JP4686682B2 - Ligand affinity complex protein acquisition method, antibody screening method, antibody screening kit, medical method, antibody drug, non-immunoreactive antibody drug, dosing method - Google Patents

Description

  The present invention relates to a method for obtaining a ligand affinity complex protein, an antibody screening method, an antibody screening kit, a medical method, an antibody drug, a non-immunoreactive antibody drug, and a dosing method.

  More specifically, the present invention quickly and in large quantities obtains one or more complex proteins selected from a very wide variety of complex proteins Xp exhibiting selective affinity for a specified ligand. The present invention relates to a method for obtaining a ligand affinity complex protein. This method for obtaining a ligand affinity complex protein can be applied to humans, and can also be applied to non-human animals in which at least antibody production by an immune reaction is observed.

  The present invention also provides an antibody screening method capable of rapidly performing high-throughput screening based on the above-described method for obtaining a ligand affinity complex protein, and a method for performing this antibody screening method. The present invention relates to an antibody screening kit.

  The present invention also relates to a medical method for a human or the above non-human animal using the antibody obtained by the above antibody screening method, and an antibody drug containing the antibody as an active ingredient.

  Furthermore, the present invention is a pharmaceutical comprising, as an active ingredient, an antibody obtained by the above-described method for obtaining a ligand-affinity complex protein targeted for human application, and is intended to be administered to the human individual targeted for application. The present invention relates to a non-immunoreactive antibody drug and a dosage method using the non-immunoreactive antibody drug.

  In the present invention, the “ligand” is a concept that includes any inorganic compound or organic compound, and various inorganic compounds or organic compounds that can be antigens, proteins, viruses, microorganisms, and the like without limitation. When the ligand is an antigen, the ligand affinity complex protein of the present invention can be an antibody or a monoclonal antibody (hereinafter also simply referred to as “Mab”).

  A protein showing selective affinity for an arbitrary ligand is important as a means for detecting, separating or purifying the ligand in a general technical field.

  On the other hand, in the field of medicine or physiology, typical examples of such affinity proteins include a very wide variety of antibodies produced in humans or at least non-human animals in which antibody production by an immune reaction is observed. it can. Antibodies are important biomolecules in immunology and can be widely applied to the prevention, diagnosis or treatment of human or non-human animal diseases. It can be widely applied to pathological research using non-human animals. Among antibodies, in particular, Mabs that can be mass-produced by so-called hybridoma technology are extremely important.

  Generally, when obtaining a ligand affinity protein, for example, a non-human animal immunized with any antigen, that is, a ligand, various infectious patients in humans, or cancer cells are used as a ligand to produce antibodies. As is typically seen in cancer patients and the like, there are often very many types of similar proteins that differ in affinity or binding power to ligands.

  For example, as in the case of using a polyclonal antibody, it is possible in some cases to use such a group of similar proteins as a mixture as it is. However, as a means for detecting, separating or purifying ligands, and as a means for preventing, diagnosing or treating diseases, excellent ligand affinity proteins for each type specified from a group of similar proteins. It is overwhelmingly advantageous to acquire only a large amount selectively.

  The hybridoma technology is important in that it opens up the possibility of selectively obtaining only one type of ligand affinity protein as a Mab. However, in this technique, although it is good to prepare a large number of B cells, which are production sources of various Mabs that are antigen-affinity proteins, using animal immune reactions, for example, the production of stable hybridomas (protein There are problems such as difficulty in preparation of production means, and much time and effort in culturing and growing hybridomas (enlarging protein production means on a large scale). Therefore, as a practical technique, it is not possible to sufficiently meet the requirements of rapid mass production and high-speed multi-sample screening.

In recent years, in view of the above-mentioned problems of hybridoma technology, in order to eliminate generation of B cells and tissue culture, for example, chimeric monoclonal antibodies (Vaquelo et al., 1999), transgenic mice (Green et al., 1994), Phage display (MacCafferty
et al., 1990) and various genetic engineering techniques such as ribosome display (Hanes & Plucthun, 1997) are being developed. However, these technologies are only elemental technologies, and cannot meet the demands of mass production of various ligand-affinity proteins and high-speed multi-analyte screening. The original and exact pairing of the H chain and L chain of the antibody has not been obtained.

Coronella, JA, Telleman, P., Truong, TD, Ylera, F., Junghans, RP, 2000. "Amplification of IgG VH and VL (Fab) fromsingle cell human plasma cells and B cells" Nucleic Acids Res. 28, e85 In addition, progress has been made in techniques for rapidly and accurately isolating B cells, kit-based laboratory reagents, sequencing and automation of analysis, single-cell reverse transcription PCR techniques, etc., for example, as described in Non-Patent Document 1 above. As described above, amplification of antibody genes derived from various cells has been reported. That is, due to the advantages of single-cell reverse transcription PCR, if the antibody L chain gene and H chain gene obtained from a single cell are amplified and an expression system using recombinant E. coli is used, Pairing with the L chain can be realized.

  However, it is necessary to clone an antibody gene, which is a reverse transcription PCR product, into a vector for expression, and the cloning and expression in a microorganism require a very troublesome work and a long time. Further, there are problems such as difficulty in handling cytotoxic antibodies, and difficulty in handling extremely large numbers of clones (very large numbers of recombinant E. coli).

  The above points point out problems related to the demand as a general technical means of rapid mass production and rapid multi-analyte screening for the identified ligand affinity proteins. From the medical or physiological standpoint, this problem is pointed out. This leads directly to the problem of providing excellent antibody screening methods, medical methods, antibody drugs and the like. Furthermore, in the case of human subjects, it is also directly related to the problem of providing non-immunoreactive antibody drugs that enable parenteral administration without rejection in diseases related to immune reactions. .

  The inventor of the present application uses human antibody-producing cells from patients with infectious diseases, etc. as a technique for quickly and mass-acquiring one or more types of proteins identified from a wide variety of ligand affinity complex proteins. Was collected by non-invasive means, or antibody-producing cells of non-human animals were prepared using an immune response, combined with single-cell reverse transcription PCR technology as an alternative to hybridoma technology, and further cloned. The idea was to combine a cell-free protein synthesis system that has been attracting attention in recent years as a technology for protein production from genes.

However, when such a production technology system is envisaged, in particular, one or two or more proteins having excellent properties from a very wide variety of ligand-affinity complex proteins are to be obtained quickly and in large quantities. In this case, the purpose must be fulfilled within the constraints of diversity that can be said to be virtually infinite due to the following a), b) and c).
B) The target protein obtained as an antibody has an extremely wide variety that can be said to be virtually infinite.
B) Each protein molecule is a complex protein composed of a plurality of fragments, and each fragment is encoded by an individual structural gene.
C) In this complex protein, there are each class such as IgG, IgA, and IgE, and each isotype such as κ chain and λ chain in the L chain.

  As far as the present inventor knows, a method for obtaining a ligand affinity complex protein that has tackled the problem of selectively and quickly obtaining a specific complex protein within the constraints of such diversity has not yet been proposed. Never before. The inventor of the present application worked to solve such an unprecedented problem and completed the method for obtaining a ligand affinity complex protein according to the present invention. Furthermore, based on this method for obtaining a ligand affinity complex protein, the inventions of antibody screening method, antibody screening kit, medical method, antibody drug, non-immunoreactive antibody drug, and administration method have also been completed.

(Configuration of the first invention)
The configuration of the first invention of the present application for solving the above-described problem is that random or through characterization is performed from among a very wide variety of complex proteins Xp exhibiting selective affinity for an arbitrarily specified ligand X. A method for obtaining one or two or more selected complex proteins, at least the following 1) production means separation step, 2) production means conversion step, 3) mass production preparation step, 4) transcription sequence addition step And 5) a method for obtaining a ligand-affinity complex protein, which includes a single-species mass production process and, if necessary, further includes the following 6) characterization process.

  1) An antibody-producing cell provided with production means Xm dedicated to production means (mRNA) for each of a wide variety of complex proteins Xp, which can be easily converted into replication means Xc (cDNA) capable of mass replication. By individually separating one or a plurality of single cells from an appropriate tissue or biological component of a specimen that is a human or non-human animal, the production means Xm is strictly separated for each type of target protein, A production means separation step for preventing the production means Xm, the replication means Xc, and the complex protein Xp in each step from being mixed in advance.

  2) A production means conversion step of converting all or part of the mRNA constituting each production means Xm to the replication means Xc by reverse transcription PCR for one or more types of production means Xm separated from each other.

  3) A mass production preparation step in which the replication means Xc obtained by the reverse transcription PCR method is mass-replicated by the PCR method to prepare a mass production capability of protein.

  4) A transcription sequence addition step for adding a transcription sequence necessary for reconversion to the production means Xm (mRNA) in a cell-free system to the replication means Xc (cDNA) replicated in large quantities.

  5) A cell-free system comprising the replication means Xc, to which the transcription sequence is added, having at least an element for reconverting the replication means Xc into the production means Xm and an element for producing a protein based on the production means Xm. A single-type mass production process in which the complex protein Xp or a complex protein Xp corresponding to a part of the mRNA defined in the production means conversion process is mass-produced for each type.

  6) A characterization process in which characteristics of the composite protein Xp produced by each of the above steps are evaluated, a composite protein Xp having excellent characteristics is screened, or sequencing of these composite proteins Xp is performed.

(Configuration of the second invention)
The configuration of the second invention of the present application for solving the above problems is presumed to be in a physiological state in which an antibody against the ligand X as an antigen has already been produced in the body when the specimen according to the first invention is a human. From the tissue or living body part, an appropriate tissue or living body part of the subject is collected by non-invasive means that are accepted by society, and 1) of 1) above is selected. This is a method for obtaining a ligand-affinity complex protein by separating single or plural single cells.

(Configuration of the third invention)
The configuration of the third invention of the present application for solving the above problem is that peripheral blood is collected as the tissue or biological component from the subject according to the second invention, and B cells that are antibody-producing cells from the peripheral blood and This is a method for obtaining a ligand-affinity complex protein, in which single cells of plasma cells are separated.

(Configuration of the fourth invention)
The configuration of the fourth invention of the present application for solving the above-described problem is that, when the specimen according to the first invention is a non-human animal, an appropriate tissue of the animal or the It is a method for obtaining a ligand affinity complex protein, wherein one or a plurality of single cells of 1) are separated from a biological component.

(Structure of the fifth invention)
The structure of the fifth invention of the present application for solving the above-described problem is that the complex protein Xp according to any of the first to fourth inventions is an immunoglobulin (antibody) comprising a pair of H chain and L chain, This is a method for obtaining a ligand-affinity complex protein, which is Fab (antigen-binding fragment), or a protein fragment equivalent thereto.

(Structure of the sixth invention)
The structure of the sixth invention of the present application for solving the above-described problem is that, in the production means separating step according to any one of the first to fifth inventions, the tissue or biological component of the specimen is introduced into a liquid and the antibody After dispersing and suspending the production cells and incubating with the magnetic microparticles bound to a certain separating antibody, the magnetic microparticles bound to the antibody producing cells via the separating antibody are collected with a magnet, whereby the suspension This is a method for obtaining a ligand affinity complex protein, in which a single cell of a target antibody-producing cell is separated from a solution.

(Structure of the seventh invention)
The structure of the seventh invention of the present application for solving the above problem belongs to a specific classification category according to the design of primers used for reverse transcription PCR in the production means conversion step according to any one of the first to sixth inventions. A method for obtaining a ligand affinity complex protein, wherein only mRNA encoding the complex protein Xp is converted into the replication means Xc and / or only mRNA encoding a specific fragment constituting the complex protein Xp is converted into the replication means Xc It is.

(Configuration of the eighth invention)
The configuration of the eighth invention of the present application for solving the above-described problem is that, in the mass production preparation process according to any one of the first to seventh inventions, mass replication by the PCR method of the replication means Xc is performed by the following two-stage PCR. This is a method for obtaining a ligand affinity complex protein.

  First stage PCR: Amplification is performed by providing the same additional sequence on the 5 'end side of each kind of primer corresponding to all kinds of cDNA constituting the replication means Xc.

  Second stage PCR: All types of DNA fragments amplified in the first stage PCR are amplified using a single kind of primer having a sequence portion complementary to the additional sequence.

(Structure of the ninth invention)
The structure of the ninth invention of the present application for solving the above-mentioned problem takes into account the cell-free derived organism of the above 5) in the additional sequence of each kind of primer used in the first step PCR according to the eighth invention. This is a method for obtaining a ligand-affinity complex protein, which contains an appropriate type of translation initiation promoting sequence.

(Configuration of the tenth invention)
The structure of the tenth invention of the present application for solving the above-described problem is that at least a DNA fragment amplified in the mass production preparation step in the transcription sequence addition step according to any of the first to ninth inventions This is a method for obtaining a ligand-affinity complex protein, in which PCR is performed to add a promoter sequence.

(Structure of 11th invention)
In order to solve the above-mentioned problems, the eleventh aspect of the present invention is a ligand which is used in the single-type mass production process according to any one of the first to tenth aspects of the present invention under the condition that the cell-free system is not reduced. This is a method for obtaining an affinity complex protein.

