Pulmonary surfactant protein d measurement method, measurement kit, monoclonal antibody, and cell

Abstract

To provide a pulmonary surfactant protein D measurement method, measurement kit, and antibody to be used in the method and kit that enable accurate quantification regardless of a size of the number of monomers constituting a polymer of the pulmonary surfactant protein D, as well as to provide a hybridoma that produces the antibody.SOLUTION: A pulmonary surfactant protein D measurement method includes the steps of: putting a sample including a pulmonary surfactant protein D in contact with an insoluble carrier having a pulmonary surfactant protein D monoclonal antibody carried; and detecting a complex of the pulmonary surfactant protein D, and at least two pulmonary surfactant protein D monoclonal antibodies, in which the insoluble carrier has only one kind of the antibody carried as the pulmonary surfactant protein D monoclonal antibody.SELECTED DRAWING: None

Description

[Technical field]

[0001] The present invention relates to a method for measuring pulmonary surfactant protein D, a measurement kit, a monoclonal antibody, and cells. This application claims priority to Japanese Patent Application No. 2023-046633, filed in Japan on March 23, 2023, the contents of which are incorporated herein by reference. [Background technology]

[0002] Pulmonary surfactant is a protein produced and secreted by type II pneumocytes in the lungs. It reduces the surface tension of the alveolar surface, making the alveoli easier to expand, and aids in breathing and gas exchange.

[0003] Pulmonary surfactants include pulmonary surfactant protein-A (hereinafter sometimes referred to as SP-A), pulmonary surfactant protein-B (hereinafter sometimes referred to as SP-B), pulmonary surfactant protein-C (hereinafter sometimes referred to as SP-C), and pulmonary 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 function and bind to specific lipids. SP-B and SP-C are hydrophobic proteins and associate with phospholipids.

[0004] Measurement of SP-D is useful for the differentiation of interstitial pneumonia, as an indicator of activity, and for predicting prognosis. SP-D is also used for monitoring the progress of treatment, as it decreases rapidly when treatment of interstitial pneumonia is effective. Measurement of SP-D in blood can be performed by sandwich ELISA (Enzyme-linked Immunosorbent Assay) using two types of antibodies, as described in Non-Patent Document 1, for example. [Prior art documents] [Non-patent literature]

[0005] [Non-Patent Document 1] Preston E. Bratcher et al., Factors Influencing the Measurement of Plasma / Serum Surfactant Protein D Levels by ELISA PLOS ONE, November 2014, Volume 9, Issue 11 Summary of the Invention [Problem to be solved by the invention]

[0006] It is known that SP-D forms a wide range of multimers in vivo, from trimers to 36-mers or more. The distribution of SP-D multimers in blood varies depending on the constitution and pathology of the sample donor. On the other hand, in the conventional SP-D measurement method using sandwich ELISA, etc., the reactivity of antibodies with SP-D of each multimer may differ due to the concentration of epitopes and the variation of avidity or the change in the exposed surface of the epitope associated with the change in the number of monomers constituting the multimer (e.g., from 12-mer to 36-mer). Therefore, if the distribution of SP-D multimers differs, the quantitative value of SP-D may change.

[0007] The object of the present invention is to provide a measurement method and measurement kit for pulmonary surfactant protein D that enable accurate quantification regardless of the number of monomers that constitute the pulmonary surfactant protein D multimer, as well as a monoclonal antibody used in these methods and a hybridoma that produces this antibody. [Means for solving the problem]

[0008] One aspect of the invention includes the following. [1] A step of contacting a sample containing pulmonary surfactant protein D with an insoluble carrier carrying an anti-pulmonary surfactant protein D monoclonal antibody; detecting a complex between said pulmonary surfactant protein D and at least two of said anti-pulmonary surfactant protein D monoclonal antibodies; A method for measuring pulmonary surfactant protein D, wherein the insoluble carrier carries only one type of antibody as the anti-pulmonary surfactant protein D antibody. [2] The measurement method according to [1], wherein the insoluble carrier is a latex particle. [3] The insoluble carrier is flat, The measurement method according to [1], wherein the complex comprises the anti-pulmonary surfactant protein D antibody supported on the insoluble carrier, and a labeled antibody comprising the anti-pulmonary surfactant protein D monoclonal antibody and a label. [4] The method according to any one of [1] to [3], wherein the sample is serum or plasma. [5] The pulmonary surfactant protein D comprises at least one basic unit which is a trimer of pulmonary surfactant protein D; The method according to any one of [1] to [4], wherein only one anti-pulmonary surfactant protein D antibody binds to one basic unit. [6] The method according to any one of [1] to [5], wherein trimer of pulmonary surfactant protein D is not detected in the detection step. [7] The method according to any one of [1] to [6], wherein in the detection step, pulmonary surfactant protein D associated with two or more of the basic units is detected. [8] A kit for measuring pulmonary surfactant protein D, comprising: a first reagent comprising an insoluble carrier carrying an anti-pulmonary surfactant protein D monoclonal antibody; and a second reagent comprising a labeled antibody comprising the anti-pulmonary surfactant protein D monoclonal antibody and a label. [9] The measurement kit described in [8], wherein the insoluble carrier and the label are the same substance.

[10] A monoclonal antibody that binds to a trimeric pulmonary surfactant protein D, wherein only one monoclonal antibody binds to one of the trimeric pulmonary surfactant protein D.

[11] A monoclonal antibody described in

[10] , which binds to at least two 12-mer pulmonary surfactant protein D molecules.

