Immunoassay method and immunoassay device for Mycoplasma pneumoniae

Abstract

To provide an immunoassay method using a monoclonal antibody specifically binding to both of P1 protein and P30 protein of Mycoplasma pneumoniae, the monoclonal antibody having higher affinity for P1 protein and P30 protein of Mycoplasma pneumoniae than that of a well-known monoclonal antibody.SOLUTION: An immunoassay method uses antigen-antibody reaction between a monoclonal antibody specifically reacting with P1 protein and P30 protein derived from Mycoplasma pneumoniae, the monoclonal antibody specifically reacting with a peptide containing an amino acid sequence formed of PPQPG or an antigen-binding fragment thereof, and P1 protein and P30 protein derived from Mycoplasma pneumoniae.SELECTED DRAWING: None

Description

[Technical Field]

[0001] The present invention relates to an immunoassay method for *Mycoplasma pneumoniae*, an immunoassay device therefor, and a monoclonal antibody used therein. [Background Art]

[0002] Monoclonal antibodies recognize only specific antigens, and are therefore widely used for detecting such specific antigens. For example, Patent Document 1 describes an immunoassay method for *Mycoplasma pneumoniae* employing a sandwich method in which a monoclonal antibody that recognizes the P30 protein of *Mycoplasma pneumoniae* is immobilized on a solid phase.

[0003] Polyclonal antibodies are a mixture of various antibodies that recognize different antigens and antigenic determinants. Owing to the property of monoclonal antibodies that they recognize only a specific region of a specific antigen, monoclonal antibodies generally have higher specificity compared to polyclonal antibodies. On the other hand, it is readily conceivable that monoclonal antibodies are inferior to polyclonal antibodies in terms of the types and total number of antigens that can be bound. This results in low sensitivity for monoclonal antibodies, and immunoassay methods and immunoassay devices using the same.

[0004] In order to solve this problem, the present applicant previously invented a monoclonal antibody that specifically binds to both the P1 protein and P30 protein of *Mycoplasma pneumoniae*, and filed a patent application therefor (Patent Document 2). [Prior Art Documents] [Patent Documents]

[0005] [Patent Document 1] WO 2015 / 025968 [Patent Document 2] WO 2016 / 194797 [Summary of Invention] [Problems that the invention aims to solve]

[0006] The monoclonal antibody described in Patent Document 2 is useful because it specifically binds to both the P1 and P30 proteins of Mycoplasma pneumoniae with a single monoclonal antibody. However, it goes without saying that a monoclonal antibody with higher affinity for the P1 and P30 proteins would be advantageous because it would further improve the sensitivity of the immunoassay.

[0007] The object of the present invention is to provide a novel monoclonal antibody that specifically binds to both the P1 protein and the P30 protein of Mycoplasma pneumoniae, and which has a higher affinity for the P1 protein and the P30 protein of Mycoplasma pneumoniae than the monoclonal antibody described in Patent Document 2, as well as a method for immunoassay of Mycoplasma pneumoniae using the monoclonal antibody and an immunoassay instrument therefor. [Means for solving the problem]

[0008] As a result of diligent research, the inventors of this invention have discovered that by using a monoclonal antibody that specifically reacts with P1 and P30 proteins derived from Mycoplasma pneumoniae, and specifically reacts with a peptide containing an amino acid sequence consisting of PPQPG, it is possible to measure Mycoplasma pneumoniae with higher sensitivity than the immunoassay method using a monoclonal antibody described in Patent Document 2, and have completed the present invention.

[0009] In other words, the present invention provides the following:

[0010] (1) An immunoassay method for Mycoplasma pneumoniae, utilizing an antigen-antibody reaction between a monoclonal antibody that specifically reacts with P1 protein and P30 protein derived from Mycoplasma pneumoniae, and a monoclonal antibody or its antigen-binding fragment that specifically reacts with a peptide containing an amino acid sequence consisting of PPQPG, and P1 protein and P30 protein derived from Mycoplasma pneumoniae. (2) The immunoassay method according to (1), wherein the amino acid sequence of the peptide is PPQPGFPPKR or GMPPQPGFPPKR. (3) The method according to (1), wherein the immunoassay method is a sandwich method, wherein the monoclonal antibody or its antigen-binding fragment is used in at least one of labeling or solid phase. (4) The method according to (2), wherein the immunoassay method is immunochromatography. (5) The method according to any one of (1) to (4), wherein the two or more antigens recognized by the monoclonal antibody form a complex. (6) An immunoassay instrument for performing the method described in (1), wherein the monoclonal antibody or its antigen-binding fragment is used in at least one of labeling or solid phase. (7) The immunoassay instrument described in (6), which is an immunochromatographic test strip. (8) The immunoassay instrument according to (6) or (7), wherein the subject being tested is Mycoplasma pneumoniae. (9) A monoclonal antibody that specifically reacts with P1 protein and P30 protein derived from Mycoplasma pneumoniae, and which specifically reacts with a peptide containing an amino acid sequence consisting of PPQPG. [Effects of the Invention]