(Configuration of the twelfth invention)
The structure of the twelfth invention of the present application for solving the above-described problem is that the characterization process according to any one of the first invention to the eleventh invention includes the ligand affinity evaluation by the ELISA method of the complex protein Xp and the complex protein Xp or A method for obtaining a ligand-affinity complex protein comprising at least one of sequencing of the gene and measurement of the Kd value of the complex protein Xp produced using the transformant.

(Structure of the thirteenth invention)
The configuration of the thirteenth invention of the present application for solving the above-described problems is the same as the method for obtaining a ligand-affinity complex protein according to any one of the first to twelfth inventions, in which an antibody against ligand X is screened in a human or non-human animal. It is an antibody screening method used for.

(Structure of the 14th invention)
The structure of the fourteenth invention of the present application for solving the above problems is a kit used for carrying out the antibody screening method according to the thirteenth invention, and comprises at least the following components for antibody screening. It is a kit.

  11) A set of a dispersion / suspension liquid according to the sixth invention and magnetic fine particles.

  12) A primer set for reverse transcription PCR according to the seventh invention.

  13) A primer set for two-stage PCR according to the eighth invention.

  14) A cell-free system according to 5) of the first invention.

(Structure of the fifteenth invention)
The structure of the fifteenth invention of the present application for solving the above-described problem is that a non-human antibody is obtained using the antibody obtained by the method for obtaining a ligand affinity complex protein according to any one of the first invention and the fourth to twelfth inventions. This is a medical method for preventing, diagnosing or treating animal diseases.

(Structure of the sixteenth invention)
In order to solve the above problems, the sixteenth invention of the present application uses an antibody obtained by the method for obtaining a ligand affinity complex protein according to any one of the first to third inventions and the fifth to twelfth inventions. Thus, it is a medical method for preventing, diagnosing or treating human diseases.

(Structure of the seventeenth invention)
The structure of the seventeenth invention of the present application for solving the above-mentioned problems is that the antibody obtained by the method for obtaining a ligand affinity complex protein according to any one of the first invention to the twelfth invention is an active ingredient, and depending on its origin It is an antibody drug that is applied to human or non-human animals.

(Structure of the 18th invention)
The structure of the 18th invention of the present application for solving the above-mentioned problem is that the antibody obtained by the method for obtaining a ligand affinity complex protein according to any of the 1st invention to the 3rd invention and the 5th invention to the 12th invention is effective. It is a non-immunoreactive antibody drug that is applied as an ingredient to a human individual from which it is derived.

(Structure of the nineteenth invention)
The composition of the nineteenth invention of the present application for solving the above problems is a method of administering the nonimmunoreactive antibody drug according to the eighteenth invention into the body of the human individual from which it is derived by a parenteral administration method. It is.

(Effect of the first invention)
In the first invention, in the production means separation step, one or a plurality of single cells of antibody-producing cells each having a production means Xm dedicated to the complex protein Xp are prepared using a human or non-human animal specimen. Therefore, it is possible to easily and quickly collectively prepare a production method for an extremely large variety of complex proteins Xp.

  Since each mRNA as the production means Xm prepared in a lump as described above is generated and separated individually in each cell unit, the production means Xm can be strictly separated and narrowed down for each type of the complex protein Xp, and the post-process Production means Xm, replication means Xc, and complex protein Xp can be completely prevented from intermingling. Moreover, since these production means Xm function as dedicated production means for each complex protein Xp, they are extremely suitable as a protein production means library. Furthermore, there is no difficulty in preparation of stable production means (hybridoma production) as in the hybridoma technology described above.

  However, in the above-described protein production means library, since each mRNA is extremely small, the production scale is very small and cannot meet the demand for mass production. Therefore, in the next production means conversion step, the product is once converted into the replication means Xc (cDNA) that does not directly function as a production means but can easily be mass-replicated. In the production means conversion step using the reverse transcription PCR method, all of the mRNA constituting the production means Xm may be converted, but by selectively converting part of the mRNA, the above-mentioned production means separation is performed. From the mRNA (mRNA set) for each type of protein narrowed down in the process, the type of mRNA can be further narrowed down.

  The replication means Xc (cDNA) obtained for the types of mRNA thus narrowed down is mass-replicated in a mass production preparation step. Since this step uses a PCR method, as in the hybridoma technique and the technique described in Non-Patent Document 1, it is troublesome to adjust the mass production scale (cultivate and propagate hybridomas and transformants). There is no difficulty of taking a long time. In addition, when the types of mRNA are narrowed down in the above-mentioned production means conversion step, since the types of primers used can be significantly reduced, PCR products with very few by-products and high specificity can be obtained. .

  In the transcription sequence addition step, a transcription sequence necessary for reconversion to the production means Xm (mRNA) in a cell-free system is added to the replication means Xc (cDNA) thus mass-replicated.

  Next, in the single-type mass production process, the replication means Xc (cDNA) to which the transcription sequence is added as described above is introduced into a cell-free system, so that the protein of each type of the complex protein Xp corresponding to the cDNA is Mass production is possible.

  As described above, the production means Xm is strictly separated for each type of the complex protein Xp in the production means separation step, and the type of mRNA is narrowed down in the production means conversion step as necessary. Therefore, a transcription sequence can be added in a state where it can function effectively, and a highly specific PCR product can be obtained in the mass production preparation step.

  Therefore, in the single-species mass production process, few by-products of the composite protein that are similar to the target single-species complex protein in terms of molecular weight, three-dimensional structure, ligand affinity, etc. are not produced. A single kind of complex protein is effectively mass-produced. As a result, high-purity separation and recovery of the target protein Xp from the cell-free system after completion of the process can be performed easily and quickly.

  Therefore, even when performing a characterization process, it is possible to perform characteristic evaluation, screening, and sequencing quickly and accurately.

  As described above, according to the first invention of the present application, one kind or two or more kinds identified from extremely many kinds of ligand affinity proteins similar to each other within the restriction of diversity that can be said to be substantially infinite. Can be obtained in high purity by rapid and large-scale cloning. In the ligand affinity complex protein thus obtained, it is confirmed that the H chain and the L chain are correctly paired.

  Regarding the point that the method for obtaining the ligand affinity complex protein of the first invention can be performed very rapidly, for example, the following evaluation is possible. That is, when acquiring one or more complex proteins that are functionally particularly excellent from such a large variety of ligand-affinity complex proteins (for example, when obtaining a Mab), usually three months or It is said that it takes longer than that. In contrast, in the first invention, antibody gene characterization from cloning, including reverse transcription PCR and other steps of about 5 hours and single species mass production steps (ie, in vitro transcription / translation) of about 1 hour. Can be completed in substantially one day.

(Effect of the second invention)
When performing the production means separation step of the first invention using a human as a specimen, the patient (subject) who produces an antibody against the ligand X from a certain pathological condition is requested to cooperate, and the antibody production is performed by a non-invasive means as much as possible. The said single cell which is a cell can be isolate | separated. In this case, the obtained ligand-affinity protein is a monoclonal antibody, and for the subject, it is a non-immunoreactive antibody derived from itself. Do not wake up. Therefore, antibody administration treatment can be performed as the optimum customized medicine for the patient.

  Such antibody administration treatment was also possible based on conventional techniques. However, there are many cases in which it is required to provide an extremely quick vaccine due to problems such as the patient's condition, and in conventional techniques, quickness has always been a bottleneck. According to the second invention, since such a problem of speediness can be solved, the medical value is extremely large.

(Effect of the third invention)
When human specimens are used, antibody-producing cells are collected by means that are as noninvasive as possible and do not place a burden on the subject. Therefore, as a preferred example, a small amount of peripheral blood of the subject is collected, and B The method of isolate | separating the single cell of a cell and / or a plasma cell can be mentioned.

(Effect of the fourth invention)
When performing the production means separation step of the first invention using a non-human animal as a specimen, a method of separating single cells of antibody-producing cells from an appropriate tissue or biological component under the immunization of the animal with ligand X It is general and convenient.

(Effect of the fifth invention)
As a suitable example of the ligand affinity complex protein in the first invention described above, an immunoglobulin (antibody) consisting of a pair of H and L chains, Fab (antigen-binding fragment) thereof, or an effective ligand equivalent thereto Mention may be made of protein fragments exhibiting affinity.

(Effect of the sixth invention)
In the production means separation step, as in the sixth invention, magnetic fine particles bound to a certain separation antibody are used, and antibody-producing cells that specifically bind to the separation antibody are separated from the cell suspension. Thus, there is an advantage that antibody-producing cells can be selectively and efficiently recovered.

(Effect of the seventh invention)
As described in the first invention, in the production means conversion step, a part of mRNA constituting the production means Xm can be converted to the replication means Xc.

  That is, the production means Xm in the production means conversion step is actually one set of mRNA encoding each fragment of the complex protein Xp. For example, there may be a protein that lacks a part of the original constituent fragment of the ligand affinity complex protein but maintains the ligand affinity, such as Fab (antigen-binding fragment) in an antibody. Moreover, for example, in the case of antibodies, there are differences for each class and each isotype in each fragment of the complex protein Xp.

  Therefore, as one advantageous embodiment in the case where a part of mRNA is converted to the replication means Xc, mRNA encoding a complex protein Xp belonging to a specific class or isotype classification category by designing a primer used in reverse transcription PCR. An embodiment is described in which only the duplication means Xc is converted. Alternatively, an embodiment in which only mRNA encoding a specific fragment constituting the complex protein Xp is converted to the replication means Xc, such as Fab, is exemplified.

  According to the seventh invention, the types of primers used for reverse transcription PCR can be remarkably reduced, and therefore the types of cDNA produced can be remarkably reduced. Can be carried out under highly advantageous conditions.

(Effect of the eighth invention)
The eighth invention is also one of the great improvements in technical significance. That is, as a general rule, in PCR, by-products are basically unavoidable due to primer dimers and non-specific annealing, it is substantially difficult to amplify cDNA as a replication means Xc by one-step PCR. It is.

  For example, once produced as a by-product DNA, there is a problem that it is easier to amplify than the target cDNA when the chain length is shorter than the target cDNA. Moreover, when starting from a very small amount of template DNA, such a negative effect is significant.

  As a usual countermeasure against this difficulty, there is a known two-stage PCR method called nested PCR using two PCR primer sets in a nested structure. However, in this method, since it is necessary to use two PCR primer sets for each type of cDNA to be amplified, it is not easy to apply to a variety of antibody genes.

  For example, when amplifying a variable region of an L chain in an antibody gene of a human B cell, a primer with 10 degenerates must be used in one step, and the same number of primers must be used in the second step. In other words, not only is the number of primers large, but the generation of by-products derived from many combinations is unavoidable, so that a large number of amplification bands are detected in addition to the target DNA, which makes the subsequent protein production process difficult. Bring. That is, for example, the synthesis amount of the target protein in a cell-free system is remarkably reduced, or cloning into Escherichia coli or the like for protein sequencing becomes extremely time consuming and time consuming.

  According to the eighth invention, although the first stage PCR uses each type of primer corresponding to each cDNA constituting the replication means Xc, the second stage PCR uses a common additional sequence provided for these primers. Then, it is possible to amplify only each cDNA constituting all the replication means Xc by a single kind of primer. Therefore, even when the replication means Xc is diverse such as an antibody gene, it can be easily applied.

  Even when a single kind of primer is used as in the second stage PCR, a certain amount of primer dimer is generated. However, the primer dimer in this case is different from the dimer due to the association of different primers, and when it is dissociated by heat and cooled, it takes a structure in which the 5 ′ end and 3 ′ end of the primer are associated (pan handle structure) No longer meet. Therefore, the amplification capability is substantially lost.

  Therefore, according to the eighth invention, it is possible to effectively prevent a decrease in the synthesis amount of the target protein in a cell-free system, or the cloning into E. coli or the like for protein sequencing is greatly simplified.

(Effect of the ninth invention)
In the first stage PCR of the eighth invention described above, by utilizing the point that an additional sequence is provided for each primer, an appropriate sequence is considered in the additional sequence (considering a cell-free derived organism used in the single-species mass production process of the first invention). A translation initiation facilitating sequence portion can be included. In that case, the translation efficiency of the target protein can be improved in the subsequent single-cell mass production process in a cell-free system.

(Effect of the tenth invention)
In the transcription sequence addition step, at least a promoter sequence can be added by PCR to the DNA fragment amplified in the mass production preparation step. This makes it possible to prepare for reconversion from DNA to mRNA in a cell-free system.

  Usually, even if such various sequences are added by PCR to a diverse template gene such as an antibody gene, it is difficult to sufficiently exert the effects of the added sequences. However, since the template genes used for such PCR are sufficiently narrowed down by the first invention, the seventh invention, the eighth invention, etc., the effect of these additional sequences is sufficiently exhibited.

