Monoclonal antibodies specific to free AIM and their applications

Abstract

To provide a monoclonal antibody capable of specifically, highly sensitively and versatilely detecting free AIM in various specimens including urine specimens.SOLUTION: By using a monoclonal antibody that recognizes an N terminal side area of an SRCR3 domain from a C terminal side area of an SRCR2 domain of AIM, it is possible to specifically, highly sensitively and versatilely detect free AIM in various samples including urine samples.SELECTED DRAWING: None

Description

Technical Field

[0001] The present invention relates to a monoclonal antibody specific for free AIM, an immunological analysis method for free AIM using the monoclonal antibody, a composition containing the monoclonal antibody, and a kit containing the composition.

Background Art

[0002] AIM (apoptosis inhibitor of macrophage; also known as CD5L, api6, Spα) is known as a secreted protein of about 50 kDa specifically produced by tissue macrophages (Non-Patent Document 1), and has an inhibitory effect on various diseases such as acute kidney injury, fatty liver, hepatocellular carcinoma, obesity, fungal peritonitis, and multiple sclerosis. It has been clarified that it has the potential to become a new therapeutic agent for a wide range of diseases.

[0003] The structure of AIM is such that three scavenger receptor cysteine-rich (SRCR) domains, which are specific sequences containing many cysteine residues, are tandemly connected. Each cysteine residue is thought to form a compact globular three-dimensional structure by forming disulfide bonds with each other within each domain.

[0004] AIM has been reported to interact with various molecules. Various molecules have been reported as its binding partners. For example, it is known to have the ability to bind to pathogen-associated molecular patterns (PAMPs) of bacteria and fungi such as lipoteichoic acid (LTA) and lipopolysaccharide (LPS) and aggregate bacteria (Non-Patent Document 2). Also, there are many cells in the body that bind AIM to the cell surface or take it up intracellularly. In addition to being taken up by the producing cell, macrophages, in adipocytes, it is taken up by endocytosis via the scavenger receptor CD36 and has been reported to induce lipolysis (Non-Patent Document 3).

[0005] Furthermore, AIM has been known to bind to IgM in the blood. In recent years, it has been reported that the binding of AIM to IgM is important for AIM to remain stable in the blood without being excreted in the urine, and that in serum samples, most AIM exists as IgM-bound AIM, and very little exists as monomers (Non-Patent Documents 4, 5).

[0006] AIM is not excreted in urine by binding to pentameric IgM, and as a result maintains a high blood concentration of approximately 5 μg / mL, but its function is inactivated. Furthermore, it has been revealed that AIM dissociates from IgM and becomes activated when disease develops, promoting the healing of the disease (Non-Patent Literature 4).

[0007] Free AIM in the blood has been reported to be particularly useful in the diagnosis of liver disease and acute kidney injury (Patent Documents 1 and 2).

[0008] Free AIM, dissociated from IgM in the blood, passes through the glomerular filtration membrane and moves into the proximal tubule. Free AIM attaches to dead cell clumps in the tubule and contributes to the healing of acute kidney injury by promoting phagocytosis by surrounding cells, at which point free AIM is excreted in the urine. In fact, it has been reported that a significant increase in blood and urinary AIM concentrations is observed in patients with acute kidney injury (Non-Patent Literature 6).

[0009] Previous reports suggested that only free AIM exists in urine because IgM-bound AIM cannot pass through the glomerular filtration membrane. Therefore, it was thought that it was sufficient to measure AIM in urine regardless of whether it was IgM-bound or free, and that free AIM could be quantified even when measuring urine samples using a measurement method that cross-reacts to IgM-bound AIM (a measurement method that reacts to both IgM-bound and free AIM).

[0010] Therefore, there are few reported methods for specifically measuring free AIM or antibodies specific to free AIM. For example, Patent Document 3 states that a monoclonal antibody (No. 12 antibody) that is said to recognize the SRCR2 domain of AIM can specifically detect free AIM in the serum of NASH-HCC patients when combined with a specific monoclonal antibody that is said to recognize the SRCR3 domain (Figure 1C, D). However, it is unclear whether the No. 12 antibody can universally and specifically recognize free AIM in combination with various antibodies, and it has not been shown that it can specifically detect free AIM in urine samples. [Prior art documents] [Patent Documents]

[0011] [Patent Document 1] International Publication No. 2017 / 022315 [Patent Document 2] International Publication No. 2017 / 538533 [Patent Document 3] International Publication No. 2020 / 158856 [Non-patent literature]

[0012] [Non-Patent Document 1] Miyazaki T. et al., J Exp Med 189:413-422, 1999 [Non-Patent Document 2] Sarrias MR et al., J Biol Chem 280:35391-35398, 2005 [Non-Patent Document 3] Kurokawa J et al., Cell Metab 11:479-492, 2010 [Non-Patent Document 4] Miyazaki et al., *Nihon Rinsho*, Vol. 71, No. 9 (2013-9), pp. 1681-1689. [Non-Patent Document 5] Arai S. et al., ScienceDirect 3(4):1187-1198, 2013 [Non-Patent Document 6] Kitada, Kento et al., Japanese Journal of Nephrology 58(8):1234-1237, 2016. [Overview of the project] [Problems that the invention aims to solve]

[0013] The present invention has been made in view of these circumstances, and its object is to provide a monoclonal antibody capable of specifically, sensitively, and universally analyzing free AIM in various samples, including urine samples. A further object of the present invention is to provide an immunological analysis method for free AIM using the monoclonal antibody, as well as compositions and kits containing the monoclonal antibody. [Means for solving the problem]

[0014] The inventors conducted diligent research to solve the above problems and found that a monoclonal antibody that recognizes the N-terminal region of the SRCR3 domain from the C-terminal region of the SRCR2 domain of AIM specifically binds to free AIM. This monoclonal antibody can specifically detect free AIM not only in serum samples but also in urine samples. Furthermore, this monoclonal antibody exhibits excellent detection sensitivity and specificity not only in immunoaggregation and enzyme immunoassay methods, particularly in sandwich ELISA and the BLEIA® method, making it usable in a wide range of immunological detection methods. Moreover, in these immunological detection methods, it was possible to combine it with various antibodies that recognize AIM (e.g., polyclonal antibodies, antibodies that recognize the N-terminal region of the SRCR2 domain of AIM, etc.). From the above, the inventors found that by using this monoclonal antibody, it is possible to specifically, sensitively, and universally detect free AIM in various samples, including urine samples, and thus completed the present invention.

