Universal biosensor system for analyte detection

Abstract

To provide a biosensor system for the detection of target analytes.SOLUTION: A biosensor system for the detection of target analytes includes: a living biological cell of a predetermined type; a signal-generating reporter associated with the living biological cell; a signal transduction pathway or other activator mechanism or means associated with the signal-generating reporter; a universal detector element associated with the activator mechanism; and an analyte binding element associated with the universal detector element, wherein the analyte binding element is specific to both the universal detector element and the target analyte.SELECTED DRAWING: None

Description

[Technical field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This patent application claims the benefit of U.S. Provisional Application No. 62 / 438,068, filed December 22, 2016, entitled "SYSTEM AND APPARATUS FOR RAPID DETECTION OF ANALYTES," and is a continuation-in-part of U.S. Provisional Application No. 15 / 642,800 (U.S. Patent No. 9,850,546), filed July 6, 2017, entitled "BIOSENSOR SYSTEM FOR RAPID DETECTION OF ANALYTES," which are hereby incorporated by reference in their entirety and made a part of the U.S. patent application for all purposes. [Background technology]

[0002] The present invention relates generally to systems, devices, reagents, and methods for detecting various analytes of interest in biological or other types of samples, and more particularly to biosensor-based systems for detecting and identifying analytes of interest in real time based on the emission of a detectable signal when the biosensor reacts with the analyte of interest in the sample being tested. The following patents provide additional background information regarding the technology of the present invention and are incorporated herein by reference in their entirety for all purposes.

[0003] In general terms, a biosensor is a system or device for the detection of an analyte that combines a sensitive biological component with a physicochemical detector component. The components of a typical biosensor system include a biological component, a transducer or detector component, and associated electronics or signal processor that displays the test results in a meaningful and useful manner. The biological components typically include biological materials such as tissues, microorganisms, organelles, cellular receptors, enzymes, antibodies, nucleic acids, etc., which can be produced by known bioengineering processes. The transducer or detector component works with physicochemical methods (e.g., optical, piezoelectric, and / or electrochemical) to convert the signal resulting from the interaction of the analyte with the biological component into another signal that can be more easily measured and quantified. Biosensors have arisen from the integration of molecular biology and information technology (e.g., microcircuits, fiber optics, etc.) to qualify or quantify biomolecule-analyte interactions, such as antibody-antigen interactions. Given the great demand for rapid, sensitive, easy-to-use, and cost-effective detection tools for detecting infectious agents, pathogens, or / and toxins in food (e.g., Mead et al., Food Related Illness and Death in the United States, Emerging Infectious Diseases; Vol. 5, No. 5, September-October 1999(607-625), which is incorporated herein by reference in its entirety), there is a continuing need for the use of biosensors, including real-time, portable devices and instruments, to detect and identify infectious agents, pathogenic microorganisms, toxins, and other contaminants in food. Summary of the Invention [Means for solving the problem]

[0004] The following provides a summary of certain exemplary embodiments of the present invention. This summary is not an extensive overview and is not intended to identify key or critical aspects or components of the present invention or to describe its scope. However, it should be understood that the use of indefinite articles in the language used to describe and claim the present invention is in no way intended to limit the described system to a single component or ingredient. Rather, the use of "a" or "an" herein should be construed to mean "at least one" or "one or more."

[0005] According to another aspect of the present invention, there is provided a first biosensor system for detecting a target analyte, the system comprising a biological cell of a predetermined type, a signal generating reporter associated with the biological cell, a signaling pathway or activation mechanism associated with the signal generating reporter, a universal detection moiety associated with the activation mechanism, and an analyte binding moiety associated with the universal detection moiety, the analyte binding moiety being specific for both the universal detection moiety and a target analyte.

[0006] According to another aspect of the present invention, there is provided a second biosensor system for detecting a target analyte, the system comprising: a biological cell of a predetermined type; a signal generating reporter in the biological cell, the signal generating reporter responsive to a predetermined change occurring in the biological cell; a signal generating pathway or activation mechanism associated with the signal generating reporter, the signal generating pathway or activation mechanism operating to induce a predetermined change in the biological cell; a universal detection component associated with the activation mechanism, the universal detection component operating to trigger the activation mechanism; and an analyte binding component associated with the universal detection component, the analyte binding component being specific to both the universal detection component and the target analyte, such that when the analyte binding component, which also binds the target analyte, binds to the universal detection component, the universal detection component triggers the activation mechanism to cause a predetermined change in the biological cell, thereby causing the signal generating reporter to generate a detectable signal.

[0007] According to another aspect of the present invention, there is provided a third biosensor system for detecting a target analyte, the system comprising: a biological cell of a predetermined type; a signal generating reporter in the biological cell, the signal generating reporter responsive to a predetermined change occurring in the biological cell; a signal generating pathway or activation mechanism associated with the signal generating reporter, the signal generating pathway or activation mechanism operating to induce a predetermined change in the biological cell; a universal detection component associated with the activation mechanism, the universal detection component operating to trigger the activation mechanism; and an analyte binding component associated with the universal detection component, the analyte binding component being specific to both the universal detection component and the target analyte, wherein when the analyte binding component, which also binds the target analyte, binds to the universal detection component, the universal detection component inhibits the activation mechanism to reduce the predetermined change in the biological cell, thereby causing the signal generating reporter to generate an attenuated signal or no signal.

[0008] Further features and aspects of the present invention will be apparent to those skilled in the art upon reading and understanding the following detailed description of the exemplary embodiments. As will be appreciated by those skilled in the art, further embodiments of the present invention are possible without departing from the scope and spirit of the present invention. Thus, the drawings and associated description should be regarded as illustrative and not limiting in nature. [Brief description of the drawings]

[0009] The accompanying drawings, which are incorporated in and form a part of this specification, illustrate one or more exemplary embodiments of the invention and, together with the general description above and the detailed description below, serve to explain the principles of the invention. [Figure 1]1a-b are diagrammatic representations of a first biosensor according to an exemplary embodiment of the invention, in which Jurkat T cells are engineered to produce aequorin and express the transmembrane non-antibody signaling component IgGbp-CD3ζ. [Diagram 2] 2a-b are diagrams of a second biosensor according to an exemplary embodiment of the invention, in which MC / 9 mast cells have been engineered to produce aequorin and express the native receptor FcεRI, which binds the soluble non-antibody signaling component IgGbp-IgE. [Diagram 3] 3a-b are illustrations of a third biosensor according to an exemplary embodiment of the invention, in which MC / 9 mast cells have been engineered to produce aequorin, and the MC / 9 cells express the native receptor FcεRI, which binds to the soluble non-antibody signaling component IgGbp-IgE excreted by the MC / 9 mast cells. [Figure 4] FIG. 4 is a diagrammatic representation of a fourth biosensor according to an exemplary embodiment of the invention, in which the biosensor cells are engineered to produce aequorin and express the transmembrane non-antibody signaling element mSA-CD3ζ that binds to a biotinylated detection element. [Diagram 5] FIG. 5 is a diagram of a fifth biosensor according to an exemplary embodiment of the invention, in which the biosensor cells are engineered to produce aequorin and express the transmembrane non-antibody signaling moiety mSA-CD3ζ that binds to a biotinylated detection moiety. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0010] Exemplary embodiments of the present invention will now be described with reference to the drawings. Although the following detailed description contains many details for purposes of illustration, those skilled in the art will appreciate that many variations and modifications to the following details are within the scope of the present invention. Thus, the following embodiments of the present invention are described without loss of generality to, and without imposing limitations on, the invention as claimed.

[0011] The present invention relates generally to systems, devices, reagents, and methods for detecting various analytes and / or other targets of interest in biological samples or other sample types, and more particularly to a biosensor-based system for detecting and identifying analytes of interest in real time based on the emission of a detectable signal when the biosensor reacts with the analyte of interest in the sample being tested. The engineered cells of the present invention are highly sensitive and effective biosensors, and because these biosensor cells have inherent detection capabilities, they provide a versatile system that can be easily adapted to detect a wide variety of infectious agents or other targets by simply selecting a soluble detector (e.g., antibody) molecule with specificity for a particular pathogen or other target of interest. Furthermore, the systems of the present invention can be easily configured for multiplex detection of several infectious agents or other analytes in a single assay, providing great flexibility and utility. The versatility of the present invention comes from the unique combination of components, particularly the combination of universal biosensor cells and specific soluble detectors (e.g., antibodies). The universal biosensor cells have the ability to respond to the presence of essentially any target molecule that can be recognized by the detector molecule. In some embodiments, the detection agent or detection antibody is added to the system as a soluble agent, so that the system can be configured to detect alternative targets simply by selecting the appropriate alternative detection agent or detection antibody. The specificity of the disclosed system is determined by the detection molecule being selected based on its specificity and affinity for a target molecule characteristic of an infectious agent or other target analyte. This combination of universal biosensor cells and soluble detectors also allows for the construction of multiplex assays by simply including multiple detection molecules (e.g., antibodies) within the test system, where the target molecule is selected based on its specificity for an alternative infectious agent or other analyte.

[0012] Genetic engineering and modification of biosensor cell types used in the present invention typically involves the use of appropriately selected gene delivery vehicles that contain genetic components that function efficiently in the selected cell type. For example, it is useful to use promoter components that direct high levels of expression of the introduced gene in the specific biosensor cell selected. In an exemplary embodiment of the present invention, such promoter components can be derived directly from the biosensor cell itself and then used to express the transgene of interest. In another embodiment of the present invention, appropriate components can be empirically determined by comparing the function of alternative promoter components in the context of alternative gene delivery vehicles to identify promoter, transgene, vector combinations that are effective for the selected cell type. Transgenes, such as genes encoding luminescent reporter proteins, can be introduced into biosensor cells using standard techniques such as electroporation or chemical transfection reagents such as lipofectamine. Other genetic engineering methods known to those skilled in the art are also compatible with the present invention.

[0013] Exemplary embodiments of the invention include a live engineered biosensor cell, which is typically a component of a mammalian immune system, a reporter protein expressed by and present in the live engineered cell, which emits a detectable signal in response to a predetermined change in the cytosol of the live engineered cell, a signal transduction pathway that controls a biological or biochemical process within the cytosol of the live engineered cell and triggers at least one biological or biochemical process when the biological or biochemical process occurs, at least one detector molecule, each detector molecule adapted to bind to a specific analyte, at least one analyte, which binds to a detector molecule specific for that analyte, and several transmembrane non-antibody signaling components expressed by the live engineered cell, each transmembrane non-antibody signaling component adapted to receive an analyte itself. When a sufficient number of analytes, which are themselves bound to the transmembrane non-antibody signaling moieties, bind to a sufficient number of detection molecules, aggregation of the signaling moieties occurs at the cell surface, a signaling pathway is activated, a biological or biochemical process occurs, and a detectable signal is emitted by the reporter protein. The system may also include an apparatus for mixing the live cells with the soluble components and a sample containing the analyte or infectious agent of interest while maintaining the viability and functionality of the live biosensor cells, and a detector for detecting the signal emitted by the biosensor cells.

[0014] Another exemplary embodiment of the invention includes live modified cells, said live modified cells being components of a mammalian immune system, said live modified cells being mast cells, and said mast cells expressing at least one predetermined receptor, a reporter protein, said reporter protein being aequorin expressed by the live modified cells, said aequorin emitting a detectable light signal in response to a predetermined change in the cytosol of the live modified cells, a signaling pathway expressed by the live modified cells, said signaling pathway controlling a biochemical process in the cytosol of the living organism, said biochemical process controlled by the signaling pathway further increasing intracellular calcium, which increase in intracellular calcium, when it occurs, causes the aequorin to emit detectable light, at least one detection molecule, each detection molecule adapted to bind to a specific analyte, at least one analyte, said at least one analyte binding to a detector molecule specific for that analyte, and a plurality of soluble non-antibody signaling components, each signaling component adapted to bind to at least one predetermined receptor and receive said detection molecule. Upon binding of a sufficient number of analytes and a sufficient number of detection molecules with a sufficient number of transmembrane non-antibody signaling components that are themselves bound to at least one type of predetermined receptor, receptor aggregation occurs at the cell surface. Upon binding of a sufficient number of analytes and a sufficient number of detection molecules with a sufficient number of transmembrane non-antibody signaling components that are themselves bound to at least one type of predetermined receptor, receptor aggregation occurs at the cell surface, a signaling pathway is activated, an increase in intracellular calcium occurs, and detectable light is emitted by aequorin. The system may also include an apparatus for mixing the live cells with the soluble components and a sample containing the analyte or infectious agent of interest while maintaining the viability and functionality of the live biosensor cells, and a detector for detecting the signal emitted by the biosensor cells.

[0015] Yet another exemplary embodiment of the present invention includes a biosensor for rapid detection, said biosensor further comprising a live modified cell, said live modified cell derived from a cellular component of a mammalian immune system (i.e., an immune cell), a reporter protein engineered and expressed within the live modified cell, and said reporter protein emits a detectable signal in response to a predetermined change in the cytosol of the live modified cell, engineered within the live modified biosensor cell that occurs naturally, said signal transduction pathway controlling a biological process within the cytosol of the live modified biosensor cell, said biological process when it occurs triggering the reporter protein, and a non-antibody signal transduction component that directly or indirectly binds to an analyte in the sample to be analyzed, said non-antibody signal transduction component then cooperating with the biosensor cell to directly or indirectly activate the signal transduction pathway.

[0016] Living cells Exemplary embodiments of the invention include live, engineered biosensor cells that are typically components of a mammalian immune system, such as immune cells. In one aspect of the invention, the biosensor cells are human or mouse B cells. B cells or B lymphocytes are a type of white blood cell of the lymphocyte subtype that function in the humoral immune component of the adaptive immune system by secreting antibodies. In another aspect of the invention, the biosensor cells are human or mouse T cells. T cells or T lymphocytes are another type of lymphocyte that plays a central role in cell-mediated immunity as part of the adaptive immune system. T cells are distinguishable from other lymphocytes due to the presence of T cell receptors on their cell surface. In another embodiment of the invention, the biosensor cells are mast cells. Mast cells are a type of white blood cell known as granulocytes that are derived from bone marrow stem cells that are part of the immune system and neuroimmune system. Other types of white blood cells and other types of cells, including basophils, which are similar in both appearance and function to mast cells, are compatible with the invention.

[0017] In another embodiment of the invention, the biological cell is a prokaryotic cell or a Ca 2+The living cell may be a eukaryotic cell that contains a signal transduction system. The living cell may be a yeast cell or an insect cell, such as an insect cell that is a Drosophila Schneider 2 (S2) cell, an sf9 cell, or an insect cell that is engineered to use aequorin as a reporter. The living cell may be a mammalian cell, such as a HEK cell, a CHO cell, a COS cell, or a 3T3 cell. The living cell may be a modified cell. The modified cell may be derived from a natural, passaged, or cultured mammalian cell. The modified cell may be derived from a non-regenerating cell, a fixed cell, a drug or chemically treated cell, an osmotically treated cell, a radiated cell, an artificial or synthetic cell, or a non-living cell, so long as it is provided that the modified cell contains a functional ligand, a signal transduction pathway, and a reporter. The cell may be an artificial or synthetic cell. The modified cells may be derived from plant cells, animal cells, insect cells or other non-mammalian cells, components of the mammalian immune system, follicular dendritic cells, natural killer cells, macrophages, monocytes, mononuclear phagocytes, neutrophils, eosinophils, or basophils. The modified cells may also be cells expressing Fc receptor types, such as B lymphocytes, follicular dendritic cells, natural killer cells, macrophages, neutrophils, eosinophils, basophils, and mast cells. In certain embodiments, the cells may be any prokaryotic or eukaryotic cell that has the appropriate receptors, signaling pathways, and signal output methods, either naturally, by genetic engineering, or by chemical addition. The cells may be artificial or non-living units, provided that they have functional receptors, signaling pathways, and signal output methods. An example of a cell useful for this system is a macrophage cell, such as the human cell line U937, which expresses Fc receptors on the cell surface. The antigen can be bound to the antibody by addition of the antibody to the target, and this antigen-antibody complex will bind to the Fc receptor on the cell and stimulate signaling that results in an increase in intracellular calcium. The cells can be fixed, frozen, dried, or lyophilized.

