Methods for producing antibodies, methods for evaluating antibodies, and related kits.

Abstract

To provide a novel method that performs screening of antibodies.SOLUTION: In one aspect, the present disclosure provides an invention as below. A method is aimed to manufacture antibodies, and the method includes a first screening step of the antibody. The first screening step is the step of screening the antibody using a solid support body. The first screening step includes: preparing the solid support body having a first substance fixed; administering antigen protein corresponding to the antibody to the solid support body, in which the antigen protein has a second substance added, and the second substance allows for binding to the first substance; administering a sample including the antibody serving as a candidate to the sold support body; and detecting the antibody serving as the candidate.SELECTED DRAWING: None

Description

Technical Field

[0001] The present disclosure relates to methods for producing antibodies, methods for evaluating antibodies, and kits related thereto.

Background Art

[0002] The immune system is a system by which vertebrates, including mammals, prevent the invasion of foreign substances from outside. The immune system is a complex system involving various cells and proteins such as cytokines. One of the proteins that play an important role in the immune system is an antibody. Antibodies are applied to antibody cocktail therapies and the like and are useful for the treatment of infectious diseases.

[0003] As a method for producing an antibody, there is a method for producing a monoclonal antibody. In this method, an antigen is injected into the body, and after a certain period of time has elapsed, cells that produce the antibody are collected. The collected cells are fused with cells such as myeloma to be immortalized and can be grown permanently. After immortalization, screening is performed to obtain an antibody having excellent functions.

[0004] Regarding screening, Patent Document 1 discloses a method for producing a monoclonal antibody, which comprises a labeling step of contacting a hybridoma with a fluorescently labeled antigen, a detection step of detecting the hybridoma after the labeling step by flow cytometry, and a seeding step of seeding one by one the hybridomas in which fluorescence has been detected in the detection step.

[0005] Also regarding screening, Patent Document 2 discloses an invention related to a detection probe for screening antibody-producing cells, which comprises an antigen protein and a fluorescent substance bound to the N-terminal or C-terminal of the antigen protein and is used for selection by flow cytometry.

[0006] Furthermore, although unrelated to screening, Non-Patent Literature 1 discloses site-specific biotinylation of proteins and protein immobilization using the binding ability of streptavidin and biotin (Abstract). Non-Patent Literature 1 also discloses an experiment in which biotinylated proteins were immobilized on plates immobilized with streptavidin using the ELISA method (Indirect ELISA with biotin-tagged antigens using mouse monoclonal or rabbit polyclonal antibodies). [Prior art documents] [Patent Documents]

[0007] [Patent Document 1] Japanese Patent Publication No. 2018-148832 [Patent Document 2] Japanese Patent Publication No. 2019-048794 [Non-patent literature]

[0008] [Non-Patent Document 1] Verma, V., Kaur, C., Grover, P., Gupta, A., Chaudhary, VK. PLoS One. (2018)13(1):e0191315 [Overview of the project] [Problems that the invention aims to solve]

[0009] When screening antibodies, the key is to select those with high practical value. However, there is a possibility that antibodies that are actually usable may be overlooked during the screening process.

[0010] For example, Western blotting is used as a method for screening antibodies. However, there was still a possibility that some antibody clones that did not test positive by Western blotting could be candidates that could be used as antibodies.

[0011] Compared to screening by Western blotting, methods using flow cytometry and labeled substances, as described in Patent Documents 1 and 2, can be useful for selecting a larger number of antibody candidates. However, when screening antibodies, accuracy is improved by performing screening in multiple phases, such as primary and secondary screening. Therefore, there is a need for new screening methods other than those described in Patent Documents 1 and 2.

[0012] In view of the above, this disclosure aims to provide a novel method for screening antibodies. [Means for solving the problem]

[0013] The inventors, through diligent research, focused on a screening method using ELISA. By binding two substances known to bind to each other to the plate and antigen sides, respectively, during ELISA screening, screening becomes possible even in cases where antibodies are not previously present.

[0014] Based on the above findings, the invention is completed, and this disclosure encompasses, in one aspect, the following invention. (Invention 1) A method for producing an antibody, the method comprising a first screening step of the antibody, the first screening step being a step of screening the antibody using a solid support, the first screening step being, • Prepare the solid support to which the first substance is fixed, · Administering an antigen protein corresponding to the antibody to the solid support, wherein the antigen protein is added with a second substance, and the second substance is capable of binding to the first substance, and said administering; · Administering a sample containing a candidate antibody to the solid support; · Detecting the candidate antibody; A method comprising the above. (Invention 2) The method of Invention 1, wherein the solid support is any one of a porous support, magnetic bead particles, and an ELISA plate. (Invention 3) The method of Invention 1 or 2, wherein the combination of the first substance and the second substance is selected from the following. Any one of streptavidin, neutravidin, and avidin, and biotin; Transition metal ions and His tag; Glutathione and GST; Maltose and MBP; FLAG tag antibody and FLAG tag; HA tag antibody and HA tag; Myc tag antibody and Myc tag; SUMO antibody and SUMO; Immunoglobulin and Protein A (Invention 4) The method according to any one of Inventions 1 to 3, wherein the method includes a second screening step of an antibody, the second screening step is a primary screening step performed before the first screening step, and the second screening step is a screening not involving heat treatment and treatment with a surfactant for the antigen protein. (Invention 5) The method of Invention 4, wherein the second screening step includes fractionating the hybridoma with a flow cytometer. (Invention 6) The method according to any one of Inventions 1 to 5, wherein the solid support is a plate for ELISA. (Invention 7) The method according to Invention 6, wherein the method includes a third screening step, The third screening step may be carried out in parallel with the first screening step or may be carried out after the first screening step. The third screening step includes at least partially denaturing the antigen protein corresponding to the antibody. The third screening step includes comparing with the result of the first screening step, and by this comparison, it becomes possible to screen an antibody that recognizes a higher-order structure. Method. (Invention 8) The method according to Invention 7, wherein at least partially denaturing the antigen protein includes carrying out at least two or more types of denaturation treatment patterns. (Invention 9) The method according to Invention 8, wherein the two or more types of denaturation treatment patterns include at least a pattern of only heat treatment and a pattern of a combination of heat treatment and treatment with a surfactant. (Invention 10) A method for evaluating an antibody, the method including a step of detecting the binding of an antibody and an antigen, and the detecting step includes: - preparing a solid support to which a first substance is fixed; - administering an antigen protein corresponding to the antibody to the solid support, wherein the antigen protein is added with a second substance, and the second substance is capable of binding to the first substance; - administering a sample containing a candidate antibody to the solid support; - administering a reagent for detecting the antibody to the solid support. The method comprising: (Invention 11) A kit for evaluating or screening an antibody, the kit comprising: • An ELISA plate on which the first substance is immobilized, • An antigen to which a second substance capable of binding to the first substance is attached, • A reagent for detecting an antibody that binds to the antigen, A kit that includes this. (Invention 12) A kit for evaluating or screening antibodies, wherein the kit is • ELISA plates and • A reagent containing a first substance for fixation to the ELISA plate, • An antigen to which a second substance capable of binding to the first substance is attached, • A reagent for detecting an antibody that binds to the antigen, A kit that includes this. [Effects of the Invention]

