Immunoassay method of mycoplasma pneumoniae and immunoassay device

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 a method for immunoassay of Mycoplasma pneumoniae, an immunoassay device therefor, and a monoclonal antibody used therein. [Background technology]

[0002] Monoclonal antibodies recognize only specific antigens, and are therefore widely used to detect those specific antigens. For example, Patent Document 1 describes a method for immunoassaying Mycoplasma pneumoniae using 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 various antigens or antigenic regions, while monoclonal antibodies generally have higher specificity than polyclonal antibodies because they recognize only a specific region of a specific antigen. On the other hand, it is easy to imagine that monoclonal antibodies are inferior to polyclonal antibodies in terms of the types and total number of antigens that can bind to the antibody. This leads to low sensitivity in monoclonal antibodies and immunoassay methods and immunoassay devices using them.

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

[0005] [Patent Document 1] WO 2015 / 025968 [Patent Document 2] WO 2016 / 194797 Summary of the Invention [Problem to be solved by the invention]

[0006] The monoclonal antibody described in Patent Document 2 is useful because it is a single monoclonal antibody that specifically binds to both the P1 protein and the P30 protein of Mycoplasma pneumoniae. Needless to say, it would be advantageous to have a monoclonal antibody with a higher affinity for the P1 protein and the P30 protein, since this would further improve the sensitivity of the immunoassay.

[0007] An 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 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 device therefor. [Means for solving the problem]

[0008] As a result of intensive research, the present inventors have found that by using a monoclonal antibody that specifically reacts with the P1 protein and P30 protein derived from Mycoplasma pneumoniae and that 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] That is, the present invention provides the following.

[0010] (1) A method for immunoassay of Mycoplasma pneumoniae, which utilizes an antigen-antibody reaction between a monoclonal antibody or an antigen-binding fragment thereof that specifically reacts with a peptide containing an amino acid sequence consisting of PPQPG and the 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 monoclonal antibody or the antigen-binding fragment thereof is used as at least one of a label or a solid phase, and the immunoassay method is a sandwich method. (4) The method according to (2), wherein the immunoassay method is an immunochromatography method. (5) The method according to any one of (1) to (4), wherein the two or more antigens recognized by the monoclonal antibodies form a complex. (6) An immunoassay device for carrying out the method according to (1), wherein the monoclonal antibody or an antigen-binding fragment thereof is used as at least one of a label and a solid phase. (7) The immunoassay device according to (6), which is an immunochromatographic test strip. (8) The immunoassay device according to (6) or (7), wherein the test subject is Mycoplasma pneumoniae. (9) A monoclonal antibody which specifically reacts with the P1 protein and P30 protein derived from Mycoplasma pneumoniae, and which specifically reacts with a peptide containing an amino acid sequence consisting of PPQPG. Effect of the Invention

[0011] The method of the present invention has improved detection sensitivity compared to the method described in Patent Document 2. Furthermore, the present invention provides an immunoassay device and a monoclonal antibody for use in the novel detection method of the present invention. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0012] The subject to be detected by the method of the present invention is Mycoplasma pneumoniae, and the antigens are P1 protein and P30 protein. Even when the subject is quantitatively or semi-quantitatively measured, the quantitative or semi-quantitative measurement necessarily involves "detection," and is therefore included in the "detection" of the present invention.

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

[0014] Antigen-binding fragments obtained by isolating only the antigen-binding site based on the monoclonal antibody of the present invention can also be used in the method of the present invention. That is, fragments (antigen-binding fragments) having specific antigen-binding ability, such as Fab, Fab', F(ab')2, and single-chain antibody (scFv), produced by known methods, are also included in the scope of the present invention. Furthermore, the class of the monoclonal antibody is not limited to IgG, and may be IgM or IgY.

