immune reaction reagents using polyclonal antibodies (E test “TOS OH” IIE 2, manufactured by Tosoh Corp.), and the black circles are immunizations using Usagi monoclonal antibody U-1 produced in the present invention. The results are obtained when each reaction reagent is used.

 Figure 8 shows an immunoreaction reagent using a polyclonal antibody (E-test “TOS OH” IIE 2) and an immunoreaction reagent using Usagi Monoclonal Antibody U-1 produced in the present invention. The result of measuring the sample and correlating it is shown.

FIG. 9 shows the results of screening the culture supernatant of 3840 clones. The vertical axis shows competitive characteristics, the horizontal axis shows specificity, and the size of each plot shows reactivity. In addition, antibodies indicated by black circles in the figure Is a clone with high competitive performance, high specificity, and high reactivity, and an antibody gene was isolated from the clone.

 Fig. 10 shows the results of preparing a calibration curve by preparing a reagent for a fully automatic immunodiagnostic system manufactured by Tosoh Corporation. The black circles in the figure are the results of using the immune reaction reagent using the rabbit monoclonal antibody UT 1 D-5 produced in the present invention, and the white squares are the results of using the immune reaction reagent using the mouse monoclonal antibody. It is.

 Figure 11 shows the results of evaluating cross-reactivity. The horizontal axis shows the concentration of T3 or T4, and the vertical axis shows the BZB 0 value.

 Figure 12 shows the results of screening of the culture supernatant of 3840 clones. The vertical axis represents fluorescence intensity, and the horizontal axis represents clones.

 Figure 13 shows the results of a sandwich assay of BNP with anti-BNP antibody (C-terminal recognition) obtained by transient expression and an antibody that recognizes the cyclic site of BNP.

 Figure 14 shows the results of ELISA evaluation of the change in the amount of antibody in the culture supernatant after cell fusion. BEST MODE FOR CARRYING OUT THE INVENTION

 Hereinafter, the present invention will be described in detail.

 (1) Types of antigen

The antigen to immunize the rabbit is not particularly limited and can be arbitrarily selected from haptens, peptides, proteins, nucleic acids, cells, lipids, sugar chains, bacteria, fluorescent substances, and biamines. The present invention is preferably applied to an antigen that requires high antibody performance in terms of affinity and specificity. Specifically, antigens for competitive methods that require high affinity in order to construct a highly sensitive measurement system based on the measurement principle, such as estrone (E 1), estradiol (E 2), Triol (E 3), steroids such as steroid hormones such as progesterone and cortisol, and thyroid hormones such as thyronine, thyroxine, and lysine thyronine. In addition to antigens for competitive methods, application to peptides that require high sensitivity, such as C peptide and brain natriuretic peptide (BNP), is also a preferable example.

 (2) Antigen immunization method

 Rabbits that immunize antigens have different immune responses depending on the type of antigen, so it is preferable to immunize as many strains and as many animals as possible. Usagi lines include, but are not limited to, Japanese white varieties that are generally available and New Zealand land moth. In addition, since there are individual differences even when using the same strain of Usagi, it is preferable to obtain antibody-producing cells derived from Usagi having a high immune response by immunizing several to about 10 individuals.

 There are no particular restrictions on the method of immunization to the rabbits, and immunization may be carried out by a commonly used method. For example, a method of immunizing by making an adjuvant and emulsion and a method of immunizing DNA encoding a protein or peptide can be mentioned.

 The place of immunization is not particularly limited. It is sufficient to immunize the rabbit at the usual place, such as subcutaneous, abdominal cavity, or footpad.

 Since the number of immunizations varies depending on the type of antigen and responsiveness to the rabbit, the antibody titer of the serum is analyzed by measuring by ELISA or BIA core (trade name) (Biacore), and It is preferable to determine by observing the progress. Usually four immunizations are enough, but in some cases, multiple immunizations (from several months to one year) may be required.

(3) Isolation of antibody-producing cells The antibody-producing cell to be isolated is not particularly limited as long as it is an antibody-producing cell, but it is preferable to use any one of spleen-derived cells, lymph node-derived cells, and terminal blood-derived cells. . In addition, isolation of antibody-producing cells may be performed by a conventional method. Although isolated cells are preferred for immediate use, they can be stored in liquid nitrogen or deep freezer, dispersed in commonly available cell storage solutions, as needed Can be used.

 (4) Selection of antibody-producing cells

 It is necessary to select cells carrying the target antibody gene from the antibody-producing cells isolated in (3) above. As a screening method, as described in Non-Patent Document 9, there is a method of selecting one cell from antibody-producing cells and isolating and amplifying an antibody gene from the selected cell. Since only the antigen-binding ability can be evaluated, it is difficult to obtain an antibody having the target ability. Therefore, in the present invention, the antibody-producing cells isolated in (3) are proliferated, and the performance such as competitive characteristics, specificity, and reactivity of the antibody expressed in the culture supernatant is evaluated, and the performance is high. It is characterized by isolating the antibody gene after selecting cells expressing the antibody.

 In the production method of the present invention, the following method can be exemplified as a method for growing antibody-producing cells.

a) A method in which antibody-producing cells are cultured in a growth medium until the antibody performance in the culture supernatant can be evaluated in a culture medium in a microwell plate. A method for evaluating antibody performance in culture supernatants by fusing with cells that impart oxidative potential. c) A method of selecting cells for antibody gene isolation by immortalizing and culturing antibody-producing cells with a virus such as Epstein-Barr virus (EBV).

Of these, the method of growing using the method a) or b) is preferred.

 As a medium used in the growth method of a), a medium for animal cells or a medium supplemented with well-known cytokines such as IL-4 and LPS can be used.

