KR20020073192A - Antibody for Detecting Mycoplasma Pneumoniae - Google Patents

Abstract

The present invention provides a method for rapidly detecting microorganisms belonging to Mycoplasma pneumoniae at a particularly high sensitivity, a detection antibody used for detection, a reagent kit for detection, and a detection antibody used for detection. It provides a manufacturing method.

That is, the present invention is an antibody against ribosomal protein of a microorganism belonging to Mycoplasma pneumoniae, an antibody that specifically reacts with the microorganism, a method for detecting the microorganism in a sample using the antibody, and comprising the antibody The present invention relates to a reagent kit for detection. Ribosome proteins include ribosomal proteins L7 / L12 proteins, which are used to detect infection of microorganisms causing pneumonia.

Description

Antibody for Detecting Mycoplasma Pneumoniae}

Diagnosis of microbial infections is usually confirmed by detection of causative bacteria at the site of infection or the like, or by antibody detection against causative bacteria in serum and body fluids. In particular, this diagnosis is important in the sense that detection of the causative organism allows for rapid treatment of the patient.

Detection of infectious agent causative organisms is usually carried out by isolation culture of causative organisms, culture identification method for identifying them based on their physiological, biochemical or structural characteristics, and polymerase chain reaction (PCR) method. Or genetic diagnostic methods for amplifying by specific nucleic acid hybridization and detecting them, and immunological methods for detecting causative bacteria using specific reactions of antibodies with antigen markers of causative bacteria.

However, when culture identification or genetic diagnostics is used, a long time is required to obtain the result. Therefore, the diagnosis by the immunological method which can detect a causative bacterium with high sensitivity in a short time and leads to rapid and appropriate patient treatment is widely used.

Conventionally, combinations of various marker antigens and antibodies have been used for the detection of the causative agent of infectious diseases by immunological methods.

Mycoplasma pneumoniae is a common causative agent of pneumonia, bronchitis and pharyngitis. It is a small, fungal, Gram-negative bacterium that selectively invades the human body and causes disease. It is believed that about 15-20% of pneumonia that collectively develops in the general society and up to 50% of pneumonia in a limited population is caused by mycoplasma pneumoniae (Ragnar Norrby 1999).

These bacteria are microorganisms that are very fine and difficult to culture, and form small colonies in the rich medium. The growth of mycoplasma pneumoniae in nutrient-rich agar medium is very slow and requires at least 21 days to identify cells in the medium (Granato, Poe, et al. 1980). Microorganisms of several mycoplasma species originating in the human body cause the same biochemical reactions. On the other hand, the absence of cell walls makes observations under bright microscopes even more difficult (Knudson and MacLeod 1979). Therefore, the gram staining method, the culture method, or the like cannot be applied as a diagnostic method for quickly detecting as a pathogen.

DNA hybridization assay probes used in the gene sequence for mycoplasma pneumoniae detection include Hyman et al., Buck et al. And Bernet et al. (Hyman, Yogev et al. 1987; Bernet, Garret et al. 1993; Buck, 0'Hara et al. 1993). Probes used to detect mycoplasma pneumoniae ribosomal RNA (rRNA) include Tilton et al. (Tilton, Dias et al. 1980), Yogev et al. (Yogev, Halachmi et al. 1988), Gobel et al. (Gobel, Geiser et al. 1987). ), Zivin and Monahan (EP0 305145) and Gobeland and Stanbridge (EPO 250662). Kai et al. (Kai, Kamiya et al. 1987) and Jensen et al. (Jensen, Sondergard-Andersen et al. 1993) describe primers of 16S rRNA sequences of Mycoplasma pneumoniae. All probes described herein are designed for DNA of Mycoplasma pneumoniae or DNA of Mycoplasma pneumoniae and Mycoplasma genitalium . Since these probes are relatively high in sensitivity, they are captured by a technique that has become very recently available, such as a PCR method. The PCR method is very sensitive and can be performed in a shorter time than hybridization. However, there is often a problem of similar positivity that is positive for asymptomatic humans that are negative for cultured mycoplasma pneumoniae and for active humans after the disease has been cured.

Japanese Patent Laid-Open No. 63-298 describes an immunodetection method based on western blot using a monoclonal antibody against a membrane antigen protein of mycoplasma pneumoniae of about 43 kD (kiloDalton). However, the antibody and the detection method using the antibody have a problem of low specificity and insufficient for specific diagnosis of species against mycoplasma pneumoniae. There is also a problem with this method that it takes at least 5 hours for results to be produced (Madsen et al. 1988),

The present invention provides antibodies useful for the detection of microorganisms belonging to Mycoplasma pneumoniae , a microorganism causing common pneumonia, a method for detecting the microorganism, a reagent kit for detecting the microorganism, and a method for producing the antibody for detecting the microorganism. It is about.

The invention is important for the diagnosis of atypical pneumonia caused by the pharmaceutical industry, in particular mycoplasma pneumoniae, and is useful in the pharmaceutical industry.

The present invention is useful for detecting mycoplasma pneumoniae, which is a microorganism included in a specimen, for example, a throat swab, a tissue sample, a body fluid, an experimental solution, and a specimen collected from a culture.

The present invention has been made to solve the above problems. That is, an object of the present invention is to provide a method for rapidly detecting microorganisms belonging to Mycoplasma pneumoniae at a particularly high sensitivity, a detection antibody for use in the detection, and a reagent kit for detection. Moreover, an object of this invention is to provide the manufacturing method of the detection antibody used for the detection.

The present inventors have found a protein that preserves the same function in all microorganisms as a useful antigenic protein. Usually, it is expected that the structural changes of such proteins are very small. Surprisingly, however, antibodies against these proteins are specific to the microorganism or genus, and antibodies to these proteins have a variety that can be used for specific identification of the microbial species or genus, while at the same time all serotypes for the target microorganism. It has been found that can detect. More specifically, the protein is a useful protein that is completely identical in structure among the same species via a change in amino acid sequence and, in other cases, species specificity accompanied by a change in structure.

