Antibody having domain having modified isoelectric point

Abstract

The purpose of the present invention is to provide an antibody capable of achieving high detection sensitivity in an immunochromatography method. The present invention provides a polypeptide including a protein domain having an antigen binding site and a protein domain having no antigen binding site, wherein the theoretical isoelectric point (pI) of the protein domain having an antigen binding site is equal to or less than the theoretical isoelectric point (pI) of the protein domain having no antigen binding site.

Description

Antibodies with domains having modified isoelectric points

[0001] The present invention relates to a polypeptide and a method for using the same, and more specifically to an antibody having a domain with an altered isoelectric point and a method for using the same, a method for producing a complex between the antibody and immunochromatographic particles, and an immunochromatographic kit using the antibody-particle complex.

[0002] In recent years, there has been a demand in the medical and food industries for technologies that can easily and quickly determine the presence or absence of diseases and pathogens. Various technologies have been developed to meet these needs. Currently, it is possible to easily and quickly perform a variety of tests, such as those for the presence or absence of pathogen infections such as influenza, pregnancy, the presence or absence of cancer markers and cardiac markers, and the presence or absence of allergens and substances that cause food poisoning in foods. Many measurement methods have been developed for these purposes, including immunochromatography, fluorescent immunochromatography, enzyme immunoassay, latex agglutination assay, and chemiluminescence assay. Among these, immunochromatography, which can be performed visually, is widely used because it does not require special equipment or specialized knowledge and allows anyone to easily and quickly perform a diagnosis. For example, pregnancy tests, which are a type of immunochromatography, have become so versatile that they are available at general pharmacies. In addition, coronavirus detection kits (Toyobo Co., Ltd.) (Patent Document 1) and influenza detection kits (Sekisui Medical Co., Ltd.) (Patent Document 2) are commercially available.

[0003] Immunochromatographic assays (hereafter referred to as "immunochromatography") utilize antigen-antibody reactions and include the sandwich method and competitive method. Measurement methods are broadly divided into lateral flow methods, in which the detected substance is spread horizontally across the membrane, and flow-through methods, in which the detected substance is spread vertically. From the perspective of simplicity, the lateral flow method using the sandwich method to capture the antigen with an antibody is widely used. The measurement procedure is as follows: (i) an antibody that reacts with the antigen or the like to be detected is immobilized at a specific site (test line) on a membrane such as nitrocellulose; (ii) a detection reagent is prepared in which a labeled substance called a color-forming particle carries an antibody that reacts with the target, and this is applied to a conjugate pad and dried, and the membrane, sample pad, and absorption pad are combined to prepare an immunochromatography kit; (iii) a developer solution mixed with a sample to be tested for the presence or absence of the antigen is dropped onto the sample pad, and the presence or absence of color on the test line (TL) is observed visually (or with a simple immunochromatography reader) to determine the presence or absence of the antigen. If the antigen is present in the sample, a line will develop on the test line.

[0004] Generally, in most cases where a visual determination is made, it is used as a qualitative analysis to determine only the presence or absence of the antigen to be detected.

[0005] Methods for binding antibodies to particles used in immunochromatography include binding through physical interaction and binding through chemical reaction. However, in either method, the binding points of the antibody moiety that binds to the particle are random, and the Fab region that captures antigens such as viruses is not necessarily oriented toward the solution, resulting in a problem of reduced antigen capture efficiency and insufficient sensitivity.

[0006] Under these circumstances, oYo-Link™ Single-Biotin (AlphaThera) discloses a method for improving antibody orientation by specifically modifying the Fc region of an antibody with biotin and then binding the antibody to particles carrying streptavidin. However, this method has problems such as the need to irradiate the antibody with 365 nm UV light, which may cause denaturation of the antibody, and the need to carry streptavidin on particles, which makes the process complicated.

[0007] As described above, a method for improving the sensitivity of immunochromatography by controlling the orientation of antibodies using a simple procedure has not yet been realized, and its development is highly desired.

[0008] JP 2023-016530 A JP 2020-63911 A

[0009] Yu Tang et al., MAbs (2021); 13(1): 1993768Doudou et al., Langmuir(2019) 35:4860-4867;Vadim V. Sumbayev et al, Small (2013) 9:472-477K. Murakami et al, Analytical Biochemistry (2021) 632:114337Aleksandr E. et al, Chemistry & Biology (2012)19:449-455

[0010] In view of the above-mentioned current state of the prior art, the problem to be solved by the present invention is to provide an antibody that can achieve high detection sensitivity in immunochromatography.

[0011] As a result of extensive research and experiments conducted by the inventors to solve the above-mentioned problems, they confirmed that by modifying the isoelectric point of the domains that make up the antibody, the antibody can be bound to negatively charged particles in an oriented manner, thereby increasing the detection sensitivity in immunochromatography, and thus completed the present invention.

[0012] That is, the present invention is as follows: [1] A polypeptide comprising a protein domain having an antigen-binding site and a protein domain not having an antigen-binding site, wherein the theoretical isoelectric point (pI) of the protein domain having the antigen-binding site is equal to or lower than the theoretical isoelectric point (pI) of the protein domain not having an antigen-binding site. [2] The polypeptide according to [1], wherein the polypeptide is an intact antibody or a fragment thereof. [3] The polypeptide according to [2], wherein the antibody fragment is selected from the group consisting of Fab, F(ab')2, Fab', a single-chain antibody, and a diabody linked to the protein domain not having an antigen-binding site via a linker. [4] The polypeptide according to [2] or [3], wherein the protein domain having the antigen-binding site is Fab and / or the protein domain not having an antigen-binding site is CH2 or CH3. [5] The polypeptide according to any of [1] to [4], wherein at least one amino acid residue in the protein domain not having an antigen-binding site is modified. [6] The polypeptide according to any one of [2] to [5], wherein the antibody is IgG, and at least one amino acid residue at amino acid positions 345, 355, 358, 359, 360, 361, 375, 386, 387, 389, 395, 413, 415, 416, 418, 419, 421, 433, 436, 438, and 441, as specified by EU numbering, is modified to lysine (K) or arginine (R). [7] The polypeptide according to [6], wherein the IgG is derived from human, mouse, or rabbit, and the amino acid residue at position 386 or 433 is modified to lysine (K) or arginine (R). [8] A particle-polypeptide complex comprising a particle having a negative zeta potential and a polypeptide defined in any one of [1] to [7]. [9] The particle-polypeptide complex according to [8], wherein the degree of orientation of the particle-polypeptide complex is 1.0 or more.

[10] The particle-polypeptide complex according to [8] or [9], wherein the particles having a negative zeta potential are cellulose-colored particles.

[11] A conjugate pad containing the particle-polypeptide complex defined in any one of [8] to

[10] .

[12] An immunochromatographic diagnostic kit comprising the conjugate pad defined in

[11] .

[13] A method for producing a particle-polypeptide complex defined in any of [8] to

[11] , comprising a step of reacting particles having a negative zeta potential with a polypeptide, wherein the theoretical isoelectric point (pI) of a protein domain having an antigen-binding site of the polypeptide is equal to or lower than the theoretical isoelectric point (pI) of a protein domain not having an antigen-binding site.

[14] The method according to

[13] , wherein the step is a step of reacting particles having a negative zeta potential with a polypeptide in a buffer solution that satisfies the following condition: pH of the buffer solution ≦ the theoretical isoelectric point (pI) of the protein domain not having an antigen-binding site.

[15] The method according to

[13] or

[14] , wherein the step is a step of reacting particles having a negative zeta potential with a polypeptide in a buffer solution that satisfies the following condition: theoretical isoelectric point (pI) of the protein domain having an antigen-binding site ≦ pH of the buffer solution ≦ the theoretical isoelectric point (pI) of the protein domain not having an antigen-binding site.

[0013] The antibody according to the present invention can achieve high detection sensitivity in immunochromatography.

[0014] 1 shows an example of an immunochromatographic diagnostic kit to which the antibody of the present invention can be applied. 2 is a conceptual diagram illustrating the antibody of the present invention.

[0015] The present inventors have surprisingly found that when the theoretical isoelectric point pI(A) of a protein domain having an antigen-binding site for an antigen is equal to or lower than the pI(B) of a protein domain not having an antigen-binding site, and the protein domain is bound to particles with a negative zeta potential in a buffer solution, the antigen detection sensitivity in an immunochromatography kit can be improved.

[0016] For example, as disclosed in Non-Patent Document 1 (Yu Tang et al., MAbs (2021);13(1):1993768), in many antibodies that exist in nature, the theoretical isoelectric point pI(A) of the Fab, which is the protein domain that has the antigen-binding site for the antigen, is equal to or higher than the pI(B) of the Fc, which is the protein domain that does not have the antigen-binding site. Therefore, when attempting to bind an antibody to a negatively charged particle through electrostatic interaction, adjusting the pH of the buffer solution used for binding does not allow the domain charge state that achieves the desired orientation, "the Fab domain region is negative and the Fc domain region is positive," to be achieved.

[0017] Furthermore, in Non-Patent Document 2 (Doudou et al., Langmuir (2019) 35:4860-4867), the orientation of the antibody on negatively charged magnetic particles is improved by binding the antibody in an acidic pH environment (buffer pH ≦ pI(B) ≦ pI(A)). However, because both the Fab and Fc domains are positively charged in this environment, it is believed that control of the orientation on the negatively charged particles is insufficient. Therefore, the inventors hypothesized that by modifying the amino acid sequence of the antibody to achieve pI(A) ≦ pI(B), which is the isoelectric point relationship that allows the Fab and Fc domains to be negatively and positively charged, respectively, the negatively charged particles and the positively charged Fc domain of the antibody would be attracted to each other, improving the orientation of the entire antibody, allowing for more efficient antigen capture and increasing the sensitivity of the immunochromatography kit. Based on this hypothesis, we conducted numerous experiments and surprisingly found that by binding the antibody to particles with a negative zeta potential in a buffer solution, it was possible to improve the antigen detection sensitivity in an immunochromatography kit.

