WO2009072708A1

WO2009072708A1 - Recombinant proteins for the diagnosis of diseases infected with mycoplasma pneumoniae and diagnostic kits comprising the same

Info

Publication number: WO2009072708A1
Application number: PCT/KR2008/003092
Authority: WO
Inventors: Chom Kyu Chong; Nam Jeong Cho; Jong Jin Park; Kwang Myun Cheong; Jai Cheol Yoo; Sang Oh Lee; Ha Na Oh; Sun Ock Park
Original assignee: Bioland Ltd.
Priority date: 2007-12-04
Filing date: 2008-06-02
Publication date: 2009-06-11
Also published as: KR100905066B1; KR20090058327A

Abstract

Disclosed are a recombinant protein specific for antibodies against Mycoplasma pneumoniae, and a composition for the diagnosis of Mycoplasma pneumoniae infection, comprising the same. Also, a kit is provided for the diagnosis of Mycoplasma pneumoniae infection with the composition. A method for the diagnosis of Mycoplasma pneumonia using the kit is also disclosed.

Description

RECOMBINANT PROTEINS FOR THE DIAGNOSIS OF

DISEASES INFECTED WITH MYCOPLASMA PNEUMONIAE

AND DIAGNOSTIC KITS COMPRISING THE SAME

Technical Field

[1] The present invention relates to recombinant proteins that specifically react with antibodies against Mycoplasma pneumoniae and methods for the diagnosis of My- coplasma pneumoniae infection and Mycoplasma pneumonia using the same. Also, the present invention is concerned with hybridoma cells capable of producing antibodies against the recombinant proteins. Background Art

[2] Pneumonia can result from a variety of causes, mainly infection with bacteria and viruses. Viral pneumonia is commonly caused by RSV (respiratory syncytial virus), adenovirus, and parainfluenza virus. Influenza viruses are the most common cause of pneumonia in children. As for bacterial pneumonia, it is also caused by various bacteria, including Mycoplasma spp.

[3] There are 16 recognized species of the genus Mycoplasma isolated from the human body. Of them, Mycoplasma pneumoniae is the predominant cause of respiratory tract infection, while Ureaplasma urealyticum and Mycoplasma hominis cause genitourinary tract infection and prenatal infection, respectively.

[4] Of patients who have mycoplasma infection, about 20% show no symptoms, 75% are afflicted with slight respiratory diseases, such as bronchitis, pharyngitis, etc., and 3 ~ 10% suffer from severe diseases such as pneumonia. In children, Mycoplasma pneumoniae is generally responsible for 10 ~ 40% of pneumonia cases. More cases of pneumonia occur during the late summer or the early fall months, rather than at other times of the year. Incidence differs from one year to another, with an epidemic cycle of four years.

[5] Lacking a cell wall, Mycoplasma is unaffected by some antibiotics such as penicillin or other beta-lactam antibiotics that target cell wall synthesis. For this reason, Mycoplasma infection must be treated with antibiotics that inhibit the synthesis of proteins or DNA, such as microlide, quinolone, and tetracycline. Although Mycoplasma pneumoniae is one of the most frequent causes of respiratory tract infection in children, the pneumonia caused thereby is one of the most seldom diagnosed diseases because of the difficult diagnosis thereof. As a matter of fact, the pneumonia caused by Mycoplasma pneumoniae is frequently misdiagnosed, resulting in the administration of inappropriate antibiotics. [6]

[7] Standard diagnostic methodologies for diseases of Mycoplasma pneumoniae include the identification of bacteria through culture, serological assay, and genetic analysis by PCR. The identification of bacteria is difficult to apply in practice because it takes 7 ~ 21 days to identify bacteria through culture, and they are cultured at a rate of only 40 ~ 90%. Serological diagnosis of Mycoplasma pneumoniae infection is most widely used. However, a CF test (complement fixation test) for serological diagnosis requires a long period of time and is not sensitive enough. Recently, a gelatin microparticle agglutination test (e.g., SeroDia MycoII, Fujirebio) and ELISA (e.g., Platelia Myco IgM/ IgG, Bio-Rad) have been generally applied to the determination of Mycoplasma pneumoniae infection. These two immunoassay methods, however, are difficult to conduct, require 3 - 4 hours for the completion thereof, and employ very expensive apparatus. Moreover, these methods do not allow point-of-care testing (POCT). Further, the concordance rate between the test results of the two methods is low, and thus the user is frequently confused as to the basis on which the determination is to be made. Therefore, active attempts have been made to develop POCT kits that effectively allow the serological detection of antibodies against Mycoplasma pneumoniae.