[0015] In one aspect of the above invention, a first substance is immobilized on a solid support. Furthermore, a second substance is attached to the antigen protein, and the second substance is capable of binding to the first substance. This enables antibody screening, and the method becomes useful in combination with other screening methods.

[0016] Furthermore, the above principle makes it possible to screen for antibodies against antigens against which no antibodies currently exist. In relation to this, while the sandwich ELISA method requires capture antibodies (antibodies to be fixed to the bottom of the plate), this method requires that antibodies against the target antigen already exist. [Brief explanation of the drawing]

[0017] [Figure 1] In one embodiment, an expression vector incorporated into Escherichia coli to express EGFP is shown. The upper vector was used for injection as an antigen during hybridoma creation and for primary screening by flow cytometry. In the lower vector, a biotinylation site (BirA Target) is fused to EGFP. The lower vector was used for secondary screening by ELISA. [Figure 2] In one embodiment, the results of a gel shift assay are shown. These results indicate the interaction between biotinylated EGFP and streptavidin, among other things. [Figure 3] In one embodiment, the reproducibility of ELISA results is demonstrated. [Figure 4] In one embodiment, the results of each screening are shown. Specifically, the percentage of positive clones obtained from each screening is shown. [Figure 5] In one embodiment, the verification results of the antibody isotypes obtained in each screening are shown. [Figure 6] In one embodiment, the results of Western blotting using each antibody clone are shown. [Figure 7] In one embodiment, the results of flow cytometry using a subset of antibody clones that were negative by Western blotting are shown. [Figure 8] In one embodiment, the results of analyzing the function of an antibody by immunoprecipitation are shown. [Figure 9] In one embodiment, the results of an analysis combining modified SAST-ELISA and other methods are shown. [Modes for carrying out the invention]

[0018] The following describes specific embodiments for carrying out the invention. The following description is intended to facilitate understanding of the invention and is not intended to limit the scope of the present invention.

[0019] 1. Method for producing antibodies In one embodiment, the disclosure relates to a method for producing an antibody. The method includes a first antibody screening step, which is a step of screening an antibody using a solid support. This screening step includes at least the following substeps: • Prepare a solid support to which the first substance is fixed. The administration of an antigen protein corresponding to an antibody onto a solid support, wherein the antigen protein has a second substance attached to it, and the second substance is capable of binding to the first substance. • Administering a sample containing candidate antibodies onto a solid support. • Detecting candidate antibodies

[0020] Here, the solid support is not particularly limited and can be any material that can adhere to the first substance. Examples of solid supports include ELISA plates (e.g., the bottom of each well in an ELISA plate), porous supports, and magnetic bead particles.

[0021] The following sections detail each sub-step when using an ELISA plate.

[0022] 1-1. Preparation of an ELISA plate to which the first substance has been immobilized. ELISA plates are not particularly limited, and any known in this field can be used. Therefore, the plate size, well size, number of wells, and coating material are not particularly limited. A typical example is a 96-well plate.

[0023] ELISA plates (more specifically, the bottom of each well) have a primary substance immobilized on them. Examples of primary substances include streptavidin, neutraavidin, avidin, transition metal ions (e.g., divalent metal ions such as nickel, cobalt, iron, and copper), maltose, glutathione, antibodies corresponding to various tags (e.g., FLAG tag, HA tag, Myc tag, SUMO tag), and immunoglobulins for binding to Protein A.

[0024] ELISA plates with the first substance already immobilized can be obtained by purchasing commercially available plates, or they can be prepared immediately before the start of the experiment. For example, a solution containing the first substance may be dispensed into the wells of an ELISA plate and incubated for a certain period of time to allow it to solidify.

[0025] 1-2. Administer the antigen protein corresponding to the antibody to the ELISA plate. Once the ELISA plate described above is prepared, a solution containing the antigen protein corresponding to the antibody is added to the plate (more specifically, to at least a portion of each well of the plate).

[0026] The purpose of this method is to immobilize the target antigen protein onto the plate. To achieve this, a second substance corresponding to the first substance is attached to the antigen protein. This second substance has the property of being able to bind to the first substance. Through the binding of the first and second substances, the antigen protein is indirectly immobilized onto the plate. Although antibodies to be screened and their corresponding antigens also have the property of being able to bind, these combinations are not included in the first and second substances.