[0018] The following describes embodiments in detail. As used herein, the term "antibody" refers to a protein that recognizes and binds to a specific antigen or substance, and is sometimes referred to as immunoglobulin (Ig). A typical antibody typically consists of two identical structural components (sometimes referred to herein as "A chain" and "B chain"), which are connected by two disulfide bonds. Each component further comprises two light chains (sometimes referred to herein simply as "light chains") and two heavy chains (sometimes referred to herein simply as "heavy chains") interconnected by disulfide bonds. There are two types of light chains, called λ chains and κ chains, and five types of heavy chains, called γ chains, μ chains, α chains, δ chains, and ε chains. IgG is further classified into subclasses depending on the type of heavy chain; when the heavy chain is γ1, it is called IgG1; when the heavy chain is γ2, it is called IgG2a or IgG2b; when the heavy chain is γ3, it is called IgG3; and when the heavy chain is γ4, it is called IgG4.

[0019] The antibody of the present invention may also be an antibody fragment and / or derivative. Examples of antibody fragments include F(ab')2, Fab, and Fv. Examples of antibody derivatives include antibodies with artificially introduced amino acid mutations in the light and / or heavy chain constant region portions, antibodies with modified domain configurations of the light and / or heavy chain constant regions, antibodies with two or more Fc regions per molecule, glycosylated antibodies, bispecific antibodies, antibody conjugates in which an antibody or antibody fragment is bound to a protein other than an antibody, antibody enzymes, tandem scFvs, bispecific tandem scFvs, and diabodies. Furthermore, when the above-mentioned antibody or a fragment or derivative thereof is derived from a non-human animal, chimeric antibodies or humanized antibodies in which part or all of the sequences other than the CDRs have been replaced with corresponding sequences of a human antibody are also included in the antibody of the present invention. Unless otherwise specified, the simple term "antibody" used in the present invention also includes antibody fragments and / or derivatives. The term "diabody" refers to a small antibody fragment prepared by constructing an sFv fragment with a short linker (approximately 5-10 residues) between the VH and VL domains, such that the V domains pair inter-chain rather than intra-chain, thereby generating a bivalent fragment, i.e., a fragment with two antigen-binding sites. According to the present invention, diabodies linked to a protein domain that does not have an antigen-binding site via an appropriate linker can also be used. Examples of linkers that can be used include, but are not limited to, flexible linkers (see Bird et al. (1988) Science 242:423-426; Huston et al. (1988) Proc. Natl. Acad. Sci. USA 85:5879-5883; McCafferty et al., Nature (1990) 348:552-554).

[0020] As used herein, the term "domain" refers to a protein (or polypeptide) or a part of its structure, which is a stable unit part in a protein structure that folds autonomously, and a protein may be formed by binding multiple domains via covalent bonds. The length of a domain is not limited, and may be, for example, from about 20 amino acid residues to about 800 amino acid residues, and may be of various lengths. Note that the hinge region is not considered to be part of a domain.

[0021] As used herein, the term "domain having an antigen-binding site" refers to a domain having an antigen-binding site (CDR, paratope) in a polypeptide chain, and specific examples include, but are not limited to, Fab, sc-FV, and VHH. As described above, "antigen-binding site" is a term used interchangeably with "CDR" and "paratope," and refers to the part of an antibody that recognizes and binds to an antigen. "CDR" or "complementarity-determining region" refers to each region of an antibody variable domain that is hypervariable in sequence and contains antigen-contact residues. Generally, antibodies contain six CDRs: three in the VH domain (CDR-H1, CDR-H2, and CDR-H3) and three in the VL domain (CDR-L1, CDR-L2, and CDR-L3). Unless otherwise specified, CDR residues and other residues of the variable domain (e.g., FR residues) are numbered herein according to the definition of Chothia & Lesk J. Mol. Biol. 196:901-917 (1987) and Chothia et al., Nature 342:878-883 (1989). The "paratope" is formed by several individual amino acid residues from the heavy and light chain variable domains of an antibody, which are positioned in close spatial proximity in the tertiary structure of the Fv region.

[0022] As used in the present invention, the term "domain without an antigen-binding site" refers to a domain that does not have an antigen-binding site (CDR, paratope) in the polypeptide chain, and specific examples thereof include CH2, CH3, etc. in the case of IgG. In this case, it is desirable that the "domain without an antigen-binding site" and the "domain with an antigen-binding site" are as far apart as possible within the entire protein. For example, in the case of IgG, it is desirable that the isoelectric point of CH3 is lower than the isoelectric point of the protein domain with the antigen-binding site. This is because positively charging a site of the antibody that is as far away as possible from the protein domain with the antigen-binding site can effectively improve the orientation of the antibody toward negatively charged particles.

[0023] The theoretical isoelectric point (pI) value in the present invention can be calculated based on the primary amino acid sequence using amino acid sequence analysis software (Genetyx, Expasy, etc.). In the present invention, it is necessary that the theoretical isoelectric point pI(A) of a protein domain having an antigen-binding site be equal to or lower than the theoretical isoelectric point pI(B) of a protein domain not having an antigen-binding site. The difference between the theoretical isoelectric point pI(A) of a protein domain having an antigen-binding site and the theoretical isoelectric point pI(B) of a protein domain not having an antigen-binding site is preferably 0.5 or more, more preferably 1.0 or more, even more preferably 1.5 or more, even more preferably 2.0 or more, and particularly preferably 2.5 or more. Furthermore, when the pIs of the respective protein domains are 14 and 1, the difference can be set to 13. In the present invention, the amino acid sequences of the full-length polypeptides of each domain constituting the polypeptide were input into these tools to calculate the theoretical pIs shown in the examples. Since the hinge region is not treated as part of the domain, the sequence of the hinge region is not used in calculating the isoelectric point.

[0024] As used herein, the term "altered amino acid sequence" includes, but is not limited to, deletion, substitution, or addition of amino acids. Methods for deleting, substituting, or adding amino acids include, but are not limited to, genetic engineering techniques such as site-directed mutagenesis (Methods in Enzymology, 154, 350, 367-382 (1987); Nucleic Acids Res., 12, 9441 (1984)), chemical synthesis methods such as the phosphate triester method and the phosphate amidite method (J. Am. Chem. Soc., 89, 4801 (1967); Science, 150, 178 (1968); Tetrahedron Lett., 22, 1859 (1981)), and combinations thereof. More specifically, DNA can be synthesized by chemical synthesis using the phosphoramidite method or triester method, or can be synthesized using a commercially available automated oligonucleotide synthesizer.

[0025] According to the present invention, a protein having enzymatic properties identical to or similar to those of a wild-type protein may have an amino acid sequence in which one to several amino acids have been deleted, substituted, added, etc. from the wild-type amino acid sequence. Here, the range of "one to several" in "one to several amino acid deletions, substitutions, or additions" of the amino acid sequence is not particularly limited, but for example, if 100 amino acids in the amino acid sequence are considered to be one unit, this means about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 amino acids per unit, preferably about 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acids, and more preferably about 1, 2, 3, 4, or 5 amino acids per unit. Furthermore, "amino acid deletion" means the absence or disappearance of an amino acid residue in an amino acid sequence, "amino acid substitution" means that an amino acid residue in an amino acid sequence is replaced with another amino acid residue, and "amino acid addition" means that a new amino acid residue is added so as to be inserted into the amino acid sequence.

[0026] Specific embodiments of the "deletion, substitution, or addition of one to several amino acids" include those in which one to several amino acids are replaced with other chemically similar amino acids. Examples include the substitution of a hydrophobic amino acid with another hydrophobic amino acid, or the substitution of a polar amino acid with another polar amino acid having the same charge. Such chemically similar amino acids are known in the art. Specific examples include nonpolar (hydrophobic) amino acids such as alanine, valine, isoleucine, leucine, proline, tryptophan, phenylalanine, and methionine. Polar (neutral) amino acids include glycine, serine, threonine, tyrosine, glutamine, asparagine, and cysteine. Positively charged basic amino acids include arginine, histidine, and lysine. Negatively charged acidic amino acids include aspartic acid and glutamic acid.

[0027] In the present invention, the protein domain without an antigen-binding site is not particularly limited, but in the case of IgG, it is desirable that the isoelectric point of CH2, preferably CH3, be equal to or lower than the isoelectric point of the protein domain with an antigen-binding site. This is because positively charging a site of the antibody as far away as possible from the protein domain with the antigen-binding site improves the orientation of the antibody relative to negatively charged particles. On the other hand, when modifying amino acid residues in the protein domain with an antigen-binding site, the site to be modified is not particularly limited, but it is preferable to modify amino acid residues in VH and VL, preferably CH1 and CL. Modifying amino acid residues in VH and VL may disrupt the three-dimensional structure important for antigen binding, potentially reducing antigen-binding activity.

[0028] As used herein, the term "surface-exposed" refers to amino acids located on the surface of a polypeptide, and amino acids located on the surface of a polypeptide are amino acids in which at least a portion of their side chains are accessible to solvent molecules. Those skilled in the art can select surface amino acids by displaying the 3D structure determined from data collected by X-ray crystallography or cryo-electron microscopy using commercially available software (PyMol, UCSF Chimera). In the present invention, there is no limitation on the amino acid residues to be modified to change the isoelectric point of a protein domain, but it is particularly desirable to modify surface-exposed amino acid residues. This is because modifying amino acid residues that are not surface-exposed, i.e., located within the 3D structure, may result in the 3D structure being unable to be maintained. Furthermore, it is desirable for the antibody surface to be positively charged in order to efficiently generate electrostatic interactions with particles.

[0029] The binding mode between the particle and the antibody of the present invention is not limited, but includes physical binding and chemical binding. In the case of chemical binding, the functional group on the particle is not limited to a COOH group, and examples thereof include NH ２ In the present invention, if the zeta potential of the whole particle is negative, the functional group of the chemical bond is not necessarily a COO group. － It is not necessary for the group to be negatively charged as shown above, but may be a basic functional group such as an NH2 group.