(Effect of the 11th invention)
Conventionally, there is a view that a cell-free system (in vitro transcription / translation coupling system) needs to be used in a reduced state. Therefore, an expensive reducing agent such as DTT (dithiothreitol) should be added. There were many. However, the inventor of the present application
It has already been confirmed that the target protein can be produced in an in vitro transcription / translation coupling system. Therefore, cost reduction can be realized by using the cell-free system under the condition without reduction.

(Effect of the twelfth invention)
The contents of the characterization process in the first invention described above are not necessarily limited. For example, ligand affinity evaluation of the complex protein Xp by ELISA, sequencing of the complex protein Xp or its gene, measurement of the Kd value of the complex protein Xp, etc. can be preferably performed. When measuring the Kd value, it is preferable to produce a composite protein Xp using a transformant into which the gene of the composite protein Xp has been introduced, and measure this composite protein Xp.

  In the single-type mass production process, since the complex protein Xp is obtained in a large amount with a very small amount of by-products, these evaluations, sequencing, and measurement of the Kd value can be performed quickly and accurately.

(Effect of the thirteenth invention)
The method for obtaining a ligand affinity complex protein can be used for various experimental purposes, research purposes, medical purposes, etc. As one of the important specific applications, screening for antibodies against ligand X in human or non-human animals It is illustrated to be used for.

(Effect of the 14th invention)
In carrying out the antibody screening method according to the thirteenth invention, the antibody screening kit according to the fourteenth invention can be preferably used. Each component of this antibody screening kit is not particularly novel, except for some primer components. However, the entire kit including the components 11) to 14) as essential components in the fourteenth invention is not possible unless the method for obtaining the ligand affinity complex protein is conceived. It has never been proposed or offered to the market.

(Effects of the fifteenth and sixteenth inventions)
A medical method similar to the medical method of the fifteenth invention or the sixteenth invention in that Mab is used in human medical care or non-human animal medical care (for example, pet animals and livestock) is based on conventional techniques such as hybridoma technology. Even it can be established. However, when based on the prior art, it is difficult to screen and produce an optimal Mab quickly and in large quantities from among a great variety of Mabs.

  According to the medical method according to the fifteenth and sixteenth inventions, an optimal Mab can be rapidly screened and produced in large quantities against antigens in infectious diseases, cancer cells in various cancers, and the like. Therefore, it has practicality in the medical field. This point has extremely important significance especially in lifesaving medical care that competes for a moment in the human medical field.

(Effect of the seventeenth invention)
An antibody drug functionally equivalent to the antibody drug according to the seventeenth invention can also be supplied based on the prior art. However, it is difficult to quickly supply an antibody drug supplied based on the conventional technique, and it is also difficult to supply it at low cost from the viewpoint of production cost. The antibody drug according to the seventeenth invention can be supplied quickly, in large quantities and at low cost.

(Effects of the 18th and 19th inventions)
In addition to the effects of the seventeenth invention, the nonimmunoreactive antibody drug according to the eighteenth invention does not induce immune rejection as a side effect when administered parenterally to a human individual from which it is derived. Therefore, the parenteral administration method as in the nineteenth invention can be safely performed. Furthermore, it is possible to dispense with the use of an immunosuppressive agent during the administration. Therefore, it is possible to reduce the therapeutic risk and the burden or pain of the patient due to the patient's reduced immune ability, and further reduce the treatment cost by saving immunosuppressive agents.

  Next, the first to nineteenth embodiments of the present invention will be described including the best mode. In the following, the term “present invention” refers to all or a part of each of the above inventions.

[Method of obtaining ligand-affinity complex protein]
The method for obtaining a ligand affinity complex protein according to the present invention is selected at random or through characterization from among a very wide variety of complex proteins Xp exhibiting selective affinity for an arbitrarily specified ligand X. This is a method for obtaining one or more complex proteins. Here, “two or more types” may be several types, several tens types, and several hundred types or more.

  The type of the complex protein Xp is not limited, but may be, for example, an immunoglobulin (antibody) composed of a pair of H chain and L chain, a Fab that is a fragment of a specific portion thereof, or an immunoglobulin or Fab Any protein fragment showing a ligand affinity according to On the other hand, the type of ligand X is not limited, and includes various inorganic compounds and organic compounds that can be antigens, proteins, viruses, microorganisms and the like without limitation.

  The method for obtaining a ligand affinity complex protein includes at least the following 1) production means separation step, 2) production means conversion step, 3) mass production preparation step, 4) transcription sequence addition step, and 5) single species mass production step. Including. Further, if necessary, a 6) characterization step described later can be included. Furthermore, it can include any pre-step for the above 1) step, can include any post-step for the above 6) step, or any intermediate step between any steps in the steps 1) -6) Can also be interposed.

[Production means separation process]
The production means separation step is a production means (mRNA) dedicated to each of a very wide variety of the complex protein Xp, and includes production means Xm that can be easily converted into replication means Xc (cDNA) capable of mass replication. Separate the production means Xm for each type of target protein by individually separating one or more single cells of the antibody-producing cells from appropriate tissues or biological components of specimens that are human or non-human animals. Thus, the production means Xm, the replication means Xc, and the complex protein Xp in each of the following steps is a step for preventing in advance mixing between different species. By this step, the production means Xm is strictly separated for each type of the complex protein Xp.

  Here, the production means Xm is specifically mRNA, and more specifically, one set of each mRNA encoding each protein fragment constituting the complex protein Xp.

  In this production means separation step, when the specimen is a human, it is not allowed to artificially generate an antibody against the ligand X in the body. Therefore, a patient who is in a certain disease state, that is, a subject who is presumed to be in a physiological state in which an antibody against ligand X as an antigen has already been produced in the body is selected and produced under the consent of the subject. A means separation step needs to be performed. Specifically, an appropriate tissue or biological component of the subject is collected by non-invasive means that are accepted by society, and the one or more single cells are separated from the tissue or biological component. To do.

  Although the kind of patient as described above is not necessarily limited, for example, an infectious disease patient infected with ligand X, various cancer patients producing antibodies against cancer cells as a ligand, and the like can be exemplified. Tissue or biological structure containing antibody-producing cells only from non-invasive means that are accepted from the patient's perspective as long as it is permitted to be collected from medical ethics and related laws Part can be collected. “Tissue or biological component” includes blood and other body fluids.

  As a specific example of collection of such a tissue or biological component and separation of antibody-producing cells therefrom, peripheral blood is collected from a subject, and B cells and / or antibody-producing cells are collected from this peripheral blood. Or the case where the single cell of a plasma cell is isolate | separated is mentioned.

  In addition, as another specific example, in a patient who has undergone surgery or separation of a tissue or biological component containing antibody-producing cells for a separate therapeutic purpose, with the consent of the person concerned, The case where a tissue or a biological component (for example, spleen, body fluid, bone marrow, etc.) is used is mentioned.

  When the sample is a non-human animal in this production means separation step, one or more single cells that are antibody-producing cells from an appropriate tissue or biological component of the animal under immunization with the ligand X Can be separated. It should be noted that it is not necessary to immunize artificially with ligand X in non-human animals that have already produced antibodies against ligand X.

  The kind of non-human animal is not limited as long as it is a non-human animal in which antibody production by an immune reaction is observed. Specifically, various experimental animals that can be easily obtained and used, and domestic animals or pet animals that are considered necessary for antibody medical treatment can be preferably exemplified. More specifically, for example, mammals such as mice, rats, rabbits, monkeys, cows, horses, pigs, dogs, cats and the like can be considered, but non-human animals other than mammals can also be targeted.

  In the step of separating production means for non-human animals, antibody-producing cells, types of tissues or biological components containing the same, and methods for collecting the same under the condition of complying with animal welfare-related laws and ethics Is not limited. As an antibody producing cell and a tissue or a biological component containing the same, when the specimen is human, a B cell as an antibody producing cell, a bone marrow tissue containing a plasma cell, and the like can be preferably exemplified.

  In the production means separation step, the method for separating and obtaining one or more single cells of antibody-producing cells from the collected tissue or biological component is not limited. Preferably, these tissue or biological components are put into a liquid (preferably a liquid medium having an appropriate composition) to disperse and suspend the antibody-producing cells, together with magnetic fine particles bound to a certain separating antibody. After the incubation, there is a method in which single particles of target antibody-producing cells are separated from the suspension by collecting magnetic fine particles bound to antibody-producing cells via a separating antibody with a magnet.

  As the separation antibody, any antibody such as an anti-mouse CD19 antibody in the case of a mouse can be used.

[Production means conversion process]
The production means conversion step refers to production means Xm contained in each isolated antibody-producing cell, that is, a set of mRNA encoding each protein fragment of the complex protein Xp is eluted out of the cell, and by reverse transcription PCR, This refers to the step of converting all or part of these mRNAs into replication means Xc (cDNA).

  Here, as one form in the case of converting a part of mRNA into cDNA, there is a form in which only mRNA encoding a complex protein Xp belonging to a specific classification category is converted into cDNA. For example, a case where only mRNA that encodes a protein belonging to a specific class or isotype in the complex protein Xp that is an antibody is converted into cDNA is exemplified.

  As another form of converting a part of mRNA into cDNA, there is a form in which only mRNA encoding a specific fragment (for example, Fab) constituting the complex protein Xp is converted into cDNA.

  Reverse transcription PCR, which converts the entire mRNA set that constitutes the production means Xm into cDNA, or converts part of it into cDNA in various forms as described above, can be easily done by selecting the primers to be used. You can choose to do it.

  In addition, the means for eluting the production means Xm contained in the antibody-producing cells out of the cell is not limited, but any known appropriate means, for example, a method of destroying B cells with a surfactant or a low osmotic pressure solution or the like is arbitrary. Can be adopted.

[Mass production preparation process]
The mass production preparation step refers to a step of preparing mass production capability of a protein by mass replicating the replication means Xc (cDNA) obtained by the reverse transcription PCR method by the PCR method.

  The content of the PCR method in this step, the implementation conditions or the kind of primer to be used is not particularly limited, but it is preferably performed by two-step PCR, and particularly by the two-step PCR having the following content, It is preferable for the reason described in the “Operation / Effect of Invention” column.

(First stage PCR)
In each type of primer corresponding to all types of cDNA constituting the replication means Xc, amplification is performed by providing the same additional sequence on the 5 ′ end side thereof. Furthermore, this additional sequence can contain an arbitrary base sequence that works advantageously in the next single-type mass production process, for example, a translation initiation promoting sequence sequence considering a cell-free derived organism described later.

  Examples of the translation initiation promoting sequence include rbs (ribosome binding site), SD (Shine Dalgarno) sequence when the cell-free derived organism is E. coli, and cell-free derived organism is eukaryotic. Kozak sequences, 5 'UTR sequences derived from viruses, and the like.

The conditions for carrying out this first stage PCR are not limited, but it is preferable to carry out the following program, for example.
94 ° C-3 minutes;
94 ° C-30 seconds / 50 ° C-45 seconds / 72 ° C-45 seconds 30 cycles;
72 ° C-7 minutes;
(Second stage PCR)
All types of DNA fragments amplified by the first step PCR are amplified using a single type of primer having a sequence portion complementary to the additional sequence.

The conditions for performing the second-stage PCR are not limited, but for example, the following program is preferable.
94 ° C-3 minutes;
65 cycles of 96 ° C-5 seconds / 45 ° C-10 seconds / 72 ° C-45 seconds;
72 ° C-7 minutes [Transfer sequence addition step]
The transcription sequence adding step is a step of adding a transcription sequence necessary for reconversion to the cell-free production means Xm (mRNA) to the replication means Xc (cDNA) replicated in large quantities. is there. The transcription sequence is not limited, but preferably includes at least a promoter sequence. It is further preferred to include a terminator sequence in addition to the promoter sequence.

  A method for performing this step is not necessarily limited, but preferably, the transcription sequence is added to the DNA fragment amplified in the mass production preparation step by PCR, for example, overlapping PCR.

[Single-type mass production process]
The single-type mass production process is an element for reconverting the replication means Xc (DNA) to which the transcription sequence is added by the above-described process into at least the production means Xm (mRNA) of the replication means Xc, and the production means Xm. Are added to a cell-free system equipped with an element for protein production based on the above, so that each of the complex protein Xp or the complex protein Xp corresponding to a part of the mRNA defined in the production means conversion step The process of mass production.

  In this cell-free system, examples of elements that convert DNA into mRNA include transcriptase. As the transcriptase, T7 RNA polymerase, T3 RNA polymerase, SP6 RNA polymerase, RNA polymerase derived from microorganisms such as Escherichia coli, and the like can be used. Examples of elements that produce proteins based on mRNA include ribosomes and various dNTPs. Such cell-free systems are known as in vitro transcription / translation coupling systems. For example, kits such as those derived from Escherichia coli, wheat germ, and rabbit reticulocytes are commercially available. These can be used.

   As an in vitro transcription / translation coupling system, it is possible to use a reduced state by adding expensive DTT, etc., but the in vitro transcription / translation coupling system in an oxidized state without the addition of this function is comparable. Therefore, it is cheaper and more advantageous to use this.