[0027] The combination of the first and second substances only needs to have properties that allow them to bond together. For example, the following combinations can be given. • Streptavidin (or avidin or neutraavidin) and biotin, • Transition metal ions and histidine (His) tags, Glutathione and GST (glutathione S-transferase), • Maltose and MBP (maltose-binding protein), FLAG-tagged antibodies and FLAG tags, HA-tagged antibodies and HA (hemagglutinin) tags, • Myc-tagged antibodies and Myc tags, • SUMO antibodies and SUMO (Small Ubiquitin-related (like) Modifier) • Immunoglobulins and ProteinA

[0028] Furthermore, the first and second substances may be arranged in reverse order, as long as this does not hinder their attachment to the plate. For example, if the second substance attached to the antigen protein is GST, the first substance that is directly attached to the plate will be glutathione. In this case, a fusion protein of the antigen protein and GST can be formed by applying genetic engineering techniques.

[0029] On the other hand, if, for example, the second substance to be attached to the antigen protein is glutathione, then the first substance directly immobilized on the plate will be GST. In this case, glutathione can be attached to the antigen protein by the desired method.

[0030] While other combinations are also applicable, the combinations listed above are preferred. This is because they are highly likely to not interfere with the three-dimensional structure of the antigen protein. All of the combinations listed above are used for protein purification. This means that these substances have little effect on the three-dimensional structure of the protein.

[0031] Some antibodies that recognize antigen proteins are sensitive to the three-dimensional structure of the antigen protein. If the antigen protein's structure is disrupted and denatured, these antibodies may not be able to recognize it, potentially preventing them from being detected during the screening stage. Therefore, the combinations listed above are advantageous because they do not interfere with the three-dimensional structure of the antigen protein.

[0032] Furthermore, among the above combinations, the preferred combination is streptavidin, neutraavidin, or avidin, paired with biotin. And among the above combinations, the most preferred is streptavidin and biotin. This is because the binding between the two is very strong. Another reason is that, compared to other proteins, neutraavidin, and avidin, streptavidin is very resistant to denaturation-inducing treatments.

[0033] These properties are advantageous when screening antibodies that recognize higher-order structures. For example, screening antibodies that recognize only three-dimensional structures can be achieved by comparing at least two screening results. In this case, an ELISA is performed using a sample in which the three-dimensional structure of the antigen protein is maintained and a sample in which the three-dimensional structure of the antigen protein is denatured. However, when the three-dimensional structure of the antigen protein is denatured, if either the first or second substance is a protein, that protein may also be denatured. If the binding between the first and second substances breaks down due to denaturation, detection by ELISA becomes impossible. As a result, screening becomes impossible.

[0034] However, since streptavidin and biotin are resistant to typical denaturation treatments (e.g., heat treatment, treatment with surfactants), the two screening methods described above can be compared (see the gel shift assay by SDS PAGE in Figure 2. Even in the presence of SDS, biotin and streptavidin bind and band shift occurs). This allows for the identification of antibodies that recognize higher-order structures.

[0035] In any case, by administering the antigen protein to which the second substance has been attached to the plate, the first and second substances bind together, and the antigen protein is fixed to the plate.

[0036] Furthermore, known processes in this field may be inserted as appropriate before or after the step of administering the antigen protein with the second substance attached to the plate, or before or after other steps. For example, before administering the antigen protein with the second substance attached to the plate, the wells may be washed with a desired washing solution (e.g., PBS). In addition, the plate may be treated with a blocking solution for purposes such as preventing nonspecific binding other than the binding of the first substance to the second substance. Examples of blocking solutions include BSA dissolved in TBST, skim milk, etc.

[0037] 1-3. Add the sample containing the candidate antibody to the ELISA plate. After the antigen protein has been immobilized on the plate, a sample containing candidate antibodies is added to the plate. The sample may be a cell suspension containing the hybridoma itself, or a solution containing the culture supernatant of the hybridoma. Antibodies that can bind to the antigen protein remain on the plate even after the solution is removed from the wells (more preferably after the wells are washed with washing solution). On the other hand, antibodies that have little ability to bind to the antigen protein are removed from the plate as the solution is removed from the wells. This allows for screening of antibodies that can bind to the target antigen protein.

[0038] 1-4. Detecting candidate antibodies. After the antigen protein and antibody have bound, the presence of any remaining antibodies in the wells is detected. For example, a reagent for detecting antibodies is added to the ELISA plate. The reagent for detecting antibodies is not particularly limited, and methods known in this field can be used.

[0039] For example, the reagent for detection may be a secondary antibody capable of binding to a candidate antibody (e.g., at least a portion of the constant region of the antibody). The secondary antibody may be fused with an enzyme protein. In this case, the procedure may further involve administering a substrate for the enzyme protein, and further involve detecting the reaction product of the substrate catalyzed by the enzyme protein.

[0040] Furthermore, the species of the secondary antibody is not particularly limited and may be an antibody from a mammal other than the target animal. Examples of mammals include humans, mice, rats, rabbits, and goats, but are not limited to these. For example, if screening for human antibodies, the secondary antibody may be a mouse antibody that recognizes human antibodies. In addition, the enzyme protein fused to the secondary antibody can be an enzyme protein known in this field, such as HRP (horseradish peroxidase) or AP (alkaline phosphatase).

[0041] Alternatively, instead of fusing the enzyme protein with the secondary antibody, a fluorescent dye or fluorescent protein may be fused with the secondary antibody. In this case, the fluorescent dye or fluorescent protein can be detected at a specific wavelength.

[0042] By performing the above sub-steps, antibody clones that test positive can be identified as antibodies capable of binding to the antigen. This enables antibody screening.

