Determination method, determination kit, monoclonal antibody and cell of pulmonary surfactant protein D

Abstract

A method for measuring pulmonary surfactant protein D, the method comprising: a step in which a sample containing pulmonary surfactant protein D is brought into contact with an insoluble carrier on which an anti-pulmonary surfactant protein D monoclonal antibody is supported; and a step for detecting a complex of the pulmonary surfactant protein D and at least two anti-pulmonary surfactant protein D monoclonal antibodies, the insoluble carrier supporting only one type of antibody as the anti-pulmonary surfactant protein D antibody.

Description

Technical Field

[0001] The present invention relates to a method for determining pulmonary surfactant protein D, a determination kit, a monoclonal antibody and cells.

[0002] This application claims priority from Japanese Patent Application No. 2023-046633 filed in Japan on March 23, 2023, the contents of which are incorporated herein by reference. Background Art

[0003] Pulmonary surfactant is a protein produced and secreted by type II alveolar epithelial cells in the lungs. It reduces surface tension on the alveolar surface, making it easier for the alveoli to expand and facilitating breathing and gas exchange.

[0004] Pulmonary surfactant includes surfactant protein-A (hereinafter sometimes referred to as SP-A), surfactant protein-B (hereinafter sometimes referred to as SP-B), surfactant protein-C (hereinafter sometimes referred to as SP-C), and surfactant protein-D (hereinafter sometimes referred to as SP-D). SP-A and SP-D are hydrophilic glycoproteins. SP-A and SP-D have a biological defense role and bind to specific lipids. SP-B and SP-C are hydrophobic proteins that associate with phospholipids.

[0005] SP-D measurement is useful for identifying interstitial pneumonia, as an indicator of activity, and for predicting prognosis. Furthermore, SP-D decreases rapidly when treatment for interstitial pneumonia is effective, and therefore is also used to monitor the course of treatment. Measurement of SP-D in blood can be performed, for example, using a sandwich ELISA (Enzyme-linked Immnosorbent Assay) using two antibodies, as described in Non-Patent Document 1.

[0006] Prior art literature

[0007] Non-patent literature

[0008] Non-patent document 1: Preston E. Bratcher et al., Factors Influencing theMeasurement of Plasma / Serum Surfactant Protein D Levels by ELISA PLOS ONE,November 2014, Volume 9, Issue 11 Summary of the Invention

[0009] Technical problem solved by the invention

[0010] SP-D is known to form a wide range of multimers in vivo, ranging from trimers to 36-mers, or even larger. The distribution of SP-D multimers in blood varies depending on the physical condition and medical condition of the sample donor. Meanwhile, conventional SP-D measurement methods, such as sandwich ELISA, can cause epitope concentration and affinity fluctuations, or changes in the exposed surface of the epitope, as the number of monomers constituting the multimer changes (for example, from a dodecamer to a 36-mer). This can lead to differences in antibody reactivity with each SP-D multimer. Consequently, variations in the distribution of SP-D multimers can alter the quantitative value of SP-D.

[0011] The object of the present invention is to provide a method for measuring surfactant protein D, an assay kit, a monoclonal antibody used therein, and a hybridoma producing the antibody, which can accurately quantify surfactant protein D regardless of the number of monomers constituting the surfactant protein D multimer.

[0012] Technical means to solve the problem

[0013] One embodiment of the present invention includes the following.

[0014] [1] A method for measuring pulmonary surfactant protein D, comprising:

[0015] a step of contacting a sample containing surfactant protein D with an insoluble carrier carrying an anti-surfactant protein D monoclonal antibody; and

[0016] a step of detecting a complex of the surfactant protein D and at least two anti-surfactant protein D monoclonal antibodies,

[0017] The insoluble carrier carries only one antibody as the anti-pulmonary surfactant protein D antibody.

[0018] [2] The measuring method according to [1], wherein

[0019] The insoluble carrier is latex particles.

[0020] [3] The measuring method according to [1], wherein

[0021] The insoluble carrier is in the form of a flat plate.

[0022] The complex comprises:

[0023] The anti-pulmonary surfactant protein D antibody is carried on the insoluble carrier; and

[0024] The labeled antibody comprises the anti-pulmonary surfactant protein D monoclonal antibody and a label.

[0025] [4] The measuring method according to any one of [1] to [3], wherein

[0026] The sample is serum or plasma.

[0027] [5] The measuring method according to any one of [1] to [4], wherein

[0028] The pulmonary surfactant protein D comprises at least one basic unit, and the basic unit is a trimer of pulmonary surfactant protein D.

[0029] For one of the basic units, only one of the anti-surfactant protein D antibodies binds.

[0030] [6] The measuring method according to any one of [1] to [5], wherein

[0031] Trimeric surfactant protein D was not detected in the above-mentioned detection process.

[0032] [7] The measuring method according to any one of [1] to [6], wherein

[0033] In the detecting step, surfactant protein D formed by association of two or more basic units is detected.

[0034] [8] A kit for measuring pulmonary surfactant protein D, comprising:

[0035] A first reagent comprising an insoluble carrier carrying an anti-surfactant protein D monoclonal antibody; and

[0036] The second reagent comprises a labeled antibody, wherein the labeled antibody comprises the anti-surfactant protein D monoclonal antibody and a label.

[0037] [9] The assay kit according to [8], wherein

[0038] The insoluble carrier and the marker are the same substance.

[0039]

[10] A monoclonal antibody that binds to a trimeric pulmonary surfactant protein D, wherein:

[0040] Only one monoclonal antibody binds to one trimer of surfactant protein D.

[0041]

[11] The monoclonal antibody according to

[10] , wherein

[0042] At least two of the monoclonal antibodies bind to the dodecamer of surfactant protein D.

[0043]

[12] A hybridoma that produces the monoclonal antibody described in

[10] or

[11] .

[0044]

[13] A hybridoma deposited with the National Institute of Technology and Evaluation (NITE Patent Microorganisms Depositary) under the accession number NITE BP-03825 or NITE BP-03826.