[0018] Signal generating reporters Exemplary embodiments of the invention include a reporter component, such as a reporter protein or enzyme, produced or expressed by a living, modified biosensor cell. The reporter protein emits a detectable signal in response to a specific, predetermined change in the cytosol of the living, modified biosensor cell. In certain embodiments of the invention, the reporter protein is a bioluminescent light-emitting protein, such as aequorin, derived from the aquatic animal Aequorea Victoria. Aequorin is used to engineer a biosignal sensor cell to generate a light signal in response to activation of various signaling pathways, and as such, various methods for engineering the production of aequorin in living cells are well known to those of skill in the art. In particular, those of skill in the art may select and use any suitable gene delivery means, such as, for example, a bacterial plasmid vector or a viral vector, to introduce the appropriate genetic material into the biosensor cell. The production of the reporter protein in the biosensor cell will be controlled by the expression of the introduced genetic material. Those of skill in the art will also understand that other light-emitting proteins or other types of reporter proteins, enzymes, and molecules can be incorporated and utilized in various alternative embodiments of the invention.

[0019] In various embodiments of the present invention, the reporter can be a protein with fluorescent properties that undergoes a detectable change in response to the activation of at least one biochemical pathway and the resulting change in the living cell. The reporter or reporter protein can be other calcium-sensitive luminescent or fluorescent molecules such as obelin, thalassicolin, mitrocomin (halistalin), clytin (phialidin), nemopsin, berobin, indo-1, fura-2, kin-2, fluo-3, rhod-2, calcium green, BAPTA, cameleon (A. Miyawaki et al., (1999) Proc. Natl. Acad. Sci. 96, 213540), or similar molecules. The reporter protein can be a protein that detects the activity of Ca 2+The reporter may be a chimeric protein comprising a binding domain and an associated fluorescent protein. The associated fluorescent protein may be green fluorescent protein (GFP). The protein may bind to other components of the phosphatidylinositol pathway (i.e., the pathway used in the embodiments described herein) and change its fluorescence. An example is a fluorescent protein engineered to bind diacylglycerol. The reporter may be an enzyme adapted to produce a luminescent or fluorescent signal. The reporter protein may be an enzyme such as luciferase or alkaline phosphatase, which produces a luminescent or fluorescent signal, respectively. It may also be a fluorescent protein or may include fluorescent, charged, or magnetic nanoparticles, nanodots, or quantum dots. The reporter may be a dye having fluorescent, ultraviolet, or visible properties, which undergo a detectable change upon activation of at least one biochemical pathway and the resulting changes in the living cell.

[0020] Activator Mechanism Exemplary embodiments of the invention include an activator mechanism in the form of a signaling pathway expressed by a live, engineered biosensor cell. The signaling pathway controls at least one biological process within the cytosol of the live, engineered cell, and causes a reporter protein to emit a detectable signal when the at least one biological process occurs. In certain embodiments of the invention, the signaling pathway regulates intracellular Ca in response to activation of a cell surface signaling molecule, such as a receptor protein. 2+ The biosensor cells used in the present invention are any biochemical pathway that induces an increase in cytoplasmic Ca concentration in response to activation of cell surface signaling molecules. 2+ For example, B cells, T cells, and mast cells can respond to activation of cell surface signaling molecules such as the B cell receptor, T cell receptor, and Fc epsilon receptor (mast cells) by increasing Ca 2+ It has the ability to induce an increase in concentration.

[0021] Mammalian cells grown in culture typically exhibit Ca expression in specific individual cells. 2+ This generates cell populations that may have different abilities to induce increases in Ca concentration. 2+ It is useful to select or screen subpopulations of cells or clonal cell lines with robust ability to generate a signal. This can be achieved, for example, by analyzing the induction of aequorin-induced flashes. In particular, transfectants created by the introduction of transgenes into cells are a mixed population of cells derived from multiple independent gene insertion events. Therefore, when constructing biosensor cells, it is useful to screen or select a specific subset of cells or clonal cell lines that have efficient signaling ability together with useful expression levels of the introduced transgene. It is particularly useful to use fluorescence-activated cell sorting (FACS) technology to select a subpopulation of high expressing cells or to create clonal cell lines for this purpose.

[0022] As mentioned above, aequorin is a signaling pathway that specifically regulates cytoplasmic Ca in living cells. 2+ It has previously been used to modify living biosensor cells to generate optical signals in response to the activation of a wide variety of signaling pathways when they result in an increase in ions. In a particular embodiment of the invention, biosensor cells producing aequorin as a reporter protein are loaded with coelenterazine (CTZ) prior to their use in detection assays. This charging step covalently binds aequorin to a hydrophobic prosthetic group (e.g., CTZ) and calcium (Ca 2+ Upon binding of CTZ, CTZ undergoes an irreversible reaction that involves a conformational change and emits blue light (at 469 nm).

[0023] As mentioned above, the signaling pathway can transmit a first signal by the release of calcium ions from the endoplasmic reticulum to the cytosol, and a second signal can be released by a reporter in response to calcium ions. This signaling pathway is a second messenger cascade found in B cells, T cells, mast cells, macrophages, and other immune cells, in which cross-linking of cell surface receptors activates tyrosine kinases, which in turn phosphorylates phospholipase C, which drives phosphatidylinositol 4,5-bisphosphate (PIP2) to inositol 1,4,5-trisphosphate (IP3) and diacylglycerol, which then opens calcium channels to release calcium from intracellular stores such as the endoplasmic reticulum, or to take up extracellular calcium, thereby increasing calcium concentration in the cytosol of the cell. Depending on the type of receptor, the type of cell, and the desired method of signaling, alternative second messenger cascades such as the G-protein-adenylyl cyclic-cAMP-protein kinase A cascade can be used. The signal transduction pathway can also transmit a signal by the release of diacylglycerol, ceramide, or other lipophilic second messenger molecules, and the reporter releases a second signal in response to the release of diacylglycerol, ceramide, or other lipophilic second messenger molecules. The signal transduction pathway can also transmit a signal by the release or production of nitric oxide ("NO"), cAMP, cGMP, or other cyclic nucleotides, and the reporter releases a second signal in response to this release or production. The signal transduction pathway can also transmit a signal by the release or production of superoxide, hydrogen peroxide, carbon monoxide, hydrogen sulfide, or other secondary redox messenger, and the reporter releases a second signal in response to the release or production of superoxide. Hydrogen peroxide, carbon monoxide, hydrogen sulfide, or other secondary redox messenger molecules. In certain embodiments, the activator mechanism includes a change in the pH or temperature of the cell, or a change in the electrical or magnetic properties of the cell.

[0024] Universal Detection Components Exemplary embodiments of the present invention include a variety of non-antibody signaling components that function as universal detection components for recognizing target analytes. Each signaling component is typically adapted to bind, i.e., to receive, an analyte-binding component (also referred to herein as a "detection molecule") that it is itself adapted to receive. In one embodiment, the signaling component is a transmembrane chimeric fusion protein engineered and expressed on the surface of the biosensor cell and adapted to activate a signaling pathway that ultimately results in a reporter protein that emits a detectable signal. In another embodiment, the signaling component is a soluble chimeric fusion protein engineered and expressed on the surface of the biosensor cell and adapted to bind to a cell surface signal transducer, such as a natural receptor or reporter protein, adapted to activate a signaling pathway that ultimately results in a reporter protein that emits a detectable signal. In yet another embodiment, the signaling component is a soluble chimeric fusion protein engineered and expressed within the biosensor cell. The soluble chimeric fusion protein is secreted / excreted into the extracellular space where it is coupled to a cell surface signaling pathway, such as a natural receptor or reporter protein, which is coupled to a cell surface signal transducer adapted to activate a signaling pathway, ultimately resulting in a reporter protein that emits a detectable signal.

[0025] The chimeric fusion protein of the invention may comprise (i) a protein component adapted to bind at least one type of detection molecule (e.g., a soluble antibody), and (ii) a component of a receptor complex normally expressed by a living, engineered biosensor cell. In some embodiments, the protein component adapted to bind at least one type of detection molecule may be derived from a bacterial binding protein (i.e., an antibody binding protein derived from bacteria), such as, for example, the IgG binding domain of the strepto-G protein (referred to in the figures herein as IgGbp or Igbp). To increase the affinity of the binding protein for soluble antibodies, a tandem repeat of this IgG binding domain may be included. In an alternative embodiment, the chimeric fusion protein component adapted to bind at least one type of detection molecule is an antibody binding domain derived from a receptor protein, such as, for example, the mouse Fc gamma RI (FcγRI) receptor. In various exemplary embodiments, the components of the receptor complexes normally expressed by live, modified biosensor cells are IgM (for B cell biosensors), Igα / β (for B cell biosensors), IgE (for mast cell biosensors), CD19 (for B cell biosensors), CD3 zeta (for T cell biosensors), or FcεRI (for mast cell biosensors).

[0026] The non-antibody signaling components of the present invention may comprise either complete protein sequences or engineered protein fragments, such as selected protein domains derived from larger protein molecules. Those skilled in the art will appreciate that fragments of larger molecules can be produced using standard genetic engineering techniques, such as synthetic genetic techniques. When using fragments of larger proteins to engineer antibody binding motifs as an embodiment of a chimeric fusion protein, it is important to design the engineered protein to ensure proper conformational folding of the selected protein fragment. Thus, it is useful to include (in the fusion protein) short spacer or linker components that do not readily form secondary protein structures. For example, short combinations of amino acids such as glycine, serine and alanine can be used for these spacer or linker components. In an exemplary embodiment, the amino acid sequence glycine (G), serine (S), alanine (A), serine (S), glycine (G), serine (S), glycine (G) is used to separate the binding domain from components of the receptor complex in the engineered protein molecule (see SEQ ID NO: 19). With respect to peptide linkers or spacers used to connect detection components to signaling components or to interconnect different segments of signaling components, the linker typically joins the carboxyl terminus of one component to the amino terminus of another. Peptide linkers can vary from 0 to 25 amino acids in length or any intermediate integer value and typically, but not always, contain hydrophilic amino acids such as glycine (G) and serine (S).

[0027] As mentioned above, each signaling moiety binds to a detection molecule that binds to a particular analyte of interest. The detection molecule bound to the analyte either (i) binds to a transmembrane signaling moiety, or (ii) a signaling moiety that itself binds to a cell surface signal transducer (e.g., a natural receptor). In the first situation, when a sufficient number of analytes that themselves bind to a transmembrane non-antibody signaling moiety bind to a sufficient number of detection molecules, aggregation of the signaling moieties occurs on the biosensor cell surface. A pathway is activated, a biological process occurs, and a detectable signal is emitted by the reporter protein. In the second case, when a sufficient number of analytes and a sufficient number of detection molecules and a sufficient number of non-antibody signaling moieties that themselves bind to the appropriate natural receptor bind to the cell surface, a signaling pathway is activated, an increase in intracellular calcium occurs, and detectable light is emitted by the reporter protein.

[0028] Each signaling first non-antibody signaling component according to an exemplary embodiment of the invention comprises a bacterial binding protein (IgGbp) fused to an IgM heavy chain constant domain (B cell) using a GSAGSGSG linker. SEQ ID NO:1 provides the DNA sequence of signaling component IgGbp-IgM, and SEQ ID NO:2 provides the protein sequence of signaling component IgGbp-IgM.

[0029] A second non-antibody signaling component according to an exemplary embodiment of the invention comprises a bacterial binding protein (IgGbp) fused to the Igα / β component of the B cell receptor using a GSAGSG linker. SEQ ID NO:3 provides the DNA sequence of signaling component IgGbp-Igα / β, and SEQ ID NO:4 provides the protein sequence of signaling component IgGbp-Igα / β.

[0030] A third non-antibody signaling component according to an exemplary embodiment of the invention comprises a bacterial binding protein (IgGbp) fused to the CD3 zeta chain of the T cell receptor using a GSAGSGSG linker. SEQ ID NO:5 provides the DNA sequence of signaling component IgGbp-CD3ζ, and SEQ ID NO:6 provides the protein sequence of signaling component IgGbp-CD3ζ.

[0031] A fourth non-antibody signaling component according to an exemplary embodiment of the invention comprises an FcγRI antibody binding domain fused to an IgM heavy chain constant domain (B cell) using a GSAGSG linker. SEQ ID NO:7 provides the DNA sequence of signaling component FcγRI-IgM, and SEQ ID NO:8 provides the protein sequence of signaling component FcγRI-IgM.

[0032] A fifth non-antibody signaling component according to an exemplary embodiment of the invention comprises an FcγRI antibody binding domain fused to an Igα / β component of the B cell receptor using a GSAGSG linker. SEQ ID NO:9 provides the DNA sequence of signaling component FcγRI-Igα / β, and SEQ ID NO:10 provides the protein sequence of signaling component FcγRI-Igα / β.

[0033] A sixth non-antibody signaling component according to an exemplary embodiment of the invention comprises an FcγRI antibody binding domain fused to the CD3 zeta chain of the T cell receptor using a GSAGSGSG linker. SEQ ID NO:11 provides the DNA sequence of signaling component FcγRI-CD3ζ, and SEQ ID NO:12 provides the protein sequence of signaling component FcγRI-CD3ζ.

[0034] A seventh exemplary non-antibody signaling component according to the invention comprises a bacterial binding protein (IgGbp) fused to an IgE constant domain (B cell) using a GSAGSG linker. SEQ ID NO: 13 provides the DNA sequence of signaling component IgGbp-IgE, and SEQ ID NO: 14 provides the protein sequence of signaling component IgGbp-IgE.

[0035] An eighth exemplary non-antibody signaling component according to the invention comprises an FcγRI antibody binding domain fused to an IgE constant domain (B cell) using a GSAGSG linker. SEQ ID NO: 15 provides the DNA sequence of signaling component FcγRI-IgE, and SEQ ID NO: 16 provides the protein sequence of signaling component FcγRI-IgE.

[0036] A ninth exemplary non-antibody signaling component according to the invention comprises monomeric streptavidin fused to the CD3 zeta chain of the T cell receptor using a GSAGSGSG linker. SEQ ID NO:17 provides the DNA sequence of signaling component mSA-CD3ζ, and SEQ ID NO:18 provides the protein sequence of signaling component mSA-CD3ζ. Monomeric streptavidin is a recombinant form of streptavidin that contains mutations that break down streptavidin tetramers into monomers and enhance the solubility of the resulting isolated subunits.

[0037] In various embodiments, the universal detection component comprises an antibody VDJ region, a Fab fragment or other antibody determinant. The universal detection component may comprise a T cell VJ region, a VDJ region, or other T cell receptor determinant. The universal detection component may comprise a synthetic peptide, a small non-peptide organic determinant, a protein or peptide determinant, a lectin determinant, a carbohydrate binding module or other carbohydrate binding determinant, a lipid binding determinant, or a metallothiogen determinant that binds a metal or other metal binding determinant.

[0038] The universal detection component can be covalently linked to a signal transduction pathway expressed by a living, biological cell. An example is a membrane anchored antibody where the anchor moiety is part of the signal transduction pathway, i.e., transduces a signal from outside the cell into the cell. In some embodiments, the universal detection component is not modular, but is an integral part of the signal transduction pathway, e.g., part of a chimeric protein that forms the pathway. The universal detection component can be non-covalently linked to the signal transduction pathway. An example is an antibody bound externally to an Fc receptor on a signal transduction molecule, where the molecule bearing the Fc receptor transduces a signal through the membrane. In other embodiments, the universal detection component is modular, and a cell containing a signal transduction pathway can be loaded with a universal detection component of choice. The universal detection component can include a determinant that non-covalently binds to a part of the signal transduction pathway, or the universal detection component can include an Fc determinant that non-covalently binds it to an Fc binding portion of the signal transduction pathway. The universal detector component can include a biotin or (strept)avidin determinant that non-covalently binds it to a biotin or (strept)avidin binding portion of the signal transduction pathway.