[Characterization process]
The characterization process is to evaluate the characteristics of the complex protein Xp produced by each of the above processes, to screen for the complex protein Xp having excellent characteristics, or to determine the sequence of these complex proteins Xp. Process.

  This step is not an essentially essential step from the viewpoint of a simple method for obtaining a ligand affinity complex protein. However, it is an indispensable process or an important process from the viewpoint that the method for obtaining a ligand affinity complex protein is the premise of the inventions of antibody screening, antibody medicine, antibody medicine and the like described later.

  In the characterization step, preferably, the affinity of the complex protein Xp by ELISA is evaluated, the sequence of the complex protein Xp or its gene is determined, and the Kd value of the complex protein Xp produced using the transformant is measured. And at least one item.

[Antibody screening method]
The antibody screening method according to the present invention is a method in which the above-described method for obtaining a ligand affinity complex protein is used for screening an antibody against ligand X in a human or non-human animal.

  That is, among the two or more types of ligand affinity complex proteins obtained by the single-type mass production process of 1) to the 5) single-member mass production process in the method for obtaining a ligand affinity complex protein, It is a method for screening particularly excellent anti-ligand X antibody through a characterization process.

  Therefore, the optimal anti-ligand X antibody can be obtained easily, rapidly and in large quantities as compared with various conventional antibody screening methods.

[Antibody screening kit]
The kit for antibody screening according to the present invention is a kit used for carrying out the above antibody screening method, and comprises at least the following components.
11) A set of the dispersion / suspension liquid according to the sixth invention and magnetic fine particles.
12) The primer set for reverse transcription PCR according to the seventh invention.
13) A primer set for two-stage PCR according to the eighth invention.
14) A cell-free system as defined in 5) of the first invention.

  Each component of said 11) -14) can be provided in the dosage form normally used as this kind of goods, or a packaging form. For example, the primer set of 12) or 13) can be provided in the form of a dried product or the like in consideration of storage stability and convenience in use.

  In addition, in the antibody screening kit, in addition to the above components 11) to 14), for example, a vector used for adding a promoter sequence or a terminator sequence in a transcription sequence addition step, used for any purpose Any useful additional components such as buffers and pH adjusters may be included.

[Medical method]
The medical method according to the present invention includes a medical method for preventing, diagnosing or treating a disease in a non-human animal using a ligand affinity complex protein (antibody) obtained from the non-human animal as a specimen, and a human as a specimen. And a medical method for preventing, diagnosing or treating a human disease using the obtained ligand affinity complex protein (antibody).

  In these medical methods, the types of diseases of non-human animals or humans are not limited, but representative examples include infectious diseases caused by ligand X, various cancers in which cancer cells are ligand X, and the like.

[Antibody drug]
The antibody drug according to the present invention comprises an antibody obtained by the above-described method for obtaining a ligand affinity complex protein as an active ingredient, and depends on the origin of the antibody (human or non-human animal used as a specimen). Applied to human or non-human animals. The administration method of this antibody drug is not limited, but is preferably a parenteral method.

  The dosage form of the antibody drug is not limited, and can be provided, for example, as a lyophilization agent, a powder formulation, a solution formulation with a pH-adjusted buffer, a microcapsule for injection, and the like. These antibody pharmaceuticals can contain any beneficial components (eg, bulking agents, pH adjusters, storage stabilizers, etc.) that may be included in this type of antibody pharmaceutical.

[Non-immunoreactive antibody drug and administration method]
The non-immunoreactive antibody drug according to the present invention comprises a ligand affinity complex protein (antibody) obtained using human as a specimen, and is administered to a human individual from which it is derived. About the dosage form and content component of this non-immunoreactive antibody medicine, the same composition as the case of the above-mentioned antibody medicine is possible.

  The administration method of the non-immunoreactive antibody drug depends on the parenteral administration method. Although the kind of parenteral administration method is not limited, For example, intravenous injection, subcutaneous injection, intramuscular injection etc. can be illustrated. In addition, various drug delivery methods that have been attracting attention in recent years, such as a method in which antibody molecules are bound to magnetic microparticles and collected effectively around cancer cells by magnetic force, and finally target cells are captured by antibodies, etc. These administration methods can also be employed.

  Examples of the present invention will be described below. Of course, the technical scope of the present invention is not limited by these examples.

[Example 1 using mouse as specimen]
(Summary of Example 1)
An outline of the first embodiment will be described. First, B cells were isolated from the spleen of a mouse immunized with the antigen CBP40 (any other antigen may be used) using a magnetic microbead conjugated with an anti-mouse CD19 antibody. The isolated B cell L chain (Lc) gene and H chain Fd region (VH domain and CH1 domain) genes were separately amplified by reverse transcription PCR (RT-PCR).

  Next, in order to obtain DNA fragments suitable for in vitro synthesis, sequences of T7 promoter, ribosome binding site (rbs) and T7 terminator were added to these two genes by overlapping PCR. This pair of full-length DNA fragments of the H chain gene and L chain gene was put into the same reaction tube and transcribed and translated in vitro to obtain Fab fragments, followed by screening by ELISA. Through such a series of processes, antigen-specific monoclonal antibodies can be obtained from various cells and tissues.

  The overall flow of the above experimental operation (process) is conceptualized in FIG.

(Example 1-1: Isolation of B cells)
BALA / c mice were immunized with 50 μg of CBP40 (Calcium Binding Protein 40). After 5 weeks, the spleen was removed and dispersed in cell units in F-10 HAM nutrient medium (manufactured by Sigma-Aldrich, UK). Next, centrifugation was performed at 4 ° C. and 1000 rpm for 3 minutes to collect splenocytes and washed once with PBS (phosphate buffered saline).

CD19MACS, a magnetic particle that combines a suspension of splenocytes with anti-mouse CD19 antibody
The cells were incubated with microbeads (Miltenyi Biotec), and B cells expressing CD19 were adsorbed to the microbeads. The cells were then collected with a magnet and the supernatant was removed. The cells were further washed with PBS (2.5% BSA), and then the cells were collected with a magnet, and B cells bound to the microbeads were collected.

  The isolated B cells were counted under a microscope (LX70 manufactured by Olympus, Japan), diluted to accommodate one cell per PCR tube, and immediately subjected to cDNA synthesis.

(Example 1-2: Synthesis of single-cell reverse transcription PCR-cDNA)
Using the mRNA of each cell as a template, cDNA synthesis was performed using a reverse transcription kit. In this case, the SUPERSCRIPT First Strand synthesis system for reverse transcription PCR of Invitrogen was used, but other manufacturers may be used.

First, prepare B cells with PBS buffer to an average of 2 cells / μL, add 1.5 μL of DEPC-treated (RNase-inactivated) sterilized water, and add a total volume of 5 μL of reaction solution [0.25 μL according to Procol. dNTP, 0.50 μL 10 × RT buffer: 200 mM Tris-hydrochloric acid (pH 8.4), 500 mM KCl, 1.0 μL 25 mM MgCl ₂ , 0.5 μL 0.1 M DTT, 0.25 μL RNase inhibitor, 0.25 μL Reverse trriptase: SUPERSCRIPT II RT, 0.25 μL, 2 μM each primer] was added, treated at 42 ° C. for 90 minutes, and then treated at 70 ° C. for 15 minutes.

This synthesis reaction yields all the cDNAs of interest, that is, all cDNAs corresponding to the B cell L chain gene (κ chain) and the H chain (γ chain) Fd part (VH domain and CH1 domain) gene. For this purpose, a mixture containing 2 μM of each of the following primers 1) to 5) required was used. In order to check for contamination, a control synthesis reaction was separately performed in a system in which B cells were not present.
1) Primer C _K- J1 (shown in SEQ ID NO: 1 in Table 1 and Sequence Listing)
2) Primer C _H- IgG1-1 (shown in SEQ ID NO: 2 in Table 1 and Sequence Listing)
3) Primer C _H- IgG2A-1 (shown in SEQ ID NO: 3 in Table 1 and Sequence Listing)
4) Primer C _H- IgG2B-1 (shown in SEQ ID NO: 4 in Table 1 and Sequence Listing)
5) Primer C _H- IgG3-1 (shown in SEQ ID NO: 5 in Table 1 and Sequence Listing)
In addition, although the base sequence of each primer of said 1) -5) and all the below-mentioned primers is shown in a table / diagram and a sequence table, respectively, the base sequence shown in a table / diagram and the base sequence shown in a sequence table should be mentioned. If there is a mismatch, the base sequences shown in the table / figure are correct sequences.

（実施例１−３：ＤＮＡの増幅−第１段階のＰＣＲ）
上記により得られたｃＤＮＡの混合物を第１段階のＰＣＲ反応のテンプレートとして使用した。その際、発現されるマウスＩｇ遺伝子の９５％はＬ鎖がκアイソタイプである（Honjo and Alt, 1995 ）ため、λアイソタイプのＬ鎖は無視することにし、抗体Ｌ鎖のκアイソタイププライマーのみを用いた。又、高い抗原結合能を持つ抗体を得ると言う目的から、Ｈ鎖についてはＩｇＧ（イムノグロブリンＧ）のみを標的とした。第１段階のＰＣＲで用いた下記の縮重プライマーは Kabatのデータベース（ http://immuno.bme.nwu.edu）に基づいて設計した。

(Example 1-3: DNA amplification-first stage PCR)
The cDNA mixture obtained above was used as a template for the first stage PCR reaction. At that time, 95% of the expressed mouse Ig genes have the κ isotype in the L chain (Honjo and Alt, 1995), so the λ isotype L chain is ignored and only the antibody L chain κ isotype primer is used. It was. For the purpose of obtaining an antibody having a high antigen-binding ability, only the IgG (immunoglobulin G) was targeted for the H chain. The following degenerate primers used in the first stage PCR were designed based on the Kabat database (http://immuno.bme.nwu.edu).

  It is considered that reducing the number of primers by such a design would be beneficial in reducing unwanted amplification. Since these degenerate primers are not sufficiently ideal as templates, the amplification reaction solution lacks 3 ′ exonuclease activity and is degenerate as compared to other DNA polymerases as described below. Taq DNA polymerase (Wang et al., 2000), which has great resistance to primer and template mismatches, is used.

The light chain genes are all degenerate primers 6) Primer V _K- M2 (shown in SEQ ID NO: 6 in Table 1 and Sequence Listing), and 7) Primer C _K- His6M2 (SEQ ID NO: in Table 1 and Sequence Listing) 7).

The heavy chain genes are degenerate primers 8) Primer V _H- M2 (shown in SEQ ID NO: 8 in Table 1 and Sequence Listing) and 9) Primer V ′ _H- M2 (in Table 1 and SEQ ID NO: 9 in the Sequence Listing) And a constant region primer 10) Primer C _H- IgG1-M2
(Shown in SEQ ID NO: 10 in Table 1 and Sequence Listing), 11) primer C _H- IgG2A-M2 (shown in SEQ ID NO: 11 in Table 1 and Sequence Listing), 12) primer C _H- IgG2B-M2 (shown in Table 1 and Table 1) Amplification was performed using a mixture of primer C _H- Ig G3-M2 (shown in SEQ ID NO: 13 in Table 1 and Sequence Listing).

The amplification reaction between these L chain gene and H chain gene was carried out using 0.25 units of Taq DNA polymerase (manufactured by Takara Shuzo, Japan), 0.2 mM of each dNTP, 0.05 μM of each primer, and 0.5 μL of the above cDNA. The reaction was carried out in 5 μL of a reaction mixture consisting of the mixture and reaction buffer. PCR was performed with the following program.
94 ° C-3 minutes;
94 ° C-30 seconds / 50 ° C-45 seconds / 72 ° C-45 seconds 30 cycles;
72 ° C-7 minutes;
(Example 1-4: DNA amplification-second stage PCR)
One-tenth the amount of the first-stage PCR product was amplified in the second-stage PCR using a single species of 14) primer SP2 (shown in SEQ ID NO: 14 in Table 1 and Sequence Listing).

This amplification reaction was carried out in 5 μL of a reaction solution consisting of 0.25 units of Pfu Turbo ™ DNA polymerase (Stratagene), 0.125 mM of each dNTP, 0.5 μM of primer SP2, and reaction buffer. PCR was performed with the following program.
94 ° C-3 minutes;
65 cycles of 96 ° C-5 seconds / 45 ° C-10 seconds / 72 ° C-45 seconds;
72 ° C.-7 minutes FIG. 2 shows the amplification results of the H chain gene (indicated by “Hc”) and the κ type L chain gene (indicated by “Lc”). These results are obtained by analyzing 2 μL of the PCR product on a 1.0% agarose gel. The λ-DNA marker digested with EcoT14I is shown on the left side of the figure.

  As shown in FIG. 2, the genes encoding the light chain and heavy chain of the Fab fragment derived from a single B cell are amplified separately under these reaction conditions, and no by-product is generated. Light chain PCR products were obtained from 39 of 72 samples of single B cells (39/72 = 54%). For the heavy chain gene, specific PCR products were obtained from 28 of 72 samples of a single B cell (28/72 = 39%). As a whole, Fabs in which L chain and H chain were paired were obtained from 20 samples out of 72 samples, and the recovery rate was 28%.