 The cell that provides the immortalizing ability used in the proliferation method of b) is a cell that can obtain an antibody-secreted antibody after fusion, and after the growth, the antibody performance is evaluated in the fused cell. The cell is not particularly limited as long as the antibody gene remains in the cell, but a cell from which a fused cell capable of maintaining the antibody-producing ability for a long time can be obtained is particularly preferable. P 3 — X 6 3— A g 8 (AT CCTIB 9), P 3-NS 1-1 -A g 4-1 (JCRB 0 0 0 9), P 3-X 6 3-A g 8-U 1 (JCRB 9 0 8 5), P 3— X 6 3 — A g 8, 6 5 3 (JCRB 0 0 2 8), SP 2 ZO-A g— 1 4 (JCRB

Mouse myeloma related such as 0 0 2 9), NSO / (RCB 0 2 1 3), S 1 9 4 X 5 XX〇, BUI (AT CCCRL-8 8 3 7), FO (AT CCCRL-1 6 4 6) , Human myeloma cell lines such as SK 0-0 0 7 and HO — 3 2 3, human B cell lines such as GM 1 5 0 0 6 GT-A 1 2, Y 3 — A gl · 2 · Examples include rat myeloma cell lines such as 3, rabbit-transformed cell lines of TP-3, and rabbit cancer cells such as VX-2. In addition, a cell line starting from the mouse myeloma cell line (for example, 2-3A described in JP-A No. 10 1 2 6 2 6 5 7 (Patent Document 1)) is also preferred immortalizing ability in the present invention. It is an example of the cell which gives. The cell fusion method used in the propagation method of b) may be carried out by a commonly used method. Examples thereof include a polyethylene glycol method, an electrofusion method, and a method using Sendai virus.

 c) Proliferation method is well known to those skilled in the art, infecting a human lymphocyte with a virus such as EBV (C o 1 eeta 1., “T he EBV-Hybridoma T echniqueand I ts A pplicationto Human Lung C ancer "M onoclonal A ntibodiesand C ancer T herapy, Reisfeldeta 1., N ew Y ork: A 1 an R. L iss I nc. It is a method in which a rabbit such as EBV is infected with a virus such as EBV and immortalized temporarily by a technique similar to that of Patent Document 10). The virus that infects rabbit B cells is not particularly limited to E B V as long as it can achieve the above-mentioned purpose.

 (5) Evaluation method of antibody performance

 The antibody performance evaluation method for selecting cells having the antibody gene may be appropriately selected according to the evaluation contents of the target antibody. For example, the contents of evaluation include antigen binding ability, competitive characteristics, specificity, and antibody productivity, and examples of evaluation methods include the ELISA method, the RIA method, and the fluorescence polarization method.

 (6) Isolation period of antibody gene

The optimal time for isolating the heavy chain (H chain) and light chain (L chain) antibody genes from the cells selected in (4) above differs depending on the antigen and the rabbit strain. There is no particular limitation as long as a sufficient amount of antibody for evaluation can be secured and the antibody gene is not lost. However, as in Example 1 2 Isolation of antibody genes is not possible because isolation of antibody genes 16 days after the start of production cell growth reduces the efficiency of antibody gene amplification and the availability of expression vectors with antigen-binding activity. It is preferably performed within 14 days after the start of production cell growth.

 (7) Antibody gene amplification method

 As a method for amplifying the antibody gene, a commonly used RT-PCR method can be used. In addition, commercially available enzymes can be used for reverse transcriptase and DNA polymerase used in RT-PCR reactions. In particular, DNA polymerase avoids introducing mutations during DNA amplification. Therefore, Phismion High Fidelity DNA ρ ο l ym erase (trade name) (Daiichi Chemicals Co., Ltd.) and P rimes TAR HS DNA pol ym erase (trade name) (Yukara Bio Inc.) It is preferable to use the enzyme with the highest fidelity.

The primer for amplifying the antibody gene is not particularly limited as long as it is designed at a position where the region encoding the variable region of the antibody is amplified. Primer combinations used to amplify antibody genes include primer combinations designed in the untranslated region upstream from the start codon and downstream from the codon for the purpose of amplifying the entire length of the antibody gene, antibody Examples include a combination of primers designed inside the antibody gene for the purpose of amplifying the variable region of the gene, or a system in which the above primers are appropriately combined. If the amplification efficiency of the antibody gene is low in a single PCR reaction, the antibody gene can be efficiently amplified by performing the second PCR reaction using the first PCR reaction product as a saddle. In this case, it is often possible to amplify the gene more efficiently by creating a new primer slightly inside the first amplification region. Is not something

 The Hagi chain is 80% or more of VH 1 (J. Immu nol., 1 5 2, 3 9 3 5; 1 9 94: Non-patent document 1 1), and L chain Are reported to be 80% or more of κ chain (J. Mo 1. B iol., 3 2 5, 3 2 5; 2 0 0 3: non-patent document 1 2) Amplification is preferably carried out using a primer set specific for the gene of interest. Furthermore, as an embodiment of a primer for amplifying an antibody gene, Non-Patent Document 10; Biotechno, 1 3, 6 1 7 1; 1 995 (Non-Patent Document 13); Nuc 1. A cids R es., 1 5, 6 1 7 1; 1 9 8 7 (Non-Patent Document 1 4); N uc 1. A cids R es., 1 0, 1 5 3 5; 1 9 8 2 (Non-patent The primer sequence described in Reference 15) and the primer sequence (SEQ ID NO: 1 to 1 1) designed with reference to Gen Bank are disclosed. You can use.

 (8) Expression vector

 The expression vector is not limited as long as it is a generally reported vector, but in particular, p ECE dhfr (J. Bioche m., 1 0 8, 6 7 3; 1 9 90: Non-patent document 1 6, Figure 4) is preferably used as an expression vector. In addition, the region of the antibody to be expressed may be expressed so as to include the variable region of the antibody, and the expression forms include Ig G, F (ab ') 2, Fab, FV, and sc FV. It can be illustrated. In addition, it is possible for those skilled in the art to easily change the performance of an antibody by adding various amino acid mutations using the isolated antibody gene as a starting material.