[8]

[9] A prerequisite to an effective assay for antibodies to Mycoplasma pneumoniae is to secure a protein which is of high antigenicity and evolutionarily well conserved among homologous species. Attachment sites of Mycoplasma pneumoniae include epidermal cells of the respiratory tract. For the attachment, various proteins, including adhesin Pl (170 kDa), adhesin P30 (30 kDa), adhesin P40 (40 kDa) and adhesin P90 (90 kDa), are collectively responsible. Of them, adhesin Pl is known to evoke potential antigenicity in humans and animals. However, adhesin Pl is difficult to recombinantly produce in E. coli because it has a large molecular weight and its gene contains 21 copies of a UGA sequence, which is recognized as a stop codon in E. coli. In fact, commercial diagnostic kits in the current market take advantage of an exudate of cultured My- coplasma pneumoniae, suffering from the problem of being low in specificity.

[10]

[11] Leading to the present invention, intensive and thorough research into the diagnosis of mycoplasma pneumonia, conducted by the present invention, resulted in the finding that a recombinant, highly antigenic, and small protein can be expressed from a fragment of the gene encoding adhesion Pl of Mycoplasma pneumoniae and allow the preparation of a fused cell producing a monoclonal antibody thereto. Based on the antibody, a kit showing selectivity and specificity for Mycoplasma pneumoniae can be provided for the diagnosis of Mycoplasma pneumonia. [12]

Disclosure of Invention

Technical Problem

[13] It is an object of the present invention to provide a recombinant protein of the adhesion Pl of Mycoplasma pneumoniae.

[14] It is another object of the present invention to provide a fused cell line capable of producing monoclonal antibodies to the recombinant adhesion Pl protein.

[15] It is a further object of the present invention to provide a composition for the diagnosis of Mycoplasma pneumoniae infection, comprising the antibody produced by the fused cell line and a kit for the diagnosis of Mycoplasma pneumoniae infection, comprising the composition.

[16]

Technical Solution

[17] In accordance with an aspect thereof, the present invention provides a recombinant adhesin Pl protein having high antigenicity to Mycoplasma pneumoniae.

[18] Adhesin Pl shows high antigenicity in humans and animals, but has a very large molecular weight and as many as 21 copies of a UGA sequence, which is recognized as a stop codon in E. coli. Selected in the present invention is a region of adhesion Pl, which shows the following features: 1) no truncation upon protein expression because of the absence of UGA codons, 2) many epitopes with high antigenicity, as predicted by epitope prediction software, and 3) a net pi less than 7 and an average hy- drophobicity index less than zero, allowing the expression thereof in a water-soluble form. In more detail, a region of adhesin Pl which 1) was predicted to have 20 or more MHC peptide binding motifs by the HLA peptide binding prediction program developed by Harvard University, 2) was found to have a molecular weight of 38.24 kDa and a pi of 4.72, as measured by the ExPASy system developed by the Swiss Institute of Bioinformatics, Switzerland, and 3) was found to have no transmembrane domains and an average hydrophobicity of -0.535, as measured by the SOSUI system (Nagoya University, Japan), was selected. The recombinant protein has the amino acid sequence of SEQ ID NO.1, encoded by a base sequence of SEQ ID NO. 2.

[19] In this regard, a gene was amplified using a pair of primers of SEQ ID NOS. 3 and 4 and was then cloned to a pGEM-T Easy vector. For the expression of the gene, a pGEX expression system (GE Healthcare, Uppsala, Sweden) was used. That is, a gene of adhesin Pl was sub-cloned to a pGEX-4T-l expression vector, which was then transformed into E. coli BL21 (DE3). The recombinant adhesin Pl protein was over- expressed by inducing the resulting transformed E. coli with 1 mM of IPTG (isopropyl-β-D-thiogalactopyranoside), followed by purification through affinity chro- matography using GSH-Sepharose gel to afford a 64.0 kDa, pure recombinant adhesin Pl protein (FIG. 3).

