TWI282368B - Antibody for detecting mycoplasma pneumoniae - Google Patents

Abstract

A method of specifically, quickly and highly sensitively detecting a microorganism belonging to Mycoplasma pneumoniae; an antibody to be used in the detection; a detection reagent kit; and a process for producing the antibody to be used in the detection. Namely, an antibody against the ribosomal protein of a microorganism belonging to Mycoplasma pneumoniae which reacts specifically with this microorganism; a method of detecting this microorganism in a specimen by using this antibody; and a detection reaction kit containing this antibody. The ribosomal protein is exemplified by Ribosomal Protein L7/L12 employed in detecting the infection with a microorganism causative of pneumonia.

Description

1282368 A7 B7 Ministry of Economic Affairs Intellectual Property Office Employees' Consumption Cooperatives Printed V. Description of the Invention (1) The present invention relates to an antibody effective for detecting microorganisms belonging to Mycoplasma pneumoniae, which is a microorganism of general pneumonia, and a method for detecting the microorganism The test kit for detecting the microorganism, and the method for detecting the antibody for the microorganism. The present invention is extremely important for the diagnosis of non-type pneumonia caused by the pneumococcal bacteria in the pharmaceutical industry, and is extremely useful in the pharmaceutical industry. The invention is effective for detecting microorganisms, pneumococcal bacteria contained in samples taken from specimens such as: welded cotton swabs, tissue samples, body fluids, experimental solutions and cultures. In the prior art, the diagnosis of a microbial infection is detected by a cause of infection in a general infectious part or the like, or is determined after blood stasis, the cause of the bacteria in the body fluid is detected. In particular, the detection of the cause of the diagnosis means that it is extremely important for the patient to be treated promptly. Detection of infectious agents The bacteria are usually classified and cultured according to their physiological, biochemical or structural characteristics, and the same bacteria are subjected to polymerase chain reaction. After the PCR method or the specific nucleic acid mixture is used for amplification, the genetic diagnosis method and the immunological method for detecting the cause of the bacteria are detected by using the genetic diagnosis method and the antigen-identification of the antibody and the causative bacteria. However, when using the same method or genetic diagnosis method, it takes a long time to obtain results. Therefore, the diagnosis of immunological methods related to rapid and appropriate treatment of patients with short-term and high-sensitivity detection causes is widely used. In the case of the detection of infectious diseases by the prior immunological method, the Chinese National Standard (CNS) A4 specification (210X297 mm)-4 - (Please read the back of the note first? Write this page) - loaded ·

,tT 1282368 A7

Ministry of Economic Affairs Intellectual Property Bureau employee consumption cooperative printing 5, invention description (2 strains of various combinations of antigens and antibodies are used. Pneumococcal pneumonia, bronchitis and pharyngitis, the general cause of bacteria. Similar to small fungi The gram-negative bacteria selectively invade the human body and cause disease. In general, the group's pneumonia caused by the group is about 15~2 〇%' and the 50% of the pneumonia in the limited group can be said to be caused by pneumonia. Caused by moldy sputum (Ragnar Norrby 1999). This bacterium is a very small microbe that is difficult to culture. It forms small colonies in a thick medium. In the nutrient-enriched agar medium, the growth of Pneumococcal bacteria is extremely slow. At least 21 days (0^1^1: 〇, 〇6, 61 & 1.1 980) is required to make the cells in the culture medium. There are several microorganisms originating from the moldy species in the human body causing the same life. Chemical reactions. In addition, because there is no cell wall, observation under a bright microscope is more difficult (Knudson and MacLeod 1 979). Because it is a diagnostic method for rapid detection of pathogens, it is not suitable for Gram. Dyeing method, culture method, etc. The DNA mixture analysis and detection method for detecting the gene arrangement of Pythium oxysporum is based on 11^11, etc.; 61^]^ and 86]:1^1; etc. ^11,丫〇26¥61 al. 1 987; Bernet, Garret et al. 1 993; Buck, O'Hara et al. 1 9 9 3) Reported by the tester. Detection of Pneumoniae R ib 〇soma 1 The detection used for RNA (r RNA) is contained in T i 11 ο n et al (T i 11 ο n, D iaset al. 1980), Yogev et al (Yogev, Halachmi et al. 1 988), Gobel et al ( Gebel, Geiser et al. 1 987), Zivin and Monahan (EP〇30 5145) and Gobeland and Stanbridge (EP 〇2 5 0 6 6 2), etc. Kai et al. (Kai, Kamiya et al. 1987) and Jensen et al. (Jensen, Sondergard et al. 1993) and other departments record the lungs (please read the note on the back - JT. π write this page) • Install · - Line · This paper scale applies to China National Standard (CNS) A4 specification (210X297 5) 2 1282368 A7 B7 Ministry of Economic Affairs, Intellectual Property Bureau, Staff Consumer Cooperatives, Printing 5, Inventions (3) Inflammatory agents of 1 6 S r RNA. The whole test system is designed for D N A of P. oxysporum, or D N A of Mycoplasma pneumoniae and Mycoplasma genitalium. These detections are highly sensitive and therefore highly compliant with recently available technologies such as the collection of such methods using the p C R method. The P C R method has a very high sensitivity and can be carried out in a shorter time than the mixture. However, the culture of pneumococcal negative was asymptomatic, and the carrier showed positive after the illness, and occasionally the problem of quasi-positive. JP-A-63-209 discloses an immunoassay based on a wet-labeling method using a monoclonal antibody for a membrane antigen protein of 431α1〇 (1α1ΐ〇η P. pneumoniae). The antibody and the detection method using the same have a problem in that the specific diagnosis of the Pseudomonas pneumoniae with low specificity is insufficient, and the time required for the diagnosis to obtain the result is at least 5 hours, which is a great problem (M adseneta) 1. 1 9 8 8) The present invention is to solve the above problems. That is, the present invention provides a method for rapidly detecting microorganisms belonging to Pythium oxysporum with high specificity and high sensitivity. The antibody for detection and the reagent kit for detection are intended. The present invention further provides a method for producing a test antibody for use in the test. The present inventors have found that a protein having the same function can be effectively used in all microorganisms. The antigenic protein. It is generally foreseeable that the structure of this protein becomes a little Arctic. It is even more surprising that the antibody against the protein is a specific microbial species or genus. The antibody against the protein can be identified by the species or genus of the specific microorganism, and the microorganisms can be detected for all the blood sputum types. The more detailed is the protein passage (please read the back first) Note _ 5 write this page) • Install. - Line paper size applies to China National Standard (CNS) A4 size (210X297 mm) _ 6 - 1282368 A7 ___ B7 __ V. Description of invention (4) Amino acid arrangement After the change, the structure of the same species is completely the same, and when the species is different, the species-specific useful protein is accompanied by the structural change. The present inventors have a molecule which functions as the same function in all microbial cells, and an amine group thereof The Ribosomal Protein L7/L12 protein with an acid structure at a certain degree of difference between microorganisms, especially a Ribosomal Protein L7/L12 protein of ribonucleoprotein protein, is the focus of discussion. The Ribosomal Protein L7/L12 protein is known. A protein with a molecular weight of about 13 kilodalton, as a necessary ribonucleoprotein protein present in protein synthesizers. The total amino acid sequence of the Ribosomal Protein L7/L12 protein in several microorganisms was analyzed. The inventors of the present invention paid attention to the fact that even if the molecules are similar to each other, the part still has the structural part inherent to each microorganism, and it is found that the antibody is utilized. The Ribosomal Protein L7/L12 protein against the P. pneumoniae can detect various microorganisms, the species of the bacteria are specific, and all the bloody types in the same species, the inventors found that Pneumocystis may be The present invention has been accomplished by obtaining a specific antibody in a protein and detecting the specificity of M. pneumoniae using the antibody. The present inventors have found that M. pneumoniae has been developed for the specific monoclonal antibody of the Rib〇s〇mal Protein (ribosomal) L7/L12 protein. This antibody is novel and, unlike the previously known antibodies, has a specific reaction property to the protein. The number 1 and 2 in the matching list is Ribosomal of Pythium oxysporum. The paper size applies to the Chinese National Standard (CNS) A4 specification (210X297 mm). (Please read the note on the back first. 4% write this page) Ministry of Economics Intellectual Property Bureau employee consumption cooperative printed 1282368 A7 B7 V. Description of invention (5) Protein (ribosomal) L7/L12 gene DNA arrangement and corresponding amino acid ligand (NCBI database accession #NC 000912). Further, the left and right ends of the amino acid arrangement listed in the list are respectively an amine terminal (hereinafter referred to as an N terminal) and a carboxyl terminal (hereinafter referred to as a c terminal), and the left and right ends of the salt base are respectively 5 End and 3 > end. Cross-typed amino acid-matched amino acids represent a one-word reporter. Further, in the cross-type, the symbol "+" represents a different amino acid and its hydrophobicity is similar to that of an amino acid. The blank mark of "" represents an amino acid of a completely different nature. Further, a series of molecular biological tests performed by the present invention can be carried out by the methods described in the general test. As a general experimenter, for example, Molecular Cloning, A Uboratory manual, Cold spring Harbor Laboratory Press, Shambrook, J. et al. (1 989). Table 1 Cross-type test Μρ·· 1 MAKLDKNQX.IESLKEMTrM£XDEIIKXV££AFGVSATPWAA6AVGGTQEAASEVTVKVT 60 1 M KLDK QUESLKEMTI+EIOEIIKAVEEAFGV^ATP+VAAGA g tqeaasev+vkvt Mg: 1 MGKLDKKQLIESLKEMTIVEIDEIIKAVEEATGVTATPIVAAGAASATQEAASSVSVKVT 60 (Please read the note on the back: write this page ) - Installed - Ordered - Ministry of Economic Affairs Intellectual Property Bureau Consumer Cooperatives Μ :: 61 GYTDNAKLAVLKLYREIAGVGLMEAKTAVEKLPCWKQDIKPEEXEELKKRFVEVGATVE 120 GY DNAKLAVLKLYREI GVGLMEAKTAVEKLPCWKQDXKPEEAEELKKHFVEVGArVE Mg: 61 GYADNAKLAVLKLYREITGVGLMEXKTAVEKLPCWKQDIKPEEAEELKKRFVEVGATVE 120