[0015] The monoclonal antibody used in the method of the present invention can be obtained by immunizing an animal to be immunized with a complex or extract containing the above two types of antigens, or one of the two types of antigens or a partial peptide thereof, using a known immunological technique, and preparing a hybridoma using the cells of the immunized animal. The length of the peptide used for immunization is not particularly limited, but a peptide of preferably 5 amino acids or more, more preferably 10 amino acids or more, can be used as an immunogen. The immunogen can be obtained from the culture medium, but can also be obtained by incorporating DNA encoding an arbitrary antigen into a plasmid vector and introducing it into a host cell for expression. The arbitrary antigen or its partial peptide to be used as an immunogen can be expressed as a fusion protein with a protein such as those exemplified below, and can be used as an immunogen after purification or as it is. To prepare a 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 / purification tags", can be used. The fusion protein with these is preferably used as an immunogen after cleaving the antigen or its partial peptide portion and the remaining tag portion with a digestive enzyme, and separating and purifying it.

[0016] Preparation of monoclonal antibodies from immunized animals can be easily performed by the well-known method of Kohler et al. (Kohler et al., Nature, vol. 256, p. 495-497 (1975)). That is, antibody-producing cells such as spleen cells and lymphocytes are collected from the immunized animal, and these are fused with mouse myeloma cells by a conventional method to prepare hybridomas. The obtained hybridomas are cloned by limiting dilution or the like, and from the monoclonal antibodies produced by each cloned hybridoma, a monoclonal antibody that undergoes an antigen-antibody reaction 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 types of antigens selected in this manner 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 types of antigens obtained in this manner, 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 in the screening is a peptide containing an amino acid sequence consisting of PPQPG, and for example, a peptide whose amino acid sequence consists of PPQPGFPPKR (SEQ ID NO: 2) and a peptide whose amino acid sequence consists of GMPPQPGFPPKR (SEQ ID NO: 3) can be used.

[0018] Monoclonal antibodies can be purified from ascites or culture supernatant by known immunoglobulin purification methods, such as fractionation by salting out using ammonium sulfate or sodium sulfate, PEG fractionation, ethanol fractionation, DEAE ion exchange chromatography, and gel filtration. Purification can also be performed by affinity chromatography using a carrier bound to Protein A, Protein G, or Protein L, depending on the immunized animal species and the class of the monoclonal antibody.

[0019] In the immunoassay method of the present invention, the monoclonal antibody or its antigen-binding fragment that specifically reacts with the two types of antigens prepared as described above (hereinafter, in the description before the examples, unless otherwise clear from the context, "antibody" means "antibody or its antigen-binding fragment") is used to perform the immunoassay using the antigen-antibody reaction between Mycoplasma pneumoniae in the sample. As the immunoassay method for this purpose, any method well known to those skilled in the art, such as the competitive method, the agglutination method, the Western blot method, the immunostaining method, and the sandwich method, can be used. In the present invention, "measurement" includes any of quantitative, semi-quantitative, and detection.

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

[0021] In the sandwich method, two types of antibodies that recognize antigens (an antibody immobilized on a solid phase and a labeled antibody) are used, but in the method of the present invention, at least one of these two types of antibodies is a monoclonal antibody that recognizes the above-mentioned two types of antigens. As described below, the amount of antibody that can be immobilized on a solid phase per unit area is limited, so in order to better achieve the object of the present invention, which is to improve sensitivity, it is preferable to use a monoclonal antibody that recognizes the above-mentioned two types of antigens as at least the immobilized antibody. Note that, when at least two types of antigens recognized by the monoclonal antibody exist in a single molecule or a single complex, it is also possible to carry out the sandwich method using a single type of monoclonal antibody as the immobilized antibody and the labeled antibody.

[0022] In immunoassays using the sandwich method as the detection principle, any solid phase on which an antibody can be immobilized by known techniques can be used, and any known substance can be selected, such as a porous thin film (membrane) with capillary action, a particulate substance, a test tube, or a resin plate. In addition, substances that can be used to label an antibody include enzymes, radioisotopes, fluorescent substances, luminescent substances, colored particles, colloidal particles, and the like. Among the immunoassays using the various materials described above, a lateral flow immunoassay using a membrane is preferred, particularly from the viewpoint of the simplicity and speed of clinical testing.

[0023] The present invention also provides an immunoassay device capable of performing lateral flow immunoassay using a monoclonal antibody that recognizes two or more antigens. The immunoassay device provided by the present invention is an immunoassay device that comprises a support having a detection region where an antibody (antibody 1) that captures the object to be measured (antigen) is immobilized, a label region having a mobile labeled antibody (antibody 2), a sample pad for adding a drop of a specimen, an absorption band that absorbs the developed specimen liquid, and a backing sheet for bonding these components together, in which at least one of antibody 1 and antibody 2 is a monoclonal antibody that recognizes two or more antigens of the present invention.