[20]

[21] In accordance with another aspect thereof, the present invention provides a hybridoma cell line producing a monoclonal antibody to the recombinant adhesion Pl protein.

[22] In a preferable embodiment of the present invention, the spleen cells from a mouse immunized with the recombinant adhesin Pl protein were fused with myeloma cells to make a hybridoma cell line. To accomplish this, the pure recombinant adhesin Pl protein was peritoneally injected into a 6-8 week-old mouse from which the spleen was then excised. Spleen cells isolated from the spleen were fused with myeloma cells to make hybridoma cells. This mixture of cells was then diluted, and clones were grown from single parent cells. After tests for ability to bind to the antigen, the most productive and stable clone was then grown in culture medium to a high volume. The hybridoma cell line thus obtained was deposited at an international depositary authority, KCTC (Korean Collection for Type Cultures; Genetic Resources Center, Korean Research Institute of Bioscience and Biotechnology (KRIBB), Yusong-ku, Taejon, Korea) on November 15, 2007, and assigned accession number KCTC 11242 BP.

[23]

[24] In accordance with a further aspect thereof, the present invention provides novel monoclonal antibodies specific for the mycoplasma adhesin Pl for use in the diagnostic kit of the present invention.

[25] As used herein, the term "monoclonal antibodies" is intended to refer to highly specific antibodies directed to a single antigenic site. Whereas polyclonal antibodies bind to different epitopes on one antigen, monoclonal antibodies are directed to a single epitope on one antigen. Monoclonal antibodies have high selectivity and specificity suitable for use in diagnosis and analysis based on antigen- antibody conjugation, and enjoy the advantage of not being contaminated by other immunoglobulins because they are produced by fused cells. Monoclonal antibodies can be produced using well-known techniques, such as a hybridoma method (Kohler and Mislstein (1976) European Journal of Immunology 6:511-519) or a phage antibody library technique (Clackson et al, Nature, 352:624-628, 1991).

[26] In a preferable embodiment of the present invention, mice 6 - 8 weeks old were immunized by injecting the hybridoma cell line into the peritoneal cavity. From the ascites fluid thus formed, monoclonal antibodies against Mycoplasma pneumoniae were isolated. In brief, the ascites fluid was extracted, added with ammonium sulfate, and centrifuged to isolate antibodies. These were purified through dialysis and then through column chromatography. The monoclonal antibodies thus obtained were called monoclonal antibody 2G6D7.

[27] This pure antibody, prepared according to the present invention, is useful in the preparation of a diagnostic kit. The diagnostic kit comprises a strip on which a test line (T) for showing a diagnosis result and a control line (C) for determining test validity are provided. Protein A and the anti-adhesin Pl monoclonal antibody are completely immobilized at the test line and the control line, respectively, on a nitrocellulose membrane.

[28]

[29] On the other hand, the purified monoclonal antibody may be applied to the direct detection of an antigen of Mycoplasma pneumoniae.

[30] Accordingly, the present invention also provides a composition for the diagnosis of

Mycoplasma pneumoniae infection, comprising the monoclonal antibody.

[31] In accordance with still a further aspect thereof, the present invention provides a kit for the diagnosis of Mycoplasma pneumoniae infection, using the monoclonal antibody produced by the hybridoma cell line described above.

[32] In the diagnostic kit of the present invention, an antigen- antibody conjugate is detected using a color particle agglutination assay. Examples of the color particles include colloidal gold particles and color glass or plastic (e.g., polystyrene, polypropylene, latex, etc.) beads, with preference for gold colloid.

[33] In the diagnostic kit of the present invention, as mentioned above, a capture is provided for binding to the antigen- antibody conjugate. As the capture, Protein A, Protein G or anti-human IgG may be used. In an embodiment of the present invention, Protein A served as a capture for binding an antigen- antibody conjugate.