[0015] More specifically, the present invention provides the following:

[0016] [1] A method for immunologically analyzing free AIM using a monoclonal antibody specific for free AIM, which does not bind to a polypeptide consisting of positions 1 to 229 of AIM and binds to a polypeptide consisting of positions 1 to 259 of AIM.

[0017] [2] The method according to [1], wherein the analysis is performed by any one of immunoprecipitation, enzyme immunoassay, RIA, CLIA, and immunochromatography.

[0018] [3] The method according to [1], wherein the analysis is performed by the sandwich method.

[0019] [4] The method according to [1], wherein the analysis is performed by immunoprecipitation.

[0020] [5] The method according to any one of [1] to [4] for immunologically analyzing free AIM in a biological sample selected from the group consisting of serum, plasma, whole blood, and urine. ​​​​​​​​​​​​​​​​​​​​​​

[11] A kit for immunological analysis of free AIM in a sample, comprising the composition described in [9] or the solid phase described in

[10] . [Effects of the Invention]

[0027] The present invention makes it possible to specifically analyze free AIM in various samples. According to the present invention, for example, free AIM excreted in urine due to kidney disease can be measured more accurately than conventional methods. The antibody of the present invention has for the first time revealed the presence of IgM-bound AIM in urine in addition to free AIM, but this IgM-bound AIM may originate from diseases other than kidney damage (such as bleeding from the urinary tract). Therefore, by using the immunoassay method of the present invention, which can specifically recognize free AIM in urine, it becomes possible to accurately diagnose or assist in the diagnosis of kidney disease (especially acute kidney injury) by distinguishing it from diseases other than kidney damage (such as bleeding from the urinary tract). [Brief explanation of the drawing]

[0028] [Figure 1] This figure shows the results of detecting rAIM fractionated by gel filtration chromatography using a sandwich ELISA method with a specific combination of monoclonal antibodies (reagent 1). [Figure 2] This figure shows the results of detecting AIM in serum fractionated by gel filtration chromatography using a sandwich ELISA method with various antibody combinations (reagents 1-5). [Figure 3A] This figure shows the results of epitope mapping for antibody clones (clone 2, clone 8, and clone 9). [Figure 3B] This figure shows the results of epitope mapping of the antibody clone (clone 24). [Figure 4] This figure shows the results of detecting AIM in a urine sample using a sandwich ELISA method with a specific antibody combination (reagents 1 and 2). [Figure 5]This figure shows the results of detecting AIM in serum samples using the BLEIA method with a specific antibody combination (reagent 3, reagent 7) and the sandwich ELISA method with a specific antibody combination (reagent 1). [Modes for carrying out the invention]

[0029] This invention provides a monoclonal antibody specific to free AIM.

[0030] In this invention, "AIM" is a secreted blood protein of approximately 40-50 kDa produced by tissue macrophages. The typical amino acid sequence of human-derived AIM is shown in Sequence ID No. 1. Human-derived AIM contains three cysteine-rich SRCR domains: the SRCR1 domain corresponds to positions 24-124 of Sequence ID No. 1, the SRCR2 domain corresponds to positions 138-238, and the SRCR3 domain corresponds to positions 244-346. "Free AIM" refers to AIM that exists in a state not bound to IgM, and in this invention, it is used in contrast to complex AIM (hereinafter also referred to as IgM-bound AIM) that exists in a complex state with IgM.

[0031] In the present invention, "specific to free AIM" means that the antibody does not substantially cross-react with IgM-bound AIM, that is, its reactivity to IgM-bound AIM is sufficiently low compared to its reactivity to free AIM. Sufficiently low means that the ratio of reactivity to IgM-bound AIM to reactivity to free AIM is 20% or less, preferably 10% or less, more preferably 6% or less, and even more preferably 5% or less. This reactivity to AIM can be evaluated, for example, by a sandwich ELISA method using the monoclonal antibody to be evaluated. In the sandwich ELISA method, there are no particular restrictions on the antibody to be combined with the monoclonal antibody to be evaluated, as long as it can recognize AIM, and it may be a polyclonal antibody or a monoclonal antibody. It is preferable that the monoclonal antibody to be combined does not compete with the monoclonal antibody to be evaluated for binding to AIM (i.e., recognizes different epitopes).

[0032] The sandwich ELISA method can be performed, for example, in accordance with the method described in Example 1. Specifically, first, a solid-phase plate is prepared on which the monoclonal antibody to be evaluated is immobilized, and the sample (a sample containing free AIM or a sample containing IgM-bound AIM) is added to the solid-phase plate and reacted. Next, after washing, a polyclonal anti-AIM antibody is added and reacted, after washing, a labeled secondary antibody is added and reacted, after washing, and finally the signal intensity of the label is measured. When horseradish peroxidase (HRP) is used as the label, the signal can be measured using a microplate reader after adding the chromogenic substrate. As a result of the measurement, for example, if the reactivity to free AIM is 100 and the reactivity to IgM-bound AIM is 4, the monoclonal antibody to be evaluated can be determined to be a free AIM-specific antibody because its reactivity to IgM-bound AIM is 4% of its reactivity to free AIM.

[0033] A preferred embodiment of the monoclonal antibody of the present invention is a monoclonal antibody that does not bind to the polypeptide consisting of positions 1 to 229 of AIM, but binds to the polypeptide consisting of positions 1 to 259 of AIM. The recognition site of the monoclonal antibody is typically the region from the C-terminal side of the SRCR2 domain (positions 138 to 238) to the N-terminal side of the SRCR3 domain (positions 244 to 346) of AIM. Particularly preferred monoclonal antibodies are those that recognize the region of AIM from positions 230 to 259, more preferably from positions 230 to 244. Specific examples include clone 2 and clone 24 described in the examples of this application.