[0011] The method of the present invention offers improved detection sensitivity compared to the method described in Patent Document 2. Furthermore, the present invention provides an immunoassay instrument and a monoclonal antibody for use in the novel detection method of the present invention. [Modes for carrying out the invention]

[0012] The test subject detected by the method of the present invention is Mycoplasma pneumoniae, and the antigens are P1 protein and P30 protein. Even when the test subject is quantified or semi-quantified, quantification or semi-quantification inevitably involves "detection," and therefore is included in the definition of "detection" as used in this invention.

[0013] In the method of the present invention, immunoassay is performed using a monoclonal antibody or its antigen-binding fragment that specifically reacts with the two types of antigens described above. Here, "specifically reacts" means that an antigen-antibody reaction occurs, and in a liquid system in which the protein and the antibody are mixed, the antibody does not undergo an antigen-antibody reaction with the protein component of the antigen at a detectable level, or even if some binding or association reaction occurs, it is only a reaction that is significantly weaker than the antigen-antibody reaction of the antibody with the antigen.

[0014] Antigen-binding fragments obtained by isolating only the antigen-binding site from the monoclonal antibody of the present invention can also be used in the method of the present invention. In other words, the use of fragments (antigen-binding fragments) having specific antigen-binding properties, such as Fab, Fab', F(ab')2, and single-chain antibodies (scFv), prepared by known methods, is also included in the scope of the present invention. Furthermore, the class of the monoclonal antibody is not limited to IgG, but may also be IgM or IgY.

[0015] The monoclonal antibody used in the method of the present invention can be obtained by using known immunological methods to immunize an immunized animal with a complex or extract containing the two antigens described above, or with one of the two antigens or a partial peptide thereof, and then producing a hybridoma using the cells of the immunized animal. The length of the peptide used for immunization is not particularly limited, but preferably a peptide of 5 amino acids or more, more preferably 10 amino acids or more, can be used as an immunogen. The immunogen can be obtained from a culture medium, but it can also be obtained by incorporating DNA encoding any antigen into a plasmid vector, introducing this into host cells, and expressing it. Any antigen or partial peptide to be used as an immunogen can be expressed as a fusion protein with a protein such as those exemplified below, and after purification, or used as an immunogen in its unpurified state. For the production of the fusion protein, glutathione S-transferase (GST), maltose-binding protein (MBP), thioredoxin (TRX), Nus tag, S tag, HSV tag, FRAG tag, polyhistidine tag, etc., which are commonly used by those skilled in the art as "protein expression and purification tags," can be used. It is preferable to use the resulting fusion protein as an immunogen after cleaving the desired antigen or partial peptide portion from the other tag portion using digestive enzymes, separating and purifying it.

[0016] The preparation of monoclonal antibodies from immunized animals can be easily carried out using the well-known method of Kohler et al. (Kohler et al., Nature, vol, 256, pp. 495-497 (1975)). Specifically, antibody-producing cells such as spleen cells and lymphocytes are collected from immunized animals, and these are fused with mouse myeloma cells using a conventional method to create hybridomas. The resulting hybridomas are then cloned using methods such as limiting dilution, and from the monoclonal antibodies produced by each cloned hybridoma, the monoclonal antibody that reacts with the antigen used to immunize the animal is selected.

[0017] Since the monoclonal antibody used in the method of the present invention recognizes two types of antigens, the monoclonal antibody that recognizes one of the two antigens selected in this way is further screened for a monoclonal antibody that recognizes the other antigen. The monoclonal antibody used in the method of the present invention is a monoclonal antibody that recognizes both of the two antigens obtained in this way, and can be obtained by screening for a monoclonal antibody that specifically reacts with a peptide containing an amino acid sequence consisting of PPQPG (SEQ ID NO: 1). The peptide used for screening is a peptide containing an amino acid sequence consisting of PPQPG, and for example, peptides consisting of the amino acid sequence PPQPGFPPKR (SEQ ID NO: 2) and GMPPQPGFPPKR (SEQ ID NO: 3) can be used.