[34] Examples of the assay system suitable for use in the diagnostic kit of the present invention include, but are not limited to, an ELISA plate, a dipstick device, an im- munochromatography and radioimmunoassay device, and a flow-through device. Preferable is a diagnostic kit in a form of a strip or a device using immuno- chromatography.

[35] A kit for the diagnosis of Mycoplasma infection using immuno-chromatography in accordance with the present invention is composed essentially of a nitrocellulose membrane, on which two invisible lines are drawn, and a glass-fiber pad in which a recombinant protein-gold conjugate is stored in a dry state. At the positions of the two invisible lines, respective different proteins are immobilized on the nitrocellulose membrane. Protein A is immobilized at a lower test line (T), while an anti-adhesin Pl monoclonal antibody is immobilized at a lower control line (C). In the glass fiber pad, an antigen-gold conjugate, made of the recombinant adhesion Pl protein and gold particles, is absorbed and dried. [36] When a specimen is loaded onto a sample window of the kit, the dry antigen-gold conjugate, stored in the glass fiber pad, is hydrated and bound to the antibody of the specimen, with concomitant migration to the nitrocellulose membrane through micropores by capillary action. Subsequently, as the Protein A, immobilized at the invisible line, is associated with the antibody bound to the antigen-gold conjugate, the line turned red because of the red color of the antigen-gold conjugate. As for the anti- adhesin Pl monoclonal antibody immobilized at the upper control line, it always reacts with an unbound antigen-gold conjugate, even if it passes without reaction to the antibody of the specimen or cannot bind to the antibody because it is absent in the specimen. Therefore, the control line appears red in every test, indicating whether the test has been conducted properly or not. In other words, when an antibody to Mycoplasma is present in the specimen, the test line and the control line appear red simultaneously. By contrast, when the specimen contains no antibodies to Mycoplasma, the control line is visible while the test line remains invisible.

[37] In accordance with a modification of the embodiment, the present invention can determine whether the human antibody to Mycoplasma is IgG or IgM. In greater detail, a kit is constructed in such a manner that anti-human IgG, anti-human IgM and an anti- adhesin Pl monoclonal antibody are immobilized at line 1, line 2 and a control line, respectively. When a specimen is developed on the glass fiber pad, the anti-adhesin Pl antibody of the specimen is bound to the antigen-gold conjugate and then reacted at line 1 when the antibody is IgG or at line 2 when the antibody is IgM. As a result, when the specimen contains IgG against adhesin Pl, line 1 appears red, while when the specimen contains IgM against adhesin Pl, line 2 appears red.

[38]

[39] The diagnostic kit of the present invention is found to show 90% or higher sensitivity and specificity for Mycoplasma pneumonia compared to commercially available kits (e.g., SeroDia Myco II kit, Fujirebio, Japan). It can diagnose mycoplasma pneumonia more rapidly and conveniently than can conventional kits (see Example 7). Accordingly, the diagnostic kit of the present invention can be conveniently and easily applied to the diagnosis of pulmonary paragonimiasis.

[40]

[41] In accordance with still another aspect thereof, the present invention provides a method for diagnosing mycoplasma infection using the diagnostic kit described above.

[42] In detail, a biological specimen (e.g., serum or plasma) suspected to be infected with

Mycoplasma pneumoniae is loaded onto the sample window of the diagnostic kit of the present invention, and after the completion of reaction, a decision is made about the infection of Mycoplasma pneumoniae with reference to the appearance of the lines of the kit. [43]

[44] As used herein, the term "biological specimen", is intended to refer to a blood sample(e.g., serum or plasma) from mammals suspected of being infected with pulmonary paragonimiasis, or to be tested therefor, including humans. For convenience, it is also expressed just as "specimen" or "sample". A biological specimen may or may not be diluted before being brought into contact with the sample window of the diagnostic kit according to the present invention.

Advantageous Effects

[45] The diagnostic kit of the present invention can be more effectively used to diagnose

Mycoplasma pneumoniae infection than can conventional techniques such as gelatin particle agglutination assay and ELISA because it takes a shorter time period using the diagnostic kit. In addition, the present invention promises an improvement in therapeutic effect because it significantly reduces misdiagnosis rates and thus prevents the misuse and overuse of antibiotics.

