FIELD OF THE INVENTION
The present invention in general relates to field of immunoassays. Particularly, the present invention relates to a method of detection of cells using immunofluorescence assay. More particularly the present invention relates to a method of direct and rapid detection of cells.
BACKGROUND ART
Rapid and accurate detection of trace amounts of organisms such as pathogenic bacteria is important in food safety mechanism, clinical diagnosis, military/civilian warfare etc. Escherichia coli 0157:H7 (E. coli 0157:H7) is one of the most dangerous food borne bacterial pathogens. It is commonly found in raw beef, fruits, vegetables, salad bar items, salami and other food products. Outbreaks of E. coli 0157:H7 infections have caused serious illnesses and led to a significant number of deaths. Therefore, in order to prevent accidental outbreaks or intentional terrorist acts, early detection of trace amounts of E, coli 0157:H7 as well as other pathogenic microorganisms is critical.
Some approaches for the detection of pathogens already known in the art include culturing methods (Silk and Donnelly 1997), biochemical tests (Adams and Moss 1995), ELISA (Gehring et al 1999), Fluorescent bacteriophage assay (Goodridge et al 1999), Chemiluminescence enzyme immunoassay (Kovacs and Rasky 2001), Capillary immunoassay (Czajka and Batt 1996), Time resolved fluorescence immunoassay (Yu et al. 2002), PCR (Uyytendaele et al 1999), Multiplex FOR (Hu et al 1999), RT-PCR (Uyttendaele et al 1999), Laser-induced fluorescence (Johnson et al 2002), Fibre-optic biosensor (Demarco and Lim 2002), SPR biosensor (Fratamico et al 1997), Microarrays (Call et al 2001), Molecular beacon (Fortin et al 2001), Integrated systems (lab-on-chip) (Belgraderet al, 1998) etc.
The key requirements for a detection technique to be used for the early detection of microorganisms are specificity, speed, and sensitivity. Conventional detection methods as shown in Figure 1 provide qualitative and quantitative information about pathogens. However, the method by which detection is measured is relatively long.
Immunological methods are widely used to detect pathogens in clinical, agricultural and environmental samples, immunological detection tests for bacterial pathogens including

latex agglutination, immunomagnetic separation, lateral flow immunoassays and enzyme linked immunosorbent assay (ELISA) are frequently used. Some sensitive immunological methods have been developed using electro-chemiluminescence and rapid flow-through assay systems.
Enzyme linked immunosorbent assay is a biochemical test used mainly in immunology to detect the presence of an antibody or an antigen in a given sample by enzyme linked technology. There is indirect, sandwich and competitive ELISA technique wherein either the antigen or the antibody is coated on the immobilized solid support that interacts with its specific antigen or antibody. The complex is detected using a secondary antibody that is labeled which would detect the presence of the antigen-antibody complex by means of calorimetric, turbidity etc.
There are several other methods that have been developed using fluorescence immunoassay principle to detect the target antigen of interest from a biological sample and measure the concentration levels of the target antigen, in particular pathogenic microorganisms. Immunofluorescent detection is a method in which the cells are captured by specific antibody attached on a solid support. Primary antibody is added to these cells, which makes a sandwich assay. A secondary antibody is tagged with fluorescence probe. The antibody is directly tagged to the flurophore or chemically modified to bind to the flurophore. The secondary antibody is specific to the primary antibody. The fluorescence emitted by the secondary antibody is measured using a fluorescent microscopy or a fluorimeter.
The disadvantage of using the above techniques is that it requires an antibody tagged to either an enzyme or fluorescent dye. Often antibodies are modified to attach the flurophore which in turn reduces the ability to specifically bind the target pathogen. Moreover, this is an indirect method of detection of the captured cells. The tagging of fluorescent dye to the antibody is expensive and low fluorescence signal in antibody labeled systems as it requires overnight grown culture and sensitive fluorescence microscopy for detection of pathogenic cells.
Therefore, to overcome the above limitations the present invention describes a method which is relatively direct and rapid to detect cells stained using membrane specific fluorescence dye using fluorescent microscopy or a fluorimeter.