[0030] As used herein, the term "colored cellulose particles" refers to particulate cellulose that is insoluble in water, buffer solutions, etc. and that carries a pigment, dye, etc., and includes color-developing particles in which the coloring component is directly granulated, such as colored cellulose particles obtained by coloring cellulose. The colored cellulose particles may also be fluorescent particles.

[0031] Regarding "particles," the "average particle size" of particles refers to the volume-average median diameter measured by dynamic light scattering, and the volume-average median diameter is in the range of 100 to 650 nm. When the average particle size is within this range, the particles have a large surface area, resulting in a denser TL when used as an immunochromatographic diagnostic kit, i.e., higher analytical sensitivity. If the average particle size is too small, the surface area will be small, resulting in reduced analytical sensitivity and possible particle aggregation. Therefore, the average particle size is preferably 150 nm or more, and more preferably 200 nm or more. If the particle size is too large, the particles may clog the pores of chromatographic media such as nitrocellulose, resulting in coloration of areas that would normally turn white after testing, adversely affecting the interpretation of test results and reducing the detection limit. Therefore, the average particle size is preferably 600 nm or less, and more preferably 550 nm or less. Note that the average particle size described here is merely an average value, and it is acceptable for part of the particle size distribution to fall outside the above range. The reason for using the volume average to evaluate particle size is that in immunochromatographic diagnostic kits, particles that are too large will clog chromatographic media such as nitrocellulose, and even the presence of a small number of particles with a large volume average will have an impact. There are various methods for evaluating particles other than the volume average, such as the number average and area average, and different evaluation methods will naturally result in different particle size values.

[0032] Generally, the driving force for binding between particles and proteins includes electrostatic interactions, hydrophobic interactions, van der Waals forces, hydrogen bonds, etc. However, in the present invention, it is preferable that the driving force for binding between particles and antibodies includes electrostatic interactions. This is because the principle of antibody orientation control is that domains without antigen-binding sites are more positively charged than domains with antigen-binding sites, which selectively attracts negatively charged particles through electrostatic interactions. As disclosed in Non-Patent Document 3 (Vadim V. Sumbayev et al., Small (2013) 9:472-477), the driving force for binding between gold colloids and proteins includes hydrophobic interactions, interactions between gold and SH groups in proteins, and electrostatic interactions, which are particles that exhibit the effects of the antibodies of the present invention. Furthermore, as disclosed in Non-Patent Document 4 (K. Murakami et al., Analytical Biochemistry (2021) 632:114337), it has been elucidated that the main driving force for the interaction between colored cellulose particles and antibodies is electrostatic interactions. Therefore, such particles are a preferred embodiment for achieving the effects of the antibodies of the present invention.

[0033] In the present invention, the "method of producing colored cellulose particles" is not particularly limited. Examples include a method of first molding organic particles and then supporting a coloring component such as a pigment or dye; a method of molding particles and then supporting smaller color-forming particles such as metal colloids or pigments; and a method of adding a coloring component such as a pigment, dye, pigment, or metal colloid during particle molding. Among these, a method of first molding particles and then supporting a coloring component such as a pigment or dye is preferred because it allows for easy adjustment of particle characteristics such as particle size, color intensity, color type, and particle surface condition. Furthermore, dyes are preferred as the coloring component to be supported because of their ease of support. When dyes are used as coloring components, reactive dyes are preferred. When dyes such as direct dyes, metal-containing dyes, acid dyes, basic dyes, disperse dyes, sulfide dyes, vegetable dyes, naphthol dyes, and fluorescent dyes are used, high color intensity cannot be obtained, and even if strong dyeing is achieved, there is a risk of discoloration. However, when a reactive dye is used, it may be combined with other dyes such as direct dyes, metal dyes, acid dyes, basic dyes, disperse dyes, sulfur dyes, vegetable dyes, naphthol dyes, and fluorescent dyes.

[0034] In the present invention, a "conjugate pad" refers to a pad containing a conjugate containing color-forming particles, such as color-forming particles bound to an antibody that binds to a test substance. The material of the conjugate pad is not particularly limited, but common glass fibers or resin fibers can be used. Resin fibers include, but are not limited to, polyolefins such as polyethylene, polyesters, polyamides, and acrylics, as well as composite fibers of these resin fibers. Resin fibers are preferred from the perspective of the working environment. Among resin fibers, materials with a certain degree of hydrophobicity are more preferable from the perspective of ease of release of color-forming particles. If the material is too hydrophobic, it may be pretreated with a surfactant or the like. A polyethylene fiber conjugate pad pretreated with a surfactant is more preferred.

[0035] In the present invention, an "immunochromatographic diagnostic kit" is a kit that utilizes an antigen-antibody reaction to easily detect the presence or absence of a test substance in various samples. The diagnostic kit can be of a lateral flow type or a flow-through type. While there are no particular limitations on the type as long as it uses color-forming particles or a sample pad, the lateral flow type is preferred. Furthermore, among lateral flow types, there are dipstick and cassette types, but these types are not particularly limited. The configuration of the diagnostic kit is not particularly limited, and any configuration commonly used in the relevant field may be used. The types of components other than the conjugate pad (b) containing antibody-sensitized color-forming particles and the sample pad (a) are not particularly limited as long as they are used in the relevant field, and examples include (e) a chromatographic medium, (f) an absorbent pad, and (g) a backing sheet as shown in Figure 1. Furthermore, some of these components may be omitted as necessary. An example of the structure is shown in Figure 1. Figure 1 attached to this specification is merely an example and does not limit the present embodiment in any way.

[0036] Method for Identifying Antibody Sequences Structural information and amino acid sequence information of wild-type antibodies before amino acid sequence modification can be obtained from the Protein Data Bank. Methods for modifying amino acid sequences to achieve a desired isoelectric point for a domain include, but are not limited to, Rosetta Commons. For example, by performing in silico stability analysis using Rosetta software (version 3.1, https: / / www.resettacommons.org / software), it is possible to select the positions of each amino acid to be modified for a target domain without damaging salt bridges that are important for maintaining the three-dimensional structure. In the present invention, the amino acid residues to be modified were identified using the algorithm disclosed in Non-Patent Document 5 (Aleksandr E. et al., Chemistry & Biology (2012) 19:449-455). The specific method is as follows. First, an arbitrary Netcharge (effective charge) such as "+10" is input, and candidate amino acid modification methods (the number of amino acid residues to be modified, the modified positions, and the amino acid residues after modification) that can achieve the Netcharge, as well as the Rosetta score, which is an index of the energy value of the three-dimensional structure folded by the amino acid sequence, can be calculated. Note that a smaller Rosetta score means a more stable three-dimensional structure. This operation was repeated 10 times, and from the 10 candidate modification methods, the modification method with the smallest Rosetta score for the three-dimensional structure of the antibody after amino acid residue modification was selected.

[0037] Methods for increasing the pI of a protein include, for example, decreasing the number of acidic amino acids (aspartic acid and glutamic acid) or increasing the number of basic amino acids (arginine (R), lysine (K), histidine). Methods for decreasing the pI of a protein include, for example, increasing the number of acidic amino acids (aspartic acid and glutamic acid) or decreasing the number of basic amino acids (arginine, lysine, histidine).

[0038] Methods for Cultivating and Purifying Antibodies Methods for preparing proteins such as antibodies are known in the art, and generally involve incorporating a nucleic acid encoding the antibody into a carrier (e.g., an expression vector), introducing the carrier into host cells, and culturing the cells to produce the antibody. The expressed or secreted antibody is then recovered from the host cells or a culture of the host cells (e.g., a culture medium) by a common purification step, thereby obtaining the desired antibody.

[0039] Although there are no particular limitations on the expression vector for expressing the antibody of the present invention, a suitable vector is generally constructed to contain: (i) a promoter capable of transcription in a host cell; (ii) a nucleic acid (polynucleotide) encoding the antibody of the present invention that is operably expressed by the promoter; and (iii) an expression cassette that contains, as components, signals that function in a host cell regarding the transcription termination and polyadenylation of the RNA molecule.

[0040] Methods for producing expression vectors include, but are not limited to, methods using plasmids, viruses, phages, cosmids, or the like, or DNA molecules having the necessary components.

[0041] The specific type of vector is not particularly limited, and a vector capable of expression in host cells can be appropriately selected. That is, a promoter sequence can be appropriately selected depending on the type of host cell to ensure expression of a nucleic acid encoding an antibody of the present invention, and a vector incorporating this promoter sequence and a nucleic acid encoding an antibody of the present invention into various plasmids or the like can be used as an expression vector. For example, when Escherichia coli is used as the host cell, commonly used expression vectors include pBluescript, pUC118, pUC18, pUC19, and pBR322. When Chinese hamster ovary cells (CHO cells) are used as the host cell, pCHO vectors, pTT5 vectors, and the like are used.

[0042] Such expression vectors contain expression control regions (e.g., promoter, terminator, and / or replication origin, etc.) depending on the type of host cell to be introduced. Conventional promoters (e.g., the promoter of the cytomegalovirus (CMV) IE (immediate early) gene, the SV40 early promoter, retrovirus promoters, metallothionein promoters, heat shock promoters, SRα promoters, Moloney murine leukemia virus promoters and enhancers, trc promoters, tac promoters, lac promoters, etc.) are used for bacterial expression vectors. Examples of promoters for animal cell hosts include viral promoters (e.g., the SV40 early promoter, the SV40 late promoter, etc.). Examples of promoters that are inducibly activated by external stimuli include the mouse mammary tumor virus (MMTV) promoter, a tetracycline-responsive promoter, a metallothionein promoter, and a heat shock protein promoter.

[0043] The expression vector preferably contains at least one selection marker, such as a drug resistance gene (neomycin resistance gene, DHFR gene, puromycin resistance gene, blasticidin resistance gene, hygromycin resistance gene, or cycloheximide resistance gene (Japanese Patent Laid-Open Publication No. 2002-262879)), or a fluorescent or bioluminescent marker gene (e.g., green fluorescent protein (GFP)).