[46]

Brief Description of the Drawings

[47] FIG. 1 is a schematic view illustrating the membrane-associated proteins of My- coplasma pneumoniae.

[48] FIG. 2 is a flow chart showing processes of preparing a recombinant protein of the adhesin Pl of Mycoplasma pneumoniae.

[49] FIG. 3 is a photograph of SDS-PAGE (SDS-polyacrylamide gel electrophoresis) showing the purification of the recombinant adhesin Pl of the present invention, in which a cell lysate obtained after protein expression (lane 1), a cell lysate obtained before protein expression(lane 2), and a purified recombinant adhesin Pl (lane 3) are run, along with a marker (lane M).

[50] FIG. 4 shows the structure of a strip-type kit for diagnosing Mycoplasma pneumoniae infection based on rapid immunochromatography, in schematic diagrams.

[51] FIG. 5 shows positive and negative diagnosis results from a strip-type kit for diagnosing Mycoplasma pneumoniae infection based on rapid immunochromatography in photographs.

[52] FIG. 6 shows the structure of a device-type kit for diagnosing Mycoplasma pneumoniae infection based on rapid immunochromatography, in schematic diagrams.

[53] FIG. 7 shows positive and negative diagnosis results of a device-type kit for diagnosing Mycoplasma pneumoniae infection based on rapid immunochromatography, in photographs.

[54]

Mode for the Invention [55] A better understanding of the present invention may be obtained through the following examples which are set forth to illustrate, but are not to be construed as the limit of the present invention.

[56]

[57] EXAMPLE 1: Gene Cloning, Expression and Purification of Adhesion Pl of My- coplasma pneumoniae

[58]

[59] 1 - 1. Gene Cloning of Adhesin P 1 of Mycoplasma pneumoniae

[60] The genomic DNA of Mycoplasma pneumoniae was purchased from Advanced

Biotechnologies Inc, U.S.A. A fragment of the adhesion Pl gene, 1053 bp in length, represented by SEQ ID NO. 2, was amplified with the genomic DNA serving as a template. That is, the DNA fragment was selected for the reason that when it was expressed, the resulting recombinant protein was predicted 1) not to be in a truncated form due to the lack of UGA sequences therein, 2) contain many epitopes with high antigenicity, as simulated by epitope prediction software, and 3) be water-soluble with a pi less than 7 and an average hydrophobicity index less than zero. PCR was performed using a pair of primers APlF (SEQ ID NO. 3) and APlR (SEQ ID NO. 4) to amplify a 1053 bp-long DNA fragment corresponding to a region of the adhesin Pl gene. The PCR product thus obtained was cloned to a pGEM-Easy T vector (Promega, U. S. A.). The clone was analyzed for base sequence by Bioneer Corporation, Korea, and was found to be incident with a 1053 bp-long, initial adhesion P gene sequence. Comparison with the base sequence of Mycoplasma pneumoniae searched with the NCBI GenBank database (GenBank Accession Number: AF290001) indicated that the amplified clone is part of the adhesion Pl gene of Mycoplasma pneumoniae. Thereafter, the DNA fragment was subcloned to a pGEX-4T-l expression vector (GE Healthcare, Sweden) after being digested with EcoRI in the 5' direction and with Xhol in the 3' direction. The expression of the gene was performed in E. coli BL21 (DE3).