SUMMARY OF THE INVENTION
It is the primary object of the present invention to describe a method of direct and rapid detection of cells using immunofluorescence assay.
Accordingly, the present invention provides a method of direct and rapid detection of cells comprising steps of immobilization of antibody in or on a solid support, culturing the cells, staining the cells with membrane specific fluorescent dye, interaction between the bound antibody and stained cells, detection of the emitted fluorescence using fluorescent microscopy or a fluorimeter.
Accordingly, the present invention relates to the method of direct and rapid detection of cells wherein the antibodies specific to the cell are immobilized on the solid support.
Accordingly, the present invention relates to the method of direct and rapid detection of cells wherein the cell culture is stained with a lipophilic or fluorescent dye which is membrane dependent.
Accordingly, the present invention relates to the method of direct and rapid detection of cells wherein the lipophilic or fluorescent dye used is DiOC and/or Dil
Accordingly, the present invention relates to the method of direct and rapid detection of ceils wherein the stained cells is added to the antibody coated on a solid support.
Accordingly, the present invention relates to the method of direct and rapid detection of cells wherein the antibody coated on the solid support is either a primary or secondary antibody.
Accordingly, in the present invention the cell stained using the fluorescent dye is added to the primary antibody to form a complex that is added to a secondary antibody immobilized on the solid support.
Accordingly, in the present invention antibody are immobilized on magnetic iron particles, polystyrene Beads, glass, polystyrene, polypropylene, silica or any other surface.

Accordingly, the present invention relates to the method of direct and rapid detection of cells wherein the cells are bacterial or mammalian cells wherein the cell can be pathogenic or non-pathogenic in nature.
Accordingly, the present invention relates to the method of direct and rapid detection of cells wherein the fluorescence emitted by the stained cell bound to the antibody on the solid support is detected using fluorescent microscope and/or measured using fluorimeter.
It is advantageous of the method described in the present invention for direct and rapid detection of cells that does not require secondary antibody labeled for detection and more than one cell is detected.
BRIEF DESCRIPTION OF ACCOMPANYING DRAWINGS
The present invention can be better understood with reference to the following figures. The components described in the figures are not necessarily to scale, emphasis instead being placed upon clearly illustrating the principles of the invention. Moreover, in the figures, like reference numerals, designate corresponding parts throughout the different views. It will also be understood that certain components and details may not appear in the figures to assist in more clearly describing the invention.
Figure 1 shows the conventional techniques to detect the cells;
Figure 2 shows the scheme for detection of the cells in accordance to the present invention;
Figure 3 shows an alternate scheme for detection of the cells in accordance to the present invention;
Figure 4 shows immobilization of antibody using magnetic particles in accordance to the present invention;

Figure 5 depicts the graph measuring the sensitivity of fluorescence emission in accordance to the present invention;
Figure 6 depicts the graph measuring the fluorescence emitted by specific and non¬specific cells in accordance to the present invention; and
Figure 7 shows a fluorescence image of DIOC stained cells against FITC labeled secondary antibody bound cell complex in accordance to the present invention.
DETAILED DESCRIPTION OF THE INVENTION
The preferred embodiments of the present invention will now be explained with reference to the accompanying drawings. It should be understood however that the disclosed embodiments are merely exemplary of the invention, which may be embodied in various forms. The following description is not to be construed as limiting the invention and numerous specific details are described to provide a thorough understanding of the present invention, as the basis for the claims and as a basis for teaching one skilled in the art how to make and/or use the invention. However, in certain instances, well-known or conventional details are not described in order not to unnecessarily obscure the present invention in detail.
Many modifications and other embodiments of the inventions set forth herein will come to the mind of one skilled in the art to which these inventions pertain having the benefit of the teachings presented in the foregoing descriptions and the associated drawings. Therefore, it is to be understood that the inventions are not to be limited to the specific embodiments disclosed and that modifications and other embodiments are intended to be included within the scope of the appended claims. Although specific terms are employed herein, they are used in a generic and descriptive sense only and not for purposes of limitation.