[0039] Analyte-binding component / detection molecule Exemplary embodiments of the invention include at least one analyte-binding component, also referred to herein as a "detection molecule," each analyte-binding component adapted to bind to a specific target analyte. The analyte-binding component may be a soluble antibody that is never expressed by the biosensor cells. A particular analyte-binding component for use with the invention is selected based on its ability to unambiguously identify a target analyte of interest. In an exemplary embodiment, the analyte-binding component is a soluble antibody, such as a commercially available IgG, specific for a particular analyte, such as an infectious agent. In another exemplary embodiment, the analyte-binding component is a biotinylated molecule (or a streptavidin-based molecule) specific for a given analyte, such as, for example, a biotinylated autoantigen molecule specific for an anti-autoantigen antibody. A detector or target molecule according to the invention may include an autoantigen or an autoantibody associated with an autoimmune disease. Representative autoimmune diseases or disorders include rheumatoid arthritis (RA), juvenile RA (JRA), type 1 diabetes, systemic lupus erythematosus, Hashimoto's disease, Graves' disease, scleroderma, celiac disease, Crohn's disease, ulcerative colitis, Sjogren's syndrome, multiple sclerosis, Goodpasture's syndrome, Addison's disease, Wegener's granulomatosis, primary biliary cirrhosis, sclerosing cholangitis, autoimmune hepatitis, polymyalgia rheumatoid arthritis, temporal arteritis / giant cell arteritis, and Guillain-Barre syndrome. The detection or target molecule may also include antibodies against antigens such as tumor-specific or tumor-associated antigens, or biologically active molecules such as EGF, peptide hormones including insulin and growth hormone, cytokines, interleukins, interferons, TNF, etc., or antibodies against such biologically active molecules.

[0040] The system of the invention can further include an analyte-binding component comprising an IgG fragment, which can be a single-chain antibody or a single-chain diabody. The detector can also be an affibody (i.e., an engineered binding protein), an aptamer (e.g., a DNA or RNA molecule engineered to bind to a ligand), or a soluble receptor, such as a soluble receptor for an infectious virus.

[0041] Target Analytes and Test Samples The intended use of the present invention is the detection of various analytes that are present or may be present in a sample to be tested. In an exemplary embodiment of the present invention, the analyte to be detected binds to a detection molecule, such as a soluble antibody, specific for that analyte. The sample to be tested may be taken from a number of food sources, including (i) meats, such as beef, pork, lamb, bison, poultry, and seafood, and (ii) plants and vegetables. The sample to be tested may also be taken from many other sources, such as water, consumable fluids, preserved fluids, and bodily fluids, such as blood. Analytes that may be detected include virtually anything that specifically binds to a detection substance or detection molecule, such as chemicals, toxins, and infectious agents, such as viruses, bacteria, and other biological materials or substances. In an exemplary embodiment of the present invention, the specific infectious agent is E. coli, although other infectious agents (such as Salmonella, Listeria, and Campylobacter) and contaminants may also be detected with the present invention. E. coli O157 H7, O26, O45, O103, O111, O121, and O145 can all be detected using the present invention, either in separate or multiplexed assays.

[0042] The present invention can detect many different analytes, including meat pathogens, as well as those found in spinach, lettuce, and other vegetables and foods. The analyte may include one or more epitopes of an antigen or allergen, including both linear or conformational epitopes, which may also include one or more ligands or mutual receptors or receptors recognized by a ligand. Exemplary analytes include bacteria such as Bacillus (e.g., Bacillus anthracis), Enterobacteriaceae (e.g., Salmonella, E. coli, Klebsiella, and Shigella), Yersinia (e.g., Yersinia pestis or Enterococcus faecalis), Staphylococcus aureus, Staphylococcus aureus, Streptococcus gonorrhoeae, Enterococcus faecalis (e.g., E. coli), Listeria monocytogenes, Brucella (e.g., B. abortus, B. melitensis, or other bacteria). or B. suis), Vibrio (e.g. B. suis), Cholera Bacterium, Corynebacterium diphtheriae, Pseudomonas (e.g. Pseudomonas or Pseudomonas aeruginosa), Burkholderia (e.g. Bacillus malayi or Pseudomalea), Shigella (e.g. S. dysenteriae), Rickettsia (e.g. R. rickettsii, R. prorowazekii, or R. typhi), Francisella tularensis, Chlamydia psittaci, Coxiella burnetii, Mycoplasma (e.g. M. mycoides), etc. Allergens such as peanut dust, mycotoxins, mold spores; or Clostridium botulinum and C.bacterial spores, such as C. perfringens; toxins, such as ricin, mycotoxins, tetrodotoxin, anthrax toxin, botulinum toxin, Staphylococcal enterotoxin B, or saxitoxin; viruses from the Adenoviridae family (e.g., adenoviruses), Arenaviruses (e.g., Machupo virus), Bunaviruses (e.g., Hantaviruses or Rift Valley fever viruses), Coronaviruses, Orthomyxoviruses (e.g., influenza viruses), Filoviridae (e.g., Ebola virus and Marburg virus), Flaviviridae (e.g., Japanese encephalitis virus and yellow fever virus), Hepadnaviridae (e.g., hepatitis B virus), Herpesviridae (e.g., herpes simplex virus), Papovaviridae (e.g., papillomavirus), Paramyxoviridae (e.g., respiratory syncytial virus), viruses such as rabies virus, measles virus, mumps virus, or parainfluenza virus), Parvoviridae (e.g., poliovirus), Poxviridae (e.g., poliovirus), Reoviridae (e.g., rotavirus), Retroviridae (e.g., human T-cell lymphotropic virus (HTLV) and human immunodeficiency virus (HIV), Rhabdoviridae (e.g., rabies virus), and Togaviridae (e.g., encephalitis virus, yellow fever virus, and rubella virus); Cryptosporidium parvum, encephalitis, Plasmodium, Toxoplasma gondii, Acanthamoeba, Entamoeba histolytica, Giardia lamblia, Trichomonas vaginalis, Leishmania, or Trypanosoma (e.g., T. brucei and T.parasitic helminths such as cestodes (tapeworms), trematodes (trematodes) or nematodes (roundworms, e.g., Ancaris lumricoides, Trichuris trichura, Necator americanus, or Ancylostosta duodenale); parasitic helminths such as parasites (e.g., a protozoan or helminth described herein); fungi such as Aspergilli, Candidae, Coccidioides immitis, Cryptococci; environmental contaminants water additives; agricultural markers; nucleic acids (e.g., oligonucleotides, polynucleotides, nucleotides, nucleosides, molecules of DNA, or molecules of RNA, including chromosomes, plasmids, viral genomes, primers, or genes); proteins (e.g., glycoproteins, metalloproteins, enzymes, prions, or immunoglobulins); metabolites; sugars; lipids; lipopolysaccharides; salts; or ions. Targets also include foodborne pathogens such as Salmonella (e.g., Salmonella typhimurium), pathogenic E. coli (e.g., O157:H7), Clostridium botulinum (e.g., B. cereus), Clostridium botulinum, Listeria monocytogenes, Yersinia (e.g., Y. enterocolitica), Norovirus (e.g., Norwalk virus), Shigella, Staphylococcus aureus, Toxoplasma gondii, Vibrio (e.g., V. vulnificus, V. cholera, V. parahaemolyticus), Campylobacter jejuni, and Clostridium perfringens, Bacillus anthracis, Yersinia pestis, Brucella (e.g., B. suis), Burkholderia mallei, Burkholderia pseudomallei, Shigella, Clostridium botulinum, Variola (e.g., V.major), Filoviridae viruses (e.g., Filovirbolaelae) (e.g., filovirus), and the like, as well as Marburg virus, Arenaviridae (e.g., Lassa virus and Machupo virus), Clostridium perfringens, any foodborne pathogen (e.g., Salmonella spp., E. coli O157:H7, or Shigella), Chlamydia psittaci, Coxiella burnetii, Staphylococcus aureus, Staphylococcus aureus (e.g., R. prorowazekii or R. rickettsii), Alphavirus (e.g., Venezuelan equine encephalitis virus, Eastern equine encephalitis virus, or Western equine encephalitis virus), Vibrio cholerae, Cryptosporidium parvum, Henipavirus (e.g., Nipah virus), Bonyaviridae (e.g., Hantavirus or Rift Valley fever virus), Flaviviridae (e.g., Japanese encephalitis virus and Yellow fever virus), and Coccidioides spp.

[0043] An epitope that can be detected as an analyte or part of an analyte is typically an antigenic determinant site on an antigen to which an immunoglobulin (or an antigen-binding fragment thereof) can specifically bind. Epitopes can be formed from both contiguous or non-contiguous amino acids juxtaposed by tertiary folding of a protein. Epitopes can be found in the Fab (variable) region of an immunoglobulin (called an "idiotypic determinant"), comprising the "idiotype" of the immunoglobulin. Epitopes and antigens can be naturally occurring or artificially produced. Depending on the nature of the epitope or antigen, the epitope or antigen can be, for example, isolated or purified from a matrix or material of origin, synthesized, or recombinantly produced. Epitopes and antigens useful as analytes can be derived from human or non-human animals, plants, bacteria, protozoa, parasites, viruses, and the like. In some embodiments, the analyte is a polypeptide, a nucleic acid molecule, a carbohydrate, a glycoprotein, a lipid, a lipoprotein, a glycolipid, or a small molecule. In some embodiments, the analyte is selected from among cancer antigens, autoantigens, allergens, endogenous antigens, infectious agent antigens, drug (small molecule) antigens, toxins, toxins, biological antigens, environmental antigens, transplantation antigens, and transplantation antigens.

[0044] The analyte may include an epitope of a cancer antigen. In some embodiments, the analyte is a tumor-associated antigen. In some embodiments, the analyte is a tumor-specific antigen. In some embodiments of the invention, the analyte is a tumor-associated antigen (TAA), where the TAA is a carbohydrate antigen with one or more post-translational modifications that differ from the wild-type protein, includes a fusion region of a protein resulting from a gene fusion that is present in malignant cells but not present in non-malignant cells, and / or the TAA includes a receptor tyrosine kinase (RTK) that is deregulated and / or dysfunctional in tumor cells due to autocrine activation, chromosomal translocation, overexpression of an RTK, or a gain-of-function mutation in an RTK gene or protein. In some embodiments of the invention, the analyte is an immunoglobulin expressed by a B-cell malignancy. Examples of B-cell malignancies include, but are not limited to, non-Hodgkin's lymphoma, Hodgkin's lymphoma, chronic lymphocytic leukemia, mantle cell lymphoma, and multiple myeloma. Additional B cell malignancies include, for example, B cell prolymphocytic leukemia, lymphoplasmatic leukemia, splenic marginal zone lymphoma, marginal zone lymphoma (extranodal and nodal), plasma cell neoplasms (e.g., plasma cell myeloma, plasmacytoma, monoclonal immunoglobulin deposition disease, heavy chain disease), and follicular lymphoma (e.g., grade I, II, III, or IV).

[0045] In some embodiments, the analyte is a tumor-associated antigen derived from a tumor cell obtained from the subject. In some embodiments, the tumor-associated antigen is 17-1A, 707-AP, AFP, Annexin II, ART-4, BAGE, BAGE-1, β-catenin, BCG, bcr / abl, Bcr / abl e14a2 fusion junction, bcr-abl(b3a2), bcr-abl p190(e1a2), bcr-abl p210(b2a2), bcr-abl p210(b3a2), bcr-abl p210(b3a2)p210(b3a2), bullous pemphigoid antigen-1, CA19-9, CA125, CA215, CAG-3, CAMEL, cancer testis antigen, caspase-8, CCL3, CCL4, CD16, CD20, CD3, CD30, CD55, CD63, CDC27, CDK-4, CDR3, CEA, cluster 5, cluster 5A, cyclin-dependent kinase-4, Cyp-B, DAM-10, DAM-6, Dek-cain, E7, EGFR, EGFRvIII, EGP40, ELF2 M, EpCAM, FucGM1, G250, GA733, GAGE, GAGE-1-8, gastrin cancer-associated antigen, GD2, GD3, globoH, glycophorin, GM1, GM2, GM3, GnTV, Gn-TV, gp100, Her-2 / neu, HERV-K-ME, high molecular weight associated antigen, high molecular weight proteoglycan (HMPG), HPV-16 E6, HPV-16 E7, HPVE6, HSP70-2M, HST-2, hTERT, human chorionic gonadotropin (HCG), human milk fat globule (HMFG), iCE, KIAA0205, KK-LC-1, KM-HN-1, L6, LAGE-1, Lcose4Cer, LDLR / FUT, Lewis A, Lewis v / b, M protein, MAGE-1, MVC, MAGE-A1-12, MAGE-C2, MAHGE-3, MART-1 / Melan -A, MC1R, ME491, MUC1, MUC2, mucin, MUM-1, MUM-2, MUM-3, mutant p53, myosin, MZ2-E, N9 neuraminidase, NA88, NA88-A, nasopharyngeal carcinoma antigen, NGA, NK1 / c-3, novel bcr / ablk fusion BCR exon 1, 13, 14, ABL exon 4, NY-ESO-1 / LAGE-2, NY-ESO-1b, OC125, osteosarcoma-associated antigen-1, P15,The antigens are one or more selected from p190minimal bcr-abl(e1a2), p53, Pm1 / RARa, polysialic acid, PRAME, PSA, PSM, RU1, RU2, SAGE, SART-1, SART-2, SART-3, Sialyl-LeA, Sp17, SSX-2, SSX-4, surface immunoglobulin, TAG-1, TAG-2, TEL / AML1, TPI, TRAG-3, TRP-1(gp75), TRP-2, TRP2-INT2, hTRT, tumor-associated glycoprotein-72 (TAG-72), tyrosinase, u-PA, WT1, and XAGE-1b, or an immunogenic fragment of any of the foregoing antigens. In some embodiments, tumor-associated antigens are identified by a SEREX (serological analysis of recombinant cDNA expression libraries) approach or based on serological screening of cDNA expression libraries made from tumor tissues or cancer cell lines of various origins, identifying immunogenic tumor proteins based on reactivity with autologous serum. In some embodiments, the analyte is a tumor-associated antigen that is a carbohydrate antigen with one or more post-translational modifications that differ from the wild-type protein. In some embodiments, the tumor-associated antigen comprises a fusion region of a protein resulting from a gene fusion found in malignant cells but not in non-malignant cells. In some embodiments, the tumor-associated antigen comprises a receptor tyrosine kinase that is deregulated and / or dysfunctional in tumor cells due to autocrine activation, chromosomal translocation, RTK overexpression, or gain-of-function mutations in RTK genes or proteins.

[0046] Analytes may include epitopes of antigens of infectious or non-infectious agents that may be either pathogenic or non-pathogenic to the subject. Analytes may be derived from commensal, parasitic, or symbiotic microorganisms, including any microorganism in an animal or plant biome, such as probiotic or commensal microorganisms in the human digestive tract, mucosal surfaces, or epithelia. In some embodiments, the bacterial pathogen is Acinetobacter baumannii (formerly Acinetobacter calcoaceticus), Actinobacillus, Actinomyces pyogenes (formerly Corynebacterium pyogenes), Actinomyces israelii, Nocadia asteroids, Aeromonas hydrophila, Amycolata autotrophica, Archanobacterium haemolyticum (formerly Corynebacterium haemolyticum), Arizona hinshawii - all serotypes, Bacillus anthracis, Bacteroides fragilis, Bordetella including B. quintana, B. vinsoni, B. pertussis, Borrelia recurrentis, B. burgdorferi, Burkholderia (BSL III), Campylobacter coli, C. fetus, C. jejuni, Chlamydia psittaci, C. trachomatis, C. pneumoniae, Clostridium botulinum (neurotoxin-producing species), Clostridium botulinum neurotoxin, Cl. chauvoei, Cl. haemolyticum, Cl. histolyticum, Cl. novyi, Cl. septicemia, Cl. tetani, Cl. Perfirngens epsilon toxin, Corynebacterium diphtheriae, C. pseudotuberculosis, C. renale, Dermatophilus congolensis, Edwardsiella tarda, Erysipelothrix rhusiopathiae, Escherichia coli - all enteropathogenic, enterotoxinogenic, toxigenic, enteroinvasive and K1 antigen-bearing strains including E. coli O157, Haemophilus ducreyi, Haemophilus influenzae, Helicobacter pylori, Klebsiella - all species except K. oxytoca (RG1), L.Legionella including pneumophila - Leptospira interrogans - all serotypes, Listeria, Moraxella, Mycobacterium (except those listed in BSL III) avium complex, M. asiaticum, M. bovis BCG vaccine strains, M. chelonii, M. kansasii, M. leprae, M. marmosens, M. marinum, MM simiae, M. surugai, M. ulcerans, M. xenopi, Mycoplasma, N. meningitides, N. courdia asteroides, Salmonella including N. braziliensis, N. otitis scabiei, N. arizonae, Vibrio cholerae, Salmonella enteritidis, Galenarium pulmorum, Salmonella paratyphi, Shigella including Salmonella A, B, C, S, Salmonella typhimurium, S. boydii, Shigella type 1, S. flexneri, S., B. abortus, B. canis, B. suis, B. melitensis, B. pseudomallei, Coxiella burnetii, Francisella tularensis, Mycobacterium bovis (including BSG II-), Yersinia enterocolitica, Bartonella, Brucella, Chlamydia, Mycobacterium tuberculosis, Mycobacteria including Mycobacteria and others (MOTT), Pasteurella multocida type B- "Buffalo" and other virulent strains, Rickettsia akari, R. Selected from R. australis, R. canada, R. prowazekii, R. rickettsii, R. siberica, R. tsutsugamushi, R. typhi (R. mooseri), and Yersinia pestis.

