Patent Grant
8642275
This application is a §371 national stage of PCT International Application No. PCT/PL2008/05015, filed Sep. 16, 2008, and claims priority of Polish Patent Application Nos. P.383361, P.383362, P.383363, P.383364, P.383365 and P.383366, filed Sep. 16, 2007, the contents of all of which are hereby incorporated by reference into this application.
The subject of the present invention is the use of a bacterial surface antigen obtained from bacterial cells in the production of antibodies for the manufacture of immunological tests for detecting bacterial cells, particularly of Clavibacter michiganensis subsp. sepedonicus.
Clavibacter michiganensis subsp. sepedonicus (Spickermann et Kotthoff) Davis et al. (Cms), the cause of potato bacterial ring rot, is one of the foremost pathogens of the potato (Lee et al. 1997, OEPP/EPPO 2006). Synonymous names of the pathogen include: Corynebacterium michiganensis subsp. sepedonicus (Spieckermann et Kotthof) Carlson et Vidaver, Corynebacterium michiganensis pv. sepedonicus (Spieckermann et Kotthof) Dye et Kemp as well as Corynebacterium sepedonicus (Spieckermann et Kotthof) Skaptason et Burkholder (OEPP/EPPO, 2006).
Clavibacter michiganensis subsp. sepedonicus is a gram-positive, rod-shaped or coccal bacteria (OEPP/EPPO, 2006) which, along with the genus Rathayibacter, form the phytopathogenic group Corynebacteria with a common phylogenetic ancestry (Lee et al. 1997). The colonies of most cultured strains are mucoidal, though intermediate and non-mucus producing strains also occur (Bishop et al. 1988, Baer and Gudmestadt 1993).
Cms virulence factors know to date are:
To date, no successful method has been found of direct chemical or biological action against the pathogen. Surface disinfection of tubers does little, since the bacteria reside in vascular bundles, to which the disinfectant has no access.
One of the most effective methods of limiting or eliminating the causal agent of potato bacterial ring rot is its early detection. This is essential during production, processing and distribution of plant material. Thus effective detection methods should be appropriately sensitive and specific, as well as being simple, fast, reliable and repeatable (Waleron et al. 2003).
According to phyto-sanitary requirements, the effective diagnostics of the causal agent of potato bacterial ring rot require the use of at least two laboratory tests based on different biological processes, along with a pathogenicity test (OEPP/EPPO, 2006). Depending on the expression of symptoms of potato bacterial ring rot, appropriate protocols are chosen: physiological, biochemical, serological, molecular and others (EC Directive 2006/56).
Physiological and biochemical tests used in routine controls facilitate the phenotyping of Cms. According to the latest EC directive, these tests must be performed using bacterial isolates obtained through passaging on agar media with glucose. Morphological comparisons must be performed in cultures derived following growth on agar media with glucose in the presence of a known Cms control (EC Directive 2006/56).
In the case of plants with evident symptoms, bacterial isolation on semi-selective NCP-88 media is performed (De la Cruz et al. 1992) as well as MTNA (Jansing and Rudolph 1998), whereas routine cultures of pure Cms bacteria make us of growth media such as nutrient agar medium (NA), dextrose nutrient agar (NDA), yeast peptone glucose agar (YPGA) as well as a mineral medium with glucose and yeast extract (YGM) (De Boer and Copeman 1980, EC Directive 2006/56).
One of the main problems with the detection and identification of Cms bacteria is the slow growth rate of this pathogen. With direct inoculation onto universal medium, exogenous saprophytic bacteria characterised by notably higher growth rates overgrow the entire surface of the medium, often making it impossible to successfully isolate pure Cms strains (OEPP/EPPO, 2006). Pathogen isolation on semi-selective media containing antibiotics such as polymyxin sulphate B, nalidixylic acid and cycloheximide (NCP-88) as well as trimetoprim, and amphotercine B (MTNA), makes it possible to decrease the amount of other bacteria. The most effective isolations on semi-selective media can be obtained following inoculation of aubergine plants with pathogenic cell extracts. Aubergine constitutes a good environment for selectively culturing the causal agent of potato bacterial ring rot (OEPP/EPPO 2006).
The pathogenicity test on young aubergine plants inoculated with a three-day culture of the tested isolate is used as a final test of latent infection as well as for the evaluation of virulence of cultures identified as Cms. This test is characterised by a relatively low sensitivity (106 CFU/ml) as well as being time-intensive, ca. 40 days. To shorten this time, tobacco is also used as an indicator plant. This test, however, has the drawback of lower sensitivity (108-109 CFU/ml) (OEPP/EPPO 2006).
One of the long used methods of identifying Cms bacteria has been Gramm staining of smears from the vascular bundles and tubers (Glick et al. 1944, Slack 1987). However, unequivocal diagnoses with this method are difficult due to the occurrence of other gram+ bacteria such as Clostridium ssp, Bacillus ssp., as well as saprophytic bacteria of the genus Corynebacterium (Crowley and De Boer 1982).
Some laboratories have made use of the latex agglutination test, making it possible to detect 107 cells in 1 ml (Slack et al. 1979). Reverse passive hemagglutination assays with ovine erythrocytes coated with anti-Cms antibodies from rabbit serum or chicken egg yolk make it possible to discover the presence of Cms within 90 minutes at concentrations of 6×106 per 1 ml (Kohm and Eggers-Schumacher 1995).
The serological detection of the causal organism of potato bacterial ring rot continues to cause many difficulties due to the morphological differentiation of the forms of the pathogen (Mills and Russell 2003). Commonly used serological tests are immunofluorescence, IF (Slack et al. 1979, De Boer and Copeman 1980), as well as immunoenzymeatic tests, ELISA (Enzyme Linked Immunoabsorbent Assay) (Drennan et al. 1993, De Boer et al. 1994, 2005). Although ELISA assays are disallowed by the EPPO as screening assays, they are much more popular in North America. Using monoclonal MAb 9A1 antibodies in an IF test ensures high specificity (De Boer and Wieczorek 1984) and detection of Cms bacteria in the range of 104 cells per ml of potato tissue extract (Baer and Gudmestad 1993). Often, sensitivity is increased through the use of cheaper, but less specific polyclonal antibodies. However, this solution has the drawback often yielding false positive results due to cross-reactions with other bacteria (Crowley and De Boer 1982) as well as not detecting non-mucus forming strains of Cms bacteria (Baer and Gudmestad 1993). The main antigen detected are not Cms cells, but soluble exopolysaccharides whose amounts in relation to cell numbers may vary (Lewosz 1997). Both methods, IF and ELISA, make it possible to detect Cms bacteria in potato tubers and stalks, wherein the immunofluorescent method exhibits higher specificity in tubers, whereas ELISA detects the pathogen with greater specificity in stalks (De Boer et al. 1994).