[12] A hybridoma producing the monoclonal antibody described in

[10] or

[11] .

[13] A hybridoma deposited at the Patent Microorganisms Depositary Center of the National Institute of Technology and Evaluation under accession number NITE BP-03825 or NITE BP-03826. Effect of the Invention

[0009] According to the above-mentioned aspects, it is possible to provide a measurement method and measurement kit for pulmonary surfactant protein D that enable accurate quantification regardless of the number of monomers that constitute the polymeric pulmonary surfactant protein D, as well as a monoclonal antibody used therein and a cell that produces this antibody. [Brief description of the drawings]

[0010] [Figure 1] 1 is a graph showing the reactivity of anti-SP-D monoclonal antibodies with SP-D derived from biological samples. [Diagram 2] This is a chromatogram obtained by analyzing rSP-D manufactured by Genscript by gel filtration chromatography. [Diagram 3] This is a chromatogram obtained by analyzing rSP-D manufactured by R&D and rSP-D manufactured by Genscript by gel filtration chromatography. [Figure 4] 1 is a graph showing the measured SP-D concentration values ​​of each fraction obtained in Analysis 5 in Example 2. [Diagram 5] 1 is a graph showing the SP-D concentration measurement values ​​of each fraction obtained in Analysis 5 in Comparative Example 3. [Figure 6] 1 is a graph showing the SP-D concentration measurement values ​​of each fraction obtained in Analysis 5 in Comparative Example 4. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0011] <Method of measuring pulmonary surfactant protein D>

[0012] In one embodiment of the present invention, a method for measuring pulmonary surfactant protein D (hereinafter referred to as SP-D) comprises the steps of contacting a sample containing SP-D with an insoluble carrier carrying an anti-SP-D monoclonal antibody, and detecting a complex between the SP-D and at least two of the anti-SP-D monoclonal antibodies, wherein the insoluble carrier carries only one type of anti-SP-D antibody.

[0013] A method for measuring SP-D in one aspect of the present invention will be described below. However, the embodiment described below is merely an example, and various modifications and applications of techniques not explicitly stated in the embodiment are not excluded. In other words, the present embodiment can be modified in various ways without departing from the spirit of the present invention.

[0014] (sample) The "sample" in this specification can mainly include body fluids derived from a living body. Examples of samples include blood, serum, plasma, and amniotic fluid, with serum and plasma being preferred. The subject from which the sample is collected includes humans or animals (e.g., monkeys, dogs, or cats), and is preferably a human. The sample may be a sample from a subject itself, or may be a sample that has been subjected to a commonly performed treatment such as dilution or concentration of a collected sample. The person who collects or prepares the sample used in the present invention may be the same person as the person who performs the measurement method of the present invention, or may be a different person. The sample used in the measurement method of the present invention may be collected or prepared when the measurement method of the present invention is performed, or may be collected or prepared in advance and stored.

[0015] (SP-D) SP-D is a monomer, but forms a trimer in which three monomers are associated. In this specification, this trimer may be referred to as a basic unit. In SP-D, four basic units are associated to form a dodecamer. Furthermore, this dodecamer is further associated to form a 36-mer, etc. In this specification, an SP-D multimer refers to one formed by the association of two or more basic units. It is preferable that the SP-D multimer is one in which four or more basic units are associated. In this specification, when simply referring to SP-D, it means a collective term including the monomer, trimer, and each multimer of SP-D.

[0016] (Monoclonal antibody) As used herein, the term "monoclonal antibody" refers to an antibody or antibody molecule obtained from a hybridoma derived from a single antibody-producing cell. In the measurement method of the present invention, an antibody fragment having the function of this monoclonal antibody can also be used as long as the effect of the present invention can be obtained. Examples of antibody fragments having the function of a monoclonal antibody include functional fragments containing the Fab portion of the monoclonal antibody obtained by enzymatic digestion of the monoclonal antibody, functional fragments containing the Fab portion of the monoclonal antibody produced by genetic recombination, and functional fragments containing scFv produced by phage display.

[0017] (Method of preparing monoclonal antibodies) The monoclonal antibody in one embodiment of the present invention can be prepared by dissolving SP-D as an antigen (also called immunogen) in a solvent such as phosphate-buffered saline, and administering the solution to a non-human animal for immunization. If necessary, an appropriate adjuvant may be added to the solution, and then the emulsion may be used for immunization. As the adjuvant, in addition to commonly used adjuvants such as water-in-oil emulsions, water-in-oil-in-water emulsions, oil-in-water emulsions, liposomes, and aluminum hydroxide gel, proteins or peptide substances derived from biological components may be used. For example, Freund's incomplete adjuvant or Freund's complete adjuvant can be suitably used. The administration route, dosage, and administration time of the adjuvant are not particularly limited, but it is desirable to select them appropriately so as to enhance the desired immune response in the animal immunized with the antigen.

[0018] The type of animal used for immunization is not particularly limited, but mammals, such as mice, rats, cows, rabbits, goats, sheep, alpacas, mice, or rats, are preferred, and mice or rats are more preferred. Animal immunization may be performed according to a general method, for example, by injecting a solution of an antigen, preferably a mixture with an adjuvant, subcutaneously, intradermally, intravenously, or intraperitoneally into an animal. Since immune responses generally vary depending on the type and lineage of the animal to be immunized, it is desirable to set an immunization schedule appropriately depending on the animal to be used. It is preferable to repeat antigen administration several times after the first immunization.