We focus on intracellular molecules, particularly ribosomal protein L7 / L12 protein, which are present as molecules of the same function in all microbial cells and whose amino acid structure differs from microorganism to microorganisms, in particular one of ribosomal proteins. It was. Ribosome Proteins L7 / L12 proteins are proteins having a molecular weight of about 13 kD and are known to exist as ribosomal proteins essential for protein synthesis. In particular, some microorganisms, including mycoplasma pneumoniae, have interpreted the entire amino acid sequence of the ribosomal protein L7 / L12 protein.

The inventors note that although these molecules are similar among microorganisms, in part they have their own structural moieties, and by using antibodies against the mycoplasma pneumoniae ribosomal protein L7 / L12 protein, It was found that specific to the bacterial species and at the same time detectable serotypes in all the same species.

The present inventors have found that an antibody specific for the protein of mycoplasma pneumoniae can be obtained, and that the antibody can be used for specific detection of mycoplasma pneumoniae, thereby completing the present invention.

Monoclonal antibodies specific for mycoplasma pneumoniae ribosomal protein L7 / L12 protein have been discovered and developed by the present invention. This antibody is novel and different from any conventionally known antibody and has the property of specifically reacting with the protein.

SEQ ID NOS: 1 and 2 in the sequence table are the DNA sequence of the mycoplasma pneumoniae ribosomal protein L7 / L12 gene and the corresponding amino acid sequence (NCBI database accession # NC 000912). In addition, the left and right ends of the amino acid sequence described in the sequence table are amino group terminal (hereinafter, N-terminal) and carboxyl terminal (hereinafter, C-terminal), respectively, and the left and right ends of the base sequence are 5 'terminal and 3' terminal, respectively. . Amino acids in the amino acid sequence of the cross match test are indicated by one letter abbreviation. In addition, "+" notation in a cross-match test means an amino acid which is a different amino acid, but has a flexible relationship in properties such as hydrophobicity, and the blank of "" indicates completely different amino acids including the property. In addition, a series of molecular biological experiments of genetic manipulation described in the present invention can be carried out by a method for describing a conventional experiment. As said normal test book, Molecular Cloning, A laboratory manual, Cold Spring Harbor Laboratory Press, Sambrook, J etc. (1989) is mentioned, for example.

In the present invention, "microorganism" refers to mycoplasma pneumoniae, and particularly refers to a microorganism having pathogenicity in the respiratory tract and having high diagnostic significance as a causative agent of mycoplasma infection. In the present invention, "an antibody that specifically reacts with a microorganism" refers to an antibody that specifically reacts with a microorganism or a genus, but an antibody that specifically reacts with a microbial species is particularly useful in the diagnosis of microbial infections.

In the present invention, the antibody refers to a polyclonal antibody or a monoclonal antibody, and can be prepared using the full length of the ribosomal protein L7 / L12 protein or a partial peptide thereof. The length of the peptide for preparing the antibody is not particularly limited, but for antibodies to ribosomal protein L7 / L12 protein, the length characterizing this protein is sufficient, preferably at least 5 amino acids, particularly preferably at least 8 amino acids Peptides can be used. The peptide or full-length protein is intact or crosslinked with carrier proteins such as keyhole-limpet hemocyanin (KLH) and bovine serum albumin (BSA), and then inoculated into the animal with an adjuvant, if necessary, to recover the serum ribosomal protein L7. Antisera comprising an antibody (polyclonal antibody) that recognizes / L12 protein can be obtained. The antibody can also be purified from antiserum. Examples of animals to be inoculated include sheep, horses, goats, rabbits, mice, and rats. Sheep, rabbits and the like are particularly preferable for producing polyclonal antibodies. It is also possible to obtain monoclonal antibodies by known methods for producing hybridoma cells, but in this case, mice are preferred.

Further, an amino acid sequence of at least 5 residues or more, preferably at least 8 residues, of the protein may be purified as a fusion protein with glutathione S-transferase (GST) or the like, or may be used as an antigen without purification.

To a recombinant antibody expressed in cells cultured using immunoglobulin genes isolated by various methods and gene cloning methods described in Antibodies (A laboratory manual, E. Harlow et al., Cold Spring Harbor Laboratory Press) It can also manufacture.

The antibody against ribosomal protein L7 / L12 that can be used as the marker antigen of the present invention can be obtained by the following method or other similar methods, but is not limited to these methods.

a) For microorganisms in which the gene sequence and amino acid sequence of the ribosomal protein L7 / L12 protein are known, peptide fragments are synthesized in a region having a low similarity with the amino acid sequence of the protein in other microorganisms, and the polyclonal as an immunogen. By producing an antibody or monoclonal antibody, a desired antibody can be obtained.

Further, the full length sequence of the gene can be obtained by using conventional gene manipulation methods such as gene amplification by a PCR method using probes of DNA sequences at both ends of the gene, and hybridization method using homologous partial sequences as a template probe. It can be acquired.

Subsequently, a fusion gene with another protein gene is constructed, E. coli and the like are inserted into the host by a gene introduction method known as a host, and then expressed in large quantities. The desired protein antigen can be obtained by purifying the expressed protein by the chemical conversion column method or the like. In this case, since the full-length protein of ribosomal protein L7 / L12 becomes an antigen, even if it acquires the antibody with respect to the amino acid part preserve | saved between microorganisms, it does not meet the objective of this invention. Therefore, with respect to the antigen obtained by this method, a hybridoma producing monoclonal antibody can be obtained by a known method, and a desired antibody can be obtained by selecting a clone producing an antibody that reacts only with the corresponding microorganism. Can be.

b) For microorganisms in which the amino acid sequence of ribosomal protein L7 / L12 protein is unknown, the amino acid sequence of ribosomal protein L7 / L12 protein is 50-60% homologous between species, and thus homologous to the amino acid sequence of the ribosome protein L7 / L12 protein. The protein gene can be easily obtained by using conventional genetic manipulation methods such as gene amplification of a specific sequence portion by PCR and hybridization method using homologous partial sequence as a template probe based on the sequence of the portion.

