JP2023107630A - Method for manufacturing antibody, and method for evaluating antibody, as well as kit related to the same - Google Patents

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

There is an application for the application of Article 30, Paragraph 2 of the Patent Act. Publication date: May 19, 2021 Publication: Journal of Bioscience and Bioengineering Vol.131, Issue 6, June 2021, Pages 696-702 Posting date: November 17, 2021 Address: https://mbsj. gakkai. online/dashboard Contents: Abstracts of the 44th Annual Meeting of the Molecular Biology Society of Japan 3. Date: December 2, 2021 Name of meeting: The 44th Annual Meeting of the Molecular Biology Society of Japan Venue: Pacifico Yokohama

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

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

As a method for producing antibodies, there is a method for producing monoclonal antibodies. This involves injecting an antigen into the body, and then recovering antibody-producing cells after a certain period of time has passed. The harvested cells are immortalized by fusing with cells such as myeloma, allowing them to proliferate permanently. After immortalization, screening is performed to obtain antibodies with superior function.

In relation to screening, Patent Document 1 discloses 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 fluorescence detection in the detection step. and a seeding step of seeding the hybridomas obtained cell by cell.

In addition, in relation to screening, Patent Document 2 describes a screening method that includes an antigen protein and a fluorescent substance bound to the N-terminus or C-terminus of the antigen protein, and is used for selection by flow cytometry. An invention relating to a detection probe for screening antibody-producing cells and the like characterized by being

In addition, although the content is unrelated to screening, Non-Patent Document 1 describes site-specific biotinylation of proteins, immobilization of proteins using the binding ability of streptavidin and biotin, and the like. (Abstract). In addition, Non-Patent Document 1 discloses an experiment in which a biotinylated protein was immobilized on a streptavidin-immobilized plate by applying the ELISA method (Indirect ELISA with biotin-tagged antigens using mouse monoclonal or rabbit polyclonal antibodies).

JP 2018-148832 A JP 2019-048794 A

Verma, V., Kaur, C., Grover, P., Gupta, A., Chaudhary, VK. PLoS One. (2018) 13(1):e0191315

In antibody screening, it is important to select highly practical antibodies. However, otherwise usable antibodies may escape selection in screening.

For example, Western blotting is mentioned as a means of antibody screening. However, there was still the possibility that antibody clones that were not positive in Western blotting contained candidates that could be used as antibodies.

Compared to screening by Western blotting, techniques using flow cytometry and labeling substances, etc., as described in Patent Documents 1 and 2, can be useful means for picking up more antibody candidates. However, when antibody screening is performed, accuracy is enhanced by performing multiple phases of screening such as primary screening and secondary screening. Therefore, there is a need for new screening methods other than the techniques of Patent Documents 1 and 2.

In view of the above points, an object of the present disclosure is to provide a new method for antibody screening.

As a result of intensive studies, the present inventor focused on a screening method using ELISA. When screening by ELISA, binding two substances that are known to bind to each other to the plate side and the antigen side, respectively, makes it possible to screen in cases where antibodies have not existed so far. .

The invention has been completed based on the above findings, and the present disclosure includes the following inventions in one aspect.
(Invention 1)
A method for producing an antibody, said method comprising a first antibody screening step, wherein said first screening step is a step of screening said antibody using a solid support, said first screening step teeth,
- providing said solid support with a first substance attached thereto;
- administering an antigenic protein corresponding to said antibody to said solid support, said antigenic protein having a second substance attached thereto, said second substance being capable of binding to said first substance; the administering;
- administering a sample containing the candidate antibody to said solid support;
- detecting said candidate antibody;
A method, including
(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,
A method, wherein the combination of said first substance and said second substance is selected from:
any one of streptavidin, neutravidin, and avidin and biotin;
transition metal ions and His tags,
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 antibodies and SUMO,
Immunoglobulin and Protein A
(Invention 4)
The method according to any one of inventions 1 to 3, wherein the method comprises a second antibody screening step,
The second screening step is a primary screening step performed before the first screening step,
The method, wherein the second screening step is screening without heat treatment and detergent treatment of the antigen protein.
(Invention 5)
The method of Invention 4, wherein the second screening step comprises fractionating hybridomas with a flow cytometer.
(Invention 6)
The method according to any one of inventions 1-5, wherein the solid support is an ELISA plate.
(Invention 7)
The method of invention 6, wherein the method comprises a third screening step,
The third screening step may be performed in parallel with the first screening step, or may be performed after the first screening step,
The third screening step comprises at least partially denaturing the antigen protein corresponding to the antibody;
The third screening step includes comparing with the results of the first screening step, and the comparison allows screening for antibodies that recognize higher-order structures.
Method.
(Invention 8)
The method of invention 7, wherein at least partially denaturing the antigenic protein comprises performing at least two or more denaturation treatment patterns.
(Invention 9)
The method according to invention 8, wherein the two or more denaturation treatment patterns include at least a pattern of heat treatment only and a pattern of combination of heat treatment and treatment with a surfactant.
(Invention 10)
A method for evaluating an antibody, said method comprising detecting binding of an antibody to an antigen, said detecting step comprising:
- providing a solid support to which the first substance is attached;
- administering to said solid support an antigenic protein corresponding to said antibody, said antigenic protein having a second substance attached thereto, said second substance being capable of binding to said first substance; the administering;
- administering a sample containing the candidate antibody to said solid support;
- administering to said solid support a reagent that detects said antibody;
A method, including
(Invention 11)
A kit for evaluating or screening an antibody, said kit comprising:
- An ELISA plate to which the first substance is adhered;
- an antigen to which a second substance capable of binding to the first substance is added;
- a reagent for detecting an antibody that binds to the antigen;
kit, including
(Invention 12)
A kit for evaluating or screening an antibody, said kit comprising:
- an ELISA plate;
- a reagent comprising a first substance for adhering to the ELISA plate;
- an antigen to which a second substance capable of binding to the first substance is added;
- a reagent for detecting an antibody that binds to the antigen;
kit, including