[0019] To obtain the monoclonal antibody of the present invention, the following procedures can be carried out, but are not limited thereto. Methods for producing monoclonal antibodies themselves are well known and widely used in the art. Thus, those skilled in the art can prepare the monoclonal antibody of the present invention by using the above antigens (see, for example, Antibodies, A Laboratory Manual (Cold Spring Harbor Laboratory Press, (1988) Chapter 6, etc.).

[0020] After the final immunization, spleen cells or lymph node cells, which are antibody-producing cells, are extracted from the immunized animal, and hybridomas can be produced by cell fusion with a myeloma-derived cell line having high proliferation ability. For cell fusion, it is preferable to use cells with high antibody production ability (quality and quantity). It is more preferable that the myeloma-derived cell line is compatible with the animal from which the antibody-producing cells to be fused are derived. Cell fusion can be performed according to a method known in the art. For example, the polyethylene glycol method, a method using Sendai virus, or a method using electric current can be adopted. The obtained hybridomas can be grown according to general conditions in the art. The desired hybridomas can be selected while confirming the properties of the produced antibodies. Hybridoma cloning can be performed by well-known methods such as limiting dilution and soft agar method.

[0021] After the cloning step, the binding ability of the produced monoclonal antibody to SP-D can be assayed using methods such as ELISA, RIA, or fluorescent antibody technique, etc. By these procedures, it can be confirmed whether the selected hybridoma produces a monoclonal antibody with the desired properties.

[0022] A monoclonal antibody having the desired characteristics can be produced by mass-culturing the hybridoma selected as described above. The mass-cultivation method is not particularly limited, but examples thereof include a method in which the hybridoma is cultured in an appropriate medium to produce a monoclonal antibody in the medium, and a method in which the hybridoma is injected into the abdominal cavity of a mammal to grow and produce a monoclonal antibody in the ascites.

[0023] The anti-SP-D monoclonal antibody of the present invention is preferably an antibody derived from cells obtained by subcutaneous and peritoneal immunization of Balb / c mice or F344 / Jc1 rats using recombinant human SP-D manufactured by Genscript as an immunogen. For example, the anti-SP-D monoclonal antibody is preferably S21208 and S21202 produced from hybridomas deposited at the National Institute of Technology and Evaluation Patent Microorganism Deposit Center (Room 122, 2-5-8 Kazusa Kamatari, Kisarazu City, Chiba Prefecture, Japan) on February 14, 2023 under the accession numbers NITE BP-03826 and NITE BP-03825, respectively, and more preferably S21208.

[0024] In the method for measuring SP-D in the present invention, only one type of monoclonal antibody that binds to SP-D, i.e., an anti-SP-D monoclonal antibody having the same amino acid sequence, is used, and a sandwich system is constructed. In this specification, the sandwich system refers to a method in which the substance to be detected, i.e., SP-D, is sandwiched between at least two monoclonal antibodies.

[0025] When constructing a sandwich system, it is preferable 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. In this specification, a solid-phase antibody means a monoclonal antibody that is directly or indirectly immobilized on a solid phase, i.e., an insoluble carrier. In this specification, a labeled antibody means a monoclonal antibody that is directly or indirectly labeled with a labeling substance well known and commonly used by those skilled in the art, which will be described later. In the present invention, the solid-phase antibody and the labeled antibody are anti-SP-D monoclonal antibodies of the same type, i.e., having the same amino acid sequence.

[0026] Only one anti-SP-D monoclonal antibody binds to a trimer of SP-D (i.e., the basic unit). The mechanism behind this is thought to be that the epitope is concentrated at one location on the basic unit, and when the first antibody binds, the second antibody is sterically hindered by the first antibody and cannot approach the epitope. Therefore, the basic unit cannot be sandwiched between the two anti-SP-D monoclonal antibodies, and a complex cannot be formed. Therefore, the measurement method of this embodiment cannot detect trimer of SP-D.

[0027] On the other hand, in the case of SP-D containing two or more basic units, such as a 12-mer or a 36-mer, two different anti-SP-D monoclonal antibodies can bind to each of the two different basic units. Therefore, the measurement method of this embodiment can detect SP-D containing two or more basic units, such as a 12-mer or a 36-mer.

[0028] A solid-phase antibody can be produced by physically adsorbing or chemically binding (optionally via a suitable spacer) a monoclonal antibody to an insoluble carrier. As the insoluble carrier, a solid phase made of a polymer substrate such as 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 can be selected, such as a flat plate (e.g., microplate and membrane), beads or particles (e.g., latex particles and magnetic particles), or a cylindrical shape (e.g., test tube).

[0029] The amount of SP-D can also be measured by using a labeled antibody that can directly bind to the monoclonal antibody used in the method for measuring SP-D of the present invention. In this specification, an antibody that binds to the monoclonal antibody of the present invention and has a label bound thereto is referred to as a secondary antibody. The label may be indirectly bound to the monoclonal antibody of the present invention by using this secondary antibody.

[0030] The amount of SP-D in a sample can be measured by measuring the intensity of the signal emitted by the labeling substance or the turbidity caused by the aggregation of the labeling substance. Examples of labeling substances for preparing labeled antibodies include metal complexes, enzymes, insoluble particles, fluorescent substances, chemiluminescent substances, biotin, avidin, radioisotopes, gold colloid particles, and colored latex. Methods for binding the labeling substance to the monoclonal antibody include physical adsorption, glutaraldehyde method, maleimide method, pyridyl disulfide method, and periodic acid method available to those skilled in the art. When an enzyme such as horseradish peroxidase (HRP) or alkaline phosphatase (ALP) is used as the labeling substance, the enzyme activity can be measured using a specific substrate for the enzyme. 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 the labeling substance, a monoclonal antibody can be labeled with biotin and reacted with avidin or streptavidin labeled with an enzyme, dye, or fluorescent label (preferably HRP).