Modern diagnostics make use of traditional, well established methods of detecting microorganisms which have many advantages. They are usually time-intensive and require specialised laboratory equipment. This precludes field testing, where an almost instantaneous result is needed, and the sample should be analyzed on-site (oś and W
grzyn, 2005). Since to-date the most effective method of preventing bacterial pathogen dispersion has been early detection, such conditions must be met in an increasing number of procedures. The need exists for fast, sensitive and direct methods of detection, both specific and inexpensive, and which would facilitate real time control during plant growth, production and storage, as well as food distribution (De Boer and Beumer 1999).
Thus, the problem rests in finding a method of quantifying Clavibacter michiganensis subsp. sepedonicus (Cms) with immunological methods using polyclonal anti-Cms antibodies.
Unexpectedly, this problem has been solved through the use of the method according to the present invention.
The first subject of the present invention is the use of a bacterial surface antigen obtained from mucoidal bacteria through washing the bacteria in a buffer selected from among glycine-HCl or glycine-NaOH to produce an immunological tests for detecting mucoidal and non-mucoidal bacteria. Preferentially, the glycine-HCl solution is used at a concentration of 0.001-1 M, preferentially 0.05-0.5 M, as well as at a pH of 1.5-3.5. preferentially 2-3. Equally preferentially the glycine-NaOH solution is used at a concentration of 0.001-1 M, preferentially in the range 0.05-0.5 M, pH 9.5-12. preferentially in the range 10-11. Preferentially, the antibody is tagged radioactively or colloidally, where the antibody is preferentially coated with gold.
The second subject of the present invention is a method of detecting the presence of bacteria in an analysed sample, characterised in that the analysed sample is suspended in a buffered solution, the resulting solution is put into contact with the tagged antibody specific against the surface antigen of the bacterium to be detected, whereafter the solution is filtered on a filter impermeable to the bacteria, wherein the presence of tagged bacteria on the filter is evidence of said bacteria in the analysed sample. Preferentially, the antibodies are tagged with colloidal gold. Equally preferentially, antibodies specific against mucoidal bacteria are obtained through immunization using the bacterial antigen obtained from mucoidal bacteria through washing bacterial cells in buffered solutions of glycine-HCl or glycine-NaOH. Preferentially, the antibacterial membranes are cellulose, polycarbonate or other membranes. Preferentially, the presence of Cms bacteria is detected, whereas the presence of antibodies tagged with colloidal gold is detected with the reduction of silver ions on colloidal gold. Equally preferentially the method, is characterised in that the filtration makes use of activated large-pore polycarbonate membranes, permeable to bacteria and modified with glutaryl aldehyde, containing immobilised anti-Cms antibodies. In another equally preferential embodiment of the present invention, the method is characterised in that the membranes facilitate the selective immuno-trapping of Cms bacteria, wherein antibodies specific against the bacteria are obtained through immunization using the bacterial antigen obtained by rinsing bacteria with a buffered solution selected from among glycine-HCl or glycine-NaOH. In the next preferential embodiment of the present invention, the method is characterised in that the presence of Cms bacteria is detected. In the next equally preferential embodiment of the present invention, the method is characterised in that the presence of bacteria other than Cms is detected, wherein the detection of the presence on the filter of these bacteria depends on the antibodies used. In the next equally preferential embodiment of the present invention a method is characterised in that the presence of bacteria immuno-trapped on the filter is ascertained using antibodies. Preferentially, a method according to the present invention is characterised in that antibodies tagged with colloidal gold are used. Equally preferentially, a method according to the present invention is characterised in that antibodies tagged with colloidal gold are detected using the reduction of silver ions on the colloidal gold. In the next equally preferential embodiment of the present invention, the method is characterised in that antibodies tagged with a fluorochrome are used. In the next equally preferential embodiment of the present invention, the method is characterised in that a fluorochrome such as carbocyanin Cy3 or any other is used. Equally preferentially, a method according to the present invention is characterised in that the presence of bacteria immuno-trapped on the filter is evaluated through culturing the cells. Equally preferentially, a method according to the present invention is characterised in that the culturing of the bacterial cells occurs after placing the immobilising filter on the surface of the medium containing nutrients for the immuno-trapped bacterial cells. In the next equally preferential embodiment of the present invention, the method is characterised in that the medium may be a selective, semi-selective or mineral medium. Equally preferentially, a method according to the present invention is characterised in that the activation of the membranes encompasses the following: coating of the membranes with an aniline polymer, and chemical modification of the polyanilin.
The third subject of the present invention is a kit for detecting the presence of bacteria in the analysed sample, characterised in that it contains plates possessing surfaces coated with activated aniline modified with glutaryl aldehyde with immuno-trapped antibodies specific against the selected bacterial antigen. Preferentially, the plates are in the form of Petri dishes or others, composed of material selected from among: polystrene, polyethylene, polycarbonate or glass. Preferentially, the antibodies specific against mucoidal bacteria are obtained via immunization using the bacterial antigen obtained from bacterial cells through rinsing bacteria with a buffered solution selected from among glycine-HCl or glycine-NaOH. Preferentially, the antibodies used are tagged, wherein preferentially the tag is selected from among: colloidal gold, enzyme, or fluorochrome. Preferentially, a kit according to the present invention additionally contains nutrients for the immuno-trapped bacterial cells. Preferentially, the nutrients are selected from among: selective media, semi-selective media and mineral media. The subject of the present invention is also the use of the kit according to the present invention in detecting or live isolation of selected bacteria.
The fourth subject of the present invention is a kit for detecting the presence of bacteria in the analysed sample, characterised in that it contains an essentially granular immunoabsorbent possessing a chemically activated surface with colloidal gold and immobilised antibodies specific against the selected bacterial antigen. Preferentially, immunoabsorbent is in the form of a polycarbonate or mineral carrier, preferentially glass microspheres. Preferentially, antibodies specific against mucoidal bacteria are produced via immunisation with the bacterial antigen obtained bacterial cells via rinsing bacteria with a buffered solution selected from among glycine-HCl or glycine-NaOH. Preferentially, the antibodies are additionally tagged with a marker, wherein, preferentially, the marker is selected from among: colloidal gold, enzyme, a fluorescent tag or a fluorochrome. Preferentially, a kit according to the present invention additionally contains nutrients for the immuno-trapped bacterial cells, which are preferentially selected from among: selective, semi-selective or mineral media. The subject of the present invention is also the use of a kit according to the present invention for detecting or live isolation of selected bacteria.