[0045] Effects of the Invention

[0046] According to the above embodiment, a method for measuring surfactant protein D, an assay kit, a monoclonal antibody used therein, and cells producing the antibody can be provided, which can accurately quantify surfactant protein D regardless of the number of monomers constituting the multimer. BRIEF DESCRIPTION OF THE DRAWINGS

[0047] [ Figure 1 ] is a graph showing the reactivity of anti-SP-D monoclonal antibodies with SP-D derived from biological samples.

[0048] [ Figure 2 ] is a chromatogram obtained by gel filtration chromatography analysis of rSP-D manufactured by Genscript.

[0049] [ Figure 3 ] is a chromatogram obtained by gel filtration chromatography analysis of rSP-D manufactured by R&D and rSP-D manufactured by Genscript.

[0050] [ Figure 4 ] is a graph showing the measured values ​​of SP-D concentration of each fraction obtained in Analysis 5 in Example 2.

[0051] [ Figure 5 ] is a graph showing the SP-D concentration measurement values ​​of each component obtained in Analysis 5 in Comparative Example 3.

[0052] [ Figure 6 ] is a graph showing the SP-D concentration measurement values ​​of each component obtained in Analysis 5 in Comparative Example 4. DETAILED DESCRIPTION

[0053] <Measurement Method of Pulmonary Surfactant Protein D>

[0054] A method for measuring pulmonary surfactant protein D (hereinafter referred to as SP-D) in one embodiment of the present invention comprises: a step of contacting a sample containing SP-D with an insoluble carrier carrying an anti-SP-D monoclonal antibody; and a step of detecting a complex of the SP-D and at least two of the anti-SP-D monoclonal antibodies, wherein the insoluble carrier carries only one antibody as the anti-SP-D antibody.

[0055] The following describes a method for measuring SP-D according to one embodiment of the present invention. However, the following embodiments are merely illustrative and do not exclude the application of various modifications and techniques not explicitly described in the embodiments. In other words, various modifications may be made to this embodiment without departing from its spirit.

[0056] (sample)

[0057] As " sample " in this specification, body fluid of biological origin can be mainly mentioned. As sample, blood, serum, plasma and amniotic fluid can be mentioned, preferably serum and plasma. The object of collecting sample comprises human or animal (such as monkey, dog or cat), preferably human. Sample can be the sample itself derived from the object, or it can be the sample obtained by carrying out the dilution or concentration etc. treatments normally carried out on the sample collected. It should be noted that, the person who collects and prepares the sample for the present invention can be the same person as the person who carries out the assay method of the present invention, or it can be a different person. In addition, the sample used in the assay method of the present invention can be the sample collected or prepared when implementing the assay method of the present invention, or it can be the sample collected or prepared in advance and preserved.

[0058] (SP-D)

[0059] SP-D is a monomer that forms a trimer formed by the association of three monomers. In this specification, this trimer is sometimes described as a basic unit. In SP-D, four basic units associate to form a dodecamer. In addition, this dodecamer further associates to form a hexadecamer, etc. In this specification, a multimer of SP-D means a multimer formed by the association of two or more basic units. A multimer of SP-D is preferably a multimer formed by the association of four or more basic units. In this specification, when simply described as SP-D, it refers to a general term for SP-D that includes monomers, trimers, and individual multimers.

[0060] (monoclonal antibody)

[0061] As used herein, the term "monoclonal antibody" refers to an antibody or antibody molecule produced by a hybridoma derived from a single antibody-producing cell. In the assay methods of the present invention, antibody fragments that possess the functions of the monoclonal antibody may also be used, as long as the effects of the present invention are achieved. Examples of antibody fragments that possess the functions of a monoclonal antibody include functional fragments comprising the FAb portion of a monoclonal antibody obtained by enzymatic cleavage of the monoclonal antibody, functional fragments comprising the FAb portion of a monoclonal antibody produced by genetic recombination, and functional fragments comprising scFv produced by phage display.

[0062] (Method for preparing monoclonal antibodies)

[0063] The monoclonal antibody in one embodiment of the present invention can be prepared by dissolving SP-D as an antigen (also referred to as an immunogen) in a solvent such as phosphate buffered saline, and applying the solution to non-human animals for immunization. After adding a suitable adjuvant as needed to the solution, an emulsion can be used for immunization. As adjuvants, in addition to general adjuvants such as water-in-oil emulsions, water-in-oil-in-water emulsions, oil-in-water emulsions, liposomes, aluminum hydroxide gels, proteins or peptide substances derived from biological components can also be used. For example, Freund's incomplete adjuvant or Freund's complete adjuvant can preferably be used. The route of administration, dosage, and administration period of the adjuvant are not particularly limited, and it is desirable to appropriately select the mode of the immune response desired by the animal that can enhance the immune antigen.

[0064] The type of animal used in the immunization is also not particularly limited, and is preferably a mammal, such as a mouse, rat, cattle, rabbit, goat, sheep, alpaca, mouse or rat, and more preferably a mouse or rat can be used. Animal immunization can be carried out according to a general method, for example, by injecting a solution of the antigen, preferably a mixture with an adjuvant, into the subcutaneous, intradermal, intravenous or intraperitoneal cavity of the animal. The immune response generally varies depending on the type and system of the immunized animal, so the immunization schedule is preferably appropriately set according to the animal used. Antigen administration is preferably repeated several times after the initial immunization.

[0065] To obtain the monoclonal antibody of the present invention, the following operations may be performed, but are not limited thereto. Methods for preparing monoclonal antibodies themselves are well known in the art and are universal. Therefore, those skilled in the art can prepare the monoclonal antibody of the present invention by using the antigen (e.g., see Antibodies, A Laboratory Manual (Cold Spring Harbor Laboratory Press, (1988) Chapter 6, etc.).