(Example 1-5: Overall description of PCR)
All the above PCR reactions were performed using the GeneAmp® PCR system (PE Applied Biosystems). In order to confirm the sequence of the PCR product, several L chain genes and H chain genes were cloned into pBluescriptSKII ⁺ vector (Stratagene) digested with EcoRV. For cloning region sequencing, Thermo Sequenase II ™ dye terminator cycle sequencing premix kit (Amersham Pharmacia Biotech) and ABIPRISM ™ 310 Genetic
Analyzer was used according to the manufacturer's recommended protocol.

(Example 1-6: Overlapping PCR)
In Example 6, two fragments containing the T7 promoter, rbs and T7 terminator were combined with the L chain gene and the H chain gene amplified by overlapping PCR, respectively, as described below. The analysis result of the overlapping PCR product is shown in FIG. FIG. 3 shows the result of analyzing 2 μL of the overlapping PCR product on a 1.0% agarose gel. The L chain gene is shown in lanes 1 to 3, and the H chain gene is shown in lanes 4 to 6, respectively.

   As a primer set for amplifying the T7 promoter and the SD sequence, 15) primer In-F (5′-CCAATACGCAAACCGCCTCTCC-3 ′) shown in SEQ ID NO: 15 in the sequence listing and 16) primer shown in SEQ ID NO: 16 in the sequence listing Using T7PR-SD (5′-ATAATCTCCTTCTTATCTAATAACAAAATTATTTCTAGAGGGAAACCG-3 ′), a fragment containing the T7 promoter and ribosome binding site was amplified from the pRSET-B vector (Invitrogen).

  Moreover, as a primer set for amplifying the T7 terminator, 17) shown in SEQ ID NO: 17 in the sequence listing 17) primer T7T-NFM2 (5'-TAATCAATAATCTCCTTCTTATCTAATTCCGGCTGCTAACAAAGCCCG-3) and 18) primer In- shown in SEQ ID NO: 18 in the sequence listing The transcription terminator fragment was amplified from the pRSET-B vector using R (5'-TACAGGGCGCGTCCCATTCG-3 ').

By overlapping PCR, the L chain or H chain according to the second round of PCR was inserted between the two kinds of DNA fragments to generate a full-length DNA fragment suitable for in vitro protein synthesis. Overlapping PCR is the following PCR in 15 μL of a reaction solution consisting of 0.75 units of Ex taq DNA polymerase (Takara Shuzo), 0.2 mM of each dNTP, 0.15 μL of each DNA fragment, and reaction buffer. Followed program.
94 ° C-5 minutes;
94 cycles of 94 ° C-30 seconds / 48 ° C-20 seconds / 72 ° C-90 seconds;
Then, 15 μL of the PCR solution was added to the reaction buffer recommended by the manufacturer (Ex taq
A DNA polymerase (1.5 units), dNTP (0.2 mM each), primers In-F and In-R (0.5 mM each) was prepared to 30 μL, and amplified by the following PCR program.
94 ° C-5 minutes;
94 ° C-30 seconds / 55 ° C-30 seconds / 72 ° C-90 seconds 25 cycles;
72 ° C-7 minutes (Example 1-7: in vitro transcription / translation coupling system)
Of the 30 μL reaction solution (overlapping PCR mixture of H chain and L chain gene derived from the same cell), 3 μL was used for in vitro transcription / translation coupling reaction. This reaction was performed according to a report by Jiang et al. (Jiang et al., 2001) using an in vitro transcription / translation system containing the following elements.
Trisacetic acid (pH 7.4) 56.4 mM;
ATP 1.2 mM;
GTP, CTP and UTP each 1.0 mM;
Creatine phosphate 40 mM;
A total of 20 unlabeled amino acids, each 0.32 mM;
Polyethylene glycol 6000 4% (W / V);
Folinic acid 34.6 mg / mL;
E. coli tRNAs 0.17 mg / mL;
Ammonium acetate 36 mM;
Mg (OAc) ₂ 8 mM;
KOAc 100 mM;
Rifampicin 10 mg / mL;
Creatine kinase 0.15 mg / mL;
T7 RNA polymerase 7.7 mg / mL;
E. coli S30 extract 28.3% (V / V);
The in vitro transcription / translation coupling reaction was performed as follows. That is, 3 μL each of the overlapping PCR product was subjected to a transcription / translation reaction at 30 ° C. for 60 minutes, and then cooled on ice to stop the reaction. At the same time, the control reaction was carried out in a similar system that differs only in that it did not contain a DNA template. To the reaction system, 0.016 mM of leucine labeled with ¹⁴ C was added for autoradiography.

(Example 1-8: SDS-PAGE and autoradiography)
5 μL of sample was boiled with the same amount of loading buffer (pH 6.8, 50 mM Tris-HCl, 10% glycerol, 2% SDS, 0.1% bromophenol blue) for non-oxidizing conditions. The sample after boiling was electrophoresed on SDS-12.5% -polyacrylamide gel, and then the gel was placed on Whatman 3M filter paper and dried with a gel dryer. Next, after exposure to an imaging plate (Fuji Film), analysis was performed with BAS-2000. The result is shown in FIG. 4, and a Fab fragment band (46.0 kDa) is observed on the gel.

(Example 1-9: ELISA analysis)
In this example, an ELISA analysis for an antigen was performed as described below to determine which Fab fragment synthesized in the in vitro system had antigen affinity.

  A 96-well plate (manufactured by Nunc, Denmark) was coated with 5 μg / mL CBP40 to 50 μL / well. After incubating these wells at 4 ° C overnight, they were blocked with 1/4 × Block Ace (manufactured by Dainippon Pharmaceutical Co., Ltd.) dissolved in distilled water at 37 ° C for 30 minutes. The plate was washed twice with 10 × Block Ace, 0.05% Tween20).

  30 μL of the cell-free reaction mixture was diluted 5-fold with PBS (pH 7.4). 50 μL of this diluted solution was added to the coated plate, incubated at 25 ° C. for 2 hours, and then washed twice with the same washing solution as described above. At the same time, a reaction solution containing no DNA template was treated in the same manner as a control.

  150 μL of anti-mouse IgG (5 μg / 1 mL of distilled water) was added to each well and incubated at 25 ° C. for 1 hour. Then, after washing twice, enzyme immunoreaction (ELISA) was performed. A solution containing o-phenylenediamine and hydrogen peroxide was used as a substrate for the peroxidase reaction. The color reaction was stopped using 2M sulfuric acid. The absorbance at 492 nm was measured. In addition, Vectastain ABC kit (made by Vector Laboratories) was used for color development of ELISA.

  According to FIG. 5 showing the results of the ELISA analysis, the ELISA signal of the clone No. 9 is remarkably stronger than the other clones, strongly suggesting that this clone can bind the immunizing antigen in the synthetic Fab.

(Example 1-10: Determination of gene sequence)
Therefore, sequencing of the L chain and H chain (Fd part) of the No. 9 clone described above was performed. The amino acid sequence and gene base sequence of the L chain are shown in FIG. 6 and SEQ ID NO: 19 in the sequence table, and the amino acid sequence and gene base sequence of the H chain (Fd part) are shown in FIG.

  The sequence determined above was analyzed by the IgBLAST database (http: //www.ncbi, nlm, nih.gov/igblast) of National Center for Biotechnology Information. As a result, the L chain sequence had 98% identity with the ce9 germline gene (GenBank accession Nos. AJ239197). The H chain sequence had 91% identity with the V130 germline gene (GenBank accession Nos. M11700). From these results, it was confirmed that a novel mouse L chain gene and H chain gene were obtained, and the Fab fragment formed from these two genes was named “JM9-Fab”.

(Example 1-11: host expression, refolding, Fab fragment purification)
The L chain and H chain of the positive Fab fragment in the overlapping PCR product were digested with XbaI-NaeI and inserted into the XbaI-NaeI site of the pRSET-B vector (Invitrogen). The thus obtained plasmid containing the L chain (named pRSETB-Lc) and the plasmid containing the H chain (named pRSETB-Hc) were used for the expression of the L chain and the H chain, respectively.

  Plasmid pRSETB-Lc and plasmid pRSETB-Hc were separately introduced into competent E. coli BL21 (DE3) plysS cells (Stratagene). The expression and refolding of the Fab fragment in E. coli was carried out in substantially the same manner as in Wibbenmeyer et al.

  That is, each peptide fragment was expressed as inclusion bodies, and then collected and washed by centrifugation, and purified. After solubilization with a solubilization buffer (6M guanidine hydrochloride, 0.4M L-arginine, 100M Tris-HCl, pH 8.0, 2.5 mM EDTA), 100 mM DTT (dithiothreitol) was added, and the L chain and K The chains were mixed to equimolarity. And after diluting 50 times with the solubilization buffer which does not contain DTT, 4 mM oxidized glutathione was added, guanidine hydrochloride, EDTA, etc. were removed gradually by dialysis, and it was made to refold.

The Fab fragment was purified by the following process. That is, 1.0 mL of 0.1 M NiSO ₄ solution was passed through a HiTrap chelate column (1 mL) (Pharmacia Biotech), washed with 10 mL of pure water, and then added with binding buffer (0.5 M NaCl, 5 mM imidazole in PBS). The above Fab solution was allowed to flow and bind to the column equilibrated with the above. After washing with a total of 20 mL of binding buffer, elution was performed with elution buffer (PBS with 0.5 M NaCl and 500 mM imidazole added).

(Example 1-12: Kd value of expressed Fab fragment)
In order to evaluate the affinity of JM9-Fab, the sequences of the L chain gene and the H chain gene were respectively cloned into the pRSET-B vector to obtain pRSETB-JM9Lc and pRSETB-JM9Hc. These cloning vectors pRSETB-JM9Lc and pRSETB-JM9Hc were separately introduced into competent E. coli BL21 (DE3) plysS cells (Stratagene). JM9-Fab was expressed, refolded and purified.

The Kd value of JM9-Fab by Stevevs method was 1.7 (± 0.3) × 10 ⁻⁸ . This result indicates that this Fab fragment has a high antigen-binding activity equivalent to that of an antibody by a conventional hybridoma method.

[Example 2 using human as a specimen]
In this Example 2, a healthy adult female was used as a sample, with the consent of the person, 2 mL of the peripheral blood was collected, and antibody-producing cells (B cells) separated from this peripheral blood were used. Each step according to the same procedure as 1 was performed.

Among the steps and contents of Example 2, points different from Example 1 are the following (a) to (d). Therefore, in the following, first, an outline of the second embodiment will be described based on FIG. 8, and then (a) to (d) will be sequentially described. Except for these points, the same procedure as in Example 1 is performed.
(A) B cell isolation step (b) Composition of monoclonal antibody (c) Primer used in PCR in each step (d) ELISA analysis, sequencing, antigen binding activity (Summary of Example 2)
As conceptualized in FIG. 8, first, B cells were isolated from peripheral blood. The isolated B cell L chain (Lc) gene and H chain (Hc) gene were separately amplified by reverse transcription PCR (RT-PCR). Subsequently, in order to obtain DNA fragments suitable for in vitro synthesis, sequences of T7 promoter, ribosome binding site (rbs) and T7 terminator were added to these genes by overlapping PCR. This pair of full-length DNA fragments of the H chain gene and L chain gene was put into the same reaction tube and allowed to perform transcription and translation in vitro to obtain Mabs, followed by screening by ELISA.

(B cell isolation step)
A healthy adult female was used as a sample, and with the consent, 2 mL of the peripheral blood was collected. Blood collection was performed in the presence of heparin to prevent blood clotting. Next, peripheral blood monocytes were separated by a centrifugal density gradient (400 × g, 40 minutes, 18 ° C.) using Ficllo-Plaque Plus (Amersham Pharmacia Biotech Inc.).

The collected cells were washed twice with PBS buffer, the coagulated body was removed by nylon (trademark) net (41 μm mesh), and CD19 ⁺ cells were isolated by MACS magnetic beads (Miltenyi Biotech, Germany). After removing the magnetic beads, staining was performed with an anti-human CD19 antibody PE conjugate (Santa Cruz Biotechnology, Inc., USA). After washing twice with PBS, only fluorescently labeled cells were collected by preparative flow cytometry (EPICA ELITE, Beckman Coulter, Japan).

  The isolated B cells were counted under a microscope (LX70, manufactured by Olympus, Japan), diluted to contain one cell per PCR tube, and immediately subjected to cDNA synthesis as the next step.

(Composition of monoclonal antibody)
In Example 1, the L chain (Lc) gene of the isolated mouse B cell and the Fd region gene of the H chain were separately amplified by reverse transcription PCR. In Example 2, the isolated human B cell Hab chain gene (Hc), κ type L chain gene (Lc (kappa)) and λ type L chain gene (Lc (lamda)) are amplified separately by reverse transcription PCR, and finally, a Mab comprising these fragments Acquired.