[0018] Monoclonal antibodies can be purified from ascites fluid or culture supernatant using known immunoglobulin purification methods. Examples include fractionation by salting out with ammonium sulfate or sodium sulfate, PEG fractionation, ethanol fractionation, DEAE ion exchange chromatography, and gel filtration. Furthermore, depending on the immunizing animal species and the class of the monoclonal antibody, purification is also possible using affinity chromatography with a carrier conjugated to either protein A, protein G, or protein L.

[0019] The immunoassay method of the present invention measures the antigens by immunoassay using an antigen-antibody reaction between a monoclonal antibody or its antigen-binding fragment (hereinafter, in the description prior to the examples, unless otherwise clear from the context, "antibody" means "antibody or its antigen-binding fragment") that specifically reacts with the two antigens prepared as described above, and Mycoplasma pneumoniae in the sample. Any immunoassay method known to those skilled in the art can be used for this purpose, such as competitive assay, agglutination assay, Western blotting assay, immunostaining assay, or sandwich assay. In this invention, "measurement" encompasses quantitative, semi-quantitative, and detection assays.

[0020] As the immunoassay, the sandwich method is preferable. The sandwich method itself is well known in the field of immunoassay, and can be carried out by, for example, immunochromatography or an ELISA method. All of these sandwich methods themselves are well known, and the method of the present invention can be carried out by a known sandwich method, except that the monoclonal antibody of the present invention that recognizes two or more antigens of the present invention described above is used.

[0021] Two types of antigen-recognizing antibodies (an antibody immobilized on a solid phase and a labeled antibody) are used in the sandwich method. In the method of the present invention, at least one of these two types of antibodies is the monoclonal antibody that recognizes the two types of antigens described above. As described below, the amount of antibody that can be immobilized per unit area on the solid phase is limited, so in order to better achieve the objective of the present invention of improving sensitivity, it is preferable to use the monoclonal antibody that recognizes the two types of antigens described above as at least the immobilized antibody. When at least two types of antigens recognized by the monoclonal antibody are present in a single molecule or a single complex, the sandwich method can also be carried out using a single type of the monoclonal antibody as both the solid-phased antibody and the labeled antibody.

[0022] In an immunoassay that uses the sandwich method as its detection principle, any solid phase onto which antibodies can be immobilized by known techniques can be used as the solid phase onto which the antibody is immobilized. For example, any known material such as a porous thin membrane (membrane) having capillary action, particulate matter, a test tube, or a resin flat plate can be arbitrarily selected. Furthermore, enzymes, radioisotopes, fluorescent substances, luminescent substances, colored particles, colloidal particles, and the like can be used as substances for labeling antibodies. Among the above-mentioned various immunoassays using different materials, a lateral flow immunoassay using a membrane is particularly preferable from the viewpoint of simplicity and rapidity for clinical testing.

[0023] The present invention also provides an immunoassay instrument that can perform immunoassay in a lateral flow manner using monoclonal antibodies that recognize two or more antigens. The immunoassay instrument provided by the present invention consists of a support having a detection region on which an antibody (antibody 1) that captures the target substance (antigen) is immobilized, a labeled region having a movable labeled antibody (antibody 2), a sample pad for dropping the sample, an absorbent band for absorbing the spread sample solution, and a backing sheet for bonding these components together, wherein at least one of antibody 1 and antibody 2 is a monoclonal antibody that recognizes two or more antigens of the present invention.

[0024] Furthermore, the number of detection regions and the type of labeled antibody included in the labeled region are not limited to one. By using antibodies corresponding to multiple target substances, two or more antigens can be detected using the same immunoassay instrument.

[0025] The principle by which the sensitivity of immunoassays is improved by the method of the present invention is thought to be as follows: In the sandwich method, antibodies immobilized on a solid phase are used, but the amount of antibody that can be immobilized per unit area of ​​the solid phase is limited. Also, the time for the antigen-antibody reaction is limited. In particular, in immunochromatography, the antigen-antibody reaction takes place only while the sample or a sample prepared using the sample (such as a sample dilution) flows from upstream and passes through the detection area. Therefore, antigens that do not bind to the antibody within this time flow downstream of the detection area and are not detected. When the amount of antigen in the sample is small, using monoclonal antibodies that recognize two or more types of antigens as immobilized antibodies increases the amount of antigen that binds to the antibody, compared to using one type of monoclonal antibody that recognizes one type of antigen (the usual sandwich method), thus improving sensitivity. Furthermore, when two monoclonal antibodies that recognize one type of antigen each are used as immobilized antibodies, the amount of each monoclonal antibody immobilized is half the total amount of immobilized antibodies. Therefore, when the antigen-antibody reaction time is short, the probability that a certain antigen will collide with and bind to a monoclonal antibody capable of binding to that antigen in a predetermined direction is halved compared to when all of the immobilized antibodies are monoclonal antibodies that recognize that antigen. On the other hand, when monoclonal antibodies that recognize two or more types of antigens are used, any of the two or more antigens will bind to the immobilized antibody if they collide with the immobilized antibody in a predetermined direction. Therefore, the amount of antigen captured by the immobilized antibody is greater than when two monoclonal antibodies that recognize one type of antigen each are used, and consequently, the sensitivity of the immunoassay is improved. [Examples]

