KR100602843B1 - Legionella species-common lipoprotein antigen PAL for diagnosis of Legionnaire's disease - Google Patents

Abstract

The present invention relates to a common lipoprotein antigen of Legionella, antibodies specific thereto, and a diagnostic reagent and diagnostic kit for detecting Legionella.

According to the present invention, based on the detection of common lipoprotein antigens of Legionella species, first, there is an advantage that can efficiently detect not only Legionella pneumophila species but also non-Pneumophila species, and secondly, in urine samples of infected patients By detecting the common antigen, it can be expected to improve the diagnosis rate of Legionella pneumonia patients in the hospital, and third, it can be applied to the detection of Legionella bacteria in environmental samples including cooling towers. The development of a diagnostic kit using this principle is superior to the existing Legionella urinary antigen diagnostic kit developed and commercialized, and it will bring economic benefits through the entry into the world market and the import substitution effect of foreign diagnostic kits in Korea. It is expected to be.

Description

Legionella species-common lipoprotein antigen PAL for diagnosis of Legionnaire's disease}

1 is an immunoblot demonstrating that the anti-Legionella PAL IgG antibody to lipoprotein antigens of Legionella type 1 serotype commonly responds to lipoprotein antigens of about 19 kDa size in various Legionella species. The result of the analysis.

Figure 2 shows the results of ELISA test using polyvalent antibodies against lipoprotein antigen of Legionella, showing the reactivity with urine samples collected from animals infected with various Legionella bacteria as absorbance values (optical density at 450 nm). .

Figure 3 is an amino acid sequence of the recombinant lipoprotein portion used in the preparation of polyvalent and monoclonal antibodies against lipoprotein antigen of the Legionella pneumophila first serotype.

The present invention relates to a common lipoprotein antigen of Legionella, antibodies specific thereto, and a diagnostic reagent and diagnostic kit for detecting Legionella.

By Legionella spp (Legionella species) Gram-negative bacilli, which are hydrophilic and widely distributed in the natural environment serotypes (serogroup) of about 70 species known so far, and about half of it is causing human infection. Legionella pneumophila serogroup has been identified from Legionella type 1 to 15, and the rest is classified as non-pneumophila serogroup. Legionella is inhaled into the human body in the form of droplets, especially when it is contaminated with environmental water, the number of cooling towers in large buildings such as hospitals or hotels, water supply facilities, and air conditioning equipment. It has emerged as one of the leading pathogens that cause fatal intravenous pneumonia in patients with community-acquired pneumonia and immunocompromised patients. Legionella pneumonia is ineffective in the beta lactam family and aminoglycoside family commonly used as the primary treatment for acute bacterial pneumonia, and should be treated with marcrolide, an optional treatment for legionella pneumonia, with an accurate diagnosis. Early treatment is important because failure to treat can result in fatal consequences.

The diagnosis of Legionella pneumonia is dependent on laboratory diagnostic methods because the clinical symptoms and radiologic findings are nonspecific (Eldestein PH, 1993, Clin Inf Dis 16, 741-749). However, in the laboratory diagnosis, Legionella bacteria culture using the patient's sputum sample is difficult to obtain a sample because the patient's culture conditions are difficult, special medium and special culture conditions, as well as the sputum discharge of patients with Legionella pneumonia is low. Serological diagnostics are late in the production of serum antibodies and are virtually unhelpful for diagnosis. In recent years, research has been actively conducted on a diagnostic method of amplifying and detecting Legionella bacteria in sputum using polymerase chain reaction (PCR). It is known as a very sensitive and unusual detection method that can effectively detect even up to. These include 16S rRNA genes (Fry et al., J Gen Microbiol, 1991, 137, 1215-1222; Birtles et al., 1996, Microbiology 142, 3525-3530) or mip genes (Ratcliff et al. 1998, J Clin Microbiol 36, 1560- Detection methods using primers for 1567) have been attempted. However, in general, the detection method by PCR has a high risk of cross contamination during sample processing, and requires careful consideration and attention at all times, and it is a method that requires expensive reagents, consumables, and equipment. It is difficult.