Μρ: 121 IK 122 +K

Mg: 121 VK 122 This paper size is applicable to the national standard (CNS) A4 specification (210X297 mm)-8 - 1282368 A7 B7 Ministry of Economic Affairs Intellectual Property Bureau employee consumption cooperative printing 5, invention description (6) In the present invention Microbial refers to the meaning of M. pneumoniae, especially in the respirator as a microbiologist with a high diagnostic value for pathogenic fungal infections. In the present invention, the antibody and the specific reaction of the microorganism are specific antibodies of the microorganism species or the genus, and the antibody specific for the microbial species in the diagnosis of the microbial infection is particularly effective in the present invention. The anti-system refers to a polyclonal antibody, or a monoclonal antibody, which can be made using the total length of the Ribosomal Protein L7/L12 protein or a partial peptide thereof. The length of the peptide to be made into the antibody is not particularly limited, and for the antibody of the Ribosomal Protein L7/L12 protein, as long as it represents the characteristic length of the protein, it is generally preferred to use a 5-amino acid or more, particularly It is preferred to use a peptide above 8 amino acids. Directly cross-linking the peptide or the full-length protein or the protein carrying KLH (keyhole-limpet hemocyanin) and BSA (bovine serum albumin), and if necessary, inoculation with the auxiliary agent, and recovering the blood stasis, the obtained protein can be obtained. Anti-blood staining of Ribosomal Protein L7/L12 protein antibody (polyclonal antibody). Furthermore, it can also be used after anti-blood sputum antibody preparation. As vaccinated animals, sheep, cattle, goats, 'rabbit' mice, experimental mice, etc., in the production of ρ 〇 1 y c 1 q n a 1 antibody, particularly sheep, rabbits and the like are preferred. Further, a monoclonal antibody can also be obtained by a known method for producing a mixed cell, and at this time, a mouse is preferred. Further, the total length of the protein or 5 residues or more, preferably the amino acid of 8 or more residues, is classified as a glutathione S-converting enzyme (GST) or the like, and is not refined. Can be used as an antigen. (Please read the note on the back to write this page) ·. h !i Jr . • Loading · Setting up _ This paper scale applies to China National Standard (CNS) A4 specification (210X297 public shame) 1282368 A7 B7 Ministry of Economic Affairs Intellectual Property Bureau Employee Consumption Cooperatives Printing 5, Inventions (7) Immune spheres isolated by various methods and gene clomng methods shown in the book (Antibodies; A laboratory manual, E. Harlow et al., Cold Spring Harbor Laboratory Press) The protein gene can also be produced by using a gene-swapping antibody found by the cultured cells after use. The antibody to Ribosomal Protein L7/L12 which can be used as the antigen for the present invention can be obtained by the following method or the like, but is not limited thereto. a) Ribosomal Protein L7/L12 protein gene assignment and amino acid alignment for the known microorganisms in their microorganisms, so that the peptide amino acid with the similarity of the amino acid of the protein is synthesized, By making a polyclonal antibody or a monoclonal antibody as an immunogen, an antibody of interest can be obtained. In addition, after the PCR method in which the DNA is arranged at both ends of the gene as a detection method, the gene is amplified, and the same portion is arranged as a mixed method for mold detection, and the full length of the gene can be obtained by a general genetic manipulation method. After forming a fusion gene with another protein gene, the coagulation gene is inserted into the host by a known gene introduction method, and the protein used as the molten protein is found in a large amount by the antibody affinity column method. After purifying and discovering the protein, the protein antigen of interest can be obtained. At this time, the full-length protein of the Ribosomal Protein L7/L12 protein is an antigen. Therefore, it is not the object of the present invention to obtain an antibody against the amino acid moiety stored between microorganisms. Therefore, the antigen obtained by the method is obtained by a known method to obtain the mixture produced by monoclonal (please read the back of the page first) ^ Install the page. The order of the paper - the paper size applies to the Chinese National Standard (CNS) A4 specification ( 210X297 mm) -10 1282368 A7 B7 Ministry of Economic Affairs Intellectual Property Office Employees' Consumption Cooperatives Printed 5, Inventions (8) After the body, select the clone produced by the antibody that reacts with the microorganism to obtain the antibody of interest. b) When the amino acid of Ribosomal Protein L7/L12 protein is classified as an unknown microorganism, the amino acid of Ribosomal Protein L7/L12 protein of 1 is assigned to 5 0~6 0 % is the same, and the same part of the amino acid is used as a base to increase the gene of the specific part by PCR, and the same part is used as a mixed method such as mold detection. This protein gene can be easily obtained. Then, after forming a fusion gene with another protein gene, the coagulation gene is used as a host, and the fusion gene is inserted into the host by a known gene introduction method. After a large amount of discovery, the protein used as a molten protein is borrowed. After the protein is purified by the antibody affinity column method or the like, the protein antigen of interest can be obtained. At this time, the full-length protein of the Ribosomal Protein L7/L12 protein is an antigen, and therefore, it is not in accordance with the object of the present invention to obtain an antibody against the amino acid moiety stored between the microorganisms. Therefore, by obtaining a mixture of monoclonal antibodies by a known method by the antigen obtained by the present method, the antibody produced by the antibody which reacts with the microorganism can be selected to obtain the desired antibody. c) or use an unknown method as the Ribosomal Protein L7/L12. The amino acid of the protein is formulated to prepare the known Ribosomal Protein L7/L12 protein amino acid in the column. A synthetic peptide of 5 to 30 amino acids co-provided between microorganisms, and a Polyclonal antibody is prepared by a known method for the peptides thereof. The paper is applicable to the Chinese National Standard (CNS) A4 specification (210X297 mm) - 11 - (Please read the note on the back to write this page) Loading · Booking 1282368 Ministry of Economic Affairs Intellectual Property Bureau employee consumption cooperative printed A7 _ B7 __ five, invention description (9) monoclonal antibodies. The highly purified Ribosomal Protem (ribosomal) L7/L12 protein can be obtained by affinity chromatography using the antibody to purify the desired microbial cell disruption solution. When the protein essence system is insufficient, the ion exchange chromatography method of the purification method, the hydrophobic chromatography method, the gel filtration method, and the like are purified, and the Ribosomal Protein is determined by the wet mapping of the prepared antibody. After the L7/L1 2 protein trade in Shibuya, the refined protein was obtained. The desired antibody can be obtained by obtaining a mixture of a purified Ribosomal Protem (ribosomal) L7/L 1 2 protein antigen by a known method and selecting a specific reaction mixture of the target microorganism. The specific antibodies of the present invention in the various microorganisms obtained by the methods a), b) and c) can provide various diagnostic reagents and k i t specific to the microorganisms of interest by using various immunological assays. For example, the antibody has a coagulation reaction for absorbing the antibody on the particles of the polystyrene latex of the known assay, and an ELISA method using a well-known technique in a microdenier plate, that is, immunochromatography, colored particles or coloring energy All of the known immunoassays, such as sandwich assays used for magnetic microparticles coated with an antibody or a phosphor coated with an antibody which is simultaneously collected by the antibody, can be used. The microbial diagnostic method using an antibody refers to an ELISA method in which a coagulation reaction for adsorbing the antibody on a particle of a polystyrene latex is carried out in a microfibrillary plate, and an existing immunochromatographic method, colored particles or having a coloring property The particle, or the enzyme, or the antibody to which the fluorescent substance is labeled, is a diagnostic method for all known immunoassay methods such as sandwich assay used for collecting magnetic microparticles coated with an antibody. This paper scale is applicable to China National Standard (CNS) A4 specification (210X297 mm) (please read the note on the back first ¥ π write this page) Loading · Book 1282368 A7 B7 Ministry of Economic Affairs Intellectual Property Bureau employee consumption cooperative printing 5, invention (10) In addition, as a microbiological diagnostic method useful for the use of antibodies, the optical film formed by polyfluorene, yttrium nitride, etc., as disclosed in Japanese Patent Publication No. 7-5509, The optical assay (〇IA, Optical Immunoassay) detected by the principle of light interference for performing an antibody reaction can be effectively used as a diagnostic method for high sensitivity. In addition, in the method of detecting, the method for extracting the intracellular labeling antigen by the necessary microorganisms is to use Triton X-100, Tween-20 as a starting point to use various surfactants by the method of extracting the reagent, using an appropriate protease, etc. The enzyme treatment method of the enzyme is used by a method of pulverizing a known cell structure which is started by microbial cell disruption by a physical method. It is preferable to set the optimum extraction conditions for each microorganism according to different reagents by combining surfactants, etc. In the present invention, the reagent kit for detecting microorganisms using an antibody is equivalent to the test using the detection method. The drug k 1 t. The Ribosomal Protein of the Pneumoniae Pneumoniae L7/L12 protein is shown in the list and the DNA is shown in the list. Therefore, this microorganism can make Ribosomal Protein L7/L12 The amino acid of the protein is listed in the list to compare the similar microbial proteins described in the "cross-type". After the synthesis of a part of the peptide having the same low identity, the polyclonal or monoclonal antibody can be omitted for the specificity of the microorganism. In particular, in the case of a polyclonal antibody, an animal which has been purified by a protein A column or the like is anti-blood, and after obtaining an I g G score, it is preferable to carry out affinity purification by using a synthetic peptide used for animal immunization. This paper scale applies to China National Standard (CNS) A4 specification (210X297 mm) =13- one (please read the note on the back--"I π write this page" Order 1282368 A7 B7 V. Invention description (11 Further, a DNA heuristic agent is prepared from the DNA of the Ribosomal Protein L7/L12 protein of the microorganism based on the arrangement of the N-terminus and the C-terminus. Using the identity of this P C R heuristic, the D N A section was propagated by the P C R method using the granule D N A. After the extraction, the Ribosomal Protein L7/L12 gene of P. pneumoniae was obtained by the usual method. The total length of the Rib osomal Protein L7/L12 gene of 71/M. pneumoniae can be known by analyzing the information of the D N A of these slices. The Ribosomal Protein L7/L12 gene line of the obtained Pneumoniae bacterium is composed of GST and the like, and after the fusion protein gene is formed, the cytoplasmic body is appropriately used to form a discovery medium, and after the shape conversion of the coliform, etc., The protein was found in large quantities. After culturing the form-transformed Escherichia coli in an appropriate amount, the purified cell suspension is obtained by using the affinity column of G S T to obtain the molten protein of Ribosomal Protein L7/L12 protein and GST of M. pneumoniae. After directly cutting the protein, or cutting a part of GST, a complex mixture of clones is established by a known method as an antigenic protein, and it is selected to show that the fungus of the pneumocystis or the fungus is broken or the pneumonia After the Ribosomal Protein L7/L 1 2 protein-specific antibody, the specificity of the target m ο η 〇c 1 ο na 1 antibody can be obtained. The antibody prepared based on the present invention can be adsorbed by the adsorption reaction of the antibody on the polystyrene latex particles of the known measurement method, and the ELISA method of the known technique is carried out in the microdenier plate, and the existing immunochromatographic method is used for coloring. Law or particles with chromogenic energy, or enzymes or phosphors are labeled to the Chinese National Standard (CNS) A4 specification (210X297 mm) _ 14 _ (please read the note on the back first) \ Kenting Ministry of Economic Affairs Intellectual Property Bureau Staff Consumer Cooperative Printed 1282368 A7 B7 V. Inventive Note (12) 2 This antibody is also coated with all known immunoassays such as the clamp® test used for collecting magnetic particles such as antibodies. The antibody produced based on the present invention is used in all immunoassays, and the antigen protein is used as a collection antibody function which can be collected in a solid phase or a liquid phase, and the peroxidase and the base are modified by a known method. After the enzyme such as phospholipase is used as an enzyme-labeled antibody, it can also be used as a test for the antibody b. [Best form of invention] The following example is Specifically, the present invention is not limited to any range. [Example 1] Cloning of the Ribosomal Protein L7/L12 gene of P. oxysporum was isolated from A TCC 15531, ATCC, and purchased. ) After adding appropriate amount of bacteria to PPL〇 agar medium (DIFC〇, 〇4 1 2 - 1 7 3) added to Mycoplasma supplement (DIFC 〇, 0 836 — 68-9), at a constant temperature of C〇2, 3 7 Incubate for 5 hours at a temperature of 5 % C〇2. Let the fern clone finally be suspended in TE buffer at a rate of 5 X 1 0 9 CFU / m. (made by Wako Pure Chemical Industries, Ltd.) 1. 5 4 After the suspension was transferred to a microcentrifuge tube, it was centrifuged at 10 00 rpm for 2 minutes to remove the supernatant. The precipitate was resuspended in 5 6 7 J of TE buffer. Add 3 0 // 1 10% SDS and 3 // 1 20 This paper scale applies to China National Standard (CNS) A4 Regulation Fu 210X297 mm 1 -15 "~~1 (Please read the note on the back first π - Install - I jc write This page) Ministry of Economic Affairs, Intellectual Property Bureau, Staff Consumer Cooperatives, Printing 1282368 A7 B7 Ministry of Economic Affairs, Intellectual Property Bureau The consumer consumption cooperative printed five, the invention (η) mg / m £ proteinasek solution, after mixed mixing, at 1 7 °c for 1 hour constant temperature. At 5 6 ° C, the suspension was kept at constant temperature for 1 hour. . Further, 80 0 / 1 1 〇 % hexadecyltrimethylammonium bromide / 〇 · 7 Μ N a C 1 solution was added and mixed thoroughly, and then kept at 65 ° C for 10 minutes. Add 700/1 of the same volume of 24:1 chloroform-isoamyl alcohol mixture and mix well. The solution was centrifuged at 1 2,0 0 〇 r p m for 5 minutes at 4 °C, and the aqueous layer was transferred to a new microcentrifuge tube. A sufficient precipitation of D N A was formed by adding a tube of 0.6 ppm of isopropanol to the vibration. After inhaling the white D N A precipitate with a glass rod, it was transferred to another microcentrifuge tube containing lm £ 70% ethanol (cooled at 20 ° C). Subsequently, the tube was centrifuged at 10,0 0 0 r p m for 5 minutes, and the supernatant was carefully removed. After adding 1 70% ethanol, the mixture was further centrifuged for 5 minutes. The D N A solution was obtained by removing the precipitate of the supernatant solution and dissolving it in 1 0 0 //1 T E buffer. The concentration of this genomic D N A solution was quantified according to Moleculan Cloning. Alaboratory manual, 1989, Eds. Shambrook, J., Fritsch, E.F. and Maniatis, T., Cold Spring Barbor Laboratory Press E5, spectrophotometric Determination of the Amount of DNAorRNA. In this genomic D N A, 10 μg was used to carry out p C R (polymerase chain reaction ). P C R is a Taq plymaerase (manufactured by Takara Co., Ltd., code R 0 0 1 A). Add 5 Μ 1 added enzyme buffer to the enzyme, 4 // 1 add enzyme NTP mix (please read the note on the back first. Install - write this page) Set _ This paper scale applies to Chinese national standard (CNS A4 size (210X297 mm) _ 16 - 1282368 Α7 Β7 V. Description of invention (14) and each oligonucleotide of 2 0 0 pm ο 1 (with list number: 3 and 4). Add purified water to make the total capacity 5 0 // 1. This mixture was subjected to TakaRa PCR Thermal Cycler 480 at 9 5 ° C for 1 minute, 50 ° C for 2 minutes, 7 2 ° C for 3 minutes, and then at 95 ° C for 1 minute, 6 2 ° cycle at 0 °C for 2 minutes, 7 2 t for 3 minutes. After a portion of this P C R product was used, electrophoresis was carried out in a 1.9 % agarose gel. After dyeing with ethidium bromide (manufactured by Zeen, Japan), after observation under ultraviolet light, c D N A having an increase of about 400 b was determined. After cutting with restriction enzymes B a m Η I and X h ο I, staining was carried out by electrophoresis with ethidium bromide in a 1.5% agarose gel. A band of about 400 b P was cut from the gel. This was purified by Sup re col (manufactured by Takara Co., Ltd.), and then inserted into a general vehicle P GEX - 6 P - 1 (manufactured by Pharmacia Co., Ltd.). The same kind of media system allows the target gene slicing device to appropriately restrict the enzyme cell body, and can have a function of finding a vehicle function as a molecular molecule of the fusion protein of G S T protein. Specifically, the molar ratio of the vehicle P GEX - 6 P - 1 to the above D N A was 1:3, and then D N A was placed in the vehicle under a T 4 D N A ligase (manufactured by In vitro). The DNA-loaded vehicle p GEX — 6 P — 1 is in the oneshot c 〇mpetentce 11 of Escherichia coli and then inoculated into the ampicillin containing 50 // g/m £ (Σ) A semi-solid culture plate LBL-agar culture solution (manufactured by Takara Co., Ltd.). At 3 7. (: After the culture plate is kept at a constant temperature for 12 hours, the growth bacteria are randomly selected. The paper size is applicable to the Chinese National Standard (CNS) A4 specification (210X297 mm) · " ' (Please read the note on the back first) Page) . Loading · , 1Τ Ministry of Economic Affairs Intellectual Property Bureau Staff Consumer Cooperative Printed 1282368 A7 B7 V. Invention Description (15 ) Inoculated in L-fermentation culture solution containing ampicillin at the same concentration. 