[0066] After the final immunization, spleen cells or lymph node cells as antibody-producing cells are extracted from the immunized animal and cell fusion is performed with a cell line derived from a myeloma with high proliferation capacity to prepare hybridomas. Cell fusion preferably uses cells with high antibody production capacity (quality and quantity). In addition, the cell line derived from myeloma is more preferably compatible with the animal from which the antibody-producing cells of the fusion are derived. Cell fusion can be performed according to methods known in the art. For example, a polyethylene glycol method, a method using Sendai virus, or a method utilizing electric current can be used. The hybridoma obtained can be propagated according to conditions common in this area. The desired hybridoma can be selected while confirming the properties of the antibody to be produced. The cloning of hybridomas can be performed by known methods such as limiting dilution method and soft agar method.

[0067] After the cloning step, the ability of the produced monoclonal antibodies to bind to SP-D can be tested using methods such as ELISA, RIA, or fluorescent antibody assays. These procedures can confirm whether the selected hybridomas produce monoclonal antibodies with the desired properties.

[0068] Monoclonal antibodies with desired properties can be produced by mass-culturing the hybridomas selected as described above. The mass-culturing method is not particularly limited, and examples thereof include: culturing the hybridomas in an appropriate culture medium to produce the monoclonal antibody in the culture medium; and injecting the hybridomas into the peritoneal cavity of a mammal to allow them to proliferate and produce the monoclonal antibody in the ascites.

[0069] The anti-SP-D monoclonal antibodies of the present invention are preferably cell-derived antibodies obtained by subcutaneously or intraperitoneally immunizing Balb / c mice or F344 / Jc1 rats with recombinant human SP-D manufactured by Genscript as an immunogen. For example, the anti-SP-D monoclonal antibodies are preferably S21208 and S21202 produced by hybridomas deposited with the National Institute of Technology and Evaluation (NITE Patent Microorganisms Depositary, Room 122, 2-5-8 Kazusa Kamata, Kisarazu-shi, Chiba Prefecture, Japan) on February 14, 2023, under the deposit numbers NITE BP-03826 and NITE BP-03825, respectively. S21208 is more preferred.

[0070] In the SP-D measurement method of the present invention, only one monoclonal antibody binds to SP-D, namely, an anti-SP-D monoclonal antibody with the same amino acid sequence, and a sandwich system is constructed. As used herein, a sandwich system refers to a method in which the target substance, SP-D, is sandwiched between at least two monoclonal antibodies.

[0071] When constructing a sandwich system, it is preferred that at least one of the at least two anti-SP-D monoclonal antibodies is a solid-phase antibody, and at least one is a labeled antibody. As used herein, a solid-phase antibody refers to a monoclonal antibody that is directly or indirectly immobilized on a solid phase, i.e., an insoluble carrier. As used herein, a labeled antibody refers to a monoclonal antibody that has been directly or indirectly labeled with a labeling substance commonly used and known to those skilled in the art. In the present invention, the solid-phase antibody and the labeled antibody are the same anti-SP-D monoclonal antibody, i.e., an anti-SP-D monoclonal antibody having the same amino acid sequence.

[0072] An anti-SP-D monoclonal antibody binds only one anti-SP-D monoclonal antibody to a trimer of SP-D (i.e., a basic unit). The mechanism is believed to be that the epitope is concentrated at a single location on the basic unit. When the first antibody binds, the second antibody is sterically hindered by the first antibody, preventing it from accessing the epitope. Consequently, the two anti-SP-D monoclonal antibodies cannot sandwich the basic unit, preventing the formation of a complex. Therefore, the assay method of this embodiment cannot detect trimers of SP-D.

[0073] On the other hand, SP-D composed of two or more basic units, such as dodecamers and hexahexamers, can bind to two different anti-SP-D monoclonal antibodies on two different basic units. Therefore, the assay method of this embodiment can detect SP-D composed of two or more basic units, such as dodecamers and hexahexamers.

[0074] Solid phase antibodies can be produced by physically adsorbing the monoclonal antibody or chemically bonding it (possibly via an appropriate spacer) to an insoluble carrier. As an insoluble carrier, a solid phase comprising a polymer substrate such as a polystyrene resin, an inorganic substrate such as glass, or a polysaccharide substrate such as cellulose or agarose can be used. The shape of the insoluble carrier is not particularly limited, and any shape such as a flat plate (e.g., microplate and membrane), a bead or particle (e.g., latex particles and magnetic particles), or a cylindrical (e.g., test tube) can be selected.

[0075] The amount of SP-D can be measured using a labeled antibody that directly binds to the monoclonal antibody used in the SP-D measurement method of the present invention. In this specification, an antibody that binds to the monoclonal antibody of the present invention and to which a labeled substance is bound is referred to as a secondary antibody. This secondary antibody can also be used to indirectly bind a labeled substance to the monoclonal antibody of the present invention.

[0076] The intensity of the signal emitted by the labeling substance, the turbidity based on the aggregation of the labeling substance, and the amount of SP-D in the sample can be measured. As the labeling substance for preparing the labeled antibody, for example, a metal complex, an enzyme, an insoluble particle, a fluorescent substance, a chemiluminescent substance, biotin, avidin, a radioactive isotope, a colloidal gold particle or a colored latex can be enumerated. As the binding method of the labeling substance and the monoclonal antibody, physical adsorption method, glutaraldehyde method, maleimide method, pyridine disulfide method or periodic acid method available to those skilled in the art can be used. When enzymes such as horseradish peroxidase (HRP) or alkaline phosphatase (ALP) are used as labeling substances, the specific substrate of the enzyme can be used to measure the enzyme activity. For example, when the enzyme is HRP, O-phenylenediamine (OPD) or 3,3',5,5'-tetramethylbenzidine (TMB) can be used, and when the enzyme is ALP, p-nitrophenyl phosphate can be used to measure the enzyme activity. When biotin is used as a labeling substance, the monoclonal antibody may be labeled with biotin and reacted with avidin or streptavidin labeled with an enzyme, a dye, or a fluorescent marker (preferably HRP).