[0024] The number of detection regions and the type of labeled antibody contained in the label region are not limited to one, and by using antibodies corresponding to multiple objects to be measured, two or more antigens can be detected using the same immunoassay device.

[0025] The principle by which the sensitivity of immunoassay is improved by the method of the present invention is believed to be as follows. In the sandwich method, an antibody immobilized on a solid phase is used, but the amount of antibody that can be immobilized per unit area of ​​the solid phase is limited. In addition, the time for the antigen-antibody reaction is also limited. In particular, in the immunochromatography method, the antigen-antibody reaction occurs only while the specimen or a sample prepared using the specimen (such as a dilution of the specimen) flows from upstream and passes through the detection area, so that the antigen that cannot bind to the antibody within this time flows directly downstream of the detection area and is not detected. When the amount of antigen in the specimen is small, the amount of antigen that binds to the antibody increases when a monoclonal antibody that recognizes two or more antigens is used as the solid-phase antibody, compared to the case of using one monoclonal antibody that recognizes one antigen (the conventional sandwich method), and therefore the sensitivity is improved. Furthermore, when two monoclonal antibodies each recognizing one type of antigen are used in combination as the solid-phase antibodies, the amount of each monoclonal antibody immobilized is half of the total amount of the solid-phase antibodies, so that when the time for the antigen-antibody reaction is short, the probability that an antigen will collide with a monoclonal antibody capable of binding to this antigen in a predetermined direction and bind to it is half compared to when all the solid-phase antibodies are monoclonal antibodies recognizing that antigen. On the other hand, when a monoclonal antibody recognizing two or more antigens is used, any of the two or more antigens will bind to the antibody if it collides with the solid-phase antibody in a predetermined direction, so the amount of antigen captured by the solid-phase antibody is greater than when two monoclonal antibodies each recognizing one type of antigen are used in combination, and therefore the sensitivity of the immunoassay is improved. EXAMPLES

[0026] The present invention will be described in more detail below with reference to examples, although the present invention is not limited to the following examples.

[0027] Example 1: Preparation 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 and used as an antigen.

[0028] 2. Preparation of Anti-Mycoplasma pneumoniae Monoclonal Antibodies BALB / c mice were immunized with the inactivated mycoplasma antigen described above, and the spleens were removed from the mice after a certain period of time and fused with mouse myeloma cells (P3×63) by 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 the cells were purified (monocloned) while checking the antibody activity of the supernatant by ELISA using a plate immobilized with Mycoplasma pneumoniae P1 antigen and a plate immobilized with Mycoplasma pneumoniae P30 antigen.

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

[0030] [Table 1]

[0031] The 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 was collected about 2 weeks later. IgG was purified from the ascites by affinity chromatography using a protein A column, and multiple purified anti-Mycoplasma pneumoniae monoclonal antibodies were obtained.

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

[0033] (Synthetic peptides) 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) Peptide 1-6: APRPGFPPQP (SEQ ID NO: 9) Peptide 1-7: APRPGMQPPR (SEQ ID NO: 10) Peptide 1-8: GMQPPRPGMP (SEQ ID NO: 11) Peptide 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 to the above synthetic peptides by the ELISA method shown below. For the measurement by the ELISA method, a 96-well plate (NUNC) with the synthetic peptide immobilized was used. The synthetic peptide (2.0 μg / ml) was added to the above plate at 100 μl / well and reacted overnight. Thereafter, the plate was washed with 1×TBS Buffer, and Blocking Buffer (1% BSA / TBS) was added at 200 μL / well. The Blocking Buffer was discarded, and the antibody diluted in Example 1 2. was added at 100 μL / well and reacted for 30 minutes. After that, the plate was 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, and 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 plate was washed with Buffer II, and 100 μl / well of a substrate solution containing OPD, a substrate for HRP, was added, and the plate was left to stand for 10 minutes. The reaction was stopped by adding 100 μl / well of 2 N sulfuric acid. 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 were found to react with the amino acid repeat sequence of P30. Monoclonal antibody P1-30Aab (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 the above results, it was confirmed that monoclonal antibody P1-30Aab has reactivity with peptide sequences containing "PPQPG (Pro-Pro-Gln-Pro-Gly)". On the other hand, monoclonal antibody P1-30Bab reacts with P1, but PPQPG is not present in the amino acid sequence of P1, and instead, PPHP is present as a similar sequence.