[0034] The monoclonal antibody of the present invention can be produced using generally known methods. For example, as described in the examples of this application, first, hybridomas can be produced using recombinant AIM (rAIM) as an immunogen, and from these, hybridomas that produce monoclonal antibodies showing high reactivity to the AIM can be selected. Furthermore, epitope analysis of the monoclonal antibody produced by the selected hybridoma can be performed to identify the clone that produces the monoclonal antibody having the above characteristics. Alternatively, a partial peptide of a specific region of AIM (for example, from the C-terminal region of the SRCR2 domain to the N-terminal region of the SRCR3 domain) may be used as the immunogen.

[0035] A representative example of the hybridoma method is the Kohler and Milstein method (Kohler & Milstein, Nature, 256:495 (1975)). The antibody-producing cells used in the cell fusion step of this method are spleen cells, lymph node cells, peripheral blood leukocytes, etc., from animals (e.g., mice, rats, hamsters, rabbits, monkeys, goats, sheep, donkeys, camels, alpacas, chickens) immunized with an antigen (target protein, its partial peptide, or cells expressing these). Antibody-producing cells obtained by treating the above cells or lymphocytes, etc., previously isolated from unimmunized animals with the antigen in a culture medium can also be used. Various known cell lines can be used as myeloma cells. The antibody-producing cells and myeloma cells may be of different animal species origins, provided they are fused, but preferably they are of the same animal species origin. Hybridomas can be produced, for example, by cell fusion between spleen cells obtained from antigen-immunized mice and mouse myeloma cells. Subsequent screening can then yield hybridomas that produce antigen-specific monoclonal antibodies. Monoclonal antibodies against antigens can be obtained by culturing hybridomas or from ascites fluid of mammals administered hybridomas.

[0036] Furthermore, if the DNA encoding the target monoclonal antibody can be obtained, it can also be produced by recombinant DNA. This method involves cloning the DNA encoding the antibody from hybridomas or B cells, incorporating it into a suitable vector, and introducing this vector into host cells (e.g., mammalian cell lines, E. coli, yeast cells, insect cells, plant cells, etc.) to produce recombinant antibodies (e.g., PJ Delves, Antibody Production: Essential Techniques, 1997; WILEY, P. Shepherd and C. Dean, Monoclonal Antibodies, 2000, OXFORD UNIVERSITY PRESS; Vandamme AM et al., Eur.J. Biochem. 192:767-775, 1990). In the expression of the antibody-encoding DNA, the DNA encoding the heavy chain or light chain may be incorporated separately into expression vectors to transform the host cells, or the DNA encoding both the heavy chain and light chain may be incorporated into a single expression vector to transform the host cells (see International Publication No. 94 / 11523). Recombinant antibodies can be obtained in a substantially pure and homogeneous form by culturing the host cells mentioned above, and then isolating and purifying them from within the host cells or from the culture medium. Antibody isolation and purification can be performed using methods commonly used for the purification of polypeptides. By using transgenic animal production technology to create transgenic animals (such as cows, goats, sheep, or pigs) into which antibody genes have been incorporated, it is also possible to obtain large quantities of monoclonal antibodies derived from the antibody genes from the milk of these transgenic animals.

[0037] The monoclonal antibody of the present invention may be a complete antibody or an antibody fragment, as long as it can recognize the antigen. Examples of antibody fragments include, but are not limited to, F(ab')2, Fab', Fab, Fv, and single-chain antibodies.

[0038] The present invention also provides a method for immunologically analyzing free AIM in a sample, the method using the monoclonal antibody described above.

[0039] The "sample" used in the method of the present invention is not particularly limited as long as it is a sample that may contain AIM (free AIM, IgM-bound AIM). Tissues or body fluids may be used, and examples of tissues include, but are not limited to, the ovaries, uterus, breasts, thyroid gland, brain, esophagus, tongue, lungs, pancreas, stomach, small intestine, duodenum, large intestine, bladder, kidneys, liver, prostate, gallbladder, pharynx, muscles, bones, and skin. Examples of body fluids include serum, plasma or whole blood, lymph, tissue fluid, body cavity fluid, digestive fluid, nasal secretions, and urine. However, when the purpose is to assist in the diagnosis of diseases related to free AIM, generally, due to the ease of collection, body fluid samples such as serum, plasma or whole blood, or urine, collected from the subject of diagnosis (preferably a human), are used.

[0040] In the method of the present invention, "analysis" includes various analyses targeting free AIM, such as quantitative analysis of free AIM, detection of the presence of free AIM, and analysis of the localization of free AIM.

[0041] A method for immunologically analyzing free AIM involves contacting the free AIM in a sample with the antibody of the present invention to induce an antigen-antibody reaction, and detecting or measuring the free AIM in the sample based on the formed immune complex. Examples of immunological analysis methods include, but are not limited to, labeled immunoassays using antigens or antibodies labeled with labeling substances, such as immunoaggregation methods (latex agglutination, gold colloid agglutination, etc.), enzyme immunoassays (EIA) using enzymes as labels, RIA (radioimmunoassay) using radioisotopes as labels, CLIA (chemiluminescent immunoassay) using chemiluminescent compounds as labels, electrochemiluminescence immunoassay, fluorescence immunoassay, and sandwich methods such as immunochromatography, Western blotting, and immunoblotting. Examples of enzyme immunoassays include ELISA, CLEIA (chemiluminescent enzyme immunoassay), and bioluminescent enzyme immunoassay, and an example of a bioluminescent enzyme immunoassay is BLEIA. Immunological measurements may be non-competitive or competitive.

[0042] There are no particular restrictions on the labeling substance as long as it can be detected by binding to the antibody, but examples include enzymes such as horseradish peroxidase (HRP), alkaline phosphatase (ALP), β-galactosidase (β-gal), and firefly luciferase; fluorescent dyes such as fluorescein isothiocyanate (FITC) and rhodamine isothiocyanate (RITC); and fluorescent proteins such as allophycocyanin (APC) and phycoerythrin (R-PE). 125 Examples include radioactive isotopes such as 1, latex particles, gold colloid particles, avidin, and biotin.