3 7 °C After shaking for a few hours, after cultivating the bacteria, use the Wizard Mimprep and separate the cytoplasmic genetics according to the instructions. The cytoplasmic genetic system is cut off by the restriction enzyme B am Η I / X h ◦ I. The cut is about 370 bp. After the DNA, the insertion of the PCR product is determined. The salt-based arrangement of the inserted DNA is determined by the use of the fluorescent sequence of the inserted DNA fragment of the clone. The determination is performed by using the fluorescent sequence of Applied Biosystems. This was carried out by using PRISM, Ready Reaction Dye Terminator Cycle Sequencing Kit (Applied Biosystems). First, adding 9 · 5 // 1 reaction to 0 · 5 microtubes , 4 · 0# 1 of 0. 8pmo 1/// 1 of T7 Promoter (Gibco BRL), and 6.5//1 of 0.16//g/ # 1 model DNA for mixing. With 2 layers of 1 0 0 / After the oil coating of /1 is carried out, 25 5 PCR amplification treatment is carried out, wherein 1 cycle is treated at 9 6 ° C for 30 seconds, at 5 5 ° C for 15 seconds, and at 60 ° C for treatment 4 In the minute, the product was maintained at 5 ° C for 5 minutes. After the reaction was completed, 80 0 / 1 sterile purified water was added and stirred. The product was centrifuged and extracted with phenol chloroform. Layer 3 times. Add 1 〇// 1 3 Μ sodium acetate ρ Η 5 · 2 and .3 0 0 // 1 ethanol to the 1 〇〇// 1 water layer and stir. After that, 14,0 The mixture was centrifuged at room temperature for 15 minutes at room temperature to recover the precipitate. The precipitate was washed with 75% ethanol, and then allowed to stand under vacuum for 2 minutes, then dried, and then sampled for Sequence. Equence (This paper scale applies to China National Standard (CNS) A4 specification (210X297 mm) (please read the note on the back of the book π π write this page) Ministry of Economic Affairs Intellectual Property Office staff The fee cooperative printed 1282368 A7 B7 Ministry of Economic Affairs Intellectual Property Bureau employee consumption cooperative printing 5, invention description (16) Procedure) The sampling system is dissolved in the EDTA containing 4 // 1 10 m Μ after the methamine 90 ° C Denaturation was carried out for 2 minutes. This person is cooled in ice and supplied to S e q u e n c e ο. Two of the five clones are randomly selected to have the same identity as the detection using Ρ C R . Further, the D N A arrangement identical to the Ribosomal Protein L7/L12 protein gene is extremely obvious. The full-salt arrangement of the structural part of the structure and the corresponding amino acid arrangement show the matching list number: 1 and 2. This gene section was clearly encoded as a gene for the Ribosomal Protein L7/L12 protein of M. pneumoniae. [Example 2] A large amount of discovery and purification in Escherichia coli of the Ribosomal Protein L7/L12 gene of M. pneumoniae. The coliforms in which the vehicle was found were taken in 50 B of L B medium, and cultured at 37 ° C for 1 day and 1 night. 5004 2 times the concentration of Y T medium was heated at 37 ° C for 1 hour. The 504 coliform solution cultured for 1 night was placed in the above-mentioned medium of 500. After 1 hour, 5 5 OmM isopropyl/3-D (-) monothiogalactopyranoside (I P T G ) was introduced and cultured for 4 hours. After recovering the product, it was transferred to a centrifuge tube of 250 ml, and centrifuged at 70 〇 〇 r p m for 10 minutes. After discarding the supernatant, it was dissolved in 2 5 lysis buffer containing 50 m Μ r 1 s buffer ρ Η 7.4, 2 5 % Sucrose. Add 1 · 2 5 10% N P — 4〇, 125// 1,1M MgC 12, then move into the plastic tube. Under cold weather, this paper scale applies to China National Standard (CNS) A4 specification (210X 297 mm)-19 - (Please read the note on the back first]||||fill this page. Install. Order 1282368 Ministry of Economics Intellectual Property Bureau employee consumption cooperative printed A7 B7 V. Invention description (17) 5 times 1 minute super first wave processing. Thereafter, the mixture was centrifuged at 1 2,0 0 0 r p m ' for 15 minutes to recover the supernatant. The supernatant was then adsorbed by P B S in the adjusted glutathione agarose column. The column was washed in a 2 bed volume by a washing solution containing 20 m Μ T r i s buffer ρ Η 7 · 4, 4 . 2 m M MgC 12 ' ImM dithio thretol ( D T T ). Dissolution treatment was carried out in a buffer containing 5 m M glutathione 50 m Μ T r i s buffer ρ Η 9 · 6. After the pigment binding method (B r a d f or d method; B i 〇 R a d C 0 ·) determines the protein content of the dissolved fraction, the main drawing score is obtained. The purified G S T melted Ribosomal Protein (ribosomal) L7/L12 protein purity is determined by the electrophoresis method to ensure the purity of about 75% of the immune source is sufficient. [Example 3] Monoclonal antibody against Ribosomal Protein L7/L12 protein of Mycoplasma pneumoniae was firstly immunized against mice. The GST fusion of Ribosomal Protein (ribosomes) of 1 0 0 // g of M. pneumoniae The L7/L12 protein antigen was dissolved in 200//1 PBS and then mixed with 200//1 of Freund's complete adjuvant. After emulsification, 200 A 1 was injected into the abdominal cavity. The same emulsified antigen was injected into the abdominal cavity at 2 weeks, 4 weeks, and 6 weeks later. Further, at 10 weeks, a 2-fold concentration of the emulsified antigen was injected into the abdominal cavity after 14 weeks. Three days after the end of the final immunization, the spleen was removed and cell fusion was performed. Take 2 X 107 bone marrow when removing 10 8 mouse spleen cells aseptically (please read the notes on the back _; write this page) - Pack - Set the paper size to the Chinese National Standard (CNS) A4 specifications (210 X 297 mm)-20 1282368 A7 B7 Ministry of Economic Affairs Intellectual Property Bureau Staff Consumer Cooperative Printed V. Inventive Note (ΐδ) After swollen cells are placed in a glass tube and mixed thoroughly, 1 500 edge in P in After centrifugation for 5 minutes, discard. After discarding the clear solution, mix the cells thoroughly. The myeloid cell line used for cell fusion was cultured with R Ρ Μ I 1 60 40 medium containing 10% of bovine fetal blood sputum using NS-1 cell line, and then fused with cells 2 weeks before cell fusion. .13 mM azoguanine, 〇.5//g/m £2MC — 210, 10% fetal calf RPMI 1640 medium was cultured for 1 week, and more than 10% bovine fetal blood sputum R Ρ Μ I 1 6 4 0 medium was cultured for 1 week. 50 liters of R Ρ Μ I 1 6 4 0 culture solution maintained at 37 ° C was added to the mixed cell sample, and centrifugation was performed at 1,500 rpm. After the supernatant was removed, 1% of the polyethylene glycol maintained at 3 7 ° C was added, and the mixture was stirred for 1 minute. Then, add 1 〇 2 of R Ρ Μ I 1 6 4 0 culture solution maintained at 3 7 ° C, and sterilize the suction tube to attract the mixture for about 5 minutes, and vigorously stir it after discharge. After centrifugation at 1,0 0 0 r p m for 5 minutes, after removing the supernatant, a 30 m £H A T culture solution was added to bring the cell concentration to 5 X 1 0 6 / m £. After the mixture was stirred until homogeneous, each was poured into a 96-well culture plate with 〇.1, and cultured at 37 ° C under a carbon dioxide atmosphere of 7%. After the addition of the H A T medium on the first day, the first week, and the second week, the desired antibody cells were produced by the E L I S A method; Divide 1 0 0 // 1 to 1 0 // g / concentration separately and dissolve in 0 · 0 5 % containing azide soda Ρ BS GST solution _ (please read the back note first) Page) Loading and binding - The paper size is applicable to China National Standard (CNS) A4 specification (210X297 mm) 21 - 1282368 A7 B7 V. Invention description (19)