On the other hand, the method of detecting soluble antigen in urine specimens of Legionella pneumonia was first described in 1979 (Berdal et al., 1979, J Clin Microbiol 9: 575-578; Tilton et al., 1979, Ann Intern Med 90 : 697-). 698) It has been extensively studied using enzyme immunoassay and radioimmunoassay for many years, and has proven to be the most powerful diagnostic method with 100% specificity and 70-100% sensitivity. , 1984, J Clin Microbiol 20: 478-482; Birtles et al., 1990, J Clin Pathol 43: 685-690; Kohler et al., 1981, Ann Intern Med 94: 601-605; Sathapatayavongs et al., 1982, Am J Med 72: 576-582; Tang et al., 1986, J Clin Microbiol 24: 556-558) (Kashuba et al., 1996, Diagn Microbiol Infect Dis 24: 129-139). The advantage of this method is that urine samples can be collected easily, antigens can be detected even after the start of antibiotic therapy, and results can be obtained quickly. Commercially available diagnostic reagents for detecting urine antigens include Binax EIA (Binax, Portland, Maine) and Biotest EIA (Biotest AG, Dreieich, Germany), which were commercially available in 1996 and 1997 respectively, and the recently developed Bartels EIA ( Bartels, Inc., Trinity Biotech Company, Wicklow, Ireland). These diagnostic reagents have been reported as sensitive and specific diagnostic methods in many clinical trials. Many researchers, however, point out that these reagents have very good sensitivity to L. pneumophila serogroup 1 antigens but vary the sensitivity of detection of L. pneumophila serogroups and non-pneumophila serogroups other than serogroup 1 (Benson 2000, J Clin Microbiol 38: 2763-2765; Dominguez et al., 2001, Diagn Microbiol Infect Dis 41: 199-203; Dominguez et al., 1998, J Clin Microbiol 36: 2718-2722; Harrison et al., 1998, Clin Microbiol Infect 4: 359-365; Kazandjian et al., 1997, J Clin Microbiol 35: 954-956). These diagnostic reagents are based on direct sandwich assays using polyvalent antibodies against the specific polyvalent antibodies of L. pneumophila serogroup 1 or soluble antigens of other L. pneumophila serogroups and non-pneumophila species. Although L. pneumophila serogroup 1 is known as a major causative agent of pneumonia, the number of different Legionella species is identified as a causative agent in some regions. Therefore, the usefulness of these diagnostic reagents for the detection of a wide range of Legionella species is unknown, and alternatively, the development of urine antigen diagnostic reagents using the Legionella common surface antigen is required.

TECHNICAL FIELD The present invention belongs to the development of a diagnostic reagent for urinary legionella urine antigen, characterized by detecting peptidoglycan associated lipoprotein (PAL), a common lipoprotein antigen of Legionella species, using an antibody against lipoprotein antigen of L. pneumophila serogroup 1 and by a wide range of detection of Legionella species in the target lipoprotein antigen it invented that Legionella pneumonia caused by a variety of Legionella species more efficiently diagnosed.

Legionella lipoprotein antigen PAL, which has been found to be suitable for the development of diagnostic reagents for Legionella urine antigen, is an outer membrane component commonly present in Legionella species, and is a component of Legionella soluble extract, and inoculates lipoprotein alone without immunoadjuvant in rabbits. It showed very good immunogenicity by inducing a strong antibody response (Kim Min-ja et al., 2003, J Clin Microbiol 41: 2974-2979; Yoon Won-suk et al., 2002, DNA Cell Biol 21: 99-107). Based on this, we hypothesized that lipoprotein PAL can be excreted in the urine of Legionella- infected patients as a soluble antigen, and also non- other other serogroups of L. pneumophila , including L. pneumophila serogroup 1 as a common antigen. Since the infection by pneumophila Legionella species will be excreted in the urine as well, the development of urine antigen diagnostic reagent using this lipoprotein antigen was considered to be very useful for the diagnosis of Legionella pneumonia regardless of the causative Legionella species. The world's first to prove this.

For those skilled in the art, in the future, the development of a urine antigen diagnostic reagent using Legionella lipoprotein antigen PAL may be applied or combined with any detection method such as immunoassay, radioimmunoassay, immunochromatography, and the like. It will be clear that this corresponds to the application of diagnostic system development.