[0044] Host cells into which nucleic acids encoding the antibodies of the present invention are introduced may be either cell lines or primary culture cell lines, but mammalian cells that can be subcultured and stably express the antibody (protein) of interest are preferred. Examples of host cells include CHO cells, HeLa cells, HEK293 cells, NIH3T3 cells, PER.C6 cells, human leukemia Namalwa cells, monkey COS cells, rat myeloma cells YB2 / 3HL.P2.G11.16Ag.20 (also referred to as YB2 / 0), mouse myeloma cells NS0, mouse myeloma cells SP2 / 0-Ag14, and Syrian hamster cells BHK.

[0045] Transformation of host cells (nucleic acid introduction) can be performed using commonly used known methods, such as, but not limited to, electroporation (Mackenxie, DA et al., Appl. Environ. Microbiol., vol. 66, pp. 4655-4661, 2000), heat shock (U.S. Pat. No. 2,765,299), lipofection (PNAS, 1989, 86: 6077; PNAS, 1987, 84: 7413), particle delivery (JP 2005-287403 A), spheroplast method (Proc. Natl. Acad. Sci. USA, vol. 75, pp. 1929-1978), and lithium acetate method (J. Bacteriology, vol. 153, pp. 163, 1983).

[0046] For other general molecular biology techniques, reference can be made to "Sambrook & Russell, Molecular Cloning: A Laboratory Manual Vol. 3, Cold Spring Harbor Laboratory Press 2001" and "Methods in Yeast Genetics, A laboratory manual (Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY)".

[0047] After culturing transformed host cells, the antibodies of the present invention are secreted into or outside the host cells and can be isolated as pure, homogeneous antibodies by undergoing a purification process. Purification methods include filtration and ultrafiltration, which separate antibodies based on molecular weight or size, and chromatography, which separates antibodies based on molecular properties. Specific examples of chromatography include Ni columns, ion exchange chromatography, gel filtration chromatography, hydrophobic chromatography, and affinity chromatography. Furthermore, purified antibodies can be concentrated to the concentration required for experiments by methods such as ultrafiltration.

[0048] The term "immunochromatographic diagnostic kit" refers to an item that utilizes an antigen-antibody reaction to easily detect the presence or absence of a test substance in various samples. Immunochromatographic diagnostic kits include lateral flow and flow-through types. While there are no particular limitations on the type of kit as long as it uses color-forming particles and a sample pad, lateral flow types are preferred. Among lateral flow types, dipstick and cassette types are also available, but these types are not particularly limited. The configuration of the immunochromatographic diagnostic kit is not particularly limited, and any configuration commonly used in the relevant field may be used. The types of components other than the color-forming particles and sample pad are not particularly limited as long as they are commonly used in the relevant field, and examples include a conjugate pad (including antibody-sensitized color-forming particles), a membrane such as nitrocellulose, an absorbent pad, and a backing. Furthermore, some of these components may be omitted as necessary.

[0049] A "diagnostic method" using an immunochromatographic diagnostic kit refers to various diagnoses performed using the immunochromatographic diagnostic kit. The diagnostic target is not particularly limited and can be a variety of diagnostic targets, including human, animal, food, plant, and other environmental tests. A typical diagnostic procedure involves collecting a specimen from the test target, pre-treating it (e.g., extraction or filtration) if necessary, dropping it onto a sample pad, waiting a predetermined time from the start of the test, and determining the diagnostic result based on the color change that varies depending on the presence or absence of the test target substance. Of course, this procedure is not limited to this, and similar procedures and principles can also be used for diagnoses. Preferably, the specimen can be pre-filtered to remove excess foreign matter and impurities, which can further shorten the diagnostic time and improve diagnostic accuracy.

[0050] There are no particular limitations on the subjects that can be diagnosed using immunochromatographic diagnostic kits, but specific examples include the following: cancer markers, hormones, infectious diseases, autoimmunity, plasma proteins, TDM, coagulation / fibrinolysis, amino acids, peptides, proteins, genes, and cells. More specifically, CEA, AFP, ferritin, β2 micro, PSA, CA19-9, CA125, BFP, elastase 1, pepsinogen 1 and 2, fecal occult blood, urinary β2 micro, PIVKA-2, urinary BTA, insulin, E3, HCG, HPL, LH, HCV antigen, HBs antigen, HBs antibody, HBc antibody, HBe antigen, HBe antibody, HTLV-1 antibody, HIV antibody, toxoplasmosis antibody, syphilis, ASO, influenza A antigen, influenza A antibody, influenza B antigen, influenza B antibody, rotavirus antigen, adenovirus antigen, rota-adenovirus antigen, group A streptococcus, group B streptococcus, Candida antigen, CD bacteria, cryptolocus antigen, Vibrio cholerae, meningococcus antigen, granulococcus elastase, Helicobacter pylori, Helicobacter pylori antibody, O157 antibody, O157 antigen, Leptospira antibody, Aspergillus antigen, MRSA, RF, total IgE, LE test, CRP, IgG, A, M, IgD, transferrin, urinary albumin, urinary transferrin, myoglobin, C3 / C4, SAA, LP(a), α1-AC, α1-M, haptoglobin, microtransferrin, APR score, FDP, D-dimer, plasminogen, AT3, α2PI, PIC, PAI-1, protein C, coagulation factor X3, type IV collagen, hyaluronic acid, GHbA1c, other various antigens, various antibodies, various viruses, various bacteria, various amino acids, various peptides, various proteins, various DNA, various cells, various allergens, various pesticide residues, various harmful substances.

[0051] The present application also provides a particle-polypeptide complex comprising a particle having a negative zeta potential and an antibody of the present invention. The complex of the present invention has, for example, a degree of orientation of 1.0 or more, preferably 1.2 or more, more preferably 1.3 or more, and most preferably 1.5 or more. In the present invention, the term "degree of orientation" refers to the direction of the antibody adsorbed on the color-developing particle, and refers to the value calculated by the following formula: degree of orientation = (accessibility to a protein domain having an antigen-binding site) / (accessibility to a protein domain not having an antigen-binding site). Here, the accessibility to a protein domain having an antigen-binding site and the accessibility to a protein domain not having an antigen-binding site refer to the values ​​calculated below, respectively. Accessibility to a protein domain having an antigen-binding site = (color intensity when an HRP-modified anti-IgG antibody (Fab-specific) is reacted with a particle-polypeptide complex) / (amount of particle-polypeptide complex) Accessibility to a protein domain not having an antigen-binding site = (color intensity when an HRP-modified anti-IgG antibody (Fc-specific) is reacted with a particle-polypeptide complex) / (amount of particle-polypeptide complex) To increase the detection sensitivity in immunochromatography, the degree of orientation should be 1.0 or more, preferably 1.3 or more, and more preferably 1.5 or more.

[0052] The present invention will be described in more detail below with reference to examples, but the present invention is not limited to these examples.

[0053] First, the methods used to measure various physical properties, and the methods used to evaluate the performance of the immunochromatographic diagnostic kit and measure the degree of orientation, which are common to all examples and comparative examples, will be described.

[0054] [Measurement of Zeta Potential] The zeta potential of the particles of the present invention in an aqueous dispersion state is preferably 0 mV or less. Here, a method for measuring the zeta potential will be described. A 0.005 mass % particle dispersion was subjected to ultrasonic treatment (manufactured by SMT Corporation, trade name "UH-50") for 1 minute, and then the zeta potential was measured using a zeta potential meter (manufactured by Otsuka Electronics Co., Ltd., trade name "ELS-Z2"). In the present invention, in order to selectively attract positively charged domains that do not have a function, this zeta potential needs to be negative, and is preferably -20 mV or less, more preferably -30 mV or less, and particularly preferably -40 mV or less.

[0055] [Measurement of antibody adsorption rate] 3.8 μl of a dispersion solution (1.06 wt%) of colored cellulose particles (NanoAct (RE1AA) manufactured by Asahi Kasei Corporation) was added so that the particle weight was 40 μg, and 34 μl of 10 mM PB (pH 9.0) and 4 μg of the purified antibody described above were added. The mixture was stirred by vortexing and allowed to stand at 37°C for 2 hours. The mixture was then centrifuged at 18,000 g for 20 minutes, and 20 μl of the supernatant was collected. The antibody concentration in this supernatant was measured using a BCA assay (Sigma-Aldrich QuantiPro™ BCA Assay Kit). The difference between the amount of antibody added (4 μg) and the amount of antibody in the supernatant was taken as the amount of antibody adsorbed to the particles, and this value was divided by the amount of antibody added to calculate the antibody adsorption rate to the particles.

[0056] [Antibody Sensitization of Colored Cellulose Particles] 6.0 μl of a dispersion solution (1.06 wt%) of colored cellulose particles (Asahi Kasei Corporation, NanoAct (RE1AA)), 54 μl of 10 mM PB (pH 9.0), and 2.9 μl of the purified antibody (0.22 wt%) described above were mixed, vortexed, and allowed to stand at 37°C for 2 hours. Subsequently, 720 μl of casein (1% casein, 100 mM boric acid, pH 8.5) was added for blocking, and the mixture was allowed to stand at 37°C for 60 minutes. The mixture was then centrifuged at 20,000 g for 20 minutes, the supernatant was removed, and 720 μl of 50 mM boric acid (pH 10.0) was added as a wash solution, followed by ultrasonic dispersion. Subsequently, the mixture was centrifuged at 20,000 g for 20 minutes, and the entire supernatant was discarded. After centrifugation, 158 μl of a storage solution (0.4% casein, 10% trehalose, 4% histidine, 50 mM boric acid, pH 10.0) was added to the precipitate so that the final particle weight was 0.038%, and the particles were dispersed in the solution.