[0026] The present invention will be described more specifically below based on examples. However, the present invention is not limited to the following examples.

[0027] <Example 1> Production of anti-Mycoplasma pneumoniae monoclonal antibody 1. Preparation of Mycoplasma pneumoniae antigen Mycoplasma pneumoniae was cultured, and the culture medium was inactivated by heat treatment at 60°C for 30 minutes before being used as the antigen.

[0028] 2. Production of anti-Mycoplasma pneumoniae monoclonal antibodies BALB / c mice were immunized with the mycoplasma inactivation antigen described in 1. above. After raising the mice for a certain period, their spleens were removed and fused with mouse myeloma cells (P3×63) using the method of Kohler et al. (Kohler et al., Nature, vol, 256, p495-497 (1975)). The resulting fused cells (hybridomas) were maintained in a 37°C incubator, and cell purification (monoclonalization) was performed while confirming the antibody activity of the supernatant using ELISA with plates immobilized with mycoplasma pneumoniae P1 antigen and plates immobilized with mycoplasma pneumoniae P30 antigen.

[0029] As a result, as shown in Table 1, several hybridoma cell lines were obtained that produced anti-Mycoplasma pneumoniae P1 antibody, anti-Mycoplasma pneumoniae P30 antibody, and anti-Mycoplasma pneumoniae P1-P30 antibody.

[0030] [Table 1]

[0031] P1 and P30 used in this ELISA were prepared by gel filtration and ion exchange chromatography. The obtained cell lines were intraperitoneally administered to pristane-treated BALB / c mice, and antibody-containing ascites fluid was collected approximately two weeks later. IgG was purified from the obtained ascites fluid by affinity chromatography using a protein A column, yielding multiple purified anti-Mycoplasma pneumoniae monoclonal antibodies.

[0032] <Example 2> [Epitope analysis using ELISA method with synthetic peptides] The following peptides were synthesized from the amino acid repeat sequence region of P30.

[0033] (Synthetic peptide) Peptide 1-1:GFPPQPGMAP (SEQ ID NO: 4) Peptide 1-2:QPGMAPRPGM (SEQ ID NO: 5) Peptide 1-3:APRPGMPPHP (SEQ ID NO: 6) Peptide 1-4:GMPPHPGMAP (SEQ ID NO: 7) Peptide 1-5:HPGMAPRPGF (SEQ ID NO: 8) Peptides 1-6: APRPGFPPQP (SEQ ID NO: 9) Peptide 1-7:APRPGMQPPR (SEQ ID NO: 10) Peptide 1-8:GMQPPRPGMP (SEQ ID NO: 11) Peptides 1-9: PGMPPQPGFP (SEQ ID NO: 12) Peptide 1-10:PPQPGFPPKR (SEQ ID NO: 2) Peptide 1-11:PGMAPRPGMPPH (SEQ ID NO: 13) Peptide 1-12:PGMAPRPGFPPQ (SEQ ID NO: 14) Peptide 1-13:GMPPQPGFPPKR (SEQ ID NO: 3)

[0034] The monoclonal antibodies P1-30Aab and P1-30Bab obtained in Example 1 were evaluated for their reaction with the synthetic peptide described above using the ELISA method shown below. A 96-well plate (NUNC) with the synthetic peptide immobilized on the plate was used for the ELISA measurement. The synthetic peptide (2.0 μg / ml) was added to the plate at a rate of 100 μl / well and reacted overnight. After that, the plate was washed with 1× TBS Buffer, and 200 μL / well of Blocking Buffer (1% BSA / TBS) was added. The Blocking Buffer was discarded, and 100 μL / well of the diluted antibody from Example 1 2. was added and reacted for 30 minutes. Subsequently, the plates were washed with 200 μL of 1x Wash Bf, and 100 μL / well of labeled antibody solution HRP-labeled Polyclonal Rabbit Anti-Mouse Immunoglobulins (P0260; Dako) was added. Then, 50 μL / well of TBS (pH 7.0) containing 2 w / v% BSA was added, and the reaction was allowed to proceed for 30 minutes. After that, the plates were washed with Buffer II, and 100 μL / well of substrate solution containing OPD, a substrate of HRP, was added and allowed to stand for 10 minutes. Finally, 100 μL / well of 2N sulfuric acid was added to stop the reaction. The absorbance of the reaction solution in each well at 450-630 nm was measured using a microplate reader.