The development of the urine antigen diagnostic reagent according to the present invention shows excellent superiority compared to the existing urine antigen diagnostic reagents that are already commercialized. First, the characteristics of the conventional techniques, the diagnostic system using the Legionella pneumophila serotype-specific lipopolysccharide antigen-antibody, the Legionella pneumophila serogroup except for the first serotype, because Legionella lipopolysaccharide shows the species specificity 15 serogroup) and non pneumophila Legionella spp. In addition, as a technique developed to compensate for this disadvantage, including various Legionella strains, all strains are individually cultured to collect sonic extracts or soluble extracts from individual strains, and antibodies to the soluble extracts. By manufacturing Legionella Since it is used for the detection of urine antigens, it may cause various detection results according to the properties of various antigens that may be included in the extract, and the detection sensitivity may be lowered for some Legionella species, resulting in false negative results. In addition, a lot of effort and time is required to prepare a diagnostic reagent, including passage and maintenance of more than 30 pathogenic Legionella species. On the other hand, since the present invention uses a lipoprotein antigen that is commonly conserved in Legionella species, it is possible to detect several Legionella species by using a lipoprotein antigen-antibody diagnostic agent of Legionella pneumophila serotype. In addition, it is possible to easily separate a large amount of recombinant lipoprotein antigens using genetic recombination techniques without Legionella culturing or antigen extraction process, thereby minimizing time and effort. It has excellent superiority such as point.

Therefore, the main object of the present invention is a 19 kDa sized lipoprotein component, a soluble antigen commonly present on the surface of various Legionella species, the causative agent of Legionella pneumonia, is excreted through urine samples of animals or patients infected with Legionella. It is to provide evidence that can be efficiently detected by the diagnostic agent of the invention.

Another object of the present invention is to overproduce the recombinant protein of the common lipid protein Legionella species using genetic recombination techniques, using the specific polyvalent antibody and monoclonal antibody prepared for the Legionella lipoprotein antigen or Legionella bacteria in the sample It is to provide a diagnostic agent or diagnostic system capable of detecting.

Another object of the present invention is to clone a gene encoding a 19 kDa lipoprotein from Legionella pneumophila serogroup 1, and to use a recombinant lipoprotein antigen and a specific antibody obtained continuously as diagnostic agents. 1 shows that it is possible to detect the remaining Legionella pneumophila serogroups (Sections 2-15) and non-Pneumophila Legionella spp. Including serotypes, providing evidence that the diagnostic agents of the present invention are broad spectrum diagnostic agents. will be.

It is another object of the present invention to provide an amino acid sequence used for generating specific antibodies against the common lipoprotein antigens of the Legionella species.

In order to achieve the object of the present invention, the present invention provides a peptidoglycan associated lipoprotein (PAL) antigen common to the Legionella species comprising the amino acid sequence of SEQ ID NO: 2 or a functional equivalent thereof.

In the present invention, the antigen is characterized by a common lipoprotein antigen of 19 kDa of the remaining Legionella pneumophila hematotypes and non pneumophila serotypes, including Legionella pneumophila serotype 1.

In the present invention, the antigen is characterized by being encoded by a DNA comprising a nucleotide sequence of SEQ ID NO: 1 or a functional equivalent thereof.

The amino acid sequence described in SEQ ID NO: 2 is an amino acid sequence of an antigen protein encoded by the nucleotide sequence of SEQ ID NO: 1. Also, functionally equivalent sequences refer to amino acid or base sequences of proteins that can exhibit the same antigenicity despite modifications such as substitutions, deletions or insertions of amino acids or bases.

In order to achieve another object of the present invention, the present invention is PCR amplification of the PAL coding gene using a primer comprising the nucleotide sequence of SEQ ID NO: 3 and 4 as a template of the gene of Legionella serogroup 1 ( L. pneumophila serogroup 1) A peptidoglycan associated lipoprotein (PAL) antigen according to claim 1 or 2, comprising inserting the amplified gene into a plasmid to form a recombinant plasmid and expressing the recombinant plasmid in a host cell. It provides a method of manufacturing.

In order to achieve another object of the present invention, the present invention provides an antibody specific for the peptidoglycan related lipoprotein (PAL) antigen common to the Legionella species according to the present invention.

In the present invention, the antibody is characterized in that the polyvalent antibody or monoclonal antibody, the polyvalent antibody and monoclonal antibody can be prepared according to the production method of antibodies well known in the art.

In order to achieve another object of the present invention, the present invention provides a diagnostic reagent for detecting antibodies of Legionella bacteria comprising the antigen according to the present invention.

In order to achieve another object of the present invention, the present invention provides a diagnostic reagent for antigen detection of Legionella bacteria comprising the antibody according to the present invention.

The diagnostic reagent according to the present invention includes peptidoglycan-associated lipoprotein (PAL), a common lipoprotein antigen of Legionella, and polyvalent and monoclonal antibodies as specific antibodies thereto. In addition, the approach of diagnosing Legionella pneumonia is firstly, the 19 kDa lipoprotein of Legionella is a soluble antigen component commonly conserved in various Legionella species, and secondly, the lipoprotein antigen is used for urine samples of patients with Legionella pneumonia. Third, the present invention provides a method for developing a diagnostic reagent for Legionella pneumonia by inventing that the common lipoprotein antigen of Legionella spp. Excreted in the urine of Legionella pneumonia patients can be detected using specific antibodies against the lipoprotein antigen. .