[0057] [Preparation of HRP-modified antibodies] HRP-modified anti-human IgG (Fab-specific) antibody and HRP-modified anti-human IgG (Fc-specific) antibody were prepared by peroxidase-labeling anti-human IgG (Fab-specific) antibody (Sigma-Aldrich I5260) and anti-human IgG (Fc-specific) antibody (Sigma-Aldrich I2136) using Peroxidase Labeling Kit-NH2 (Dojindo Laboratories LK11).

[0058] [Measurement of Orientation Degree] 49 μL of D-PBS and HRP-modified anti-human IgG (Fab-specific) antibody were added to 8 μL of the antibody-sensitized colored cellulose particle dispersion prepared by the above method, and the mixture was allowed to stand at 37°C for 1 hour. The mixture was then centrifuged at 17,900 g for 20 minutes, the supernatant was removed, and 300 μL of D-PBS was added as a washing solution, followed by dispersion by vortex mixing. This washing procedure was repeated four times, followed by centrifugation at 17,900 g for 20 minutes. 40 μL of D-PBS was added to the precipitate from which the supernatant was removed, and the mixture was dispersed by ultrasonication. The resulting dispersion was diluted 1,000-fold with D-PBS, and 100 μL was added to a 96-well plate (Sumitomo Bakelite Co., Ltd. MS-8296F). A TMB substrate solution prepared by mixing KPL TMB Peroxidase Substrate (product number 5120-0048, manufactured by Seracare) and KPL Peroxidase Substrate Solution B (product number 5120-0037, manufactured by Seracare) at a 1:1 volume ratio was diluted 5-fold with D-PBS, and then 50 μL of this solution was added to each well of the 96-well plate at 10-second intervals. After incubation at room temperature for 30 minutes, 100 μL of 1 M hydrochloric acid (product number 083-01095, manufactured by Fujifilm Wako Pure Chemical Industries, Ltd.) was added to each well. The absorbance at 450 nm was measured using an INFINITE 200 PRO M NANO plate reader (manufactured by Tecan). The absorbance was divided by the amount of sensitized particles to obtain the accessibility to the protein domain having the antigen-binding site. The same procedure was performed using an HRP-modified anti-human IgG (Fc-specific) antibody instead of an HRP-modified anti-human IgG (Fab-specific) antibody to obtain the accessibility to the protein domain not having the antigen-binding site. The degree of orientation was calculated by dividing the accessibility to the protein domain having the antigen-binding site by the accessibility to the protein domain not having the antigen-binding site.

[0059] [Impregnation of Conjugate Pad with Antibody-Sensitized Colored Cellulose Particles and Drying] A polyethylene conjugate pad (6613 manufactured by Ahlstorm) was immersed in a large excess of 0.05 wt% Tween-20 (registered trademark) (T2700 manufactured by Sigma-Aldrich), and after removing the excess liquid, it was dried at 50°C for 60 minutes. Subsequently, this conjugate pad was cut into a shape with a width of 10 mm and a length of 50 mm. Next, 132 μl of a 0.038 wt% antibody-sensitized colored cellulose particle dispersion was evenly applied using a micropipette, and the pad was dried at 37°C for 60 minutes.

[0060] [Pretreatment of Sample Pad] A cellulose sample pad (Millipore, C08 3) prepared by a known method was immersed in a PBS buffer solution (66 mM, pH 7.4) containing a large excess of 2.0 wt % BSA (Sigma-Aldrich, A7906) and 2.0 wt % Tween-20. After removing excess liquid, the pad was dried at 50°C for 60 minutes and then cut into a shape of 20 mm in height and 300 mm in length.

[0061] Preparation of Capture Antibody-Coated Nitrocellulose Membrane: A nitrocellulose membrane (Sartorius, CN95) was cut to a shape of 25 mm in height and 300 mm in length. Using a liquid applicator (Musashi Engineering, 300DS), a PBS solution (4% sucrose, 1000 ppm BSA, 66 mM, pH 7.4) containing 0.1 wt% anti-SARS-CoV-2 Spike S2 ​​Subunit Mab antibody (R&D) was applied at a rate of 0.1 μl / mm to a section 9 mm from the upstream end. Subsequently, a PBS solution (66 mM, pH 7.4) containing 0.1 wt% anti-human goat antibody (Sigma-Aldrich, I5260) was applied at a rate of 0.1 μl / mm to a section 15 mm in height, followed by drying at 50°C for 24 hours.

[0062] [Preparation of Immunochromatographic Diagnostic Kit] The prepared capture antibody-coated nitrocellulose membrane, an absorbent pad (manufactured by Millipore), a conjugate pad containing antibody-sensitized colored cellulose particles, and a regenerated cellulose continuous long fiber nonwoven fabric sample pad were attached to a backing card (manufactured by Adhesives Research, AR9020) in the layout shown in Figure 1, and then cut into a width of 4 mm using a cutter to obtain an immunochromatographic diagnostic kit with a width of 4 mm and a height of 60 mm.

[0063] [Performance Evaluation of Immunochromatographic Diagnostic Kit] The performance of the obtained immunochromatographic diagnostic kit was evaluated, and the results are shown in Table 1 below.

[0064]

[0065] [Measurement of color intensity of immunochromatographic diagnostic kit] The immunochromatographic diagnostic kit was cut to a width of 4 mm and placed in a plastic housing. 80 μl of antigen (200 ng / ml) was applied to the resulting diagnostic kit in the housing, and after 30 minutes, the color intensity of the test line was measured using an immunochromatographic reader (TOR500, manufactured by Trust Medical Co., Ltd.). SARS-CoV-2 spike protein (Z03481, manufactured by GenScript) was used as the test substance.

[0066] [Antibody Sequence Information] The structures and sequence information of the wild-type antibodies and mutant antibodies used in the following examples are shown below.

[0067]

[0068] Example 1: Preparation of an expression vector for wild-type human IgG1 To prepare an expression vector for a mutant (pos14) in which the amino acid sequence was modified to increase the isoelectric point of CH3, we first prepared an expression vector for a wild-type anti-SARS-CoV-2 RBD antibody (wild-type human IgG1) to serve as a template for mutation (Comparative Example 1).

[0069] [HiFi Host Gene PCR] A forward primer anneals 22 bases from the 5' end of the human IgG1 constant region gene, a forward primer anneals 22 bases from the 5' end of the human Ig kappa constant region gene, and a reverse primer (common) anneals 25 bases from the 3' end of the nucleotide sequence encoding the secretory signal sequence in mammalian cell expression were prepared (Thermo Fisher Scientific). Forward primer (G1 constant region): 5'-GCGTCGACTAAGGGTCCGTCGG-3' (SEQ ID NO: 29) Forward primer (kappa constant region): 5'-AGAACAGTGGCCGCCCCTTCTG-3' (SEQ ID NO: 30) Reverse primer: 5'-ACACTGGACACCTTTTGAGCACAGCG-3' (SEQ ID NO: 31)

[0070] Adalimumab heavy or light chain-encoded mammalian cell expression vector pcDNA3.4 TOPO (Thermo Fisher Scientific) was prepared at 20 ng / μL and mixed at 1 μL, and the two corresponding primers (30 μM each) were added at 0.5 μL each, and 25 μL of KOD One (registered trademark) PCR Master Mix (Toyobo) was added, and the reaction solution was adjusted to 50 μL with sterile water. After incubation at 98 ° C for 1 minute, 20 cycles of reaction consisting of 98 ° C for 10 seconds, 60 ° C for 5 seconds, and 68 ° C for 40 seconds were performed to amplify the vector sequence containing the human IgG1 or Igκ constant region.

[0071] [HiFi Insert Gene PCR] The insert gene for the mouse IgG1κ-derived anti-SARS-CoV-2 RBD antibody 3C1 (Zhang et al., Nat. Commun. 12:264, A forward primer (5'-AAAGGTGTCCAGTGTGAAGTCCAGTTGCAGGAATCCGGACCTAGTC-3' (SEQ ID NO: 32)) that anneals approximately 40 bases from the 5' end of the VH domain gene of IgG1 (SEQ ID NO: 32), a forward primer (5'-AAAGGTGTCCAGTGTACATTGTCATGACTCAGTCACACAAATTCATGTCCACTTCTGTG-3' (SEQ ID NO: 33)) that anneals approximately 40 bases from the 5' end of the VL domain gene of IgG1 (SEQ ID NO: 33), and a forward primer (5'-AAAGGTGTCCAGTGTACATTGTCATGACTCAGTCACACAAATTCATGTCCACTTCTGTG-3' (SEQ ID NO: 33)) that anneals approximately 15 bases from the 5' end of the human IgG1 constant region. A reverse primer (5'-ACCCTTAGTCGACGCGCTGGATACAGTAAGTGTTGTTCCTTGTCC-3' (SEQ ID NO: 34)) containing the sequence AA15001 and annealing approximately 30 bases from the 3' end of the 3C1 antibody VH domain gene, and a reverse primer (5'-GGCGGCCACTGTTCTCTTGATCTCCAGCTTGGTGCCGC-3' (SEQ ID NO: 35)) containing 15 bases after the 5' end of the Igκ constant region and annealing approximately 30 bases from the 3' end of the 3C1 antibody VL domain gene were prepared (Thermo Fisher Scientific). The mammalian cell expression vector pcDNA3.4 TOPO encoding the 3C1 antibody heavy or light chain, the two corresponding primers, KOD One® PCR Master Mix, and sterile water were mixed in the same ratio as above to make a 50 μL reaction solution. After incubation at 98°C for 1 minute, 40 cycles of 98°C for 10 seconds, 60°C for 5 seconds, and 68°C for 2 seconds were performed to amplify the 3C1 antibody VH or VL gene sequence.