[0043] Furthermore, since the ELISA screening method described above does not involve the denaturation of antibody proteins, it is possible to pick up more antibodies, including those that recognize higher-order structures, during the screening process. In addition, compared to other screening methods (e.g., porous supports, magnetic beads, Western blotting, and immunoprecipitation), the ELISA screening method allows for the evaluation of many antibody clones at once in a short amount of time.

[0044] 1-5. Preparation of candidate antibody clones In the above method, it is necessary to prepare candidate antibody clones in advance. The method for creating antibody clones is not particularly limited and can be done according to methods known in this field. For example, an antigen protein may be injected into an animal, antibody-producing cells may be collected, these cells may be immortalized by cell fusion or the like to prepare a hybridoma that produces monoclonal antibodies. Furthermore, a culture supernatant or the like may be prepared from the hybridoma.

[0045] 1-6. Multi-stage screening 1-6-1. Primary Screening Another screening step (second screening) may be combined before or after the screening step described above. For example, the above screening step may be considered a secondary screening, and a primary screening may be combined with it. There are no particular limitations on specific examples of primary screening, but preferably, a screening method that does not involve denaturation of the antigen protein (e.g., heat treatment, treatment with a surfactant, etc.) is preferred. This is to avoid compromising the advantages of the secondary screening described above.

[0046] More preferably, in the case of primary screening, a method with high throughput is preferred because there may be many candidate clones. From this viewpoint, a preferred primary screening method is flow cytometry (e.g., FACS®). In an example using flow cytometry, a fluorescent substance may be fused to the antigen protein, then the hybridoma and antigen protein may be mixed, and the hybridomas from which fluorescence is detected may be fractionated by flow cytometry.

[0047] 1-6-2.3rd Screening Furthermore, a further screening (third screening) may be performed after or in parallel with the first screening step described above. In one embodiment, this third screening is a modified version of the first screening step described above. Therefore, it may include the following sub-steps, similar to the first screening step. • Prepare a solid support (e.g., an ELISA plate) on which the first substance is immobilized. The method of administering an antigen protein corresponding to an antibody onto a solid support (e.g., an ELISA plate), wherein the antigen protein has a second substance attached to it, and the second substance is capable of binding to the first substance. • Administering a sample containing candidate antibodies onto a solid support (e.g., an ELISA plate). • Detecting candidate antibodies

[0048] However, this third screening differs from the first screening step in at least one respect, which is that it involves at least partially denaturing the antigen protein corresponding to the antibody.

[0049] Methods known in the art can be used to modify the material, and include heat treatment and surfactant treatment. Heat treatment includes, for example, treatment at a temperature of 40°C or higher, preferably 60°C or higher, and more preferably 90°C or higher. Surfactant treatment typically includes treatment with anionic surfactants or cationic surfactants. An example of anionic surfactant is SDS (sodium lauryl sulfate). An example of cationic surfactant is CTAB (Cetyl Trimethyl Ammonium Bromide).

[0050] Furthermore, multiple modification methods may be combined. For example, heat treatment and SDS treatment may be combined.

[0051] The resistance to denaturation varies depending on the individual antigen protein. Therefore, the denaturation conditions should be determined according to the antigen protein. For example, for proteins whose three-dimensional structure is easily disrupted, a temperature of around 60°C can induce sufficient denaturation. On the other hand, as an extreme example, proteins found in thermophilic bacteria (e.g., polymerase used in PCR) have high resistance to heat treatment, so it is preferable to combine this with treatment using a surfactant.

[0052] The significance of performing a separate screening process that includes denaturation, as described above, is as follows: By comparing the results of the first screening process with the results of the third screening, the properties of the antibody can be evaluated. For example, suppose that in the first screening process, antibody clone A and another antibody clone B are positive. In this case, if in the third screening, antibody clone A is still positive, but antibody clone B is negative, then antibody clone B can be evaluated as an antibody that recognizes the higher-order structure of the antigen protein. This is because the reason why antibody clone B was negative in the third screening is highly likely to be that the protein was denatured and its higher-order structure was destroyed.

[0053] In another embodiment, this third screening may be performed by Western blotting. For example, the antigen protein is loaded into each lane of an SDS-PAGE and electrophoresis is started. After blotting onto a membrane, it is reacted with the antibody clone sample described above. In this case, the antigen protein is denatured by the sample processing of the SDS-PAGE (boiling and treatment with the surfactant SDS). Therefore, the antigen protein blotted onto the membrane is also in a denatured state (of course, there are some exceptions as some proteins have extremely high structural stability). By comparing the results of this Western blotting with the results of the first screening described above, antibody clones with a high probability of recognizing higher-order structures can be extracted. For example, if the Western blotting is negative and the first screening is positive, it can be determined to be an antibody clone with a high probability of recognizing higher-order structures.

[0054] 1-6-3. First and second substances suitable for tertiary screening Considering such denaturation treatments, the combination of the first and second substances described above is preferably one that is highly resistant to denaturation. That is, a combination in which the bond between the two substances does not break down due to denaturation treatment is preferred. In this regard, streptavidin is known to be highly resistant to denaturation. Therefore, a preferred combination of the first and second substances is streptavidin and biotin.

[0055] 1-6-4. Multi-stage evaluation of higher-order structures by tertiary screening Proteins, after being manufactured according to the central dogma, undergo transformations through primary, secondary, tertiary, and sometimes quaternary structures to achieve their desired function.

[0056] Therefore, by creating multiple stages of degeneration conditions in the tertiary screening, it is possible to evaluate characteristics corresponding to these higher-order functions. For example, the degeneration conditions in the tertiary screening may be set as follows. First modification condition: Modification by heat treatment Second modification condition: Heat treatment + treatment with surfactant

[0057] The significance of setting denaturation conditions in such a multi-stage manner is as follows: Although it varies depending on the properties of the protein, we can assume that for a certain protein, the tertiary structure collapses under the first denaturation condition, but the secondary structure is maintained, and under the second denaturation condition, the secondary structure also collapses. In this case, an antibody clone that is positive under the first denaturation condition and negative under the second denaturation condition may produce an antibody that recognizes the secondary structure.