[0077] In this specification, the physical or chemical support of an antigen or antibody on a solid phase, or the state where the antigen or antibody is physically or chemically supported on a solid phase, may be referred to as "immobilization" or "solid-phased." Furthermore, the terms "analysis," "detection," or "measurement" include the quantification of SP-D.

[0078] The SP-D measurement method of the present invention utilizes the reaction between an antigen and an antibody to measure the concentration of SP-D contained in a sample. Examples of SP-D measurement methods of the present invention include, but are not limited to, enzyme immunoassay (ELISA) and latex immunoturbidimetry (LTIA). The immunoassay method of the present invention is preferably LTIA.

[0079] The SP-D assay method of the present invention can be an in vivo or in vitro immunoassay. Furthermore, a sensitizer may be used to enhance sensitivity. In the SP-D assay method of the present invention, the order in which the anti-SP-D monoclonal antibody and the sample are added to the assay system is not limited as long as the effects of the present invention are achieved. Specifically, the anti-SP-D monoclonal antibody may be added to the assay system before, simultaneously with, or after the sample is added.

[0080] The following describes the measurement steps and principles for each measurement method employed. The following merely illustrates the measurement steps and principles in one embodiment of the present invention and does not limit the scope of the present invention in any way.

[0081] In each of the immunoassay methods described below, specific methods such as the method of immobilizing the monoclonal antibody on a solid phase, the method of binding the monoclonal antibody to the labeling substance, and the type of the labeling substance include those described above, and methods known to those skilled in the art can be used without limitation.

[0082] (Latex immunoturbidimetry)

[0083] Latex immunoturbidimetry is an immunoassay method that utilizes the agglutination of latex particles caused by the binding of anti-SP-D monoclonal antibodies supported on the surface of latex particles to SP-D. The latex particles are not particularly limited, as long as they are commonly used in in vitro diagnostics. The concentration of the latex particles used in the agglutination assay, the average particle size of the latex particles, and other parameters can be appropriately set based on sensitivity and performance. The SP-D assay method of the present invention, using the latex immunoturbidimetry assay, includes the following steps and principles.

[0084] (1) Latex particles loaded with anti-SP-D monoclonal antibodies are brought into contact with a sample.

[0085] (2) SP-D in the sample forms a complex with at least two anti-SP-D monoclonal antibodies, causing latex particles to agglutinate.

[0086] (3) Irradiate the sample with near-infrared light (e.g., wavelength 600 nm), measure the absorbance or scattered light, and calculate the concentration of SP-D based on the measured value.

[0087] When latex immunoturbidimetry is used as the SP-D measurement method of the present invention, the latex particles serve as an insoluble carrier and also as a labeling substance. Only one type of monoclonal antibody binds to the SP-D supported on the latex particles, namely, an anti-SP-D monoclonal antibody having the same amino acid sequence.

[0088] As a measuring apparatus, a biochemical automatic analyzer (for example, Hitachi Automatic Analyzer 3500 manufactured by Hitachi, Ltd.) can be used.

[0089] (ELISA)

[0090] As used herein, ELISA refers to a method in which SP-D, the substance to be detected, is captured in a sample using an anti-SP-D monoclonal antibody, followed by detection using an enzymatic reaction. The solid phase is preferably a plate (also called an immunoplate). HRP or ALP can be used as a label. The SP-D assay method of the present invention, using a sandwich ELISA, includes the following steps and principles.

[0091] (1) A sample is brought into contact with a solid phase on which an anti-SP-D monoclonal antibody is immobilized, and SP-D in the sample is bound to the anti-SP-D monoclonal antibody, i.e., the solid phase antibody.

[0092] (2) When a labeled anti-SP-D monoclonal antibody, i.e., labeled antibody, is added to a solid phase and reacted, the labeled antibody binds to SP-D to form a solid phase antibody-SP-D-labeled antibody complex, i.e., a sandwich.

[0093] (3) After washing, the labeled substance is allowed to develop color and the absorbance is measured.

[0094] The amount of SP-D in the sample can be determined based on the measured amount of the labeling substance.

[0095] In the sandwich ELISA method, a secondary antibody can also be used. By using a secondary antibody, the reaction can be amplified and the detection sensitivity can be improved. In the following example, the secondary antibody is an antibody that specifically recognizes the anti-SP-D monoclonal antibody. When using a secondary antibody, the following steps (1) to (5) can be used.

[0096] (1) A sample is added to a solid phase immobilized with anti-SP-D monoclonal antibodies, followed by incubation, removal of the sample, and washing.

[0097] (2) Anti-SP-D monoclonal antibody was added for incubation and washing.

[0098] (3) Further add enzyme-labeled secondary antibody for incubation.

[0099] (4) Add substrate for color development.

[0100] (5) The amount of SP-D is determined by measuring color development using a microplate reader or the like.

[0101] When ELISA is used as the method for measuring SP-D of the present invention, the solid phase antibody and the labeled antibody that bind to SP-D are the same type, ie, anti-SP-D monoclonal antibodies having the same amino acid sequence.

[0102] According to the above embodiment, accurate quantification of SP-D can be performed regardless of the number of monomers of SP-D constituting the multimer.

[0103] <Assay Kit>

[0104] A kit for measuring SP-D in one embodiment of the present invention comprises: a first reagent comprising an insoluble carrier supporting an anti-surfactant protein D monoclonal antibody; and a second reagent comprising a labeled antibody comprising the anti-surfactant protein D monoclonal antibody and a label.

[0105] The insoluble carrier and the labeling substance may be the same substance or latex particles. The SP-D monoclonal antibody supported on the insoluble carrier and the SP-D monoclonal antibody used as the labeling antibody are of the same species, ie, antibodies with the same amino acid sequence.

[0106] The SP-D monoclonal antibody binds to a trimeric surfactant protein D, binding only one trimer of surfactant protein D. At least two SP-D monoclonal antibodies bind to a dodecamer or hexadecamer of SP-D.

