MXPA06007541A - Method of enhancing signal detection of cell-wall components of cells - Google Patents

Abstract

The invention relates to methods of enhancing signal detection of components of cell walls, wherein the methods involve lysing cells to form cell-wall fragments and analyzing the cell-wall fragments.

Description

METHOD OF IMPROVEMENT OF SIGNAL DETECTION OF CELLULAR WALL CELL COMPONENTS BACKGROUND OF THE INVENTION The emergence of bacteria that exhibit resistance to the commonly used antibiotics is a growing problem with serious implications for the treatment of infected individuals. Accordingly, it is of primary importance to determine the presence of such bacteria at an early stage and in a relatively rapid manner to have a better control over said bacteria. This also applies to a variety of different microbes. One such microbe of important interest is Staphylococcus ("S. aureus"). This is a pathogen that causes a wide spectrum of infections including: superficial injuries such as small cutaneous abscesses and wound infections; systemic and life-threatening conditions such as endocarditis, pneumonia and septicemia; as well as toxinsis such as food poisoning and toxic shock syndrome. Some strains (for example those resistant to methicillin S. aureus) are resistant to all antibiotics except some selected ones. REF. : 174205 Current techniques for the detection of microbes, particularly bacteria resistant to antibiotics, generally consume time and typically involve culturing the bacteria in pure form. One such technique for the identification of pathogenic staphylococci associated with an acute infection, ie S. aureus in humans and animals and S. intermedius and S. hyicus in animals, is based on the ability of the microbe to coagulate plasma. At least two different coagulase tests have been described: a tube test for free coagulase and a coagulase slide test attached to a coagulation factor. The tube coagulase test typically involves mixing a culture obtained overnight in brain-heart infusion broth with reconstituted plasma, incubating the mixture for 4 hours and observing the tube to determine clots by slowly tilting the tube to observe formation of clots Incubation of the test during the night has been recommended for S. aureus since a small amount of strains may require a longer time or 4 hours for the formation of the clot. The slide coagulase test is typically faster and more economical; however, 10% to 15% of the strains of S. aureus can provide a negative result, which requires that the isolate be retested by means of the test tube test.

Although methods of detecting S. aureus as well as other microbes have been described in the art, improved detection methods would be an advantage.

SUMMARY OF THE INVENTION The invention provides methods for improving signal detection of cell wall components, wherein the methods involve lysing cells to form cell wall fragments and analyzing the cell wall fragments in search of a component of interest. In particular, the methods are useful for detecting one or more cell wall components that are characteristic of a microbe, particularly Staphylococcus aureus. In one embodiment, the present invention provides a method for improving signal detection of cell wall components of cells. The method includes: providing a test sample that includes cells; lysing the cells to form a lysate including cell wall fragments, and analyzing the cell wall fragments in search of a cell wall component; wherein the cell wall component has an increased signal relative to the same component in unused cells. In another embodiment, a method for improving signal detection of a cell wall component of cells characteristic of Staphylococcus aureus is provided. The method includes, providing a test sample that includes uncultivated cells; lysing non-cultured cells to form a lysate including cell wall fragments; and analyzing the cell wall fragments in search of a cell wall component characteristic of Staphylococcus aureus; wherein the characteristic cell wall component of Staphylococcus aureus has an increased signal relative to the same component in unused cells. In another embodiment, a method for improving signal detection of a cell wall component of cells characteristic of Staphylococcus aureus is provided. The method includes: providing a test sample that includes uncultivated cells; contacting the uncultivated cells with lysostaphin to form a Used that includes fragments of cell wall; and analyzing the cell wall fragments in search of protein A; wherein protein A in the cell wall fragments presents an increased signal in relation to protein A in the cell walls of unused cells. The terms "comprising" and variations thereof do not have a limiting meaning in which those terms appear in the description and the claims. As used herein, "a", "an", "the", "at least one" and "one or more" are used interchangeably. The above summary of the present invention is not intended to describe each modality described or each implementation of the present invention. The description that follows exemplifies in a more particular way the illustrative modalities. In several places through the application a guide is provided regarding lists of examples, examples which can be used in various combinations in each case, the list mentioned serves only as a representative group and should not be interpreted as an exclusionary list.