[0031] In this specification, the physical or chemical support of an antigen or antibody on a solid phase or the state of being supported thereon may be expressed as "immobilization" or "solid-phased". In addition, the terms "analysis", "detection", and "measurement" include quantification of SP-D.

[0032] The method for measuring SP-D in the present invention is a method for measuring the concentration of SP-D contained in a sample by utilizing the reaction between an antigen and an antibody. Examples of the method for measuring SP-D in the present invention include, but are not limited to, enzyme-linked immunosorbent assay (ELISA) and latex turbidimetric immunoassay (LTIA). The immunoassay method of the present invention is preferably LTIA.

[0033] The method for measuring SP-D of the present invention may be an in vivo or in vitro immunoassay method. A sensitizer may also be used to enhance sensitivity. In the method for measuring SP-D of the present invention, the order in which the anti-SP-D monoclonal antibody and the sample are added to the measurement system is not limited as long as the effects of the present invention can be obtained. That is, the anti-SP-D monoclonal antibody can be added to the measurement system before, simultaneously with, or after the addition of the sample.

[0034] The measurement procedures and principles are described below for each measurement method employed. The following is merely an example of the measurement procedures and principles in one embodiment of the present invention, and is not intended to limit the scope of the present invention in any way.

[0035] In each of the immunoassay methods described below, specific methods such as the method for immobilizing the monoclonal antibody on a solid phase, the method for binding the monoclonal antibody to a labeling substance, and the type of labeling substance can be any method known to those skilled in the art, including those described above, without any restrictions.

[0036] (Latex immunoturbidimetric assay) The latex turbidimetric immunoassay is an immunoassay that utilizes the agglutination of latex particles caused by binding of anti-SP-D monoclonal antibodies carried on the surface of latex particles with SP-D. There are no particular limitations on the latex particles, so long as they are latex particles that are generally used in in vitro diagnostics. The concentration of latex particles during agglutination reaction measurement, the average particle size of latex particles, and the like can be appropriately set depending on sensitivity or performance. The measurement procedure and principle when using the latex turbidimetric immunoassay as the method for measuring SP-D of the present invention are as follows. (1) A sample is brought into contact with latex particles carrying anti-SP-D monoclonal antibodies. (2) SP-D in the sample forms a complex with at least two anti-SP-D monoclonal antibodies, causing latex particles to agglutinate. (3) Irradiate the sample with near-infrared light (e.g., wavelength 600 nm) and measure the absorbance or scattered light. Based on the measured value, calculate the concentration of SP-D.

[0037] When the latex immunoturbidimetric method is used as the method for measuring SP-D of the present invention, the latex particles act as an insoluble carrier and as a labeling substance. The monoclonal antibody that binds to SP-D carried by the latex particles is only one type, that is, an anti-SP-D monoclonal antibody having the same amino acid sequence.

[0038] As a measuring device, a biochemical automatic analyzer (for example, Hitachi Automated Analyzer 3500 manufactured by Hitachi Ltd.) can be used.

[0039] (ELISA) In this specification, ELISA refers to a method in which SP-D, a substance to be detected contained in a sample, is captured using an anti-SP-D monoclonal antibody and then detected using an enzyme reaction. The solid phase is preferably a plate (also called an immunoplate). HRP or ALP can be used as a label. The measurement procedure and principle when sandwich ELISA is used as the SP-D measurement method of the present invention are as follows. (1) A sample is brought into contact with a solid phase on which an anti-SP-D monoclonal antibody has been immobilized, and SP-D in the sample binds to the anti-SP-D monoclonal antibody, i.e., the solid phase antibody. (2) When a labeled anti-SP-D monoclonal antibody, i.e., the labeled antibody, is added to the solid phase and allowed to react, the labeled antibody binds to SP-D to form a solid phase antibody-SP-D-labeled antibody complex, i.e., a sandwich. (3) After washing, the labeling substance is allowed to develop color and the absorbance is measured.

[0040] The amount of SP-D in the sample can be determined according to the amount of the labeled substance measured.

[0041] In the sandwich ELISA method, a secondary antibody can also be used. By using a secondary antibody, the reaction is amplified, and the detection sensitivity can be increased. In the following example, the secondary antibody is an antibody that specifically recognizes the anti-SP-D monoclonal antibody. When a secondary antibody is used, the following procedures (1) to (5) can be adopted. (1) A sample is added to the solid phase on which anti-SP-D monoclonal antibody is immobilized, followed by incubation, followed by removal of the sample and washing. (2) Add anti-SP-D monoclonal antibody, then incubate and wash. (3) An enzyme-labeled secondary antibody is then added and incubated. (4) Add substrate to develop color. (5) The amount of SP-D is measured by measuring the color development using a plate reader or the like.

[0042] When ELISA is used as the method for measuring SP-D of the present invention, the solid-phase antibody that binds to SP-D and the labeled antibody are of the same type, that is, anti-SP-D monoclonal antibodies having the same amino acid sequence.

[0043] According to the above embodiment, accurate quantification of SP-D is possible regardless of the number of monomers constituting the multimeric SP-D.