"Sample suspected of containing microorganism" shall mean any sample that is reasonably suspected of containing a microorganism can be analyzed by the method of the present invention. Such samples can include human, animal or man-made samples. The sample can be prepared in any convenient medium which does not interfere with the

assay. Typically, the sample is an aqueous solution or biological fluid as described in more detail below.
The fluid sample may be a biological fluid such as, but not limited to, whole blood, serum, plasma, nasal secretions, sputum, urine, sweat, saliva, transdermal exudates, pharyngeal exudates, bronchoalveolar lavage, tracheal aspirations, cerebrospinal fluid, vaginal or urethral secretions, or the like. Herein, fluid homogenates of cellular tissues such as, hair, skin and nail scrapings, meat extracts and skins of fruits and nuts are also considered biological fluids. Fluid samples also include non-biological fluids such assoil extracts, water supplies etc. Multiple organisms may be detected from a single fluid sample.
"Antibody" shall mean an immunoglobulin having an area on its surface or in a cavity that specifically binds to and is thereby defined as complementary with a particular spatial and polar organization of another molecule. The antibody can be polyclonal or monoclonal. Antibodies may include a complete immunoglobulin or fragments thereof, which immunoglobulins include the various classes and isotypes, such as IgA (IgAI and lgA2), IgD, IgE, IgM, and IgG (lgG1, lgG2, lgG3, and lgG4) etc. Fragments thereof may included Fab, Fv and F(ab')2, Fab', and the like.
"Cell" shall mean any compound capable of binding to an antibody, or against which antibodies can be raised.
"Microorganism" shall mean a member of one of the following classes: bacteria, fungi, algae, protozoa or viruses.
The present invention provides a method of direct and rapid detection of cells that exhibits antibody specificity wherein the antibody specific to the cell is immobilized on the solid surface that captures the cell (pathogenic microorganism) from the sample.
In one embodiment of the present invention, a method is provided wherein the nature of membrane to accept certain type of molecules on its surface is utilized. The molecules that are being inserted are fluorescently labeled lipid inserting dyes such as 3, 3'-dihexyloxacarbocyanine iodide (DiOC), 1, 1'-dioctadecyl-3, 3, 3', 3'-tetramethylindocarbocyanine perchlorate (Dil) etc. The stained cell (2) interacts with the primary antibody (4) coated on the solid surface of the ELISA plate. The primary

antibody-cell tagged with a fluorphore complex is directly and rapidly detected using fluorescent microscope and/or measured using fluorimeter as shown in Figure 2.
The use of fluorescent dyes as indicators of cell viability is widespread. Dyes such as ethidium bromide, acridine orange, propidium iodide and fluorescein diacetate have all been used successfully by analyzing through fluorescence microscopy and flow cytometry.
Selective staining of cell walls with fluorescent dyes has been useful in morphological and developmental studies. Cell membranes provide a convenient conduit for loading live and fixed cells with lipophilic dyes. The cells can not only tolerate a high concentration of the lipophilic dye, but also lateral diffusion of the dye within the membrane can serve to stain the entire cell, even if the dye is applied locally. These properties have made lipophilic carbocyanine and aminostyryl dyes particularly important for anterograde and retrograde tracing in neuronal cells. Lipophilic tracers are used to label cells, organelles, liposomes, viruses and lipoproteins in a wide variety of long-term tracing applications, including cell transplantation, migration, adhesion, and fusion studies.
In another embodiment of the present invention, the sample containing more than one cell is stained using a fluorescently labeled lipid inserting dye. The cells are added to the primary antibody. A secondary antibody is coated on to the solid surface of the microtitre plate. The fluorescently labeled cell (2) bound to the primary antibody (4) is added to microtitre plate coated with secondary antibody (6). This method is used in case the sample contains various types of cells (like E. coli and Salmonella) are to be captured by their specific primary antibody and finally added onto secondary antibody immobilized on a plate as shown in Figure 3.