[0107] The SP-D monoclonal antibody, insoluble carrier, labeling substance, and labeled antibody are as described above, and their contents are applicable.

[0108] The SP-D measurement kit in one embodiment of the present invention may contain at least one of a standard antigen substance used in SP-D measurement, other test reagents such as an antigen sample for quality control, and a sample diluent.

[0109] It should be noted that the above-described methods for producing the monoclonal antibodies of the present invention, nucleic acid molecules encoding the monoclonal antibodies of the present invention, vectors or plasmids containing the nucleic acid molecules, cells containing the nucleic acid molecules, vectors or plasmids, hybridomas producing the antibodies of the present invention, etc. are also subject of the present invention.

[0110] Example

[0111] The present invention is described in more detail below with reference to examples, but the present invention is not limited to the following examples.

[0112] <Preparation of Monoclonal Antibodies>

[0113] Recombinant human SP-D (hereinafter referred to as rSP-D) manufactured by Genscript was used as the immunogen. For the primary immunization, rSP-D was mixed with Freund's Complete Adjuvant (Difco Laboratories) in a 1:1 ratio. For the secondary immunization and subsequent immunizations, rSP-D was mixed with Freund's Incomplete Adjuvant (Difco Laboratories) in a 1:1 ratio. Balb / c mice or F344 / Jcl rats were subcutaneously immunized every other week with 25 μg of the immunogen for the primary immunization and 20 μg (diluted in PBS) for the secondary and subsequent immunizations. After three immunizations, blood antibody titers were assessed using an antigen-immobilized ELISA. Individuals demonstrating a sufficient titer increase were intraperitoneally immunized with the immunogen diluted in PBS one to three days prior to autopsy. Then, spleen cells, iliac lymph node cells, and mouse cervical lymph node cells were recovered and fused with myeloma SP2 / 0 cells by electrofusion. The fused cells (i.e., hybridomas) were cultured in 96-well plates, and the culture supernatant was recovered after 7 or 8 days of fusion. Then, screening was performed based on the antigen immobilized ELISA described below to select strains that showed reactivity to rSP-D. It should be noted that the culture medium was replaced the day before the screening.

[0114] <Screening of anti-SP-D antibodies>

[0115] In a 96-well ELISA plate (NUNC442404), dispense rSP-D (1 μg / mL in PBS) at 50 μL / well and let it stand at room temperature for 2 hours. After washing three times with PBST, dispense blocking solution (1% BSA-PBST) at 100 μL / well and let it stand at room temperature for 1 hour or overnight at 4°C. After removing the blocking solution, dispense cell culture supernatant and 1000-fold and 10,000-fold diluted antiserum at 50 μL / well, respectively, and let it stand at room temperature for 1 hour. After washing three times with PBST, Goat anti-Mouse IgG (H+L) PAb-HRP (manufactured by Southern Biotech, 9500-fold dilution) or Goat anti-Rat IgG (H+L) PAb-HRP (manufactured by Southern Biotech, 8500-fold dilution) was dispensed at 50 μL / well and allowed to stand at room temperature for 1 hour. After washing three times with PBST, OPD colorimetric solution was dispensed at 50 μL / well and allowed to stand at room temperature for 10 minutes. Stop solution was dispensed at 50 μL / well, and after the reaction was stopped, the reaction was measured using a microplate reader (Abs. 492 nm). In this way, antibodies reactive with rSP-D were selected. The resulting anti-SP-D monoclonal antibodies are shown in Table 1.

[0116] [Table 1]

[0117]

[0118] <Analysis Example 1> Confirmation of Specificity of Anti-SP-D Monoclonal Antibody

[0119] A mixture of rSP-D, recombinant human surfactant A (SP-A), recombinant human Collectin Liver 1 (CLL1), and recombinant human Mannan-Binding Lectin (MBL) antigens (each 1 μg / mL in PBS) was dispensed at 50 μL / well into a 96-well ELISA plate (NUNC442404) and allowed to stand at room temperature for 2 hours. After washing three times with PBST, blocking solution (1% BSA-PBST) was dispensed at 100 μL / well and allowed to stand at room temperature for 1 hour or overnight at 4°C. After removing the blocking solution, each anti-SP-D monoclonal antibody solution (5 μg / mL) listed in Table 2 was dispensed at 50 μL / well and allowed to stand at room temperature for 1 hour. After washing three times with PBST, goat anti-mouse IgG (H+L) PAb-HRP (Southern Biotech, 1031-05, 9500-fold dilution) or goat anti-rat IgG (H+L) PAb-HRP (Southern Biotech, 3050-15, 8500-fold dilution) was dispensed at 50 μL / well and allowed to stand at room temperature for 1 hour. After washing three times with PBST, OPD colorimetric solution was dispensed at 50 μL / well and allowed to stand at room temperature for 10 minutes. Stop solution was dispensed at 50 μL / well, and after stopping the reaction, the plate was measured using a microplate reader (Abs. 492 nm). The measured value of each well was subtracted from the measured value of the well without the anti-SP-D monoclonal antibody, and the detection value was calculated. The results are shown in Table 2. All anti-SP-D monoclonal antibodies reacted strongly only with rSP-D and had no or very low reactivity with antigens other than SP-D.

[0120] [Table 2]

[0121]

[0122] <Analysis Example 2> Evaluation of Reactivity of Anti-SP-D Monoclonal Antibodies with SP-D Derived from Biological Samples

[0123] The reactivity of anti-SP-D monoclonal antibodies with biologically derived SP-D contained in amniotic fluid was evaluated by intermolecular interaction measurement using Octet (Sartorius, Red384). Anti-SP-D monoclonal antibodies biotinylated using Biotin Labeling Kit-NH2 (Dongren Chemical Research Institute) according to conventional methods were bound to a streptavidin sensor and blocked. The reactivity of amniotic fluid diluted 5 times with PBST (phosphate buffered saline with Tween (registered trademark) added) containing 1% BSA, or rSP-D adjusted to 1μg / mL or 10μg / mL, with anti-SP-D monoclonal antibodies was evaluated. The results are shown in Figure 2. Figure 1 As shown. Figure 1 As shown, all anti-SP-D monoclonal antibodies obtained reacted with rSP-D and SP-D derived from organisms in amniotic fluid.