  FIG. 9 shows the results of amplification of these genes by the second-stage PCR performed in the same manner as in Example 1. In each figure of “Hc”, “Lc (kappa)”, and “Lc (lamda)” in FIG. 9, the λ-DNA marker digested with EcoT14I is shown on the left side of the figure.

  As shown in FIG. 9, the Fab chain H chain gene, κ type L chain gene and λ type L chain gene derived from a single B cell are amplified separately, and no by-product is observed.

(Primers used in PCR in each step)
In Example 2, since the primers used in PCR in each step were different from those in Example 1, a list thereof is shown in Table 2, and their base sequences are also described in the sequence table. Each primer shown in Table 2 is briefly described below.

（逆転写ＰＣＲ用プライマー）
前記実施例１−２において使用した１）〜５）の各プライマーに相当するものが、次の２１）〜２３）のプライマーである。

(Primer for reverse transcription PCR)
The following primers 21) to 23) correspond to the primers 1) to 5) used in Example 1-2.

  21) Primer HuCH-1: Primer for H chain gene synthesis. The base sequence is shown in SEQ ID NO: 21 in the sequence listing.

  22) Primer HuCκ-1: A primer for synthesizing a κ type L chain gene. The nucleotide sequence is shown in SEQ ID NO: 22 in the sequence listing.

  23) Primer HuCλ-2: Primer for synthesizing λ type L chain gene. The nucleotide sequence is shown in SEQ ID NO: 23 in the sequence listing.

(Primer for first stage PCR)
The following primers 24) to 37) correspond to the primers 6) to 13) used in Example 1-3.

  24) Primer HuVH-1: Primer for the heavy chain variable region, and the base sequence is shown in SEQ ID NO: 24 in the sequence listing.

  25) Primer HuVH-2: Primer for heavy chain variable region, the base sequence of which is shown in SEQ ID NO: 25 in the Sequence Listing.

  26) Primer HuVH-3: Primer for the heavy chain variable region, the base sequence of which is shown in SEQ ID NO: 26 in the Sequence Listing.

  27) Primer HuVH-4: Primer for the heavy chain variable region, and the nucleotide sequence is shown in SEQ ID NO: 27 in the sequence listing.

  28) Primer HuVκ-1: Primer for the variable region of the L chain κ gene, whose base sequence is shown in SEQ ID NO: 28 in the sequence listing.

  29) Primer HuVκ-2: A primer for the variable region of the L chain κ gene, whose base sequence is shown in SEQ ID NO: 29 in the sequence listing.

  30) Primer HuVκ-3: Primer for the variable region of the L chain κ gene, whose base sequence is shown in SEQ ID NO: 30 in the sequence listing.

  31) Primer HuVκ-4: A primer for the variable region of the L chain κ gene, whose base sequence is shown in SEQ ID NO: 31 in the sequence listing.

  32) Primer HuVλ-1: Primer for the variable region of the L chain λ gene. The nucleotide sequence is shown in SEQ ID NO: 32 in the sequence listing.

  33) Primer HuVλ-2: Primer for the L chain λ gene variable region, the base sequence of which is shown in SEQ ID NO: 33 in the Sequence Listing.

  34) Primer HuVλ-3: Primer for the L chain λ gene variable region, and the nucleotide sequence is shown in SEQ ID NO: 34 in the sequence listing.

  35) Primer HuCH-2: Primer for the heavy chain constant region, and the nucleotide sequence is shown in SEQ ID NO: 35 in the sequence listing.

  36) Primer HuCκ-2: A primer for the constant region of the L chain κ gene, whose base sequence is shown in SEQ ID NO: 36 in the sequence listing.

  37) Primer HuCλ-2: Primer for the constant region of the L chain λ gene. The nucleotide sequence is shown in SEQ ID NO: 37 in the sequence listing.

(Primer for second stage PCR)
The primer corresponding to 14) used in Example 1-4 is the primer of 38) below.

  38) Primer HuSP1: a single kind of primer for second-stage PCR, whose base sequence is shown in SEQ ID NO: 38 in the sequence listing.

(ELISA analysis, sequencing, binding activity to antigen)
In order to prove that an active antibody molecule (Fab) was synthesized from the antibody gene amplified in the same manner as in Example 1, it was examined by ELISA reaction against blood group antigen B type. Since the sample adult is a blood group of type A, it is assumed that it naturally has a blood group antibody against the type B antigen. This is the same situation as the presence of antibodies against the pathogen causing the infection in the blood of a patient infected with the infection.

  FIG. 10 shows the results of ELISA performed using B-type antigen BSA as an antigen. The experimental method was substantially the same as in Example 1. The difference from the case of Example 1 is that Blood Group B Trisaccharide-BSA 10-atom spacer (Destra laboratoriesm Ltd.) was used instead of CBP40 as an antigen.

  The antibody groups obtained as a result were found to have different binding activities for B-type antigens. Considering that the human body produces various types of antibodies and has them in the body, this result is fully expected, suggesting that this experiment has proceeded as expected.

  Of these, the L chain and H chain sequences of No. 2 and No. 10 clones with high ELISA signals were determined. The amino acid sequence and gene base sequence of the L chain of the No. 2 clone are shown in FIG. 11 and SEQ ID NO: 39 of the sequence table, and the amino acid sequence and gene base sequence of the No. 2 clone are shown in FIG. No. 40 shows the amino acid sequence and gene base sequence of the L chain of the No. 10 clone in FIG. 13 and SEQ ID NO: 41 of the sequence table, and FIG. 14 shows the amino acid sequence and gene base sequence of the No. 10 clone of the H chain. It shows in SEQ ID NO: 42 in the sequence listing.

  For these sequences, a homology search was performed against the human antibody database IgBlast. As a result, 95% homology with the V1-18 germline gene (GeneBank accession Nos. D187018) was detected in the L chain of the No. 10 clone. The H chain of the No. 10 clone was found to be 91% homologous with the VH2-70 germiline gene GeneBank accession Nos. AB019437). A similar search was performed for the No. 2 clone. The L chain was 100% homologous with V1-5germline (GeneBank accession Nos. D87015), and the H chain was 90% with VH4-4 (GeneBank accession Nos. AB019441). Homology was observed for each.

  From these results, it was proved that by using the method of the present invention, a human Mab capable of binding to the target antigen and its cDNA can be obtained.

According to the present invention, one or two or more proteins can be obtained rapidly, in large quantities and with high purity from a very wide variety of ligand affinity complex proteins showing affinity for a specific ligand. .


[Sequence Listing]

SEQUENCE LISTING

<110> National University Corporation Nagoya University

<120> Ligand affinity complex protein obtaining method, antibody screening method, antibody screening kit, medical method, antibody drug, non-immunoreactive antibody drug, dosing method

<130> POK-04-029

<160> 42

<210> 1
<211> 21
<212> DNA
<213> Artificial Sequence

<400> 1
ttaacactca ttcctgttga a 21

<210> 2
<211> 21
<212> DNA
<213> Artificial Sequence

<400> 2
aattttcttg tccaccttgg t 21

<210> 3
<211> 21
<212> DNA
<213> Artificial Sequence

<400> 3
aattttcttg tccaccttgg t 21

<210> 4
<211> 21
<212> DNA
<213> Artificial Sequence

<400> 4
aagttttttg tccaccgtgg t 21

<210> 5
<211> 21
<212> DNA
<213> Artificial Sequence

<400> 5
gattctcttg atcaactcag t 21

<210> 6
<211> 51
<212> DNA
<213> Artificial Sequence

<400> 6
attagataag aaggagatta ttgaatgga (c / t) attgtg (a / c) t (c / g) a 40
c (a / c) ca (a / g) (a / t) ct (a / c) ca 51

<210> 7
<211> 66
<212> DNA
<213> Artificial Sequence

<400> 7
attagataag aaggagatta ttgattagtg gtggtggtgg tggtgacact cattcctgtt 60
gaagct 66

<210> 8
<211> 47
<212> DNA
<213> Artificial Sequence

<400> 8
attagataag aaggagatta ttgaatg (c / g) a (a / g) gt (a / c / g / t) (a / c) agctg (c / g) 40
ag (c / g) agtc 47

<210> 9
<211> 50
<212> DNA
<213> Artificial Sequence

<400> 9
attagataag aaggagatta ttgaatg (c / g) a (a / g) gt (a / c / g / t) (a / c) agctg (c / g) 40
ag (c / g) agtc (a / t) gg 50

<210> 10
<211> 54
<212> DNA
<213> Artificial Sequence

<400> 10
attagataag aaggagatta ttgattaaat tttcttgtcc accttggtgc tgct 54

<210> 11
<211> 52
<212> DNA
<213> Artificial Sequence

<400> 11
attagataag aaggagatta ttgattaaat tttcttgtcc accttggtgc tg 52

<210> 12
<211> 52
<212> DNA
<213> Artificial Sequence

<400> 12
attagataag aaggagatta ttgattaaag ttttttgtcc accgtggtgc tg 52

<210> 13
<211> 54
<212> DNA
<213> Artificial Sequence

<400> 13
attagataag aaggagatta ttgattagat tctcttgatc aactcagtct tgct 54

<210> 14
<211> 24
<212> DNA
<213> Artificial Sequence

<400> 14
attagataag aaggagatta ttga 24

<210> 15
<211> 22
<212> DNA
<213> Artificial Sequence

<400> 15
ccaatacgca aaccgcctct cc 22

<210> 16
<211> 48
<212> DNA
<213> Artificial Sequence

<400> 16
ataatctcct tcttatctaa taacaaaatt atttctagag ggaaaccg 48

<210> 17
<211> 48
<212> DNA
<213> Artificial Sequence

<400> 17
taatcaataa tctccttctt atctaattcc ggctgctaac aaagcccg 48

<210> 18
<211> 20
<212> DNA
<213> Artificial Sequence

<400> 18
tacagggcgc gtcccattcg 20

<210> 19
<211> 219
<212> PRT
<213> Mus musculus

<400> 19
Asp Ile Val Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Leu Gly 16
GAC ATT GTG ATG ACC CAG TCT CCA TCC TCC CTG TCT GCC TCT CTG GGA 48

Asp Arg Val Thr Ile Ser Cys Arg Ala Ser Gln Asp Ile Ser Asn Tyr 32
GAC AGA GTC ACC ATC AGT TGC AGG GCA AGT CAG GAC ATT AGC AAT TAT 96

Leu Asn Trp Tyr Gln Gln Lys Pro Asp Gly Thr Val Lys Leu Leu Ile 48
TTA AAC TGG TAT CAG CAG AAA CCA GAT GGA ACT GTT AAA CTC CTG ATC 144

Tyr Tyr Thr Ser Arg Leu His Ser Gly Val Pro Ser Arg Phe Ser Gly 64
TAC TAC ACA TCA AGA TTA CAC TCA GGA GTC CCA TCA AGG TTC AGT GGC 192

Ser Gly Ser Gly Thr Asp Tyr Ser Leu Thr Ile Ser Asn Leu Glu Gln 80
AGT GGG TCT GGA ACA GAT TAT TCT CTC ACC ATT AGC AAC CTG GAG CAA 240

Glu Asp Ile Ala Thr Tyr Phe Cys Gln Gln Gly Asn Thr Leu Tyr Thr 96
GAA GAT ATT GCC ACT TAC TTT TGC CAA CAG GGT AAT ACG CTG TAC ACG 288

Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys Arg Ala Asp Ala Ala Pro 112
TTC GGA GGG GGG ACC AAG CTG GAA ATA AAA CGG GCT GAT GCT GCA CCA 336

Thr Val Ser Ile Phe Pro Pro Ser Ser Glu Gln Leu Thr Ser Gly Gly 128
ACT GTA TCC ATC TTC CCA CCA TCC AGT GAG CAG TTA ACA TCT GGA GGT 384

Ala Ser Val Val Cys Phe Leu Asn Asn Phe Tyr Pro Lys Asp Ile Asn 144
GCC TCA GTC GTG TGC TTC TTG AAC AAC TTC TAC CCC AAA GAC ATC AAT 432

Val Lys Trp Lys Ile Asp Gly Ser Glu Arg Gln Asn Gly Val Leu Asn 160
GTC AAG TGG AAG ATT GAT GGC AGT GAA CGA CAA AAT GGC GTC CTG AAC 480

Ser Trp Thr Asp Gln Asp Ser Lys Asp Ser Thr Tyr Ser Met Ser Ser 176
AGT TGG ACT GAT CAG GAC AGC AAA GAC AGC ACC TAC AGC ATG AGC AGC 528

Thr Leu Thr Leu Thr Lys Asp Glu Tyr Glu Arg His Asn Ser Tyr Thr 192
ACC CTC ACG TTG ACC AAG GAC GAG TAT GAA CGA CAT AAC AGC TAT ACC 576

Cys Glu Ala Thr His Lys Thr Ser Thr Ser Pro Ile Val Lys Ser Phe 208
TGT GAG GCC ACT CAC AAG ACA TCA ACT TCA CCC ATT GTC AAG AGC TTC 624

Asn Arg Asn Glu Cys His His His His His His 219
AAC AGG AAT GAG TGT CAC CAC CAC CAC CAC CAC 657