[0058] Furthermore, there are several representative examples of secondary structures, such as α-helices and β-sheets. Each has different levels of stability; for example, α-helices are known to be more stable than β-sheets.

[0059] Therefore, if the denaturation conditions under which both the α-helix and β-sheet collapse in an antigen protein, and the denaturation conditions under which only the β-sheet collapses, are known in advance, the following applications become possible. Specifically, denaturation treatment is performed under conditions such that both the α-helix and β-sheet collapse under the second denaturation condition, but only the β-sheet collapses under the first denaturation condition. In this case, an antibody clone that is negative under the second denaturation condition and positive under the first denaturation condition may produce an antibody that recognizes the β-sheet structure.

[0060] Similar applications are possible when proteins have the property of forming oligomers (quaternary structures) in vivo. For example, by comparing screening results using a complex of oligomers that maintain their quaternary structure and biotin with screening results using a complex of oligomers that have lost their quaternary structure and biotin, antibodies that recognize quaternary structures can be screened.

[0061] By setting two or more denaturation treatment patterns in this way, it becomes possible to perform not only a simple evaluation of whether or not the antibody being screened recognizes higher-order structures, but also a detailed evaluation of what level of higher-order structure it recognizes.

[0062] 2. Other methods using solid supports The above embodiment describes a state in which the solid support is an ELISA plate. However, the solid support may be something other than an ELISA plate.

[0063] In another embodiment, the solid support may be magnetic beads. In the case of magnetic beads, the first material is fixed to the magnetic beads. Such fixing methods are known in the art. Alternatively, streptavidin magnetic beads (e.g., Magnosphere) may be used. TMMagnetic beads with the first substance attached, such as MS160 / Streptavidin (JSR Life Sciences), may already be commercially available. Subsequently, an antigen protein to which the second substance has been attached is prepared, similar to the embodiment of the ELISA method described above. Then, the antigen protein corresponding to the antibody can be administered to the magnetic beads. For example, a suspension containing magnetic beads may be placed in a container, and while stirring, a solution of the antigen protein may be added to the container. After a certain period of time, the magnetic beads can be recovered using magnetic force or the like and resuspended in a suitable solvent. Then, a sample containing the antibody is added, causing the antibody bound to the antigen on the surface of the magnetic beads to bind. After a certain period of time, the magnetic beads can be recovered using magnetic force or the like. Subsequently, the presence of the antibody on the surface of the magnetic beads can be detected (for example, by using a secondary antibody, similar to the ELISA method described above).

[0064] In yet another embodiment, the solid support may be a porous support. Porous supports are materials also used for protein purification and the like. They have numerous pores, allowing the sample to pass through sufficiently. The material is not particularly limited; for example, the porous support may be made of resin. The first substance can be fixed to the outer and / or inner surface of the porous support using known methods. Then, an antigen protein to which the second substance has been attached is prepared, similar to the embodiment of the ELISA method described above. The antigen protein corresponding to the antibody can then be administered to the porous support. For example, the porous support may be packed into an affinity column, and a solution containing the antigen protein may be introduced into the column. After a certain period of time, an eluent that disrupts the binding of the antibody and antigen can be introduced into the column, and the antibody bound to the antigen can be detected. Antibody detection may be performed, for example, by using a secondary antibody, similar to the ELISA method described above.

[0065] 3. Methods for evaluating antibodies In one embodiment, the present disclosure relates to a method for evaluating antibodies. For example, if samples of multiple candidate antibodies that may recognize the same antigen are available in advance, the antibody evaluation method of the present disclosure can be used.

[0066] The antibody evaluation method in one embodiment may include the same steps as the antibody production method described above. Furthermore, various variations of the antibody production method described above can be applied to the antibody evaluation method in one embodiment.

[0067] 4. Kit In one embodiment, the disclosure relates to a kit for evaluating or screening antibodies. The use of the kit is not particularly limited and may be for research or diagnostic purposes.

[0068] The kit includes at least the following instruments and reagents. • Plates for ELISA • An antigen to which a second substance capable of binding to a first substance has been attached. • Reagents for detecting antibodies that bind to antigens

[0069] The primary antibody that recognizes the antigen is the antibody that is being evaluated or screened for.

[0070] In addition to the above, other reagents, manuals, etc. may be included in the kit as appropriate. Examples of other reagents include a solution for washing the well plate and a blocking solution to prevent nonspecific binding when binding antibodies.

[0071] Alternatively, the first substance may be pre-fixed to the bottom of the wells in the ELISA plate. Alternatively, although this would increase the number of steps, a reagent containing the first substance may be included separately in the kit. In this case, a separate step is required to fix the first substance to the bottom of the wells.

[0072] Furthermore, the reagent used to detect antibodies that bind to an antigen may be a single reagent or a combination of multiple reagents. A typical example of a reagent used to detect antibodies that bind to an antigen is a secondary antibody. If, for example, a fluorescent dye or fluorescent protein is bound to this secondary antibody, detection can be performed using the reagent alone. On the other hand, if an enzyme protein (for example, HRP or AP as mentioned above) is bound to the secondary antibody, a reagent that serves as a reaction substrate for the enzyme protein is provided separately in combination with the secondary antibody. [Examples]

[0073] In relation to the embodiments described above, further specific embodiments are disclosed below, but, as with the embodiments described above, these do not limit the scope of the present invention.