In one aspect of the above invention, the first substance is adhered to the solid support. Also, the antigen protein has a second substance added thereto, and the second substance is capable of binding to the first substance. This enables antibody screening, making the technique useful in combination with other screenings.

In addition, the above principle enables antibody screening targeting antigens for which antibodies have not existed so far. In this regard, the sandwich ELISA method requires a capture antibody (an antibody to stick to the bottom of the plate), but this method requires that antibodies to the antigen of interest are already present. be a condition.

In one embodiment, an expression vector that integrates into E. coli to express EGFP is shown. The upper vector was used for injection as an antigen when creating hybridomas and for primary screening by flow cytometry. In the lower vector, EGFP is fused with a biotinylated site (BirA Target). The lower vector was used in secondary screening by ELISA method. In one embodiment, the results of a gel shift assay are shown. The results indicate the interaction between biotinylated EGFP and streptavidin. In one embodiment, reproducibility of ELISA results is demonstrated. In one embodiment, the results of each screening are shown. Specifically, the percentage of positive clones from each screening is shown. In one embodiment, the antibody isotype validation results from each screen are shown. In one embodiment, Western blot results using each antibody clone are shown. In one embodiment, flow cytometry results using some antibody clones that tested negative on Western blot are shown. In one embodiment, the results of functional analysis of antibodies by immunoprecipitation are shown. In one embodiment, the results of analysis using a combination of denatured SAST-ELISA and the like are shown.

Specific embodiments for carrying out the invention will be described below. The following description is intended to facilitate understanding of the invention. it is not intended to limit the scope of the invention.

1. Methods of Producing Antibodies In one embodiment, the present disclosure relates to methods of producing antibodies. The method includes a first screening step for antibodies, and the first screening step for antibodies is screening for antibodies using a solid support. This screening step includes at least the following substeps:
- Preparing a solid support to which a first substance is attached - Administering an antigen protein corresponding to the antibody to the solid support, wherein the antigen protein is attached with a second substance, and the second substance is administering a sample containing the candidate antibody to a solid support, which is capable of binding to a first substance; detecting the candidate antibody.

Here, the solid support is not particularly limited as long as it can adhere to the first substance. Examples of solid supports include ELISA plates (eg, the bottom portion of each well of an ELISA plate), porous supports, magnetic bead particles, and the like.

Each sub-step when using an ELISA plate will be described in detail below.

1-1. Preparation of ELISA plate to which the first substance is adhered The ELISA plate is not particularly limited, and those known in the art can be used. Therefore, plate size, well size, number of wells, coating material, etc. are not particularly limited. A typical example is a 96-well plate.

A first substance is affixed to the ELISA plate (more specifically, to the bottom of each well). Examples of the first substance include streptavidin, neutravidin, avidin, transition metal ions (e.g., divalent metal ions such as nickel, cobalt, iron, copper, etc.), maltose, glutathione, tags (e.g., FLAG tag, HA tag, Myc tag, SUMO tag), an immunoglobulin for binding to Protein A, and the like.

The ELISA plate to which the first substance is attached may be obtained by purchasing a commercially available product, or may be prepared just before the start of the experiment. For example, a solution containing the first substance may be dispensed into wells of an ELISA plate and incubated for a period of time to allow it to adhere.

1-2. Administering an antigen protein corresponding to the antibody to the ELISA plate After the ELISA plate is prepared as described above, an antigen corresponding to the antibody is added to the plate (more specifically, to at least a portion of each well of the plate). A solution containing protein is administered.

The purpose is to immobilize the antigen protein of interest to the plate. To achieve this purpose, a second substance corresponding to the first substance is added to the antigen protein. This second substance has the property of being able to bond with the first substance. The binding of the first substance and the second substance indirectly fixes the antigen protein to the plate. Although the antibody to be screened and the corresponding antigen also have the property of being able to bind, the combination of these is not included in the first substance and the second substance.