[0044] <Measurement kit> In one embodiment of the present invention, the SP-D measurement kit comprises a first reagent containing an insoluble carrier carrying an anti-pulmonary surfactant protein D monoclonal antibody, and a second reagent containing a labeled antibody comprising the anti-pulmonary surfactant protein D monoclonal antibody and a label.

[0045] The insoluble carrier and the label may be the same substance, or may be latex particles. The SP-D monoclonal antibody carried on the insoluble carrier and the SP-D monoclonal antibody of the label are of the same species, i.e., antibodies with the same amino acid sequence.

[0046] The SP-D monoclonal antibodies bind to trimeric pulmonary surfactant protein D, and only one of the SP-D monoclonal antibodies binds to one trimeric pulmonary surfactant protein D. At least two of the SP-D monoclonal antibodies bind to 12-mer or 36-mer SP-D.

[0047] The SP-D monoclonal antibody, the insoluble carrier, the label and the labeled antibody are as described above, and the contents thereof can be applied.

[0048] In one embodiment of the present invention, the SP-D measurement kit may include at least one of other test reagents and specimen dilution solutions, such as a standard antigen substance used in measuring SP-D and an antigen sample for quality control.

[0049] The present invention also covers the above-described method for preparing the monoclonal antibody of the present invention, the nucleic acid molecule encoding the monoclonal antibody of the present invention, a vector or plasmid containing this nucleic acid molecule, a cell containing this nucleic acid molecule, vector or plasmid, and a hybridoma producing the antibody of the present invention. EXAMPLES

[0050] The present invention will be described in more detail below with reference to examples, but the present invention is not limited to the examples described below.

[0051] <Preparation of monoclonal antibodies> Recombinant human SP-D (hereinafter, rSP-D) manufactured by Genscript was used as the immunogen. rSP-D was mixed 1:1 with Freund's Complete Adjuvant (Difco Laboratories) for the first immunization and with Freund's Incomplete Adjuvant (Difco Laboratories) for the second immunization and thereafter. Balb / c mice or F344 / Jcl rats were subcutaneously immunized every other week with 25 μg of the immunogen for the first immunization and 20 μg (diluted with PBS) for the second immunization and thereafter. After three immunizations, the blood antibody titer was evaluated by antigen-immobilized ELISA. For individuals in which a sufficient increase in titer was confirmed, the immunogen diluted with PBS was intraperitoneally immunized 1 to 3 days before dissection. Spleen cells, iliac lymph node cells, and inguinal lymph node cells were then collected and fused with myeloma cells SP2 / 0 by electric fusion. The fused cells (i.e., hybridomas) were cultured in a 96-well plate, and the culture supernatant was collected 7 or 8 days after the fusion. Screening was then performed by antigen-immobilized ELISA, as described below, to select strains that showed reactivity to rSP-D. The medium was replaced the day before screening.

[0052] <Screening for anti-SP-D antibodies> rSP-D (1 μg / mL in PBS) was dispensed at 50 μL / well into a 96-well ELISA plate (NUNC442404) and left to stand at room temperature for 2 hours. After washing three times with 400 μL / well of PBST, blocking solution (1% BSA-PBST) was dispensed at 100 μL / well and left to stand at room temperature for 1 hour or overnight at 4°C. After removing the blocking solution, cell culture supernatant and 1000- and 10000-fold diluted antisera were dispensed at 50 μL / well and left to stand at room temperature for 1 hour. After washing three times with PBST, Goat anti-Mouse IgG(H+L)PAb-HRP (SouthernBiotech, 9500-fold dilution) or Goat anti-Rat IgG(H+L)PAb-HRP (SouthernBiotech, 8500-fold dilution) was dispensed at 50μL / well and left to stand at room temperature for 1 hour. After washing three times with PBST, OPD coloring solution was dispensed at 50μL / well and left to stand at room temperature for 10 minutes. Stop solution was dispensed at 50μL / well to stop the reaction, and the plate was read (Abs.492nm). Antibodies showing reactivity to rSP-D were selected. The anti-SP-D monoclonal antibodies obtained are shown in Table 1.

[0053] [Table 1]

[0054] <Analysis Example 1> Confirmation of specificity of anti-SP-D monoclonal antibody A mixture of rSP-D, recombinant human surfactant A (SP-A), recombinant human Collectin Liver1 (CLL1) and recombinant human Mannan-Binding Lectin (MBL) antigens (1 μg / mL each in PBS) was dispensed into a 96-well plate for ELISA (NUNC442404) at 50 μL / well and left at room temperature for 2 hours. After washing three times with 50 μL / well of PBST, a blocking solution (1% BSA-PBST) was dispensed at 100 μL / well and left 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) shown in Table 2 was dispensed at 50 μL / well and left at room temperature for 1 hour. After washing three times with PBST, Goat anti-Mouse IgG(H+L)PAb-HRP (SouthernBiotech, 1031-05, 9500-fold dilution) or Goat anti-Rat IgG(H+L)PAb-HRP (SouthernBiotech, 3050-15, 8500-fold dilution) was dispensed at 50μL / well and left to stand at room temperature for 1 hour. After washing three times with PBST, OPD color development solution was dispensed at 50μL / well and left to stand at room temperature for 10 minutes. Stop solution was dispensed at 50μL / well to stop the reaction, and the plate was measured with a plate reader (Abs.492nm). The detection value was calculated by subtracting the measurement value of the well without dispensing anti-SP-D monoclonal antibody from the measurement value of each well, and the results are shown in Table 2. All of the anti-SP-D monoclonal antibodies reacted strongly only with rSP-D, and showed no or very low reactivity with antigens other than SP-D.