[61]

[62] 1-2. Bacterial Expression and Purification of Recombinant Adhesin Pl Protein

[63] The E. coli BL21 (DE3) carrying the recombinant pGEX-4T-l-Pl expression vector was cultured to an OD of 0.6 ~ 0.8 at 600 nm. After treatment with 1 mM of IPTG (isopropyl-β-D-thiogalactopyranoside), culturing for additional 4 - 5 hrs induced the overexpression of the recombinant adhesin Pl protein. The E. coli cells were collected by centrifugation and lysed using an ultrasonicator. After the centrifugation of the cell lysate, the supernatant was subjected to affinity chromatography using GSH-bound Sepharose. In this regard, the supernatant was loaded on a column with GSH-bound Sepharose to attach GST-adhesin Pl fusion protein to the Sepharose, followed by washing off unbound materials with a buffer. Thereafter, the GST-adhesin Pl fusion protein was eluted from the column with a Tris buffer (pH 7.0) containing 100 mM of GSH (glutathione). The eluted fractions were dialyzed to concentrate the GST-adhesin Pl fusion protein, which was then used in the present invention without additional treatment. If necessary, GST was removed by treatment with thrombin (GE healthcare, Sweden) to isolate only the recombinant adhesion Pl protein before use in the present invention.

[64]

[65] EXAMPLE 2: Production of Monoclonal Antibody for Recombinant Adhesin Pl

[66]

[67] 2-1. Immunization with the Recombinant Adhesin Pl Protein

[68] A solution of 100 μg of the recombinant adhesin Pl, protein prepared in Example 1, was injected, along with an equal volume of a complete Freund's adjuvant suspension, into the peritoneal cavity of a female mouse 6 - 8 weeks old (BALB/C, Dae Han BioLink Co. Ltd., Korea). An incomplete Freund's adjuvant was injected once into the mouse two weeks later and once more an additional two weeks later. Two days after the final immunization, a serum sample was taken from the tail of the mouse, 1/1000 diluted in PBS and analyzed for antibody titer using ELISA (enzyme-linked immunosorbent assay). If the antibody titer was low, immunization was again performed two weeks later.

[69]

[70] 2-2. Fusion of Splenocyte with Myeloma Cells

[71] The spleen was excised from the mouse immunized in the same manner as in

Example 2- 1 and homogenized using a tissue homogenizer. The cell suspension was centrifuged to recover splenocytes which were then counted. Myeloma cells were removed from a cell culture flask, suspended in an RPMI 1640 medium and counted. Ix 10⁷ myeloma cells and IxIO⁸ splenocytes were mixed and suspended in an RPMI 1640 medium in a 50 ml flask, followed by centrifugation at 200xg for 5 min to recover the cells. These cells were resuspended in an RPMI 1640 medium and added with 1 ml of a PEG solution (50% polyethylene glycol) over 1 min with slow shaking. 5 min later, an RPMI 1640 medium was slowly added in an amount of 1 ml over 30 sec, again in an amount of 3 ml over 30 sec, again in an amount of 17 ml over 1 min, and finally in an amount of 20 ml. The cells were allowed to stand for 5 min. After centrifugation at 200xg for 5 min, the medium was removed and the cells were carefully suspended in 50 ml of an RPMI 1640 1% HAT

(hypoxathine-aminopterin-thymidine) and 100 μl of the cell suspension was layered on feeder cells in each well of 96- well plates before incubation at 37⁰C in a 5% CO₂ incubator.

[72] [73] 2-3. Cloning of Fused Cell Line and Antibody Production Using Ascites Fluid

[74] When the fused cells of Example 2-2 gave rise to colonies during incubation for 10 days, 100 μl of the medium was taken from each well and assayed for antibody activity against adhesion Pl through ELISA. After each test for antibody activity, the cells, which were identified to produce the antibodies, were transferred to 24- well cell culture plates and incubated. The cloning procedure was repeated until stable monoclonal cell lines were obtained. The cell lines were cultured in T flasks. As soon as enough cells were present, they were harvested, frozen and stored in liquid nitrogen.

[75]

[76] 0.5 ml of an incomplete Freund's adjuvant was injected into the peritoneal cavity of a mouse (BALB/C) 6 ~ 8 weeks old. On Day 7 after the injection, the fused cells were suspended at a density of 1.5xlO⁶ cells in 0.5 ml of PBS, and this cell suspension was peritoneally injected into the mouse. After 1 ~ 2 weeks, the ascites fluid was sampled with a syringe and freeze- stored.