The combination of the first substance and the second substance only needs to have properties that allow the two to bind together, and examples thereof include the following combinations.
- streptavidin (or avidin or neutravidin) and biotin,
- Transition metal ions and histidine (His) tags,
- glutathione and GST (glutathione S-transferase),
- maltose and MBP (maltose binding protein),
- FLAG tag antibody and FLAG tag,
- HA tag antibody and HA (hemagglutinin) tag,
- Myc tag antibody and Myc tag,
- SUMO antibody and SUMO (Small Ubiquitin-related (like) Modifier),
・Immunoglobulin and Protein A

Also, the first substance and the second substance may be reversed as long as they do not prevent them from sticking to the plate. For example, if the second substance to be added to the antigen protein is GST, the first substance directly adhered to the plate will be glutathione. In this case, gene recombination technology or the like can be applied to form a fusion protein of an antigen protein and GST.

On the other hand, for example, when the second substance to be added to the antigen protein is glutathione, the first substance directly adhered to the plate is GST. In this case, glutathione can be added to the antigen protein by any desired method.

Although other combinations are applicable, the combinations listed above are preferred. The reason for this is that there is a high probability that it will not interfere with the three-dimensional structure of the antigen protein. Any of the combinations listed above find use in protein purification. This means that these substances have little effect on the three-dimensional structure of proteins.

Some antibodies that recognize antigen proteins are sensitive to the three-dimensional structure of antigen proteins. Such antibodies cannot recognize antigen proteins that are denatured due to collapse of the three-dimensional structure, and thus may not be picked up at the screening stage. Therefore, the combinations listed above are advantageous in the sense that they do not interfere with the three-dimensional structure of the antigen protein.

Among the above combinations, any one of streptavidin, neutravidin, and avidin is preferably combined with biotin. More preferred among the above combinations are streptavidin and biotin. The reason for this is that the bond between the two is very strong. A further reason is that compared to other proteins, neutravidin and avidin, streptavidin is very resistant to denaturation-inducing treatments.

Such properties are advantageous in screening for antibodies that recognize higher-order structures. For example, screening for antibodies that recognize only conformation can be achieved by comparing at least two screening results. In this case, ELISA is performed by preparing 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, if either the first substance or the second substance is a protein or the like when the three-dimensional structure of the antigen protein is denatured, the protein may also be denatured. When the binding between the first substance and the second substance is destroyed by denaturation, detection by ELISA becomes impossible. As a result, screening becomes impossible.

However, since the binding of streptavidin and biotin is resistant to typical denaturation treatments (e.g., heat treatment, detergent treatment, etc.), a method for comparing the above two screenings becomes feasible (SDS in FIG. 2). See gel shift assay by PAGE.Band shift occurs due to binding of biotin and streptavidin even in the presence of SDS). Antibodies that recognize higher-order structures can then be picked up.

In any case, when the antigen protein to which the second substance is added is administered to the plate, the first substance and the second substance bind and the antigen protein adheres to the plate.

Moreover, before and after the step of administering the antigen protein to which the second substance is added to the plate, or before and after other steps, a treatment known in the art may be appropriately inserted. For example, before the step of administering the antigen protein to which the second substance is added to the plate, the inside of the wells may be washed with a desired washing solution (eg, PBS, etc.). Furthermore, for the purpose of preventing non-specific binding other than the binding between the first substance and the second substance, etc., it may be treated with a blocking solution. Blocking solutions include, for example, BSA dissolved in TBST, skim milk, and the like.

1-3. Applying Samples Containing Candidate Antibodies to the ELISA Plate After the antigen proteins have been attached to the plate, samples containing the candidate antibodies are applied to the plate. The sample may be a cell suspension containing the hybridomas themselves, or a solution containing the hybridoma culture supernatant. Antibodies capable of binding to the antigen protein remain on the plate even after the solution has been removed from the wells (more preferably after the wells have been washed with a wash solution). Antibodies with poor ability to bind to the antigen protein, on the other hand, are removed from the plate as the solution is removed from the wells. This allows screening for antibodies capable of binding to the antigen protein of interest.

1-4. Detecting Candidate Antibodies After the binding of antigen proteins and antibodies, the presence of antibodies remaining in the wells is detected. For example, reagents for detecting antibodies are applied to ELISA plates. Reagents for antibody detection are not particularly limited, and means known in the art can be used.

For example, the reagent for detection can be a secondary antibody capable of binding to the candidate antibody (eg, at least a portion of the constant region of the antibody). The secondary antibody may then be fused with an enzyme protein. This may further comprise administering a substrate for the enzyme protein and may further comprise detecting a reaction product of the substrate catalyzed by the enzyme protein.

Species of the secondary antibody are not particularly limited, and antibodies of mammals other than the target animal may be used. Mammals include, but are not limited to, humans, mice, rats, rabbits, goats, and the like. 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 the art, such as HRP (horseradish peroxidase), AP (alkaline phosphatase), and the like.

Alternatively, instead of fusing the enzyme protein and the secondary antibody, a fluorescent dye or fluorescent protein may be fused with the secondary antibody. In this case, fluorochromes or fluorescent proteins can be detected by specific wavelengths.

By performing the above sub-steps, antibody clones that are positive can be determined as antibodies capable of binding to the antigen. This allows antibody screening.

In addition, since the screening by ELISA does not involve denaturation of the antibody protein, more antibodies including those recognizing higher-order structures can be picked up during the screening. In addition, compared to other screening methods (e.g., porous supports, magnetic beads, Western blotting, and immunoprecipitation), the ELISA screening described above allows many antibody clones to be evaluated at once in a short period of time. do.