 (9) Host cells

Expression vectors can be introduced into appropriate host cells to Recombinant antibodies can be produced. SV40 large T antigen protein-expressing cells are preferably used as host cells for transiently high expression of recombinant antibodies, and examples include Examples 7, 1 3, and 14 C o S 1 cells used in Biotechno 1. Lett., 1 7, 1 3 5; 1 9 95: Non-patent literature 1 7; J. Ferm. B ioeng., 7 9, 40 5; 1 9 9 5: Non-patent literature 1 8), 2 9 3 T cells and COS 7 cells can be used. In order to stably express recombinant antibodies, Although expression using plants is possible, animal cells and microorganisms typified by yeast and Escherichia coli are preferably used as phosphine cells, and animal cells, particularly CH 0 cells are preferably used. A more preferred stable production strain is a phosphine vector system in which a vector incorporating a dhfr gene augmentation system capable of amplifying an antibody gene is introduced into CH cells.

The method for producing a rabbit monoclonal antibody according to the present invention allows an antibody-producing cell isolated from a rabbit immunized with an antigen to proliferate without using a generally non-circular rabbit-myeloma cell, and then possesses an antibody gene. This method is characterized in that the antibody gene is isolated and amplified from the selected cell, and it is possible to produce a rabbit antibody with the knowledge of normal laboratory facilities and molecular biology. Is. In addition, since the production method of the present invention is difficult in the prior art, a monoclonal antibody having a combination of heavy chain and light chain originally produced by Usagi can be easily produced. Usagi Monoclonal Antibodies with high affinity that can only be handled in Japan can be easily handled at the normal laboratory level. Example

 EXAMPLES Hereinafter, although this invention is demonstrated further in detail using an Example, this invention is not limited to these.

 Example 1 Preparation of immune antigen and screening antigen

 E 2 —B S A (trade name) (manufactured by Sigma) was used as an immunizing antigen. In addition, an antigen for screening with ELIISA was prepared by the following method.

 (1) BCP (Blue Carrier Protein: manufactured by Pierce) 10 O mg of PBS was added to 1 O mg ZmL, and the 6th position was activated with N-hydroxysuccinimide. Tradiol (E 2) 1. 2 5 mg was mixed with DM F 1 mL.

 (2) After stirring at room temperature for 2 hours, E 2 — B CP was obtained by dialysis against P B S (1 L X 3 times).

 Example 2 Animal and antigen immunization method

 As the immunized animal, a 12-week-old female of Usagi (Japanese white species) was used. Immediately after the first immunization, Freund's complete adjuvant is used. After the second immunization, Freund's incomplete adjuvant is used. After mixing the antigen solution (500 gZmL) with the same amount of adjuvant, immunization is performed at intervals of 2 weeks. did. The amount of immunizing anti-antigen is 50 g at the first time and 25 g after the second immunization.

 Next, a sufficient increase in the antibody titer was confirmed by the following ELISA (Enzyme lin medI mmunnosor rbentAssay).

 (1) E 2—B C P (0. b g / m L) was immobilized on an E L I S A plate and blocked with 1% skim milk.

(2) Reacted with blood collected from a rabbit. Anti-E bound to E 2 2 Usagi monoclonal antibody is bound with alkaline phosphatase-labeled anti-usagi IgG antibody, unreacted enzyme-labeled antibody is separated by BZF, and enzyme substrate 4-methylumbelliferyl phosphate (4 One MU P) was dispensed and detected by measuring fluorescence intensity.

 Example 3 Proliferation of antibody-producing cells using spleen cells (culture in medium)

)

 The spleen of a rabbit with a sufficiently increased antibody titer was removed, and spleen cells were isolated according to a conventional method. Spleen cells were suspended in GIT medium (manufactured by Nissui Pharmaceutical Co., Ltd.) containing 10% F C S, and cultured on a microplate. One week later, the culture supernatant was evaluated by the method described in Example 2 for the antibody in the culture supernatant of 192 clones. The results are shown in FIG. Thus, the production of antibodies could be confirmed in the supernatant of the microwell plate. Example 4 Proliferation of antibody-producing cells using lymphocytes derived from peripheral blood cells (culture using cell fusion)

 Peripheral blood cell-derived lymphocytes were fused with immortalizing cells by the method described below.

 (1) Fusion of lymphocytes collected from the peripheral blood of a rabbit with a sufficiently high antibody titer and a cell (2-3A, Patent Document 1) in which the mouse myeloma cell gene is partially integrated did. This cell (2-3A) was established by Eiken Chemical Co., Ltd. and was transferred with the courtesy of Eiken Chemical Co., Ltd.

 (2) According to a standard method (PEG method), 2-3 A cells and lymphocytes derived from peripheral blood cells were fused.

 (3) The cell suspension after the fusion was suspended in GIT medium (manufactured by Nissui Pharmaceutical Co., Ltd.) containing 10% FCS and 1 XHAT and placed on a microtiter plate.

The antibodies in the culture supernatant of 3 8 4 clones were evaluated by the method described in Example 2. The calculated results are shown in FIG. In this way, antibody production could be confirmed in the supernatant of the microwell plate.

 Example 5 Proliferation of antibody-producing cells using spleen cells (culture using cell fusion)

 Spleen cells were fused with immortalizing cells by the method described below.

 (1) The spleen of a rabbit with a sufficiently increased antibody titer was removed, and spleen cells were prepared according to a conventional method. The cells used in Example 4 (2-3A, Patent Document 1) were used as the cells that fused with the rabbit spleen cells.

(2) According to the standard method (PEG method), 2-3 A cells were fused with rabbit spleen cells.

 (3) The cell suspension after the fusion was suspended in a GIT medium (manufactured by Nissui Pharmaceutical) containing 10% FCS and 1 XHAT and spread on a microtie plate.

 (4) The performance of the anti-E2 antibody in the culture supernatant was evaluated by the following three ELISA.