[0043] When an enzyme is used as a labeling substance, various detection methods can be performed depending on the substrate by adding peroxides and / or chromogenic substrates (for example, 3,3',5,5'-tetramethylbenzidine (TMB) which develops color by an oxidation-catalyzed reaction by peroxidase in the presence of hydrogen peroxide), fluorescent substrates, or chemiluminescent substrates.

[0044] In addition to the method of directly detecting free AIM using the monoclonal antibody of the present invention conjugated with a labeling substance, it is also possible to use a method of indirect detection by not conjugating the monoclonal antibody of the present invention with a labeling substance, but instead using a secondary antibody conjugated with a labeling substance. Furthermore, in the sandwich method described later, when using the monoclonal antibody of the present invention as the capture antibody and another antibody as the detection antibody, the other antibody can be labeled. If the other antibody is not labeled, a labeled secondary antibody can be used in a similar manner. Here, "secondary antibody" refers to an antibody that shows reactivity to an antibody that directly binds to an antigen (primary antibody). For example, if the primary antibody is a mouse antibody, an anti-mouse IgG antibody can be used as the secondary antibody. Labeled secondary antibodies that can be used for antibodies derived from various biological species such as rabbits, goats, and mice are commercially available, and an appropriate secondary antibody can be selected and used depending on the biological species from which the primary antibody originates. It is also possible to use protein G or protein A conjugated with a labeling substance instead of a secondary antibody.

[0045] The biotin-avidin system can also be used to bind antibodies to labeling substances. In this method, for example, an antibody is biotinylated, and then an avidin-modified labeling substance is applied to it. The interaction between biotin and avidin is then used to bind the labeling substance to the antibody.

[0046] In the method of the present invention, the sandwich method is preferred as the detection principle for free AIM because it allows for the construction of a highly sensitive detection system. In the sandwich method, for example, as in the sandwich ELISA method, the target substance is captured with a solid-phase capture antibody, recognized by a detection antibody bound to a labeling substance, and after B / F separation (washing), detection is performed according to the type of labeling substance. Alternatively, as in the immunochromatography method, the target substance may be recognized by a detection antibody bound to a labeling substance, B / F separation may be performed while capturing the target substance with a solid-phase capture antibody, and detection may be performed according to the type of labeling substance. As the solid phase, for example, plates such as plastic plates, fibrous materials such as nitrocellulose, and particles such as magnetic particles and latex particles can be used.

[0047] The capture antibody may be directly immobilized on the solid phase, or it may be immobilized indirectly. For example, the capture antibody can be indirectly immobilized on the solid phase by immobilizing a substance that binds to the capture antibody on the solid phase and then binding the capture antibody to that substance. Examples of substances that bind to the capture antibody include, but are not limited to, the secondary antibody, protein G, and protein A mentioned above. Furthermore, if the capture antibody is biotinylated, an avidin-containing solid phase can be used.

[0048] In the sandwich method, at least one of the antigen-capturing antibody and the detection antibody is the monoclonal antibody of the present invention. The other antibody may be any antibody capable of binding to AIM (anti-AIM antibody), and antibodies other than the monoclonal antibody of the present invention may be used. The other antibody may be a monoclonal antibody or a polyclonal antibody. It is preferable that the monoclonal antibody combined with the monoclonal antibody of the present invention does not compete for binding to AIM (i.e., recognizes different epitopes). In fact, in the examples of this application, free AIM was successfully detected specifically and with high sensitivity in both combinations of the monoclonal antibody of the present invention (clone 2, clone 24) and monoclonal antibodies that recognize other regions on AIM (clone 8, clone 9), and in combinations of the monoclonal antibody of the present invention (clone 2, clone 24) and a polyclonal antibody.

[0049] The amount of free AIM obtained from the measured values ​​can generally be quantified by comparison with measurements from a standard sample. In this case, for example, the amount of free AIM in the sample can be determined by examining where the actual measured value falls on a standard curve created based on measurements from a standard sample.

[0050] In the method of the present invention, immunoaggregation methods such as immunoturbidimetry and immunoturbidimetry (e.g., latex agglutination, gold colloid agglutination) are preferred as analytical methods for free AIM, as they do not require a B / F separation step before detection of free AIM and allow for simple and rapid detection of free AIM. Immunoglutination methods use insoluble carrier particles (solid phase) to which antigen-specific antibodies are immobilized (bound) in a liquid phase. By utilizing the property that the insoluble carrier particles agglutinate due to the formation of an immune complex between the insoluble carrier particles and the antigen, agglutination of the insoluble carrier particles is detected by measuring turbidity, visual inspection, or absorbance. As the insoluble carrier particles, metals or magnetic particles can be used, but latex particles are preferred, and generally, polystyrene latex is used. Antibodies can be immobilized on the surface of the solid phase by known techniques, such as physical adsorption or chemical bonding.

[0051] The insoluble carrier particles used in the immunoaggregation method, to which capture antibodies are immobilized, preferably contain at least two types of antibodies, and one or more monoclonal antibodies may be bound to one type of insoluble carrier particle. Examples include combinations of latex particles to which the monoclonal antibody of the present invention is immobilized and latex particles to which monoclonal antibodies other than the monoclonal antibody of the present invention are immobilized. It is preferable that the monoclonal antibodies combined with the monoclonal antibody of the present invention do not compete for binding to AIM (i.e., recognize different epitopes). In fact, in the examples of this application, free AIM was successfully detected with the same level of specificity and sensitivity as the sandwich ELISA method by combining the monoclonal antibody of the present invention (clone 2) with a monoclonal antibody that recognizes other regions on AIM (clone 8). When quantifying free AIM by agglutination of latex particles, for example, the absorbance of the liquid phase can be measured using an optical device, and the change (increase) can be used as an indicator.

[0052] The method of the present invention can be used for the diagnosis of diseases associated with free AIM (assessment of disease incidence and risk thereof). "Diseases associated with free AIM" are not limited to any disease for which free AIM can be used as a marker. Suitable diagnostic targets include, but are not limited to, kidney diseases (especially acute kidney injury), infections, inflammation, arteriosclerosis, obesity-related inflammatory diseases, COPD, cancer, liver disease, asthma, pulmonary tuberculosis, osteoarthritis, rheumatoid arthritis, and sepsis.