[77]

[78] 2-4. Purification of Monoclonal Antibody

[79] Ammonium sulfate was added at a concentration of 10% to the ascites fluid of

Example 2-3 and mixed for 30 min. After centrifugation at 15,000xg for 30 min, ammonium sulfate was added to the supernatant at a concentration of 50% and the mixture was left at 4⁰C for 30 min. After an additional centrifugation at 15,000xg for 30 min, the pellet was suspended in a 20 mM phosphate buffer solution (pH 7.0). The suspension was dialyzed in 20 mM PBS for 18 hrs or longer and loaded on a protein G-coupled column equilibrated with 20 mM PBS (pH 7.0), followed by washing with PBS to remove unattached materials. The antibodies bound to the column were eluted with a 10 mM glycine solution (pH 2.8). In this regard, 1 M Tris (pH 9.0) was added at a 1/10 volume of the eluent so as to adjust the pH to 7.0 ~ 7.5. After dialysis in 150 mM PBS, the antibody fraction was quantified for antibody activity using a Bradford assay and frozen and stored until use. The purified monoclonal antibodies were named monoclonal antibody 2G6D7.

[80]

[81] EXAMPLE 3: Construction of Strip-Type Diagnostic Kit Using Rapid ImmunoChro- matography Assay with Recombinant Antigen

[82]

[83] 3-1. Immobilization of Protein A and Anti-Adhesin Pl Monoclonal Antibody

[84] Protein A and anti-adhesin Pl monoclonal antibody 2G6D7 were allocated at respective specific positions, that is, a test line (T) and control line (C), on a nitrocellulose membrane layered on a plastic card, followed by incubation under dry conditions at 3O⁰C for 2 days in an incubator to completely immobilize the protein A and the monoclonal antibody onto the membrane.

[85]

[86] 3-2. Preparation of Gold Conjugate and Gold Conjugate Pad

[87] The recombinant adhesin Pl protein was incubated along with colloidal gold particles 40 nm in size in a 37⁰C water bath for 1 hr to associate them with each other. Thereafter, BSA (bovine serum albumin) and sucrose were added at concentrations of 3% and 1%, respectively, followed by incubation for an additional 1 hr so as to associate them with the colloidal gold particles which remained unbound. The recombinant protein-gold conjugates were harvested by centrifugation at 10,000xg for 20 min and analyzed for absorbance at 540 nm before cold storage. The gold conjugate was infused into a plastic microwell plate and dried to prepare a gold conjugate well.

[88]

[89] 3-3. Assembly of Strip Kit

[90] As illustrated in FIG. 4, a strip-type plastic card was overlaid with the protein A and monoclonal antibody-immobilized nitrocellulose membrane for a half region thereof while an absorption pad capable of absorbing a specimen and a buffer was attached onto the other half region of the plastic card to prepare a strip-type kit.

[91]

[92] EXAMPLE 4: Construction of Device-Type Diagnostic Kit Using Rapid Im- munoChromatography with Recombinant Antigen

[93]

[94] 4-1. Immobilization of Protein A and Anti- Adhesin Pl Monoclonal Antibody

[95] Protein A and anti-adhesin Pl monoclonal antibody 2G6D7 were allocated at respective specific positions, that is, a test line (T) and a control line (C), on a nitrocellulose membrane layered on a plastic card, followed by incubation under dry conditions at 3O⁰C for 2 days in an incubator to completely immobilize the protein A and the monoclonal antibody on the membrane, as shown in FIG. 4.

[96]

[97] 4-2. Preparation of Gold Conjugate and Gold Conjugate Pad

[98] The recombinant adhesin Pl protein was incubated along with colloidal gold particles 40 nm in size in a 37⁰C water bath for 1 hr to associate them with each other. Thereafter, BSA (bovine serum albumin) and sucrose were added at concentrations of 3% and 1%, respectively, followed by incubation for an additional 1 hr so as to associate them with the colloidal gold particles that remained unbound. The recombinant protein-gold conjugates were harvested by centrifugation at 10,000xg for 20 min and analyzed for absorbance at 540 nm before cold storage. The gold conjugate was infused into a glass fiber pad and dried to prepare a gold conjugate pad.