1-5. Preparation of Candidate Antibody Clones In the above method, candidate antibody clones must be prepared in advance. The method of cloning an antibody is not particularly limited, and can be prepared according to methods known in the art. For example, a protein that serves as an antigen may be injected into an animal, antibody-producing cells may be collected, and the cells may be immortalized by cell fusion or the like to prepare a monoclonal antibody-producing hybridoma. Furthermore, a culture supernatant or the like may be prepared from the hybridoma.

1-6. multi-stage screening
1-6-1. Primary Screening Before and after the screening process described above, another screening process (second screening) may be combined. For example, the screening steps described above may be considered secondary screening and combined with primary screening. Specific examples of the primary screening are not particularly limited, but screening methods that do not involve denaturation of the antigen protein (eg, heat treatment, treatment with a surfactant, etc.) are preferred. The reason for this is to avoid impairing the advantages of the secondary screening described above.

More preferably, in the case of primary screening, high-throughput techniques are preferred because there may be a large number of candidate clones. From this point of view, preferred primary screening includes flow cytometry (eg, FACS (registered trademark)). In an example using flow cytometry, the antigen protein may be fused with a fluorescent substance, then the hybridomas and the antigen protein may be mixed, and the hybridomas for which fluorescence is detected may be fractionated by flow cytometry.

1-6-2. Tertiary Screening 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, the following sub-steps can be included in the same way as the first screening step.
- Preparing a solid support (e.g., ELISA plate) to which the first substance is adhered - Administering an antigen protein corresponding to the antibody to the solid support (e.g., ELISA plate), wherein the antigen protein is attached with a second substance, wherein the second substance is capable of binding to the first substance;・Detecting candidate antibodies

However, this third screening differs from the first screening step in at least one respect, which includes at least partially denaturing the antigenic protein corresponding to the antibody.

Methods known in the art can be used as means for denaturation, and treatment with heat, treatment with surfactants, and the like can be used. The heat treatment includes, for example, heat treatment at 40° C. or higher, preferably 60° C. or higher, more preferably 90° C. or higher. In the case of treatment with a surfactant, typically treatment with an anionic surfactant, treatment with a cationic surfactant, or the like can be included. Examples of anionic surfactants include SDS (sodium lauryl sulfate) and the like. Examples of cationic surfactants include CTAB (Cetyl Trimethyl Ammonium Bromide) and the like.

Also, a plurality of denaturing means may be combined. For example, heat treatment and SDS treatment may be combined.

Different antigen proteins have different resistance to denaturation treatment. Therefore, denaturation conditions may be determined according to the antigen protein. For example, a temperature of about 60° C. can induce sufficient denaturation in the case of a protein that tends to lose its three-dimensional structure. On the other hand, as an extreme example, proteins found in thermophilic bacteria (for example, polymerase used in PCR) are highly resistant to heat treatment, so it is preferable to combine treatment with a surfactant separately.

The significance of separately performing the screening step including the treatment for denaturation as described above is as follows. That is, the properties of the antibody can be evaluated by comparing the results of the first screening step and the results of the third screening step. For example, assume that one antibody clone A and another antibody clone B were positive in the first screening step. At this time, in the third screening, if the antibody clone A is still positive and the antibody clone B is negative, the antibody clone B can be evaluated as an antibody that recognizes the higher-order structure of the antigen protein. can. The reason for this is that the reason why the antibody clone B was negative in the third screening is highly likely that the protein was denatured and its higher-order structure collapsed.

In another embodiment, this third screening may be Western blotting. For example, an antigen protein is loaded onto each lane of SDS PAGE and electrophoresis is started. Then, after blotting onto a membrane, it is reacted with the antibody clone sample described above. In this case, the proteins serving as antigens have been denatured by sample treatment for SDS PAGE (boiling and treatment with detergent SDS). Therefore, the antigen protein blotted on the membrane is also denatured (of course, there are some exceptions because some proteins have extremely high structural stability). Therefore, by comparing the results of the Western blotting with the results of the first screening, it is possible to extract antibody clones with a high probability of recognizing higher-order structures. For example, when Western blotting is negative and the first screening is positive, it can be determined as an antibody clone with a high probability of recognizing a higher-order structure.

1-6-3. First substance and second substance suitable for tertiary screening Considering such denaturation treatment, the combination of the first substance and second substance described above is preferably a combination with high resistance to denaturation. In other words, a combination that does not break the bond between the two by modification treatment is preferred. In this regard, streptavidin is known to be highly resistant to denaturation. Therefore, a preferred combination of the first substance and the second substance includes a combination of streptavidin and biotin.

1-6-4. Multistage evaluation of higher-order structure by tertiary screening Proteins, after being produced according to the central dogma, undergo changes to primary structure, secondary structure, tertiary structure, and sometimes quaternary structure. , the desired function can be realized.