 (4-1) Reactivity: E 2 — B C P (0.5 g ZmL) was immobilized on an ELIISA plate and then blocked with 1% skim milk. Thereafter, the culture supernatant after cell fusion was reacted. Anti-E 2 Usagi monoclonal antibody bound to E 2 binds alkaline phosphatase-labeled anti-rabbit Ig G antibody, B / F separation of unreacted enzyme-labeled antibody, and 4-methylumbelliferous enzyme substrate. Ferryl phosphate (41-MU P) was dispensed and detected by measuring fluorescence intensity.

(4 1-2) Competitive characteristics: An experiment was conducted in which E 2 (500 pg / mL) coexists in the experimental system for evaluating reactivity. By dividing the value (B) obtained here by the value (B 0) obtained by reactivity, the competitive property (B ZB (0 value) was calculated.

 (4-3) Specificity: In the experimental system to evaluate the reactivity, various steroids (S 卜 ron: El, Estriol: E3, ethynyl estradiol: E t E2, testosterone: TS, Each experiment was conducted in the presence of 500 pg / m L). The specificity (BZB 0 value) was calculated by dividing the value (B) obtained here by the value obtained by reactivity (B) 0).

 The three data obtained above were graphed (Fig. 3). Specificity (BZB 0 value) is plotted on the horizontal axis, competitive characteristics (BZB 0 value) on the vertical axis, and reactivity is plotted in circle size. In this graph, clones filled with black circles are clones with high competitive characteristics, high specificity, and high reactivity.

 Example 6 Isolation / Amplification of antibody gene and preparation of expression vector As a result of Example 5, after selecting clones filled with black circles in Fig. 3, antibody genes were isolated from the clones by the following method, amplified and expressed A vector was fabricated.

 (1) MagNAxTM-96 1 Tota 1 RN A 1 So 1 tiob iori) was used to isolate RNA from cells according to the attached protocol.

 (2) The isolated R N A is used as a saddle, and R e a d y-T o-Go Y u u-P r i m e F i r s t-S t r a n d B e a d s (trade name

) (Manufactured by GE HEALTH CARE) was used for reverse transcription to synthesize cDNA.

(3) Primer specific to the rabbit antibody gene (primer specific to heavy chain (H chain) and light chain (L chain), SEQ ID NOs: 1 to 4, SEQ ID NOs: 1 and 2 are H chains Sequences 3 and 4 are primers specific to the L chain, respectively) P husion H igh— Fidelity D The antibody gene was amplified by PCR reaction using NA polymerase (trade name) (Daiichi Chemical Co., Ltd.). The conditions are shown below.

 (Reagent composition)

 The buffer solution supplied with the kit was used with d N T P 0.2 M and the primer 0.5.

 (Reaction conditions)

 After holding for 30 seconds at 98, the cycle of 10 seconds at 98, 10 seconds at 72, and 50 seconds at 72 was repeated 35 cycles, and then at 72, the cycle was held for 10 minutes.

 (4) Gene amplification was confirmed by electrophoresis.

 (5) The obtained H-chain and L-chain PCR fragments were treated with restriction enzymes BglII and XbaI, and then the animal cell expression vector (pECE dhfr: Fig. 4) BglIiZXbal Introduced into the site, expression vectors for H and L chains were prepared.

 Example 7 Expression of recombinant antibody (transient expression by COS cells) and purification

 Using the expression vector for H chain and L chain obtained in Example 6, recombinant antibodies were produced in COS 1 cells (Non-patent Documents 17 and 18). The antibody gene isolation procedure in Example 6 was performed 8 days after cell fusion.

 (1) To COS cells in the logarithmic growth phase on 96 well plates, add 20 ng each of the H chain expression vector and L chain expression vector, ribofectamine 2 200 (trade name) (Invitrogen) Introduced). As a medium for gene introduction, Opti-M E M (trade name) (manufactured by Invitrogen) was used.

(2) Three days after gene transfer, the culture supernatant was collected and the presence of the antibody was confirmed by ELISA. (3) COS cells having antibody gene pairs with confirmed antigen binding ability were cultured at an increased culture scale, and the antibody was transiently expressed.

 (4) The anti-E2 antibody was purified from the culture supernatant using a Protein η G. column (manufactured by GE HEALTHEARE) according to a conventional method. The performance of the antibody was evaluated, and the antibody with the highest performance in terms of competitive characteristics, specificity, and productivity was selected.

 Example 8 Production of stable production strain of Usagi monoclonal antibody

 An antibody expression vector selected in Example 7 was introduced into CHO cells to produce a stable antibody-producing strain.

 In a logarithmic growth phase, C HO cells (DXB 11: dhfr-deficient strain) were transfected with a gene under the same conditions as in Example 7, and alpha 1 ME M (—) was added with 10% dialyzed serum. Cells were cultured. Thereafter, the concentration of MTX (mesotrexate) was gradually increased from 5 nM to 500 nM to obtain rabbit monoclonal antibody U-1-expressing CHO cells.

 Example 9 Affinity measurement of expressed antibody

 The affinity of the following three types of monoclonal antibodies was measured using BIAcore T-1100 (trade name) (Biacore).

 (a) Mouse monoclonal antibody isolated by Tosoh Corporation Ma 1

 (b) Rat monoclonal antibody Ra-1 isolated by Tosohichi Corporation

 (c) The affinity of the rabbit monoclonal antibody U-1 produced in the present invention was measured by the following method.

(1) An E2 derivative (in which an amino group is introduced at the 6-position) bonded to the sensor chip (CM5) by the amine force printing method. used.

 (2) Analysis was performed under the following conditions: buffer solution: H B S—EP, temperature: 25, flow rate 30 L Zmin, binding time: 1 minute, dissociation time: 10 minutes

(3) Using the analysis software (Biacore T 100 Evaluation Evaluation software, version 1.1), the obtained data was analyzed with the bivalent model, and the value of K d (M) was calculated. .