Subsequently, a fusion gene with another protein gene is constructed, the fusion gene is inserted into the host by a known gene introduction method using E. coli, etc., and expressed in large quantities. The desired protein antigen can be obtained by purifying the expressed protein by the chemical conversion column method or the like. In this case, since the full-length protein of ribosomal protein L7 / L12 becomes an antigen, even if it acquires the antibody with respect to the amino acid part preserve | saved between microorganisms, it does not meet the objective of this invention. Therefore, for the antigen obtained by this method, a hybridoma that produces a monoclonal antibody is obtained by a known method, and a target antibody can be obtained by selecting a clone that produces an antibody that reacts only with the corresponding microorganism. Can be.

c) or another method in which the amino acid sequence of the ribosomal protein L7 / L12 protein is unknown, wherein the amino acid sequence of the known ribosomal protein L7 / L12 protein corresponds to a consensus sequence portion that is conserved among microorganisms in the amino acid sequence of 5 to 30; Synthetic peptides of amino acids are prepared and polyclonal antibodies or monoclonal antibodies are prepared by known methods for the peptide sequences. Highly purified ribosomal protein L7 / L12 protein can be obtained by purifying the desired microbial cell lysate using affinity column chromatography using this antibody. If the degree of purification of the protein is insufficient, it is purified by a known purification method such as ion exchange chromatography, hydrophobic chromatography, gel filtration, and the like, and then ribosomal protein L7 / L12 protein by a method such as western blot using the prepared antibody. Elution fractions can be identified to obtain purified protein. Based on the obtained purified ribosomal protein L7 / L12 protein antigen, hybridomas can be obtained by a known method, and a desired antibody can be obtained by selecting a hybridoma that specifically reacts with a desired microorganism.

The antibodies of the present invention specific for various microorganisms obtained by the methods a), b) and c) can be used in various immunological assays to provide various diagnostic reagents and kits specific for the microorganism of interest. For example, the antibody is characterized by agglutination reaction in which the antibody is adsorbed onto polystyrene latex particles, which is a known measurement method, ELISA method, which is a known technique performed in a micro titer plate, conventional immunochromatography method, colored particles, or color developing ability. It can be used for all the known immunoassay methods, such as sandwich analysis using the particle which has, or the magnetic fine particle coat | covered with a capture antibody with the said antibody labeled with the enzyme or the fluorescent substance.

The microorganism diagnostic method using an antibody includes an aggregation reaction in which the antibody is adsorbed onto polystyrene latex particles, an ELISA method known in the art in a micro titer plate, a conventional immunochromatography method, colored particles, or particles having a color developing ability, Or a diagnostic method using all known immunoassay methods such as sandwich analysis using magnetic particles coated with a capture antibody together with the antibody labeled with an enzyme or phosphor.

In addition, as a useful microbial diagnostic method using an antibody, so-called optical immunity detected by optical interference principle by performing an antibody reaction on an optical thin film formed of silicon, silicon nitride or the like described in Japanese Patent Application Laid-Open No. 7-509565. Analysis (OIA, Optical Immunoassay) is useful as a high sensitivity diagnostic method.

In addition, as a method of extracting the intracellular marker antigen from the microorganisms required in the detection method, treatment with an extraction reagent using various surfactants including Triton X-100, Tween-20, and enzymes such as appropriate proteases are used. Known cell structure disruption methods are used, including disruption of microbial cells by an enzyme treatment method and a physical method. It is preferable to set the optimum extraction conditions by the reagent for each microorganism by combination of surfactants and the like.

In addition, the reagent kit for microorganism detection using the antibody in this invention is corresponded to the reagent kit for detection using the said detection method.

The amino acid sequence and DNA sequence of mycoplasma pneumoniae ribosomal protein L7 / L12 protein are shown in the sequence table. Thus, in the case of this microorganism, it is possible to compare the amino acid sequence of the ribosomal protein L7 / L12 protein with a homologous protein of a similar microorganism described as "cross match" in the sequence table. Synthesis of peptides of low homology segments, and the preparation of polyclonal or monoclonal antibodies against them, may omit the choice of having specificity for the microorganism.

In particular, in the case of polyclonal antibodies, it is preferable to obtain an IgG fraction by purifying antiserum of an immunized animal with a protein A column or the like, and further carrying out affinity purification with a synthetic peptide used for animal immunity.

In addition, PCR primers were prepared based on the N- and C-terminal sequences from the DNA sequence of the ribosomal protein L7 / L12 protein of the microorganism.

Using the homology of the PCR primers, DNA fragments can be amplified by the PCR method using genomic DNA, and the fragments of the Mycoplasma pneumoniae ribosomal protein L7 / L12 gene can be obtained by conventional methods. . The total length of mycoplasma pneumoniae ribosomal protein L7 / L12 gene can be determined by analyzing the DNA sequence information of these fragments.

The obtained mycoplasma pneumoniae ribosomal protein L7 / L12 gene, for example, constitutes a fusion protein gene such as GST and the like, constructs an expression vector using an appropriate expression plasmid, and then transforms E. coli and the like to transform the protein. Can be expressed in large quantities. A suitable amount of transformed E. coli is cultured, and the cell lysate is purified by an affinity column using GST to obtain a fusion protein of mycoplasma pneumoniae ribosomal protein L7 / L12 protein and GST.

After the protein is cut as it is or the GST portion is cleaved, a plurality of hybridoma clones are established by a method known as an antigen protein, and the mycoplasma pneumoniae antibody or cell lysate or the mycoplasma pneumoniae ribosomal protein L7 It is also possible to obtain the specific monoclonal antibody of interest by selecting the antibody which shows a specific reaction to / L12 protein.