[0055] [Table 2]

[0056] <Analysis Example 2> Evaluation of reactivity between anti-SP-D monoclonal antibody and SP-D derived from biological samples Using Octet (Sartorius, Red384), the reactivity of anti-SP-D monoclonal antibodies with SP-D derived from living organisms contained in amniotic fluid was evaluated by intermolecular interaction measurement. Anti-SP-D monoclonal antibodies biotinylated according to the standard method using Biotin Labeling Kit-NH2 (Dojindo Laboratories) were bound to a streptavidin sensor and subjected to blocking treatment. The reactivity of anti-SP-D monoclonal antibodies with amniotic fluid diluted 5-fold with PBST (phosphate buffered saline containing Tween (registered trademark)) containing 1% BSA, or rSP-D adjusted to 1 μg / mL or 10 μg / mL, was evaluated. The results are shown in Figure 1. As shown in Figure 1, all of the anti-SP-D monoclonal antibodies obtained reacted with rSP-D and SP-D derived from living organisms in amniotic fluid.

[0057] <Analysis Example 3> Detection of SP-D derived from biological samples by sandwich ELISA We investigated whether SP-D in serum could be detected by combining the anti-SP-D monoclonal antibodies obtained. Anti-SP-D monoclonal antibodies as solid-phase antibodies, prepared with PBS at 5 μg / mL, were dispensed into each well of an ELISA plate at 50 μL / well and left to stand at room temperature for 2 hours. Each well was washed three times with TBST (washing solution) containing 5 mM calcium chloride at 400 μL / well, and 1% BSA / TBST (blocking solution) was dispensed into each well at 100 μL / well and left to stand at room temperature for 1 hour. The blocking solution was removed from each well, and a solution of serum diluted with 1% BSA / TBST containing 5 mM calcium chloride to an SP-D concentration of 100 ng / mL was dispensed into each well at 50 μL / well and left to stand at room temperature for 1 hour. Each well was washed three times with 400μL / well of washing solution, and a solution of 0.2μg / mL of biotinylated anti-SP-D monoclonal antibody prepared by Biotin Labeling Kit-NH2 (Dojindo Laboratories) in 1% BSA / TBST containing 5mM calcium chloride was dispensed at 50μL / well and left at room temperature for 1 hour. Each well was washed three times with 400μL / well of washing solution, and then 50μL / well of HRP-Streptavidin prepared at 0.2μg / mL in TBS containing 5mM calcium chloride was dispensed and left at room temperature for 30 minutes. After washing three times with washing solution, 50μL / well of OPD color development solution was dispensed and left at room temperature for 10 minutes. 50μL / well of stop solution was dispensed, and the absorbance at a wavelength of 492nm was measured using a plate reader.

[0058] The results are shown in Table 3. Antibody combinations with an absorbance of less than 0.1 were rated as -, monoclonal antibody combinations with an absorbance of 0.1 or more and less than 0.5 were rated as +, monoclonal antibody combinations with an absorbance of 0.5 or more and less than 1.0 were rated as ++, and monoclonal antibody combinations with an absorbance of 1.0 or more were rated as +++. SP-D in serum was detectable with multiple antibody combinations, including combinations of the same monoclonal antibodies. In particular, the combination of S21202, S21205, and S21208 antibodies enabled high-sensitivity detection of SP-D derived from biological samples.

[0059] [Table 3]

[0060] <Analysis Example 4> Analysis of rSP-D polymer distribution 1 A solution containing 55 μg of rSP-D (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. The eluate was dispensed in 400 μL portions into tubes that had previously been dispensed with 100 μL of TBS containing 5% BSA. The fractions were mixed well, dispensed, and stored at -30°C.

[0061] The chromatogram of rSP-D (Genscript) analyzed by gel filtration chromatography is shown in Figure 2. The 12-mer SP-D was mainly eluted in fraction 14, and the trimer SP-D was mainly eluted in fraction 23.

[0062] <Detection of rSP-D gel filtration fractions using anti-SP-D monoclonal antibody 1> Using the anti-SP-D monoclonal antibodies S21208, S21205 and S21202, rSP-D in each fraction obtained in Analytical Example 4 was measured using four ELISA systems: 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) according to the procedure described below.

[0063] The solid-phase antibody prepared at 5 μg / mL with PBS was dispensed at 50 μL / well on an ELISA plate and allowed to stand at room temperature for 2 hours. After washing three times with TBST containing 5 mM calcium chloride at 400 μL / well, 1% BSA / TBST (blocking solution) was dispensed at 100 μL / well and allowed to stand at room temperature for 1 hour. After removing the blocking solution, each fraction obtained in Analysis Example 4, diluted 1000-fold with 1% BSA / TBST containing 5 mM calcium chloride, was dispensed at 50 μL / well and allowed to stand at room temperature for 1 hour. After washing three times with 400μL / well of washing solution, a solution of biotinylated antibody biotinylated according to the standard method with Biotin Labeling Kit-NH2 (Dojindo Laboratories) was prepared at 0.2μg / mL with 1% BSA / TBST containing 5mM calcium chloride, dispensed at 50μL / well, and left at room temperature for 1 hour. After washing three times with 400μL / well of washing solution, HRP-Streptavidin prepared at 0.2μg / mL with TBS containing 5mM calcium chloride was dispensed at 50μL / well, and left at room temperature for 30 minutes. After washing three times with 400μL / well of washing solution, OPD color development solution was dispensed at 50μL / well, and left at room temperature for 10 minutes. Stop solution was dispensed at 50μL / well, and the absorbance at a wavelength of 492nm was measured with a plate reader.