[0053] Furthermore, the present invention provides a composition for detecting free AIM in a sample, comprising the monoclonal antibody of the present invention. The monoclonal antibody contained in the composition of the present invention may be conjugated with a labeling substance, as described above. In addition to the antibody component, the composition of the present invention may optionally contain other components such as sterile water, physiological saline, buffers, and preservatives.

[0054] Furthermore, the present invention provides a solid phase for detecting free AIM in a sample, wherein the monoclonal antibody of the present invention is bound to the solid phase. Examples of the monoclonal antibody-bound solid phase include plates, fibrous materials, and particles bound to the monoclonal antibody of the present invention when using a sandwich method such as sandwich ELISA as the detection principle. When immunochromatography is used as the detection principle, examples include an immunochromatographic device in which the monoclonal antibody of the present invention is bound to a labeled reagent zone (in the case of a detection antibody) or a detection zone (in the case of a capture antibody). When immunoaggregation is used as the detection principle, examples include insoluble carrier particles bound to the monoclonal antibody of the present invention, such as latex particles.

[0055] Furthermore, the present invention provides a kit for immunological analysis of free AIM in a sample. The kit of the present invention includes a composition comprising the monoclonal antibody of the present invention or a solid phase to which the monoclonal antibody of the present invention is conjugated. If necessary, it can also include standard reagents (reagents containing free AIM at various concentrations), control reagents, sample diluents, dilution cartridges, washing solutions, etc. If enzyme labeling is used for detection, the kit can include substrates and reaction stop solutions necessary for detecting the label. If free AIM is detected indirectly, the kit can include a label containing a substance that binds to the primary antibody (secondary antibody, protein A, etc.). If the monoclonal antibody of the present invention is biotinylated, an avidinated label can be included. The kit of the present invention can also include instructions for use of the kit.

[0056] The compositions, solid phases, and kits of the present invention can be used, for example, as in vitro diagnostic pharmaceuticals for measuring free AIM concentrations, which form the basis for diagnosing diseases related to free AIM. [Examples]

[0057] [Reference Example 1] Production of anti-AIM antibody <Animal sensitization> An emulsion was prepared by mixing human rAIM (2 mg / mL) as the antigen with an equal volume of TiterMaxGold (G-1 Funakoshi). Two 6-week-old female Jcl:Wistar rats (CREA Japan Co., Ltd.) were used as immunizing animals, and 50 μL of the emulsion was administered to the soles of their hind feet. The same administration was performed two weeks later, and after a further two weeks or more, 50 μg of the antigen solution was administered to the soles of the feet in preparation for cell fusion three days later.

[0058] <Myeloma cells> Myeloma cells were prepared using mouse P3U1 cells. For proliferation culture, glutamine and pyruvate were added to RPMI1640 (11875-119 GIBCO), and FBS (S1560 BWT) was added to a 10% medium. Appropriate amounts of penicillin and streptomycin were added as antibiotics.

[0059] <Cell fusion> Popliteal lymph nodes were aseptically extracted from rats under anesthesia, placed in a beaker with a #200 mesh filter, and a cell suspension was prepared by pressing with a silicone rod. The cells were washed twice by centrifugation using an RPMI1640, and the number of cells was counted. Myeloma cells in the logarithmic growth phase were collected by centrifugation, washed, and then adjusted to a 5:1 ratio to lymphocytes before mixed centrifugation.

[0060] Cell fusion was performed using PEG1500 (783641 Roche). Specifically, 1 mL of PEG solution was reacted with the cell pellet for 3 minutes, followed by serial dilution, washing by centrifugation, and then culture medium was added. 200 μL of each mixture was placed into 15 96-well plates and cultured for 1 week. The culture medium used was myeloma cell medium with HAT supplement (21060-017 GIBCO) added, resulting in an FBS concentration of 15%.

[0061] <Mouse ascites fluid collection> After thawing and culturing cryopreserved cells, 1 x 10¹⁶ cells were injected into the peritoneal cavity of nude mice (BALB / cAJcl-nu / nu CREA Japan) that had been intraperitoneally administered 0.5 mL of pristane (42-002 CosmoBio) more than one week prior. 7After administering the drug, 4-12 mL of ascites fluid was obtained approximately two weeks later. After removing solid material by centrifugation, the fluid was cryopreserved.

[0062] <Electrophoretic Analysis> After thawing and processing the ascites fluid through a 5 μm filter, the monoclonal antibody bands present in the ascites fluid were confirmed by cellulose acetate membrane electrophoresis.

[0063] Electrophoresis was performed using 0.05M varbital Na Buffer pH 8.6 (020-13415 Wako Pure Chemical Industries), SELECA-V (ADVANTEC), 1 mA / cm, and for 25 minutes. 0.1% nigrosine (2% acetic acid) was used for fixation and staining.

[0064] Antibodies that react with the human rAIM antigen (hereinafter referred to as clone 2, clone 8, clone 9, and clone 24) were obtained from each cell (clone names: clone 2, clone 8, clone 9, and clone 24).

[0065] [Reference Example 2] Creation of rAIM rAIM was prepared using the method described in the literature (Biochemistry, Vol. 84, No. 7, Purification of functional rAIM protein, pp. 588-591, 2012).

[0066] [Example 1] Rabbit polyclonal anti-AIM antibodies obtained by immunizing rabbits with rAIM using the standard method, and each monoclonal antibody obtained in Reference Example 1, were used to measure the reactivity with free AIM or IgM-bound AIM obtained by the following method.

[0067] The sample was prepared by dissolving the serum sample in a buffer solution (0.05 M phosphate buffer: pH 7.0, sodium chloride: 0.3 M), adding it to a column, and performing fractionation by gel filtration chromatography under the following conditions.

[0068] Gel filtration chromatography conditions Equipment used: SHIMADZU SPD-20AV Column: Phenomenex SEC-3000 Mobile phase: 0.05M phosphate buffer, 0.3M NaCl, pH 7.0 Flow rate: 0.5mL / min Fraction: 0.5 mL / fraction Fractions with elution times of 10-17 minutes (eluate volume of 5-8.5 mL) were quantified and prepared using an AIM measurement kit (manufactured by IBL Corporation) to obtain a sample with IgM-bound AIM at 20 ng / mL, and similarly, fractions with elution times of 19-23 minutes (eluate volume of 9.5-11.5 mL) were prepared to obtain a sample with free AIM at 20 ng / mL.