DETAILED DESCRIPTION OF THE ILLUSTRATIVE MODALITIES The present invention provides methods for improving the signal detection of cell wall components of cells from prokaryotic and eukaryotic organisms, for example. Such methods involve the lysis of cells (which can be cultured or not cultured) in a test sample to form fragments of cell wall and analyze the cell wall fragments in search of a component of interest. In particular, the methods of the present invention are useful for detecting one or more cell wall components that are characteristic of a species of interest (e.g., a microbe of interest) and optionally one or more internal components of the cell that are additionally characteristic. of a species of interest (for example an antibiotic resistant microbe, which is of interest). In the present, it is considered that the cell wall fragments analyzed are solid pieces of cell wall. That is, it is considered that they are not solubilized by the lysate; rather, that the cell wall has simply broken into pieces. In addition, the cell wall component that is analyzed is still part of (i.e., it is inside or on top of) the cell wall fragments. That is, they have not been solubilized by the lysate. Significantly, this improves the signal of the cell wall component relative to the same component in an unused cell. Microbes (ie, microorganisms) of particular interest include gram-positive bacteria, gram-negative bacteria, fungi, protozoa, mycoplasmas, yeasts, viruses and even viruses enveloped in lipids. Particularly relevant organisms include members of the family Iditerojbacteriaceae or the genera Staphylococcus spp., Streptococcus spp., Pseudomonas spp., Enterococcus spp., Escherichia spp., Bacillus spp., Listeria spp., Vibrio spp., As well as herpes virus, Aspergillus spp., Fusarium spp., And Candida spp. Particularly virulent organisms include Staphylococcus aureus (which includes resistant strains such as methicillin-resistant Staphylococcus aureus (MRSA)), S. epidermidis, Streptococcus pneumoniae, S. agalactiae, s. pyogenes, Enterococcus faecalis, Vancomycin Resistant Enterococcus (VRE), vancomycin-resistant Staphylococcus aureus (VRSA), resistant to vancomycin intermediary Staphylococcus aureus (VISA), Bacillus anthracis, Pseudomonas aeruginosa, Escherichia coli, Aspergillus niger, A. fumigatus, A. clavatus, Fusarium solani, F. oxysporum, F. chlamydosporum, Listeria monocytogenes, Vibrio cholera, V. parahemolyticus, Salmonella cholerasuis, S. tiphi, S. typhimurium, Candida albicans, C glabrata, C. krusei and gram-negative bacilli resistant to multiple medications (MDR). Gram-positive and gram-negative bacteria are of interest. Gram-positive bacteria such as Staphylococcus aureus are of particular interest. Typically, these can be detected by detecting the presence of a cell wall component characteristic of the bacterium, such as a cell wall protein. In addition, antibiotic resistant microbes including MRSA, VRSA, VISA, VRE and MDR are also of particular interest. Typically, these can be detected by additionally detecting the presence of a component of the internal cell, such as a membrane protein.