The Ribosomal Protein L7/L12 protein and G S T protein were injected into a 96-well culture plate and adsorbed for 1 night at 4 °C. After the supernatant was removed, a solution of 2 0 0 // 1 1 % bovine blood albumin (in P B S) was added, and the reaction was terminated at room temperature for 1 hour. After removing the supernatant, the product was washed with a washing solution (0 · 0 2 % Tween 20, P B S ). A culture solution of 1 〇 2 fused cells was added thereto, and the reaction was carried out for 2 hours at room temperature. After removing the supernatant, the precipitate is washed with a washing solution. Further, 10 0 / 1 of a peroxidase-labeled peroxidase of g o a t a n t i - m o u s e I g G antibody solution was added, and the reaction was carried out for 1 hour at room temperature. After removing the supernatant liquid, the product was washed again with a washing solution. A 100//1 TMB solution (manufactured by KPL Co., Ltd.) was separately added, and the mixture was allowed to react at room temperature for 20 minutes. After the coloring was added to .1 0 0 // 1 1 N sulfuric acid, the reaction was stopped, and the absorbance at 450 n m was measured. As a result, it was judged that the Ribosomal Protein L7/L12 protein which is found to be unreactive in the G S T protein and reacted with the G S T melted Ribosomal Protein L7/L12 protein is an antibody-containing antibody. Therefore, it was cultured in a 24 μm plastic plate in which the cells in the positive Walle were separately recovered in H A Y medium. The culture medium was cultured in a number of cells of about 20 cells/min. After dilution in T medium, 50 μl of the thymocytes of 6-week-old mice suspended in Η Τ medium were cultured in 1 6 cells in 96 cells. Mix in the plate. After mixing, culture at 2 7 °C for 7 weeks under 7 % C 0 2 conditions. This paper scale applies to Chinese National Standard (CNS) A4 size (210 X 297 mm) _ 22 - (Please read the back of the note first) π • Install l· — 3⁄4舄 Page) Ministry of Economic Affairs Intellectual Property Bureau Staff Consumer Cooperative Printed 1282368 Ministry of Economic Affairs Intellectual Property Bureau Staff Consumer Cooperative Printed A7 B7 V. Invention Description (20) 同样 Same as the above ELISA method After culturing the antibody activity in the supernatant, the positive cells reactive with Ribosomal Protein L7/L12 protein were recovered. Perform the same dilution test and repeat the cloinng operation to obtain a mixture of Μ P R B — 1 to 5 for 5 clones. [Example 4] Selection of Monoclonal Antibody for Ribosomal Protein L7/L12 Protein of M. pneumoniae The positive mixed cells obtained as described above were used for production recovery of monoclonal antibodies according to the predetermined method. Specifically, the subcultured cells were intraperitoneally injected with 0·5 mC octadecane in the peritoneal cavity of Blab/C mice by using R Ρ Μ I 1 6 4 0 medium (plus 10% FCS). Inject 5 x 106 (in PBS). After recovering the ascites for 3 weeks, the supernatant was obtained by centrifugation. After the antibody-containing solution was absorbed in a Protein A column (5, manufactured by Pharmacia), it was washed with 3 times the amount of P B S . Further, the solution was eluted with citric acid buffer at ρ Η 3 . After the antibody was collected, the monoclonal antibody produced was obtained by each of the conjugates. Using the monoclonal antibodies of the above 5 conjugates, § was determined by the E L I S A method. A sandwich assay is used in the evaluation of monoclonal antibodies. The monoclonal antibody is used as a detection antibody when it binds to peroxidase. The enzyme identification system uses horseradish peroxidase, and (Sigma gled VI) is combined with this paper scale to apply Chinese National Standard (CNS) A4 specification (210X297 mm). )-23 - (Please read the note on the back 7" Loading and setting 1282368 A7 B7 V. Invention description (21) After using the reagent S-acetamidine thioacetic acid N-hydroxysuccinamine, by biological Chemical analysis 132 (1983), 68-73. The ELISA method was carried out in a 96-well culture plate diluted with 1 0 0 // 1 at a concentration of 10 // g / respectively. After the Pneumococcal polyclonal antibody (Biodesign, rabbit), the adsorption was carried out for 1 night at 4 times. After removing the supernatant, add 2 0 0 # 1 1 % bovine blood albumin solution (in PBS), room temperature After the reaction was completed for 1 hour, the supernatant was removed, and the supernatant was washed with a washing solution (0 · 0 2 % Tween 2 〇, PBS), and each microbial culture solution was added thereto at a concentration of 3%. After Trhon X-100, at room temperature, add extraction for 5 minutes. 1 0 0 // 1 antigen solution, react at room temperature for 2 hours. After removing the supernatant solution, wash the product again with a washing solution. Add 10 0 / 1 concentration 5 A g / rn £ peroxidation After identifying the anti-Ribosomal Protein L7/L12 protein antibody solution, the enzyme was reacted for 1 hour at room temperature. After removing the supernatant solution, the product was washed again with a washing solution. Each was added to 1 0 0 // 1 Τ Μ B solution (KPL company), the mixture was reacted at room temperature for 20 minutes. After adding 100//1 1 Ν sulfuric acid under coloring, the reaction was stopped. The absorbance at 450 nm was measured. When monoclonal antibody is used as an enzyme-labeled antibody, all strains of M. pneumoniae are tested at a sensitivity of 108 per sensitivities, and other influenza bacilli, pneumonia, pneumonia, and cerebral Microorganisms such as cocci are still not reactive at a concentration of 108 / for Ribosomal Protein L7/L12 (please read the note on the back. 3⁄4-- π write this page) Ding Economics Department Intellectual Property Bureau employee consumption cooperative printed this paper scale applicable to China Quasi (CNS) A4 size (210X297 mm) _ 24-- after five 1282368 A7 B7 described (22) using monoclonal antibody proteins invention, may be made with a specific determined Qing Chu logo pneumoniae bacteria of the paddle are reactive. This antibody is called A Μ Μ P - 1. Table 2 g represents the result of using A Μ Μ P - 1. This does not mention the results of using other antibodies shown by cross-reacting with other microorganisms. Table 2 Results of detection (1 06 cells/m£) pneumoniae (proteobacteria) + detection results (10 cells/m£) N.meningitides (meningococcal) - N. 1 actamice (lactose naphthyl) Bacteria - N.mucosa - N. sicca - H. influenzae - B. catarrharis - N. gonorrhoeae (Neisseria gonorrhoeae) ) - E.coli (E. coli) - K.pneumoniae (K. pneumoniae) - (+; positive, -; negative) Printed by the Ministry of Economic Affairs Intellectual Property Office Staff Consumer Cooperative [Example 5] Using Ribosomal Protein L7 /L12 Protein Immobilized Affinity Antibody Obtains Specific Reaction with Ribosomal Protein L7/L12 Protein Polyclonal Antibody The Pneumocystis Pneumoniae Paper Size obtained by the method of Example 1 is applicable to China National Standard (CNS) A4 Specification (210X297 mm) -25 - 1282368 A7 B7 Ministry of Economic Affairs Intellectual Property Office Staff Consumer Cooperative Printed V. Description of Invention (23) Ribosomal Protein L7/L12 Protein or Triton X-100 At Qing was on the cell as an antigen use it. About 1 · 2 m of physiological saline containing 1 〇 〇 // g antigen was simultaneously emulsified with 1 · 5 m of Freund's adjuvant. The rabbits were subcutaneously injected with an emulsifier in S P F Japanese white rabbits for immunization. After 5 to 6 immunizations every 2 weeks, the antibody price was determined. The determination of the antibody price is based on the E L I s A method. The Ribosomal Protein L7/L12 protein dissolved in 0. 