[99] [ 100] 4-3. Assembly of Device-Type Kit

[101] As shown in FIG. 6, the nitrocellulose membrane, immobilized with protein A and the monoclonal antibody, was attached onto a mid-region of the plastic card while the opposite end regions of the plastic card were overlaid respectively with an absorption pad, and a sample pad serving to load a sample thereon. This strip construct was assembled with a plastic housing, as shown in FIG. 6, to prepare a diagnostic kit.

[102]

[103] EXAMPLE 5: Diagnosis with Strip-Type Kit

[104] One drop of a washing buffer was placed in the gold conjugate well provided along with the kit. This was mixed with four microliters of a blood sample using a pipette. The absorption pad attached to the strip-type kit was put into the gold conjugate well for 10 min so as to absorb the sample solution sufficiently, and then into a washing well containing three drops of an assay buffer for 10 min so as to wash the membrane. Infection with Mycoplasma pneumoniae could be read with reference to the positions at which red lines appear, as illustrated in FIG. 5.

[105]

[106] EXAMPLE 6: Diagnosis with Device-Type Kit

[107] A device-type kit was unpacked from an aluminum pouch and placed on a flat surface. Four microliters of a blood sample was loaded on a sample window, as shown in FIG. 6a, so that it was absorbed by the absorption pad. Thereafter, three drops of an assay buffer were loaded. 15 min later, the presence of the antibody to Mycoplasma pneumoniae could be read according to the red appearance of the test line.

[108]

[109] EXAMPLE 7: Efficacy Test of Strip- and Device-Type Diagnostic Kits Using Rapid Immunochromatography

[HO]

[111] 7- 1. Efficacy Test for Positive Sensitivity

[112] The diagnostic kits of Examples 3 and 4 were tested for sensitivity for Mycoplasma antibody-positive clinical specimen. This test was conducted with 95 positive clinical samples provided by Chungju St. Mary's Hospital, Korea. For comparison, the same samples were tested with SeroDia Myco II Kit (Fujirebio, Japan). The results are summarized in Table 1, below.

[113]

[114] Table 1 [Table 1]

[115] [116] 7-2. Efficacy Test for Negative Selectivity [117] The diagnostic kits of Examples 3 and 4 were tested for specificity for Mycoplasma antibody-negative clinical specimens. This test was conducted with 120 negative clinical samples provided by the Parasitology Lab of Medical College, SungKyunKwan University, Korea. For comparison, the same samples were tested with SeroDia Myco II Kit (Fujirebio, Japan). The results are summarized in Table 2, below.

[118] [119]

[120]

[121] 7-3. Conclusion

[122] As described hitherto, the diagnostic kits of the present invention are more convenient for use and allow more rapid diagnosis without the aid of expensive in- struments, compared to conventional diagnostic kits. In addition to these advantages, the diagnostic kits of the present invention are of high selectivity and specificity, and thus show clinically highly useful applicability.

Claims

Claims[1] A composition for use in the diagnosis of Mycoplasma pneumoniae infection, comprising a monoclonal antibody specific for a recombinant adhesin Pl protein of Mycoplasma pneumoniae. [2] The composition according to claim 1, wherein the recombinant adhesin Pl protein of Mycoplasma pneumoniae has an amino acid sequence of SEQ ID NO.

1. [3] The composition according to claim 1, wherein the monoclonal antibody specific for a recombinant adhesin Pl protein of Mycoplasma pneumoniae is monoclonal antibody 2G6D7. [4] The composition according to claim 3, wherein the monoclonal 2G6D7 is produced by a fused cell of Deposition No. KCTC 11242BP.

[5] A diagnostic kit for Mycoplasma pneumoniae infection, comprising the composition of one of claims 1 to 4. [6] The diagnostic kit according to claim 5, which is in a form of a strip type using immunochromatography for diagnosis. [7] The diagnostic kit according to claim 5, which is in a form of a device type using immunochromatography for diagnosis. [8] The diagnostic kit according to claim 5, which uses a color particle agglutination assay in detecting an antibody- antigen conjugate.

[9] Antibody 2G6D7, produced by a fused cell of Deposition No. KCTC 11242BP.

[10] A fused cell of Deposition No. KCTC 11242BP.