[0074] 1. Preparation of E. coli expression vector The fluorescent protein EGFP was used as an antigen. This was incorporated into an E. coli expression vector. The vector maps of the E. coli expression vectors used (His-EGFP-pColdII, EGFP-BirHis-pET23(+)) are shown in Figure 1.

[0075] 2. Immunity and Cell Fusion 100 μL of modified complete Freund's adjuvant and 100 μg (1 μg / μL) of His-EGFP protein were emulsified and intraperitoneally immunized BALB / cAJcl mice. Eighteen days later, these mice were additionally immunized intraperitoneally with 100 μg (1 μg / μL PBS) of His-EGFP protein (pH 7.2). Three days after the final immunization, splenocytes were harvested from the mice and electrically fused with myeloma cells SP2 / 0.

[0076] 3. Purification of hybridomas by density gradient centrifugation. After cell fusion, selection was performed using HAT. Live cells were isolated 5-7 days later using 13% Optiprep (AXS). Cell counts were measured and the cells were cultured in RPMI1640 medium (+1 ng / mL IL-6, 10% NBS) containing HT.

[0077] 4. Primary screening using flow cytometry (MIHS method) The MIHS method (Membrane Immunoglobulin directed Hybridoma Screening and cloning method) is one of the antibody screening methods. Rather than extracting antibodies from hybridomas and screening those antibodies, this method screens for antibodies present on the cell membrane surface of hybridomas (and, if necessary, the screened hybridomas may be further cloned). Specifically, hybridomas were screened using the following procedure.

[0078] The purified hybridomas were fluorescently labeled the day before use in a 24-well plate (Sumitomo Bakelite) at a rate of 2.5 × 10⁶ per well. 5 Cells were seeded and cultured overnight at 5% CO2 and 37°C. The following day, 2 nmol of His-EGFP recombinant protein was mixed with 1 ml of RPMI1640 medium to the hybridomas, and the cells were incubated at 5% CO2 and 37°C for 2 hours. The cells were centrifuged at 800 rpm for 3 minutes in RPMI1640 (+10% NBS, +IL6) medium, washed three times, and finally resuspended in 500 μl of RPMI1640 medium. 5 μl of 7AAD was added to this cell suspension and mixed by inversion. After filtering this suspension, it was analyzed by FCM (Flow Cytometer) and sorted. Sorting was performed by considering cells with an intensity approximately three times or greater than the mode of the unlabeled population as positive. Cells were sorted individually into 96-well plates (Sumitomo Bakelite) with 200 μl / well of HT(+)RPMI1640 (+10% NBS, +IL6) medium added. Additionally, hybridomas that were not fluorescently labeled on the same day were sorted into 1-cell portions in a 96-well plate for conventional screening.

[0079] 5. Preparation of biotin-labeled antigens for use in secondary screening (SAST-ELISA) SAST-ELISA (Stereo-specific Antibody Screening Technique based on ELISA) is a screening method based on the ELISA method that allows for the detection of stereospecific antibodies.

[0080] Biotin labeling of purified EGFP-BirHis protein was performed according to the protocol described in Fairhead and Howarth (Methods Mol Biol. 2015; 1266: 171-184.). 20 μM EGFP-BirHis protein was added to 500 μl PBS containing 5 mM MgCl2, 2 mM ATP, 1 μM BirA (Biotinligase expressed and purified in E. coli), and 150 μM MD-biotin, and gently mixed at 30°C. After 1 hour, another 150 μM MD-biotin and 1 μM BirA were added, and the mixture was gently mixed for another hour. Subsequently, the mixture was dialyzed twice with 1 L of PBS to remove unreacted biotin.

[0081] Next, a supershift assay using streptavidin was performed to confirm the biotin labeling of the EGFP-BirHis protein. 5 μl of a 10 μM biotin-labeled EGFP-BirHis protein solution was added to 2.5 μl of 5× sample buffer for SDS-PAGE and 7.5 μl of PBS, and boiled for 5 minutes. After returning to room temperature, 3.75 μg of streptavidin was added and allowed to stand at room temperature for 5 minutes. The gel, electrophoresed using 12% SDS-PAGE, was stained with CBB, and the change in molecular weight due to the binding of streptavidin to the biotin-labeled antigen was confirmed.

[0082] Samples were prepared by adding streptavidin to both unlabeled EGFP-BirHis and biotin-labeled EGFP-BirHis, and without adding streptavidin. The results of electrophoresis by SDS-PAGE are shown in Figure 2. Comparing samples 4 and 5, in sample 5, when streptavidin was added to biotin-labeled EGFP-BirHis, no band was observed at all for the molecular weight of EGFP-BirHis alone (approximately 28 kDa). Instead, a band was observed at a higher position than the streptavidin band, which was not observed in the other samples. These bands were determined to be complexes of EGFP-BirHis and streptavidin, confirming that biotin labeling was almost completely successful.

[0083] 6. Optimization of SAST-ELISA method conditions 50 μl of streptavidin solution prepared to 4 μg / ml in PBS was added to a 96-well ELISA plate and allowed to stand overnight at 4°C. The following day, 50 μl of 10% skim milk dissolved in TBST (final concentration 5%) was added, and the plate was shaken at room temperature for 1 hour. Next, the solution in the wells was removed, biotin-labeled EGFP recombinant protein (200 ng / well) was added to the wells, and the plate was shaken for 10 minutes, after which the wells were washed three times with PBS. Next, 100 μl of the culture supernatant of 94 clones of anti-EGFP monoclonal antibody, diluted in TBST, was added, and the plate was shaken at room temperature for 1 hour. After washing the wells three times with PBS, 50 μl of goat anti-mouse IgG(H+L)-HRP diluted 10,000-fold in TBST was added, and the plate was shaken at room temperature for 1 hour. After washing the wells three times with PBS, the reaction was visualized using the TMB microwell peroxidase system, as in the ELISA protocol. To ensure reproducibility, the same operation was performed twice to verify its reproducibility.