Therefore, by setting the denaturation conditions in the tertiary screening in multiple stages, it is possible to evaluate the characteristics according to such higher-order functions. For example, denaturation conditions for the tertiary screening may be set as follows.
First denaturation condition: denaturation by heat treatment Second denaturation condition: heat treatment + treatment with surfactant

The significance of setting the denaturation conditions in such multiple stages is as follows. In other words, although it varies depending on the properties of the protein, in the case of 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. Assume that there is a property In this case, antibody clones that are positive under the first denaturing condition and negative under the second denaturing condition are likely to produce antibodies that recognize the secondary structure.

In addition, the secondary structure includes several representative examples such as α-helix and β-sheet. And each has different stability. For example, α-helix is known to be more stable than β-sheet.

Therefore, if the denaturation conditions under which both the α-helix and β-sheet collapse and the denaturation conditions under which only the β-sheet collapses in the antigen protein are known in advance, the following applications are possible. Specifically, the denaturation treatment is performed under each condition such that both the α-helix and the β-sheet are collapsed under the second denaturation condition, but only the β-sheet is collapsed under the first denaturation condition. In this case, antibody clones that are negative under the second denaturing condition and positive under the first denaturing condition are likely to produce antibodies that recognize the β-sheet structure.

Similar applications are possible when proteins have the property of forming oligomers (quaternary structures) in vivo. For example, by comparing the results of screening using an oligomer-biotin conjugate that maintains the quaternary structure with those using an oligomer-biotin conjugate with a disrupted quaternary structure, it is possible to recognize the quaternary structure. Antibodies can be screened for

Thus, by setting two or more types of denaturation treatment patterns, it is possible to not only simply evaluate whether the antibody to be screened recognizes a higher-order structure, but also what level of higher-order structure it recognizes. It is possible to make a detailed evaluation of whether or not

2. Methods Using Other Solid Supports The above embodiments describe situations where the solid support is an ELISA plate. However, the solid support may be other than an ELISA plate.

In another embodiment, the solid support may be magnetic beads. In the case of magnetic beads, the first substance is attached to the magnetic beads. Such anchoring techniques are known in the art. Alternatively, magnetic beads to which the first substance is attached may already be commercially available, such as streptavidin magnetic beads (eg Magnosphere ^™ MS160/Streptavidin JSR Life Sciences). After that, an antigen protein to which a second substance is added is prepared as in the embodiment of the ELISA method described above. Antigen proteins corresponding to the antibodies can then be administered to the magnetic beads. For example, a suspension containing magnetic beads may be charged into a container, and an antigen protein solution may be further charged into the container while stirring. After a period of time has passed, the magnetic beads can be recovered using magnetic force or the like and resuspended in a suitable solvent. Then, an antibody-containing sample is introduced, whereby the antibody bound to the antigen on the surface of the magnetic beads binds. After a certain period of time has elapsed, the magnetic beads can be collected using magnetic force or the like. The presence of antibodies on the surface of the magnetic beads can then be detected (eg, using a secondary antibody, similar to the ELISA method described above, etc.).

In yet another embodiment, the solid support may be a porous support. A porous support is a material that is also used for protein purification and the like. It has a large number of holes, allowing the sample to pass through. The material is not particularly limited, and for example, the porous support may be made of resin. The first substance can be adhered to the outer surface and/or inner surface of the porous support using known techniques. Thereafter, an antigen protein to which a second substance has been added is prepared in the same manner as in the embodiment of the ELISA method described above. An antigenic 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 or the like, and a solution containing the antigen protein may be introduced into the column. After a certain period of time has passed, an eluent that destroys the binding between the antibody and the antigen is applied to the column, and the antibody bound to the antigen can be detected. Antibody detection may be performed, for example, by using a secondary antibody as in the ELISA method described above.

3. Antibody Evaluation Methods In one embodiment, the present disclosure relates to antibody evaluation methods. For example, the antibody evaluation method of the present disclosure can be used when samples of a plurality of candidate antibody groups that are likely to recognize the same antigen are available in advance.

The antibody evaluation method in one embodiment can include the same steps as the antibody production method described above. Various variations of the antibody production method described above can be applied to the antibody evaluation method in one embodiment.

4. In one kit 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 testing.

The kit includes at least the following instruments, reagents, and the like.
・ELISA plate ・Antigen to which a second substance that can bind to the first substance is added ・Reagent for detecting antibody that binds to the antigen

The antibody to be evaluated or screened corresponds to the primary antibody that recognizes the antigen.

In addition to the above, other reagents, manuals, and the like may be included in the kit as appropriate. Examples of other reagents include solutions for washing well plates, blocking solutions for preventing non-specific binding when binding antibodies, and the like.

Also, the first substance may be immobilized in advance on the bottom of the wells of the ELISA plate. Alternatively, although the number of steps increases, a reagent containing the first substance may be separately attached to the kit. In this case, it is necessary to perform a separate step of fixing the first substance to the bottom of the well.

A reagent for detecting an antibody that binds to an antigen may be a single reagent or a combination of multiple reagents. A typical example of a reagent that detects antibody binding to an antigen is a secondary antibody. For example, when a fluorescent dye or fluorescent protein is bound to this secondary antibody, the reagent alone can be used for detection. On the other hand, when an enzyme protein (for example, the above-mentioned HRP, AP, etc.) is separately bound to the secondary antibody, a reagent serving as a reaction substrate for the enzyme protein is separately provided in combination.