Dissociation constant (K d) on the horizontal axis and vertical axis on the competitive property (value obtained when E 2 of l OOO pg Zm L coexists with the value when E 2 does not exist. As a result of measurement using AIA-6200 II, it can be seen that antibodies with higher affinity have higher competitive properties and a highly sensitive measurement system can be constructed (Fig. 5). Mouse monoclonal antibody M a- 1 (K d = 8. 1 X 1 0 - 1 0 M), rat Tomonokuro one monoclonal antibody R a _ l (K d = 5. 2 X 1 0- 1 1) respectively generalization Is classified as a high-affinity antibody, but the rabbit monoclonal antibody U-1 (K d = 3.3 X 1 0-1 ³ M) produced in the present invention is approximately 2 5 0 than M a 1 1 The friendliness was improved by 0 times, approximately 160 times that of Ra-1.

 Example 10 Evaluation of Isolated Antibody by Fluorescence Depolarization Method

The binding of the isolated antibody was evaluated by fluorescence depolarization (Figure 6). Using E 2 labeled with tetramethylrhodamine, the antibody concentration is plotted on the horizontal axis and the degree of fluorescence polarization (mP) is plotted on the vertical axis. When the mP value of tetramethylrhodamine labeled E 2 is 20, the change in the degree of polarization is shown when the antibody concentration is increased. As the antibody concentration increases, E 2 labeled with tetramethylrhodamine binds to the antibody, resulting in a higher degree of polarization. Usagimo made in the present invention The monoclonal antibody U-1 has an increased degree of polarization at a concentration that is at least 100 times lower than that of the mouse monoclonal antibody Ma-1 and the rat monoclonal antibody Ra-1. This indicates that U-1 has a much higher affinity than M a-1 and R a _ 1.

 Example 1 1 Evaluation of Monoclonal Antibody in AIA Reagent Form The immune reaction reagent using the rabbit antibody monoclonal antibody U-1 produced in the present invention was changed to the immunoreaction reagent using the polyclonal antibody (E test “T〇”). S〇H ”IIE 2, manufactured by Tosohichi Co., Ltd.) Fig. 7 shows the calibration curves for both immune reaction reagents. The detection limit for E 2 is 25.2 pg ZmL for immunoreactive reagents using polyclonal antibodies, whereas 2 2.4 pg ZmL for immunoreactive reagents using U-1 is It has been clarified that a measurement system with higher sensitivity than an immunoreaction reagent using a null antibody can be constructed.

 In addition, when cross-reactivity (l O n gZmL) with estrone (E 1) and estriol (E 3) to similar steroids was measured, E 1 (commercially available AIA reagent) : 4.7 7%, AIA Reagent with Usagi Monoclonal Antibody U_1: 0.92%) and E 3 (Commercial AIA Reagent: 4. 19%, Usagi Monoclonal Antibody 1; — 1 8) Reagents: 0.28%), both of which significantly improved the cross-reactivity to sterolides.

 Next, FIG. 8 shows the results of examining the correlation between both measurement systems using 40 actual samples. The regression coefficient was 0.92 4, y-intercept—2.3 8 7 pg / mL, and the correlation coefficient r = 0.99 8, indicating a very high correlation. Example 1 2 Gene isolation time and percentage of isolated genes

Although there is information that the heterohyperidoma is likely to lose the gene, detailed analysis such as how long it disappears has not been reported. The present inventors also used the culture supernatant after cell fusion as EL. After evaluating by ISA and confirming that the antibody was expressed in the culture supernatant, even when attempting to amplify the antibody gene from the cell, the antibody gene could not be amplified. As a result of investigating why the gene cannot be amplified from various viewpoints such as PCR conditions and primer sequences, it is the same as handling normal mouse monoclonal antibodies when subculturing heterohybridomas obtained after cell fusion. When it is handled with a sense, it was found that it is difficult to specify when the antibody is not expressed. Normally, when cells are subcultured, the cell suspension is transferred to a new medium of about 10 times volume and the culture is continued, so the first culture supernatant is diluted by transferring to a new medium. Go. In other words, when cells do not stably express antibodies, the culture supernatant is diluted, and the signal of the culture supernatant after subculture is drastically reduced. However, this theory did not hold for the rabbit monoclonal antibody. Usagi monoclonal antibody has extremely high affinity compared to mouse monoclonal antibody, and sufficient signal can be obtained by ELISA even if the culture supernatant is diluted about 10 times or 100 times by subculture. Therefore, the antibody gene has already dropped from the heterohybridoma, and the ELISA is positive even though the antibody is not expressed. This is because, in Example 10, the binding properties of the rabbit monoclonal antibody isolated in this patent and the mouse monoclonal antibody were compared. As a result, the rabbit monoclonal antibody was more than 100 times lower than the mouse monoclonal antibody. This is supported by the fact that antigen-binding ability was observed even at concentrations.

Therefore, in Example 6, the time for isolating the antibody gene after cell fusion was changed, and the optimum time for isolation was examined. The results are shown in Table 1. table 1

When the gene was amplified on the 8th day after cell fusion, the H chain gene was amplified with a probability of 86.1% and the L chain gene with a probability of 94.4% on average of three experiments. Then, when an expression vector was prepared from the amplified gene and the antibody expressed in COS cells was evaluated, 75.0% of the antibodies had antigen-binding ability. In addition, a stable amplification rate and antibody acquisition rate were demonstrated through three experiments, indicating that a vector with antibody binding activity can be stably acquired by amplification during this period.

 On the other hand, when the genes were isolated on the 16th day after cell fusion, the respective values decreased to 25.0%, 88.9%, and 9.52%. From this, it can be seen that when the isolation period is late, rapid loss of the antibody gene occurs particularly in the H chain gene.

 Example 1 3 Isolation of Anti-Triyoidothyronine (T 3) Antibody Anti-triyodothyronine (T 3) Usagi monoclonal antibody was isolated by the following method.