[0069] The measurement method involved first dispensing solid-phase antibodies into each well of a 96-well microplate, washing them, and then immobilizing 50 μL of each solid-phase antibody to prepare a solid-phase plate. After washing, a solution containing 1% BSA was added and the plate was allowed to stand at room temperature for 2 hours. 50 μL of the sample was added to the solid-phase plate and reacted at room temperature for 1 hour. Subsequently, the solution in the wells was aspirated and washed, and 50 μL of rabbit polyclonal anti-AIM antibody was added and reacted at room temperature for 1 hour. After further aspirating and washing the solution in the wells, 50 μL of anti-rabbit IgG-HRP was added and reacted at room temperature for 1 hour. After further aspirating and washing the solution in the wells, 50 μL of o-phenylenediamine-containing substrate solution was added as a chromogenic substrate and reacted, followed by the addition of 50 μL of 2N sulfuric acid as a stop solution. The results were measured using a microplate reader at wavelengths of 492 / 650 nm.

[0070] The measurement results show, for each antibody, the ratio of the reactivity of IgM-bound AIM to the reactivity of free AIM (with the measured value of free AIM set as the baseline (100)) is shown in Table 1. Antibody clones 2 and 24 were shown to be specific to free AIM.

[0071] [Table 1]

[0072] In the table, the measured value of free AIM is set as the baseline (100), and the ratio of the measured value of IgM-bound AIM to the measured value of free AIM is shown (in %).

[0073] Furthermore, the polyclonal antibody used in this example was confirmed to react with the peptides at positions 20-128, 124-240, and 236-337 of AIM using a sandwich ELISA method.

[0074] [Example 2] Measurement of rAIM by sandwich ELISA method The rAIM obtained in Reference Example 2 was fractionated by gel filtration chromatography according to elution time (eluate volume), and its reactivity was measured.

[0075] <Sample Preparation> rAIM was fractionated by gel filtration chromatography in the same manner as in Example 1, and fractions of 0.5 mL each with elution times from 8 minutes to 28 minutes (eluate volume 4-14 mL) were used as samples.

[0076] <Measurement method> The obtained samples were combined with antibodies obtained from clones AIM-CL-6 (accession number: NITE BP-1092) (hereinafter also referred to as CL-6 or clone 6) and AIM-CL-7 (accession number: NITE BP-1093) (hereinafter also referred to as CL-7 or clone 7), which are anti-AIM antibodies described in International Publication No. 2011 / 145725 and International Publication No. 2017 / 022315, respectively, deposited at the Patent Microorganism Depository Center of the National Institute of Technology and Evaluation, as shown in Table 2, and measured using the following sandwich ELISA method.

[0077] First, 50 μL of solid-phase antibody was dispensed into each well of a 96-well microplate, washed, and then each solid-phase antibody was immobilized. After washing, 1% BSA solution was added to prepare the immobilized plate. In addition, each labeling antibody was biotinylated using the biotin labeling reagent Biotin(AC5)2Slufo-osu (Dojin Chemical Research Institute).

[0078] 50 μL of standard solution or sample was added to a solid-phase plate and reacted at room temperature for 1 hour. Subsequently, the solution in the wells was aspirated and washed, and 50 μL of biotin-labeled antibody was added and reacted at room temperature for 1 hour. After further aspirating and washing the solution in the wells, 50 μL of streptavidin-HRP was added and reacted at room temperature for 1 hour. After further aspirating and washing the solution in the wells, 50 μL of o-phenylenediamine-containing substrate solution was added as a chromogenic substrate and reacted, and then 50 μL of 2N sulfuric acid was added as a reaction stop solution. The reaction was then measured at a measurement wavelength of 492 / 650 nm using a microplate reader.

[0079] The measurement results are shown in Figure 1. In reagent 1, a main peak was observed in the fractionated sample around 20 minutes (10 mL).

[0080] [Example 3] Measurement of serum samples using the sandwich ELISA method Except for using a fractionated serum sample instead of rAIM, the monoclonal antibodies obtained in Reference Example 1 were combined as shown in Table 2, and measurements were performed in the same manner as in Example 2.

[0081] [Table 2]

[0082] The measurement results are shown in Figure 2. With Reagent 1, in addition to a peak around 20 minutes (10 mL) of elution time (flow rate), a peak around 12 minutes (6 mL) was observed. With Reagents 2-4 using antibody clone 2, only the peak of the fractionated sample around 20 minutes (10 mL) was observed in all cases, indicating that they were specific to free AIM. With Reagent 5, a peak was observed only around 12 minutes (6 mL).

[0083] [Example 4] Epitope mapping of anti-AIM antibody Epitope analysis was performed on antibody clones 2, 8, and 9 used in reagents 2-5, as well as antibody clone 24 used in reagent 7 of Example 12 described later.

[0084] <Preparation of AIM Deficient Variants> Expression vectors (pcDNA3.3) of the full-length protein of AIM or each deletion variant of SEQ ID NO: 1 were prepared (Table 3) and transfected into HEK293T cells.

[0085]

Table 3

[0086] Two days after transfection, the cells were harvested and protein extraction buffer was added. The mixture was shaken at 25 °C for 10 minutes, centrifuged at 4 °C, 10000 rpm for 10 minutes, and the supernatant was collected.

[0087] <Dot Blot> 5 μL of the collected supernatant was spotted onto a nitrocellulose membrane for each. As a negative control (NC), a protein extract of HEK293T cells without vector introduction was spotted in the same manner. This was shaken at 25 °C for 1 hour in 5% skim milk / PBS. Subsequently, after washing 4 times with PBS-T, it was reacted with antibody clone 2 at 4 °C overnight. Further, after washing 4 times with PBS-T, it was reacted in an anti-mouse IgG-HRP conjugate solution at 25 °C for 1 hour. Finally, after washing 4 times with PBS-T, it was reacted with a chemiluminescence detection reagent for 1 minute, and chemiluminescence was detected with a CCD camera.