In order to achieve another object of the present invention, the present invention provides a diagnostic kit for detecting Legionella bacteria in a sample containing the diagnostic reagent of the present invention.

In the present invention, the sample is a clinical patient sample including sputum, bronchial aspirate, bronchial lavage fluid, lung tissue, or the like, or an environmental sample including a cooling tower water.

Preferably, the detection method used in the diagnostic kit of the present invention is characterized by ELISA method, EIA method or Disp-stick assay method. In the case of the ELISA kit, the first antibody was reacted with a plate coated with the antigen of the present invention, and then the secondary antibody labeled with peroxidase was bound, followed by OPD (orange), ABTS (green) or TMB (pink). Configured to react with the substrate. In the case of the disp-stick assay kit, the antigen of the present invention was adsorbed and fixed on the gold, and then, dipped into a membrane on the glass pad, a second antibody bound to the horseradish peroxidase or alkaline phosphatase was added thereto, and then the sample was glass. It is configured to react by dropping on the pad.

In the present invention, the antigenic protein can be produced by recombinant technology by PCR amplifying a gene encoding the antigenic protein, inserting the same into a vector, expressing it in a host cell, and recovering the expressed antigenic protein. For recombinant production, host cells are transfected with a vector encoding an antigenic protein and then cultured in a nutrient medium suitably modified to activate a promoter and select a transformant or amplify the gene. Suitable vectors are those that are viable and replicable in the selected host, and are derived from a combination of chromosomes, non-chromosomal and synthetic DNA sequences such as vector plasmids, phage DNA, baculoviruses, yeast plasmids, plasmids and phage DNA. Include. The polypeptide sequence is incorporated into the vector at the appropriate site using restriction enzymes such that it is operably linked to an expression control region comprising a promoter, ribosomal binding site (common region or Shine-Dalgarno sequence), and optionally an operator (control element). Can be.

Those skilled in the art will appreciate that individual components of expression control regions suitable for a given host and vector according to established molecular biology principles (incorporated by reference to Sambrook et al, Molecular Cloning: A Laboratory Manual, 2nd ed., Cold Spring Harbor, NY, 1989). Can be selected. Suitable promoters include, but are not limited to, LTR or SV40 promoter, E. coli lac, tac or trp promoter and phage lambda P _L promoter. The vector will preferably incorporate not only the origin of replication but also a selection marker, i.e. an ampicillin resistance gene. Suitable bacterial vectors include pET, pQE70, pQE60, pQE-9, pbs, pD10 phagescript, psiX174, pbluescript SK, pbsks, pNH8A, pNH16a, pNH18A, pNH46A, ptrc99a, pKK223-3, pKK233-3, pDR540, pRIT5 and eukaryotic Vectors pBlueBacIII, pWLNEO, pSV2CAT, pOG44, pXT1, pSG, pSVK3, pBPV, pMSG and pSVL. Host cells include bacteria such as E. coli , Bacillus subtilis , Streptomyces ; Fungals , namely Aspergillus niger , Aspergillus nidulins ; Yeast, ie Saccharomyces or eukaryotic, ie CHO, COS.

Upon expression of the antigenic protein in culture, cells are typically harvested by centrifugation and then milled by physical or chemical means (if the expressed polypeptide is not secreted into the medium) and the resulting crude extract Are maintained to isolate the antigenic protein. Purification of the antigenic protein from the culture medium or lysate is carried out by techniques established according to the nature of the protein, ie ammonium sulfate or ethanol precipitation, acid extraction, anion and cation exchange chromatography, phosphocellulose chromatography, hydrophobicity It can be achieved using interactive chromatography, hydroxylapatite chromatography and lectin chromatography. Final purification can be accomplished using HPLC. Antigen proteins can be expressed with or without leader or secretory sequences. In the former case, the leader may be removed using post-translational processing (including US 4,431,739; 4,425,437; and 4,338,397 references) or chemically removed after purification of the expression polypeptide.