[0047] Analytes may be from viral pathogens. For example, in some embodiments, analytes may be from Adenoviruses, Human-All Types, Alphaviruses (Togaviruses), Eastern Equine Encephalomyelitis Virus, Eastern Equine Encephalomyelitis Virus, Venezuelan Equine Encephalomyelitis Vaccine Strain TC-83, Western Equine Encephalomyelitis Virus, Arenaviruses, Lymphocytic Choriomeningitis Virus (Non-Neurotropic Strains), Tacaribe Virus Complex, Bunyaviruses, Bunyamwera Virus, Rift Valley Fever Virus Vaccine Strain MP-12, Calciviruses, Coronaviruses. Flaviviruses (Togaviruses)-Group B Arboviruses, Desmoviruses, and the like. Influenza virus serotypes 1, 2, 3, and 4, yellow fever virus vaccine strain 17D, hepatitis A, B, C, D, and E, cytomegalovirus, Epstein-Barr virus, herpes simplex types 1 and 2, human herpesviruses 6 and 7, influenza viruses A, B, and C, Newcastle disease virus, measles virus, mumps virus, parainfluenza virus types 1, 2, 3, and 4, polyomaviruses (JC virus, BK virus), respiratory syncytial virus, human parvovirus (B19), Coxsackieviruses A and B, echoviruses, polioviruses, rhinoviruses, astrim (Variola minor virus), smallpox (Variola major virus), whitepox reovirus, Coltivirus, human rotavirus, and orbivirus (Colorado tick fever virus), rabies virus, vesicular stomatitis virus, Rubivirus (rubella), Semliki Forest virus, Saint Louis encephalitis virus, Venezuelan equine encephalitis virus, Venezuelan equine encephalomyelitis virus, Arenaviruses (also known as South African Encephalitis viruses including erythropoietin virus (ERV), erythropoietin virus (YV), Lymphocytic choriomeningitis virus (LCM) (neurotrophic strains), Hunter virus, Rift Valley virus, Yellow fever virus, Monkeypox virus, Human immunodeficiency virus types 1 and 2 (HTLV), Human immunodeficiency virus types 1 and 2 (SIV), Vesicular stomatitis virus, Guanarito virus, Lassa fever virus, Junin virus, Machupo virus, Sabia, Crimean-Congo hemorrhagic fever virus, Ebola virus, Marburg virus, Tick-borne encephalitis including Far Eastern Dakar virus, Herpes simplex virus (Herpes B or Simian B virus), Cherkopithesin herpesvirus 1 (Herpes B virus), Equine morbillivirus (Hendra and Hendra-like virus), Nipah virus, Variola major virus (Small pox virus), Variola minor virus (Alastrim), African swine fever virus, African horse virus, Akabane virus, Avian influx virus, The virus is derived from a viral pathogen selected from influenza virus (highly pathogenic), blue tongue virus, camel pox virus, classic swine fever virus, cowdry aluminant (heartwater), foot and mouth disease virus, goat pox virus, Japanese encephalitis virus, lumpy skin disease virus, malignant catarrhal fever virus, menangle virus, Newcastle disease virus (VVND), peste de petilluminant virus, linder plague virus, sheep pox virus, swine vesicular virus, and vesicular stomatitis virus (exotic).

[0048] The analyte may be from a parasite. For example, in some embodiments, the analyte may be from Anklostoma human hookworm, including A. duodenale, Ascaris, including Ascaris lumbricoides suum, Babesia, including B. divergens, malayi, B. timori, Coccidia, Cryptosporidium, including C. parvum, Cysticercus cellulosae (larvae of T. solium), Echinoccus, including E. granulosis, E. multilocularis, E. vogeli, Entamoeba histolytica, Enterobius, Giardia, including Fasciola liglia, Giardia, including G. lamblia, Hymenolepis, including H. diminuta, H. nana, Isospora, Le eishmania, including L. braziliensis, L. donovani, L. ethiopica, L. major, L. mexicana, L. peruvania, L. tropica, Loa loa and from a parasite selected from the group consisting of firria worms, Microsporidium, Naegleria fowleri, N. americanus, O. vivax, S. isis, including S. suihominis, Schistosoma, including S. haematobium, Strongyloides, including S. japonicum, S. mansoni, S. mekongi, S. stercoralis, Taenia solium, Toxocara, including T. canis, T. trichinella, T. brucei, T. brucei gambiense, T. brucei rusesiense, T. cruzi, or Wuchereria bancrofti filarial worms.

[0049] The analyte can be a fungal pathogen. For example, in some embodiments, the analyte is selected from the group consisting of Aspergillus fumigates, Blastomyces dermatitidis, Cladosporium bantianum, Candida albicans, C. (Xylophyfa) trichoides, Cryptococcus neoformans, Dactylia gallopaba (Octromynia gallopa), Epidermophyton, Exophiala (Wangiella) dermatitidis, Fonsecaea pedrosoi, Microsporum, Paracoccidioides braziliensis, Penicillium marneffei, Pneumocystis carinii, Sporothrix schenckii, Trichophyton, Coccidioides immitis, Coccidioides posadasii, and the like. posadasii), Histoplasma capsulatum, and H. capsulatum var. duboisii.

[0050] The analyte can be a toxin. In some embodiments, the analyte is a toxin selected from abrin, botulinum neurotoxin, Clostridium perfringens epsilon toxin, conotoxin, diacetoxyscirpenol, ricin, saxitoxin, shiga-like ribosome-inactivating protein, shiga toxin, staphylococcal enterotoxin, T-2 toxin, and tetronic toxin.

[0051] In some embodiments, the analyte is Hepatitis B surface antigen (HBsAg), B. burgdorferi OspA, HPV L1, RSV Selected from F protein, influenza hamanglutanin, influenza stem loop region, influenza M2, P. falciparum merozoite surface protein 1-10, GLURP, SERA, S antigen, 6-cys family, AMA1, EBA175, 140, 181, MTRAP, PTRAMP, ASP, Rh1, 2a, 2b, 4, 5, RAP1, 2, 3, RAMA, RHOPH1, 2, 3, P. vivax circumnavigating sporozoite protein, sporozoite surface protein 2, SSP2 / TRAP, CSP-N, CSP-R, CSP-1, MSP-1, MSP-9, DBPRIII, AMA-1, Pvs25, Pvs28, Staphylococcus aureus capsular polysaccharide, poly-N-acetylglucosamine, HIV gp120, gp41, and Dengue virus conserved region.

[0052] In another embodiment, the analyte comprises at least one epitope of an allergen. The allergen may be naturally occurring or man-made, such as the allergens included in allergy vaccines. Examples of allergens include animal products (e.g. Fel d 1, fur dander, cockroach calyx, wool, dust mite excretion), drugs (e.g. penicillin, sulfonamides, salicylates, local anesthetics), foods (e.g. celery and celeriac, corn, eggs (e.g. albumin), fruits, legumes (e.g. beans, peas, peanuts, soy), milk, seafood (e.g. shellfish), sesame, soy, nuts (e.g. pecan, almond), wheat, insect venom, and the like. These include, but are not limited to, grasses (e.g., ryegrass, timothy grass, weeds (e.g., ragweed, plantain, nettle, Artemisia vulgaris, Polypodium album, sorrel), and trees (e.g., birch, alder, hazel, hornbeam, willow, poplar, sycamore, tilia, olea, ash juniper).

[0053] In some embodiments, the analyte is an allergen derived from latex proteins, such as unprocessed latex liquor, raw latex containing ammonia, or finished latex products in which the proteins have been exposed to chemicals and high temperatures. In some embodiments, the allergen is a mite allergen, such as Dermatophagoides farinae, Dermatophagoides pteronyssinus, Acarus siro, Blomia tropicalis, Chortoglyphus arcuatas, Euroglyphus cannei, Lepidoglyphus destructor, Tyrophagus putrescentiaeme, or Glyphapus gous. In some embodiments, the allergen is derived from venom, such as Bombus spp., Vespa crabro, Apis mellifera, Dolichovespula spp., Polistes spp., Vespula spp., Dolichovespula maculata, or Dolichovespula arenaria. In some embodiments, the analyte is an allergen derived from an insect, such as Camponotus pennsylvanicus, Solenopsis invicta, Solenopsis richteri, Periplaneta Americana, Blattella germanica, Blatta orientails, Tebanus spp., Musca domestica, Ephemeroptera sp., Culicidae sp., or Heterocera spp.

[0054] In some embodiments, the allergen analyte is epithelium, dander or hair from an organism such as, for example, Serinus canaria, Felis catus (domestic), Bos taurus, Gallus gallus (domestic), Canis familiaris, Allias platyrhynchus, Meriones ugicras, Capra hecus, Anser domesticus, Cavia porcellus (cobaya), Mesocrietus auratus, Sus scrofa, Equus caballus, Psittacidae, Columba fasciata, Oryctolagus cuniculus, Rattus norvegicus, or Ovis aries.

[0055] In some embodiments, the allergen analyte is of fungal origin, such as Cephalosporium acremonium, Alternaria tenuis, Aspergillus glaucosus, Aspergillus flavus, Aspergillus fumigatus, Aspergillus nidulans, Aspergillus niger, Aspergillus terreus, Aspergillus versicolor, Aureobasidium pullulans, Drechslera sorokiniana, Helminthosporium bean, Botrytis cinerea, Candida albicans, Chaetomium globosum, Cladosporium herbarum, Cladosporium sphaerospennum (Homodendrum hordei), Drechslera spicifera (Curvularia spicifera), Epicoccum nigrum (Epicoccum purpurascens), Epidermophyton floccosum, Fusarium moniliforme, Fusarium solani, Geotrichum candidum, Gliocladium viride, Helminthosporium solani, Microsporum canis, Mucor circinelloidesf circinelloides, Mucor circinelloidesf lusitanicus, Mucor plumbous, Mycogone perniciosa, Neurospora intermedia, Nigrospora oryzae, Paecilomyces variotii, Penicillum brevicompactum, Penicillum camembertii, Penicillum chrysogenum, Penicillum digitatum, Penicillum expansum, Penicillum notatum, Penicillum roquefortii.Phoma betae, Phoma herbarum, Rhizopus oryzae, Rhizopus stolonifer, Rhodotorula mucilaginosa, Saccharomyces cerevisiae, Scopulariopsis brevicaulis, Serpula lacrymans, Setosphaeria rostrata, Stemphylium botryosum, Stemphylium solani, Trichoderma harzianum, Trichophyton mentagrophytes, Trichophyton rubrum, or Trichothecium roseum. In some embodiments, the allergen is from a smut, such as Ustilago nuda, Ustilago synodontis, Ustilago candis, Sporisorium culentum, Ustilago avenae, or Ustilago tritici.

[0056] In some embodiments, the allergen analyte is from a grass such as, for example, Paspalum notatum, Cynodon dactylon, Poa compressa, Bromus inennis, Phalaris arundinacea, Zea cans, Elytrigia repens (Agropyron repens), Sorghum haelpense, Poa pratensis, Festuca pratensis (Festuca pratensis), Avena sativa, Dactylis glomerata, Agrostis gigantea (Alba), Secale cereale, Leymus (Elymus) condensatus, Lolium perenne ssp. multiflorum, Lolium perenne, Anthoxanthum odoratum, Phleum pratense, Holcus lanatus, Triticum aestivum, or Elymus (Agropyron) smithii.

[0057] In some embodiments, the allergen analyte is selected from the group consisting of weeds, e.g., Atriplex polycarpa, Baccharis halimifolia, Baccharis sarothroides, Hymenoclea salsola, Amaranthus hybridus, Xanthium strumarium (commune), Rumex crispus, Eupathium capillifolium, Solidagoss pulpuslamba (Acnida tamariscina), Allenrolfea occidentalis, Chenopodium botrys, Cochopodium album, Iva xanthifolia, Iva angustifolia, Chenopodium ambrosioides, Artemisia vulgaris, Artemisia ludovician, Urtica dioica, termite weevils, paramecium, bark beetles, bark beetles, Asteraceae, Ambrosia trifida, Ambrosia artemisiifolia, Ambrosia bidentata, Ambrosia psilostachya, Salsola kali(pestifer), Artemisia californica, Artemisia frigida, Artemisia tridentata, Atriplex wrightii, Atriplex and other items.

[0058] In some embodiments, allergen analysis is carried out from trees, for example, acacia seed, arnus·glutinosa, arnus·rubra, arnus·incana seed, rugosa, Alnus rhombifolia, Fraxinus velutina, Fraxinus pennsylvanica, Fraxinus latifolia, Fraxinus americana, Populus tremuloides, Myrica cerifera, Fagus grandifolia (America), Birch birch, Birch pendula, Black birch, Birch occidentalis (fontinalis), Birch populifolia, Acer negundo, Cryptomeria japonica, Juniperus ashei (sabinoides), Juniperus virginiana, Tamarix gallica, Populus balsamifera ssp. crassifolia、Ulm pumila、Eucalyptus globulus, Celtis occidentalis, Corylus americana, Corylus avellana, Carya ovata, Carya laciniosa, Carya alba, Juniferus monosperma, Juniperus princhotii, Juniperus scopulorum, Juniperus occidentalis, Robinia pseudoacacia, Mangifera indica, Acer macrophyllum, Acer rubrum, Acer saccharum, Melaleuca quinquenervia (leucadendron), Prosopisglandulosa (juliflora), Broussonetia papyrifera, Morus rubra, Morums alba, Quercus gambelii, Quercus velutina, Quercus macrocarpa, Quercus kelloggii, Quercus agrifolia, Quercus lobata, Quercus ilex, Quercus stellata, Quercus rubra, Quercus dumosa, Quercus virginiana, Quercus nigra, Quercus garryana, Quercus alba, Olea europaea, Elaegnus angustifolia, Citrus sinensis, Arecastrum romanzoffianum(Cocos plumosa), Carya illnoensis, Schinus molle, Schinus terebinthifolius, Pinus taeda, Pinus strobus, Pinus palustris, Pinus ponderosa, Pinus elliottii, Pinus virginiana, Pinus monticola, Pinus echinata, Populus nigra, Populus alba, Ligustrum vulgare, Liquidambar styraciflua, Platanus occidentalis, Platanus orientalis, Platanus racemosa, Platanus acerifolia, Juglans nigra, Juglans californica, Juglans regia, Salix lasiolepsis, Salix nigra, or Salix discolor. In some embodiments, the allergen is from a flower, such as Chrysanthemum eucanthum, Taraxacum officinale, or Helianthus annuus. In some embodiments, the allergen is from an agricultural crop, such as Medicago sativa, Ricinus communis, Trifolium pratense, Brassica species, or Beta vulgaris.