<210> 20
<211> 213
<212> PRT
<213> Mus musculus

<400> 20
Glu Val Lys Leu Gln Gln Ser Gly Ala Glu Phe Val Arg Pro Gly Ala 16
GAA GTA AAG CTG CAG CAG TCT GGG GCT GAG TTT GTA AGG CCA GGG GCC 45

Leu Val Lys Leu Ser Cys Lys Thr Ser Gly Phe Asn Ile Lys Asp Tyr 32
TTA GTC AAG TTG TCC TGC AAA ACT TCT GGC TTC AAC ATT AAA GAC TAC 90

Tyr Met His Trp Val Lys Gln Arg Pro Glu Gln Gly Leu Glu Trp Ile 48
TAT ATG CAC TGG GTG AAG CAG AGG CCT GAA CAG GGC CTG GAG TGG ATT 135

Gly Trp Ile Asp Pro Glu Asn Asp Asp Thr Ile Tyr Asp Pro Lys Phe 64
GGA TGG ATT GAT CCT GAG AAT GAT GAT ACT ATA TAT GAC CCG AAA TTC 180

Gln Gly Lys Ala Ser Ile Thr Ala Asp Thr Ser Ser Asn Thr Ala Tyr 80
CAG GGC AAG GCC AGT ATA ACA GCT GAC ACA TCC TCC AAC ACA GCC TAC 225

Leu Gln Leu Ser Ser Leu Thr Ser Glu Asp Thr Ala Val Tyr Phe Cys 96
CTG CAG CTC AGC AGC CTG ACA TCT GAG GAC ACT GCC GTC TAT TTC TGT 270

Ala Gly Asp Gly Tyr Ser Gly Asn Tyr Trp Gly Gln Gly Thr Ser Val 112
GCT GGT GAT GGT TAC TCC GGG AAC TAC TGG GGT CAA GGA ACC TCA GTC 315

Thr Val Ser Ser Ala Lys Thr Thr Pro Pro Ser Val Tyr Pro Leu Ala 128
ACC GTC TCC TCA GCC AAA ACG ACA CCC CCA TCT GTC TAT CCA CTG GCC 360

Pro Gly Ser Ala Ala Gln Thr Asn Ser Met Val Thr Leu Gly Cys Leu 144
CCT GGA TCT GCT GCC CAA ACT AAC TCC ATG GTG ACC CTG GGA TGC CTG 405

Val Lys Gly Tyr Phe Pro Glu Pro Val Thr Val Thr Trp Asn Ser Gly 160
GTC AAG GGC TAT TTC CCT GAG CCA GTG ACA GTG ACC TGG AAC TCT GGA 450

Ser Leu Ser Ser Gly Val His Thr Phe Pro Ala Val Leu Gln Ser Asp 176
TCC CTG TCC AGC GGT GTG CAC ACC TTC CCA GCT GTC CTG CAG TCT GAC 495

Leu Tyr Thr Leu Ser Ser Ser Val Thr Val Pro Ser Ser Thr Trp Pro 192
CTC TAC ACT CTG AGC AGC TCA GTG ACT GTC CCC TCC AGC ACC TGG CCC 540

Ser Glu Thr Val Thr Cys Asn Val Ala His Pro Ala Ser Ser Thr Lys 208
AGC GAG ACC GTC ACC TGC AAC GTT GCC CAC CCG GCC AGC AGC ACC AAG 585

Val Asp Lys Lys Ile 213
GTG GAC AAG AAA ATT 639

<210> 21
<211> 18
<212> DNA
<213> Artificial Sequence

<400> 21
gctggcctct caccaact 18

<210> 22
<211> 18
<212> DNA
<213> Artificial Sequence

<400> 22
acactctccc ctgttgaa 18

<210> 23
<211> 18
<212> DNA
<213> Artificial Sequence

<400> 23
tgaacattcy gyaggggc 18

<210> 24
<211> 50
<212> DNA
<213> Artificial Sequence

<400> 24
gaaacaagaa tagaaggaga tattgtaatg caggtgcagc tggtgcagtc 50

<210> 25
<211> 53
<212> DNA
<213> Artificial Sequence

<400> 25
gaaacaagaa tagaaggaga tattgtaatg caggtcaact taagggagtc tgg 53

<210> 26
<211> 50
<212> DNA
<213> Artificial Sequence

<400> 26
gaaacaagaa tagaaggaga tattgtaatg gaggtgcagc tgstgsagtc 50

<210> 27
<211> 50
<212> DNA
<213> Artificial Sequence

<400> 27
gaaacaagaa tagaaggaga tattgtaatg caggtrcagc tgcagsagtc 50

<210> 28
<211> 51
<212> DNA
<213> Artificial Sequence

<400> 28
gaaacaagaa tagaaggaga tattgtaatg gacatcswga tgacccagtc t 51

<210> 29
<211> 53
<212> DNA
<213> Artificial Sequence

<400> 29
gaaacaagaa tagaaggaga tattgtaatg gatattgtgm tgactcagtc tcc 53

<210> 30
<211> 53
<212> DNA
<213> Artificial Sequence

<400> 30
gaaacaagaa tagaaggaga tattgtaatg gaaattgtgt tgacgcagtc tcc 53

<210> 31
<211> 52
<212> DNA
<213> Artificial Sequence

<400> 31
gaaacaagaa tagaaggaga tattgtaatg gaaacgacac tcacgcagtc tc 52

<210> 32
<211> 50
<212> DNA
<213> Artificial Sequence

<400> 32
gaaacaagaa tagaaggaga tattgtaatg cagtctgtgc tgactcagcc 50

<210> 33
<211> 50
<212> DNA
<213> Artificial Sequence

<400> 33
gaaacaagaa tagaaggaga tattgtaatg cagtctgccc tgactcagcc 50

<210> 34
<211> 50
<212> DNA
<213> Artificial Sequence

<400> 34
gaaacaagaa tagaaggaga tattgtaatg tcttatgagc tgacwcagcc 50

<210> 35
<211> 73
<212> DNA
<213> Artificial Sequence

<400> 35
gaaacaagaa tagaaggaga tattgtatta gtggtggtgg tggtggtgaa ctbtcttgtc 60
caccttggtg ttg 73

<210> 36
<211> 52
<212> DNA
<213> Artificial Sequence

<400> 36
gaaacaagaa tagaaggaga tattgtatta acactctccc ctgttgaagc tc 52

<210> 37
<211> 52
<212> DNA
<213> Artificial Sequence

<400> 37
gaaacaagaa tagaaggaga tattgtatta tgaacattcy gyaggggcma ct 52

<210> 38
<211> 27
<212> DNA
<213> Artificial Sequence

<400> 38
gaaacaagaa tagaaggaga tattgta 27

<210> 39
<211> 213
<212> PRT
<213> Homo sapiens

<400> 39
Gln Ser Ala Leu Thr Gln Pro Pro Lys Val Leu Gly Leu Gln Ala Arg 16
CAG TCT GCC CTG ACT CAG CCT CCC AAA GTG TTG GGA TTA CAG GCG CGA 48

Ala Thr Thr Pro Gly Tyr Leu Phe His Phe Ile Leu Met Thr Thr Leu 32
GCC ACC ACG CCT GGT TAT TTA TTT CAT TTT ATA CTT ATG ACA ACC CTG 96

Gly Ser Pro Ser Val Ile Leu Cys Gln Pro Leu Thr Leu Ser Phe Leu 48
GGA AGT CCC TCT GTC ATT CTA TGC CAG CCG TTG ACA TTG TCA TTC TTG 144

Pro Phe Tyr Thr Gly Arg Asn Lys Leu Arg Glu Lys Val Met Ser Ile 64
CCA TTT TAT ACA GGA CGA AAT AAA CTC AGA GAG AAG GTA ATG AGC ATA 192

Ile Cys Leu Arg Ser Gln Asp Thr Trp Gln Ser Gly Asn Ser Asn Pro 80
ATC TGT CTG AGG TCA CAG GAT ACG TGG CAG AGC GGA AAT TCG AAT CCT 240

Ser Ile Leu Thr Ser Glu Pro Val Ser Trp Thr Leu Trp Leu Leu Ser 96
AGC ATT CTG ACT TCA GAA CCT GTT TCC TGG ACA CTC TGG CTT CTC TCG 288

Met Trp Ser Pro Ala Arg Gln His Lys Thr Leu Val Gln Pro Lys Ala 112
ATG TGG TCC CCG GCC CGG CAG CAT AAG ACA CTA GTC CAG CCC AAG GCT 336

Ala Pro Ser Val Thr Leu Phe Pro Pro Ser Ser Glu Glu Leu Gln Ala 128
GCC CCC TCG GTC ACT CTG TTC CCG CCC TCC TCT GAG GAG CTT CAA GCC 384

Asn Lys Ala Thr Leu Val Cys Leu Ile Ser Asp Phe Tyr Pro Gly Ala 144
AAC AAG GCC ACA CTG GTG TGT CTC ATA AGT GAC TTC TAC CCG GGA GCC 432

Val Thr Val Ala Trp Lys Ala Asp Ser Ser Pro Val Lys Ala Gly Val 160
GTG ACA GTG GCT TGG AAA GCA GAT AGC AGC CCC GTC AAG GCG GGA GTG 480

Glu Thr Thr Thr Pro Ser Lys Gln Ser Asn Asn Lys Tyr Ala Ala Ser 176
GAG ACC ACC ACA CCC TCC AAA CAA AGC AAC AAC AAG TAC GCG GCC AGC 528

Ser Tyr Leu Ser Leu Thr Pro Glu Gln Trp Lys Ser His Arg Ser Tyr 192
AGC TAT CTG AGC CTG ACG CCT GAG CAG TGG AAG TCC CAC AGA AGC TAC 576

Ser Cys Gln Val Thr His Glu Gly Ser Thr Val Glu Lys Thr Val Ala 208
AGC TGC CAG GTC ACG CAT GAA GGG AGC ACC GTG GAG AAG ACA GTG GCC 624

Pro Thr Glu Cys Ser 213
CCT ACA GAA TGT TCA 639

<210> 40
<211> 219
<212> PRT
<213> Homo sapiens

<400> 40
Gln Val Gln Leu Gln Gln Ser Gly Tyr Pro Ser His Glu His Pro Ile 16
CAG GTA CAG CTG CAG CAG TCC GGA TAT CCC TCC CAT GAG CAT CCT ATC 48

Leu Trp Thr Ser Asp Val Leu Tyr Lys Glu Ala Pro Ser Asn Phe Leu 32
CTC TGG ACT TCT GAT GTT CTA TAT AAA GAG GCA CCC TCA AAT TTT CTT 96

Phe Ser Glu Ala Lys Pro Trp Arg Glu Trp Glu Tyr Pro Tyr Glu Glu 48
TTC TCC GAA GCT AAG CCT TGG AGA GAA TGG GAG TAC CCC TAC GAA GAG 144

Phe Leu Leu Ser Val Ser Arg Leu Ser Glu Cys Asn Tyr Gln Ser His 64
TTT CTA CTC TCA GTG TCC AGG TTA TCT GAG TGC AAT TAC CAG TCC CAT 192

Asn Ile Pro Ile Pro Glu Ser Tyr Val Arg Leu Phe Pro Glu Glu His 80
AAC ATA CCA ATT CCA GAA TCA TAT GTG CGT CTC TTT CCA GAA GAA CAT 240

Arg Ser Tyr Leu Lys Gly Cys Cys Asp Thr Asn Ser Tyr Asp Met Leu 96
AGA AGT TAT CTC AAA GGG TGT TGT GAC ACT AAC AGC TAC GAT ATG TTA 288

Arg Tyr Leu Ala Glu Tyr Gln Val Glu Gly Glu Thr Leu Tyr Asn Arg 112
AGG TAT TTG GCA GAA TAT CAG GTA GAA GGT GAA ACA CTT TAT AAC AGG 336

Leu Leu Ser Val Ser Thr Lys Gly Pro Ser Val Phe Pro Leu Ala Pro 128
CTA TTA TCA GTA TCC ACC AAG GGC CCA TCC GTC TTC CCC CTG GCG CCC 384

Cys Ser Arg Ser Thr Ser Glu Ser Thr Ala Ala Leu Gly Cys Leu Val 144
TGC TCC AGG AGC ACC TCC GAG AGC ACA GCC GCC CTG GGC TGC CTG GTC 432

Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly Ala 160
AAG GAC TAC TTC CCC GAA CCG GTG ACG GTG TCG TGG AAC TCA GGC GCC 480

Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu Gln Ser Ser Gly 176
CTG ACC AGC GGC GTG CAC ACC TTC CCG GCT GTC CTA CAG TCC TCA GGA 528

Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser Leu Gly 192
CTC TAC TCC CTC AGC AGC GTG GTG ACC GTG CCC TCC AGC AGC TTG GGC 576

Thr Lys Thr Tyr Thr Cys Asn Val Asp His Lys Pro Ser Asn Thr Lys 208
ACG AAG ACC TAC ACC TGC AAC GTA GAT CAC AAG CCC AGC AAC ACC AAG 624