In the present invention, any method known in the art can be used to produce the antibody. For example, the antigenic protein is administered to an animal to induce antibodies to the antigen. Methods of administering the antigenic proteins and the animals used are well known in the art and are not particularly limited. Animals used for antibody production can be, for example, but not limited to mice, rabbits, goats and chickens. The antigenic protein is bound to a carrier protein such as hemocyanin and can be administered in combination with an adjuvant. An antibody that specifically binds to the antigen is then collected from the animal immunized with the antigen protein. In the process of collecting antibodies, blood is collected from the immunized animal, from which blood is isolated using purification methods known in the art such as salting out, ion exchange chromatography, gel permeation chromatography, affinity chromatography, and ultrafiltration. It may include, but is not limited to

Hereinafter, the present invention will be described in more detail with reference to the following examples. These examples are only for illustrating the present invention in more detail, it will be apparent to those skilled in the art that the scope of the present invention is not limited to these examples.

Example 1: Preparation of Antigen Protein

A pair of primers (5'-TCTAGATTGTGGAATGAAAGCCGGATCGT-3 'and 5'-GTCCACCCATGAGGCGAAA GGAAGCATC-3') were prepared to prepare a recombinant protein of peptidoglycan associated lipoprotein (PAL), a common surface protein antigen of Legionella species. The template was obtained from Legionella pneumophila serogroup 1 and amplified and isolated from the gene encoding PAL using PCR technique. Sequencing the amplified gene with an automatic sequencer confirmed that it has the nucleotide sequence of SEQ ID NO: 1. It was then inserted into the pT7 Blue plasmid, amplified in E. coli XL1 Blue, plasmids were separated, and the gene fragment obtained through restriction enzyme treatment was inserted into the pGEX-KG plasmid to make a pGEX-KG (PAL) recombinant plasmid. Recombinant plasmid pGEX-KG (PAL) was subsequently inserted into Escherichia coli DH5-α and Escherichia coli BL21 (DE3) pLys, and IPTG was added to overexpress lipoproteins while culturing Escherichia coli BL21 (DE3) pLys. After incubation, the cells were obtained by centrifugation, and the cells were subjected to ultrasonic disruption, followed by centrifugation to obtain glutathione sulfur transferase (GST) -PAL fusion protein.

After solubilizing 7M urea for solubilization of insoluble GST-PAL protein, centrifugation to obtain soluble supernatant, 1: 1 mixing with glycerol for re-folding of GST-PAL, followed by 100-fold volume dilution buffer Dropped. Recombined GST-PAL protein was subjected to affinity chromatography using Glutathione-Sepharose 4B having affinity for GST to obtain purified GST-PAL protein. The GST-PAL protein obtained here was treated with thrombin to liberate the GST portion, and then passed through the newly prepared Glutathione-Sepharose 4B (Pharmacia Co.) to purify the 19 kDa Legionella lipoprotein antigen (PAL) in the filtrate. The purified antigenic protein has the amino acid sequence of SEQ ID NO.

Example 2: Preparation of Antibodies

For a control experiment of the diagnostic agent of the present invention, a known Legionella soluble antigen and an antibody thereof were prepared and used as a standard test system. The preparation of soluble antigens used the method described by other researchers previously (Berdal et al., 1979, J Clin Microbiol 9: 575-578; Tang et al., 1986, J Clin Microbiol 24: 556-558). Among the L. pneumophila serogroups, 1, 3, 4, 5, 6 serotypes and L. micdadei , L. dumoffi , L. gormanii , L. sainthelensi , L. anisa , L. jordanis, L Strains of oakridgensis and the like were cultured for 48 hours in a buffered charcoal-yeast extract agar-α-ketoglutarate ( BCYE -α) medium under 5% CO 2, collected, suspended, and suspended in 0.05 M PBS (pH 7.4). To precipitate the cells. The precipitated 0.5 ml of cells were sterilized at 100 ° C. for 1 hour and left at 4 ° C. for 10 days, and then the suspension was centrifuged at 10,000 × g for 10 minutes to obtain a supernatant and used as the soluble antigen.

Antiserum was obtained after immunization by injection of 100 μg of New Zealand rabbits at 2 week intervals to prepare antibodies against PAL antigen and soluble antigen from the recombinant PAL protein and soluble antigen of Legionella obtained above. In the serum of IgG, rabbit serum was injected into protein A-Superose HR 10/2 column (Pharmacia LKB), and IgG for each recombinant PAL antigen and IgG for soluble antigen were separated using high performance liquid chromatography.

The preparation of immunoglobulin-enzyme conjugates was purified by mixing horseradish peroxidase and IgG purified from the above method to make a conjugate, followed by Sephadex G-200 gel filtration chromatography. Legionella antigen was detected using ELISA method for urine samples of guinea pigs infected with Legionella of various strains and urine samples of Legionella pneumonia patients using purified immunoglobulin-enzyme conjugates (Anti-PAL IgG-peroxidase). Legionella PAL lipoprotein antigen was detected and showed 85% sensitivity and 98.8% specificity.