 (1) Preparation of immune antigen

 After adding boric acid buffer (0.05 M, pH 8.5) so that 5 mg of BSA (manufactured by Sigma) is 1 O mg / mL, and protecting the carboxylic acid as a methyl ester The linker was extended from the amino group. Next, the solution was mixed with a solution obtained by dissolving T 37 mg activated with N-hydroxysuccinimid in DMSO 100 / L. After stirring at room temperature for 30 minutes, dialysis against PBS (50 mL x 3 times) gave T3-BSA as an immunizing antigen.

 (2) Preparation of screening antigen

BCP (Blue Carrier Protein: manufactured by Pierce) was used in the same manner as (1). Boric acid buffer (0.05 M, pH 8.5) was added so that 5 mg of BCP was 1 O mg ZmL. In the solution, N-hydroxysucci A solution obtained by dissolving 7 mg of T 3 activated with medium in DMSOIOOL was mixed. After stirring at room temperature for 30 minutes, dialysis against PBS (50 mL × 3 times) yielded ELISA screening antigen T 3 —BCP.

 (3) Animals and antigen immunization methods

 As the immunized animal, a 12-week-old female of Usagi (Japanese white species) was used. Freund's complete adjuvant is used for the first immunization, and Freund's incomplete adjuvant is used for the second and subsequent immunizations. After mixing the antigen solution (500 / g mL) with the same amount of adjuvant, the emulsion is prepared and separated for 2 weeks. Immunized with. The amount of immunizing anti-antigen is 1 mg for the first time and 500 g after the second immunization.

 Next, a sufficient increase in antibody titer was confirmed with the following ELISA.

(3-1) T 3 — B C P (0.5 / I g / mh) was immobilized on E L I S A plate and then blocked with 1% skim milk.

 (3-2) Reacted with sera collected from rabbits. Anti-T3 Usagi monoclonal antibody bound to T3 binds alkaline phosphatase-labeled anti-rabbit IgG antibody, unreacted enzyme-labeled antibody is separated by BZF, and then enzyme substrate 4-methyl It was detected by dispensing umbelliferyl phosphate (4 — MUP) and measuring the fluorescence intensity.

 (4) Growth of antibody-producing cells (culture in medium)

 The spleen of a rabbit with a sufficiently high antibody titer is removed, and the cell cryopreservation solution

(Cell banker 1 (trade name), manufactured by Nippon Zenyaku Kogyo Co., Ltd.) In a state of being dispersed in, it was stored in the deep freezer (180). The preserved cells were thawed according to a standard method, suspended in GIT medium (manufactured by Nissui Pharmaceutical Co., Ltd.), and spread on a micromouth plate. The performance of anti-T3 antibody in the culture supernatant after 5 days of culture was evaluated by the following three ELISA.

(4-1) Reactivity: A nti — R abbit I g G (0.5 ^ g ZmL) was immobilized on an ELISA plate and blocked with 1% skim milk. Thereafter, the culture supernatant and alkaline phosphatase-labeled T3 were reacted at the same time. Unreacted enzyme labeled T3 was isolated from the Usagi monoclonal antibody by BZF separation, and the enzyme substrate 41-methylmethylphosphorylphosphate (41-MUP) was dispensed and detected by measuring the fluorescence intensity.

 (4 1-2) Competitive characteristics: An experiment was conducted in which T 3 (2 n M) coexists in the experimental system for evaluating reactivity. The competitive property (B / B 0 value) was calculated by dividing the value (B) obtained here by the value (B 0) obtained from the reactivity.

 (4-3) Competitive characteristics: An experiment was conducted in which T 4 (20 nM) coexists in the experimental system for evaluating reactivity. The specificity (BZB 0 value) was calculated by dividing the value (B) obtained here by the value (B 0) obtained from the reactivity.

 The three data obtained above were graphed (Fig. 9). Specificity (BZB 0 value) is plotted on the horizontal axis, competitive characteristics (BZB 0 value) on the vertical axis, and reactivity is plotted in circle size. In this graph, clones filled with black circles are clones with high competitive performance, high specificity, and high reactivity.

 (5) Isolation of antibody genesAmplification and production of expression vectors

 As a result of (4), the clones filled with black circles in FIG. 9 were selected, and the antibody gene was isolated by the following method on the next day after selection.

 (5-1) RNA was extracted from cells according to the attached protocol using RNeasy Plu s Microkit (trade name) (QIAGEN).

(5-2) Using the RNA obtained in (5-1) as a saddle and using Sensiscript RTK it (trade name) (QIAGEN) The reverse transcription reaction was performed to synthesize cDNA.

 (5-3) Usagi 'Phsion High— Fidelity DNA pol ym erase (Primers specific to antibody genes (primers specific to H and L chains; SEQ ID NOs: 1 to 3, 5 to 7)) (Product name) (Daiichi Chemical Co., Ltd.) and the antibody gene was amplified. The conditions are shown below. The second P C R when amplifying the H chain is carried out under the same reagent composition and reaction conditions as the first P C R except that the first P C R product (2 L) is a saddle type.

 (Reagent composition)

 The buffer supplied with the kit, d NT P 0.2 M> Primer at 0.5 M each.

 (Reaction conditions)

 After holding for 30 seconds at 98, the cycle of 10 seconds at 98, 10 seconds at 72, and 50 seconds at 72 was repeated 35 cycles, and then at 72, the cycle was held for 10 minutes.

 (Primer)

 H chain: Sequences 2 and 5 for the first PCR, sequences 1 and 6 for the second PCR

 L chain: SEQ ID NOs: 3 and 7

 (5-4) Amplification of the gene was confirmed by electrophoresis.

 (5-5) The obtained H-chain and L-chain PCR fragments were treated with restriction enzymes BglII and XbaI, and then the Bgl of the expression vector for animal cells (p ECE dhfr: Fig. 4) was used. Introduced into the II / Xbal site, expression vectors for H chain and L chain were prepared.