05% sodium azide PBS was diluted to a concentration of 1 〇//g/2 to 1 0 0 // g After injecting into a 96-well culture plate, adsorption was carried out for 1 night. After the supernatant was removed, a 1% 0% bovine blood albumin solution (in P B S) was added, and the reaction was carried out at room temperature for 1 hour. After removing the supernatant, the product was washed with a washing solution (0 · 0 2 % Tween 20, P B S ). Add 1 〇 〇 // 1 to dilute the normal rabbit blood sputum and immunize the rabbit's anti-blood solution, and react at room temperature for 2 hours. After removing the supernatant liquid, the product is washed with a washing solution. Further, a peroxidase-labeled anti-rabbit I g G antibody solution having a concentration of 50//1 of 50//1 was added, and the mixture was reacted at room temperature for 1 hour. After removing the supernatant liquid, the product was washed again with a washing solution. Each of 1 0 0 // 1 〇 P D solution (manufactured by Sigma) was added, and the mixture was allowed to react at room temperature for 20 minutes. After the coloring, 1 0 0 // 1 1 N sulfuric acid was added and the reaction was stopped. The absorbance at 4 9 2 n m was measured. After confirming that the antibody price has risen, a large amount of blood is collected. After taking blood from the ear artery in a glass centrifuge tube, it was allowed to stand at 37 ° C for 1 hour, and then allowed to stand at 4 ° C for 1 night. Then centrifuge at 3000 rpm for 5 minutes (please read the note on the back of this page first) - Packing and binding - The paper size applies to China National Standard (CNS) A4 specification (210X297 mm) _ 26 - 1282368 A7 B7 V. Description of invention (24), recovery of sputum. Obtain anti-blood stasis and store at 4 °C. An affinity column for the Ribosomal Protein L7/L12 protein of the immobilized P. pyogenes was prepared. The column was activated by Hi Trap NHS (1, manufactured by Phamacia). Immediately after replacing the column with 1 m Μ H C 1 , the Ribosomal Protein L7/L12 protein P B S solution (1 m g /2) was added. After the column was allowed to stand for 30 minutes, the adhesion test was added, and then equilibrated at P B S. After the affinity column was immobilized using the Ribosomal Protein L7/L12 protein of M. pneumoniae, the anti-blood obtained by purifying the sputum of the cells treated with Triton X-100 of M. pneumoniae as an antigen was performed. Polyclonal antibody in sputum. After the anti-blood sputum was diluted 5 times with PBS, the Ribosomal Protein L7/L12 protein was immobilized by the P. oxysporum strain at a flow rate of 0 · 5 / mi η after passing through a filter of 〇······· Column. Thereafter, it was eluted from the column with P i Μ glycine buffer in P Η 2 · 1 and immediately neutralized with ρ Η 9.0 in 1 Μ T ris buffer, by the same antibody valence assay. The ELIS method is used to recover the antibody for the purpose of dissolution. The poly clonal anti-system thus obtained is evaluated by the 〇I A method contained in the special publication No. 7-5 5 95 5 5 . The refined anti-system is used as a collection antibody user of the 〇I A method. Further, as the antibody to be detected, the enzyme marker was used as the peroxidase using the AM C T-1 monoclonal antibody described in Example 4. The enzyme identification is based on the biochemical analysis of the use of wasabi peroxidase (Σ gled VI) combined with the test drug S-acetamidine thioacetate N-pyridyl succinimide (please read the notes on the back π • -- π Write this page) Customs Ministry of Economic Affairs Intellectual Property Bureau Staff Consumer Cooperatives Printed Paper Size Applicable to China National Standard (CNS) Α4 Specifications (210X297 mm) -27 - 1282368 A7 _ B7 _ V. Invention Description (25) The method described in 1 3 2 (1 9 8 3 ), 6 8 - 7 3 is carried out. In the 〇IA reaction, the purified polyclonal antibody containing 〇·〇5 % sodium azide PBS was diluted to 10° with 0.10 HEPES buffer ρΗ8·0 on each polyoxynide wafer. After the reaction was carried out at room temperature for 30 minutes, it was washed with distilled water, and coated with a coating liquid containing sucrose and alkali-treated casein. After adding the Triton X-100 at a concentration of 0.5% to each of the microbial culture solutions obtained in the above operation, after extracting at room temperature for 5 minutes, an antigen solution of 15 // 1 was added to the polyfluorene oxide wafer. At room temperature, the reaction was carried out for 10 minutes. Add 1 5 // 1 of 2 0 // g / peroxidase to identify monoclonal antibody and react for 1 〇 minutes. After washing with distilled water, a solution of 1 5 // 1 Τ Μ B (manufactured by K P L) was added, and the mixture was reacted at room temperature for 5 minutes. The product was washed with distilled water, and the cyan color produced by the enzyme reaction was visually observed. As a result, as shown in Table 3, after the purified polyclonal antibody A Ρ Μ Ρ -1 was used as the antibody to be collected, the sensitivity of 1 8 8 /j was clearly displayed to detect the pneumocystis pneumoniae and the inability to detect other microorganisms. Reactive. By immobilizing the affinity column of the Ribosomal Protein L7/L12 protein of P. falciparum, it was confirmed that the polyclonal antibody against P. pneumoniae was obtained. This paper scale applies to China National Standard (CNS) A4 specification (210x297 mm) (please read the note on the back: write this page) Ministry of Economic Affairs Intellectual Property Bureau Staff Consumer Cooperative Printed 1282368 A7 B7 V. Invention Description (26 Table Results (10 8 cells/mC ) M. pneumoniae (H. pneumoniae) + H. influenzae (E. coli) ATCC10211 - E.coli (E. coli) ATCC25922 - E.faecalis (fecal bacillus) ATCC19433 - K. Pneumoniae (K. pneumoniae) ATCC 1 3 8 8 3 - N. gonorrhoeae (Neisseria gonorrhoeae) IID821 - Nl act amice (N. lactis) ATCC 2 3 970 N. meningitides ATCC13090 - P. Aeruginosa (Pseudomonas aeruginosa) ATCC27853 - GroupB Streptococcus (Group B Streptococcus) ATCC12386 _ S.aureus (Streptococcus mutans) ATCC25923 - S.pneumoniae (Streptococcus pneumoniae) ATCC27336 Which S. pyogenes (Streptococcus pyogenes) ATCC19615 - (Please read the note on the back of this page first) - Installed and edited by the Ministry of Economic Affairs, Intellectual Property Bureau, employee consumption cooperative (+; positive, -; negative) [industrial availability] According to the present invention, when a microorganism is used, an antibody can be specifically detected for an intracellular molecule that is effectively retained, and not only a microorganism of a specific species can be specifically detected, but also all blood-type microorganisms which can detect good precision in the same type. Ribosomal protein, Ribosomal Protemi This paper scale applies to Chinese National Standard (CNS) A4 specification (210X297 mm) -29 - 1282368 A7 B7 V. Description of invention (27) Ribosomal L7/L12 protein antibody After the use of the antibody, the detection of P. pneumoniae with good precision can be performed. Further, when the reagent for detecting microorganisms k i t is used as a component constituting the antibody, it is possible to detect microorganisms having a wider and better precision. (Please read the note on the back: write this page) - Install ·