[0124] <Analysis Example 3> Detection of SP-D from Biological Samples Based on Sandwich ELISA

[0125] The anti-SP-D monoclonal antibodies obtained by combining them were used to investigate their ability to detect SP-D in serum. A solid-phase anti-SP-D monoclonal antibody prepared in PBS at 5 μg / mL was dispensed into each well of an ELISA plate at a concentration of 50 μL / well and allowed to stand at room temperature for 2 hours. Each well was washed three times with TBST (washing solution) containing 5 mM calcium chloride at a concentration of 400 μL / well. 1% BSA / TBST (blocking solution) was dispensed into each well at a concentration of 100 μL / well and allowed to stand at room temperature for 1 hour. The blocking solution was removed from each well, and serum was diluted with 1% BSA / TBST containing 5 mM calcium chloride to a SP-D concentration of 100 ng / mL and dispensed into each well at a concentration of 50 μL / well. The plate was then allowed to stand at room temperature for 1 hour. Each well was washed three times with 400 μL / well of the wash solution. A 0.2 μg / mL solution of biotinylated anti-SP-D monoclonal antibody, biotinylated using the Biotin Labeling Kit-NH2 (Dojindo Chemical Research Institute) was dispensed at 50 μL / well and allowed to stand at room temperature for 1 hour. After washing each well three times with 400 μL / well of the wash solution, HRP-Streptavidin was dispensed at 0.2 μg / mL in TBS containing 5 mM calcium chloride at 50 μL / well and allowed to stand at room temperature for 30 minutes. After washing three times with the wash solution, OPD color development solution was dispensed at 50 μL / well and allowed to stand at room temperature for 10 minutes. Stop solution was dispensed at 50 μL / well and the absorbance at 492 nm was measured using a microplate reader.

[0126] Antibody combinations with absorbances below 0.1 were assigned a value of -, monoclonal antibody combinations with absorbances between 0.1 and 0.5 were assigned a value of +, monoclonal antibody combinations with absorbances between 0.5 and 1.0 were assigned a value of ++, and monoclonal antibody combinations with absorbances above 1.0 were assigned a value of +++. The results are shown in Table 3. Multiple antibody combinations, including combinations of the same monoclonal antibodies, can detect SP-D in serum. In particular, the combination of S21202, S21205, and S21208 antibodies enables highly sensitive detection of SP-D from biological samples.

[0127] [Table 3]

[0128]

[0129] <Analysis Example 4> Analysis of rSP-D Multimer Distribution 1

[0130] A solution containing 55 μg of rSP-D (manufactured by Genscript) was added to a Superdex 200 Increase 10 / 300GL column (Cytiva) equilibrated with TBS, and gel filtration chromatography was performed at a flow rate of 0.7 mL / min. The UV absorbance was recorded. 400 μL of the eluate was aliquoted into a tube pre-filled with 100 μL of TBS containing 5% BSA. The fractions were thoroughly mixed, aliquoted, and stored at -30°C.

[0131] Figure 2 This is a chromatogram obtained by gel filtration chromatography analysis of rSP-D manufactured by Genscript. Dodecamer SP-D was mainly eluted in fraction 14, while trimer SP-D was eluted in fraction 23.

[0132] <Detection of rSP-D gel filtration fractions using anti-SP-D monoclonal antibodies 1>

[0133] Using anti-SP-D monoclonal antibodies S21208, S21205 and S21202, and using four ELISA systems, namely S21208 solid phase-S21208 liquid phase (Example 1), S21202 solid phase-S21202 liquid phase (Example 2), S21205 solid phase-S21208 liquid phase (Comparative Example 1), and S21202 solid phase-S21205 liquid phase (Comparative Example 2), the rSP-D of each component obtained in Analysis Example 4 was determined according to the steps described below.

[0134] The solid phase antibody prepared with PBS at 5 μg / mL was dispensed into the ELISA plate in a manner of 50 μL / well and allowed to stand at room temperature for 2 hours. After washing 3 times with TBST containing 5 mM calcium chloride at 400 μL / well, 1% BSA / TBST (blocking solution) was dispensed in a manner of 100 μL / well and allowed to stand at room temperature for 1 hour. After removing the blocking solution, each component obtained in Analysis Example 4 diluted 1000 times with 1% BSA / TBST containing 5 mM calcium chloride was dispensed in a manner of 50 μL / well and allowed to stand at room temperature for 1 hour. After washing 3 times with a cleaning solution at 400 μL / well, the biotinylated antibody biotinylated according to the conventional method using BiotinLabeling Kit-NH2 (Dongren Chemical Research Institute) was prepared into a solution of 0.2 μg / mL in 1% BSA / TBST containing 5 mM calcium chloride and dispensed in a manner of 50 μL / well and allowed to stand at room temperature for 1 hour. After washing three times with a 400 μL / well volume of the washing solution, dispense HRP-Streptavidin at 0.2 μg / mL in TBS containing 5 mM calcium chloride at a 50 μL / well volume, and let it stand at room temperature for 30 minutes. After washing three times with a 400 μL / well volume of the washing solution, dispense OPD colorimetric solution at a 50 μL / well volume, and let it stand at room temperature for 10 minutes. Dispense the stop solution at a 50 μL / well volume, and measure the absorbance at a wavelength of 492 nm using a microplate reader.