[0072] [HiFi DNA Assembly] After PCR, 1 μL of DpnI (New England Biolabs) was added to the entire reaction solution, mixed, and incubated at 37°C for 1 hour to cleave the template DNA plasmid. The reaction solution was purified using a FastGene Gel / PCR Extraction Kit (Nippon Gene Co., Ltd.) according to the method described in the attached instructions. The concentration of the purified gene was measured using a NanoDrop 2000c (Thermo Fisher Scientific). The vector containing the heavy chain or light chain gene was mixed to approximately 100 ng, and the 3C1 antibody VH or VL gene was added to approximately 17 ng. 10 μL of NEBuilder HiFi DNA Assembly Master Mix (New England Biolabs) was then added, and the reaction solution was adjusted to 20 μL with sterile water. The reaction solution was incubated at 50°C for 1 hour, and then transformed with Escherichia coli JM109 strain (Toyobo Co., Ltd.). The resulting colonies were inoculated into LB medium and cultured. DNA was extracted using the FastGene Plasmid Mini Kit (Nippon Gene Co., Ltd.) according to the instructions in the accompanying manual. The base sequence of the extracted DNA was determined by Sanger sequencing (outsourced to FASMAC), and vectors were prepared containing a heavy chain gene with a secretion signal, 3C1VH, and human IgG1 constant region, and a light chain gene with a secretion signal, 3C1VL, and human IgGκ constant region. Hereinafter, the antibody of Comparative Example 1 expressed by this gene will be referred to as "wild-type human IgG1."

[0073] Design of expression vectors for mutant antibodies (pos11, pos14) using Rosetta To change the isoelectric point of the CH3 domain of human IgG1, the amino acid sequence was designed. First, using the crystal structure of the human IgG1 Fc domain (PDB ID: 4W4N) from the Protein Data Bank (PDB), structure optimization was performed in the Rosetta force field using relax in Rosetta (version 2021.16.61629). Next, supercharge in Rosetta (same version) was performed according to the published protocol (Miklos at al., Chem. Biol. 19:4, 2020) with the following settings. The amino acid residue number of the CH2 domain (from the beginning to EU numbering 341) was entered in the resfile, and the mutation site was limited to the CH3 domain. The net charge was set to +10 relative to the wild-type sequence. The above supercharge calculation was repeated 10 times, and the sequence with the smallest score was A chain: E345R, Q419K, H433K, Q438K, B chain: E345R, Q386K, N389K, Q419K, H433K, a total of 9 mutations, resulting in a wild-type net charge of 0 vs. 11, and this mutant was designated pos11 (Example 2). In addition, all the mutations presented were integrated, and the sequence with a net charge of 14 due to the 6 mutations of E345R, Q386K, N389K, Q419K, H433K, Q438K in both chains was designated pos14 (Example 1).

[0074] Preparation of mutant antibody (pos14) An antibody having the amino acid sequence designed by the above method was prepared by the following procedure.

[0075] [Preparation of Expression Vector by PCR] A synthetic oligo-DNA was prepared (Integrated DNA Technologies) that encodes EU numbering 345-438, a portion of the human IgG1 CH3 domain, and contains six mutations: E345R, Q386K, N389K, Q419K, H433K, and Q438K. A forward primer with the 18-base sequence at the 3' end of the synthetic oligo-DNA and annealing 23 bases after EU numbering 439 of human IgG1, and a reverse primer with the 18-base sequence at the 5' end of the synthetic oligo-DNA and annealing 26 bases before EU numbering 344 were also prepared (Thermo Fisher Scientific). Forward primer: 5'-AAAAACCACTACACAAAGAAGAGCCTCTCCCCTGTCTCCGGG-3' (SEQ ID NO: 36) Reverse primer: 5'-CGTGTAGACCTGAGGTCTTCGGGGCTGCCCTTTGGCTTTGGAGA-3' (SEQ ID NO: 37)

[0076] Subsequently, 1 μL of wild-type human IgG1 heavy chain DNA plasmid vector prepared at 10 ng / μL was mixed, and 0.5 μL of each of the above two primers (30 μM each) was added to 25 μL of KOD One (registered trademark) PCR Master Mix, and the reaction solution was adjusted to 50 μL with sterile water. After 1 minute of incubation at 98 ° C, 18 cycles of reaction consisting of 10 seconds at 98 ° C, 5 seconds at 60 ° C, and 40 seconds at 65 ° C were performed to amplify the gene of the vector sequence containing the wild-type human IgG1 heavy chain gene. After PCR, the gene was treated with DpnI and purified in the same manner as above. The purified gene was mixed to approximately 100 ng, and 1.7 μL of synthetic oligo DNA prepared at 10 ng / μL was added, and the NEBuilder HiFi DNA Assembly reaction was performed in the same manner as above. Thereafter, transformation, inoculation, cultivation, and DNA extraction were carried out in the same manner as above, and the introduction of the Fc pos14 mutation into wild-type human IgG1 was confirmed by sequence analysis by the Sanger method (outsourced to FASMAC).

[0077] [Antibody Expression] Expression of IgG1κWT and pos14 mutants was carried out as follows: ExpiCHO-S™ cells (Thermo Fisher Scientific) were suspended in ExpiCHO™ expression medium (Thermo Fisher Scientific) preheated to 37°C, and 6 × 10 ６ At a cell density of 100 cells / mL, 25 mL of the medium was added to an unbaffled Erlenmeyer flask (125 mL, CORNING). Using the ExpiFectamine™ CHO Transfection Kit (Thermo Fisher Scientific), 13 μg each of wild-type human IgG1 heavy chain WT or pos14 gene plasmid and wild-type human IgG1 light chain gene plasmid were transfected according to the Max Titer protocol described in the user guide, and the cells were cultured. On day 14, the culture medium was collected and centrifuged (5000 g, 10 minutes, 4°C) to remove the cells.

[0078] [Antibody purification] The culture supernatant obtained by the method described above was passed through a 0.8 μm filter (ADVANTEC) and then loaded onto a chromatography column (Bio-Rad) packed with 1 mL of rProtein A Sepharose™ Fast Flow (Cytiva) resin equilibrated with phosphate-buffered saline (PBS) at pH 7.4. The resin was washed with 10 mL of PBS (pos14 was further washed with 6 mL of 20 mM Tris-HCl pH 8, 100 mM Arg-HCl, 500 mM NaCl), and the antibody was eluted with Pierce™ IgG Elution Buffer (Thermo Fisher Scientific). The eluate was dialyzed overnight (50 times at 4°C) against 20 mM Tris-HCl pH 8, 200 mM Arg-HCl, 500 mM NaCl and purified by size exclusion chromatography using HiLoad™ 16 / 600 Superdex™ 200 pg (Cytiva).

[0079] [Quantification of antibody concentration] The concentration of the purified antibody was measured as follows. The antibody sequence was entered into ProtParam tool (Expasy) to obtain the molar extinction coefficient and molecular weight. The absorbance at a wavelength of 280 nm was measured using NanoDrop 2000c, divided by the molar extinction coefficient, and multiplied by the molecular weight to calculate the concentration (unit: mg / mL).

[0080] Example 2 An antibody having the amino acid sequence (pos11) designed in Example 1 was prepared by the following method.

[0081] [Preparation of expression vector by PCR] In addition to the nine mutations of the mutant pos11 determined by Rosetta calculation in Example 1, in order to combine heterogeneous Fc domains between different types of heavy chain genes, disulfide-linked knobs-into-holes (dKiH) (Akiba at al., Antibody Therapeutics 2:3, 2019) was introduced. A total of 15 mutations were introduced by the following method: A chain: E345R, S354C, T366W, Q419K, H433K, Q438K, B chain: E345R, Y349C, T366S, L368A, Q386K, N389K, Y407V, Q419K, H433K (EU numbering).

[0082] Next, synthetic oligo-DNAs were prepared (Integrated DNA Technologies): a nucleotide sequence encoding positions 345 to 438 (EU numbering), which is a part of the human IgG1 CH3 domain, and containing six mutations: E345R, S354C, T366W, Q419K, H433K, and Q438K; and a nucleotide sequence encoding positions 345 to 433, and containing nine mutations: E345R, Y349C, T366S, L368A, Q386K, N389K, Y407V, Q419K, and H433K. A forward primer was prepared, which has an 18-base sequence at the 3' end of a synthetic oligo DNA and anneals to 23 or 27 bases after EU numbering position 439 or 434 of human IgG1, and a reverse primer was prepared, which has an 18-base sequence at the 5' end of a synthetic oligo DNA and anneals to 26 bases before EU numbering position 344 (Thermo Fisher Scientific). Forward primer: 5'-AAGAATCACTATACAAAAAAGAGCCTCTCCCTGTCTCCGGG-3' (SEQ ID NO: 38) Forward primer: 5'-ATGCACGAGGCACTGAAAAACCACTACACGCAGAAGAGCCTCTCC-3' (SEQ ID NO: 39) Reverse primer: 5'-CGTGTAAACCTGTGGCCGTCGGGGCTGCCCTTTGGCTTTGGAGA-3' (SEQ ID NO: 40) Reverse primer: 5'-CGTGTAGACCTGAGGTCTTCGGGGCTGCCCTTTGGCTTTGGAGA-3' (SEQ ID NO: 41)

[0083] Subsequently, PCR, DpnI treatment, purification, HiFi DNA assembly, transformation, inoculation, culture, DNA extraction, and sequence analysis were carried out in the same manner as in Example 1 to confirm the introduction of the pos11 and dKiH mutations.

[0084] [Antibody Expression, Purification, and Quantitation of Antibody Concentration] Expression, purification, and quantitation of antibody concentration of pos11 were carried out in the same manner as in Example 1.

[0085] [Example 3] A sequence in which the net charge of the CH3 domain is 36, compared with the wild-type net charge of 0, was obtained in the same manner as in Example 1, and this mutant was designated pos36. Preparation of an expression vector by PCR, expression, purification, and quantification of the antibody concentration were also performed in the same manner as in Example 1.

[0086] Example 4 To prepare an antibody (c-10 / pos14) in which the isoelectric point of the CH3 domain of human IgG1 was increased and the isoelectric point of the Fab domain was decreased, a sequence was obtained in which the net charge of the Fab domain was −7, compared with the net charge of the wild-type Fab domain of +4, using the same method as in Example 1.