In another embodiment of the present invention magnetic particles that have been increasingly used as carriers for binding proteins, enzymes, and drugs are used. The immobilized biomolecules can be used directly for a bioassay or as affinity ligands to capture or modify target molecules or cells. The magnetic iron particles (8) (Fe203.Y) are
used to immobilize the primary antibody (4)-stained cell (2) complex. Similarly, polystyrene Beads, glass, polystyrene, polypropylene, silica, or any other surface can be used for Immobilization of the antibody of interest as shown in Figure 4.

In the present invention the antibody that is immobilized is a primary or secondary antibody specific to the target cell that is detected using immunofluorescence assay. This technique does not require the use of fluorescently tagged antibodies and thereby, their does not exist a need to engineer the secondary protein against the antigen is essential.
The present invention will now be described in more detail by the way of examples which should not construed as limiting the invention thereto:
Example 1
Method for detecting a target bacterium cell in an environmental fluid sample was prepared according to the following procedure: E. coli 0157 cells were inoculated into 5 ml sterile Escherichia coli (EC) broth for enrichment. The E. coli 0157 cells are incubated for 8 hours at 120 rpm at 37 °C in a shaker. This was done for the selective growth of the gram negative E. coli cells thereby preventing the growth of gram positive cells. After the incubation, 2 ml of cell culture was centrifuged at 7000 rpm for about 2 minutes. The pellet obtained after centrifugation was washed with 2 ml PBS buffer of pH 7.4. The pellet was re-suspended in 200 micro liter of DiOCis dye in dimethyl sulfoxide (DMSO) (50pM) for staining. It is incubated for 20 minutes at room temperature. 1 ml of PBS buffer of pH 7.4 is added to it and further centrifuged at 7000 rpm for 2 minutes. The pellet obtained was washed with 2 ml PBS buffer of pH 7.4 twice. Finally, the washed pellet was re-suspended in 2 ml of PBS buffer of pH 7.4 to give a cell solution.
Example 2
The coating of specific antibody on solid support was prepared according to the following procedure: The wells of polystyrene ELISA plate are coated with Escherichia coli (E. coli) 0157 antibody (1:1000 dilutions in carbonate buffer of pH 9). The coated ELISA plate is incubated for 2 hours at 37°C. The plate is then washed with Phosphate buffered saline (PBS)-Tween buffer (PBS of pH 7,4 with 0.05% Tween) twice to remove the unbound antibody. 1% solution of non-interacting protein, such as bovine serum albumin (BSA) in carbonate buffer of pH 9 is added. The plate is incubated for 2 hours at 37°C. The incubated plate is further washed with PBS-Tween buffer twice. This step is known as blocking, because the serum proteins block non-specific adsorption of other proteins to the plate.

Example 3
Interaction of antibody with stained cell according to the following procedure: 200pl of the cell solution (typically having 10^ to 10^ cells/ml) as explained in Example 1 is added to each well of the E. Coli 0157 antibody coated ELISA plate as explained in Example 2. The plate is covered and incubated for 20 minutes at 37 °C. The incubation between immobilized antibody and stained cell is achieved by this process.
Example 4
The elution of antibody-cell complex was prepared according to the following procedure: The incubated plates obtained from Example 3 were washed with PBS-Tween thrice. 250 pi of DMSO was dispensed to each well. The plates are incubated for 20 minutes. The well solution was vigorously mixed with pipette and recovered in 4ml cuvette.
Example 5
Detection of the fluorescence emission from the captured cells bound to the immobilized antibody on the solid support as obtained from Example 4 is measured in fluorimeter or fluorescence microscopy. For the purpose of quantification, fluorescence detection was achieved using the excitation filter (Ex 450-490, Nikon) and emission filter (BA 520 Nikon). Fluorescence emission is collected at 90 degrees to the excitation beam through a photodiode-preamplifier module.
Example 6
Fluorescence signal from the DiOCi8 captured cells bound to the immobilized antibody on the solid support at various concentrations is calibrated using non-specific cells as positive control and buffer solution as blank as shown in Figure 5. The normalized signal between the E. Coli 0157:H7 cells are compared to non-specific control cells which quantitatively depicts the strength of detection of specific signal of the cells present in the given sample as shown in Figure 6. Quantitative data on number of cells detected can be obtained by comparing absolute signal with calibration data given in Figure 5.