In connection with the above-described embodiments, more specific examples are disclosed below, but like the above-described embodiments, they are not intended to limit the scope of the invention.

1. The prepared fluorescent protein EGFP in E. coli expression vector was used as antigen. This was incorporated into an E. coli expression vector. A vector map of the E. coli expression vector (His-EGFP-pColdII, EGFP-BirHis-pET23(+)) used is shown in FIG.

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

3. Purification of Hybridomas by Density Gradient Centrifugation After cell fusion, selection was performed with HAT. Viable cells were separated with 13% Optiprep (AXS) after 5-7 days. Cells were counted and cultured in RPMI1640 (+1 ng/mL IL-6, 10% NBS) medium containing HT.

4. Primary Screening by Flow Cytometry (MIHS Method) MIHS method (Membrane Immunoglobulin directed Hybridoma Screening and Cloning method) is one of antibody screening methods. The method is a method of screening for antibodies present on the cell membrane surface of hybridomas rather than extracting antibodies from hybridomas and screening for the antibodies (and, if necessary, screened hybridomas may be further cloned). Specifically, hybridomas were screened by the following procedure.

The purified hybridomas were seeded at 2.5×10 ⁵ cells per well of a 24-well plate (Sumitomo Bakelite) on the day before fluorescent labeling, and cultured overnight at 37° C. in 5% CO ₂ . The next day, the hybridomas were mixed with 2 nmol of His-EGFP recombinant protein in 1 ml RPMI1640 medium and the cells were incubated at 37° C. in 5% CO ₂ for 2 hours. Cells were centrifuged in RPMI1640 (+10% NBS, +IL6) medium at 800 rpm for 3 minutes, 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 passing this suspension through a filter, it was analyzed by FCM (Flow Cyto Meter) and sorted. Sorting considered positive cells with an intensity greater than or equal to about three times the mode of the non-fluorescently labeled population. Each cell was sorted into a 96-well plate (Sumitomo Bakelite) containing 200 μl/well of HT(+) RPMI1640 (+10% NBS, +IL6) medium. On the same day, hybridomas that were not fluorescently labeled were sorted into a 96-well plate (1 cell) for conventional screening.

5. Preparation of biotin-labeled antigen used in secondary screening (SAST-ELISA) SAST-ELISA method (Stereo-specific Antibody Screening Technique based on ELISA) is based on ELISA method so that stereospecific antibodies can be picked up. It is a method of screening.

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

Next, a supershift assay using streptavidin was performed to confirm the biotin labeling of the EGFP-BirHis protein. To 5 μl of a 10 μM Biotin-labeled EGFP-BirHis protein solution, 2.5 μl of 5×Sample buffer for SDS-PAGE and 7.5 μl of PBS were added and boiled for 5 minutes. After returning to room temperature, 3.75 µg of Streptavidin was added and left at room temperature for 5 minutes. The gel run on 12% SDS-PAGE was stained with CBB to confirm the change in molecular weight due to the binding of streptavidin to the biotin-labeled antigen.

Samples of EGFP-BirHis not labeled with biotin and EGFP-BirHis labeled with biotin were prepared with/without streptavidin, and electrophoresed by SDS-PAGE. The results are shown in FIG. Comparing samples 4 and 5, in sample 5, when streptavidin was added to biotin-labeled EGFP-BirHis, no band was observed at the molecular weight of EGFP-BirHis alone (approximately 28 kDa). Instead, a band can be confirmed at a position higher than the streptavidin band that is not observed in other samples. These bands could be judged to be complexes of EGFP-BirHis and streptavidin, and it was confirmed that they were almost completely labeled with biotin.

6. Investigation of Conditions for SAST-ELISA Method 50 μl of a streptavidin solution adjusted to 4 μg/ml with PBS was added to a 96-well ELISA plate and allowed to stand at 4° C. overnight. The next day, 50 μl of 10% skim milk dissolved in TBST was added (final concentration 5%) 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, shaken for 10 minutes, and then the wells were washed three times with PBS. Next, 100 μl of the obtained anti-EGFP monoclonal antibody hybridoma culture supernatant of 94 clones diluted with TBST was added, and the plate was shaken at room temperature for 1 hour. After washing the wells with PBS three times, 50 μl of goat anti-mouse IgG (H+L)-HRP diluted 10,000 times with TBST was added, and the plate was shaken at room temperature for 1 hour. After washing the wells three times with PBS, reactions were visualized using the TMB microwell peroxidase system as in the ELISA protocol. In order to obtain reproducibility, the same operation was performed twice to confirm reproducibility.

The reproducibility of the SAST-ELISA method was confirmed using the culture supernatant of anti-EGFP mAb 94 clones obtained so far. The results of the same experiment conducted twice were plotted on a graph in which the vertical axis represents the absorbance values of the first time and the horizontal axis represents the absorbance values of the second time (Fig. 3). Linear approximation reveals that y=0.9672x and that y=x. Therefore, it was confirmed that the SAST-ELISA method developed this time has high reproducibility.