Antibodies prepared on the basis of the present invention are agglutination reaction in which the antibody is adsorbed onto polystyrene latex particles, which are known measurement methods, ELISA method, which is a known technique performed in a micro titer plate, conventional immunochromatography method, and colored particles. Or any known immunoassay method, such as a sandwich assay using particles having a color developing ability or magnetic particles coated with a capture antibody together with the antibody labeled with an enzyme or phosphor.

In addition, the antibody produced based on the present invention can function as a so-called capture antibody that captures the antigenic protein in a solid or liquid phase in all immunoassay methods, and at the same time, enzymes such as peroxidase and alkaline phosphatase are known. Modified by the method, so-called enzyme-labeled antibody can also function as an antibody for detection.

The following example is for demonstrating this invention concretely, and does not limit the range at all about this invention.

<Example 1>

Cloning of Ribosome Protein L7 / L12 Gene from Mycoplasma Pneumoniae

Mycoplasma pneumoniae (ATCC15531, sold and purchased from ATCC) was placed on PPLO agar medium (DIFCO, 0412-17-3) to which Mycoplasma Supplement (DIFCO, 0836-68-9) was added. After incubating a suitable amount, the cells were cultured in a CO ₂ incubator for 5 hours under 37 ° C and 5% CO ₂ . The grown colonies were suspended in TE buffer (manufactured by Wako Pure Chemical Industries, Ltd.) to finally reach around 5 × 10 ⁹ CFU / ml. About 1.5 ml of this suspension was transferred to a microcentrifuge tube, centrifuged at 10,000 rpm for 2 minutes, and the supernatant was removed. The precipitate portion was resuspended in 567 μl TE buffer. In addition, 30 µl of 10% SDS and 3 µl of 20 mg / ml proteinase K solution were added thereto, mixed well, and incubated at 37 ° C for 1 hour. The suspension was incubated for another 1 hour at 56 ° C. Subsequently, 80 µl of 10% cetyltrimethylammonium bromide / 0.7 M NaCl solution was added and mixed well, followed by incubation at 65 ° C for 10 minutes. 700 mu l of the same volume of a 24: 1 chloroform-isoamyl alcohol mixture was added and stirred well.

The solution was centrifuged at 12,000 rpm for 5 minutes at 4 ° C. in a microcentrifuge and the aqueous layer fractions were transferred to a new microcentrifuge tube. To this was added 0.6 times of isopropanol and the tubes were shaken well to form a precipitate of DNA. The white DNA precipitate was floated on a glass rod and transferred to a separate microcentrifuge tube containing 1 ml of 70% ethanol (cooled to -20 ° C). The tube was then centrifuged at 10,000 rpm for 5 minutes and the supernatant was carefully removed. 1 ml of 70% ethanol was added and the mixture was further centrifuged for 5 minutes.

After removing the supernatant again, the precipitate was dissolved in 100 μl of TE buffer to obtain a DNA solution. The concentration of this genomic DNA solution is described in Molecular Cloning, A laboratory manual, 1989, Eds. Shambrook, J., Fritsch, E. F., and Maniatis, T., Cold Spring Harbor Laboratory Press E5, Spectrophotometric Determination of the Amount of DNA or RNA].

PCR (polymerase chain reaction) was performed using 10 ng of the genomic DNA. PCR was performed using Taq polymerase (manufactured by Takara Shuzo Co., Ltd., code R001A). 5 μl of the buffer attached to the enzyme, 4 μl of the dNTP mixture attached to the enzyme and 200 pmol of oligonucleotides (shown in SEQ ID NOS: 3 and 4) each were added to the enzyme. Purified water was added to bring the total volume to 50 μl.

The mixture was subjected to 5 cycles of 1 minute at 95 ° C, 2 minutes at 50 ° C, and 3 minutes at 72 ° C using TaKaRa PCR Thermal Cycler 480, followed by 1 minute at 95 ° C, 2 minutes at 60 ° C, and 3 at 72 ° C. 25 cycles were performed. Part of this PCR product was used for electrophoresis in a 1.5% agarose gel. After staining with ethidium bromide (manufactured by Nippon Jinsa), it was observed under ultraviolet light to confirm that about 400 bp of cDNA was amplified. After cleavage using restriction enzymes BamHI and XhoI, electrophoresis and ethidium bromide staining were performed in a 1.5% agarose gel. A band of about 400 bp was cut from the gel. This band was purified by Suprec01 (manufactured by Takara Shuzo Co., Ltd.) and inserted into pGEX-6P-1 (manufactured by Pharmacia), which is a general vector. This vector can function as an expression vector of the target molecule capable of expressing a fusion protein with a GST protein by inserting the desired gene fragment into an appropriate restriction enzyme site.

Specifically, the vector pGEX-6P-1 and the DNA were mixed so that the molar ratio was 1: 3, and the DNA was inserted into the vector with a T4 DNA ligase (Invitrogen). The vector pGEX-6P-1 into which DNA was inserted was introduced into E. coli one-shot compact cells by genetic method, followed by LB L-, a semi-solid culture plate containing 50 μg / ml of ampicillin (Sigma). Broth agar (Takara Shuzo Co., Ltd.) was inoculated. The plates were incubated at 37 ° C. for 12 hours, and the grown colonies were randomly selected and inoculated in L-broth cultures containing the same concentration of ampicillin. After culturing and incubating for 8 hours at 37 ° C, the plasmid was separated using Wizard Miniprep according to the attached instructions. Plasmids were digested with the restriction enzyme BamHI / XhoI. Insertion of the PCR product was confirmed by cleaving about 370 bp of DNA. The base sequence of the inserted DNA was determined using this clone.

Base sequence determination of the inserted DNA fragment was carried out using a fluorescent sequencer manufactured by Applied Biosystems.