Val Asp Lys Arg Val His His His His His His 219
GTG GAC AAG AGA GTT CAC CAC CAC CAC CAC CAC 657

<210> 41
<211> 213
<212> PRT
<213> Homo sapiens

<400> 41
Gln Ser Val Leu Thr Gln Pro Tyr Pro Gly Glu Gly Phe Leu Ile Tyr 16
CAG TCT GTG CTG ACT CAG CCT TAT CCA GGT GAA GGC TTT CTG ATA TAT 48

Gln Ser Ala Tyr Leu Ala Pro Phe Cys Leu Ser Tyr Tyr Thr Val Lys 32
CAA TCT GCT TAT TTG GCT CCA TTC TGT CTC TCA TAC TAT ACT GTG AAG 96

Arg Arg Pro Cys Leu Ile His Val Asp Thr Thr His Ser Arg Ile Met 48
CGA AGA CCA TGT CTC ATT CAT GTA GAC ACT ACA CAC AGT AGG ATA ATG 144

His Val Glu Leu Asn Trp Leu Met His Glu Tyr Ala Ser Tyr Gln Arg 64
CAT GTT GAG TTA AAT TGG CTT ATG CAT GAA TAT GCT TCC TAC CAG CGA 192

Gly Val Asn Gly Leu Ser Lys Asp Glu His His Leu Phe Leu Tyr Ile 80
GGA GTC AAT GGT CTA AGT AAG GAT GAA CAT CAT CTG TTT CTG TAC ATT 240

Tyr Phe Gln Thr Val Val Arg Leu Gly Val Pro Arg Ser Lys Asp Tyr 96
TAC TTT CAA ACA GTA GTG AGG CTG GGA GTA CCT AGA AGC AAG GAC TAT 288

Val Leu Thr Gln Val Ser Lys Phe Ile Phe Tyr Ser Phe Pro Lys Ala 112
GTT TTA ACC CAA GTT TCA AAA TTT ATC TTT TAT TCG TTT CCC AAG GCC 336

Asn Pro Thr Val Thr Leu Phe Pro Pro Ser Ser Glu Glu Leu Gln Ala 128
AAC CCC ACG GTC ACT CTG TTC CCG CCC TCC TCT GAG GAG CTC CAA GCC 384

Asn Lys Ala Thr Leu Val Cys Leu Ile Ser Asp Phe Tyr Pro Gly Ala 144
AAC AAG GCC ACA CTA GTG TGT CTG ATC AGT GAC TTC TAC CCG GGA GCT 432

Val Thr Val Ala Trp Lys Ala Asp Gly Ser Pro Val Lys Ala Gly Val 160
GTG ACA GTG GCT TGG AAG GCA GAT GGC AGC CCC GTC AAG GCG GGA GTG 480

Glu Thr Thr Lys Pro Ser Lys Gln Ser Asn Asn Lys Tyr Ala Ala Ser 176
GAG ACG ACC AAA CCC TCC AAA CAG AGC AAC AAC AAG TAC GCG GCC AGC 528

Ser Tyr Leu Ser Leu Thr Pro Glu Gln Trp Lys Ser His Arg Ser Tyr 192
AGC TAC CTG AGC CTG ACG CCC GAG CAG TGG AAG TCC CAC AGA AGC TAC 576

Ser Cys Gln Val Thr His Glu Gly Ser Thr Val Glu Lys Thr Val Ala 208
AGC TGC CAG GTC ACG CAT GAA GGG AGC ACC GTG GAG AAG ACA GTG GCC 624

Pro Thr Glu Cys Ser 213
CCT ACA GAA TGT TCA 639

<210> 42
<211> 219
<212> PRT
<213> Homo sapiens

<400> 42
Gln Val Asn Leu Arg Glu Ser Gly Gln Leu Met Pro Ala Ser Ser Pro 16
CAG GTC AAC TTA AGG GAG TCT GGA CAA TTA ATG CCT GCC TCC TCT CCC 48

Tyr Pro Thr Asn Val His Ile Leu Ile Leu Arg Thr Cys Glu Tyr Gly 32
TAC CCC ACA AAT GTT CAC ATC CTA ATT CTT AGA ACC TGT GAA TAT GGT 96

Glu Tyr Val Glu Tyr Val Thr Tyr His Gly Gln Glu Val Leu Tyr Arg 48
GAA TAT GTT GAA TAT GTT ACC TAC CAT GGT CAA GAG GTA CTT TAC AGA 144

Tyr Glu Gln Leu Arg Thr Leu Arg Glu Ala Gln Ser Ser Arg Leu Leu 64
TAT GAG CAA TTA AGG ACC CTG AGA GAG GCA CAA TCA TCT AGA TTA CTC 192

Arg Gln Ala Gln Ser Gly His Gln Gly Pro Cys Lys Arg Lys Val Gly 80
AGG CAA GCC CAA AGT GGC CAC CAG GGT CCC TGT AAG AGG AAG GTA GGG 240

Arg Val Lys Arg Gln Gln Ile Lys Ser Arg Asp Glu Ser Asp Ala Leu 96
AGG GTT AAG AGA CAG CAG ATA AAG AGC AGA GAC GAG AGT GAT GCA CTT 288

Gly Lys Met Lys Glu Gly His Glu Val Lys Glu Cys Trp Trp Pro Leu 112
GGA AAG ATG AAA GAA GGT CAT GAG GTT AAG GAA TGC TGG TGG CCT CTA 336

Glu Ala Arg Lys Ser Thr Lys Gly Pro Ser Val Phe Pro Leu Ala Pro 128
GAA GCT AGA AAG TCC ACC AAG GGC CCA TCG GTC TTC CCC CTG GCA CCC 384

Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly Cys Leu Val 144
TCC TCC AAG AGC ACC TCT GGG GGC ACA GCG GCC CTG GGC TGC CTG GTC 432

Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly Ala 160
AAG GAC TAC TTC CCC GAA CCG GTG ACG GTG TCG TGG AAC TCA GGC GCC 480

Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu Gln Ser Ser Gly 176
CTG ACC AGC GGC GTG CAC ACC TTC CCG GCT GTC CTA CAG TCC TCA GGA 528

Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser Leu Gly 192
CTC TAC TCC CTC AGC AGC GTG GTG ACC GTG CCC TCC AGC AGC TTG GGC 576

Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys Pro Ser Asn Thr Lys 208
ACC CAG ACC TAC ATC TGC AAC GTG AAT CAC AAG CCC AGC AAC ACC AAG 624

Val Asp Lys Arg Val His His His His His His 219
GTG GAC AAG AGA GTT CAC CAC CAC CAC CAC CAC 657

It is a figure which conceptualizes and shows the whole flow of Example 1.

It is a figure which shows the amplification result of the H chain gene and L chain gene in Example 1.

FIG. 3 is a diagram showing the analysis results of overlapping PCR products of Example 1.

It is a figure which shows the result of the autoradiography in Example 1. FIG.

It is a figure which shows the result of the ELISA analysis in Example 1. FIG.

It is a figure which shows the result of the sequencing of the L chain in Example 1.

It is a figure which shows the result of the sequencing of the H chain in Example 1.

It is a figure which conceptualizes and shows the whole flow of Example 2.

It is a figure which shows the amplification result of the H chain gene and L chain gene in Example 2.

It is a figure which shows the result of the ELISA analysis in Example 2. FIG.

It is a figure which shows the result of the L chain sequencing of No. 2 clone in Example 2. FIG.

It is a figure which shows the result of the H chain sequencing of No. 2 clone in Example 2. FIG.

It is a figure which shows the result of the L chain sequencing of No. 10 clone in Example 2. FIG.

It is a figure which shows the result of the H chain sequencing of No. 10 clone in Example 2. FIG.

Claims (12)

One or two or more kinds of complex proteins Xp as immunoglobulins (antibodies) that exhibit an antigen-antibody reaction with ligand X as an arbitrarily specified antigen , randomly or through characterization A method for obtaining a complex protein, comprising at least the following 1) production means separation step, 2) production means conversion step, 3) mass production preparation step, 4) transcription sequence addition step, and 5) single species mass production step . A method for obtaining a ligand-affinity complex protein comprising the following 6) characterization step as necessary .
1) An antibody-producing cell provided with production means Xm dedicated to production means (mRNA) for each of a wide variety of complex proteins Xp, which can be easily converted into replication means Xc (cDNA) capable of mass replication. By individually separating one or a plurality of single cells from an appropriate tissue or biological component of a specimen that is a human or non-human animal, the production means Xm is strictly separated for each type of target protein, A production means separation step for preventing the production means Xm, the replication means Xc, and the complex protein Xp in each step from being mixed in advance.
2) A production means conversion step of converting all or part of the mRNA constituting each production means Xm to the replication means Xc by reverse transcription PCR for one or more types of production means Xm separated from each other.
3) A mass production preparation step in which the replication means Xc obtained by the reverse transcription PCR method is mass-replicated by the following two-stage PCR to prepare a protein mass production capability.
First stage PCR: Amplification is carried out by providing the same additional sequence on the 5 ′ end side of each kind of primer corresponding to all kinds of cDNA constituting the replication means Xc.
Second stage PCR: All types of DNA fragments amplified in the first stage PCR are amplified using a single kind of primer having a sequence portion complementary to the additional sequence.
4) A transcription sequence addition step for adding a transcription sequence necessary for reconversion to the production means Xm (mRNA) in a cell-free system to the replication means Xc (cDNA) replicated in large quantities.
5) A cell-free system comprising the replication means Xc, to which the transcription sequence is added, having at least an element for reconverting the replication means Xc into the production means Xm and an element for producing a protein based on the production means Xm. A single-type mass production process in which the complex protein Xp or a complex protein Xp corresponding to a part of the mRNA defined in the production means conversion process is mass-produced for each type.
6) A characterization process in which characteristics of the composite protein Xp produced by each of the above steps are evaluated, a composite protein Xp having excellent characteristics is screened, or sequencing of these composite proteins Xp is performed. In the case where the specimen is a human, a subject presumed to be in a physiological state in which an antibody against the ligand X, which is an antigen, has already been produced in the body is selected, and the subject is allowed by social convention. 2. Acquisition of a ligand-affinity complex protein according to claim 1 , wherein one or a plurality of single cells of 1) above are separated from an appropriate tissue or biological component collected by a non-invasive means. Method. 3. The ligand-affinity complex according to claim 2, wherein B cells and / or plasma cells that are antibody-producing cells are isolated from peripheral blood collected as the tissue or biological component from the subject. Protein acquisition method. In the case where the specimen is a non-human animal, after immunizing the animal with the ligand X, separating one or more single cells of 1) from an appropriate tissue or biological component of the animal The method for obtaining a ligand affinity complex protein according to claim 1. In the production means separation step, the tissue or biological component of the specimen is put into a liquid to disperse / suspend antibody-producing cells, and incubated with magnetic microparticles bound to a certain separation antibody, followed by separation by recovering the magnetic particles bound to antibody-producing cells via antibody with a magnet, one of the claims 1 to 4, characterized in that the separation of single cell antibody producing cells of interest from the suspension method of obtaining the ligand affinity complex protein according to or. In the production means conversion step, by design of primers used for reverse transcription PCR, only mRNA encoding a complex protein Xp belonging to a specific classification category is converted to replication means Xc and / or a specific protein constituting the complex protein Xp 6. The method for obtaining a ligand affinity complex protein according to any one of claims 1 to 5, wherein only the mRNA encoding the fragment is converted to the replication means Xc. During the addition sequence of each type of primer used in the first step PCR, claims, characterized in that to keep including appropriate types of translation initiation promoting sequence portion in consideration of the biological origin of the cell-free system of the 5) The acquisition method of the ligand affinity complex protein in any one of Claims 1-6 . The ligand according to any one of claims 1 to 7, wherein in the transcription sequence addition step, PCR is performed to add at least a promoter sequence to the DNA fragment amplified in the mass production preparation step. A method for obtaining an affinity complex protein. The method for obtaining a ligand-affinity complex protein according to any one of claims 1 to 8 , wherein the cell-free system is used in the single species mass production step without being reduced. The characterization step includes the evaluation of the ligand affinity of the complex protein Xp by ELISA, the sequencing of the complex protein Xp or its gene, and the measurement of the Kd value of the complex protein Xp produced using the transformant. The method for obtaining a ligand-affinity complex protein according to any one of claims 1 to 9 , wherein at least one item is included. An antibody screening method, wherein the method for obtaining a ligand affinity complex protein according to any one of claims 1 to 10 is used for screening an antibody against ligand X in a human or non-human animal. The kit for antibody screening used for implementing the antibody screening method of Claim 11 , Comprising: At least the following components are comprised, The kit for antibody screening characterized by the above-mentioned.
11) A set of a dispersion / suspension liquid as defined in claim 5 and magnetic fine particles.
12) A primer set for reverse transcription PCR as defined in claim 6 .
13) A primer set for two-stage PCR as defined in claim 1.
14) A cell-free system as defined in 5) of claim 1.

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