[0087] Design of an expression vector for mutant antibody (c-10 / pos14) using Rosetta. Using the crystal structure (PDB ID: 5WUV) of Fab Certolizumab, which has the same sequence as the human IgG1κ constant region CH1 and CL domains, structural optimization was performed in the Rosetta force field using relax in Rosetta. Next, Rosetta supercharge was performed according to the published protocol with the following settings. The amino acid residue numbers of the Certolizumab VH and VL domains were entered in the resfile, and the mutation sites were limited to the CH1 and CL domains. The net charge was set to -7 compared to +4 for the wild type. The above supercharge calculation was repeated 10 times, and the sequence with the best score was obtained by using a total of seven mutations: CH1 domain: K133D, S160D, S165E, S191D, K205E, and CL domain: K145E, Q199E (EU numbering), which resulted in an effective charge of -6, a reduction of 10 from the wild-type effective charge of +4. This mutant was designated c-10, and a mutant incorporating the pos14 mutation was designated c-10 / pos14.

[0088] [Preparation of Expression Vector] Primers were prepared for both the sense and antisense strands (Thermo Fisher Scientific), annealing to 6 bases before and approximately 18 bases after the mutation site and having 3 bases encoding the above amino acid mutations. CH1 K133D sense strand (5'-TCGTCCGACTCTACCTCGGGCGGCACTG-3' (SEQ ID NO: 42)) antisense strand (5'-GGTAGAGTCGGACGATGGAGCCAGTGG-3' (SEQ ID NO: 43)) CH1 S160D S165E sense strand (5'-TGGAACGACGGTGCTTTGACCGAGGGC-3' (SEQ ID NO: 44)) antisense strand (5'-AGCACCGTCGTTCCAGCTCACAGTAACAG-3' (SEQ ID NO: 45)) CH1 S191D sense strand (5'-CCCTCGGACTCTCTCGGTACCCAAACA-3' (SEQ ID NO: 46)) Antisense strand (5'-GAGAGAGTCCGAGGGCACTGTGACCAC-3' (SEQ ID NO: 47)) CH1 K205E Sense strand (5'-AACCACGAGCCCAGCAACACTAAGGTC-3' (SEQ ID NO: 48)) Antisense strand (5'-GCTGGGGCTCGTGGTTCACGTTGCAGATG-3' (SEQ ID NO: 49)) CL K145E Sense strand (5'-GAGGCCGAGGTGCAGTGGAAAGTGG-3' (SEQ ID NO: 50)) Antisense strand (5'-CTGCACCTCGGCCTCGCGTGGGGTAGAAG-3' (SEQ ID NO: 51)) CL Q199E Sense strand (5'-ACCCACGAGGGCCTGAGCAGCCCAGTTAC-3' (SEQ ID NO: 52)) Antisense strand (5'-CAGGCCCTCGTGGGTCACTTCGCAGGC-3' (SEQ ID NO: 53))

[0089] The wild-type human IgG1 heavy chain pos14 mutant or wild-type human IgG1 light chain gene plasmid prepared in Example 1 was prepared at 5 ng / μL and mixed at 1 μL, and the corresponding primers (30 μM each) were added at 0.5 μL each, and 25 μL of KOD One (registered trademark) PCR Master Mix was added, and the reaction solution was adjusted to 50 μL with sterile water. After 1 minute of incubation at 98 ° C, 20 cycles of reaction consisting of 10 seconds at 98 ° C, 5 seconds at 55 ° C, and 5 seconds at 68 ° C were performed. After DpnI treatment in the same manner as in Example 1, transformation with E. coli JM109 strain, inoculation, culture, DNA extraction, and sequence analysis were performed to confirm the introduction of mutations. The process from PCR using primers for mutations that could not be introduced to sequence analysis was repeated to prepare wild-type human IgG1 heavy chain c-10, pos14 mutant, and wild-type human IgG1 light chain c-10 mutant gene plasmids.

[0090] [Antibody Expression, Purification, and Quantitation of Antibody Concentration] Expression, purification, and quantitation of antibody concentration of c-10 / pos14 were carried out in the same manner as in Example 1.

[0091] Example 5 To prepare an antibody (c-10) with a lowered isoelectric point of the Fab domain of human IgG1, a sequence was obtained in which the net charge of the Fab domain was −7, compared to the net charge of the wild-type, +4, using the same method as in Example 1.

[0092] [Preparation of Wild-Type Human IgG1 c-10: Transplantation of the Neg Mutation Site of CH1 into Wild-Type Human IgG1] [HiFi Host Gene PCR] A forward primer annealing to 23 bases behind the second base of EU numbering 214 in the wild-type human IgG1 heavy chain gene and a reverse primer annealing to 22 bases before the second base of EU numbering 124 were prepared (Thermo Fisher Scientific). Forward primer: 5'-AGGTCGAGCCTAAGAGCTGCGAC-3' (SEQ ID NO: 54) Reverse primer: 5'-GACGGACCCTTAGTCGACGCGC-3' (SEQ ID NO: 55)

[0093] Subsequently, the wild-type human IgG1 heavy chain gene plasmid prepared in Example 1 was prepared to 5 ng / μL and mixed at 1 μL, and the two corresponding primers (30 μM each) were added at 0.5 μL each, and KOD One (registered trademark) PCR Master Mix 25 μL was added, and the reaction solution was adjusted to 50 μL with sterile water. After incubation at 98 ° C for 1 minute, 18 cycles of reaction consisting of 98 ° C for 10 seconds, 57 ° C for 5 seconds, and 68 ° C for 45 seconds were performed to amplify the vector sequence containing the base sequence of 3C1 VH, human IgG1 CH2,3 domain.

[0094] [HiFi Insert Gene PCR] A forward primer annealing 22 bases behind EU numbering 118 and a reverse primer annealing 30 bases before EU numbering 220 were prepared (Thermo Fisher Scientific). Forward primer: 5'-GCGTCGACTAAGGGTCCGTCGG-3' (SEQ ID NO: 56) Reverse primer: 5'-GCAGCTCTTAGGCTCGACCTTCTTATCGAC-3' (SEQ ID NO: 57)

[0095] Subsequently, in the same mixing ratio as above, wild-type human IgG1 c-10_pos14 heavy chain, the two corresponding primers above, KOD One (registered trademark) PCR Master Mix, and sterile water were used to adjust the reaction solution to 50 μL. After 1 minute of incubation at 98 ° C, 40 cycles of reaction consisting of 10 seconds at 98 ° C, 5 seconds at 60 ° C, and 1 second at 68 ° C were performed, and the CH1 domain gene sequence into which the c-10 mutation had been introduced was amplified. PCR, DpnI treatment, purification, HiFi DNA Assembly, transformation, inoculation, culture, DNA extraction, and sequence analysis were performed in the same manner as in Example 1, and it was confirmed that the wild-type human IgG1 c-10 heavy chain gene sequence.

[0096] [Antibody Expression, Purification, and Quantitation of Antibody Concentration] Expression, purification, and quantitation of antibody concentration of c-10 were carried out in the same manner as in Example 1.

[0097] [Example 6] Amino acid modifications are carried out to change the isoelectric point of the CH2 domain of human IgG1.

[0098] [Example 7] Amino acid modifications are carried out to change the isoelectric point of the CH3 domain of human IgG2.

[0099] [Example 8] Amino acid modifications are carried out to change the isoelectric point of the CH3 domain of human IgG4.

[0100] [Example 9] Amino acid modifications are carried out to change the isoelectric point of the CH3 domain of mouse IgG1.

[0101] [Example 10] Amino acid modifications are carried out to change the isoelectric point of the CH3 domain of mouse IgG2a.

[0102] [Example 11] Amino acid modifications are carried out to change the isoelectric point of the CH3 domain of mouse IgG2b.

[0103] [Example 12] Amino acid modifications are carried out to change the isoelectric point of the CH3 domain of rabbit IgG.

[0104] Example 13 Immuno-assessment is carried out under the same conditions as in Example 1, except that the zeta potential is −10 mV.

[0105] Example 14 Immuno-assessment is carried out under the same conditions as in Example 1, except that the zeta potential is -15 mV.

[0106] Example 15 Immuno-evaluation was carried out under the same conditions as in Example 1, except that the sensitization conditions were pH<pI(A)<pI(B).

[0107] Example 16 Immuno-evaluation was carried out under the same conditions as in Example 1, except that the sensitization conditions were pI(A)<pI(B)<pH.

[0108] Example 17 Immuno-evaluation was carried out under the same conditions as in Example 1, except that the binding mode with the particles was chemical bonding.