 (6) Expression of recombinant antibodies (transient expression by COS cells

)

(5) From the H chain and L chain expression vectors obtained in (5), COS 1 cells ( Non-patent literature 1 7, 1 8)

(6-1) 96 COS cells in logarithmic growth on a well plate, 16.6 ng of the H chain expression vector, 8.3 ng of the L chain expression vector, and lipofuctamine 20 0 0 (trade name) (Invitrogen). As a medium at the time of gene introduction, Opti-M E M (trade name) (Invitrogen) was used.

 (6-2) Three days after gene transfer, the culture supernatant was collected and the presence of the antibody was confirmed by ELISA.

 (6-3) COS cells having antibody gene pairs with confirmed antigen-binding ability were cultured at an increased culture scale, and the antibody was transiently expressed.

 (7) Evaluation of monoclonal antibodies in AIA reagent form

 Using the rabbit-derived anti-T3 monoclonal antibody (UT 1 D-5) prepared in (6), a test cup for AI was prepared. A I A is a fully automated immunodiagnostic system sold by Tosoh Corporation. Using the antibody prepared this time, an immune reaction reagent for A I A was prepared and evaluated.

 Fig. 10 shows the calibration curves of the immune reaction reagent using the isolated rabbit antibody UT 1 D-5 and the immune reaction reagent using the mouse-derived anti-T 3 monoclonal antibody. As shown in Fig. 10, it became clear that the use of the isolated antibody makes it possible to construct a measurement system that is more sensitive than mouse-derived reagents.

Figure 11 shows the results of cross-reactivity evaluation using an immunoreaction reagent using UT 1 D-5 prepared this time. Figure 11 shows that the antibody isolated this time has no cross-reactivity to the thyroxine (T 4), a similar compound of T 3, and has a very high specificity for T 3. Being I understand.

 Example 14 4 Isolation of anti-brain natriuretic peptide (BNP) antibody Anti-brain natriuretic peptide (BNP) Usagi monoclonal antibody was isolated by the following method.

 (1) Preparation of immune antigen

 A peptide corresponding to 7 amino acids on the C-terminal side of BNP (C KV L R R H, SEQ ID NO: 12) was synthesized. Using the cysteine at the N-terminus of the synthesized peptide, it was reacted with maleimide K L H (manufactured by PIERC) and used as an immunizing antigen. The reaction method was according to the attached protocol, and 1 mg of peptide was reacted with 1 mg of maleimide K L H.

 (2) Preparation of screening antigen

 A peptide with a linker sequence (GGGSGGGS, SEQ ID NO: 13) added to the N terminus of the peptide corresponding to the C-terminal 7 amino acids of BNP (C KV LRRH, SEQ ID NO: 12), and then the N-terminus is piotinized. (Biotin-GGGS GG GSC KV LRRH, SEQ ID NO: 14; hereinafter referred to as Bio-BNC) was prepared and used as a screening antigen in ELISA.

 (3) Animals and antigen immunization methods

 As the immunized animal, a 12-week-old female of Usagi (Japanese white species) was used. The first immunization is Freund's complete adjuvant, the second and subsequent immunizations are Freund's incomplete adjuvant, mixed with an antigen solution (500 ^ gZmL) and the same amount of adjuvant, and then immunized at intervals of 2 weeks after creating the emulsion. did. The amount of immunizing anti-antigen is 50 g at the first time and 25 z after the second immunization.

Next, the increase in antibody titer was confirmed by ELISA shown below. (3-1) Streptavidin (0. S zg ZmL) was immobilized on an ELISA plate and blocked with 1% skim milk.

 (3-2) BIO—BNC (0.5 g / ml) was reacted with an ELISA plate. After that, the rabbit serum immunized with the antigen was reacted. Bio—Anti-BNP Usagi monoclonal antibody conjugated to BNC binds alkaline phosphatase-labeled anti-rabbit Ig G antibody, and unreacted enzyme-labeled antibody is separated by BZF, followed by the enzyme substrate 4-methylumbelliferyl phosphorus. It was detected by dispensing acid (4-MU P) and measuring fluorescence intensity.

 (4) Growth of antibody-producing cells (culture in medium)

 The spleen of a rabbit with a sufficiently high antibody titer is removed, and the cell cryopreservation solution

(Cell banker 1 (trade name), manufactured by Nippon Zenyaku Kogyo Co., Ltd.) In a state of being dispersed in, it was stored in the deep freezer (180). The preserved cells were thawed according to a standard method, suspended in GIT medium (manufactured by Nissui Pharmaceutical), and spread on a microtiter plate. The performance of anti-BNP antibody in the culture supernatant after 5 days of culture was evaluated by the following ELISA.

 (4-1) Streptavidin (0. S g ZmL) was immobilized on an ELISA plate, blocked with 1% skim milk, and then reacted with Bio- BNC (0. bg / mL). A rate was made.

 (4-2) Reacted with the culture supernatant. Anti-BNP Usagi monoclonal antibody bound to Bio_BNC binds alkaline phosphatase-labeled anti-rabbit Ig G antibody, and unreacted enzyme-labeled antibody is separated by B / F, followed by enzyme substrate 4-methylun It was detected by dispensing berylferyl phosphate (4-MU P) and measuring the fluorescence intensity.

The obtained results are shown in Fig. 12. The signal is stronger than the result of Fig. 1 2. Clones were selected and antibody genes were isolated.

 (5) Isolation of antibody gene and production of expression vector

 The antibody gene was isolated from the clone selected in (4) by the following method.

 (5-1) RNA was extracted from cells according to the attached protocol using RNeasyPlu sMiccrokit (trade name) (manufactured by QIAGEN).

 (5-2) Using the obtained RNA as a saddle type, reverse transcription reaction was carried out using Sensiscript RTKit (trade name) (manufactured by QIAG EN) to synthesize cDNA.