[0088] The results are shown in Figures 3A and 3B. Antibody clone 2 and antibody clone 24 did not react with AIM1 - AIM8 respectively, reacted with AIM9, and reacted more strongly with AIM10. From this, it was confirmed that antibody clone 2 and antibody clone 24 react with the region from the C-terminal side of the SRCR2 domain to the N-terminal side of the SRCR3 domain (positions 230 - 259) in AIM. On the other hand, since it was confirmed that antibody clones 8 and 9 react with AIM4, it was confirmed that they react with the region of the N-terminal side of the SRCR2 domain (positions 155 - 169) in AIM.

[0089] [Example 5] Measurement of urine samples by sandwich ELISA method Except for using urine samples (17 cases) diluted five-fold with phosphate buffer (pH 7.4) as the sample, the measurements were performed using either Reagent 1 or Reagent 2 from Example 3.

[0090] The measurement results are shown in Figure 4. Reagent 1 and Reagent 2 showed a good correlation, but Reagent 1 showed high values ​​in two samples (sample numbers #74 and #77). As for the reason why the results for Reagent 1 and Reagent 2 differed in these two samples, it is possible that the discrepant samples contained IgM-bound AIM, which is not normally thought to be present in urine, and therefore showed high values ​​for Reagent 1.

[0091] [Example 6] Measurement of IgM concentration by sandwich ELISA method The urine sample #74 from Example 5 and six other urine samples (a total of 7 samples) were each diluted five-fold with phosphate buffer (pH 7.4), and measurements were performed in the same manner as in Example 5. The measurement results are shown in Table 4.

[0092] [Table 4]

[0093] The IgM concentration of each sample was measured using the Human IgM ELISA Quantitation Set (Betyl, E80-100) according to the kit's instructions, with each sample diluted 20-fold. In samples with high IgM concentrations, the discrepancy between the AIM concentrations measured by reagent 1 and reagent 2 was large, while in samples with low IgM concentrations, the measured values ​​from reagent 1 and reagent 2 did not deviate and showed a good correlation. These results confirmed the presence of IgM in urine and suggested the possibility of IgM-bound AIM being present in urine.

[0094] [Example 7] Measurement of IgM-bound AIM by sandwich ELISA method Since CL-6 was thought to bind to both IgM-conjugated AIM and free AIM based on the results of Example 1, IgM-conjugated AIM was measured in the same manner as in Example 2, except that CL-6 was used as the solid-phase antibody, the anti-IgM antibody from Example 6 (Human IgM ELISA Quantitation Set (Betyl, E80-100)) was used as the labeling antibody, and the samples from Example 6 were each diluted 20-fold with phosphate buffer (pH 7.4). The measurement results are shown in Table 5.

[0095] [Table 5]

[0096] In Example 6, samples with high IgM concentrations (samples with a large discrepancy between AIM concentrations measured by Reagent 1 and Reagent 2) showed high absorbance, while samples with low IgM concentrations (samples with good correlation where AIM concentrations measured by Reagent 1 and Reagent 2 did not discrepancy) showed low absorbance. This confirmed that the cause of the discrepancy was urinary IgM-bound AIM. This demonstrated that IgM-bound AIM, which was previously thought not to exist in urine, is present in urine. In urine samples, it was found that Reagent 1 measures both IgM-bound AIM and free AIM, while Reagent 2 specifically measures free AIM.

[0097] [Example 8] Measurement of serum samples by sandwich ELISA method The measurement was performed using the sandwich ELISA method with reagents 1-3, in the same manner as in Example 3, except that serum samples diluted with phosphate buffer (pH 7.4) were used as the samples.

[0098] The results are shown in Table 6.

[0099] [Table 6]

[0100] Since most AIMs in serum samples exist as IgM-bound AIMs, the measurements obtained with reagent 1, which measures both IgM-bound and free AIMs, differed significantly from the measurements obtained with reagents 2 and 3, which measure only free AIMs.

[0101] According to reagents 2 and 3, which are one embodiment of the present invention, it has been shown that accurate measurement is possible even when free AIM is present in the blood.

[0102] [Example 9] Measurement of urine samples using the sandwich ELISA method The urine sample was measured using the sandwich ELISA method with reagents 1 and 2, in the same manner as in Example 3, except that the urine sample was diluted fivefold with phosphate buffer (pH 7.4).

[0103] The results are shown in Table 7.

[0104] [Table 7]

[0105] Since most AIM exists as free AIM in urine samples, the measured values ​​of reagent 1, which measures both IgM-bound AIM and free AIM, were slightly higher, but reagents 1 and 2 showed a good correlation.

[0106] [Example 10] Measurement of serum samples by latex agglutination method Serum samples (6 samples) were measured using the latex agglutination method with the antibody combination (clone 8, clone 2) from reagent 3.

[0107] <Preparation of latex reagents> Each anti-human AIM monoclonal antibody was mixed with latex particles having a carboxyl group, and the anti-human AIM monoclonal antibody was immobilized on the surface of the latex particles using a known chemical bonding method with 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride (hereinafter referred to as WSC (manufactured by Dojin Chemical Co., Ltd.)) to obtain latex carrying each monoclonal antibody. Then, a solution of each monoclonal antibody-carrying latex (reagent 1) containing 50 mM HEPES buffer (pH 7.2) was prepared.

[0108] <Measurement method> A sample was prepared by mixing 16.0 μL of serum sample, diluted 100-fold with phosphate buffer (pH 7.4), with 120 μL of buffer containing 50 mM HEPES (pH 7.3) (second reagent). 40 μL of monoclonal antibody-loaded latex solution was mixed with the sample and reacted at 37°C. The absorbance change at a wavelength of 660 nm was measured using a TBA120FR pearl (Canon Medical). Furthermore, serum samples diluted with phosphate buffer (pH 7.4) using reagents 2 and 3 from Example 3 were measured using the sandwich ELISA method in the same manner as in Example 3. The measurement results are shown in Table 8.

[0109] [Table 8]

[0110] No discrepancies were observed in the measured values, demonstrating that serum samples can be measured using the latex agglutination method.