[0059] In some embodiments, the allergen analyte is derived from a plant food (edible plant), e.g. Plum (Prunus dulcis), Malus pumila, Plum (Prunus armeniaca), Musa paradisiaca (Musa paradisiaca), Hordeum vulgare, Phaseolus lanatus, Phaseolus vulgaris, Phaseolus sp. botrytis、Fagopyrum esculentum、Brassica oleracea var. capitata, Theobroma cacao, Cucumis melo, Daucus carota, Brassica oleracea var.botrytis, Apium graveolens var.Dulce, Prunus sp., Cinnamomum verum, Coffea arabic, Zea cans, Vaccinium macrocarpon, Cucumis sativus, Allium sativum, Zingiber officinale, Vitis sp., Citrus paradisi, Humulus lupulus, Citrus limon, Lactuca sativa, Agaricus campestris, Brassica sp., Myristica fragrans, Avena sativa, Olea europaea, Allium cepa var.cepa, Citrus sinensis, Vigna unguiculata, Pisum sativum, Prunus persica, Pyrus communis, Piper nigrum, Capsicum annuum var.annuum, Ananas comosus, Ipomoea batatas, Solanum tuberosum, Rubus idaeus var.idaeus、Oryza sativa、Secale cereale、Oriental sesame(indicum)、Glycine max、Spinacia oleracea、Cucurbita pepo var.melopepo、Fragaria chiloensis、Lycopersicon esculentum(lycopersicon)brassica rapacum var.rapa, Vanilla planifolia, Citrullus lanatus var.lanatus, Triticun aestivum.

[0060] In some embodiments, the allergen analyte is from a fish or crustacean, such as Micropterus sp., Ictalurus punctatus, Mercenaria mercenaria, Gadus morhua, Callinectes sapidus, Platichthys sp., Hippoglossus sp., Homarus americanus, Scomber scombrus, Crassostrea virginica, Sebastes marinus, Salmo salar, Clupeiformes, Pecten magellanicus, Penaeus sp., Salvelinus sp., or Thunnus sp. In some embodiments, the allergen is an animal food, such as from Bos taurus, Ovis aries, or Sus scrofa. In some embodiments, the allergen is a poultry product, such as a chicken (Gallus gallus) product or a turkey (Meleagris gallopavo) product. In some embodiments, the allergen is from dairy products, such as bovine casein or bovine milk.In some embodiments, the allergen is from nuts, such as Bertholletia excelsa, Anacardium oceidentale, Cocos nucifera, Corylus americana, Arachis hypogaea, Carya illinoensis, Juglans nigra, or Juglans regia.In some embodiments, the allergen is from dust, such as barley flour, corn flour, house dust, mattress flour, oat flour, wheat grain, upholstery flour, or latex flour.

[0061] In some embodiments, the antigen analyte is an autoantigen associated with an autoimmune disease. In some embodiments, the autoimmune disorder is a cell- or organ-specific autoimmune disorder, and the autoantigen analyte is an acetylcholine receptor (myasthenia gravis), actin (chronic active hepatitis, primary biliary cirrhosis), adenine nucleotide translocator (ANT) (dilated cardiomyopathy, myocarditis), beta-adrenergic receptor (dilated cardiomyopathy), aromatic L-amino acid decarboxylase (autoimmune polyendocrine syndrome type I (APS-1)), asialoglycoprotein receptor (autoimmune hepatitis), bactericidal / permeability enhancing protein (Bpi) (cystic fibrosis vasculitis), calcium sensing receptor (acquired hypoparathyroidism), cholesterol side-chain cleavage enzyme (CYPIIa) (APS-1), collagen type IV alpha 3 chain (Goodpasture's syndrome), cytochrome P450 2D6 (CYP2D6) (autoimmune hepatitis), desmin (Crohn's disease, coronary artery disease), desmoglein 1 (pemphigus vulgaris), desmoglein 3 (pemphigus vulgaris), F-actin (autoimmune hepatitis), GM ganglioside (Guillain-Barré syndrome), glutamic acid decarboxylase (GAD65) (type 1 diabetes, stiff man syndrome), glutamate receptor (GLUR) (Rasmussen's encephalitis), H / K ATPase (autoimmune gastritis), 17-α-hydroxylase (CYP17) (APS-1), 21-hydroxylase (CYP21) (Addison's disease), IA-2 (ICA512) (type 1 diabetes), insulin (type 1 diabetes), insulin-hypoglycemic syndrome (Hirata's disease), type B insulin resistance, acanthosis nigricans, systemic lupus erythematosus (SLE)), intrinsic factor type 1 (pernicious anemia), leukocyte function-associated antigen (LFA-1) (treatment-resistant Lyme arthritis), myelin-associated glycoprotein (MAG) (polyneuropathy), myelin basic protein (multiple sclerosis, demyelinating diseases)), myelin oligodendrocyte glycoprotein (MOG) (multiple sclerosis), myosin (rheumatic fever), p-80-coilin (atopic dermatitis), piluve dehydrogenase complex-E2 (PDCE2) (primary biliary cirrhosis), sodium iodide symporter (NIS) (Graves' disease, autoimmune hypothyroidism), SOX-10 (vitiligo), shared protein of thyroid and eye muscle (autoimmune thyroiditis), thyroid peroxidase (autoimmune Hashimoto's thyroiditis), thyrotropin receptor (Graves' disease), tissue transglutaminase (celiac disease), transcription activator p75 (atopic dermatitis), tryptophan hydroxylase (APS-1), tyrosinase (vitiligo, metastatic melanoma), with the associated autoimmune disorder being listed in parentheses immediately following each autoantigen analyte.

[0062] In some embodiments, the autoimmune disorder is a systemic autoimmune disorder and the autoantigen analyte is selected from ACTH (ACTH deficiency), aminoacyl-tRNA histidyl synthetase (myositis, dermatomyositis), aminoacyl-tRNA synthetase (polymyositis, dermatomyositis), cardiolipin (SLE), carbonic anhydrase II (SLE, Sjogren's syndrome, systemic sclerosis), collagen (rheumatoid arthritis (RA), SLE, progressive systemic sclerosis), centromere-associated protein (systemic sclerosis), DNA-dependent nucleosome-stimulated ATPase (dermatomyositis)), fibrillarin (scleroderma), fibronectin (SLE, RA, morphea), glucose-6-phosphate isomerase (RA), β2-glycoprotein I (β2-GPI) (primary antiphospholipid syndrome), golgins (95, 97, 160, and / or 180) (Sjögren's syndrome, SLE, RA), heat shock proteins (various immune-related disorders), hemidesmosomal protein 180 (bullous pemphigoid, pemphigus herpetiformis, histone H2A-H2B-DNA (SLE), IgE receptor (chronic idiopathic urticaria), keratin (RA), Ku-DNA-protein kinase (SLE), Ku-nuclear protein (connective tissue syndrome), La phosphoprotein (La Autoantigens were selected from among 55-B), myeloperoxidase (necrotizing and focal glomerulonephritis (NCGN), systemic vasculitis), proteinase 3 (PR3) (Wegener's granulomatosis, Churg-Strauss syndrome), RNA polymerase I-III (RNP) (systemic sclerosis, SLE), signal recognition protein (SRP54) (polymyositis), topoisomerase-1 (Scl-70) (scleroderma, Raynaud's syndrome), tubulin (chronic liver disease, visceral leishmaniasis), and vimentin (systemic autoimmune diseases), and the associated autoimmune disease is listed in parentheses immediately following each autoantigen.

[0063] In some embodiments, the autoimmune disorder is a plasma protein autoimmune disorder or a cytokine autoimmune disorder, and the autoantigen analyte is selected from C1 inhibitor (autoimmune C1 deficiency), C1q (SLE, membranoproliferative glomerulonephritis (MPGN)), cytokines (for example). The autoantigens selected from the following autoimmune diseases are selected: IL-1α, IL-1β, IL-1, IL-10, LIF) (RA, systemic sclerosis), factor II (prolonged clotting time), factor V (prolonged clotting time), factor VII (prolonged clotting time), factor VIII (prolonged clotting time), factor IX (prolonged clotting time), factor X (prolonged clotting time), factor XI (prolonged clotting time), factor XII (prolonged clotting time), thrombin (prolonged clotting time), vWF (prolonged clotting time), glycoproteins IIb / IIIg and Ib / IX (autoimmune thrombocytopenic purpura), IgA (immunodeficiency), and oxidized LDL (OxLDL) (atherosclerosis), and the associated autoimmune disease is mentioned in parentheses immediately following each autoantigen analysis.

[0064] In some embodiments, the autoimmune disorder is cancer or a paraneoplastic autoimmune disorder and the autoantigen analyte is selected from amphiphysin (neuropathy, small cell lung cancer), cyclin B1 (hepatocellular carcinoma), DNA topoisomerase II (liver cancer), desmoplakin (paraneoplastic pemphigus), gephyrin (paraneoplastic stiffness syndrome), Hu protein (paraneoplastic encephalomyelitis), neuronal nicotinic acetylcholine receptor (subacute autonomic neuropathy, cancer), p53 (cancer, SLE), p62 (IGF-II) mRNA binding protein) (hepatocellular carcinoma), ribeykin (cancer-associated retinopathy), R1 protein (paraneoplastic opsoclonus-myoclonus ataxia), beta IV spectrin (lower motor neuron syndrome), synaptotagmin (Lambert-Eaton myasthenic syndrome), voltage-dependent calcium channel (Lambert-Eaton myasthenic syndrome), and Yo protein (paraneoplastic cerebellar degeneration).

[0065] In some embodiments, the antigen analyte is an endogenous antigen that is an abnormally expressed polypeptide. Examples of such endogenous antigens include amyloid beta (Abeta), alpha synuclein, cystatin C, tau, ABri, ADan, superoxide dismutase (SOD), mutant huntingtin, PrPsc, or any fragment thereof.

[0066] In some embodiments of the present invention, the analyte comprises at least one epitope of an implant introduced into a subject, a metabolite or decomposition product of an implant material, or a substance that specifically binds to an epitope of an implant material, such as an antibody. Such implants may include, for example, powered implants (e.g., artificial pacemakers), bioimplants (biological materials surgically implanted in a subject's body to replace damaged tissue (e.g., orthopedic reconstructive prostheses)), cardiac prostheses (artificial valves), skin, cornea), contraceptive implants, dental implants, orthopedic implants, and adhesion prevention devices. Examples of implant materials that can carry epitopes include metals such as latex, silicone, cobalt-chromium (Co-Cr) alloys, titanium, and titanium alloys, polymers such as ultra-high molecular weight polyethylene (UHMWPE) and polymethylmethacrylate cement (PMMA), and bioceramics such as hydroxyapatite and bioglass.

[0067] In certain embodiments, the non-antibody binding component can be a bacteria-binding protein or an antibody-binding domain. The analyte of interest can be selected from beneficial enterobacteria, pathogenic bacteria, protein toxins, protein biomarkers, small molecule toxins, metabolites, or chemical warfare agents. For such analytes, in some embodiments, the assay is modified to a competitive format in which the small molecule analyte is bound to a protein or other polymeric carrier such that antibodies against the free analyte also recognize the immobilized analyte (if there are no antibodies). They can be generated by immunizing animals with the immobilized analyte. The immobilized analyte will agglutinate cellular receptors that give a luminescent signal that are bound to the anti-analyte antibodies. If the immobilized analyte is mixed with the free analyte to be measured and it is unable to bring about agglutination, the luminescent signal will decrease. The activator can be a receptor, and the non-antibody binding component can be a ligand that is specific for the receptor and that causes a conformational change in the receptor (rather than agglutination) upon binding to the receptor. Only after it has bound to the analyte of interest. The ligand can be fused to a detector, which acts to prevent the ligand from binding to the receptor unless it has first bound to the analyte of interest. The activator may also be a receptor designed to bind to a given analyte, where the receptor undergoes a conformational change upon binding to the given analyte. Also, this variant does not rely on aggregation effects. However, in other embodiments, the aggregation event may be mediated by a carrier molecule, such as serum albumin, that binds to multiple copies of a target, such as a physiological or drug metabolite.

[0068] Biosensor Example I 1a-1b, a first biosensor 100 according to an exemplary embodiment of the present invention includes a Jurkat T cell 102 that is engineered to produce aequorin 104 and loaded with CTZ 106 to form an aequorin / CTZ complex. This particular biosensor is also engineered to express a transmembrane non-antibody signaling component 108 that is IgGbp-CD3ζ (SEQ ID NOS: 5-6), although a transmembrane non-antibody signaling component FcγRI-CD3ζ (SEQ ID NOS: 11-12) can also be used with the biosensor 100. The biosensor cell 102 also includes at least one signaling pathway 110, the activation of which is mediated by intracellular Ca 2+ As shown in FIG. 1b, when a sufficient amount of a detection molecule 114 (e.g., a soluble antibody) with bound target analyte 116 (e.g., E. coli 0157) binds to the transmembrane non-antibody signaling component 108, a signaling pathway 110 is activated, resulting in an increase in Ca 2+ 112 increases, the aequorin / CTZ complex undergoes a conformational change emitting a light 118 signal (photons) that is detected by a photomultiplier tube 120, causing a spike 122 to be displayed graphically on the test device (see below), indicating the presence of the target analyte 116 in the sample being tested. This display can be both qualitative and quantitative with respect to the target analyte 116.

[0069] Biosensor Example II 2a-b, as described above, a second biosensor 200 according to an exemplary embodiment of the present invention includes MC / 9 (ATCC® CRL-8306™) mast cells 202 that are engineered to produce aequorin 204 and loaded with CTZ 206 to form an aequorin / CTZ complex. This particular biosensor expresses a native Fc epsilon receptor (i.e., FcεRI) 207 that binds to a soluble non-antibody signaling component 208 that is IgGbp-IgE (SEQ ID NOS: 13-14), although non-antibody signaling components FcγRI-IgE. (SEQ ID NOS: 15-16) may also be used with the biosensor 200. As shown in FIG. 2b, when a sufficient number of detection molecules 214 (e.g., soluble antibodies) with bound target analytes 216 (e.g., E. coli 0157) bind to non-antibody signaling moieties 208 already bound to natural Fc epsilon receptors 207, a signaling pathway 210 is activated, leading to an increase in intracellular Ca 2+ 212 increases, the aequorin / CTZ complex undergoes a conformational change emitting a light 218 signal (photons) that is detected by a photomultiplier tube 220, causing a spike 222 to be displayed graphically on the test device (see below), indicating the presence of the target analyte 216 in the sample being tested. This display can be both qualitative and quantitative with respect to the target analyte 216.

[0070] Biosensor Example III 3a-b, as described above, a third biosensor 300 according to an exemplary embodiment of the invention includes MC / 9 (ATCC® CRL-8306™) mast cells 302 that are engineered to produce aequorin 304 and loaded with CTZ 306 to form an aequorin / CTZ complex. This particular biosensor expresses a native Fc epsilon receptor (i.e., FcεRI) 307 that binds to a non-antibody signaling component 308 that is IgGbp-IgE (SEQ ID NOS: 13-14), although a non-antibody signaling component FcγRI-IgE. (SEQ ID NOS: 15-16) can also be used with the biosensor 300. In this particular embodiment, the biosensor cells 302 are further engineered to express IgGbp-IgE and to export this non-antibody signaling component to the extracellular space, which binds to the native FcεRI expressed on the cell surface. As shown in FIG. 3b, when a sufficient amount of a detection molecule 314 (e.g., a soluble antibody) with bound target analyte 316 (e.g., E. coli 0157) binds to a non-antibody signaling component 308 already bound to a natural Fc epsilon receptor 307, a signaling pathway 310 is activated, leading to an increase in intracellular Ca 2+ 312 increases, the aequorin / CTZ complex undergoes a conformational change emitting a light 318 signal (photons) that is detected by a photomultiplier tube 320, causing a spike 322 to be displayed graphically on the test device (see below), indicating the presence of the target analyte 316 in the sample being tested. This display can be both qualitative and quantitative with respect to the target analyte 316.

[0071] Example of Biosensor IV Referring to FIG. 4, a fourth biosensor 400 according to an exemplary embodiment of the invention includes a biosensor cell 402 designed to produce aequorin and express a transmembrane non-antibody signaling component 408 that is mSA-CD3ζ (SEQ ID NOs: 17-18). The non-antibody signaling component mSA-CD3ζ (monomeric streptavidin-CD3ζ) binds to a biotinylated detection molecule 414, which specifically binds to a target molecule 416, such as epidermal growth factor (EGF). An anti-target molecule antibody 417, such as anti-EGF, clusters multiple signaling components and generates target multimers that induce signaling, as described above. In other embodiments, the monomeric streptavidin component is replaced with a biotinylated component and alternative linking means may be used.