 (5 — 3) Use primers specific to the rabbit antibody gene (primers specific to the H and L chains; see SEQ ID NOs: 2, 3, 6 to 1 1). Ph sion High— Fidelity DNA The antibody gene was amplified with po 1 ymerase (Daiichi Chemical Co., Ltd.). The conditions are shown below. When performing the second PCR (50 L reaction volume), if the H chain is amplified, the reagents are the same as for the first PCR except that the first PCR product (2 L) is in a saddle shape. When the L chain is amplified under the composition reaction conditions, the same as the first PCR, except that the first PCR product is diluted 100-fold with TE (2 L) in a bowl shape. Reagent composition Performed under Z reaction conditions.

 (Reagent composition)

 The buffer solution supplied with the kit was used with d N T P 0.2 M and the primer 0.5 L each.

 (Reaction conditions)

After holding for 30 seconds at 98, the cycle of 10 seconds at 98 and 10 seconds at 7 8 and 35 seconds at 7 2: was repeated 35 cycles, and then at 72, the cycle was held for 10 minutes. (Primer)

 H chain: Sequences 2 and 6 for the first PCR, sequences 8 and 9 for the second PCR

 L chain: Sequences 3 and 7 for the first P C R, sequences 10 and 1 1 for the second P C R

 (5-4) Gene amplification was confirmed by electrophoresis.

 (5-5) The obtained H and L chain PCR fragments were digested with B g 1 I i ZX bal and treated with B gl II ZX of the animal cell expression vector (p ECE dhfr: Fig. 4). Infection C 1 oningkit (trade name) ^ C 1 ontech) was introduced into the bal site to prepare expression vectors for H and L chains.

(6) Expression of recombinant antibodies (transient expression by Cº S cells

)

 Recombinant antibodies were produced in C O S 1 cells (Non-patent Documents 17 and 18) from the expression vectors for H and L chains obtained in (5)

(6-1) 9 6 COS cells in logarithmic growth on a well plate, 2.0 ng of H chain expression vector, 20 ng of L chain expression vector, and lipofuctamine 200 Name) (manufactured by Invitrogen). As a medium for gene introduction, Opti-MEM (trade name) (manufactured by Invitrogen) was used.

 (6-2) Three days after gene transfer, the culture supernatant was collected and the presence of the antibody was confirmed by ELISA. Two clones with confirmed antigen-binding ability

(2 7, 4 5).

 (7) Evaluation of monoclonal antibodies in the sandwich assembly system

Usagi-derived anti-BNP mono obtained from the clone obtained in (6) The clonal antibody (27, 45) was evaluated by ELISA according to the following method.

 (7-1) The culture supernatant after the antibody was transiently expressed from the clone obtained in (6) was reacted with an ELISA plate immobilized with anti-rabbit antibody (lO / z gZmL). BNP (0.1 g / mL) was reacted.

(7-2) Furthermore, an antibody (A 28 _Qnm = 0.003) as a protein concentration that recognizes the cyclic part of BNP labeled with alkaline phosphatase was reacted.

 (7-3) After unreacted enzyme-labeled antibody was separated by BZF, the enzyme substrate 4-methylumbelliferyl phosphate (4-MUP) was dispensed and detected by measuring the fluorescence intensity. .

 The obtained results are shown in FIG. From the results in Fig. 13, there was a clear difference in reactivity in the presence or absence of BNP, confirming the isolation of a rabbit monoclonal antibody that recognizes the C-terminus of BNP. Example 1 5 Change in signal value of ELISA after cell fusion

 The experiment shown below was conducted using the frozen spleen cells of rabbits immunized with E 2 — B SA.

 (1) According to the method shown in Example 5, cell fusion was performed, and the fused cells were spread on a microtiter plate.

 (2) On the first day of culture 1, 2, 3, 4, 7, and 11, the same amount of culture supernatant was added. When taking the culture supernatant, the same amount of medium with the same composition is added to maintain the amount of medium.

(3) The results of evaluation of the obtained culture supernatant by ELISA shown in Example 2 are shown in FIG. The signal intensity increased from day 1 to day 7 after cell fusion, but almost no signal intensity was observed after day 7. There was no change. From the above results, it can be seen that it is preferable to isolate the antibody gene from the culture supernatant cultured 7 days after cell fusion. Industrial applicability

 According to the production method of the present invention, a rabbit antibody having high affinity characteristics can be produced with knowledge of ordinary laboratory facilities and molecular biology. Furthermore, by using the rabbit monoclonal antibody produced by the method of the present invention as an immunodiagnostic drug, a highly sensitive measurement system that is not affected by foreign substances can be constructed.

Claims

The scope of the claims

1. Isolate antibody-producing cells from rabbits immunized with antigen, select cells carrying the target antibody gene, isolate and amplify heavy and light chain antibody genes from the selected cells, In a method for producing a recombinant antibody that expresses a recombinant rabbit monoclonal antibody by introducing it into a phosphine cell after introduction into an expression vector, an antibody-producing cell is selected in selecting a cell carrying the target antibody gene. A method for producing a genetically engineered antibody, comprising:

2. The operation of isolating heavy and light chain antibody genes from the sorted cells is performed within a period in which the antibody genes do not fall out of the sorted cells. A method for producing a recombinant antibody.

 3. The genetic recombination according to claim 1, wherein the operation of isolating heavy and light chain antibody genes from the selected cells is carried out within 14 days after the start of antibody-producing cell growth. Antibody production method.

 4. The method for producing a recombinant antibody according to any one of claims 1 to 3, wherein the antibody-producing cells are any of spleen cells, lymph node cells, and peripheral blood cells of a rabbit immunized with an antigen.

 5. The method for producing a gene recombinant antibody according to any one of claims 1 to 4, wherein the method for growing antibody-producing cells is a method for culturing antibody-producing cells in a medium.

 6. Production of a recombinant antibody according to claims 1 to 4, characterized in that the method of growing antibody-producing cells is a method of fusing and culturing cells capable of immortalizing. Method.

7. The antigen that immunizes the rabbit is a hapten or peptide. The method for producing a recombinant antibody according to claim 1, wherein:

 8. The method for producing a recombinant antibody according to any one of claims 1 to 7, wherein the host cell is an animal cell.