Therefore, the Legionella PAL lipoprotein antigen-antibody detection system designed in the present invention proves that the lipoprotein antigen is excreted in the urinary tract by confirming the presence of urine antigens that react with specific antibodies of the lipoprotein antigen in urine samples of animals infected by various Legionella species. In addition, we confirmed the successful detection of lipoprotein antigens of various Legionella species, and these results showed a high correlation with the results of the Legionella Soluble Extracted Antigen-Antibody Diagnostic System, which was used as a control test. And Legionella urine antigen detection diagnostic system.

Example 3: Confirmation of reactivity of anti-lipoprotein antibody (anti-PAL IgG) to Legionella soluble antigen

Legionella bacteria common lipid protein antigen (PAL) prepared in rabbits were confirmed by immunoblotting method to have a specific reactivity to Legionella strains.

Anti-Legionella lipid protein antibody (Anti-rPAL IgG antibody) prepared from antiserum collected after immunizing purified recombinant lipoprotein antigen (rPAL) to rabbits was prepared. Soluble protein samples of each Legionella strain were centrifuged in the culture of each strain, and then the cells were suspended in buffer and freed from the cells by sonication. Free soluble protein samples were observed by electrophoresis on 12% SDS-polyacrylamide gel and stained. Immunoblotting assays were performed on the same gel, followed by electrophoresis, transfer to nitrocellulose membrane, antilipoprotein antibody as primary antibody, secondary antibody reaction and ECL detection reagent (Amersham Co.) with nitrocellulose membrane. After confirming the photosensitive X-ray film.

1 demonstrates that the anti-Legionella PAL IgG antibody against lipoprotein antigens of Legionella type 1 serotype responds in common to lipoprotein antigens of about 19 kDa size in various Legionella species. As a result of electrophoresis of soluble antigens extracted from Legionella strains, A shows that the soluble extract contains various proteins, and B reacts the same soluble antigen components of Legionella as A as polyvalent antibodies to this lipoprotein antigen. The results of immunoblot analysis showed responsiveness to about 19 kDa antigens in common in various strains.

As a result of immunoblotting according to the above method, as shown in FIG. 1, the lipoprotein antibody derived from the anti-Regionella pneumophila 1 serotype is L. pneumophila SG4 (sequence 1, ATCC) in addition to the Legionella pneumophila 1 serotype. 33156), L. pneumophila SG6 (SEQ ID NO: 33, ATCC 33215), L. pneumophila SG5 (SEQ ID NO: 33, ATCC 33216), and also the non pneumophila species L. micdadei ( SEQ ID NO : 4, ATCC 33204), L. jordanis (5). Sequence, ATCC 33623), L. oakridgensis ( sequence 6, ATCC 35761), L. anisa ( sequence 7, ATCC 35292), L. gormanii ( sequence 8, ATCC 33297), L. sainthelensi ( sequence 9, ATCC 35248) It was confirmed that the reaction also. In addition, it was confirmed that the Legionella PAL lipoprotein antigen was a conserved antigen in common among Legionella strains.

Example 4: Confirmation of Correlation between PAL Lipoprotein Antigen and Legionella Soluble Antigen in Guinea Pick Urine Samples Infected with Legionella

To identify positive urine samples infected with Legionella, anti-PAL IgG antibodies and anti-soluble antigens IgG antibodies obtained from soluble antigens of 12 Legionella strains were used. The sandwich ELISA was performed at the same time and the results were compared. At this time, the positive determination was limited to the case where the absorbance is 0.22 (0.12 ± 0.10SD) or more. Urine samples were collected from guinea pigs infected with eight different Legionella strains, and a total of 17 urine samples collected once to three times per strain were tested. The ELISA results using anti-soluble antigen immunoglobulin antibody showed a relatively high absorbance of 0.47-3.0, which was dark yellow in all samples. From these results, the validity of the urine sample and ELISA method for Legionella soluble antigen were verified by the standard test method.

Figure 2 shows the results of ELISA test using polyvalent antibodies against lipoprotein antigen of Legionella, showing the reactivity with urine samples collected from animals infected with various Legionella bacteria as absorbance values (optical density at 450 nm). . The absorbance values of the antigen-antibody response to PAL antigen in each urine sample are determined by the absorbance values of the antigen-antibody response to soluble antigens used in the control test. Show the correlation.