The present invention is advantageous with respect to conventional techniques for analyzing samples for said microbes because the signal for the cellular wall component characteristic of the microbe is increased. Such cell wall components include, for example, cell wall proteins such as protein A and microbial surface components that recognize adhesive matrix molecules (MSCRAMM), such as fibrinogen-binding proteins (e.g. coagulation), fibronectin-binding proteins, collagen-binding proteins, heparin-binding proteins and heparin-related polysaccharides, and the like. Protein A and coagulation factors such as fibrinogen binding factors and coagulation factors A, B and Efb are also particularly useful in methods for detecting the presence of Staphylococcus aureus. Other cell wall components of interest include capsular polysaccharides and cell wall carbohydrates (e.g., teichoic acid and lipoteichoic acid). Such microbes or other species of interest can be analyzed in a test sample that can be derived from any source, such as a physiological fluid, for example blood, saliva, lens fluid, synovial fluid, cerebrospinal fluid, pus, sweat, exudates , urine, mucus, lactation milk or similar. In addition, the test sample can be derived from a site of the body, for example wounds, skin, nasal passages, scalp, nails, etc. The technique describes various techniques of sampling a patient for the detection of microbes such as S. aureus. Such sampling techniques are suitable for the method of the present invention as well. It is common to obtain a sample of cleaned from the nostrils of a patient. A particularly preferred sampling technique includes a swab from the anterior nostrils of the subject (for example from the patient) with a sterile swab or with a sampling device. For example, a swab is used to sample each subject, that is, a swab for both nostrils. Sampling may be performed, for example, by inserting the swab (such as those commercially available from Puritan, East Grinstead UK under the trade designation "re-raps"), dried or pre-moistened with an appropriate solution within the anterior tip of the swabs. The subject's nostrils should be rotated and the complete swab rotated two times around the mucosal surface of the nasal passages. The swab is typically then grown directly or extracted with an appropriate solution that typically includes water optionally in combination with a buffer and at least one surfactant. In addition to physiological fluids other test samples may include others, liquids as well as one or several solids dissolved in a liquid medium. The samples of interest may include streams of processes, water, soil, plants or other vegetation, air, surfaces (for example contaminated) and the like. The test sample (for example liquid) can be subjected to pretreatment, such as dilution of viscous fluids. The test sample (for example liquid) can be subjected to other treatment methods before injection into the sample orifice such as concentration, filtration, centrifugation, distillation, dialysis or the like; dilution, filtration, inactivation of natural components, addition of reagents, chemical treatment, etc. The signal enhancement of the cell wall components occurs as a result of lysate of the cells in the test sample. In the methods of the present invention, the lysate may include contacting the cells with a usable agent or physically lysing the cells. The lysate can be carried out under conventional conditions such as, for example, a temperature of about 5 ° C to about 37 ° C, preferably at a temperature of about 15 ° C to about 25 ° C. Significantly, the lysate can be carried out using uncultivated cells, i.e., a direct test sample, although cultured cells can also be used. As a result of the lysate of the cells to form cell wall fragments and the resulting enhancement of the signal from the cell wall components, samples having relatively low concentrations of the species of interest can be evaluated. In this way, the methods of the invention are sold with improved sensitivity. For example, for certain embodiments, the test sample may include relatively low concentrations of microbes, particularly Staphylococcus aureus. Such relatively low concentrations include, for example, less than about 5 x 104 unit forming colony (ufe) per milliliter (cfu / ml) or microbe, less than about 5 x 10 3 cfu / ml, less than about 1000 cfu / ml even a concentration as low as 500 cfu / ml. The microbes, so that S. aureus can be detected in high concentrations too, ranging from up to as large as 5 x 107 cfu / ml for example. Suitable lysate agents include, for example, enzymes such as lysostaphin, lysozyme, endopeptidase, N-acetylmuramyl-L-alanine, amidase, endo- "-N-acetyl-glucosaminidase and ALE-1. Various combinations of enzymes may be used if desired. Lysostaphin is particularly useful in methods for detecting the presence of Staphylococcus aureus. Other usable agents include salts (for example chaotropic salts), solubilizing agents (for example detergents), reducing agents (for example DTT, DTE, cysteine, N-acetylcysteine), acids (for example HCl), bases (for example NaOH). Various combinations of such lysate agents may be used if desired. The lysate can also be carried out by physically lysing the cells. Physical lysate can be carried out by swirling the test sample with glass spheres, by sonication, boiling or by submitting the test sample at high pressure, as is the case with the use of a French press. If desired, the methods of the present invention may additionally include analyzing the lysate in search of an internal component of the cell, which may or may not be associated with the cell membrane. The internal components of the cell are particularly useful for analyzing microbes resistant to antibiotics such as MRSA, VRSA, VISA, VRE and MDR. Internal cell components that may be characteristic of such microbes include membrane proteins. Examples of such membrane proteins include cytoplasmic membrane proteins, outer membrane proteins and cellular membrane proteins. Cytoplasmic membrane proteins such as penicillin-binding proteins (PBP) (e.g., PBP2 'or PBP2a) may be particularly characteristic of antibiotic-resistant microbes. For example, the cytoplasmic membrane protein PBP2 'is characteristic of MRSA. The methods of the present invention may involve not only detecting the presence of a cell wall component, but preferably identifying said cell wall component, which may lead to identifying a microbe for which that cell wall component is characteristic. In some embodiments, the analysis of cell wall fragments in search of a cell wall component includes quantifying the cell wall component. Depending on the analysis techniques used in the methods of the present invention, relatively small volumes of test sample can be used. Although test sample volumes as large as 1-2 milliliters (ml) can be used, advantageously test samples of the order of 50 •! they are enough for some methods. Depending on the analysis techniques used in the methods of the present invention, the detection time may be relatively short. For example, the detection time may be less than about 300 minutes, less than about 250 minutes, less than about 200 minutes, less than about 150 minutes, less than about 100 minutes, less than about 60 minutes, and even as short as about 30 minutes. Said analysis techniques can be any of a wide variety of conventional techniques known to those skilled in the art. For example, such methods may include the use of fluorometric immunochromatography (e.g., the rapid analyte measurement method as described in the U.S. patent number. ,753,517), acoustic wave sensors, ELISA (for example colorimetric ELISA) and other colorimetric techniques (for example colorimetric sensors that include polydiacetylene (PDA) materials) such as those described in the patent application publication of E.U.A. number 2004/0132217; patent application of E.U.A. serial number 10 / 325,276, filed on December 19, 2002; and the co-pending application of the assignee of the applicant serial number filed on the same date as in this document "Colorimetric Sensors Constructed of Dyacetylene Materials" (attorney's file number 60422US002) as well as surface plasmon resonance (SPR), using biosensors of the available type from Biacore, Upsala, Sweden). Enzyme-linked immunosorbent assays (ELISA) are based on two important biological phenomena: 1) the discriminatory ability of antibodies to differentiate between a virtually unlimited number of specific foreign compounds, and 2) the ability of enzymes to specifically catalyze detectable chemical reactions. This combination of reaction to foreign compounds, of both bound and soluble antibodies, as well as the detection of these reactions by a subsequent reaction catalyzed by an enzyme bound to the soluble antibody, provide a very sensitive and specific measurement of the foreign compounds. Such techniques are well known to those skilled in the art. Surface plasmon resonance (SPR) is an optical technique based on surface plasmon resonance that measures changes in the refractive index near the surface of the sensor. When the light moves from an optically denser medium (ie, one that has a higher refractive index) to a less dense medium (ie, one that has a lower refractive index), a total internal reflection occurs (TIR) in the boundary between the two media if the angle at which the light hits the limit is greater than a critical angle. When IRR occurs, an electromagnetic "evanescent wave" propagates from the boundary to the middle of the lower refractive index. If the boundary is covered with a thin layer of a certain conductive material (eg gold or silver), the evanescent wave can be coupled with a constellation of free electrons, called surface plasons, on the surface of the conductor. Such resonant coupling occurs at a specific angle of incident light, absorbing the light energy and causing the characteristic decrease in the intensity of light reflected at that angle. The electromagnetic surface wave generates a second evanescent wave with an improved electric field that penetrates into the less dense medium. The resonance angle is sensitive to various factors including the wavelength of the incident light and the nature and thickness of the conductive film. However, most importantly, the angle depends on the refractive index of the medium in which the evanescent wave of the surface plasmon wave propagates. When the other factors remain constant, the resonance angle in this way is a direct measure of the refractive index of the less dense medium, and the angle is very sensitive to changes in the refractive index in the medium. The evanescent wave SPR decreases exponentially with distance from the boundary and effectively penetrates a medium with a refractive index lower than a depth of one wavelength. Therefore, only changes in the index, of refraction very close to the limit, can be detected. This technique can be carried out using biosensors of the available type from Biacore, Upsala, Sweden. In some embodiments of the present invention a method of analyzing the cell wall component may involve detecting the change in at least one physical property. This may include a change in viscosity and / or a change in mass that results in a change in the wave phase or wave velocity. In some modalities, such change can be detected by a biosensor. As used herein, the term "biosensor" refers to a device that detects a change in at least one physical property and that produces a signal in response to the detectable change. The means by which the biosensor detects a change may include an electrochemical medium, an optical medium, an electro-optical medium, an acoustic mechanical means, etc. For example, electrochemical biosensors use potentiometric and perometric measurements, while optical biosensors use absorbance detection, fluorescence or visible luminescence detection and evanescent waves. For some modalities, a biosensor can be used that uses a mechanical acoustic means for detection such as a surface acoustic wave biosensor (SAW). Biosensors using acoustic mechanical means and the components of such biosensors are described, for example, in the U.S. Patents. numbers 5,076,094; 5,117,146; 5,235,235; 5,151,110; 5,763,283; 5,814,525; 5,836,203 and 6,232,139. SAW biosensors based on piezoelectric materials typically work based on their ability to detect minimal changes in mass or viscosity. As described, for example, in the patent of E.U.A. number ,814,525 (Renschler et al.), The class of piezoelectric based acoustic mechanical devices can be further subdivided into surface acoustic wave (SAW), acoustic plate mode (APM) or quartz crystal microequilibrium (QCM) devices depending on their detection mode. APM devices operate under a principle similar to SAW devices, except that the acoustic wave used can be operated with the device in contact with a liquid. Similarly, an alternating voltage applied to two opposite electrodes on a QCM (typically AT cut quartz) device induces a shear wave mode thick whose resonant frequency changes in proportion to mass changes in a coating material.