1T Ministry of Economic Affairs Intellectual Property Bureau Staff Consumer Cooperative Printed Paper Standards Applicable to China National Standards (CNS) A4 Specifications (210X297 mm) -30 - 1282368 A7 B7 Ministry of Economic Affairs Intellectual Property Bureau Staff Consumer Cooperatives Printing V. Inventions ( 28) SEQUENCE LISTING <110> ASAHI KASEI KABUSHIKI KAISHA <120> Detection of antibodies against M. pneumoniae <130>ASAHI-8 <150> JP 2000-062729 <151> 2000-01-31 <160 〉 4 <21〇> 1 <211> 369 <212> DNA <2 1 3> Mycoplasma pneumoniae <400> 1 atg gca aaa eta gat aaa aac caa tta att gaa teg ttg aag gaa atg 48 Met Ala Lys Leu Asp Lys Asn Gin Leu lie Glu Ser Leu Lys Glu Met 15 10 15 acc ate atg gaa ate gat gaat ate att ag get gta gaa gaa get ttt 96 Thr lie Met Glu lie Asp Glu lie lie Lys Ala Val Glu Glu Ala Phe 20 25 30 gga gta teg gca aca cct gta gta get get ggt get gtt ggl ggt aca 144 Gly Val Ser Ala Thr Pro Val Val Ala Ala Gly Ala Val Gly Gly Thr 35 40 ^ 45 caa gaa get get age gaa gtg act gtg Aaa gtt act ggt tac act gac 192 G In Glu Ala Ala Ser Glu Val Thr Val Lys Val Thr Gly Tyr Thr Asp 50 55 60 This paper scale applies to Chinese National Standard (CNS) A4 specification (21 OX297 mm)-31 - (Please read the note on the back: write This page) - installed · , 11 1282368 A7 B7 5 , invention description (29 ) aac get aaa tta get gig xta aag ett tac ege gaa att get ggt gtt 240