[0072] Example of Biosensor V Referring to FIG. 5, a fifth biosensor 500 according to an exemplary embodiment of the invention includes a biosensor cell 502 designed to produce aequorin and express a transmembrane non-antibody signaling component 508 that is mSA-CD3ζ (SEQ ID NOs. 17-18). The non-antibody signaling component mSA-CD3ζ (monomeric streptavidin-CD3ζ) binds to a biotinylated detection molecule 514, which in some embodiments is an autoantigen molecule. The biotinylated detection molecule 514 specifically binds to a target molecule 516, which in some embodiments is an anti-autoantigen molecule. Autoantibodies in the serum sample cluster multiple signaling components and generate target multimers that induce signaling, as described above. In other embodiments, the monomeric streptavidin component is replaced with a biotinylated component and alternative linking means may be used.

[0073] The amino acid sequence of a signaling polypeptide used to produce a chimeric protein of the invention can have at least 70%, 75%, 80%, 85%, 87.5%, 90%, 92.5%, 95%, 97.5%, 98%, 99% sequence identity or similarity to the proteins or domains identified by or identified in the following accession numbers: IgM heavy chain (GenB ank:CAC20458.1), Ig-alpha (P11912.2, GI:547896), Ig-beta (P40259.1, GI:728994), CD19 (AAA69966.1, GI:901823), CD3 zeta (P20963.2, GI:23830999), IgE alpha (1F2Q_A, GI:9257150), and Fc-εR1 subunit alpha (P123191, GI:119865).

[0074] Staphylococcus aureus protein A (P02976.3, GI:110283003) is encoded by the spa gene of Staphylococcus aureus and its structure including the Ig-binding segment, and its immunoglobulin-binding properties are well known and are incorporated herein by reference in Graille, et al., Proc Natl Acad Sci US A. 2000 May 9;97(10):5399-404; and Roben, et al. J Immunol. 1995 Jun 15;154(12):6437-45. Variants of protein A or its immunoglobulin-binding segments having at least 70%, 75%, 80%, 85%, 87.5%, 90%, 92.5%, 95%, 97.5%, 98%, 99% sequence identity or similarity to known protein A amino acid sequences and the ability to bind immunoglobulins or other analytes as disclosed by Graille et al. and Roben et al. can be produced by molecular biology techniques well known in the art including the direct synthesis of nucleic acids encoding immunoglobulin-binding amino acid sequences.

[0075] Other bacterial immunoglobulin-binding proteins, such as streptococcal protein G and engineered variants of such proteins, are well known and are incorporated herein by reference in Bailey, et al., J Immunol Methods. 2014 Dec 15;415:24-30 (doi:10.1016 / j.jim.2014.10.003) (Epub 2014 Oct 22); and Watanabe, et al., J Biol Chem. 2009 May 1;284(18):12373-8 (doi:10.1074 / jbc.M809236200) (Epub 2009 Mar 6). Variants of protein G or its immunoglobulin-binding segment having at least 70%, 75%, 80%, 85%, 87.5%, 90%, 92.5%, 95%, 97.5%, 98%, 99% sequence identity or similarity to the known protein G amino acid sequence and the ability to bind to immunoglobulins or other analytes as disclosed by Bailey et al. and Watanabe et al. can be produced by molecular biology techniques well known in the art including the direct synthesis of nucleic acids encoding the immunoglobulin-binding amino acid sequences.

[0076] Fc receptors (FcR) bind the Fc portion of immunoglobulins, and many types of Fc receptors are known, including FcγRI and FcεRI. The structural and functional binding properties of these FcRs are incorporated by reference in Fridman, FASEB J. 1991 Sep;5(12):2684-90. Variants of FcRs or their immunoglobulin-binding segments having at least 70%, 75%, 80%, 85%, 87.5%, 90%, 92.5%, 95%, 97.5%, 98%, 99% sequence identity or similarity to known FcR amino acid sequences, such as those disclosed by Fridman, can be produced by molecular biology techniques well known in the art, including direct synthesis of nucleic acids encoding immunoglobulin-binding amino acid sequences.

[0077] The signaling proteins according to the present invention can have at least 70%, 75%, 80%, 85%, 87.5%, 90%, 92.5%, 95%, 97.5%, 98%, 99% sequence identity or similarity to the chimeric signaling proteins disclosed by SEQ ID NOs: 2, 4, 6, 8, 10, 12, 14, 16, and 18, and also have the ability to bind to an analyte, such as an immunoglobulin, and then transmit a signal to a modified biosensor cell. Such variants can be constructed by methods well known in the art of molecular biology, or by chemically synthesizing a polynucleotide encoding the variant chimeric reporter protein, inserting the coding sequence into a vector, and transforming or transfecting a competent cell with the vector.

[0078] Cell sorting and cloning The design and construction of the biosensor of the present invention results in a mixed population of biosensor cells when cultured. Some cells did not express the modifier, while others expressed the factor at various levels. After successful electroporation and gene insertion, the biosensor cells were cultured and tested for biological response (flash signal) as a mixed population. Single cell sorting was performed using a flow cytometer. Cells were isolated and then expanded for analysis to select those that expressed high levels of the desired protein. For this process, fluorescently labeled antibodies were used to target different receptors on the biosensor cells, thereby enabling the sorting process. Individual clones were screened for signaling and the best clones were selected. Through this process, the most suitable clones were identified and isolated. Fluorescence-activated cell sorting (FACS) and live cell staining for extracellular proteins were performed as follows.

[0079] The modified biosensor cells were counted, gently centrifuged, and diluted to 1 × 10 in wash buffer (HBSS + 2% BSA). 7 ~1×10 8 In each experiment, either full antibodies with Fc regions or F(ab) were used. 2Either Fc receptor blocking antibody or 100 μL of Fc receptor blocking antibody was used. When using complete antibodies, 1–0.5 μg of Fc receptor blocking antibody was added to each empty 12 × 15 mm tube intended for receiving cells. To each of these tubes, 100 μL of cells (1 × 10 6 ~1×10 7 F(ab) was added on top of the Fc-blocking antibody. The cells were gently mixed and incubated for 15 minutes at 4°C or room temperature. 2 When using , the above-mentioned step of blocking Fc was omitted. A total of 1 μg of primary antibody (against the receptor of choice) was added, then the cells were gently mixed and then incubated on ice (or at 4 °C) for 20-40 min. This temperature prevented receptor internalization. The cells were intermittently gently agitated (swirled) to promote labeling. A 2 mL volume of cold wash buffer was added, then the cells were centrifuged at 4 °C and the supernatant was discarded. After repeating the wash step, the cells were resuspended in 100 μL of wash / sort buffer. A secondary FITC-labeled antibody was added to the cells (0.5-1 μg), mixed and then incubated on ice (or at 4 °C) for 20-40 min. The cells were protected from light throughout the entire process. A 2 mL volume of cold wash buffer was added, then the cells were centrifuged and the supernatant was discarded. The wash step was repeated and the cells were resuspended in 0.5-1 mL of wash buffer. The cells were incubated on ice until sorting. Sorting was performed as soon as possible (at least on the same day). Cloning and culture of the cells after single cell sorting were carried out as follows.

[0080] Biosensor cells were sorted into 96-well plates such that each well contained one cell and 100–200 μL of cell growth medium. The plates were scanned / monitored for the next 10–14 days to determine the growth rate and to determine when to transfer to 24-well plates. During scanning, different markings were used for different conditions. Some wells were marked if they contained live cells, but not ready for transfer, and contaminated wells were also marked. Cells were transferred to 24-well plates containing 1.0 mL of the appropriate medium in each well. If cells were contaminated, they were washed by adding all of the cell suspension from the wells to 5 ml of sterile 1x PBS in a 15 mL conical tube. Cells were then centrifuged at 170 RCF for 10 min and the supernatant was discarded. The pellet was resuspended in 1.0 mL of fresh medium in the 24-well plate and cultured. After continued growth, cells were transferred to 12-well plates containing 1.5 mL of fresh medium per well.

[0081] For clone screening, after growth in 12-well plates, cells were counted to determine whether they were ready for the fill and flush test. During the flush test, 25,000 cells were associated per repeat. Enough cells were grown to fit the test, and some were left to continue growing. This step represented the first period of clone screening. Selected clones were further expanded and subjected to subsequent tests depending on the desired properties. For biological response, for example, Jurkat-FcγRI-CD3ζ clones were screened using anti-CD3ε antibody (positive control) and monoclonal antibodies against bacteria with the respective bacteria, while digitonin was used for chemical response tests. MC / 9-Aeq clones were screened using anti-FcεRI antibody (biological response) and digitonin (chemical response).

[0082] In summary, fluorescence-activated cell sorting (FACS) was performed using fluorescent antibody labeling to select and isolate cells highly expressing the desired protein (in this case the modified receptor). This process resulted in a population of highly expressing biosensor cells that were further confirmed by a flash test using a PMT in the testing device. During the entire process, cells were counted using an automated cell counter to eliminate human error and increase consistency and efficiency. Different clones of Jurkat-FcγRI-CD3ζ showed different levels of biological response when tested with anti-E. coli O111 mAb and E. coli O111 bacteria. Many clones were tested in the same way, and the ones that showed the highest response were kept in a clone bank. Similarly, chemical response results obtained from testing different MC / 9-Aeq clones with the chemical digitonin showed that different clones gave different levels of chemical response depending on the level of aequorin expression. The clones with the highest signal were kept in a clone bank.

[0083] Cultivation of biosensor cells Different media formulations were used for different cell lines to ensure optimal growth conditions. MC / 9 mast cells were cultured in complete mast cell medium (DMEM-Sigma, Cat. No. D5796; 1x Pen / Strep; 10% FBS; 10% T-Stim Supplement; 50 μM β-mercaptoethanol). Jurkat T cells were cultured in complete RPMI medium (RPMI-ThermoFisher; 10% FBS; 1x Pen / Strep). Depending on the properties of the electroporated construct, different antibiotics were used for selection in cell culture. Appropriate antibiotics were added to the growth medium 2–3 days after electroporation to select for cells that had successfully integrated the linearized DNA construct. The cell concentration was 4.0 × 10 for optimal cell growth. 5 ~1.0×10 6The different cell lines and clones were processed for long-term storage and stocks were frozen in liquid nitrogen as follows: (i) cells were centrifuged at 150 RCF for 10 min and the supernatant was discarded; (ii) the cell pellet was diluted with 5.0 × 10 5 (iii) Volumes of 1 mL were aliquoted into 2 mL Nunc Cryo vials and frozen at -80°C for 24 hours before being transferred to liquid nitrogen for long-term storage.

[0084] Loading of biosensor cells The biosensor cells of the present invention were centrifuged at 150 RCF for 10 min in a 50 mL conical tube. The supernatant was discarded and the pellet was resuspended in loading medium (RPMI; 10% antibody-depleted FBS; 1x pen / strep; 0.1% Pluronic F68 and 1.5 mM coelenterazine) at a concentration of 25,000 cells / 180 μL. Additionally, cells were also loaded at different concentrations, e.g., 100,000 cells / 180 μL and 400,000 cells / 180 μL. Cells were loaded with gentle shaking / rocking at room temperature for 24-26 h. Commercially available antibody-depleted FBS may be further purified using other antibody removal systems prior to use.

[0085] Cell enrichment It was demonstrated that the biosensor cells of the present invention are more effectively loaded at low concentrations than at high concentrations. For example, loading cells at a density of 25,000 cells / 180 μL versus 400,000 cells / 180 μL showed a two-fold increase in detectable signal. Jurkat-FcγRI-CD3ζ clone P5G7 cells were loaded at both 400,000 cells / 180 μL and 25,000 cells / 180 μL, then tested at 400,000 cells / 180 μL in each reaction. An overnight E. coli O111 bacterial culture was used with 23 nM anti-E. coli O111 mAb. Biosensor cells loaded at low concentrations gave a higher signal for the same number of bacterial cells tested. The density of the biosensor cells was mostly varied by concentrating the cells after loading to allow optimal pathogen detection. When the detection molecule was a soluble antibody, different concentrations were used for pathogen detection, depending on the target pathogen and the quality of the antibody. Biosensor cells were concentrated by centrifugation at 150 RCF for 10 min, and the cell pellet was resuspended in the desired amount of test medium. The loading medium could also serve as the test medium. In certain instances, the addition of normal FBS to the medium triggered a biological response, resulting in a biosensor signal (flash) due to the high concentration of antibodies in normal FBS. Therefore, a commercially available antibody-depleted FBS was used in the loading process, which reduced the antibody-induced signal without completely eliminating it. An additional method was used to further remove trace amounts of antibodies in the commercially available antibody-depleted FBS.

[0086] Bioassay In exemplary aspects of the invention, analyte bioassays were formatted using biosensor cells and soluble monoclonal antibodies (mAbs) specific for the analyte (e.g., pathogen). In these embodiments, the specificity of the bioassay is directly related to the selective binding of the soluble antibody to the target analyte, and the specificity and sensitivity of the biosensor were determined by detecting and measuring bioluminescence. In this process, the biosensor cells were first loaded using a luminescent molecule, coelenterazine (CTZ). The selected soluble antibody and the sample under analysis were then added. If the target pathogen is present in the sample, it interacts with the soluble antibody that binds to the fusion protein expressed by the biosensor cells, triggering a signal cascade that ultimately results in light emission from the biosensor cells. The emitted light is detected by a photomultiplier tube (PMT) in the test device, and the signal emitted by the biosensor cells is displayed as the number of photons per second. As described below, various methods have been developed for detecting pathogens based on soluble antibodies and target pathogens. Three such methods, described in detail below, include: (i) immediate addition of a detection molecule (e.g., an antibody), (ii) coating of biosensor cells with a detection molecule (e.g., an antibody), and (iii) coating of the analyte (e.g., bacteria) with an antibody.

[0087] Test Unit The bioassay aspects of the invention herein can be carried out in a test subunit or test cartridge designed for use with benchtop or portable test systems and devices such as those disclosed in U.S. Pat. No. 9,023,640, which is incorporated herein by reference in its entirety. The test cartridge may be a one-time disposable item that receives both the sample and the biosensor, and mixes the sample and the biosensor in a predictable and controlled manner by introducing the biosensor into the test cartridge. The test cartridge further includes a reaction chamber for receiving the test sample and the biosensor, the reaction chamber having a predetermined internal shape and adapted to minimize or eliminate background noise for the purpose of improving the overall signal-to-noise ratio. At least one stabilizer can be disposed in the reaction chamber to minimize shear damage to the test sample and the biosensor during the mixing process.

[0088] In an exemplary embodiment, the reaction chamber in the test cartridge and the fluid channel leading to the reaction chamber are designed to achieve several objectives. The inlet channel for fluid entering the reaction chamber includes a tubular shape, with the diameter of the tube being relatively small and tapering to a smaller diameter at the inlet to the reaction chamber. This increases the velocity of the fluid entering the reaction chamber, promoting more vigorous and homogeneous mixing due to the bulk movement of the reagents in the reaction chamber. It is desirable to mix the reagents and sample in a manner that promotes mixing beyond molecular diffusion to minimize the duration of the test by ensuring that any infectious material present in the sample quickly encounters the biosensor. The inlet channel is offset from the central axis of the reaction chamber to promote clockwise or counterclockwise rotational movement of the reagents around the central axis of the test chamber as the fluids are mixed to increase the homogeneity of the mixture. The inlet channel also nearly tangents to the inner surface of the reaction chamber to allow the incoming fluid to move from the inlet channel to the reaction chamber while remaining in contact with the sides of the reaction chamber, which minimizes turbulence and minimizes the introduction of air bubbles into the mixed fluid. Air bubbles are undesirable due to the unpredictable refraction of light they cause, as light emitted by the reagents will travel through air bubbles within or on the surface of the mixed reagent. To ensure that the reagents mix with the fluid present at the bottom of the reaction chamber, the axis of the inlet channel may be tilted upward (e.g., about 30 degrees) from the horizontal to impart a partially downward direction to the incoming fluid flow. Alternatively, reagents may be introduced into the test chamber using alternative fluid delivery means, such as a vertical channel to deliver reagents to the bottom of the reaction chamber, or by delivering fluid directly onto the central axis of the test chamber to create a column of reagent that flows into the test chamber, thereby promoting mixing by entrainment.