The preparation of sequence samples was performed using PRISM, Ready Reaction Dye Terminator Cycle Sequencing Kit (manufactured by Applied Biosystems, Inc.). First, 9.5 μl of reaction solution, 4.0 μl of 0.8 pmol / μl T7 promoter primer (Gibco BRL), and 6.5 μl of 0.16 μg / μl template DNA were added to a 0.5 ml microtube and mixed. The mixture was coated with two layers of 100 μl mineral oil, followed by 25 cycles of PCR amplification treatment. Here, one cycle consists of a 30 second process at 96 degreeC, a 15 second process at 55 degreeC, and a 4 minute process at 60 degreeC. The product was held at 4 ° C. for 5 minutes. After the reaction was completed, 80 µl of sterile purified water was added and stirred. The product was centrifuged and the aqueous layer was extracted three times with phenol-chloroform mixture. 10 μl of 3 M sodium acetate pH 5.2 and 300 μl of ethanol were added to 100 μl of aqueous layer and stirred. Thereafter, the mixture was centrifuged at 14,000 rpm for 15 minutes at room temperature to recover the precipitate. The precipitate was washed with 75% ethanol, then left to stand in vacuo for 2 minutes and dried to obtain a sample for sequence. Sequence samples were dissolved in formamide containing 4 μl of 10 mM EDTA and denatured at 90 ° C. for 2 minutes. It was cooled in ice and used in sequence.

Two of the five randomly selected clones had sequence homology with the probes used for PCR. In addition, a DNA sequence consistent with the gene sequence of the ribosomal protein L7 / L12 protein was evident. The entire nucleotide sequence and corresponding amino acid sequence of the structural gene moiety were the sequences as shown in SEQ ID NOS: 1 and 2. This gene fragment certainly coded for the gene of Mycoplasma pneumoniae ribosomal protein L7 / L12 protein.

<Example 2>

Mass Expression and Purification of Ribosome Protein L7 / L12 Gene from Mycoplasma Pneumoniae in Escherichia Coli

E. coli with the expression vector was incubated overnight at 50 ml, 37 ° C in LB medium. 500 mL of 2-fold concentration of YT medium was heated at 37 ° C. for 1 hour. 50 ml of overnight E. coli solution was placed in the 500 ml medium described above. After 1 hour, 550 μl of 100 mM isopropyl β-D (−)-thiogalactopyranoside (IPTG) was introduced and incubated for 4 hours. The product was recovered, transferred to a 250 ml centrifuge tube and centrifuged at 7000 rpm for 10 minutes. The supernatant was removed and dissolved in 25 ml of Lysis buffer containing 25 mM sucrose, 50 mM Tris buffer pH 7.4. Also, 1.25 ml of 10% NP-40 and 125 µl of 1 M MgCl ₂ were added and transferred to a plastic tube. Ultrasonication was performed 5 times for 1 minute under ice cooling. Thereafter, the mixture was centrifuged at 12,000 rpm for 15 minutes to recover the supernatant.

Subsequently, the supernatant was adsorbed onto the glutathione agarose column adjusted with PBS. The column was then washed for 2 bed volumes with a wash containing 20 mM Tris buffer pH 7.4, 4.2 mM MgCl ₂ , 1 mM dithiothreitol (DTT). Elution was performed in 50 mM Tris buffer pH 9.6 containing 5 mM glutathione. The protein content of the eluted fraction was determined by the pigment binding method (Bradford method; BioRad Co.) to obtain the main fraction.

The purity of the obtained purified GST fusion ribosomal protein L7 / L12 protein was confirmed by electrophoresis, which was about 75%, ensuring sufficient purity as an immunogen.

<Example 3>

Preparation of Monoclonal Antibodies Against Mycoplasma Pneumoniae Ribosome Protein L7 / L12 Protein

First, for mouse immunity, 100 µg of Mycoplasma pneumoniae GST fusion ribosomal protein L7 / L12 protein antigen was dissolved in 200 µl PBS, and 200 µl of Freund's complete adjuvant was added and mixed. After emulsification, 200 μl was injected intraperitoneally. The same emulsified antigen was injected intraperitoneally after 2 weeks, 4 weeks and 6 weeks later. Two-fold concentrations of emulsified antigen were injected intraperitoneally after 10 and 14 weeks. Three days after the end of the final immunization, the spleens were removed and cell fused.

For 10 ⁸ splenocytes of aseptically extracted mice, 2 x 10 ⁷ myeloma cells were transferred to a glass tube, mixed well, centrifuged at 1500 rpm for 5 minutes to remove the supernatant, and then the cells were mixed well.

Myeloma cells used for cell fusion were cultured in RPMI1640 medium containing 10% fetal bovine serum using NS-1 cell lines, and 0.13 mM azaguanine, 0.5 μg / ml MC-210 from 2 weeks before cell fusion. And cultured for one week in RPMI1640 medium containing 10% fetal bovine serum, and then cultured for one week in RPMI1640 medium containing 10% fetal bovine serum.

50 mL of RPMI1640 culture medium maintained at 37 ° C. was added to the mixed cell sample and centrifuged at 1500 rpm. After removing the supernatant, 1 ml of 50% polyethylene glycol maintained at 37 ° C. was added and stirred for 1 minute. 10 ml of RPMI 1640 culture solution maintained at 37 ° C was added, and the mixed solution was agitated vigorously by suction and discharge for about 5 minutes with a sterilized pipette.

After removing the supernatant by centrifugation at 1,000 rpm for 5 minutes, 30 ml HAT culture was added so that the cell concentration was 5 × 10 ⁶ / ml. The mixture was stirred until uniform, and 0.1 ml each was poured into a 96-hole culture plate and incubated under 37 ° C. and 7% carbon dioxide gas atmosphere. 0.1 ml of HAT medium was added at 1 day, 1 week, and 2 weeks, respectively, and the cells producing the desired antibodies were screened by ELISA.

100 μl of a liquid diluted with GST fusion ribosomal protein L7 / L12 protein and GST protein, respectively, dissolved in PBS containing 0.05% of azide soda at a concentration of 10 μg / ml, was respectively separated into 96-well plates at 4 ° C. overnight. Adsorbed.