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Example 7
A fluorescence image of E. Coli 0157:H7 cells stained with DiOCia bound to primary antibody is compared against E. Coli 0157:H7 cells bound to primary Antibody that is treated with a secondary antibody labeled with Fluorescein isothiocyanate (FITC) at optimal concentration. The FITC labeled secondary antibody is used under 1:200 dilution, which is saturation binding for the cells. Primary antibody is 1:10 dilution which is above saturation binding to the cells. DiOC dye is used at optimum concentration 50 micro molar/ml. The fluorescence intensity of cell E. Coli 0157:H7 stained with DiOCia bound to primary antibody is higher than the intensity from a cell complex containing FITC labeled secondary antibody as shown in Figure 7. it clearly indicates that the fluorescence intensity and image from the E. Coli 0157:H7 stained with DiOCia is relatively simple and rapid method of detection.
It will be apparent to those skilled in the art that various modifications and variations can be made in the present invention without departing from the scope or spirit of the invention. Other embodiments of the invention will be apparent to those skilled in the art from consideration of specification and practice of the invention disclosed herein. It is intended that the specification and examples be considered as exemplary only with a true scope and spirit of the invention being indicated by the following claims.
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GLOSSARY OF THE TERMS AND ITS DEFINITIONS
PBS Phosphate Buffered Saline
DMSO Dimethyl Sulfoxide
ELISA Enzyme Linked Immunoadsorbent Assay
BSA Bovine Serum Albumin
Flic Fluorescein isothiocyanate
DiOC 3, 3'-dihexyloxacarbocyanine iodide
Dil 1, 1'-dioctadecyl-3, 3, 3', 3'-tetramethylindocarbocyanine perchlorate
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REFERENCES
Adams, M.R. and Moss, M.O. (1995) Food Microbiology. Cambridge: The Royal Society of Chemistry.
Belgrader, P., Bennett, W., Hadley, D., Long, G., Mariella, R.,Milanovich, F., asarabadi, S., Nelson, W. et al. (1998) Rapid pathogen detection using a microchip PCR array instrument. Clinical Chemistry 44, 2191-2194.
Czajka, J. and Batt, C.A. (1996) A solid phase fluorescent capillary immunoassay for the detection of Escherichia coli 0157:H7 in ground beef and apple cider. Journal of Applied Microbiology 81, 601-607
Demarco, D.R. and Lim, D.V. (2002) Detection of Escherichia coli 0157:H7 in 10- and 25-gram ground beef samples with an evanescent-wave biosensor with silica and polystyrene waveguides. Journal of Food Protection 65, 596-602.
Call, D.R., Brockman, F.J. and Chandler, D.P. (2001) Detecting and genotyping Escherichia coli 0157:H7 using multiplexed PCR and nucleic acid microarrays. International Journal of Food Microbiology 67, 71-80
Fortin, N.Y., Mulchandani, A. and Chen, W. (2001) Use of real-time polymerase chain reaction and molecular beacons for the detection of Escherichia coli 0157:H7. Analytical Biochemistry 289, 281-88.
Fratamico, P.M., Strobaugh, T.P., Medina, M.B. and Gehring, A.G. (1997) A Surface Plasmon Resonance Biosensor for Real-time Immunologic Detection of Escherichia coli 0157:H7, New Techniques in the Analysis of Foods, pp. 103-112. Washington DC: American Chemical Society.
Gehring, A.G., Brewster, J.D., Irwin, P.L, Tu, S.-l. and van Houten, L.J. (1999) 1- naphtyl phosphate as an enzymatic substrate for enzyme-linked immunomagnetic electrochemistry. Journal of Electroanalytical Chemistry 469, 27-33.
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Goodridge, L., Chen, J. and Griffiths, M. (1999a) Development and characterization of a fluorescent- bacteriophage assay for the idetection of Escherichia coli 0157:H7. Applied and Environmental Microbiology 65, 1397-1404.
