Abstract:
A set of three unique monoclonal antibodies have been produced which recognize Legionella with particular specificity, without substantial cross-reactivity with non-Legionella bacteria. These monoclonal antibodies are useful as immunodiagnostic reagents for detecting Legionella.

Description:
The present invention is related generally to immunodiagnostic reagents. More particularly, the present invention is related to novel monoclonal antibodies for genus wide detection specifically of Legionella species. 
     BACKGROUND OF THE INVENTION 
     Currently, there are about 30 recognized species containing 47 serogroups of Legionella. Present serological detection methods are based on about 48 serogroup specific antigens which require about 48 serogroup specific antisera. Sampson et al. (J. Clin. Micro., 23:92-99, 1986) described a 58-kilodalton (later 60-kilodalton) protein antigen that was present in all Legionella species examined and which was found to be immunogenic in humans, reacting with serum from 100% of the culture-confirmed cases of legionellosis that were tested. Antibody to this protein was considered as a possible probe in immunological procedures to detect all species in the genus Legionella. Polyclonal rabbit antisera to the 60-kilodalton protein was produced by the method of Plikaytis et al. (J. Clin. Micro., 25:2080-2084, 1987) for use as a detection reagent. To achieve specificity to the Legionella genus, the polyclonal antisera had to be sequentially absorbed with three heterologous organisms, thereby significantly decreasing the potency of the antisera. In contrast, the monoclonal antibodies produced by the hybridoma cell lines are not subject to the variabilities encountered in the production of polyclonal antisera. 
     A variety of diagnostic techniques for detection of Legionnaire&#39;s disease are known. Patent No. 87/05609 issued to Hoffman describes monoclonal antibodies for the detection of Legionella species. However, these antibodies have cross-reactivity with the bacteria of other genus. 
     SUMMARY OF THE INVENTION 
     It is, therefore, an object of the present invention to provide a set of at least three monoclonal antibodies which, in combination, recognize all known species of Legionella without substantial cross-reactivity with other bacteria with which heretofore known monoclonal antibodies exhibit significant cross-reactivity. 
     It is another object of the present invention to provide novel immunodiagnostic reagents for differentially detecting the presence of bacteria belonging to the genus of Legionella in biological or environmental samples and the like. 
     Other objects and advantages will become evident from the following detailed description of the invention. 
    
    
     BRIEF DESCRIPTION OF THE DRAWINGS 
     These and other objects, features and many of the attendant advantages of the invention will be better understood upon a reading of the following detailed description when considered in connection with the accompanying drawings wherein: 
     FIGS. 1A-1D show binding specificity of Hoffman&#39;s monoclonal antibody and the claimed monoclonal antibodies with the 60 kDa protein among non-Legionella bacteria using Western blot analysis. 
    
    
     Blots were probed with Hoffman&#39;s monoclonal antibody GW2X4B8B2H6 (A), Aloisio&#39;s monoclonal GB5BE8 (B), GB5AF6 (C), and CA4AF5 (D). Lane 1 contains a positive control strain of L. pneumophila serogroup 1; 2, Acinetobacter lwoffii; 3, Pseudomonas aeruginosa; 4, Bordetella bronchiseptica; and 5 B. pertussis. Note the strong reactivity in lanes 2, 4, and 5 with Acinetobacter, B. bronchiseptica, and B. pertussis when probed with the Hoffman antibody. 
     DETAILED DESCRIPTION OF THE INVENTION 
     The above and various other objects and advantages of the present invention are achieved by producing a set of three stable hybridoma clones such that each clone secretes sufficient amount of uniformly specific monoclonal antibodies which bind to specific antigenic sites (isotypic epitopes) unique to the 60-kilodalton protein of Legionella. These three monoclonal antibodies obtained in accordance with the present invention are designated herein as GB5BE8, GB5AF6 and CA4AF5. 
     A deposit of the three hybridomas secreting monoclonal antibodies GB5BE8, CA4AF5 and GB5AF6 has been made at the ATCC, Rockville, Md., on May 23, 1990, Apr. 24, 1990 and May 10, 1990, respectively under accession numbers HB 10459, HB10439 and HB10453, respectively. The deposit shall be viably maintained, replacing if it becomes non-viable during the life of the patent, for a period of 30 years from the date of the deposit, or for 5 years from the last date of request for a sample of the deposit, whichever is longer and, upon issuance of the patent, made available to the public without restriction in accordance with the provisions of the law. The Commissioner of Patents and Trademarks, upon request, shall have access to the deposit. 
     Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Although any methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present invention, the preferred methods and materials are now described. All publications mentioned hereunder are incorporated herein by reference. Unless mentioned otherwise, the techniques employed or contemplated herein are standard methodologies well known to one of ordinary skill in the art. The materials, methods and examples are illustrative only and not limiting. 
     The term &#34;substantial&#34; cross reactivity as defined herein means significant or considerable amount of cross reactivity. 