[0084] The reproducibility of the SAST-ELISA method was confirmed using the culture supernatant of 94 anti-EGFP mAb clones obtained to date. The results of the same experiment performed twice were plotted on a graph with the absorbance values ​​of the first and second experiments on the x-axis (Figure 3). Linear approximation showed that the relationship was approximately y=x, with y=0.9672x. Therefore, it was confirmed that the SAST-ELISA method developed in this study has high reproducibility.

[0085] 7. Optimization of SAST-ELISA method conditions Of the 273 clones obtained in the primary screening, 172 were evaluated by SAST-ELISA (Figure 4). Therefore, 63% of the analyzed clones were positive, and we were able to obtain positive clones with a higher probability than when screening using the ELISA-Western and MIHS-Western methods as previously shown (Sakaguchi A et.al., J Biosci Bioeng. (2021) 131(6):696-702. doi: 10.1016 / j.jbiosc.2021.02.006).

[0086] The isotypes of 72 clones obtained using the MIHS-SAST ELISA method were determined and compared with the conventional ELISA-western blotting method and the MIHS-Western blotting method (Figure 5). As a result, it was found that, as in previous trends, IgG class monoclonal antibodies can be obtained at a high frequency using the screening method via the MIHS method.

[0087] 8. Western blotting reactivity and flow cytometry of SAST-ELISA clones The reactivity of 72 clones that tested positive by SAST-ELISA to the EGFP-BirHis protein, which was overexpressed and purified in E. coli, was analyzed by Western blotting (Figure 6). Some clones were identified as positive (sample numbers 1-9, 12, 14-20, 23, 26-29, 37-39, 41, 49, 51-53, 55-56, 58, 60-72 in Figure 6). As a result, a positive band was confirmed in 63.9% (46 / 72 clones).

[0088] Here, we examined some of the clones that tested negative. Specifically, we examined Clone 50 (S2E8) and 54 (S2F1). These clones did not react to Western blotting. However, when they were fluorescently labeled (specifically, His-EGFP recombinant protein was administered to the hybridomas, as in the primary screening described above) and analyzed using a cell sorter, they were strongly fluorescently labeled as shown in Figure 7. Generally, it has been observed that monoclonal antibodies that recognize higher-order structures do not react to Western blotting. This suggests that a certain proportion of the monoclonal antibodies secreted by the clones obtained using the MIHS-SAST ELISA method contain antibodies that recognize higher-order structures.

[0089] 9. Testing of antibody function by immunoprecipitation. Immunoprecipitation was performed to investigate the structural recognition ability of monoclonal antibodies obtained by the MIHS-SAST ELISA method. 5.2 μg of biotin-labeled recombinant GFP protein was added to 500 μl of PBS containing 10 μl of streptavidin magnetic beads (JSR Life Sciences), and the mixture was periodically inverted and mixed at room temperature for 20 minutes. After removing the supernatant by adsorbing the beads with the magnet, the mixture was washed twice with 500 μl of PBS. 100 μl of culture supernatant from EGFP hybridomas obtained by the MIHS-SAST ELISA method (16 clones) and the ELISA-Western method (4 clones) was added to 50 μl of PBS, and the mixture was gently rotated at 4°C for 2 hours. After separating the mixture by adsorbing the beads with the magnet and removing the supernatant, the mixture was washed twice with 500 μl of PBS. The mixture was suspended in 20 μl of 1×SDS sample buffer, boiled for 3 minutes, and then centrifuged to collect the supernatant. The presence or absence of IgG antibodies was confirmed by Western blotting using goat anti-mouse IgG(H+L)-HRP antibody or anti-mouse IgM antibody (Figure 8).

[0090] Of the four clones obtained using the ELISA-Western blotting method, only one could be immunoprecipitated. In contrast, all 16 clones (15 IgG clones, 1 IgM clone) obtained using the MIHS-SAST-ELISA method were able to be immunoprecipitated (data for the IgM clone is not included). These results indicate that monoclonal antibodies suitable for immunoprecipitation can be obtained using the SAST-ELISA method.

[0091] 10. Principle of confirming structural recognition ability using the modified SAST-ELISA method To investigate whether monoclonal antibodies obtained by the SAST ELISA method possess structural recognition ability, a denatured SAST ELISA method was performed. This is based on the following principle: The EGFP protein used in the SAST ELISA method loses its green fluorescence when boiled, indicating that the active structure of the recombinant protein is thermally denatured. However, because the binding of biotin to streptavidin is extremely strong, the biotin-streptavidin binding ability of biotin-labeled recombinant proteins is retained even after boiling. Therefore, when comparing EGFP protein denatured by boiling using the SAST ELISA method with undenatured EGFP protein that retains green fluorescence, monoclonal antibodies that react only with the undenatured EGFP protein and not with the thermally denatured EGFP protein can be considered structural recognition antibodies whose epitopes have been lost due to thermal denaturation.

[0092] Therefore, we analyzed the reactivity of previously obtained clones (ELISA-Western blotting: 22 clones and MIHS-Western blotting: 68 clones) and newly acquired MIHS-SAST ELISA clones (72 clones) using this denatured SAST ELISA method and the non-denatured SAST ELISA method.

[0093] There are three possible patterns of reaction. (1) Negative for denatured SAST-ELISA and negative for non-denatured SAST-ELISA (2) Negative denatured SAST-ELISA, positive non-denatured SAST-ELISA (3) Positive for denatured SAST-ELISA and positive for non-denatured SAST-ELISA

[0094] The antibody corresponding to pattern (1) is thought to be, for example, an antibody that recognizes the primary structure of a protein that has been exposed due to the breakdown of its higher-order structure.