[0089] The shape of the reaction chamber (i.e., the predetermined arrangement) may be a cross section of revolution that facilitates clockwise or counterclockwise movement of the mixed fluid around the central axis of the reaction chamber. Alternatively, reaction chamber shapes other than a cross section of revolution, such as a rectangular or irregular shape, may be utilized, if desired. In one embodiment, the cross section of revolution used to form the reaction chamber is a portion of an ellipse that facilitates collection of stray light emitted by the reagents and reflects this light toward the surface of a detector, which may be a photomultiplier tube (PMT) (Hamamatsu). To enhance the light collection properties of the ellipse, the surface of the reaction chamber may be reflective. In some embodiments, the maximum diameter of the surface of the PMT is restricted to achieve the maximum signal-to-noise ratio of the output of the system. The diameter of the reaction chamber may be designed to approximately match the diameter of the PMT, which affects the elliptical shape that may be achieved in a reaction chamber designed to hold a particular volume of fluid. Due to the constrained elliptical shape, the surface color of the reaction chamber may be partially diffuse white due to the additional light collection that occurs when the light is partially diffused by the white surface unless it is directly reflected to the PMT surface, and some of this is directed to the PMT surface. Alternatively, materials such as aluminum with other surface finishes and near mirror finishes, or transparent materials can be used if desired. Additionally, it is desirable for the reaction chamber material to have minimal phosphorescence to prevent light emitted from the reaction chamber itself from drowning out any light emitted from the reagents and interfering with detection. White polymeric materials such as acrylonitrile butadiene styrene or other such polymeric materials have been found to exhibit low levels of phosphorescence, but the additional light collection provided by a combination of light reflection and diffusion has been found to be beneficial to the signal to noise ratio of the optical sensing circuit output.

[0090] In an exemplary embodiment, the test subunit provides a system for use in sample analysis, the system including a biosensor reagent, the biosensor reagent being a biological cell, a reservoir card having a long loop portion and a short loop portion, the reservoir card storing the biosensor reagent, and a test cartridge base configured to receive the reservoir card. The test cartridge base further includes: (i) a reaction chamber having a central axis, the reaction chamber having a shape of a semi-ellipsoid of revolution, and (ii) an inlet channel connected to the reaction chamber, the inlet channel being disposed above the reaction chamber at an angle of 15 to 60 degrees relative to the horizontal and offset from the central axis of the reaction chamber, and wherein upon introduction of a sample to be analyzed into the test cartridge base through the inlet channel, the sample is mixed uniformly with the biosensor reagent while minimizing damage to the biological cells.

[0091] In another exemplary embodiment, the test subunit provides a system for rapidly detecting the presence of an analyte in a biological sample. The system includes a biosensor reagent including at least one antibody specific for a given analyte and a bioluminescent agent, where the at least one antibody is expressed on the surface of live modified lymphocytes and the bioluminescent agent is expressed by the live modified lymphocytes. The biosensor reagent is operable to (i) detect the presence of a particular analyte in the sample being tested and (ii) emit a detectable light signal when the biosensor reagent reacts with the sample to detect the presence of the particular analyte in the sample. A test cartridge is also included. The test cartridge further includes: (i) a reservoir card, the reservoir card further including a biosensor reagent, and (ii) a test cartridge base, the test cartridge base configured to receive the reservoir card. The test cartridge base further includes: a) a reaction chamber having a central axis, the reaction chamber having a shape of a semi-ellipsoid of revolution; and b) an inlet channel connected to the reaction chamber, the inlet channel disposed above the reaction chamber at an angle of 15-60 degrees to the horizontal and offset from the central axis of the reaction chamber, and c) upon introduction of a sample through the inlet channel into the test cartridge base, the sample is mixed uniformly with the biosensor reagent while minimizing damage to the live modified lymphocytes and minimizing any bubbling of the mixed biosensor reagent and the sample in the reaction chamber. Also included is a test unit adapted to receive the test cartridge. The test unit includes a sensor for detecting a detectable light signal emitted by the biosensor reagent upon reaction with the sample, where detection of the emitted detectable light signal indicates the presence of an analyte in the sample, and detection of the specific analyte in the sample occurs in real time.

[0092] Bioassay Example 1: Immediate Addition of Antibody In an exemplary embodiment of the bioassay of the present invention, the detection molecule is a soluble antibody, and the soluble antibody and the sample to be tested are mixed together immediately prior to the introduction of the biosensor cells into the test sample. In this embodiment, the packed biosensor cells were centrifuged and concentrated in the packing medium to about 400,000 cells / 180 μL (sufficient for a single reaction). Then, a 180 μL (about 400,000 cells) aliquot of the packed biosensor cells was placed in the long loop portion of the reservoir card. For the positive control, 30 μL of anti-CD3ε antibody in RPMI medium was placed in the short loop portion of the reservoir card. The reservoir card was then fixed to the test cartridge base. A 2 μL volume of antibody (0.5 mg / mL) against a target pathogen, such as anti-E. coli O111 (target pathogen is E. coli O111), was mixed with 28 μL of the sample to be tested in the cartridge mixing chamber. The test cartridge base was inserted into the test device, and the packed biosensor cells were injected into the reaction chamber to initiate the reaction. The resulting signal was recorded for 4-8 min, and at the end of the test period, 30 µL of anti-CD3ε antibody was injected into the reaction vessel as a positive control reaction that was recorded for 2 min. As another positive control, 30 µL of 0.61 mM digitonin rather than anti-CD3ε antibody can be used. Negative control tests can be performed using a predetermined pathogen that is not specific for the antibody being used.

[0093] Bioassay Example 2: Coating of Biosensor Cells with Antibodies In another exemplary embodiment of the bioassay of the present invention, the detection molecule is a soluble antibody, and the biosensor cells were coated with the soluble antibody for a period of time prior to mixing the sample to be tested with the biosensor cells. In this embodiment, the packed biosensor cells were centrifuged and concentrated to about 400,000 cells / 180 μL (sufficient for a single reaction) in the packed medium. Then, a 180 μL (about 400,000 cells) aliquot of the biosensor cells was mixed with a 2 μL volume of antibody (0.5 mg / mL) against a target pathogen, such as anti-E. coli O111 (target pathogen is E. coli O111) in an Eppendorf tube. The biosensor cells mixed with the antibody were incubated at room temperature for 10 minutes and then placed in the long loop portion of the reservoir card. For the positive control, 30 μL of anti-CD3ε antibody in RPMI medium was placed in the short loop portion of the reservoir card. The reservoir card was then secured to the test cartridge base. A 30 μL volume of the sample to be tested was added to the reaction chamber. The test cartridge base was inserted into the test device and the biosensor cells were injected into the mixing chamber to initiate the reaction. The resulting signal was recorded for 4-8 min, and at the end of the test period, 30 μL of anti-CD3ε antibody was injected into the reaction vessel as a positive control reaction that was recorded for 2 min. As an alternative positive control, 30 μL of 0.61 mM digitonin can be used rather than anti-CD3ε antibody. Negative control tests can be performed using a predetermined pathogen that is not specific for the antibody being used.

[0094] Bioassay Example 3: Coating of analytes with antibodies In another exemplary embodiment of the bioassay of the present invention, the detection molecule is a soluble antibody, and the analyte (e.g., pathogenic bacteria) is coated with the soluble antibody for a period of time prior to mixing the sample to be tested with the biosensor. In this embodiment, the packed biosensor cells were centrifuged and concentrated to about 400,000 cells / 180 μL (sufficient for a single reaction) in the packing medium. An aliquot of 180 μL (about 400,000 cells) of the biosensor cells was placed in the long loop portion of the reservoir card. For the positive control, 30 μL of anti-CD3ε antibody in RPMI medium was placed in the short loop portion of the reservoir card. The reservoir card was then fixed to the cartridge base. A 2 μL volume of antibody (0.5 mg / mL) against a target pathogen, such as anti-E. coli O111 (target pathogen is E. coli O111), was mixed with 28 μL of the sample to be tested in an Eppendorf tube. The sample was incubated at room temperature for 10 minutes and then added to the cartridge mixing chamber. The cartridge was inserted into the PMT and the biosensor cells were injected into the mixing chamber to initiate the reaction. The resulting signal was recorded for 4-8 min, and at the end of the test period, 30 µL of anti-CD3ε antibody was injected into the reaction vessel as a positive control reaction that was recorded for 2 min. As another positive control, 30 µL of 0.61 mM digitonin can be used rather than anti-CD3ε antibody. Negative control tests can be performed using a predetermined pathogen that is not specific for the antibody being used.

[0095] The exemplary bioassays described herein may include other additives that reduce background noise and enhance signal. Using an anti-CD3ε antibody as a positive control, the system has been demonstrated to detect less than 10 loaded biosensor cells in a mixture of 50,000 unloaded biosensor cells. The biosensor itself has been demonstrated to detect 230 CFU of bacteria in a 30 μL sample. In the bioassays described above, a dedicated monoclonal antibody (1F11) against E. coli O111 bacteria was used to detect E. coli O111 bacteria, with E. coli O157 as a negative control. E. coli O157 was demonstrated to give a negative result, thereby proving the specificity of the system. A number of commercially available antibodies may also be used with the bioassays described herein. For the dedicated monoclonal antibody (1F11), antibody analysis and monoclonal antibody selection were accomplished as described below.

[0096] Antibody production was determined by ELISA performed in 96-well multiwell plates. Each well was coated with a different LPS (E. coli O157, E. coli O127, E. coli O111, E. coli O26, Klebsiella pneumoniae, Salmonella enterica, and Naive Sella) or bacterial cells (E. coli O157, E. coli O111, E. coli O26, and E. coli DH5α). Hybridoma supernatants from different clones of mAb O157 or mAb O111 were added to the wells. Horseradish peroxidase-conjugated (HRP) goat anti-mouse IgG was used for detection (Appendix III.A.3). Two hybridoma clones of E. coli O157 (1B10 and 6G1) recognize the LPS of E. coli O157 and only E. coli O157. Nine hybridoma clones of E. coli O111 specifically recognize E. coli O111 LPS and E. coli O111. The clones from the highest optical density (OD) readings were selected for validation, which was achieved as described below.

[0097] Hybridoma cell pellets were harvested and stored at -80°C prior to RNA extraction. Extracted RNA was used as a template for reverse transcription into cDNA followed by nested PCR amplification. All positive PCR products were cloned into a TA cloning vector and sent for sequencing. The variable regions of the light and heavy chains were determined after sequence analysis. Four single-chain antibodies (scFv) of O157 (1B10) (customized mAb from FSC) and ATCC HB10452, as well as two single-chain antibodies of O111 (1F11 and 1F2) from FSC, were recombinantly expressed and purified by immobilized metal ion affinity chromatography (IMAC). The sequence of the scFv was constructed in the following order: pel B secretion signal + alanine + histidine tag + glycine-serine-serine-glycine amino acids + TEV cleavage site + glycine-serine-serine-glycine amino acids + heavy chain variable region + linker region serine-alanine-aspartic acid-aspartic acid-alanine-lysine-lysine-aspartic acid-alanine-alanine-lysine-lysine-aspartic acid-alanine-lysine-lysine-aspartic acid-aspartic acid + light chain variable region. The purified scFvs were tested using multiwell plates coated with O157 or O111 LPS.

[0098] The aim of this study was to investigate the interaction of monoclonal antibodies (mAbs) to whole bacterial cells (E. coli O157 or E. coli O111) and to estimate the rate constants (KD) of antibody-bacteria interactions. These assays used a goat anti-E. coli O157 polyclonal antibody, a goat anti-E. coli O111 polyclonal antibody, three monoclonal antibodies against E. coli O157 (1022, 1024, and 1061), and one monoclonal antibody against E. coli O111. A CM5 sensor chip and an amine coupling kit were also used. All assays were performed on a Biacore X100 instrument. In this protocol, one polyclonal antibody (against a selected bacterium) was immobilized on a CM5 sensor chip. The selected bacterium was then allowed to bind, followed by injection of a monoclonal antibody against the same bacterium in a continuous buffer flow. The interaction was monitored in real time. The relative binding of the antibodies to each bacterium was recorded in resonance units (RU). Results of BIAcore analysis of the binding of E. coli O157-specific antibody (mAb FF754) to E. coli O157 and E. coli O111 indicated that the O157 mAb was specific for its target antigen.

[0099] Fc receptors Receptors, e.g., ligands or detectors, utilized by the present invention may include alternative Fc-containing chimeric receptors. The chimeric receptors described herein comprise an extracellular domain having binding affinity and specificity for the Fc portion of an immunoglobulin ("Fc binder"), a transmembrane domain, at least one costimulatory signaling domain, and a cytoplasmic signaling domain comprising an ITAM. When expressed on a host cell, the chimeric receptor is configured such that the extracellular ligand-binding domain is located extracellularly for binding to a target molecule (e.g., an antibody or Fc fusion protein) and the costimulatory signaling domain, and the ITAM-containing cytoplasmic signaling domain is located within the cytoplasm for triggering activation and / or effector signaling. In some embodiments, the chimeric receptor constructs described herein comprise, from N-terminus to C-terminus, an Fc binder, a transmembrane domain, at least one costimulatory signaling domain, and an ITAM-containing cytoplasmic signaling domain. In other embodiments, the chimeric receptor constructs described herein comprise, from N-terminus to C-terminus, an Fc binder, a transmembrane domain, an ITAM-containing cytoplasmic signaling domain, and at least one costimulatory signaling domain.

[0100] Any of the chimeric receptors described herein may further comprise a hinge domain that may be located at the C-terminus of the Fc binding agent and the N-terminus of the transmembrane domain.Alternatively, or in addition, the chimeric receptor constructs described herein may comprise two or more costimulatory signaling domains that may be linked to each other or separated by an ITAM-containing cytoplasmic signaling domain.The extracellular Fc binding agent, the transmembrane domain, the costimulatory signaling domain, and the ITAM-containing cytoplasmic signaling domain in the chimeric receptor construct may be linked to each other directly or via a peptide linker.

[0101] The chimeric receptor constructs described herein comprise an Fc binder, i.e., an extracellular domain capable of binding to the Fc portion of an immunoglobulin (e.g., IgG, IgA, IgM, or IgE) of a suitable mammal (e.g., human, mouse, rat, goat, sheep, monkey). Suitable Fc binders may be derived from naturally occurring proteins such as mammalian Fc receptors or certain bacterial proteins (e.g., protein A, protein G). Additionally, Fc binders may be synthetic polypeptides specifically engineered to bind with high affinity and specificity to the Fc portion of any of the Ig molecules described herein. For example, such Fc binders may be antibodies or antigen-binding fragments thereof that specifically bind to the Fc portion of an immunoglobulin. Examples include, but are not limited to, single chain variable fragments (scFv), domain antibodies, or nanobodies. Alternatively, the Fc binding agent may be a synthetic peptide that specifically binds to an Fc portion, such as a Kunitz domain, a small modular immunopharmaceutical (SMIP), an adnectin, an avimer, an affibody, a DARPin, or anticalin, which are identified by screening peptide binding libraries for binding activity to Fc.

[0102] In some embodiments, the Fc binding agent is an extracellular ligand-binding domain of a mammalian Fc receptor. As used herein, an "Fc receptor" is a cell surface-associated receptor expressed on the surface of many immune cells (including B cells, dendritic cells, natural killer (NK) cells, macrophages, neutrophils, mast cells, and eosinophils) and exhibits binding specificity for the Fc domain of an antibody. An Fc receptor typically consists of at least two immunoglobulin (Ig)-like domains that have binding specificity for the Fc (fragment crystallizable) portion of an antibody. In some instances, binding of an Fc receptor to the Fc portion of an antibody can trigger an antibody-dependent cell-mediated cytotoxicity (ADCC) effect. The Fc receptor used to construct the chimeric receptor as described herein can be a naturally occurring polymorphic variant (e.g., CD16 V158 variant) that can have increased or decreased affinity for Fc compared to its wild-type counterpart. Alternatively, the Fc receptor may be a functional variant of its wild-type equivalent having one or more mutations (e.g., substitutions of up to 10 amino acid residues) that alter its binding affinity to the Fc portion of an Ig molecule. In some instances, the mutations can alter the glycosylation pattern of the Fc receptor and thus its binding affinity to Fc.