After removing the supernatant, 200 μl of a 1% bovine serum albumin solution (in PBS) was added and allowed to react for 1 hour at room temperature to block. After removing the supernatant, the product was washed with rinse (0.02% Tween20, PBS). 100 ml of the culture solution of the fusion cells was added thereto and allowed to react at room temperature for 2 hours. The supernatant was removed and the precipitate was washed with rinse. Subsequently, 100 µl of a goat anti-mouse IgG antibody solution labeled with a concentration of 50 ng / ml peroxidase was added and allowed to react at room temperature for 1 hour. The supernatant was removed and the product was washed again with rinse. 100 µl of TMB solution (manufactured by KPL) was added, and the mixture was allowed to react at room temperature for 20 minutes. After coloring, 100 µl of 1 N sulfuric acid was added to stop the reaction, and the absorbance at 450 nm was measured.

As a result, a positive well was found that responded only to the GST fusion ribosomal protein L7 / L12 protein but not to the GST protein, and it was found that an antibody against the ribosomal protein L7 / L12 protein was included.

Therefore, the cells in the positive wells were each recovered and cultured in HAT medium in a 24-hole plastic plate.

The cultured fusion medium was diluted in HT medium so that the cell number was about 20 cells / ml, and 50 μl was mixed in 10 ^6- week-old mouse cells suspended in HT medium and 96-well culture plates. After mixing, the cells were cultured at 37 ° C. for 2 weeks under the condition of 7% CO ₂ .

Antibody activity in the culture supernatant was similarly assayed by the ELISA method described above, and cells positive for reaction with ribosomal protein L7 / L12 protein were recovered. In addition, the same dilution assay and cloning procedure were repeated to obtain a total of 5 clones of hybridoma MPRB-1-5.

<Example 4>

Selection of Monoclonal Antibodies Detecting Mycoplasma Pneumoniae Ribosome Protein L7 / L12 Protein

The positive hybridoma cells obtained as described above were used to produce and recover monoclonal antibodies according to the conventional method.

Specifically, 5 × 10 ⁶ cells (in PBS) were injected into the abdominal cavity of Blab / C mice, which were injected intraperitoneally with 0.5 ml of pristane two weeks before the cells passaged using RPMI1640 medium (10% FCS insertion). It was. Three weeks later, ascites was collected and the centrifuged supernatant was obtained.

The solution containing the obtained antibody was taken up in a Protein A column (5 ml, made by Pharmacia) and washed with 3 times PBS. Then eluted with citric acid buffer pH 3. Antibody fractions were collected and monoclonal antibodies produced by each hybridoma were obtained. The 5 strains of hybridoma-derived monoclonal antibodies were used to evaluate by ELISA.

The sandwich assay was used for the evaluation of a monoclonal antibody. The prepared monoclonal antibody was used as a detection antibody by binding to peroxidase.

Enzymatic labeling was made with horseradish peroxidase (Sigma grade VI) and for binding using reagent S-acetylthioacetic acid N-hydroxysuccinimide. Analytical Bio-chemistry 132 (1983), 68-73 ] According to the method described. In the ELISA reaction, 100 μl of liquid diluted with a concentration of 10 μg / ml of a commercially available anti-mycoplasma pneumoniae polyclonal antibody (Biodesign, Rabbit) was separated into 96-well plates. It was adsorbed overnight at 캜.

After removing the supernatant, 200 μl of a 1% bovine serum albumin solution (in PBS) was added and allowed to react for 1 hour at room temperature to block. After removing the supernatant, the product was washed with rinse (0.02% Tween20, PBS). To the culture medium of each microorganism, 100 µl of the antigen solution obtained by adding Triton X-100 in an amount of 0.3% in concentration and extracting at room temperature for 5 minutes was added and allowed to react at room temperature for 2 hours. The supernatant was removed and the product was washed again with rinse. Subsequently, 100 µl of a solution of peroxidase-labeled anti-ribosome protein L7 / L12 protein antibody having a concentration of 5 µg / ml was added and allowed to react at room temperature for 1 hour. The supernatant was removed and the product was washed again with rinse. 100 µl of TMB solution (manufactured by KPL) was added, and the mixture was allowed to react at room temperature for 20 minutes. After coloring, 100 µl of 1 N sulfuric acid was added to stop the reaction. Absorbance at 450 nm was measured.

When a monoclonal antibody derived from hybridoma MPRB-1 was used as an enzyme-labeled antibody, all strains of mycoplasma pneumoniae tested were detected at a sensitivity of 10 ⁶ / ml and other Haemophilus influenza influenzae , Klebsiella pneumoniae , Chlamydia pneumoniae , and Neisseria meningitides are not responsive at high concentrations of 10 ⁸ / ml. By using a monoclonal antibody against the ribosomal protein L7 / L12 protein, it was clearly confirmed that an antibody having specific reactivity to mycoplasma pneumoniae was obtained. This antibody was named AMMP-1. Table 2 shows only the results using AMMP-1. The results of using other antibodies that cross-react with separate microorganisms are not mentioned here.

Detection result (10 ⁶ cells / ml) M. Pneumoniae (M. pneumoniae) + Detection result (10 ⁸ cells / ml) yen. Meningitides (N. meningitides) - yen. N. lactamica - yen. Mucosa - yen. N. sicca - H. Influenza (H. influenzae) - ratio. Qatar Harris (B. catarrharis) - yen. Gonorrhoeae - this. E. coli - K. Pneumoniae (K. pneumoniae) - +; Positive,-; Negative

Example 5

Acquisition of polyclonal antibodies that specifically react with mycoplasma pneumoniae ribosomal protein L7 / L12 protein using ribosomal protein L7 / L12 protein immobilized affinity column

The supernatant of mycoplasma pneumoniae ribosomal protein L7 / L12 protein or Triton X-100 treated cells obtained by the method described in Example 1 was used as an antigen. About 1.2 ml of physiological saline containing 100 μg of antigen was emulsified with 1.5 ml of Freund's adjuvant. The rabbits were immunized by subcutaneous injection into SPF Japanese white rabbits. Antibodies were checked by immunization every 5 to 6 times for 2 weeks.