The direction of acoustic wave propagation (eg, in a plane parallel to the waveguide or perpendicular to the plane of the waveguide) can be determined by a crystal slice of the piezoelectric material from which the biosensor is constructed. SAW biosensors in which most of the acoustic wave propagates into and out of the plane (eg wave Rayleigh, most of Lamb waves) not typically semi used in sensing applications in liquids due to acoustic damping liquid in contact with the surface. For liquid sample media, a horizontal shear surface acoustic wave biosensor (SH-SAW) can preferably be used. SH-SAW sensors are typically constructed of a piezoelectric material with a crystal-cut and orientation that allows the propagation wave to be rotated to the horizontal shear mode, that is, parallel to the plane defined by the waveguide, resulting in a loss of reduced acoustic damping to a liquid in contact with the detection surface. The horizontal acoustic shear waves may include, for example, modes of 'shear thick (TSM by its acronym), mode acoustic panel (APM its acronym), volume waves skimming surface ( SSBW, Love waves, leakage acoustic waves (LSAW) and waves of Bleustein-Gulyaev (BG). In particular, Love wave sensors may include a substrate that supports an SH wave mode such as ST quartz SSBW of the 36 ° leak wave YXLiTa03. These modes, which can preferably be converted to the Love wave mode by application of a thin acoustic guide layer or a waveguide. These waves often depend and can be generated if the shear wave velocity of the waveguide layer is lower than that of the piezoelectric substrate. Materials waveguide may preferably be materials that exhibit one or more of the following properties: low acoustic losses, low electrical conductivity, robustness and stability in water and aqueous solutions, relatively low acoustic velocities, hydrophobicity, higher molecular weights, highly crosslinked , etc. In one example, they have been used as an acoustic waveguide layer on a quartz substrate. Examples of other crosslinked polymeric and thermoplastic waveguide materials include, for example, epoxy resins, polymethyl methacrylate, phenolic resin (for example NOVALAC), polyimide, polystyrene, etc. Other potentially suitable wave guide materials and constructions for use with acousto-mechanical sensors used in the detection cartridges of the present invention can be described, for example, in the application PCT of the transferee of the applicants number filed on the same date as this document, entitled "Acoustic Sensors and Methods" (attorney's file number 60209WO003). Alternatively, QCM devices can also be used as liquid sample media. Biosensors using acoustic-mechanical devices and components can be described, for example, in the U.S. Patents. numbers 5,076,094 (Frye et al.); 5,117,146 (Martin et al.); 5,235,235 (Martin et al.); 5,151,110 (Bein et al.); 5,763,283 (Cernosek et al.); 5,814,525 (Renschler et al.); 5,836,203 (Martin et al-); and 6,232,139 (Casalnuovo et al.). SAW horizontal shear devices can be obtained from various manufacturers such as Sandia Corporation, Albuquerque, New Mexico. Some SH-SAW biosensors that may be used in connection with the present invention are also described in Branch et al., "Low-level detection of a Bacillus anthracis simulantusin Love-wave biosensors on 36 ° YXLiTa03," Biosensors and Bioelectronics, 19 , 849-859 (2004). As discussed herein, the methods of the present invention can be used in various detection systems and components (such as detection cartridges including biosensors) which can be found, for example, in the patent application of E.U.A. serial number 60 / 533,169, filed on December 30, 2003; request PCT number entitled "Acousto-Mechanical Detection Systems and Methods of Use, "filed on the same date as in the present (attorney's file number 59468WO003) and the PCT application number entitled "Detection Cartridges, Modules, System, and Methods," filed on the same date as the present one (file of attorney-in-fact number 60342WO003). In some embodiments, the biosensor comprises a reagent (e.g., an antibody) that binds a S. aureus of interest to the surface of a piezoelectric biosensor. If S. aureus is present, the lysed cells in the test sample are analyzed for protein A, which is characteristic for S. aureus, and can be detected with an antibody specific for protein A immobilized on the surface of the biosensor. . Additionally, lysed cells, such as S. aureus bacteria, release protein markers from the inner portion of the cells (as opposed to the cell wall portion of the cells). Such protein markers can be detected by a molecule that reacts with S. aureus. This technique is particularly suitable for detecting S. aureus resistant to methicillin. In some cases an antibody against S. aureus is used as the reagent for S. aureus,. The term "antibody against S. aureus" refers to an immunoglobulin that has the ability to specifically bind to a given antigen including the antigen-binding fragments thereof. The one proposed to include complete antibodies of any isotype (IgG, IgA, IgM, IgE, etc.), and fragments thereof from vertebrates, for example from mammalian species which are also specifically reactive with foreign compounds, for example proteins The antibodies can be fragmented using conventional techniques and the fragments can be screened for utility, in the same manner as whole antibodies. Thus, the term includes segments of proteolytically separated or recombinantly prepared portions of an antibody molecule that are capable of selectively reacting with a certain protein. Non-limiting examples of such proteolytic and / or recombinant fragments include Fab, F (ab ') 2, Fab, Fv, and single chain antibodies (scFv) that contain a linked VL and / or VH domain by a peptide linker. The scFvs can be covalently or non-covalently bound to form antibodies having two or more binding sites. The antibodies can be labeled with any detectable portion known to those skilled in the art. In some aspects, the antibody that binds to an analyte to be measured (the primary antibody) is not labeled, but instead is detected indirectly by binding a labeled secondary antibody or other reagent that specifically binds to the primary antibody. . Various antibodies are known in the art.