[0135] These results are shown in Table 4. In Examples 1 and 2, in which the same anti-SP-D monoclonal antibody was used for sandwich detection of SP-D, the dodecamer SP-D contained in Fraction 14 was detected, while the trimer SP-D contained in Fraction 23 was not detected. In Comparative Examples 1 and 2, in which two different anti-SP-D monoclonal antibodies were used for sandwich detection of SP-D, both dodecamer and trimer SP-D were detected. These results demonstrate that sandwich detection of SP-D using the same anti-SP-D monoclonal antibody allows detection of only SP-D larger than the trimer.

[0136] [Table 4]

[0137]

[0138] <Analysis Example 5> Analysis of rSP-D Multimer Distribution 2

[0139] Gel filtration chromatography of rSP-D manufactured by R&D Corporation or Genscript Corporation was performed in the same manner as in Analysis Example 4. 217 μg of rSP-D was subjected to gel filtration chromatography.

[0140] Figure 3 Chromatograms of rSP-D manufactured by R&D and rSP-D manufactured by Genscript analyzed by gel filtration chromatography are shown. Mainly, SP-D of more than thirty-sixmers elutes in fraction 8, SP-D of dodecamers elutes in fraction 12, and SP-D of trimers elutes in fraction 20. The main constituent of rSP-D manufactured by R&D is SP-D of more than thirty-sixmers, and the main constituent of rSP-D manufactured by Genscript is SP-D of dodecamers. Hereinafter, the n-th eluted fraction when analyzing SP-D manufactured by R&D is referred to as fraction Rn, and the n-th eluted fraction when analyzing SP-D manufactured by Genscript is referred to as fraction Gn. The UV absorbance of fraction R8 containing a large amount of SP-D of more than thirty-sixmers is 24.68 mAU, and the UV absorbance of fraction G12 containing a large amount of SP-D of dodecamers is 11.53 mAU. Since the protein concentration of rSP-D is proportional to the UV absorbance, the protein amount ratio of SP-D of more than thirty-sixmers in fraction R8 to SP-D of dodecamers in fraction G12 is 2.14:1.

[0141] <Detection of rSP-D Gel Filtration Fractions 2>

[0142] Using anti-SP-D monoclonal antibodies S21208, S21205, and S21202, and using three ELISA systems of S21208 solid phase - S21208 liquid phase (Example 1), S21205 solid phase - S21208 liquid phase (Comparative Example 1), and S21202 solid phase - S21205 liquid phase (Comparative Example 2), rSP-D in each fraction obtained in Analytical Example 5 was measured by the same procedure as <Detection of rSP-D Gel Filtration Fractions 1 Based on Anti-SP-D Monoclonal Antibodies>.

[0143] These results are shown in Table 5. In Example 1 where SP-D was detected by sandwiching with the same anti-SP-D monoclonal antibody, the ratio of the absorbance when measuring fraction R8 containing SP-D of more than thirty-sixmers to the absorbance when measuring fraction G12 containing SP-D of dodecamers was 2.76:1, which was at the same level as the protein amount ratio of SP-D of more than thirty-sixmers to SP-D of dodecamers in gel filtration chromatography, i.e., 2.14:1. In addition, the trimeric SP-D contained in fraction G20 was not detected in Example 1.

[0144] In Comparative Examples 1 and 2 for sandwich detection of SP-D using two different anti-SP-D monoclonal antibodies, trimeric SP-D can be detected. On the other hand, the ratios of the absorbance when measuring Fraction R8 containing SP-D of 36-mers or more to the absorbance when measuring Fraction G12 containing SP-D of 12-mers are 1:0.87 and 1:0.44, respectively, and do not reflect the ratio of the amounts of the respective proteins of the SP-D multimers in gel filtration chromatography. More specifically, according to the results of gel filtration chromatography, the absorbance when measuring the fraction of SP-D of 12-mers, which should contain less protein than SP-D of 36-mers or more, is higher than the absorbance when measuring the fraction containing SP-D of 36-mers or more.

[0145] From the above results, it is considered that when sandwich detecting SP-D of 12-mers or more using the same anti-SP-D monoclonal antibody, regardless of the number of basic units in the SP-D multimer, it is possible to measure the amount of the substantial protein.

[0146] [Table 5]

[0147]

[0148] <Preparation of LTIA Reagent for SP-D Determination>

[0149] 1) First Reagent

[0150] As the first reagent, a solution having the following composition is prepared.

[0151] 100 mM MES-NaOH (pH 6.0)

[0152] 500 mM NaCl

[0153] 0.5% BSA

[0154] 2) Solution of Anti-Human SP-D Monoclonal Antibody-Sensitized Latex Particles

[0155] To a 1% polystyrene latex solution (manufactured by SEKISUI MEDICAL CO., LTD.) having an average particle diameter of 317 nm and a critical agglutination concentration of 280 mM (in 10 mM MOPS buffer), an anti-SP-D antibody solution diluted to 0.35 mg / mL with an equal amount of 10 mM MOPS buffer is added, and the mixture is stirred at 4°C for 2 hours. Then, 10 mM MOPS buffer containing an equal amount of 0.5% BSA is added, and the mixture is stirred at 4°C for 1 hour to prepare a solution of anti-human SP-D monoclonal antibody-sensitized latex.

[0156] 3) Second Reagent

[0157] 3-1) Example 2

[0158] The S21208 antibody-sensitized latex particle solution prepared in the step 2) was diluted with 5 mM MOPS-NaOH (pH 7.0) buffer so that the absorbance at a wavelength of 600 nm was 6.0 Abs. to prepare a second reagent.

[0159] 3-2) Comparative Example 3

[0160] 5 mM MOPS-NaOH (pH 7.0) buffer and the antibody-sensitized latex particle solution are mixed as the second reagent so that the absorbance of the S21205 antibody-sensitized latex particle solution prepared in the step described in 2) at a wavelength of 600 nm is 3.0 Abs. and the absorbance of the S21208 antibody-sensitized latex particle solution at a wavelength of 600 nm is 6.0 Abs.