The antibody value was confirmed by ELISA method. 100 μl of liquid diluted with Mycoplasma pneumoniae ribosomal protein L7 / L12 protein dissolved in PBS containing 0.05% of azide soda at a concentration of 10 μg / ml was separated into 96-well plates at 4 ° C. Absorbed overnight. After removing the supernatant, 200 μl of a 1% bovine serum albumin solution (in PBS) was added and allowed to react for 1 hour at room temperature to block. After removing the supernatant, the product was washed with rinse (0.02% Tween20, PBS). 100 µl of the solution obtained by diluting normal rabbit serum and anti-serum of immunized rabbit was added and reacted for 2 hours at room temperature. The supernatant was removed and the product was washed again with rinse. Subsequently, 100 µl of a solution of peroxidase-labeled anti rabbit IgG antibody at a concentration of 50 ng / ml was added and allowed to react at room temperature for 1 hour. The supernatant was removed and the product was washed again with rinse. 100 [mu] l of OPD solution (manufactured by Sigma) was added, and the mixture was allowed to react at room temperature for 20 minutes. After coloring, 100 µl of 1 N sulfuric acid was added to stop the reaction. Absorbance at 492 nm was measured.

After confirming the antibody value rise, a large amount of blood was collected. Blood was collected from the ear artery into a glass centrifuge tube, left at 37 ° C. for 1 hour, and then left at 4 ° C. overnight. Thereafter, the supernatant was recovered by centrifugation at 3000 rpm for 5 minutes. The obtained antiserum was stored at 4 ° C.

An affinity column immobilized with mycoplasma pneumoniae ribosomal protein L7 / L12 protein was prepared. HiTrap NHS activation column (1 ml, manufactured by Pharmacia) was used. Immediately after replacing the column with 1 mM HCl, a PBS solution of ribosomal protein L7 / L12 protein (1 mg / ml) was added. The column was allowed to stand for 30 minutes and then added with blocking reagent to equilibrate with PBS.

Using this mycoplasma pneumoniae ribosomal protein L7 / L12 protein immobilization affinity column, a polyclonal antibody in antiserum obtained as a supernatant of mycoplasma pneumoniae triton X-100 treated cells as an antigen was purified. . The antiserum was diluted five-fold with PBS, passed through a 0.45 μm filter and adsorbed onto Mycoplasma pneumoniae ribosomal protein L7 / L12 protein immobilization column at a flow rate of 0.5 ml / min. Thereafter, the mixture was eluted from the column with 0.1 M glycine buffer pH 2.1, and immediately neutralized with 1 M Tris buffer pH 9.0, and then the eluted fraction of the target antibody was recovered by the same ELISA method as the antibody value measurement method.

The polyclonal antibody thus obtained was evaluated by the OIA method described in JP-A-7-509565.

The purified antibody was used as a capture antibody of OIA method. As the detection antibody, one obtained by enzyme-labeling the AMCT-1 monoclonal antibody described in Example 4 with peroxidase was used. Enzyme labeling was made with horseradish peroxidase (Sigma grade VI) and for binding using reagent S-acetylthioacetic acid N-hydroxysuccinimide. Analytical Bio-chemistry 132 (1983), 68-73 ] According to the method described.

In the OIA reaction, 50 μl of a liquid diluted with 0.1 M HEPES buffer pH 8.0 at a concentration of 10 μg / ml of purified polyclonal antibody in PBS containing 0.05% sodium azide was added to the silicon wafer, and room temperature After reacting for 30 minutes at, it was washed with distilled water, coated with a coating solution containing saccharose and alkali treated casein, and then used.

15 µl of the antigen solution obtained by adding Triton X-100 in an amount of 0.5% to the culture solution of each microorganism obtained by the above operation and extracting for 5 minutes at room temperature was added onto the silicon wafer, and allowed to react at room temperature for 10 minutes. Subsequently, 15 µl of 20 µg / ml peroxidase-labeled monoclonal antibody was added and allowed to react for 10 minutes. After washing with distilled water, 15 µl each of a TMB solution (manufactured by KPL) was added, and the mixture was allowed to react at room temperature for 5 minutes. The product was washed with distilled water and the blue color produced by the enzymatic reaction was visually observed.

As a result, as shown in Table 3, by using the purified polyclonal antibody APMP-1 as a capture antibody, mycoplasma pneumoniae can be detected at a sensitivity of 10 ⁸ / ml and the reactivity of other microorganisms was also detected. It turned out that you can't. In this way, it was confirmed that a polyclonal antibody that specifically reacts with mycoplasma pneumoniae was obtained by an affinity column immobilized with ribosomal protein L7 / L12 protein of mycoplasma pneumoniae.

Detection result (10 ⁸ cells / ml) M. Pneumoniae + H. influenza ATCC10211 - this. Coli ATCC25922 - this. Fakalis (E. faecalis) ATCC19433 - K. Pneumoniae ATCC13883 - yen. Gonorhoea IID821 - yen. Lactmica ATCC23970 - yen. Meningite ATCC13090 - blood. P. aeruginosa ATCC27853 - Group B Streptococcus ATCC12386 - s. Aureus (S. aureus) ATCC25923 - s. Pneumoniae ATCC27336 - s. S. pyogenes ATCC19615 - +; Positive,-; Negative

According to the present invention, it is possible not only to specifically detect microorganisms of a specific species by using antibodies to intracellular molecules functionally maintained during the evolution of microorganisms, but also to provide high precision for all serotype microorganisms in the same species. Can be detected.

As such antibodies, detection of mycoplasma pneumoniae can be performed with good accuracy by using antibodies against ribosomal proteins and ribosomal proteins L7 / L12 proteins of microorganisms.