7. Investigation of conditions for SAST-ELISA method When 273 clones obtained in the primary screening were judged by SAST-ELISA, 172 clones were positive (Fig. 4). Therefore, 63% of the clones analyzed were positive and screened by 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) positive clones could be obtained with a higher probability.

Isotypes were determined for 72 clones obtained by the MIHS-SAST ELISA method and compared with the existing ELISA-Western blotting method and MIHS-Western blotting method (Fig. 5). As a result, it was found that IgG class monoclonal antibodies can be frequently obtained by the screening method using the MIHS method, as has been the case up to now.

8. Western blotting reactivity of SAST-ELISA clones and flow cytometry Using the culture supernatant of 72 clones among the positive SAST-ELISA clones, reactivity to EGFP-BirHis protein overexpressed and purified in E. coli was analyzed by Western blotting. It was analyzed by blotting (Fig. 6). Some clones were determined to be positive (sample numbers 1-9, 12, 14-20, 23, 26-29, 37-39, 41, 49, 51-53, 55-56, 58, 60-72). As a result, positive bands were confirmed in 63.9% (46/72 clones).

Here, some of the negative clones were verified. Specifically, Clone 50 (S2E8) and Clone 54 (S2F1) were verified. These clones were non-reactive clones in Western blotting. However, when fluorescence-labeled (specifically, the His-EGFP recombinant protein was administered to the hybridomas as in the primary screening described above) and analyzed by a cell sorter, the hybridomas were strongly fluorescent-labeled as shown in FIG. In general, it has been observed that monoclonal antibodies that recognize higher order structures do not react in Western blotting. It was suggested that the monoclonal antibodies secreted by the clones obtained by the MIHS-SAST ELISA method this time contain high-order structure-recognizing antibodies in a certain proportion.

9. Antibody Functional Assay by Immunoprecipitation Immunoprecipitation was performed to examine the structure recognition ability of the monoclonal antibodies obtained by MIHS-SAST ELISA. To 500 μl of PBS to which 10 μl of streptavidin magnetic beads (JSR Life Sciences) was added, 5.2 μg of biotin-labeled GFP recombinant protein was added and mixed periodically at room temperature for 20 minutes by inversion. After the beads were adsorbed to a magnet and the supernatant was removed, the beads were washed twice with 500 μl of PBS. 100 μl of culture supernatant of EGFP hybridomas obtained by MIHS-SAST ELISA method (16 clones) and ELISA-Western method (4 clones) and 50 μl of PBS were added and gently rotated at 4° C. for 2 hours. The beads were separated by being adsorbed to a magnet, and after removing the supernatant, the beads were washed twice with 500 μl of PBS. The mixture was suspended in 20 μl of 1×SDS sample buffer, boiled for 3 minutes, and 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 (Fig. 8).

Of the 4 clones obtained by ELISA-Western blotting method, only 1 clone could be immunoprecipitated, whereas 16 clones obtained by MIHS-SAST-ELISA method (15 clones: IgG, 1 clone: IgM). All were found to be immunoprecipitable (data not shown for IgM clones). These results show that the SAST-ELISA method can obtain immunoprecipitable monoclonal antibodies.

10. Principle of Confirmation of Structure Recognition Ability by Denatured SAST-ELISA Method A denatured SAST ELISA method was performed to examine whether the monoclonal antibody obtained by the SAST ELISA method has the structure recognition ability. This is based on the following principle. The EGFP protein used in the SAST ELISA loses its green fluorescence when boiled, so the active structure of the recombinant protein is thermally denatured. However, the binding of biotin to streptavidin is extremely strong, so the biotin-labeled recombinant protein retains its biotin-streptavidin binding ability even after boiling. Therefore, when the EGFP protein boiled and heat-denatured by SAST ELISA is compared with the undenatured EGFP protein having green fluorescence, a monoclonal antibody that reacts only with the undenatured EGFP protein but does not react with the heat-denatured EGFP protein was found. can be said to be a structure-recognizing antibody whose epitope has been lost by heat denaturation.

Therefore, the reactivity of the existing clones (ELISA-Western blotting: 22 clones and MIHS-Western blotting: 68 clones) and the newly obtained MIHS-SAST ELISA clones (72 clones) by this denaturing SAST ELISA method and non-denaturing SAST ELISA method was analyzed.

The following three patterns are conceivable as reactivity patterns.
(1) Denatured SAST-ELISA negative, non-denatured SAST-ELISA negative (2) Denatured SAST-ELISA negative, non-denatured SAST-ELISA positive (3) Denatured SAST-ELISA positive, non-denatured SAST-ELISA positive

An antibody corresponding to pattern (1) is, for example, an antibody that recognizes the primary structure of a protein that has been exposed due to collapse of the higher-order structure.

An antibody corresponding to pattern (2) is considered to be an antibody that recognizes a higher-order structure. Conformation in this case is believed to include tertiary structure and secondary structure, particularly weak secondary structure.

An antibody corresponding to pattern (3) is considered to be an antibody that recognizes a higher-order structure. However, as a difference from pattern (2), it is also positive in denatured SAST-ELISA. Therefore, compared with the antibody corresponding to pattern (2), the antibody corresponding to pattern (3) is considered to be an antibody that recognizes a thermodynamically stronger structure.