Johnson, P.E., Lund, M.L., Shorthill, R.W., Swanson, J.E. and Kellogg, J.L. (2001) Real time biodetection of individual pathogenic microorganisms in food and water. Biomedical Sciences Institute 37, 191-96.
Hu, Y., Zhang, Q. and Meitzler, J.C. (1999) Rapid and sensitive detection of Escherichia coli 0157:H7 in bovine faeces by a multiplex PCR. Journal of Applied Microbiology 87, 867-76.
Invitrogen Molecular Probes. Carbocyanine dyes.
Kovacs, H.D. and Rasky, K. (2001) Testing of a chemiluminescence enzyme immunoassay for selective detection of E. coli 0157 from ground beef samples. Acta Veterinaria Hungaria 49, 377-383.
Silk, T.M. and Donnelly, C.W. (1997) Increased detection of acidinjured Escherichia coli 0157:H7 in autoclaved apple cider by using nonselective repair on trypicase soy agar. Journal of Food Protection 60, 1483-1486.
Uyttendaele, M., van Boxstael, 8. and Debevre, J. (1999) PCR assay detection of the E. coli 0157:H7 eae-gene and effect of the sample preparation method on PCR detection of heat-killed E. coli 0157:H7 in ground beef. International Journal of Food Microbiology 52, 85-95.
Yu, L.S.L., Reed, S.A. and Golden, M.H. (2002) Time-resolved fluorescence immunoassay (TRFIA) for the detection of Escherichia coli 0157:1-17 in apple cider. Journal of Microbiological Methods 49, 63-68.
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WE CLAIM
1. A method of direct and rapid detection of cell comprising the steps of:
a. immobilization of antibody on a solid support;
b. staining the cell with membrane specific fluorescent dye;
c. antibody and stained cell interaction; and
d. detection of the fluorescence emission.
2. The method as claimed in claim 1 wherein the antibody is specific to the cell.
3. The method as claimed in claim 1 wherein the antibody is immobilized on a solid support.
4. The method as claimed in claim 3 wherein the antibody that is immobilized on a solid support is a primary or secondary antibody.
5. The method as claimed in claim 1 wherein the cell is a bacterial or mammalian cell.
6. The method as claimed in claim 1 wherein the cell is pathogenic or non¬pathogenic cell.
7. The method as claimed in claim 1 wherein the solid support used is made of a material selected from the group consisting of magnetic particles, polycarbonate, polystyrene beads, polypropylene, polyethylene, glass, cellulose, nitrocellulose, silica gel, surface modified with streptavidin, avidin, biotin, glass and polyvinyl chloride.
8. The method as claimed in claim 1 wherein the solid support is selected from the group consisting of PCR plates, ELISA plate, immunomagnetic particles, polystyrene beads, glass beads, microwell plate, sheets, test particles selected from beads and microspheres, test tubes, test sticks, test strips, wells and modules.
9. The method as claimed in claim 1 wherein the activated solid support is provided
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with an active functional group selected from the group consisting of halide, aldehyde, acetyl, epoxide, succinamide, isothiocyanate, acylazide and amine for covalent binding.
10. The method as claimed in claim 1 wherein the fluorescent dye is membrane
dependent.
11. The method as claimed in claim 1 wherein the dye used to stain the membrane of cell is a lipophilic dye.
12. The method as claimed in claim 1 wherein the membrane dependent lipophilic dye is selected from a group consisting of DiOC2(3), DiOC5(3), DiOC18(3), DiSC3(5), DilC1(5), DASPMI (4-Di-1-ASP), DASPEI and CoroNa Red Na.
13. The method as claimed in claim 1 wherein the fluorescent dye used is DiOC18(3)
or Dil.
14. The method as claimed in claim 1 wherein the fluorescence emission is detected
using fluorimeter or a fluorescent microscopy, imaging or photo detection.
15. A method of direct and rapid detection of cell thereof substantially described
particularly with reference to the accompanying drawings.

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