[0161] <Quantification of rSP-D Gel Filtration Fractions Using SP-D Assay Reagent>

[0162] 1) LTIA reagent-based assay

[0163] The first and second reagents were combined and the SP-D concentrations in each component obtained in Analytical Example 5 (Example 2 and Comparative Example 3) were measured and calculated using a biochemical automatic analyzer (manufactured by Hitachi, Ltd., Hitachi Automatic Analyzer 3500) by the following steps. 120 μL of the first reagent was added to 5 μL of each component and heated at 37°C for 5 minutes. 40 μL of the second reagent was added to each component and stirred. The absorbance change was measured for 5 minutes at a main wavelength of 570 nm and a secondary wavelength of 800 nm. The SP-D concentration in each component was calculated using a standard curve plotted with the absorbance change per unit time when measuring recombinant SP-D (R&D Company) of known concentration as the horizontal axis and the recombinant SP-D concentration as the vertical axis.

[0164] 2) Measurement method using CL SP-D "YAMASA" NX (YAMASA Soy Sauce Co., Ltd., certified in vitro diagnostic drug (hereinafter referred to as certified reagent))

[0165] The components obtained in Analytical Example 5 (Comparative Example 4) were assayed according to the package insert of the certified reagent. According to the package insert of the certified reagent, this reagent is a reagent for measuring SP-D in serum based on a chemiluminescent enzyme immunoassay using two different anti-human SP-D mouse monoclonal antibodies.

[0166] Table 6 shows the measured values ​​of SP-D concentration when the components R8, R12, and R20 obtained in Analysis 5 were used. Figure 4 This is a graph showing the measured values ​​of SP-D concentration of each fraction obtained in Analysis 5 in Example 2. Figure 5This is a graph showing the measured values ​​of SP-D concentration of each fraction obtained in Analysis 5 in Comparative Example 3. Figure 6 This is a graph showing the measured values ​​of SP-D concentration of each fraction obtained in Analysis 5 in Comparative Example 4.

[0167] In Example 2, SP-D was detected at a ratio equivalent to the protein ratio of SP-D (2.14:1) of 36-mer or larger and SP-D (2.14:1) in gel filtration chromatography, similar to the ELISA in Example 1. Furthermore, in Example 2, the measured value of fraction G20, which contains trimer SP-D, was comparable to the measured value of fraction G18, which had a lower protein content than fraction G20, measured in gel filtration chromatography. This suggests that substantially no trimer SP-D was detected in Example 2.

[0168] On the other hand, in Comparative Examples 3 and 4 using two different antibodies, the measured value of fraction G12 containing dodecamers of SP-D was calculated to be lower than the measured value of fraction R8 containing 36-mers or more of SP-D, unlike the results of gel filtration chromatography. In addition, both Comparative Examples 3 and 4 showed higher measured values ​​than those of fraction G18 when fraction G20 containing trimers of SP-D was measured, and trimers of SP-D ( Figure 5 、 Figure 6 ) From the above, it can be seen that when SP-D is detected using the same antibody sandwich, the LTIA reagent can be used to measure the amount of the actual protein, regardless of the number of basic units in the SP-D multimer.

[0169] [Table 6]

[0170]

[0171] Industrial Applicability

[0172] According to the above embodiment, a method for measuring surfactant protein D, an assay kit, a monoclonal antibody used therein, and cells producing the antibody can be provided, which can accurately quantify surfactant protein D regardless of the number of monomers constituting the multimer.

[0173] [Accession number]

[0174] NPMD NITE BP-03825

[0175] NPMD NITE BP-03826

[0176] Note that NPMD is an abbreviation for NITE Patent Microorganisms Depositary, National Institute of Technology and Evaluation (Room 122, 2-5-8 Kazusa Kamata, Kisarazu-shi, Chiba Prefecture, Japan).

Claims

1. A method for determining pulmonary surfactant protein D, comprising: a step of contacting a sample containing surfactant protein D with an insoluble carrier carrying an anti-surfactant protein D monoclonal antibody; and a step of detecting a complex of the surfactant protein D and at least two anti-surfactant protein D monoclonal antibodies, The insoluble carrier carries only one antibody as the anti-pulmonary surfactant protein D antibody.

2. The assay method according to claim 1, wherein The insoluble carrier is latex particles.

3. The measuring method according to claim 1, wherein The insoluble carrier is in the form of a flat plate. The complex comprises: The anti-pulmonary surfactant protein D antibody is carried on the insoluble carrier; and The labeled antibody comprises the anti-pulmonary surfactant protein D monoclonal antibody and a label.

4. The measuring method according to claim 1, wherein The sample is serum or plasma.

5. The measuring method according to any one of claims 1 to 4, wherein The pulmonary surfactant protein D comprises at least one basic unit, and the basic unit is a trimer of pulmonary surfactant protein D. For one of the basic units, only one of the anti-surfactant protein D antibodies binds.

6. The measuring method according to claim 5, wherein Trimeric surfactant protein D was not detected in the above-mentioned detection process.

7. The measuring method according to claim 6, wherein In the detecting step, surfactant protein D formed by association of two or more basic units is detected.

8. A kit for determining pulmonary surfactant protein D, comprising: A first reagent comprising an insoluble carrier carrying an anti-surfactant protein D monoclonal antibody; and The second reagent comprises a labeled antibody, wherein the labeled antibody comprises the anti-surfactant protein D monoclonal antibody and a label.

9. The assay kit according to claim 8, wherein The insoluble carrier and the marker are the same substance.

10. A monoclonal antibody that binds to trimeric pulmonary surfactant protein D, wherein: Only one monoclonal antibody binds to one trimer of surfactant protein D. The monoclonal antibody according to claim 10 , wherein At least two of the monoclonal antibodies bind to surfactant protein D in a dodecamer or larger form. A hybridoma producing the monoclonal antibody according to claim 10 or 11.

13. A hybridoma deposited with the National Institute of Technology and Evaluation (NITE Patent Microorganisms Depositary) under accession number NITE BP-03825 or NITE BP-03826.

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