In light of the above, the subject of the present invention is also an immunological assay of the ELISA, IFAS or other commonly known sort, characterised in that the antigen from bacterial cells, preferentially mucoidal bacteria such as Cms, has been produced according to a method encompassing a stage of rinsing bacteria with buffered solutions: glycine-HCl or glycine-NaOH in order to obtain the antigen for producing the antibodies. Equally preferentially, the antibody used in the immunological assay is tagged radioactively or colloidally. In the next embodiment of the present invention the antibody is coated with colloidal gold. A method of tagging bacteria using membranes, being the second subject of the present invention, particularly of polycarbonate membranes, possessing excellent physicochemical properties, yields a white background, on which the tagged bacteria are very visible. The hue of the tagged bacterial cells does not fade, as is the case for some fluorochromes. Staining with a conjugate of colloidal gold and then silver staining of tagged bacterial cells makes it possible to preserve the image for several months. The test is a simple and quick method for evaluating the presence of Cms bacteria isolated from pure cultures in over a dozen samples simultaneously. Another advantage is the universal nature of the test, which, depending on the antibody used, can be used successfully against other bacteria. In the present invention, activation using an aniline polymer was used as a method of obtaining a functional immuno-biochip. Test device surfaces subjected to chemical modification as well as immunoactivation according to the present invention, preferentially Petri dishes, may be produced from various materials (polystyrene, polyethylene, polycarbonate and glass). The present invention has made it possible to produce a functional substrate with novel chemical, optical and sorbent properties capable of activating antibodies (i.e. those directed against a new type of antigen, bacterial cells denuded of bacterial mucus to be used in the detection of Cms bacteria). The universality and functionality of this immunoabsorbent rests in the possibility of using it for every other bacteria, depending on the antibodies used. In the present invention, a facile immunoabsorbent was produced by using granular material a dextran gel (or glass microspheres), which were covalently coated with chitosan and colloidal gold following prior chemical modifications (via APTES, glutaryl aldehyde and aminocysteine). Of and in itself the dextran gel or glass microspheres are relatively inexpensive materials, easily obtained and functional. Both are in the form of microparticles, several to several dozen times larger than bacteria, and much heavier. This characteristic enables them to be used in binding and extracting bacteria and other biomolecules (viruses, proteins etc.) from a tested solution, without using additional equipment such as a centrifuge. Significant advantages gained in the present invention are: the possibility of binding various biomolecules on the surface of colloidal gold, which exhibits many advantageous characteristics as an absorbent medium, an increased absorptive surface of the medium, the possibility of covalently immobilizing antibodies and other biomolecules. A dextran hydrogel in the form of microspheres activated with antibodies against a given bacterium facilitates rapid contact between the antibodies and bacteria of interest. Unmodified dextran particles settle relatively slowly. After “weighing” with a layer of colloidal gold occluding on the surface of the dextran, they settle on the bottom within 2-3 minutes. Thus, additional procedures such as centrifugation are not necessary, to rinse and separate the microspheres from the impure solution.
Aqueous suspensions were made of the cells of the retained Cms bacterial strains, BPR-527, PD 221 as well as PD 406. The suspensions were washed thrice with ddH2O and centrifuged 15-25 min. at 7000 g on a Beckman J-21 centrifuge. The supernatant was discarded, an exopolysaccharides remaining on the bacterial cells were washed off 1-6 with 0.001-1M glycine-HCl buffer (pH 1.5-3.5), 1-6 times with 0.001-1M glicyne-NaOH buffer (pH 9.5-12), thrice with sterile H2O, centrifuging the bacteria each time as above. Bacteria were lyophilised, and a mixture of the three lyophilisates was prepared in a ratio of 1:1:1 by mass. Subcutaneous immunisation was performed on a rabbit using a 1% aqueous solution of lyophilisate with Gerbu 100 adjuvant (1:1 v/v). The suspension was administered six times at biweekly intervals in 1 ml doses. Bleeding and antibody isolation was performed according to Ball et al. (1993). The blood was collected from the peripheral vein of the ear directly into centrifuge tubes. After collection, it was left to coagulate for 30 min. at 37° C., and then overnight at 4° C. The clot was gently separated from the walls and centrifuged for 15 minutes at 1000 g at 4° Cin a Beckman J-21 centrifuge. The serum was gently decanted, NaN3 was added to a concentration of 0.02%, and then diluted tenfold with ddH2O. An equal volume of saturated ammonium sulphate was added, gently mixed and left for 60 min. at RT in order to precipitate. The mixture was centrifuged for 5 min. at 8000 g on a Sorvall RC-5B centrifuge. The supernatant was discarded and the precipitate was dissolved in 0.5×PBS pH 7.4 in a volume double that of the initial serum volume, and dialysed overnight against three changes of the same buffer with 0.02% NaN3. The dialysate was placed on a DEAE-cellulose column equilibrated with 0.5×PBS with 0.02% NaN3. which was then used to wash the column, eluting the antibody. 3 ml fractions were collected whose A280/A250 absorbance ratio was =2.5÷2.7. The fractions were pooled and then passed through an antibacterial filter 0.2 μm and stored at 4° C. Antibody solutions were brought to a concentration of 2 mg/ml using the above mentioned buffer, accepting that IgG absorbance at 1 mg/ml at λ=280 nm is 1.4. Antibodies react with Cms cells encapsulated with mucus as well as slightly with cells partially denuded of bacterial mucus (Table 5).