[0064] These results are shown in Table 4. In Examples 1 and 2, in which SP-D was subjected to sandwich detection with the same anti-SP-D monoclonal antibody, the 12-mer SP-D contained in fraction 14 was detected, but the trimer SP-D contained in fraction 23 was not detected. In Comparative Examples 1 and 2, in which SP-D was subjected to sandwich detection with two different anti-SP-D monoclonal antibodies, both the 12-mer and the trimer SP-D were detectable. From the above results, it was found that only SP-D larger than a trimer can be detected by sandwich detection of SP-D with the same anti-SP-D monoclonal antibody.

[0065] [Table 4]

[0066] <Analysis Example 5> Analysis of the multimer distribution of rSP-D 2 Gel filtration chromatography of rSP-D manufactured by Company R&D or Genscript was performed in the same manner as in Analysis Example 4. 217 μg of rSP-D was subjected to gel filtration chromatography.

[0067] Figure 3 shows the chromatograms when rSP-D manufactured by Company R&D and rSP-D manufactured by Genscript were analyzed by gel filtration chromatography. Mainly, SP-D of 36-mers or more eluted in fraction 8, 12-mer SP-D eluted in fraction 12, and 3-mer SP-D eluted in fraction 20. For rSP-D manufactured by Company R&D, SP-D of 36-mers or more was the main component, and for rSP-D manufactured by Genscript, 12-mer SP-D was the main component. Hereinafter, the fraction eluted nth when analyzing SP-D manufactured by Company R&D is defined as fraction Rn, and the fraction eluted nth when analyzing SP-D manufactured by Genscript is defined as fraction Gn. The UV absorbance of fraction R8 containing a large amount of SP-D of 36-mers or more was 24.68 mAU, and the UV absorbance of fraction G12 containing a large amount of 12-mer SP-D was 11.53 mAU. Since the protein concentration of rSP-D is proportional to the UV absorbance, the protein mass ratio of SP-D of 36-mers or more in fraction R8 to 12-mer SP-D in fraction G12 is 2.14:1.

[0068] <Detection of rSP-D gel filtration fractions 2> Using anti-SP-D monoclonal antibodies S21208, S21205, and S21202, 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) were used, and rSP-D in each fraction obtained in Analysis Example 5 was measured by the same procedure as in <Detection of rSP-D gel filtration fractions by anti-SP-D monoclonal antibody 1>.

[0069] These results are shown in Table 5. In Example 1 where SP-D was detected by sandwich using the same anti-SP-D monoclonal antibody, the ratio of the absorbance when measuring fraction R8 containing SP-D of 36-mers or more to the absorbance when measuring fraction G12 containing 12-mer SP-D was 2.76:1, which was equivalent to the protein mass ratio of SP-D of 36-mers or more to 12-mer SP-D in gel filtration chromatography of 2.14:1. Also, in Example 1, trimeric SP-D contained in fraction G20 was not detected.

[0070] In Comparative Examples 1 and 2 where SP-D was detected by sandwich using two different anti-SP-D monoclonal antibodies, trimeric SP-D was detectable. 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 12-mer SP-D were 1:0.87 and 1:0.44, respectively, and did not reflect the protein mass ratios of the respective SP-D multimers in gel filtration chromatography. More specifically, the absorbance when measuring the fraction containing 12-mer SP-D, which should have less protein than SP-D of 36-mers or more based on the results of gel filtration chromatography, was higher than the absorbance when measuring the fraction containing SP-D of 36-mers or more.

[0071] From the above results, it was considered that when sandwich detecting SP-D of 12-mers or more using the same anti-SP-D monoclonal antibody, measurement reflecting the actual protein amount was possible regardless of the number of basic units in the SP-D multimer.

[0072]

Table 5

[0073] <Preparation of LTIA reagent for SP-D measurement> 1) First reagent A solution with the following composition was prepared as the first reagent. 100 mM MES-NaOH (pH 6.0) 500 mM NaCl 0.5% BSA

[0074] 2) Anti-human SP-D monoclonal antibody-sensitized latex particle solution To a 1% polystyrene latex solution (manufactured by Sekisui Medical Co., Ltd.) (10 mM MOPS buffer) with an average particle diameter of 317 nm and a critical aggregation concentration of 280 mM, an anti-SP-D antibody solution diluted to 0.35 mg / mL with an equal volume of 10 mM MOPS buffer was added, and the mixture was stirred at 4°C for 2 hours. Then, an equal volume of 10 mM MOPS buffer containing 0.5% BSA was added, and the mixture was stirred at 4°C for 1 hour to prepare an anti-human SP-D monoclonal antibody-sensitized latex solution.

[0075] 3) Second reagent 3-1) Example 2 The S21208 antibody-sensitized latex particle solution prepared by the procedure described in 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 obtain the second reagent.

[0076] 3-2) Comparative Example 3 5 mM MOPS-NaOH (pH 7.0) buffer and the antibody-sensitized latex particle solution were mixed so that the absorbance at a wavelength of 600 nm of the S21205 antibody-sensitized latex particle solution prepared by the procedure described in 2) was 3.0 Abs. and the absorbance at a wavelength of 600 nm of the S21208 antibody-sensitized latex particle solution was 6.0 Abs. to obtain the second reagent.