[0095] Antibodies corresponding to pattern (2) are thought to be antibodies that recognize higher-order structures. In this case, higher-order structures are thought to include tertiary structures and secondary structures, especially weak secondary structures.

[0096] The antibody corresponding to pattern (3) is thought to be an antibody that recognizes higher-order structures. However, unlike pattern (2), it is also positive in denatured SAST-ELISA. Therefore, compared to the antibody corresponding to pattern (2), the antibody corresponding to pattern (3) is thought to be an antibody that recognizes thermodynamically stronger structures.

[0097] As described above, by combining denatured SAST-ELISA and non-denatured SAST-ELISA, it is possible to screen for antibodies that recognize higher-order structures.

[0098] In the above-mentioned denatured SAST-ELISA, the antigen protein EGFP is denatured by boiling. A more preferable method is to combine the denaturation pattern obtained by boiling with the denaturation pattern obtained by a combination of boiling and SDS, which makes it possible to analyze the level of higher-order structure recognized by the antibody in more detail.

[0099] For example, in the case of pattern (3) above, the antibody reaction can be verified after further denaturation treatment using a combination of boiling and SDS.

[0100] The possible patterns in this case are a negative result in the denaturation pattern obtained by the combination of boiling and SDS (pattern (3)-1) and a positive result (pattern (3)-2).

[0101] In this case, the antibody that reacts with pattern (3)-2 is able to recognize a thermodynamically stable higher-order structure compared to the antibody that reacts with pattern (3)-1.

[0102] If the denaturation treatment pattern is boiling only, the higher-order structure recognized by the antibody can be evaluated in two stages (for example, from the perspective of thermodynamic stability, pattern (2) < pattern (3)). However, by further combining the denaturation treatment patterns of boiling and SDS, it can be evaluated in three stages (for example, from the perspective of thermodynamic stability, pattern (2) < pattern (3)-1 < pattern (3)-2).

[0103] 11. Confirmation of structural recognition ability by denatured SAST-ELISA method Based on the above principles, we analyzed the reactivity of previously obtained clones (ELISA-Western blotting: 22 clones and MIHS-Western blotting: 68 clones) and newly acquired MIHS-SAST ELISA clones (72 clones).

[0104] The results are shown in Figure 9. All MIHS-SAST ELISA clones were shown to correspond to antibodies that recognize higher-order structures. Furthermore, no antibodies corresponding to pattern 2 were obtained using the other methods, ELISA-WS and MIHS-WS. This is likely because the other methods include screening using Western blotting, which excluded antibody clones corresponding to pattern 2.

[0105] As described above, by combining two or more denaturation treatment patterns, it is possible to analyze in detail the structures recognized by antibodies. Based on the analysis results, it is then possible to screen for the desired antibodies.

[0106] The above describes specific embodiments of the invention. The above embodiments are merely examples, and the present invention is not limited to these embodiments. For example, the technical features disclosed in one of the above embodiments can be applied to other embodiments. Also, unless otherwise specified, for a particular method, it is possible to swap the order of some steps with those of other steps, and further steps may be added between two specific steps. The scope of the present invention is defined by the claims.

Claims

1. A method for producing an antibody, the method comprising a first antibody screening step and a second antibody screening step, the first screening step being a step of screening an antibody using a solid support, the first screening step being, - Prepare the solid support to which the first substance is fixed, - Administering an antigen protein corresponding to the antibody to the solid support, wherein the antigen protein has a second substance attached to it, and the second substance is capable of binding to the first substance, - Administering a sample containing candidate antibodies to the solid support, - To detect the candidate antibodies mentioned above, Includes, The method wherein the second screening step is a primary screening step performed prior to the first screening step, and the second screening step includes fractionation of the hybridoma using a flow cytometer.

2. The method according to claim 1, wherein the solid support is one of a porous support, magnetic bead particles, and an ELISA plate.

3. The method according to claim 1 or 2, A method in which the combination of the first substance and the second substance is selected from the following: One of streptavidin, neutraavidin, and avidin, and biotin, Transition metal ions and His tags, Glutathione and GST, Maltose and MBP, FLAG-tagged antibodies and FLAG tags, HA-tagged antibodies and HA tags, Myc-tagged antibodies and Myc tags, SUMO antibodies and SUMO, Immunoglobulins and Protein A

4. A method according to any one of claims 1 to 3, The method wherein the second screening step is a screening that does not involve heat treatment or treatment with a surfactant on the antigen protein.

5. A method according to any one of claims 1 to 4, wherein the solid support is an ELISA plate.

6. The method according to claim 5, wherein the method includes a third screening step, The third screening step may be performed in parallel with the first screening step, or after the first screening step. The third screening step includes at least partially denaturing the antigen protein corresponding to the antibody, The third screening step includes comparing the results with those of the first screening step, and the comparison makes it possible to screen for antibodies that recognize higher-order structures. method.

7. A method according to claim 6, wherein at least partially denaturing the antigen protein includes performing at least two or more patterns of denaturation treatment.

8. A method according to claim 7, wherein the two or more modification treatment patterns include at least one pattern of heat treatment alone and one pattern of a combination of heat treatment and treatment with a surfactant.

9. A method for evaluating an antibody, the method comprising the step of detecting the binding of an antibody to an antigen, the detection step being, - Fractionation of hybridomas using a flow cytometer. - Prepare a solid support to which the first substance is fixed, - Administering an antigen protein corresponding to the antibody derived from the hybridoma onto the solid support, wherein the antigen protein has a second substance attached to it, and the second substance is capable of binding to the first substance, - Administering a sample containing candidate antibodies to the solid support, - Administering the reagent for detecting the antibody to the solid support, Methods that include...

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