In order to obtain Fab fragments, a reduction reaction was performed on the disulphide (S-S) bonds of whole IgG molecules, incubating them in EDTA-DTT in anoxic conditions. Six solutions with varying antibody concentrations were prepared, ranging from 20 do 500 μg/ml in 0.2 ml final volume of freshly prepared EDTA-DTT. The solutions, in 1.5 ml Eppendorf tubes, were incubated overnight at RT under 0.1 MPa vacuum. After opening, the solutions containing the IgG fragments were immediately combined with a standard colloidal gold solution from 4.0 at a 1:1 (v/v) ratio. The solutions were incubated for 10 minutes at RT with gentle mixing, whereafter 50 μl of each solution was collected and mixed with 50 μl of 2N NaCl. After determining optimal proportions, the colloidal gold was mixed with an appropriate amount of reduced antibodies and incubated for 5-60 minutes at RT with gentle mixing. To avoid non-specific absorption, the remaining colloidal gold surface was blocked with 1-20% BSA, whereas surplus unbound fragments were removed from the conjugate solution using 3-fold rinsing with TBS-BSA and concentration on an Eppendorf 5415C centrifuge (0.5-2 hours at 1000-4000 g). The precipitate from the last centrifugation was brought to an from OD535 nm=0.1-4.0 with the latter buffer and stored at 4° C. (IgG-Au1 solution).
Antibody solutions with concentrations of 0.01-1 mg/ml were made, which were dialysed overnight at 4° C. against 3 changes of 10 mM borate buffer pH 9.2 and brought to a concentration of 0.1 mg/ml assuming that the A280 nm of such a solution is 0.140. Whereas a standard colloidal gold solution was brought to 10 mM with borate buffer pH 9.0. An initial titration was performed by making IgG solutions with concentrations ranging from 5 do 50 rig/ml, which were combined with a double volume of colloidal gold solution from 4.0, mixed and incubated 10 minutes at RT. Next, 50 μl of each solution was sampled and mixed with 50 μl 2N NaCl. After determining the optimal proportions, the colloidal gold was mixed with an appropriate amount of reduced antibodies and incubated 5-60 minutes at RT with gentle mixing. The procedure then followed that of Example 1. The precipitate obtained from the final centrifugation was brought, using TBS-BSA buffer, to an OD535 nm of 0.1-4.0 and stored 4° C. (IgG-Au2 solution).
Aqueous suspensions were made of the cells of the retained Cms bacterial strains, BPR-527, PD 221 as well as PD 406. The suspensions were washed thrice with ddH2O and centrifuged 15-25 min. at 7000 g on a Beckman J-21 centrifuge. The supernatant was discarded, an exopolysaccharides remaining on the bacterial cells were washed off 1-6 with 0.001-1M glycine-HCl buffer (pH 1.5-3.5), 1-6 times with 0.001-1M glicyne-NaOH buffer (pH 9.5-12), thrice with sterile H2O, centrifuging the bacteria each time as above. Bacteria were lyophilised, and a mixture of the three lyophilisates was prepared in a ratio of 1:1:1 by mass. Subcutaneous immunisation was performed on a rabbit using a 1% aqueous solution of lyophilisate with Gerbu 100 adjuvant (1:1 v/v). The suspension was administered six times at biweekly intervals in 1 ml doses. Bleeding and antibody isolation was performed according to Ball et al. (1993). The blood was collected from the peripheral vein of the ear directly into centrifuge tubes. After collection, it was left to coagulate for 30 min. at 37° C., and then overnight at 4° C. The clot was gently separated from the walls and centrifuged for 15 minutes at 1000 g at 4° Cin a Beckman J-21 centrifuge. The serum was gently decanted, NaN3 was added to a concentration of 0.02%, and then diluted tenfold with ddH2O. An equal volume of saturated ammonium sulphate was added, gently mixed and left for 60 min. at RT in order to precipitate. The mixture was centrifuged for 5 min. at 8000 g on a Sorvall RC-5B centrifuge. The supernatant was discarded and the precipitate was dissolved in 0.5×PBS pH 7.4 in a volume double that of the initial serum volume, and dialysed overnight against three changes of the same buffer with 0.02% NaN3. The dialysate was placed on a DEAE-cellulose column equilibrated with 0.5×PBS with 0.02% NaN3. which was then used to wash the column, eluting the antibody. 3 ml fractions were collected whose A280/A250 absorbance ratio was =2.5÷2.7. The fractions were pooled and then passed through an antibacterial filter 0.2 μm and stored at 4° C. Antibody solutions were brought to a concentration of 2 mg/ml using the above mentioned buffer, accepting that IgG absorbance at 1 mg/ml at λ=280 nm is 1.4. Antibodies react with Cms cells encapsulated with mucus as well as slightly with cells partially denuded of bacterial mucus (Table 1).
In order to obtain Fab fragments, a reduction reaction was performed on the disulphide (S-S) bonds of whole IgG molecules, incubating them in EDTA-DTT in anoxic conditions. Six solutions with varying antibody concentrations were prepared, ranging from 20 do 500 μg/ml in 0.2 ml final volume of freshly prepared EDTA-DTT. The solutions, in 1.5 ml Eppendorf tubes, were incubated overnight at RT under 0.1 MPa vacuum. After opening, the solutions containing the IgG fragments were immediately combined with a standard colloidal gold solution from 4.0 at a 1:1 (v/v) ratio. The solutions were incubated for 10 minutes at RT with gentle mixing, whereafter 50 μl of each solution was collected and mixed with 50 μl of 2N NaCl. After determining optimal proportions, the colloidal gold was mixed with an appropriate amount of reduced antibodies and incubated for 5-60 minutes at RT with gentle mixing. To avoid non-specific absorption, the remaining colloidal gold surface was blocked with 1-20% BSA, whereas surplus unbound fragments were removed from the conjugate solution using 3-fold rinsing with TBS-BSA and concentration on an Eppendorf 5415C centrifuge (0.5-2 hours at 1000-4000 g). The precipitate from the last centrifugation was brought to an from OD535 nm=0.1-4.0 with the latter buffer and stored at 4° C. (IgG-Au1 solution).
Antibody solutions with concentrations of 0.01-1 mg/ml were made, which were dialysed overnight at 4° C. against 3 changes of 10 mM borate buffer pH 9.2 and brought to a concentration of 0.1 mg/ml, assuming that the A280 nm of such a solution is 0.140. Whereas a standard colloidal gold solution was brought to 10 mM with borate buffer pH 9.0. An initial titration was performed by making IgG solutions with concentrations ranging from 5 do 50 μg/ml, which were combined with a double volume of colloidal gold solution from 4.0, mixed and incubated 10 minutes at RT. Next, 50 μl of each solution was sampled and mixed with 50 μl 2N NaCl. After determining the optimal proportions, the colloidal gold was mixed with an appropriate amount of reduced antibodies and incubated 5-60 minutes at RT with gentle mixing. The procedure then followed that of Example 1. The precipitate obtained from the final centrifugation was brought, using TBS-BSA buffer, to an OD535 nm of 0.1-4.0 and stored 4° C. (IgG-Au2 solution).