S. aureus. For example, S. aureus antibodies are commercially available from Sigma-Aldrich and Accurate Chemical. In addition, the antibodies of S. aureus are described in the patent of E.U.A. Do not . 4,902,616. Typically, the concentration of antibody used is at least 2 nanograms / ml. Preferably, the antibody concentration is at least 100 nanograms / ml. For example, a concentration of 50 micrograms / ml can be used. Typically, a maximum of about 500 micrograms / ml is used. As previously described, it is preferred to immobilize the S. aureus antibody on the surface of the biosensor. One or more of the analysis techniques described herein can be coupled with electrical and / or electrochemical methods. Microbial metabolism currently results in an increase in both conductance and capacitance which causes a decrease in impedance. Therefore, they have been used in the literature to detect bacteria measurements that belong to these concepts. For example, a wave impedance sensor, reusable bulk acoustics has been developed for detection of microorganisms. These organisms are capable of transducing metabolic redox reactions into quantifiable electrical signals. Therefore, electrochemical methods have also been used to detect bacterial organisms. The methods include direct potentiometric detection, light assisted potentiometric detection (LAPS) and amperometric detection. An ELISA technique coupled with a redox reaction with an antibody labeled with horseradish peroxide has been electrically monitored. Other variations include immunofiltration techniques combined with amperometric detection. Such techniques are described in D. Ivinitski et al., Biosensors & amp;; Bioelectronics, 14, "599-624 (1999).

EXAMPLES The present invention has now been described with reference to several specific embodiments anticipated by the inventor for which enabling descriptions are available. The non-substantial modifications of the invention, which include modifications that are not currently anticipated, nevertheless, must be considered as constitutive equivalents to it. Thus, the scope of the present invention should not be limited by the details and structures described herein, but rather only by the following claims and equivalents thereof.

Example 1. Detection by ELISA Preparation of the plates with antibody Polystyrene micropore plates are coated (Costar, 96 well cell culture groups, flat bottomed with lid, treated with tissue culture, non-pyrogenic polystyrene plates, catalog number 3596, Corning Incorporated, Corning, NY) with rabbit IgG ChromPure (molecule complete, catalog number 011-000-003, Jackson ImmunoResearch Laboratories, West Grove, PA) as an antibody at 10 • g / ml. The antibody solution is prepared by diluting the antibody in 0.1 M sodium bicarbonate, pH 9.6 (Sigma-Aldrich, St. Louis, MO). The coated plates are incubated at 37 aC for one hour.

Washing the plates The plates are then washed by aspiration and 0.25 ml of a "PBS buffer" solution consisting of 0.02 M sodium phosphate (Sigma-Aldrich) and 0.15 M sodium chloride (Sigma-Aldrich) are supplied in each well. ), to which 0.05% volume-volume (v / v) of polyoxyethylene (20) sorbitan monolaurate (commercial designation, TWEE? 20 available from Sigma-Aldrich, St. Louis, MO) has been added, the pH of the solution is 7, 5 and the washing is repeated 5 cycles.

Blockade of the plates A concentrated solution is prepared by mixing Carnation-free dry non-fat milk (Nestle USA, Inc., Solon, OH), with the previous wash solution with a load of 2% by weight in volume (w / v). A 0.2 ml portion of this concentrated solution is added to each well and the plates are incubated at 37 aC for 1 hour. The plates are then washed as described above.