Asn Ala Lys Leu Ala Val Leu Lys Leu Tyr Arg Glu lie Ala Gly Val (Please read the note on the back first) 65 70 75 80 g:gt tta atg gaa get aaa act get gtg gaa aaa ett cct tgt gtt gtt 288

Gly Leu Met Glu Ala Lys Thr Ala Val Glu Lys Leu Pro Cys Val Val 85 90 95 aag caa gac ate aaa cct gaa gaa get gaa gaa ett aaa aag cgt ttc 336

Lys Gin Asp He Lys Pro Glu Glu Ala Glu Glu Leu Lys Lys Arg Phe 100 105 110 gtt gaa gtt gga gca act gtt gaa ate aaa taa 369

Val Glu Val Gly Ala Thr Val Glu He Lys 115 120

<210〉 2 <211> 122 <212> PRT Ministry of Economic Affairs Intellectual Property Bureau Staff Consumer Cooperative Print <2 1 3> Mycoplasma pneumoniae <400> 2 This paper scale applies to China National Standard (CNS) A4 specification (210X297 mm)-32 - Ministry of Economic Affairs Intellectual Property Bureau employee consumption cooperative printed 1282368 V. Invention description (30 Μβτ Ala Lys Leu Asp Lys Asn Gin Leu lie Glu Ser Leu Lys Glu Met 1 5 10 15

Thr He Met Glu lie Asp Glu lie lie Lys Ala Val Glu Glu Ala Pbe 20 25 30

Gly Val Ser Ala Thr Pro Val Val Ala Ala Gly Ala Val Gly Gly Thr 35 40 45

Gin Glu Ala Ala Ser Glu Val Thr Val Lys Val Thr Gly Tyr Thr Asp 50 55 60

Asn Ala Lys Leu Ala Val Leu Lys Leu Tyr Arg Glu II© Ala Gly Val 65 70 75 80

Gly Leu \lei Glu Ala Lys Thr Ala Val Glu Lys Leu Pro Cys Val Val 85 90 95

Lys Gin Asp lie Ly^ Pro Glu Glu Ala Glu Glu Leu Lys Ly$ Arg Phe 100 105 110

Val Glu Val Gly Ala Thr Val Glu lie Lys 115 120 <210〉 3

<211> 29 <212> DNA <213> Artificial Seq uence <220><223> The pcR primer DNA used for the Ribosomal Protein L7/L12 gene was obtained from Mycoplasma pneumoniae. <400〉 3 aatggatcca tggcaaaact agataaaaa 29 , This paper scale applies to Chinese National Standard (CNS) A4 specification (21 OX 297 mm)-33 -

1 -- I 1 - · 1282368 A7 B7 V. Description of invention (31 )

<210> 4 <211> 30 <212>DNA<2 1 3> Artificial Sequence <220><223> From Mycoplasma pneumoniae, the P C R primer DNA used for the Ribosomal Protein L7/L12 gene was obtained. <400〉 4

QA tgactcgagt tatttgatxt caacagttgc (please read the note on the back: write this page) - install -

, 1T line _ Ministry of Economic Affairs Intellectual Property Bureau employee consumption cooperative printing This paper scale applies China National Standard (CNS) A4 specification (210X297 mm) _ 34 -

Claims (1)

[282368 1 Announcement 4| L —^ VI. Application for patent scope Special Issue. Patent Application No. 90 1 0 1 940 Patent Application Amendment (please read the note on the back and then fill out this page) Amendment of June 7, 1995 1 · An antibody against Pneumonia Mycoplasma pneumoniae is a microbial Ribosomal Protein L7/L12 is a specific antibody that is characterized by the reaction with Rib osomal Protein L7/L12 of Mycoplasma pneumoniae' but with other Ribosomal Protein L7/L12 of the species or genus of microorganisms does not respond. 2. The antibody of claim 1, wherein the antibody is bound to an antibody to the enzyme. A method for detecting a microorganism of the genus Pneumocystis, which is characterized by the use of an antibody according to any one of claims 1 to 2. Ministry of Economic Affairs, Intellectual Property Bureau, Staff Consumer Cooperatives, Printing 4 · A detection method characterized by a) extracting ribonucleoprotein from microbes after contacting the sample with the solution, b) extracting the sample with The antibody of any one of claim 1 or 2, wherein the antibody is immobilized on a solid surface, and the antigen-antibody complex is formed between the ribonucleoprotein protein and the collected antibody, and c) The antibody according to any one of the first or the second aspect, wherein the antibody for detection is detected as a microorganism of the genus Pneumocystis in the test sample obtained by detecting the antigen-antibody complex. 5. The method of claim 4, wherein the antigen-antibody complex system is detected by a specific substrate of the enzyme. This paper scale applies to China National Standard (CNS) A4 specification (21〇X: 297 mm) -1 - 1282368 A8 B8 C8 D8 VI. Patent application scope 6 · Test for the detection of microorganisms of the genus Pneumocystis _ ^ (kit), which is characterized by the use of antibodies as in claim 1 or 2 of the patent application. 7. The method for producing an antibody according to the first aspect of the patent application, characterized in that the method is a part of the Ribosomal Protein L7/L12 protein of the microorganism belonging to the genus Pneumocystis obtained by genetic manipulation or purification and purification by microorganisms. Peptide, or synthetic peptide equivalent to its partial peptide as the source of immunity 0 li k— n 0—tnnn I — - n (Please read the notes on the back and fill out this page) Printed by the Intellectual Property Office of the Ministry of Economic Affairs, the Consumer Cooperatives. This paper scale applies to the Chinese National Standard (CNS) A4 specification (210X297 mm) -2 -