A series of suspensions of Ervinia carotovora subsp. carotovora were made in 10 mM PBS pH 7.2 in dilutions from 5*102 to 5*105 CFU/ml. 100 μl of each suspension was collected and 20 μl of the colloidal gold-tagged conjugate with anti-Ecc IgG were added to each. After 10 minutes, the mixture was filtered on polycarbonate membranes (porosity 0.4 μm) in a vacuum transfer apparatus. Following 1-6 rinses in 1×PBS pH 7.4 with 0.005-1% Tween 20, spot intensity on the membrane surface was examined or bacteria were observed under an optical microscope. To make the tagged Ecc bacteria more visible, the optical signal was amplified using reduction of silver ions on the colloidal gold. For this procedure, the membranes were placed into a freshly made solution of 0.015-0.5 M hydroquinone, 0.033-15 mM silver nitrate in 10 mM citrate buffer pH 2.5-4.0 and incubated around 2-12 min. The reaction was stopped by placing the membranes in ddH2O and drying on tissue paper. Ecc bacteria tagged with a conjugate of colloidal gold z with IgG-AP was contrasted enzymatically using BCIP/NBT as a substrate. Spots on the membrane were covered with 50 μl of freshly prepared BCIP/NBT solution and incubated 2-20 min. at room temperature. The reaction was stopped by placing the membranes in 1×PBS, pH 7.4 with 0.05% Tween 20 and drying on tissue paper. After amplifying the signal, the staining intensity of the spots on the membrane was examined using the unaided eye or bacteria were observed under an optical microscope.
A series of suspensions of Ervinia amyloria were made in 10 mM PBS pH 7.2 in dilutions from 5*102 to 5*105 CFU/ml. 100 μl of each suspension was collected and 20 μl of the colloidal gold-tagged conjugate with anti-Ea IgG were added to each.
After 10 minutes, the mixture was filtered on polycarbonate membranes (porosity 0.4 μm) in a vacuum transfer apparatus. Following 1-6 rinses in 1×PBS pH 7.4 with 0.005-1% Tween 20, spot intensity on the membrane surface was examined or bacteria were observed under an optical microscope.
To make the tagged Ea bacteria more visible, the optical signal was amplified using reduction of silver ions on the colloidal gold. For this procedure, the membranes were placed into a freshly made solution of 0.015-0.5 M hydroquinone, 0.033-15 mM silver nitrate in 10 mM citrate buffer pH 2.5-4.0 and incubated around 2-12 min. The reaction was stopped by placing the membranes in ddH2O and drying on tissue paper.
Ecc bacteria tagged with a conjugate of colloidal gold z with IgG-AP was contrasted enzymatically using BCIP/NBT as a substrate. Spots on the membrane were covered with 50 μl of freshly prepared BCIP/NBT solution and incubated 2-20 min. at room temperature. The reaction was stopped by placing the membranes in 1×PBS, pH 7.4 with 0.05% Tween 20 and drying on tissue paper.
After amplifying the signal, the staining intensity of the spots on the membrane was examined using the unaided eye or bacteria were observed under an optical microscope.
A series of suspensions of Clavibacter michiganensis subsp. michiganensis were made in 10 mM PBS pH 7.2 in dilutions from 5*102 to 5*105 CFU/ml. 100 μl of each suspension was collected and 20 μl of the colloidal gold-tagged conjugate with anti-Cms IgG were added to each.
After 10 minutes, the mixture was filtered on polycarbonate membranes (porosity 0.4 μm) in a vacuum transfer apparatus. Following 1-6 rinses in 1×PBS pH 7.4 with 0.005-1% Tween 20, spot intensity on the membrane surface was examined or bacteria were observed under an optical microscope.
To make the tagged Ea bacteria more visible, the optical signal was amplified using reduction of silver ions on the colloidal gold. For this procedure, the membranes were placed into a freshly made solution of 0.015-0.5 M hydroquinone, 0.033-15 mM silver nitrate in 10 mM citrate buffer pH 2.5-4.0 and incubated around 2-12 min. The reaction was stopped by placing the membranes in ddH2O and drying on tissue paper.
The assay made use of polycarbonate membranes produced and chemically activated as in Example 1 and then activated with antibodies against Cms bacteria. IgG were immobilised covalently on the polyaniline surface using both a directed method and random method. In the first case, previously oxidated antibodies as well as chemically unmodified polyaniline were used, whereas in the second case chemically unmodified antibodies as well polyaniline activated with glutaryl aldehyde were used.
12.1 Directed Method
Polycarbonate membranes were activated as earlier in example 1, coated with 20 μl of a solution of oxidized antibodies at a concentration of 0.001-1 mg/ml in 1×PBS pH 7.4 with 20-60% glycerol. Membranes were incubated for 15-120 min. at 37° C. and washed thrice with 1×PBS pH 7.4. Next, 20 μl of 0.1-30% NaCNBH4 in 1×PBS pH 7.4 were placed on them for 15-120 min. under a fume extractor and washed thrice in 1×PBS pH 7.4. Activated surfaces were incubated 15-120 min. at 37° C. with blocking buffer (0.1-1.5% gelatine in 1×PBS, pH 7.4), and then washed thrice with 1×PBS pH 7.4 with 0.05% Tween 20.
12.2 Random Method
Polycarbonate membranes were activated with polyaniline and chemically modified with glutaryl aldehyde. Subsequently, 1-50 μl of antibody solutions with concentrations of 0.001-1 mg/ml in 1×PBS pH 7.4 and 20-60% glycerol, were immobilised. Next, the procedure was identical as in the directed method of antibody immobilisation.