In addition, by using a reagent kit for detecting microorganisms having such an antibody as a component, detection of microorganisms can be performed more generally and with good accuracy.

Cited References:

<Patent Documents>

* U.S. Patent No. 5,552,279, Welsburg, et al., September 3, 1996 "Nucleic Acid Probes for Detection of Mycoplasma Pneumoniae and Mycoplasma Genitarium"

* EPO 305145, Application No. 88307793.5 Zivin and Monaban. March 1, 1989. Methods and probes for detecting nucleic acid.

* EPO 250662, Application No.86304919,3 Gobel and Stanbridge. January 7, 1988, Detection of Mycoplasma by DNA hybridization.

* Japanese Patent Publication (Small) 63 (1988) -298

<Other patent documents>

Bernet, C., M. Garret, et al., (1993) "Detection of Mycoplasma Pneumoniae Using Polymerase Chain Reaction" J Clin Microbiol 31 (4), 1013-5

* Buck, G. E., L. C. 0'Hara, et al., (1993) "A Rapid and Sensitive Detection of Mycoplasma Pneumoniae in Similar Clinical Samples by DNA Propagation" J Clin Microbiol 31 (4), 1013-5

* Gobel, U, B., A. Geiser, et al., (1987) "oligonucleotide probes complementary to ribosomal RNA differentiation variable regions between mycoplasma species"

Granato, P. A., L. Poe, et al., (1980) "Report on the Use of Improved New York City Medium in Mycoplasma Pneumoniae Growth" Zh Mikrobiol Epidemiol Immunobiol (8), 50-3

* Hyman, H. C., D. Yogev, et al., (1987) "DNA Probes for the Detection and Identification of Mycoplasma Pneumoniae and Mycoplasma Genitarium"

Jensen, J. S., J. Sondergard Andersen, et al., (1993) "Detection of Mycoplasma Pneumoniae in Similar Clinical Samples Using Polymerase Chain Reactions" J Med Microbiol 38 (3), 166-70

Kai, M., S, Kamiya, et al., (1987) "Quick Detection of Mycoplasma Pneumoniae Using Polymerase Chain Reaction" J Gen Microbiol 133 (Pt7), 1969-74

* Knudson, DL and R. MacLeod (1979) "Mycoplasma pneumoniae and Mycoplasma salivarium-electron micrograph of colony growth in agar" Sci RepRes Inst Tohoku Univ [Med] 26 (3-4), 71- 91

Madsen RD, Weiner LB, McMillan JA, Saeed JA, North JA, Coates SR. (1988). Direct detection of Mycoplasma pneumoniae antigen in clinical specimens by a monoclonal antibody immunoblot assay Am J Clin Pathol 89 (1): 95-9

* Ragnar Norrby, S, (1999) "Representative Pneumonia in the Nordic Countries, Causes of It and Comparative Clinical Results of Ploxacin and Doxycycline, Nordic Atypical Pneumoniae Study Group" J Med Microbiol 48 (12), 1115-22

Tilton, R. C., F. Dias, et al., (1980) "Culturing with Mycoplasma Pneumoniae Detection DNA Probes in Clinical Samples" J Clin Microbiol 12 (6), 748-52

Yogev, D., D. Halachmi, et al. (1988) "Detection of Mycoplasma and Mycoplasma Strains by rRNA Gene Probes and Genomic DNA Fingerprints" J Clin Microbiol 26 (11), 2266-9

Chan ED, Kalayanamit T, Lynch DA, Tuder R, Arndt P, WinnR. Schwarz MI. "Mycoplasma pneumoniae-associated microbronchiolitis causes serious earlobe in adults-literature review" Chest., 1999; 115 (4), 1188-94

NCBI database # NC # 000912., Himmelreich, R., Hilbelt, H., Plagens, H., Pirkl, E., Li, B. C., and Herrmann, R.

* Cimolai N. "Respiratory Infectious Disease by Mycoplasma Pneumoniae" Pediatr Rev., 1998; 19 (10), 327-31

Harlow, E., and D. Lane (1988) "Antibodies, Laboratory Manual" New York, Cold Spring Harbor Laboratory Press

* Shambrook, J., E. F. Fritsch, and T. Maniatis, (1989) "Molecular Cloning, Laboratory Manual, Second Edition" Cold Spring Harbor Laboratory Press

Claims (9)

An antibody against a ribosomal protein of a microorganism belonging to Mycoplasma pneumoniae , an antibody that specifically reacts with the microorganism. The antibody of claim 1, wherein the ribosomal protein of the microorganism belonging to mycoplasma pneumoniae is ribosomal protein L7 / L12. The antibody of claim 1 or 2, wherein the antibody is monoclonal or polyclonal. The antibody of claim 1, wherein the antibody is an antibody bound to an enzyme. The antibody of any one of Claims 1-4 is used, The detection method of the microorganism which belongs to mycoplasma pneumoniae. a) extracting the ribosomal protein from the microorganism by contacting the sample with the lysate, b) extracting the extracted sample from the capture antibody, i.e., the antibody according to any one of claims 1 to 4, Forming an antigen antibody complex between the ribosomal protein and the capture antibody by contacting; and c) detecting the antigen antibody complex using a detection antibody, i.e., the antibody according to any one of claims 1 to 4. A method for detecting microorganisms belonging to mycoplasma pneumoniae in a specimen comprising a. The method according to claim 6, wherein the detection antibody is an antibody bound to an enzyme, and the antigen antibody complex is detected by a specific substrate of the enzyme. A reagent kit for detecting microorganisms belonging to mycoplasma pneumoniae, comprising using the antibody according to any one of claims 1 to 4. An immunogen comprises a ribosome protein L7 / L12 protein of a microorganism belonging to mycoplasma pneumoniae, a partial peptide thereof, or a synthetic peptide corresponding to the partial peptide thereof, obtained by genetic engineering or by isolation and purification from a microorganism. The manufacturing method of the antibody as described in any one of Claims 1-3.