[0036] <Example 3> Immunoassay device for measuring Mycoplasma pneumoniae 1. Immobilization of anti-Mycoplasma pneumoniae antibodies onto nitrocellulose membrane The antibody prepared in Example 1 was diluted with purified water to 1.0 mg / mL, and an anti-mouse IgG antibody was prepared. The antibody was applied in a line shape to the sample pad side of a nitrocellulose membrane backed with a PET film, and the anti-mouse IgG antibody was applied in a line shape to the absorber 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 1.0 mg / mL, and colored polystyrene particles were added to this to a concentration of 0.1%. After stirring, carbodiimide was added to a concentration of 1% and further stirred. The supernatant was removed by centrifugation, and the particles were resuspended in 50 mM Tris (pH 9.0) and 3% BSA to obtain colored polystyrene particles bound to anti-Mycoplasma pneumoniae antibodies. In this example, these are called antibody-immobilized particles.

[0038] 3. Preparation of Test Pieces for Measuring Mycoplasma pneumoniae The antibody-immobilized membrane prepared in 1 was attached to other members (backing sheet, absorbent, sample pad) and cut to a width of 5 mm to prepare a Mycoplasma pneumoniae test piece. In this example, these are referred to as test pieces.

[0039] Immunochromatography Sensitivity Example 4: Comparison of sensitivity of immunoassay devices for measuring Mycoplasma pneumoniae 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 were used in the combinations shown in Table 2 to prepare three types of antibodies: P1P30, P30 test strip, and P1×P1P30, according to the procedure of Example 3.

[0040] [Table 2]

[0041] 50 μL of the specimen suspension containing the Mycoplasma pneumoniae antigen diluted arbitrarily and the antibody-immobilized particles prepared in 2 was added dropwise to each test piece, and the test piece was left to stand for 15 minutes. If color development was confirmed visually at the positions where both the anti-mouse IgG antibody and the anti-Mycoplasma pneumoniae antibody were applied, the result was judged as +. If color development was confirmed visually only at the position where the anti-mouse IgG antibody was applied, but not at the position where the anti-Mycoplasma pneumoniae antibody was applied, the result was judged as -. If color development was not confirmed visually at the position where the anti-mouse IgG antibody was applied, the result was judged as invalid.

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

[0043] [Table 3]

[0044] As shown in Table 3, the immunoassay device using P1-30Bab, which is the monoclonal antibody of the present invention, exhibited superior sensitivity compared to immunoassay devices using other antibodies.

Claims

1. A method for immunoassay of Mycoplasma pneumoniae, utilizing an antigen-antibody reaction between a monoclonal antibody or an antigen-binding fragment thereof that specifically reacts with a peptide comprising an amino acid sequence consisting of PPQPG and the P1 protein and P30 protein derived from Mycoplasma pneumoniae.

2. The immunoassay method according to claim 1, wherein the amino acid sequence of the peptide is PPQPGFPPKR or GMPPQPGFPPKR.

3. 2. The method according to claim 1, wherein the monoclonal antibody or an antigen-binding fragment thereof is used as at least one of a label and a solid phase, and the immunoassay method is a sandwich method.

4. The method according to claim 2, wherein the immunoassay method is an immunochromatography method.

5. The method according to any one of claims 1 to 4, wherein the two or more antigens recognized by the monoclonal antibodies form a complex.

6. 2. An immunoassay device for carrying out the method according to claim 1, wherein the monoclonal antibody or an antigen-binding fragment thereof is used as at least one of a label and a solid phase.

7. 7. The immunoassay device according to claim 6, which is an immunochromatographic test strip.

8. 8. The immunoassay device according to claim 6, wherein the test subject is Mycoplasma pneumoniae.

9. A monoclonal antibody which specifically reacts with P1 protein and P30 protein derived from Mycoplasma pneumoniae, and specifically reacts with a peptide comprising an amino acid sequence consisting of PPQPG.

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