A suspension was prepared of Cms bacteria in a mixture of potato juice with 1×PBS pH 7.4 in dilutions ranging from 100 to 25000 CFU/ml. A membrane with immunoactivated spots of polyaniline laid out exactly like microwells in a vacuum blotter were placed between the covering layers of a vacuum blot. 1 ml of each solution was sampled and filtered gently through the membranes. Excess unbound bacteria were rinsed off thrice with 1×PBS pH 7.4 with 0.05% Tween 20, whereas immuno-trapped Cms bacteria were evaluated using various techniques:
A mixture of potato tuber extract with 1×PBS pH 7.4 was prepared. The membrane with immunoactivated antibodies against Clavibacter michiganensis subsp. sepedonicus on spots of polyaniline laid out identically like microwells in a vacuum blotter, was placed between the halves of a vacuum blotter. 1 ml of each solution was sampled and filtered gently through the membranes. Excess unbound bacteria were rinsed off thrice with 1×PBS pH 7.4 with 0.05% Tween 20, whereas immuno-trapped Cms bacteria were evaluated by staining the bacteria with a conjugate of colloidal gold with anti-Cms antibodies and then amplifying the signal with silver ions.
A mixture of potato tuber extract with 1×PBS pH 7.4 was prepared. The membrane with immunoactivated antibodies against Clavibacter michiganensis subsp. sepedonicus on spots of polyaniline laid out identically like microwells in a vacuum blotter, was placed between the halves of a vacuum blotter. 1 ml of each solution was sampled and filtered gently through the membranes. Excess unbound bacteria were rinsed off thrice with 1×PBS pH 7.4 with 0.05% Tween 20, whereas immuno-trapped Cms bacteria were evaluated by staining using the immunofluorescent IFAS method and observing the results under a fluorescent microscope.
A mixture of potato tuber extract with 1×PBS pH 7.4 was prepared. The membrane with immunoactivated antibodies against Clavibacter michiganensis subsp. sepedonicus on spots of polyaniline laid out identically like microwells in a vacuum blotter, was placed between the halves of a vacuum blotter. 1 ml of each solution was sampled and filtered gently through the membranes. Excess unbound bacteria were rinsed off thrice with 1×PBS pH 7.4 with 0.05% Tween 20, whereas immuno-trapped Cms bacteria were evaluated live, by placing the membrane activated side down directly onto NCP-88 medium and incubating for 10 days at 21° C. live, by placing the activated side of the membrane directly onto NCP-88 medium and incubating for 10 days at 21° C.
Preparation of Media
The assay made use of Petri dishes of polycarbonate or polystyrene or polyethylene or glass activated chemically as in application No. 07. and then antibodies against Cms bacteria were activated.
IgG was immobilised covalently on the surface of polyaniline in both a directed and random method. In the first case, use was made of antibodies previously oxidated as well as chemically unmodified polyaniline, whereas the in the second case chemically unmodified antibodies as well as polyaniline activated with glutaryl aldehyde are used.
Directed Method
Petri dishes activated with polyaniline as in Example 1 were coated with 20 μl of a solution of previously oxidized anti-Cms antibodies at a concentration of 0.001-10 mg/ml in 1×PBS pH 7.4 with 5-70% glycerol. The surface of the dishes was incubated for 15-120 min. at 37° C. and rinsed thrice with 1×PBS pH 7.4. Next, 20 μl of 0.1-30% NaCNBH4 in 1×PBS pH 7.4. were loaded and placed for 15-120 min. under a fume extractor and subsequently rinsed thrice with 1×PBS pH 7.4. Activated surfaces were incubated for 15-120 min. at 37° C. in blocking buffer (0.1-1.5% gelatine in 1×PBS, pH 7.4), an then rinsed thrice in 1×PBS pH 7.4 with 0.05% Tween 20.
Random Method
Petri dishes activated with polyaniline and glutaryl aldehyde as in Example 1. 1-50 μl of anti-Cms antibody solution at a concentration of 0.001-1 mg/ml in 1×PBS pH 7.4 with 20-60% glycerol were immobilised. The Petri dishes were incubated for 15-120 min. at 37° C. and rinsed thrice in 1×PBS pH 7.4. Next, 20 μl was loaded of 0.1-30% NaCNBH4 in 1×PBS pH 7.4, placed for 15-120 min. under a fume extractor rinsed thrice with 1×PBS pH 7.4. Activated surfaces were incubated for 15-120 min. at 37° C. in blocking buffer (0.1-1.5% gelatine or 1-15% bovine albumin in 1×PBS, pH 7.4), an then rinsed thrice in 1×PBS pH 7.4 with 0.05% Tween 20.
A suspension of Cms bacteria in buffered potato tuber extract (1×PBS pH 7.4 or another) in dilutions ranging from 100 to 25000 CFU/ml was prepared. The mixture was placed in a previously prepared immunoactive Petri dish with z polyaniline and incubated from 15 min. to 24 h at 4° C., RT or 37° C. Surplus unbound bacteria were rinsed off thrice with 1×PBS pH 7.4 with 0.05% Tween 20, whereas immuno-trapped Cms bacteria were evaluated with various methods:
Said test may be realised in other variants:
Petri dishes modified chemically with polyaniline and glutaryl aldehyde as in Example 1 were coated with 25 μA of an antibody solution against bacteria Clavibacter michiganensis subsp. sepedonicus, with a concentration of 1 mg/ml in 1×PBS pH 7.4 z 20% glycerol. The Petri dishes were incubated for 30 min. at 37° C. and washed thrice in 1×PBS pH 7.4. Next, 25 μl 0.5% of NaCNBH4 in 1×PBS pH 7.4 was added and they were placed for 10 min. under a fume extractor and were rinsed thrice in 1×PBS pH 7.4. Activated surfaces were incubated for 30 min. at 37° C. with a blocking buffer with 5% bovine albumin in 1×PBS, pH 7.4, whence they were rinsed thrice in 1×PBS pH 7.4 with 0.05% Tween 20.
The potato tuber extract was diluted 1:1 with 1×PBS pH 7.4. The mixture was placed in a prepared Petri dish with polyaniline coated with anti-Cms antibodies and incubated for 2 hours at 37° C. Excess unbound bacteria were removed by rinsing thrice with 1×PBS pH 7.4 with 0.05% Tween 20, whereas immuno-trapped Cms bacteria were stained using indirect immunofluorescence using a conjugate of goat anti-rabbit antibodies tagged with indocarbpcyanine Cy3. Stained bacterial cells were observed under a fluorescent microscope.