The absorbances obtained from the sandwich ELISA results using anti-lipoprotein immunoglobulin antibodies performed in the same urine sample showed a high correlation as shown in FIG. 2 when compared with the results using anti-soluble antigen immunoglobulin antibodies (Pearson Correlation coefficient 0.86, p <0.01).

Example 5: Confirmation of the sensitivity and specificity of the Legionella anti-lipoprotein antigen antibody

The sensitivity and specificity of the ELISA method using anti-PAL antibody were investigated according to the cutoff value. The sensitivity and specificity of 17 infected urine specimens (8 pneumophila serotypes and 7 nonpneumophila species) and 67 non-infected urine specimens were examined. As shown in Table 1 below, the sensitivity was 88.2%, specificity 95.5%, false positive rate 3.0%, false negative rate 16.7% when the threshold was 0.1. Urine samples of Legionella-infected guinea pigs , except two out of three specimens of L. sainthelensi , were above the limit. On the other hand, in the case of ELISA using an antibody to the soluble antigen (anti-soluble antigen antibody), all of the samples infected with Legionella responded higher than the limit of the negative control. In the above results, Legionella lipoprotein antigen was confirmed by ELISA method to be excreted in the urine of guinea pigs or patients infected with Legionella, and confirmed the possibility of development as a urine antigen diagnostic reagent.

Table 1 Sensitivity and Specificity of ELISA using Legionella Lipoprotein Antigen

Cutoff responsiveness(%) % Specificity Positive determination (%) Negative judgment value (%) 0.11 76.5 98.5 92.9 94.3 0.10 88.2 95.5 83.3 97.0 0.09 88.2 89.6 68.2 96.8 0.08 100.0 77.6 50.0 100.0

Example 6: Excellence of ELISA using Legionella lipoprotein antigen antibody

In order to evaluate the superiority of Legionella urine antigen detection of Legionella lipoprotein antigen antibody, it was compared with Enzyme Immunoassay (EIA) for the commercially available Legionella urine antigen. As a result, as shown in Table 2 below, the biotester's EIA detected only 17 infected urine samples, whereas the ELISA method using Legionella lipoprotein antigen antibody did not detect only L. saint helensi. It also showed positive reactions against the remaining non pneumophila species. Therefore, the Legionella lipoprotein antigen antibody used in the present invention is considered to be more useful for detecting various Legionella infections.

Table 2 Comparison of Legionella Antigen Antibody ELISA and Biotest's EIA Diagnostic Tests in 17 Urine Samples of Guinea Pigs Infected with Legionella

Legionella fungus Lipoprotein Antigen ELISA Results Biotest's EIA Kit Results L. pneumophila SG1 (AA100) + + L. pneumophila SG1 (AA100) + + L. pneumophila SG1 (isolated strain) + + L. pneumophila SG1 (isolated strain) + + L. pneumophila SG1 (isolated strain) + + L. pneumophila SG3 (ATCC 33155) + - L. pneumophila SG3 (ATCC 33155) + - L. pneumophila SG6 (ATCC 33215) + - L. sainthelensi (ATCC 35248) - - L. sainthelensi (ATCC 35248) + - L. sainthelensi (ATCC 35248) _ L. anisa (ATCC 35292) + - L. anisa (ATCC 35292) + - L. oakridgensis (ATCC 35761) + - L. oakridgensis (ATCC 35761) + - L. jordanis (ATCC 33623) + L. jordanis (ATCC 33623) +

Example 7: Absorbance Range of Legionella Lipoprotein Antigen Antibody ELISA to Negative Control

ELISA was performed on 161 urine specimens in 3 groups of healthy adults, non-neumophila pneumonia patients, and urethral infection patients to determine how ELISA using Legionella lipoprotein antigen antibody responded to urine in patients with negative Legionella. As a result, the absorbance was negative in all three negative control groups as shown in Table 3 below.

TABLE 3 Confirmation of absorbance range of Legionella lipoprotein antigen (PAL) ELISA method

Target Sample Urine samples Absorbance Mean ± standard deviation Minimum Maximum value Patients with Legionella Urethral Infection 16 0.058 ± 0.015 0.006 0.076 Non-Regionella Pneumoniae Patients 59 0.047 ± 0.013 0.028 0.083 Healthy adult 86 0.053 ± 0.007 0.042 0.092

Example 8 Legionella Lipoprotein Antigen-Antibody ELISA Results in Urine Specimens of Legionella Pneumonia Patients

Comparison of absorbances obtained from sandwich ELISA method using Legionella lipoprotein antigen-antibody and commercialized Biotest EIA results in urine specimens clinically diagnosed with Legionella pneumonia was shown in Table 4 below. The ELISA results with antibodies showed higher absorbance compared to the results obtained with Biotest's EIA kit.