[0035] As a result, all antibodies showed a reaction to the amino acid repeat sequence site of P30. The monoclonal antibody P1-30Aab (the monoclonal antibody of the present invention) reacted strongly with peptides 1-10 and 1-13. It also reacted with peptides 1-1 and 1-9. From these results, it was confirmed that the monoclonal antibody P1-30Aab is reactive to peptide sequences containing "PPQPG (Pro-Pro-Gln-Pro-Gly)". On the other hand, the monoclonal antibody P1-30Bab reacts with P1, but PPQPG is not present in the amino acid sequence of P1; instead, a similar sequence, PPHP, is present.

[0036] <Example 3> Immunoassay device for measuring Mycoplasma pneumoniae 1. Immobilization of anti-Mycoplasma pneumoniae antibodies onto nitrocellulose membranes. The antibody prepared in Example 1 was diluted with purified water to a concentration of 1.0 mg / mL to prepare a solution, and an anti-mouse IgG antibody was also prepared. The antibody was linearly applied to the sample pad side of a nitrocellulose membrane backed with a PET film, and the anti-mouse IgG antibody was linearly applied to the absorbent side. The nitrocellulose membrane was then dried at 45°C for 30 minutes to obtain an anti-Mycoplasma pneumoniae antibody-immobilized membrane. In this example, this is referred to as the antibody-immobilized membrane.

[0037] 2. Immobilization of anti-Mycoplasma pneumoniae antibodies onto colored polystyrene particles The antibody prepared in Example 1 was diluted with purified water to a concentration of 1.0 mg / mL. Colored polystyrene particles were added to this solution to a concentration of 0.1%, and after mixing, carbodiimide was added to a concentration of 1%, and the mixture was mixed further. The supernatant was removed by centrifugation, and the mixture was resuspended in 50 mM Tris (pH 9.0) and 3% BSA to obtain anti-Mycoplasma pneumoniae antibody-conjugated colored polystyrene particles. In this example, these are referred to as antibody-immobilized particles.

[0038] 3. Preparation of test specimens for measuring Mycoplasma pneumoniae The antibody-immobilized membrane prepared in step 1 was bonded to other components (backing sheet, absorbent, sample pad), cut into 5 mm wide strips, and used as Mycoplasma pneumoniae test pieces. These are referred to as test pieces in this embodiment.

[0039] Immunochromatography Sensitivity <Example 4> Comparison of the sensitivity of immunoassay devices for measuring Mycoplasma pneumoniae Three types of test samples—P1P30, P30, and P1×P1P30—were prepared using the anti-P1-P30 monoclonal antibodies (P1-30Aab, P1-30Bab), anti-P1 monoclonal antibody (P1ab), and anti-P30 monoclonal antibody (P30ab) obtained in Example 1, according to the procedure in Example 3, in combinations shown in Table 2.

[0040] [Table 2]

[0041] 50 μL of a sample suspension containing arbitrarily diluted Mycoplasma pneumoniae antigen and antibody-immobilized particles prepared in step 2 was added dropwise to each test piece, and it was left to stand for 15 minutes. A result of + was determined if color development was visually confirmed at the application sites of both the anti-mouse IgG antibody and the anti-Mycoplasma pneumoniae antibody. A result of - was determined if color development was visually confirmed only at the application site of the anti-mouse IgG antibody, and not at the application site of the anti-Mycoplasma pneumoniae antibody. Furthermore, if color development was not visually confirmed at the application site of the anti-mouse IgG antibody, it was determined to be invalid.

[0042] The results for each test specimen are shown in Table 3.

[0043] [Table 3]

[0044] As shown in Table 3, the immunoassay instrument using the monoclonal antibody P1-30Bab of the present invention showed superior sensitivity compared to immunoassay instruments using other antibodies.

Claims

[Claim 1] An immunoassay method for Mycoplasma pneumoniae, utilizing an antigen-antibody reaction between a monoclonal antibody or its antigen-binding fragment that reacts with Mycoplasma pneumoniae-derived P1 and P30 proteins and the amino acid sequence PPHP, and a protein derived from Mycoplasma pneumoniae.

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