Petri dishes modified chemically with polyaniline and glutaryl aldehyde as in Example 1 were coated with 25 μA of an antibody solution against Erwinia carotovora subsp. atroseptica, with a concentration of 1 mg/ml in 1×PBS pH 7.4 z 20% glycerol. The Petri dishes were incubated for 30 min. at 37° C. and washed thrice in 1×PBS pH 7.4. Next, 25 μl 0.5% of NaCNBH4 in 1×PBS pH 7.4 was added and they were placed for 10 min. under a fume extractor and were rinsed thrice in 1×PBS pH 7.4. Activated surfaces were incubated for 30 min. at 37° C. with a blocking buffer with 5% bovine albumin in 1×PBS, pH 7.4, whence they were rinsed thrice in 1×PBS pH 7.4 with 0.05% Tween 20.
The potato tuber extract was diluted 1:1 with 1×PBS pH 7.4. The mixture was placed in a prepared Petri dish with polyaniline coated with anti-Eca antibodies and incubated for 2 hours at 37° C. Excess unbound bacteria were removed by rinsing thrice with 1×PBS pH 7.4 with 0.05% Tween 20, whereas immuno-trapped Eca bacteria were stained using indirect immunofluorescence using a conjugate of goat anti-rabbit antibodies tagged with indocarbpcyanine Cy3. Stained bacterial cells were observed under a fluorescent microscope.
Petri dishes modified chemically with polyaniline and glutaryl aldehyde as in Example 1 were coated with 25 μl of an antibody solution against Erwinia carotovora subsp. carotovora, with a concentration of 1 mg/ml in 1×PBS pH 7.4 z 20% glycerol. The Petri dishes were incubated for 30 min. at 37° C. and washed thrice in 1×PBS pH 7.4. Next, 25 μl 0.5% of NaCNBH4 in 1×PBS pH 7.4 was added and they were placed for 10 min. under a fume extractor and were rinsed thrice in 1×PBS pH 7.4. Activated surfaces were incubated for 30 min. at 37° C. with a blocking buffer with 5% bovine albumin in 1×PBS, pH 7.4, whence they were rinsed thrice in 1×PBS pH 7.4 with 0.05% Tween 20.
The potato tuber extract was diluted 1:1 with 1×PBS pH 7.4. The mixture was placed in a prepared Petri dish with polyaniline coated with anti-Ecc antibodies and incubated for 2 hours at 37° C. Excess unbound bacteria were removed by rinsing thrice with 1×PBS pH 7.4 with 0.05% Tween 20, whereas immuno-trapped Ecc bacteria were stained using indirect immunofluorescence using a conjugate of goat anti-rabbit antibodies tagged with indocarbocyanine Cy3. Stained bacterial cells were observed under an epifluorescent microscope.
This test is equally capable of performing a live evaluation, as well as an optical evaluation of the morphology of the identified cells.
Petri dishes modified chemically with polyaniline and glutaryl aldehyde as in Example 1 were coated with 25 μA of an antibody solution against bacteria Clavibacter michiganensis subsp. sepedonicus, with a concentration of 1 mg/ml in 1×PBS pH 7.4 z 20% glycerol. The Petri dishes were incubated for 30 min. at 37° C. and washed thrice in 1×PBS pH 7.4. Next, 25 μl 0.5% of NaCNBH4 in 1×PBS pH 7.4 was added and they were placed for 10 min. under a fume extractor and were rinsed thrice in 1×PBS pH 7.4. Activated surfaces were incubated for 30 min. at 37° C. with a blocking buffer with 5% bovine albumin in 1×PBS, pH 7.4, whence they were rinsed thrice in 1×PBS pH 7.4 with 0.05% Tween 20.
The potato tuber extract was diluted 1:1 with 1×PBS pH 7.4. The mixture was placed in a prepared Petri dish with polyaniline coated with anti-Cms antibodies and incubated for 2 hours at 37° C. Excess unbound bacteria were removed by rinsing thrice with 1×PBS pH 7.4 with 0.05% Tween 20, whereas immuno-trapped Cms bacteria were stained using indirect immunofluorescence using a conjugate of goat anti-rabbit antibodies tagged with indocarbocyanine Cy3. Stained bacterial cells were observed under an epifluorescent microscope.
Petri dishes modified chemically with polyaniline and glutaryl aldehyde as in Example 1 were coated with 25 μA of an antibody solution against bacteria Clavibacter michiganensis subsp. sepedonicus, with a concentration of 1 mg/ml in 1×PBS pH 7.4 z 20% glycerol. The Petri dishes were incubated for 30 min. at 37° C. and washed thrice in 1×PBS pH 7.4. Next, 25 μl 0.5% of NaCNBH4 in 1×PBS pH 7.4 was added and they were placed for 10 min. under a fume extractor and were rinsed thrice in 1×PBS pH 7.4. Activated surfaces were incubated for 30 min. at 37° C. with a blocking buffer with 5% bovine albumin in 1×PBS, pH 7.4, whence they were rinsed thrice in 1×PBS pH 7.4 with 0.05% Tween 20.
The potato tuber extract was diluted 1:1 with 1×PBS pH 7.4. The mixture was placed in a prepared Petri dish with polyaniline coated with anti-Cms antibodies and incubated for 2 hours at 37° C. Excess unbound bacteria were removed by rinsing thrice with 1×PBS pH 7.4 with 0.05% Tween 20, whereas immuno-trapped Cms bacteria were stained using a conjugate of colloidal gold with anti-Cms antibodies, and the optical signal obtained was amplified using the reduction of silver on the colloidal gold. Bacterial cells thus stained were observed under a optical microscope.
Petri dishes modified chemically with polyaniline and glutaryl aldehyde as in Example 1 were coated with 25 μA of an antibody solution against bacteria Clavibacter michiganensis subsp. sepedonicus, with a concentration of 1 mg/ml in 1×PBS pH 7.4 z 20% glycerol. The Petri dishes were incubated for 30 min. at 37° C. and washed thrice in 1×PBS pH 7.4. Next, 25 μl 0.5% of NaCNBH4 in 1×PBS pH 7.4 was added and they were placed for 10 min. under a fume extractor and were rinsed thrice in 1×PBS pH 7.4. Activated surfaces were incubated for 30 min. at 37° C. with a blocking buffer with 5% bovine albumin in 1×PBS, pH 7.4, whence they were rinsed thrice in 1×PBS pH 7.4 with 0.05% Tween 20.