     MATERIALS AND METHODS 
     Purified 60-kilodalton protein antigen was produced from Legionella pneumophilia serogroup-1. A highly enriched extract was produced by ultracentrifugation. Further purification was carried out by standard affinity chromatography and ion exchange high performance liquid chromatography. For initial immunization, a dosage of 20 micrograms of purified protein in phosphate buffered saline with incomplete Freund&#39;s adjuvant was administered to mice. This was repeated at 6 weeks. One week later, the animal was given 10 micrograms purified protein intravenously on two consecutive days. The general procedures of Kohler and Milstein (Nature, 256, 1975) were used as modified by Zola and Brooks (Monoclonal Antibodies: Techniques and Applications, J. G. R. Hurrell (ed.), CRC Press, Boca Raton, pp. 1-57, 1982). Immunized mice and antibody from hybridized cells were screened for reactivity by standard Western blot analysis using purified 60-kilodalton protein as the antigen and goat anti-mouse horseradish peroxidase conjugate. From numerous clones tested, three clones were isolated that exhibited the desired reactivity. Further analysis of the three clones was done using standard SDS-PAGE followed by immunoblots on 42 Legionella serogroups (23 species) and 62 non-Legionella bacterial strains; the results are shown in Table 1. The results indicate that the three hybridoma cell lines produce monoclonal antibody that, in combination, react with all tested strains of Legionella. 
     It should be noted that there was no reactivity with the five strains of Bordetella and one strain of Acinetobacter tested by Western blot analysis. This is a significant difference from the reactivity of the GW2X4B8B2H6 monoclonal antibody reported by Hoffman. Hoffman reported that this antibody reacts strongly with 12 strains of Bordetella and one strain of Acinetobacter when tested in an ELISA method. In comparative tests presented herein, it was found that the GW2X4B8B2H6 antibody strongly reacted with the Bordetella and Acinetobacter species in Western blot analysis (FIG. 1). This is quite significant since a monoclonal antibody or pool of monoclonal antibodies that detect most Legionella species without substantial cross-reactivity with non-Legionella species would provide an important screening tool for both clinical and environmental samples. Clearly, the strong cross-reactivity of the Hoffman monoclonal antibody makes it unfit for this type of application due to unacceptable cross-reactivity. The strong cross-reactivity of the GW2X4B8B2H6 Hoffman antibody and the lack of this cross-reactivity in the monoclonal antibodies reported herein indicate that even though these antibodies react to the same 60-kDa protein, the epitope specificity is distinctly different. 
     The unique monoclonal antibodies of the present invention now provide a reliable, rapid, simple and specific diagnostic test for Legionella. The method comprises reacting a sample suspected of Legionella infection with a part (reactive fragment) or whole of the monoclonal antibodies of the present invention, a positive immunological reaction being indicative of the presence of Legionella bacterium in the sample. Such immunodiagnostic tests, which include immunohistochemical, immunoblotting, radioimmunoassay and the like, are quite routine and standard and well known to one of ordinary skill in the art. A diagnostic kit or an immunodiagnostic reagent in accordance with the present invention comprises containers containing an immunoreactive amount of the monoclonal antibodies of the present invention, either alone or as a combined mixture in a sterile, non-toxic medium such as a buffered solution and the like. 
     It is understood that the examples and embodiments described herein are for illustrative purposes only and that various modifications or changes in light thereof will be suggested to persons skilled in the art and are to be included within the spirit and purview of this application and scope of the appended claims. 