[0077] <Quantification of rSP-D Gel Filtration Fraction by SP-D Measurement Reagent> 1) Measurement method using LTIA reagent The first and second reagents were combined, and the SP-D concentration in each fraction obtained in Analysis Example 5 was measured and calculated using a biochemical automatic analyzer (Hitachi, Hitachi Automatic Analyzer 3500) according to the following procedure (Example 2 and Comparative Example 3). 120 μL of the first reagent was added to 5 μL of each fraction, and heated at 37° C. for 5 minutes. 40 μL of the second reagent was added and stirred. The change in absorbance over 5 minutes was measured at a main wavelength of 570 nm and a sub-wavelength of 800 nm. The SP-D concentration in each fraction was calculated using a calibration curve created by plotting the amount of absorbance change per unit time when measuring recombinant SP-D (R&D) of known concentration on the horizontal axis and the recombinant SP-D concentration on the vertical axis.

[0078] 2) Measurement method using CL SP-D "Yamasa" NX (Yamasa Shoyu Co., Ltd., certified in vitro diagnostic reagent (hereinafter referred to as certified reagent)) Each fraction obtained in Analysis Example 5 was measured according to the package insert of the certified reagent (Comparative Example 4). According to the package insert of the certified reagent, the reagent measures SP-D in serum based on a chemiluminescent enzyme immunoassay method using two different types of anti-human SP-D mouse monoclonal antibodies.

[0079] Table 6 shows the SP-D concentration measurement values ​​when fractions R8, R12, and R20 obtained in Analysis 5 were used. FIG. 4 is a graph showing the SP-D concentration measurement values ​​of each fraction obtained in Analysis 5 in Example 2. FIG. 5 is a graph showing the SP-D concentration measurement values ​​of each fraction obtained in Analysis 5 in Comparative Example 3. FIG. 6 is a graph showing the SP-D concentration measurement values ​​of each fraction obtained in Analysis 5 in Comparative Example 4.

[0080] In Example 2, SP-D was detected at an amount equivalent to the protein amount ratio (2.14:1) of SP-D of 36 or more mers to SP-D of 12 mers in gel filtration chromatography, as in the ELISA of Example 1. Furthermore, in Example 2, the measured value of fraction G20, which contains trimer SP-D, was equivalent to the measured value of fraction G18, which was measured to have a lower protein content than fraction G20 in gel filtration chromatography, and it was considered that Example 2 did not substantially detect trimer SP-D.

[0081] On the other hand, in Comparative Example 3 and Comparative Example 4, which used two different antibodies, the measured value of fraction G12 containing 12-mer SP-D was calculated to be lower than the measured value of fraction R8 containing 36-mer or more SP-D, which was different from the result of gel filtration chromatography. In addition, in both Comparative Example 3 and Comparative Example 4, the measured value of fraction G20 containing trimer SP-D was higher than that of fraction G18, and trimer SP-D was detected (Figures 5 and 6). From the above, when SP-D was sandwich-detected using the same antibody, the LTIA reagent made it possible to measure the actual amount of protein regardless of the number of basic units in the SP-D multimer.

[0082] [Table 6] [Industrial Applicability]

[0083] According to the above-mentioned aspects, it is possible to provide a measurement method and measurement kit for pulmonary surfactant protein D that enable accurate quantification regardless of the number of monomers that constitute the polymeric pulmonary surfactant protein D, as well as a monoclonal antibody used therein and a cell that produces this antibody.

[0084] [Accession number] NPMD NITE BP-03825 NPMD NITE BP-03826 NPMD is an acronym for the National Institute of Technology and Evaluation's Patent Microorganism Deposit Center (Room 122, 2-5-8 Kazusa Kamatari, Kisarazu City, Chiba Prefecture, Japan).

Claims

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

2. The method according to claim 1 , wherein the insoluble carrier is a latex particle.

3. The insoluble carrier is flat, The measurement method according to claim 1, wherein the complex comprises the anti-pulmonary surfactant protein D antibody supported on the insoluble carrier, and a labeled antibody comprising the anti-pulmonary surfactant protein D monoclonal antibody and a label.

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

5. The pulmonary surfactant protein D comprises at least one basic unit which is a trimer of pulmonary surfactant protein D; The method according to any one of claims 1 to 4, wherein only one anti-pulmonary surfactant protein D antibody binds to one basic unit.

6. The method according to claim 5 , wherein trimer of pulmonary surfactant protein D is not detected in the detection step.

7. The method according to claim 6, wherein in the detection step, pulmonary surfactant protein D associated with two or more of the basic units is detected.

8. A kit for measuring pulmonary surfactant protein D, comprising: a first reagent containing an insoluble carrier on which an anti-pulmonary surfactant protein D monoclonal antibody is supported; and a second reagent containing a labeled antibody comprising the anti-pulmonary surfactant protein D monoclonal antibody and a label.

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

10. A monoclonal antibody that binds to trimeric pulmonary surfactant protein D, A monoclonal antibody that binds only to one of the trimer pulmonary surfactant protein D.

11. The monoclonal antibody according to claim 10, which binds to at least two pulmonary surfactant protein D molecules having a 12-mer or greater structure.

12. A hybridoma producing the monoclonal antibody according to claim 10 or 11.

13. An antibody produced from a hybridoma deposited at the Patent Microorganisms Depositary Center of the National Institute of Technology and Evaluation under accession number NITE BP-03825 or accession number NITE BP-03826.

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