The potato tuber extract was diluted 1:1 with 1×PBS pH 7.4. The mixture was placed in a prepared Petri dish with polyaniline coated with anti-Cms antibodies and incubated for 2 hours at 37° C. Excess unbound bacteria were removed by rinsing thrice with 1×PBS pH 7.4 with 0.05% Tween 20, whereas immuno-trapped Cms bacteria were stained immunoenzymatically using a conjugate of anti-Cms antibodies with alkaline phosphatase and using NBT/BCIP as a substrate for the enzyme, yielding an insoluble product on the surface of Cms bacteria
A portion of the purified microspheres were activated chemically at a temperature of 4-37° C. each time treating them for 15-60 min. with an triple volume of the appropriate modifying solution and rinsing five times with ten volumes of ddH2O after each step. The following were used 0.5-25% APTES in 96% ethanol, 0.06-25% aqueous glutaryl aldehyde, 0.001-1% chitosan in 0.01% acetic acid, 0.0001-1 M aqueous cystamine as well as a colloidal gold solution with an OD of 0.05-10.0. Glass microspheres were activated chemically, colloidal gold was settled, and antibodies were immobilised as above.
Dextran microspheres with colloidal gold were coated with anti-Cms IgG antibodies (0.0005-5 mg/ml) in 0.001-2M borate buffer pH 9.2 and incubated for 30 min. to 2 hours at a temperature in the range 4-37° C. After removing excess unbound antibodies, in order to block the remaining microsphere surface two volumes of 0.1-15% bovine albumin were added, rinsed thrice with 10 volumes of TBS-BSA pH 8.2 and placed in 4° C.
Extracts were made of potato tubers suspected of being infected with Cms bacteria and 1×PBS pH 7.4 was added at a ratio of 1:1. The following was placed into individual Eppendorf tubes: 2 ml of solution and 10-50 μA of immunoabsorbent (Dextran microspheres with colloidal gold coated with anti-Cms antibodies prepared as in Example 1). The whole mixture was incubated for 15 min. to 2 hours at room temperature with gentle mixing, to keep the microspheres in solution. After stopping the mixing and autologous sedimentation, the solution was removed from over the dextran grains and rinsed using 1×PBS pH 7.4 with 0.05% Tween 20, in this way removing surplus unbound bacteria. This was repeated twice, and then the bacteria trapped on the above immunoabsorbents were evaluated in two ways:
The test using glass microspheres is performed identically as for the dextran grains
0
0.218
0.458
0.111
0.225
0.604
0.658
0.166
0.492
0.835
0.973
0.215
0.702
0.865
1.302
Other embodiments of the test predict its use against other types of bacteria.
Dextran microspheres with colloidal gold were coated with anti-Cms IgG antibodies 0.5 mg/ml in 0.01 borate buffer pH 9.2 and incubated for 30 min. at a temperature in the range of 4-37° C. After removing excess unbound antibodies, in order to block the remaining microsphere surface two volumes of 10% bovine albumin were added, rinsed thrice with 10 volumes of TBS-BSA pH 8.2 and placed in 4° C.
Extracts were made of potato tubers suspected of being infected with Cms bacteria and 1×PBS pH 7.4 was added at a ratio of 1:1. The following was placed into individual Eppendorf tubes: 2 ml of solution and 30 μA of immunoabsorbent Dextran microspheres with colloidal gold coated with anti-Cms antibodies. The whole mixture was incubated for 30 min. at room temperature with gentle mixing, to keep the microspheres in solution. After stopping the mixing and autologous sedimentation, the solution was removed from over the dextran grains and rinsed using 1×PBS pH 7.4 with 0.05% Tween 20, in this way removing surplus unbound bacteria. This was repeated twice, and then the bacteria trapped on the above immunoabsorbents were evaluated immunoenzymatically using as a marker a conjugate of IgG anti-Cms with alkaline phosphatase and incubating with a substrate solution of pNPP, performing absorbance readings at λ=405 nm following 1, 2 and 4 hours of incubation.
Dextran microspheres with colloidal gold were coated with anti-Erwinia carotovora subsp. carotovora IgG antibodies 0.5 mg/ml in 0.01 borate buffer pH 9.2 and incubated for 30 min. at a temperature in the range of 4-37° C. After removing excess unbound antibodies, in order to block the remaining microsphere surface two volumes of 10% bovine albumin were added, rinsed thrice with 10 volumes of TBS-BSA pH 8.2 and placed in 4° C.
Extracts were made of potato tubers suspected of being infected with Ecc bacteria and 1×PBS pH 7.4 was added at a ratio of 1:1. The following was placed into individual Eppendorf tubes: 2 ml of solution and 30 μA of immunoabsorbent Dextran microspheres with colloidal gold coated with anti-Ecc antibodies. The whole mixture was incubated for 30 min. at room temperature with gentle mixing, to keep the microspheres in solution. After stopping the mixing and autologous sedimentation, the solution was removed from over the dextran grains and rinsed using 1×PBS pH 7.4 with 0.05% Tween 20, in this way removing surplus unbound bacteria. This was repeated twice, and then the bacteria trapped on the above immunoabsorbents were evaluated immunoenzymatically using as a marker a conjugate of IgG anti-Ecc with alkaline phosphatase and incubating with a substrate solution of pNPP, performing absorbance readings at λ=405 nm following 1, 2 and 4 hours of incubation.
| Number | Date | Country | Kind |
|---|---|---|---|
| 383361 | Sep 2007 | PL | national |
| 383362 | Sep 2007 | PL | national |
| 383363 | Sep 2007 | PL | national |
| 383364 | Sep 2007 | PL | national |
| 383365 | Sep 2007 | PL | national |
| 383366 | Sep 2007 | PL | national |
| Filing Document | Filing Date | Country | Kind | 371c Date |
|---|---|---|---|---|
| PCT/PL2008/050015 | 9/16/2008 | WO | 00 | 6/17/2010 |
| Publishing Document | Publishing Date | Country | Kind |
|---|---|---|---|
| WO2009/035357 | 3/19/2009 | WO | A |
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| 6015681 | Ralls et al. | Jan 2000 | A |
| 6602669 | Letsinger et al. | Aug 2003 | B2 |
| 20020176084 | Garini | Nov 2002 | A1 |
| 20040089079 | Engebretson | May 2004 | A1 |
| 20050244943 | Ladisch et al. | Nov 2005 | A1 |
| 20060141546 | Pugia et al. | Jun 2006 | A1 |
| 20090054254 | Throsby et al. | Feb 2009 | A1 |
| Number | Date | Country |
|---|---|---|
| 293606 | Sep 1991 | DE |
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| Number | Date | Country | |
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| 20100255511 A1 | Oct 2010 | US |