[0111] [Example 11] Measurement of urine samples by latex agglutination method The latex reagent prepared in Example 10 was used for measurement, except that the urine samples (6 cases) were used without dilution. Furthermore, the sandwich ELISA method was performed using reagent 2, in the same manner as in Example 3, except that each urine sample was diluted fivefold with phosphate buffer (pH 7.4) before being used as a sample. The measurement results are shown in Table 9.

[0112] [Table 9]

[0113] No discrepancies were observed in the measured values, demonstrating that urine samples can be measured using the latex agglutination method.

[0114] [Example 12] Measurement of serum samples by bioluminescent enzyme immunoassay (BLEIA method) Using the serum sample fractions obtained by the method described in Example 1, clones 2, 8, and 24 obtained in Reference Example 1 were combined as shown in Table 10 below and measured by bioluminescent enzyme immunoassay (BLEIA).

[0115] [Table 10]

[0116] <Measurement method (BLEIA method)> The procedure was carried out according to the method described in Japanese Patent Publication No. 10-239314 (conjugating biotin to both antibody and enzyme). Specifically, various antibody-immobilized magnetic particles were prepared by immobilizing various solid-phase antibodies onto magnetic particles, various biotin-labeled antibodies were obtained by mixing various labeling antibodies with biotinylation reagents, and streptavidin-biotinylated luciferase was prepared. Then, in combinations of reagents 3 and 7, 80 μL of biotin-labeled antibody solution, 100 μL of sample, and 20 μL of antibody-immobilized magnetic particles (1.5 mg / mL) were mixed and reacted at 37°C for 15 minutes. Furthermore, 500 μL of BL washing solution (Eiken Chemical) was added to the reaction solution containing the magnetic particles, and the BL washing solution was removed. Subsequently, 80 μL of streptavidin-biotinylated luciferase was added and reacted at 37°C for 15 minutes. 500 μL of BL washing solution (Eiken Chemical) was added to the reaction solution containing magnetic particles, and the washing solution was removed. Subsequently, 50 μL of BL luminescence reagent 1 from the BL luminescence reagent set (Eiken Chemical) and 50 μL of BL luminescence substrate solution (luciferin solution), which is a substrate solution for luciferase, were added, and the luminescence intensity was measured using a fully automated biochemical luminescence immunoassay analyzer BLEIA-1200 to calculate the AIM concentration in the sample. In addition, the same method as in Example 2 (sandwich ELISA method) was used with reagent 1 (comparative example).

[0117] The measurement results are shown in Figure 5. In both reagent 3 and reagent 7, only the peak in the fractionated sample with an elution time (flow rate) of around 20 minutes (10 mL) was observed, indicating specificity for free AIM. In other words, it was shown that antibody clone 24, like antibody clone 2, can specifically detect free AIM in the sample. Furthermore, from the commonality of epitopes in these antibody clones, it was confirmed that antibodies that do not bind to the polypeptide consisting of positions 1 to 229 of AIM, but bind to the polypeptide consisting of positions 1 to 259 of AIM, can specifically detect free AIM. It was also confirmed that the sample can be measured using the BLEIA method. [Industrial applicability]

[0118] As described above, the present invention makes it possible to detect free AIM specifically, with high sensitivity, and in a general manner. Since free AIM in urine serves as a marker for acute kidney injury and other conditions, the present invention can make a significant contribution not only to research but also to the diagnosis of diseases related to AIM.

Claims

1. A method for immunologically analyzing free AIM in a urine sample using a monoclonal antibody specific to free AIM, which does not bind to the polypeptide consisting of positions 1 to 229 of AIM, but binds to the polypeptide consisting of positions 1 to 259 of AIM.

2. A composition for immunological analysis of free AIM in a urine sample, comprising a monoclonal antibody specific to free AIM that does not bind to the polypeptide consisting of positions 1 to 229 of AIM, but binds to the polypeptide consisting of positions 1 to 259 of AIM.

3. A solid phase for detecting free AIM in urine samples, consisting of insoluble carrier particles to which a monoclonal antibody specific to free AIM is bound. This antibody does not bind to the polypeptide consisting of positions 1 to 229 of AIM, but binds to the polypeptide consisting of positions 1 to 259 of AIM.

4. A kit for immunological analysis of free AIM in a urine sample, A composition comprising a monoclonal antibody for detecting free AIM in a urine sample, or a solid phase to which a monoclonal antibody is conjugated for detecting free AIM in a urine sample, The kit is a monoclonal antibody specific to free AIM, which does not bind to the polypeptide consisting of positions 1 to 229 of AIM, but binds to the polypeptide consisting of positions 1 to 259 of AIM.

5. A composition for immunological analysis of free AIM in a urine sample, A composition comprising an antigen-capturing antibody and a detection antibody immobilized on a solid phase, wherein at least one of the antibodies is a monoclonal antibody specific to free AIM, and does not bind to the polypeptide consisting of positions 1 to 229 of AIM, but binds to the polypeptide consisting of positions 1 to 259 of AIM.

6. A method for immunologically analyzing free AIM in a urine sample, A method using an antigen-capturing antibody and a detection antibody immobilized on a solid phase, wherein at least one of the antibodies is a monoclonal antibody specific to free AIM, which does not bind to the polypeptide consisting of positions 1 to 229 of AIM, but binds to the polypeptide consisting of positions 1 to 259 of AIM.

7. The method according to claim 6, wherein the antigen-capturing antibody is a monoclonal antibody specific to free AIM, which does not bind to the polypeptide consisting of positions 1 to 229 of AIM, but binds to the polypeptide consisting of positions 1 to 259 of AIM.

8. The method according to any one of claims 1, 6, and 7, wherein the analysis is performed by immunoaggregation, enzyme immunoassay, RIA, CLIA, and immunochromatography.

9. The method according to any one of claims 1, 6, and 7, wherein the analysis is performed using a sandwich method.

10. The method according to any one of claims 1, 6, and 7, wherein the analysis is performed by immunoaggregation.

11. A method for testing acute kidney injury, comprising immunologically analyzing free AIM in a urine sample by the method described in any one of claims 1 and 6 to 10.

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