Preparation of bacteria suspension S. aureus bacteria are obtained from The American Type Culture Collection, Rockville, MD under the trade designation "ATCC 25923". The bacteria are allowed to grow overnight (17-22 hours at 37 ° C) in broth cultures prepared by inoculating 5-10 ml of sterile Tryptic Soy Broth prepared (Hardy Diagnostics, Santa Maria, CA) with the bacteria. The cultures are washed by centrifugation (8,000-10,000 rpm for 15 minutes in an Eppendorf centrifuge model number 5804R (Brinkman Instruments, Westbury, NY) and resuspended in PBS buffer containing 0.2% (w / v) of PLURONIC L64 surfactant (BASF Corporation, Mount Olive, NJ) and washed or centrifuged for 3 additional cycles with this solution.

Dilution of bacteria The washed bacterial suspensions are then diluted in the following solutions. Solution 1 is PBS buffer with 0.2% (w / v) of PLURONIC L64 surfactant (BASF Corporation). Solution 2 is a buffer made by combining 0.01 M Tris-HCl, 1 mM EGTA, 1% Triton X-100, 2.5 mM sodium pyrophosphate, 1 mM sodium phosphate and 1 μg / ml leupeptin (Sigma- Aldrich, St. Louis, MO). Solution 3 is a lysate buffer made by combining solution 2 above with lysostaphin at 3 • g / ml (catalog number L-4402, Sigma-Aldrich). The S. aureus bacteria are diluted in serial quintuple dilutions at 108, 2 x 107, 4 x 106, 8 x 10s and 1.6 x 10 * / ml within each of the three solutions. Cultures of S. epidermidis ATCC 12228 (American Culture Collection, Rockville, MD) are prepared in the same manner and the S. epidermidis bacteria are resuspended only in the 3 to 1? Vml solution as a comparator.

ELISA tests of antigen solutions Samples of each antigen preparation and dilution as well as samples of each solution containing no bacteria are added to the previously coated plates, blocked and washed. Each sample is plated in duplicate by adding 0.1 ml of the sample solution in separate microwells in the plate. The plates are incubated at 372C for 1 hour. The plates are then washed as indicated above and 0.1 ml of primary antibody solution is added to the appropriate wells. The primary antibodies are biotinylated anti-S. aureus rabbit IgG (Biotin Rabbit Anti-Staphylococcus aureus, catalog number YVS6887, Accurate Chemical and Scientific Company, Westbury, NY) and biotinylated anti-protein A mouse IgG (Monoclonal Anti-Protein A Clone SPA-27, conjugated with biotin, catalog number B-3150, Sigma-Aldrich, St. Louis, MO). These antibodies are diluted to 5 μg / ml in concentrated solution and 0.1 ml of a primary antibody solution is added to the appropriate wells. The plates are incubated at 372C for 1 hour. After incubation the plates are washed as indicated above and 0.1 ml of streptavidin-phosphate-alkaline-conjugate preparation (SA-AP, Jackson ImmunoResearch Laboratories) is added to the appropriate wells. The streptavidin-alkaline phosphatase conjugate preparation (SA-AP) is made by diluting streptavi-dine-alkaline phosphatase conjugate (catalog number 016-050-084, Jackson ImmuoResearch Laboratories) at 0.5 • g / ml in concentrated solution. The plates are incubated at 37 eC for 1 hour and then washed as in the above. After washing, a 0.1 ml portion of an alkaline phosphatase substrate preparation is added to the preparation wells. The alkaline phosphatase substrate preparation is para-nitrophenyl phosphate substrate (pNPP, product code 50-80-00, Kirkegaard and Perry Laboratories, Gaithersburg, MD) prepared as indicated by the manufacturers. The plates are then incubated at room temperature for 15 minutes. After the 15 minute incubation period, 0.1 ml disodium EDTA 5% (w / v) (Sigma-Aldrich) is added to stop the substrate development catalyzed by the enzyme. The plates are read with a plate reader Bio-Tek model EL808 Microwell (Bio-Tek Instruments, Inc., Winooski, VT) at 405 nanometers and the results are presented in Table 1 below (N / A = not applicable (en say, not measured)).

Example 2. Detection of fluorescent assay Preparation and dilution of bacteria suspension S. aureus bacteria is obtained from The American Type Culture Collection, Rockville, MD under the trade designation "ATCC 25923". The bacteria are grown overnight (17-22 hours at 37 SC) in broth cultures prepared by inoculating 5-10 ml of sterile Tryptic Soy Borth, prepared (Hardy Diagnostics, Santa Maria, CA) with the bacteria. The cultures are washed by centrifugation (8,000-10,000 revolutions per minute (rpm)) for 15 minutes in an Eppendorf centrifuge model number 5804R (Brinkman Instruments, Westbury, NY) and resuspended in PBS buffer with 0.2% weight by volume ( p / v) of PLURONIC L64 surfactant (BASF Corporation, Mount Olive, NJ) and washed by centrifugation for 3 additional cycles with this solution. The suspension of washed S. aureus 25923 is then diluted in serial serial dilutions from 105 to 103 / ml in two different diluents (E5 to E3). The first is. a RAMP No. 1 test sample buffer (Response Biomedical Corporation, Burnaby, BC, Canada) and the second is the same as the first buffer only lysostaphin (Sigma-Aldrich) is added to provide a solution of 3 • g / ml . Shock absorber samples are also run only (E0). The assays are performed in a RAMP fluorescent assay reader (Response Biomedical Corporation, Burnaby, BC, Canada) following the manufacturer's instructions. The results are given below in Table 2.