[Table 4] Comparison of Legionella Lipoprotein Antigen ELISA and Biotest's EIA Test in Urine Specimens of Legionella Pneumonia

Sample Number Patient urine Sample Collection Date Biotest EIA PAL lipoprotein ELISA One Negative control 0.144 0.122 2 Positive control 1.592 1.134 3 Patient 1-1 99/11/1 overflow 0.947 4 Patient 1-2 99/11/11 overflow 0.94 5 Patient 2-1 02/8/2 0.167 0.285 6 Patient 2-2 02/9/23 0.175 0.324 7 Patient 2-3 02/10/1 0.165 0.267 8 Patient 2-4 02/10/24 0.179 0.34 9 Patient 3-1 02/9/9 1.007 0.69 10 Patient 3-2 02/9/20 0.166 0.294 11 Patient 3-3 02/9/28 0.197 0.445 12 Patient 3-4 02/10/5 0.134 0.27

Example 9: dot blot results using Legionella lipoprotein antigen-specific monoclonal antibody

Soluble antigen extracted from Legionella pneumophila 3 and 6 non-pneumophila strains, respectively, by injecting the recombinant lipoprotein antigen of the Legionella pneumophila serotype into BALB / c mice and producing monoclonal antibodies using the myeloma cell fusion technique. Dot blot was performed to confirm the antigen-antibody reaction. The five monoclonal antibodies produced showed specific responses to the nine Legionella species tested as shown in Table 5 below.

Table 5 Reactivity of Legionella Lipoprotein Antigen Specific Monoclonal Antibodies with Soluble Antigens of Legionella Species

Soluble Antigens of Each Legionella Species Reactivity with Monoclonal Antibodies  D-1-1 D-4-1-7 D-35-5 D-41-2 C-46-1 L. pneumophila SG 4 (ATCC 33156)  + + + + + L. pneumophila SG 6 (ATCC 33215) + + + + + L. pneumophila SG 5 (ATCC 33216) + + + + + L. micdadei + + + + + L. jordanis + + + + + L. oakridgensis + + ± + + L. anisa + + + + + L. gormannii + + + + + L. sainthelensi + + ± + +

As described and demonstrated in detail above, the present invention provides the detection of Legionella by using common lipoprotein antigens of Legionella spp. The diagnostic agent for urine and the urine antigen diagnostic system of Legionella pneumonia.

Therefore, the present invention is based on the detection of common lipoprotein antigens of Legionella spp., Firstly, it has the advantage of efficiently detecting non-pneumophila spp. By detecting the antigen, it can be expected to improve the diagnosis rate of Legionella pneumonia patients in the hospital, and third, it can be applied to the detection of Legionella bacteria in environmental samples including cooling towers. The development of a diagnostic kit using this principle is superior to the existing Legionella urinary antigen diagnostic kit developed and commercialized, and it will bring economic benefits through the entry into the world market and the import substitution effect of foreign diagnostic kits in Korea. It is expected to be.

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Claims (8)

A peptidoglycan associated lipoprotein (PAL) antigen common to Legionella species consisting of the amino acid sequence of SEQ ID NO: 2. The antigen of claim 1, wherein the antigen is encoded by a DNA consisting of the nucleotide sequence of SEQ ID NO: 1. PCR amplification of the PAL coding gene using a primer containing the nucleotide sequences of SEQ ID NOs: 3 and 4 as a template of the gene of L. pneumophila serogroup 1, and the amplified gene was inserted into the plasmid to generate a recombinant plasmid. A method for producing a peptidoglycan associated lipoprotein (PAL) antigen according to claim 1 or 2, comprising making and expressing a recombinant plasmid in a host cell. An antibody specific for a peptidoglycan related lipoprotein (PAL) antigen common to the Legionella species according to claim 1. The antibody of claim 4, wherein the antibody is a polyvalent antibody or a monoclonal antibody. A diagnostic reagent for detecting antibodies of Legionella, comprising the antigen according to claim 1. A diagnostic reagent for antigen detection of Legionella bacteria comprising the antibody according to claim 4 or 5. A diagnostic kit for detecting Legionella bacteria in a sample containing the diagnostic reagent according to claim 6.

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