[0109] [Method for binding antibodies to particles with carboxyl groups introduced] A 100 mM MES buffer solution with a pH of 9.0 was prepared using 2-morpholinoethanesulfonic acid (hereinafter referred to as "MES," manufactured by Tokyo Chemical Industry Co., Ltd., M0606), caustic soda, and pure water. 54.0 μL of the resulting MES buffer solution, 6.0 μL of a 1.0 wt% dispersion of carboxyl-introduced hydrophilic colored cellulose microparticles, 0.78 μL of a 4.0 wt% solution of 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride (hereinafter referred to as "EDC," manufactured by Tokyo Chemical Industry Co., Ltd., D1601), and 1.56 μL of a 4.0 wt% solution of N-hydroxysuccinimide (hereinafter referred to as "NHS," manufactured by Tokyo Chemical Industry Co., Ltd., B0249) were placed in a container and incubated at room temperature for 15 minutes. The supernatant was then discarded by centrifugation to remove unreacted EDC and NHS. After adding 60.0 μL of MES buffer and dispersing the microparticles, the antibody of Example 1 was added to the conical tube at 10.0 wt% relative to carboxyl group-introduced colored cellulose microparticles (Asahi Kasei Corporation, NanoAct RE1CA) and allowed to react for 2 hours at 37 ° C. Then, 720.0 μL of blocking solution (100 mM borate buffer, pH = 8.5) containing 1.0 wt% casein (Wako Pure Chemical Industries, Ltd., 030-01505) was added to the conical tube and allowed to stand in a dryer at 37 ° C for 1 hour. After 1 hour, using a centrifuge (Kubota Shoji Co., Ltd., 6200) and a centrifuge rotor (Kubota Shoji Co., Ltd., AF-5008C), centrifugation was performed at 14,000 g for 30 minutes, and antibody-bound carboxyl group-introduced colored cellulose microparticles (Asahi Kasei Corporation, NanoAct RE1CA) were precipitated, and the supernatant was discarded. Next, 720.0 μL of borate buffer (50 mM, pH = 10.0) was added to the Spitz tube, and treated with an ultrasonic disperser (SMT Corporation, UH-50) for 10 seconds to disperse the antibody-bound carboxyl group-introduced hydrophilic colored cellulose microparticles. After sufficient dispersion, centrifugation was performed at 14,000 g for 20 minutes, and the supernatant was discarded. After centrifugation, 158 μl of a storage solution (0.4% casein, 10% trehalose, 4% histidine, 50 mM boric acid, pH 10.0) was added to the precipitate so that the final particle weight was 0.038%, and the particles were dispersed in the solution.Next, 132 μl of a 0.038 wt % antibody-sensitized colored cellulose particle dispersion was evenly applied using a micropipette to the above conjugate pad cut into a shape of 10 mm width and 50 mm length, and dried at 37° C. for 60 minutes.

[0110] Example 18: Immunoassay was performed under the same conditions as in Example 1, except that the particles were gold colloids. 500 μl of colloidal gold dispersion (60 nm, manufactured by BBI) was mixed with 56 μl of 100 mM phosphate buffer (pH 9.0), and 1.8 μl of the 2 mg / ml antibody prepared in Example 1 was added. The mixture was stirred and allowed to stand at 25°C for 30 minutes. Subsequently, 31 μl of 0.005 wt% SH-PEG was added for blocking, and the mixture was allowed to stand at 25°C for 30 minutes. Next, 61 μl of a 1% aqueous sodium caseinate solution was added, and the mixture was allowed to stand at 25°C for 10 minutes. The solution was centrifuged (15,000 g, 10 minutes) to precipitate the gold colloid-conjugated antibody, and the supernatant was removed to obtain the gold colloid-conjugated antibody. Next, for washing, the colloidal gold-labeled antibody was dispersed in 833 μl of 0.25% sodium caseinate, 2.5% sucrose, and 20 mM Tris-HCl buffer (pH 8.2), followed by centrifugation (15,000 g, 10 minutes) to remove the supernatant again. Next, 204 μl of 0.25% sodium caseinate, 2.5% sucrose, and 20 mM Tris-HCl buffer (pH 8.2) was added to the precipitate to disperse the particles. A 10 mm x 40 mm strip of glass fiber pad was impregnated with the colloidal gold-labeled antibody solution and dried at room temperature to prepare a conjugate pad.

[0111] An immunochromatography kit was assembled using the conjugate pad in the same manner as in Example 1. Next, 80 μl of antigen (10 μg / ml) was developed, and the color intensity of the test line after 30 minutes of development was measured using an immunochromatography reader (TOR500, manufactured by Trust Medical Co., Ltd.). SARS-CoV-2 spike protein (Z03481, manufactured by GenScript) was used as the test substance. The antigen concentration used in the development was 10 μg / ml, which is a higher concentration than when the kits using NanoAct were tested in Examples 1 to 20.

[0112] [Example 19] Immuno-evaluation was carried out under the same conditions as in Example 1, except that the particles were latex particles (manufactured by Moritex Corporation, chemically bonded type Latex RED (0.4 μm), DR1040CA). The particles with introduced carboxyl groups were chemically bonded to the antibody in the same manner as in Example 17.

[0113] [Example 20] Immunoassay is carried out under the same conditions as in Example 1, except that the amino acid residue modifications are at positions 386 and 433.

[0114] [Example 21] Immuno-evaluation was carried out under the same conditions as in Example 1, except that the sensitization pH was 7.0.

[0115] [Example 22] Immuno-evaluation was carried out under the same conditions as in Example 2, except that the sensitization pH was 7.0.

[0116] [Example 23] Immuno-evaluation was carried out under the same conditions as in Example 2, except that the sensitization pH was 5.0.

[0117] [Example 24] Immuno-evaluation was carried out under the same conditions as in Example 4, except that the sensitization pH was 5.0.

[0118] Comparative Example 1 Immuno-evaluation was carried out under the same conditions as in Example 1, except that the antibody used was a wild-type human IgG1.

[0119] Comparative Example 2 Immunoassessment is carried out under the same conditions as in Example 1, except that the antibody used is a wild-type human IgG2.

[0120] Comparative Example 3 Immunoassessment is carried out under the same conditions as in Example 1, except that the antibody used is a wild-type human IgG4.

[0121] Comparative Example 4 Immunoassessment is carried out under the same conditions as in Example 1, except that the antibody used is a wild-type mouse IgG1.

[0122] Comparative Example 5 Immunoassessment is carried out under the same conditions as in Example 1, except that the antibody used is a wild-type mouse IgG2a.

[0123] Comparative Example 6 Immunoassessment is carried out under the same conditions as in Example 1, except that the antibody used is a wild-type mouse IgG2b.

[0124] Comparative Example 7 Immuno-evaluation is carried out under the same conditions as in Example 1, except that the antibody used is a wild-type rabbit IgG.

[0125] Comparative Example 8 Immuno-evaluation was performed under the same conditions as in Example 1, except that the particles used were poly-L-lysine-modified colored cellulose particles, and the zeta potential of the particles was +30 mV. Because the particles were positively charged, the electrostatic interaction with the antibody was small, and the antibody was not adsorbed. Therefore, no color intensity was observed in immunochromatography, and thus the result was recorded as "-", indicating that measurement was impossible.

[0126] Comparative Example 9 Immuno-evaluation was carried out under the same conditions as in Comparative Example 1, except that the particles used were antibody-bound, carboxyl-group-introduced colored cellulose microparticles (NanoAct RE1CA, manufactured by Asahi Kasei Corporation).

[0127] Comparative Example 10 Immuno-evaluation was carried out under the same conditions as in Comparative Example 1, except that the particles used were a gold colloid dispersion (manufactured by BBI: 60 nm).

[0128] Comparative Example 11 Immuno-assessment is carried out under the same conditions as in Comparative Example 1, except that the particles used are latex particles (manufactured by Moritex Corporation, chemically bonded type latex RED (0.4 μm), DR1040CA).

[0129] Comparative Example 12 Immuno-evaluation was carried out under the same conditions as in Comparative Example 1, except that the pH of the buffer used to bind the particles and the antibody was 5.0.

[0130] Comparative Example 13 Immuno-evaluation was carried out under the same conditions as in Comparative Example 1, except that the pH of the buffer used to bind the particles and the antibody was 9.0.

[0131] The polypeptide according to the present invention can be suitably used as a color-developing particle in an immunochromatographic diagnostic kit that achieves high detection sensitivity.

Claims

1. A polypeptide comprising a protein domain having an antigen-binding site and a protein domain not having an antigen-binding site, wherein the theoretical isoelectric point (pI) of the protein domain having the antigen-binding site is equal to or lower than the theoretical isoelectric point (pI) of the protein domain not having the antigen-binding site.

2. The polypeptide of claim 1, wherein the polypeptide is an intact antibody or a fragment thereof.

3. The polypeptide of claim 2, wherein the antibody fragment is selected from the group consisting of Fab, F(ab')2, Fab', single-chain antibody, and diabody linked via a linker to a protein domain that does not have an antigen-binding site.

4. The polypeptide according to claim 2, wherein the protein domain having an antigen-binding site is Fab and / or the protein domain not having an antigen-binding site is CH2 or CH3.

5. The polypeptide of claim 4, wherein at least one amino acid residue in a protein domain that does not have an antigen-binding site is modified.

6. The polypeptide of claim 5, wherein the antibody is an IgG and at least one amino acid residue at amino acid positions 345, 355, 358, 359, 360, 361, 375, 386, 387, 389, 395, 413, 415, 416, 418, 419, 421, 433, 436, 438, and 441, as identified by EU numbering, is modified to lysine (K) or arginine (R).

7. The polypeptide of claim 6, wherein the IgG is derived from a human, mouse, or rabbit, and the amino acid residue at position 386 or 433 has been modified to lysine (K) or arginine (R).

8. A particle-polypeptide complex comprising a particle having a negative zeta potential and a polypeptide as defined in any one of claims 1 to 7.

9. The particle-polypeptide complex of claim 8, wherein the degree of orientation of the particle-polypeptide complex is 1.0 or greater.

10. The particle-polypeptide complex of claim 8, wherein the particles having a negative zeta potential are cellulose-colored particles.

11. A conjugate pad containing a particle-polypeptide complex as defined in claim 10.

12. An immunochromatographic diagnostic kit comprising a conjugate pad as defined in claim 11.

13. A method for producing a particle-polypeptide complex as defined in claim 8, comprising the step of reacting a particle having a negative zeta potential with a polypeptide, wherein the theoretical isoelectric point (pI) of a protein domain of the polypeptide having an antigen-binding site is equal to or lower than the theoretical isoelectric point (pI) of a protein domain not having an antigen-binding site.

14. The method according to claim 13, wherein the step is a step of reacting particles having a negative zeta potential with a polypeptide in a buffer solution that satisfies the following condition: pH of the buffer solution ≦ the theoretical isoelectric point (pI) of the protein domain that does not have an antigen-binding site.

15. The method according to claim 13, wherein the step is a step of reacting particles having a negative zeta potential with a polypeptide in a buffer solution that satisfies the following condition: the theoretical isoelectric point (pI) of the protein domain having an antigen-binding site ≦ pH of the buffer solution ≦ the theoretical isoelectric point (pI) of the protein domain not having an antigen-binding site.