Example 3. Colorimetric detection Coating of polydiacetylene liposomes on a polycarbonate membrane A formulation of (60/40) diacetylene HO (O) C (CH2) 2C (O) O (CH2) 4-C = CC = C (CH .) 40 (O) C (CH,) 12CH3 (prepared as in Example 6 of U.S. Patent Application Publication No. 2004/0132217) and 1,2-dimeristoyl-sn-glycero-3-phosphocholine (DMPC) , formula weight (FW for its acronym in English) 678, available from Sigma-Aldrich, catalog number P2663) are applied as a coating to porous polycarbonate membranes 25 mm in diameter with pores with a diameter of 200 nm (Avestin, Inc. ., Ottawa, Canada) to produce colorimetric detector samples. The membranes are coated using a portable extrusion process. The 60/40 diacetylene / DMPC mixture is weighed into glass jars and suspended in HEP? Buffer (5 mM, pH 7.2) to produce a 1 mM solution. This solution is then sonicated with a probe using a Misonix XL202 sonicator for 2 minutes and placed in a refrigerator at 4aC for about 2 hours. This procedure results in the formation of a suspension of polydiacetylene liposome (PDA). The polycarbonate membrane to be coated is placed inside a stainless steel chamber of a portable extruder system, commercial designation LIPOFAST, available from Avestin, Inc. (Ottawa, Canada). The covered membrane of the toroidal ring of the bottom of the TEFLON base. Care is taken to avoid bending and / or folding the membrane. The TEFLON toroidal ring block in the upper part is placed inside the stainless steel housing on top of the membrane. Then the chamber is sealed by manually tightening the stainless steel plugs. A Gas Tight Hamilton 500- •! Syringe is filled with a suspension of diacetylene liposomes and attached to the base and attached to a second syringe to the other cap. The liposomes of the first syringe are driven slowly through the chamber with a constant uniform pressure. The membrane retained by the liposomes on the surface allows the clear buffer to flow slowly through and into the second syringe. This action is considered as a one-pass coating. The membrane samples used as detectors in this example use two coating passes. The second pass is applied in a similar way to the first by a second portion of 0.5 ml of liposome that is applied to the already coated membrane. The second syringe contains the filtered buffer and separates and the contents are discarded. The stainless steel end cap is unscrewed and the TEFLON toroidal ring block is removed. The wet membrane is removed and placed with the coated side up on a glass slide and placed in the refrigerator at 5aC for at least 3 hours. The sample is then dried in a desiccator containing CaSO4 for 30 minutes and exposed to UV light of 254 nm for 30-90 seconds. The substrate coated with PDA (25 mm circle) is cut into four quarters. Each quarter sample is used as a sample for an experiment. The substrates are placed in wells separated from 24-well microtiter plates. A phosphate-buffered saline solution is prepared by diluting 10 times a liquid PBS lOx concentrate (commercially available from EMD Biosciences, San Diego, CA). This results in a PBS buffer solution with the following salt composition: 10 mM sodium phosphate, 137 mM sodium chloride, 2.7 mM potassium chloride. To the PBS buffer is also added 0.2% (w / v) of PLURONIC L64 surfactant (commercially available from BASF Corporation, Mount Olive,? J) which provides a PBS L64 buffer solution. Complete bacterial sample solutions are prepared by mixing 250 μl of PBS L64 buffering solution containing complete S. aureus ATCC 25923 bacteria with 250 -1 of antibody solution. The antibody solution contains rabbit anti-Staphylococcus aureus antibody (catalog number YVS6881, Accurate Chemical and Scientific Corp.) at a concentration of 100 • g / ml in a solution of PBS L6 buffer. Samples containing S. aureus lysate ATCC 25923 are prepared in PBS L64 buffer solution using a lysate buffer which consists of lysostaphin, lysostaphin at 3 • g / ml (catalog number L-4402, Sigma-Aldrich) in a PBS L64 shock absorber solution. The sample solutions of lysed bacteria consist of 250"1 of lysed bacterium of S. aureus ATCC 25923 in PBS-L64 mixed with 250" 1 of antibody solution prepared as described above. The concentration of the bacteria used in the test samples varies between 0 and 10 cfu / ml as reported in Table 3 below. The mixture of the bacterium and the antibody solution is allowed to sit for 5 minutes and then added to a 24-well plate containing the substrate coated with PDA. Control samples are also prepared for comparison.

The control sample contains no bacteria and consists simply of 250 μl of PBS-L64 buffer mixed with 250 μl of antibody solution prepared as described above. - - I know I take a picture every 5 minutes using a digital camera. The image is scanned using a program (software) from Adobe Systems Incorporated (San Jose, CA) with the commercial designation ADOBE PHOTOSHOP version 5.0, to obtain the RGB channel values (abbreviations in English for red, green and blue) for each sensor . The colorimetric response (CR) is determined using the red and blue channel values as provided by the equation CR = ((PRinicial - PRsample) / PRinicial) where PR = value in red percent of The sample is provided by the equation PR = Rvalue / (Rvalue + Bvalor) * 100, where Rvalor and Bvalor correspond to the value of the red and blue channels of the polydiacetylene sensor, respectively. The data in Table 3 below show the difference in the colorimetric response between a control sample and a sample containing bacteria (either complete or lysed) measured at 15 minutes.