As described above, by combining denatured SAST-ELISA and non-denatured SAST-ELISA, antibodies recognizing higher-order structures can be screened.

Here, in the above denatured SAST-ELISA, the antigen protein EGFP is denatured by boiling. As a more preferable method, in addition to the denaturation pattern by boiling, further combination of the denaturation pattern by the combination of boiling and SDS makes it possible to further analyze the level of the higher-order structure recognized by the antibody in detail.

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

Possible patterns at this time include a case where the denaturation pattern due to the combination of boiling and SDS is negative (pattern (3)-1) and a case where it is positive (pattern (3)-2). .

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

When the denaturation treatment pattern is only boiling, the higher-order structure recognized by the antibody can be evaluated in two stages (for example, pattern (2) < pattern (3) in terms of thermodynamic stability). . However, furthermore, by combining the pattern of denaturation treatment with boiling and SDS, three steps (for example, in terms of thermodynamic stability, pattern (2) < pattern (3)-1 < pattern (3)-2 ) can be evaluated.

11. Confirmation results of structure recognition ability by denatured SAST-ELISA method Based on the above principle, existing clones (ELISA-Western blotting: 22 clones and MIHS-Western blotting: 68 clones) and newly obtained MIHS-SAST ELISA clones (72 clone) was analyzed for reactivity.

Results are shown in FIG. All of the MIHS-SAST ELISA clones were shown to correspond to antibodies recognizing higher-order structures. In addition, no antibodies corresponding to Pattern 2 were obtained by other techniques, ELISA-WS and MIHS-WS. This is probably because antibody clones corresponding to Pattern 2 were excluded because other techniques include screening using Western blotting.

As described above, by combining two or more denaturation treatment patterns, the structure recognized by the antibody can be analyzed in detail. Then, the desired antibody can be screened based on the analysis results.

Specific embodiments of the invention have been described above. The above embodiments are only specific examples, and the present invention is not limited to the above embodiments. For example, technical features disclosed in one of the above embodiments may be applied to other embodiments. Also, unless otherwise stated, for a particular method, some steps may be interchanged with other steps, and additional steps may be added between two particular steps. The scope of the invention is defined by the claims.

Claims (12)

A method for producing an antibody, said method comprising a first antibody screening step, wherein said first screening step is a step of screening said antibody using a solid support, said first screening step teeth,
- providing said solid support with a first substance attached thereto;
- administering an antigenic protein corresponding to said antibody to said solid support, said antigenic protein having a second substance attached thereto, said second substance being capable of binding to said first substance; the administering;
- administering a sample containing the candidate antibody to said solid support;
- detecting said candidate antibody;
A method, including 2. The method of claim 1, wherein the solid support is any one of a porous support, magnetic bead particles, and an ELISA plate. 3. The method of claim 1 or 2,
A method, wherein the combination of said first substance and said second substance is selected from:
any one of streptavidin, neutravidin, and avidin and biotin;
transition metal ions and His tags,
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 antibodies and SUMO,
Immunoglobulin and Protein A The method of any one of claims 1-3, wherein the method comprises a second antibody screening step,
The second screening step is a primary screening step performed before the first screening step,
The method, wherein the second screening step is screening without heat treatment and detergent treatment of the antigen protein. 5. The method of claim 4, wherein said second screening step comprises flow cytometric fractionation for hybridomas. The method of any one of claims 1-5, wherein the solid support is an ELISA plate. 7. The method of claim 6, said method comprising a third screening step,
The third screening step may be performed in parallel with the first screening step, or may be performed after the first screening step,
The third screening step comprises at least partially denaturing the antigen protein corresponding to the antibody;
The third screening step includes comparing with the results of the first screening step, and the comparison allows screening for antibodies that recognize higher-order structures.
Method. 8. The method of claim 7, wherein at least partially denaturing the antigen protein comprises performing at least two or more denaturation treatment patterns. 9. The method of claim 8, wherein the two or more denaturation treatment patterns include at least a heat treatment only pattern and a combination heat treatment and surfactant treatment pattern. A method for evaluating an antibody, said method comprising detecting binding of an antibody to an antigen, said detecting step comprising:
- providing a solid support to which the first substance is attached;
- administering an antigenic protein corresponding to said antibody to said solid support, said antigenic protein having a second substance attached thereto, said second substance being capable of binding to said first substance; the administering;
- administering a sample containing the candidate antibody to said solid support;
- administering to said solid support a reagent that detects said antibody;
A method, including A kit for evaluating or screening an antibody, said kit comprising:
- An ELISA plate to which the first substance is adhered;
- an antigen to which a second substance capable of binding to the first substance is added;
- a reagent for detecting an antibody that binds to the antigen;
kit, including A kit for evaluating or screening an antibody, said kit comprising:
- an ELISA plate;
- a reagent comprising a first substance for adhering to the ELISA plate;
- an antigen to which a second substance capable of binding to the first substance is added;
- a reagent for detecting an antibody that binds to the antigen;
kit, including