     
                                           TABLE 1__________________________________________________________________________SPECIFICITY OF LEGIONELLA 60K MONOCLONALS                          GB5 GB5 CA4Bacteria        Serogroup                 Strain ID                          BE8 AF6 AF5__________________________________________________________________________Legionella pneumophila           1     Philadelphia 1                          +   +   +Legionella pneumophila           2     Togus 1  +   +   +Legionella pneumophila           3     Bloomington 2                          +   +   +Legionella pneumophila           4     Portland 1                          +   +   +Legionella pneumophila           5     Cambridge 2                          +   +   +Legionella pneumophila           6     Chicago 2                          +   +   +Legionella pneumophila           7     Chicago 8                          +   +   +Legionella pneumophila           8     Concord 3                          +   +   +Legionella pneumophila           9     In-23-G2-C2                          +   +   +Legionella pneumophila           10    Leiden 1 +   +   +Legionella pneumophila           11    797-PA-H +   +   +Legionella pneumophila           12    570-CO-H -   +   +Legionella pneumophila           13    82A3105  +/- +   +Legionella pneumophila           14    1169-MN-H                          + /-                              +   +Legionella bozemanii           1     WIGA     +   +   +Legionella bozemanii           2     Toronto 3                          -   +/- +/-Legionella dumoffii           1     NY-23    +/- +   -Legionella gormanii           1     LS-13    +   +   +/-Legionella micdadei           1     Tatlock  +/- +   +Legionella longbeachae           1     Long Beach 4                          +   +   +/-Legionella longbeachae           2     Tucker 1 +   +   +/-Legionella jordanis           1     BL-540   +   +   +/-Legionella oakridgensis           1     Oak Ridge 10                          -   -   +Legionella wadsworthii           1     81-716   +   +   -Legionella feeleii           1     WO-44C-C3                          -   -   +Legionella feeleii           2     691-WI-H -   -   +Legionella sainthelensi           1     Mt.St.Helen 4                          +   +   +Legionella anisa           1     WA-316-C3                          +   +   +/-Legionella santicrucis           1     SC-63-C7 -   +   +Legionella steigerwaltii           1     SC-18-C9 +   +   +/- Legionella parisiensis           1     PF-209C-C2                          +   +   +Legionella spiritensis           1     Mt.St.Helen 9                          +   +   +Legionella hackeliae           1     Lansing 2                          -   -   +/-Legionella hackeliae           2     798-PA-H -   -   +/-Legionella maceachernii           1     PX-1-G2-E2                          +/- +/- +Legionella jamestowniensis           1     JA-26-G1-E2                          +/- +   +Legionella cherrii           1     ORW      +   +   +/-Legionella rubrilucens           1     WA-270A-C2                          +   +   +Legionella erythra           1     SE-32A-C8                          +/- +/- -Legionella israelensis           1     Bercovier 4                          -   -   +Legionella birminghamensis           1     1407-AL-H                          +/- +/- +Legionella cincinnatiensis           1     72-OH-H  +   +   +/-Pseudomonas testosteroni                 KC1765   -   -   -Yersinia pseudotuberculosis                 514-84   -   -   -Shigella sonnei 0     85       -   -   -Pseudomonas aeruginosa                 5        -   -   -Streptococcus pneumoniae                 Pn-1     -   -   -Pseudomonas maltophilia                 KC1768   -   -   -Bordetella bronchisepta                 F6286    -   -   -Bordetella bronchisepta                 F6287    -   -   -Pseudomonas diminuta  KC1797   -   -   +/-Pseudomonas cepacia   KC1766   -   -   -Serratia marcescens   4391-83  -   -   -Escherichia coli      16       -   -   -Serratia marcescens   4391-83  -   -   -Escherichia coli      16       -   -   -Hemophilus influenzae a                 KC818    -   -   -Pseudomonas fluorescens                 CDC93    -   -   -Escherichia coli      013      -   -   -Klebsiella ascorbata  426-84   -   -   -Escherichia fergusonii                 1295-83  -   -   -Shigella flexneri     37       -   -   -Staphylococcus aureus 42BP     -   -   -Yersinia enterocolitica                 1149-84  +/- +/- -Hemophilus influenzae e                 KC528    -   -   +/-Hemophilus influenzae b                 1179-85  -   -   +/-Pseudomonas aeruginosa                 2        -   -   -Pseudomonas aeruginosa           0     8        -   -   -Klebsiella oxytoca    4698-84  -   -   -Escherichia hermanii  460-84   -   -   -Providencia stuartii  4007-83  -   -   -Providencia rettgeri  5317-81  -   -   -Enterobacter aerogenes                 1942-81  -   -   -Alcaligenes faecalis           -   -   -Acinetobacter lwoffii mima     -   -   -Klebsiella pneumoniae 4809-84  -   -   -Pseudomonas paucimobilis                 B3271    -   -   -Bordetella pertussis  F6324    -   -   -Bordetella pertussis  F6323    -   -   -Pseudomonas alcaligenes                 ABB50    -   -   -Pseudomonas acidovorans                 KC1769   -   -   -Neisseria meningitidis           C     KC792    -   -   -Pseudomonas fluorescens                 E.B.     -   -   -Bordetella parapertussis                 E1142    -   -   -Flavobacterium meningosepticum           0     698      -   -   -Listeria monocytogenes                 KC1775   -   -   -Listeria monocytogenes                 KC2380   -   -   -Listeria innocua      KC1783   -   -   -Listeria innocua      KC1784   -   -   -Streptococcus pyogenes                 SS482    -   -   -Streptococcus pyogenes                 SS91     -   -   -Campylobacter jejuni  D133     +/- +/- -Campylobacter jejuni  L1       +/- +/- -Campylobacter jejuni  L2       +/- +/- -Campylobacter jejuni  L9       +/- +/- -Campylobacter fetus   D223     -   -   -Campylobacter fetus   D373     -   +/- -Campylobacter fetus   D406     -   -   -Campylobacter fetus   D411     -   -   -Vibrio cholerae       01 Inata -   -   -Salmonella typhimurium                 2489-88  -   -   -Clostridium difficile           0     880-221  -   -   -Clostridium perfringens                 860386   -   -   -Clostridium septicum  70426    -   -   -Streptococcus salivarius                 SS908    -   -   -Streptococcus salivarius                 SS1062   -   - 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