Various modifications and alterations of this invention will be apparent to those skilled in the art without departing from the scope and spirit of this invention. It is to be understood that this invention is not designed to be unduly limited by the illustrative embodiments and examples provided herein and that such examples and embodiments are presented by way of example only with the scope of the invention which is deemed to be limited only by the claims set forth herein, as follows.

It is noted that in relation to this date, the best method known to the applicant to carry out the aforementioned invention, is that which is clear from the present description of the invention.

Claims (28)

CLAIMS Having described the invention as above, property is claimed as contained in the following claims:

1. A method for improving signal detection of a cell wall component of cells, characterized in that it comprises: providing a test sample comprising cells; lysing the cells to form a lysate comprising cell wall fragments; and analyzing the cell wall fragments to determine a cell wall component; wherein the cell wall component has an increased signal relative to the same component in non-lysed cells.

2. The method according to claim 1, characterized in that the cell wall component comprises a cell wall protein.

3. The method according to claim 2, characterized in that the cell wall protein is protein A.

4. The method according to claim 2, characterized in that the cell wall protein is a coagulation factor.

5. The method according to claim 1, characterized in that the cell wall component comprises a capsular polysaccharide or a cell wall carbohydrate.

6. The method according to claim 1, characterized in that the lysate of the cells comprises contacting the cells with a lysing agent. The method according to claim 6, characterized in that the lysing agent comprises an enzyme selected from the group consisting of lysostaphin, lysozyme, endopeptidases, N-acetylmuramyl-L-alanine amidase, endo-3-N-acetylglucosaminidase, ALE-1 and combinations thereof. The method according to claim 6, characterized in that the lysing agent comprises a salt, solubilizing agent, a reducing agent, an acid, a base or combinations thereof. 9. The method according to claim 1, characterized in that the lysate of the cells comprises physically lysate the cells. 10. The method according to claim 1, characterized in that the cells comprise one or more microbes. 11. The method according to claim 10, characterized in that the microbes comprise gram-positive bacteria. 12. The method according to claim 11, characterized in that the gram-positive bacteria comprise Staphylococcus aureus. The method according to claim 10, characterized in that the microbes comprise a gram-negative bacterium. 14. The method according to claim 1, characterized in that the cells are uncultivated. 15. The method according to claim 1, characterized in that it further comprises analyzing the lysate to determine an internal component of the cell. 16. The method according to claim 15, characterized in that the cells comprise microbes resistant to antibiotics. 1

7. The method according to claim 15, characterized in that the internal component of the cell comprises a cell membrane. 1

8. The method according to claim 17, characterized in that the cell membrane comprises a membrane protein. 1

9. The method according to claim 18, characterized in that the membrane protein is a cytoplasmic membrane protein. 20. The method according to claim 19, characterized in that the cytoplasmic membrane protein is PBP2 '. 21. The method according to claim 1, characterized in that the analysis of the cell wall fragments for a cell wall component comprises identifying the cell wall component. 22. The method according to claim 1, characterized in that the analysis of the cell wall fragments for a cell wall component comprises quantifying the cell wall component. 23. The method according to claim 1, characterized in that the analysis of the cell wall fragments for a cell wall component comprises analyzing with fluorometric immunochromatography. 24. The method according to claim 1, characterized in that the analysis of the cell wall fragments for a cell wall component comprises analyzing with ELISA. 25. The method according to claim 1, characterized in that analyzing "the cell wall fragments for a cell wall component comprises analyzing with an acoustic wave sensor 26. The method according to claim 1, characterized in that the analysis of the cell wall fragments for a cell wall component comprises colorimetrically analyzing 27. A method for increasing the signal detection of a cell wall component of characteristic cells of Staphylococcus aureus, is characterized in that it comprises: test comprising uncultivated cells; lysing non-cultured cells to form a lysate comprising cell wall fragments; and analyzing the cell wall fragments in search of a cell wall component characteristic of Staphylococcus aureus; wherein the characteristic cell wall component of Staphylococcus aureus has an increased signal relative to the same component in non-lysed cells. 28. The method according to claim 27, characterized in that the cell wall component comprises a cell wall protein. 2 . The method according to claim 28, characterized in that the cell wall protein is protein A. 30. The method according to claim 27, characterized in that the lysate of non-cultured cells comprises contacting the uncultivated cells with lysostaphin. . 31. The method according to claim 27, characterized in that it further comprises analyzing the lysate for an internal component of the cell. 32. The method according to claim 31, characterized in that the internal component of the cell comprises a cell membrane. 33. The method according to claim 32, characterized in that the cell membrane comprises a membrane protein. 34. The method according to claim 33, characterized in that the membrane protein is a cytoplasmic membrane protein characteristic of MRSA. 35. The method according to claim 34, characterized in that the cytoplasmic membrane protein is PBP2 '. 36. The method according to claim 27, characterized in that the analysis of the cell wall fragments for a cell wall component comprises quantifying the cell wall component. 37. The method according to claim 27, characterized in that the test sample comprises Staphylococcus aureus in a concentration lower than 5 x 104 cfu / ml. 38. A method for increasing signal detection of a cell wall component of cells, characteristic of Staphylococcus aureus, characterized in that it comprises: providing a test sample comprising uncultivated cells; contacting the non-cultured cells with lysostaphin to form a lysate comprising cell wall fragments; and analyzing the cell wall fragments in search of Ajen protein where protein A in the cell wall fragments presents an increased signal in relation to protein A in the cell walls of non-lysed cells.