[0103] The following table lists some exemplary polymorphisms in the extracellular domain of an Fc receptor (see, e.g., Kim et al., J. Mol. Evol. 53:1-9, 2001). [Table 1]

[0104] Fc receptors are classified based on the isotype of the antibody they can bind. For example, Fc gamma receptors (FcγR) generally bind IgG antibodies, such as one or more of its subtypes (i.e., IgG1, IgG2, IgG3, IgG4), Fc alpha receptors (FcαR) generally bind IgA antibodies, and Fc epsilon receptors (FccR) generally bind IgE antibodies. In some embodiments, the Fc receptor is an Fc gamma receptor, an Fc alpha receptor, or an Fc epsilon receptor. Examples of Fc gamma receptors include, but are not limited to, CD64A, CD64B, CD64C, CD32A, CD32B, CD16A, and CD16B. An example of an Fc alpha receptor is FcαR1 / CD89. Examples of Fc epsilon receptors include, but are not limited to, FcεRI and Fc epsilon RII / CD23. The following table lists exemplary Fc receptors and their corresponding binding activities for the Fc domains for use in constructing the chimeric receptors described herein. [Table 2]

[0105] The selection of the ligand binding domain of an Fc receptor for use in the chimeric receptors described herein will be apparent to one of skill in the art. For example, it may depend on factors such as the isotype of the antibody to which the Fc receptor is desired to bind and the desired affinity of the binding interaction. In some examples, (a) is an extracellular ligand binding domain of CD16 incorporating a naturally occurring polymorphism that can modulate affinity for Fc. In some examples, (a) is an extracellular ligand binding domain of CD16 incorporating a polymorphism at position 158 (e.g., valine or phenylalanine). In some embodiments, (a) is generated under conditions that alter its glycosylation state and its affinity for Fc. In some embodiments, (a) is an extracellular ligand binding domain of CD16 incorporating a modification that renders the chimeric receptor incorporating it specific for a subset of IgG antibodies. For example, mutations can be incorporated that increase or decrease affinity for IgG subtypes (e.g., IgG1).

[0106] In other embodiments, the Fc binding agent is derived from a naturally occurring bacterial protein capable of binding to the Fc portion of an IgG molecule. The Fc binding agent for use in constructing the chimeric receptors described herein can be a full-length protein or a functional fragment thereof. Protein A is a 42 kDa surface protein originally found in the cell wall of the bacterium Staphylococcus aureus. It consists of five domains, each of which folds into a three-helix bundle and can bind IgG via interactions with the Fc region of most antibodies and the Fab region of human VH3 family antibodies. Protein G is an approximately 60 kDa protein expressed in group C and group G streptococci that binds to both the Fab and Fc regions of mammalian IgG. Natural protein G also binds albumin, but recombinant mutants have been engineered that eliminate albumin binding.

[0107] Fc binders for use in chimeric receptors can also be generated de novo using combinatorial biology or directed evolution methods. Starting with a protein scaffold (e.g., scFv from IgG, Kunitz domain from Kunitz-type protease inhibitor, ankyrin repeats, Z domain from protein A, lipocalin, fibronectin type III domain, SH3 domain from Fyn, etc.), amino acid side chains for a set of residues on the surface can be randomly substituted to generate a large library of mutant scaffolds. From the large library, it is possible to isolate rare mutants with affinity for a target, such as the Fc domain, by first selecting for binding and then amplifying by phage, ribosome, or cell display. A series of repeated selections and amplifications can be used to isolate proteins with the highest affinity for the target.

[0108] Any of the Fc binding agents described herein may have suitable binding affinity for the Fc portion of a therapeutic antibody. As used herein, "binding affinity" refers to the apparent association constant or K A It means. A is the dissociation constant K D The extracellular ligand-binding domain of the Fc receptor domain of the chimeric receptor described herein is at least 10 -5 , 10 -6 , 10 -7 , 10 -8 , 10 -9 , 10 -10M , or a lower K than the Fc portion of an antibody DIn some embodiments, the Fc binding agent has a higher binding affinity for an antibody, antibody isotype, or subtype thereof compared to the binding affinity of the Fc binding agent for another antibody, antibody isotype, or subtype thereof. In some embodiments, the extracellular ligand binding domain of the Fc receptor has specificity for an antibody, antibody isotype, or subtype thereof compared to the binding of the extracellular ligand binding domain of the Fc receptor for other antibodies, antibody isotypes, or subtypes thereof. Fc gamma receptors with high affinity binding include CD64A, CD64B, and CD64C. Fc gamma receptors with low affinity binding include CD32A, CD32B, CD16A, and CD16B. An Fc epsilon receptor with high affinity binding is Fc epsilon RI and an Fc epsilon receptor with low affinity binding is Fc epsilon RII / CD23.

[0109] The binding affinity or binding specificity for an Fc receptor or chimeric receptor (e.g., the extracellular ligand binding domain of an Fc receptor) comprising an Fc binding agent can be determined by a variety of methods including equilibrium dialysis, equilibrium binding, gel filtration, ELISA, surface plasmon resonance, or spectroscopy.

[0110] In some embodiments, the extracellular ligand-binding domain of the Fc receptor comprises an amino acid sequence that is at least 90% (e.g., 91, 92, 93, 94, 95, 96, 97, 98, 99%) identical to the amino acid sequence of the extracellular ligand-binding domain of a naturally occurring Fc gamma receptor, Fc alpha receptor, or Fc epsilon receptor. The "percent identity" of two amino acid sequences can be determined using the algorithm of Karlin and Altschul Proc. Natl. Acad. Sci. USA 87:2264-68,1990, as modified in Karlin and Altschul Proc. Natl. Acad. Sci. USA 90:5873-77,1993. Such an algorithm is incorporated into the NBLAST and XBLAST programs (version 2.0) of Altschul, et al. J. Mol. Biol. 215:403-10,1990. BLAST protein searches can be performed using the XBLAST program, score=50, wordlength=3 to obtain amino acid sequences homologous to the protein molecules of the present disclosure. When gaps exist between the two sequences, Gapped BLAST can be used as described in Altschul et al., Nucleic Acids Res. 25(17):3389-3402, 1997. When utilizing BLAST and Gapped BLAST programs, the default parameters of the respective programs (e.g., XBLAST and NBLAST) can be used.

[0111] Yet another embodiment of the present invention provides a system comprising: a space or compartment for contacting a modified cell with an analyte; a modified cell comprising a ligand, a signal transduction pathway, and a reporter; and a detector, wherein a universal detection component is bound to a given detection component, the signal transduction pathway receives a first signal induced by binding of the analyte to the ligand and transmits the first signal to the reporter, and the reporter emits a second detectable signal upon receiving the first signal from the signal transduction pathway.

[0112] Yet another embodiment of the invention provides a system comprising a space or compartment for contacting the modified cell with a predetermined analyte; a modified cell comprising an assembly of a ligand and a signaling moiety that constitutively transmits a signal to a detector that emits light or another detectable signal; and a detector, where binding of the predetermined analyte to the assembly of the ligand and the signaling moiety attenuates signal transduction and attenuates emission of light or other detectable signal by the reporter. The assembly of the ligand and the signaling moiety is maintained by a sticky adaptor, and when the sticky adaptor is bound by the predetermined analyte, its ability to maintain the assembly of the ligand and the signaling moiety is weakened.

[0113] Yet another embodiment of the present invention provides a system including a space or compartment for contacting the modified cell with a predetermined analyte; a modified cell including a ligand, a signaling moiety that transmits an inhibitory signal when bound to the predetermined analyte, and a reporter that constitutively emits light or another detectable signal; and a detector, where binding of the predetermined analyte to the universal detection moiety induces an inhibitory signal that attenuates the emission of light or other detectable signal by the reporter. In this embodiment, the universal detection moiety can include an immunoreceptor tyrosine-based inhibitory motif (ITIM). ITIMs are further disclosed in Staub E, Rosenthal A, Hinzmann B (2004) "Systematic identification of immunoreceptor tyrosine-based inhibitory motifs in the human proteome". Cell Signal 16(4):435-456, which is incorporated by reference herein.

[0114] While the present invention has been illustrated by describing exemplary embodiments thereof and has been described in a certain degree of detail, it is not the applicant's intention to restrict or in any way limit the scope of the appended claims to such details. Additional advantages and modifications will be readily apparent to those skilled in the art. Thus, the invention in its broader aspects is not limited to any of the specific details, representative apparatus and methods, and / or illustrative examples shown and described. Accordingly, departures may be made from such details without departing from the spirit or scope of the applicant's general inventive concept.

Claims

1. 1. A biosensor system for detecting a target analyte, comprising: (a) a biological cell of a predetermined type; (b) a signal generating reporter associated with said biological cell; and (c) a signaling pathway or activation mechanism associated with said signal generating reporter; and (d) a universal detection moiety associated with said activation mechanism; and (e) an analyte binding moiety associated with the universal detection moiety, the analyte binding moiety being specific for both the universal detection moiety and a target analyte; A biosensor system comprising:

2. 2. The biosensor system of claim 1, wherein the biological cell is a prokaryotic cell, a eukaryotic cell, a yeast cell, an insect cell, a mammalian cell, an animal cell, a plant cell, a non-germ cell, a fixed cell, a drug-treated cell, a chemically treated cell, an osmotically treated cell, an irradiated cell, an artificial cell, a synthetic cell, a follicular dendritic cell, a natural killer cell, a macrophage, a monocyte, a mononuclear phagocyte, a neutrophil, an eosinophil, or a basophil.

3. 3. The biosensor system of claim 2, wherein the insect cells are Drosophila Schneider 2 cells or sf9 cells.

4. 3. The biosensor system of claim 2, wherein the mammalian cells are HEK cells, CHO cells, COS cells, 3T3 cells, or other cultured, immortalized, or passaged mammalian cells.

5. 2. The biosensor system of claim 1, wherein the signal generating reporter is a dye having fluorescent, ultraviolet, or visible properties; an enzyme adapted to generate a luminescent or fluorescent signal; a fluorescent, charged, or magnetic nanoparticle, nanodot, or quantum dot; a fluorescent protein; or other calcium-sensitive luminescent or fluorescent molecule.

6. 6. The biosensor system of claim 5, wherein the fluorescent protein is green fluorescent protein.

7. 6. The biosensor system of claim 5, wherein the signal generating reporter is obelin, thalassicolin, mitrocomin (halistalin), clytin (phiaridin), mnemopsin, berobin, Indo-1, Fluo-2, Quin-2, Fluo-3, Rhod-2, calcium green, BAPTA, cameleon, or other calcium-sensitive luminescent or fluorescent molecule.

8. 2. The biosensor system of claim 1, wherein the signal transduction pathway or activation mechanism is activated by a change in pH or temperature of the biological cell; a change in electrical or magnetic properties of the biological cell; activation of a G protein-coupled receptor signal transduction pathway in the biological cell; activation of a phosphatidylinositol pathway in the biological cell; activation of a signal transduction pathway in the biological cell that releases diacylglycerol, ceramide, or other lipophilic messenger molecules; activation of a signal transduction pathway in the biological cell that releases or generates nitric oxide, cAMP, cGMP, or other cyclic nucleotides; activation of a signal transduction pathway in the biological cell that releases or generates superoxide, hydrogen peroxide, carbon monoxide, hydrogen sulfide, or other secondary redox signal transduction molecules; or a conformational change in a receptor expressed by the biological cell, the conformational change occurring only after the universal detection component binds to a target analyte.

9. The biosensor system of claim 1, wherein the universal detection component comprises an antibody variable diversity joining (VDJ) region, a Fab fragment, or other antibody determinant; a T cell variable joining (VJ), variable diversity joining (VDJ), or other T cell receptor determinant; a synthetic peptide; a non-peptide organic determinant of known size; a lectin determinant, a carbohydrate binding module, or other carbohydrate binding determinant; a lipid binding determinant; a metallothionein determinant that binds metals or other metal binding determinants; an immunoreceptor tyrosine-based inhibitory motif (ITIM); an Fc determinant that non-covalently binds to an Fc binding portion of a signal transduction pathway in the biological cell; or a biotin or (strept)avidin determinant that non-covalently binds to a biotin or (strept)avidin binding portion of a signal transduction pathway in the biological cell.

10. 10. The biosensor system of claim 1, wherein the analyte binding component is an affibody, an aptamer, or a soluble receptor, or the analyte binding component comprises an IgG fragment that is a single chain antibody or a single chain diabody.

11. 10. The biosensor system of claim 1, wherein the target analyte is a mutualistic, symbiotic, or parasitic microorganism, a pathogenic microorganism, a bioware microorganism, or other microorganism, or a pharmaceutical, drug, poison, toxin, chemical weapon, hormone, metabolite, or small molecule bound to a polymeric carrier.

12. 1. A biosensor system for detecting a target analyte, comprising: (a) a biological cell of a predetermined type; (b) a signal generation reporter within the biological cell, the signal generation reporter being responsive to a predetermined change occurring within the biological cell; and (c) a signal transduction pathway or activation mechanism associated with the signal generating reporter, the signal transduction pathway or activation mechanism operating to induce a predetermined change in the biological cell; and (d) a universal detection moiety associated with the activation mechanism, the universal detection moiety operative to trigger the activation mechanism; and (e) an analyte binding moiety associated with the universal detection moiety, the analyte binding moiety being specific for both the universal detection moiety and a target analyte; (f) when an analyte-binding component, which also binds a target analyte, binds to the universal detection component, the universal detection component triggers the activation mechanism to cause a predetermined change in the biological cell, thereby causing a signal-generating reporter to generate a detectable signal.

13. 13. The biosensor system of claim 12, wherein the biological cell is a prokaryotic cell, a eukaryotic cell, a yeast cell, an insect cell, a mammalian cell, an animal cell, a plant cell, a non-germ cell, a fixed cell, a drug-treated cell, a chemically treated cell, an osmotically treated cell, an irradiated cell, an artificial cell, a synthetic cell, a follicular dendritic cell, a natural killer cell, a macrophage, a monocyte, a mononuclear phagocyte, a neutrophil, an eosinophil, or a basophil.

14. 14. The biosensor system of claim 13, wherein the insect cells are Drosophila Schneider 2 cells or sf9 cells.

15. 14. The biosensor system of claim 13, wherein the mammalian cells are HEK cells, CHO cells, COS cells, 3T3 cells, or other cultured, immortalized, or passaged mammalian cells.

16. 13. The biosensor system of claim 12, wherein the signal generating reporter is a dye having fluorescent, ultraviolet, or visible properties; an enzyme adapted to generate a luminescent or fluorescent signal; a fluorescent, charged, or magnetic nanoparticle, nanodot, or quantum dot; a fluorescent protein; or other calcium-sensitive luminescent or fluorescent molecule.

17. 17. The biosensor system of claim 16, wherein the fluorescent protein is green fluorescent protein.

18. 17. The biosensor system of claim 16, wherein the signal generating reporter is obelin, thalassicolin, mitrocomin (halistalin), clytin (phiaridin), mnemopsin, berobin, Indo-1, Fluo-2, Quin-2, Fluo-3, Rhod-2, calcium green, BAPTA, cameleon, or other calcium-sensitive luminescent or fluorescent molecule.

19. 13. The biosensor system of claim 12, wherein the signal transduction pathway or activation mechanism is activated by at least one of: a change in pH or temperature of the biological cell; a change in electrical or magnetic properties of the biological cell; activation of a G protein-coupled receptor signal transduction pathway in the biological cell; activation of a phosphatidylinositol pathway in the biological cell; activation of a signal transduction pathway in the biological cell that releases diacylglycerol, ceramide, or other lipophilic messenger molecules; activation of a signal transduction pathway in the biological cell that releases or generates nitric oxide, cAMP, cGMP, or other cyclic nucleotides; activation of a signal transduction pathway in the biological cell that releases or generates superoxide, hydrogen peroxide, carbon monoxide, hydrogen sulfide, or other secondary redox signal transduction molecules; or a conformational change in a receptor expressed by the biological cell, wherein the conformational change occurs only after the universal detection component binds to a target analyte.

20. The biosensor system of claim 12, wherein the universal detection component comprises at least one of: an antibody variable diversity joining (VDJ) region, a Fab fragment, or other antibody determinant; a T cell variable joining (VJ), variable diversity joining (VDJ), or other T cell receptor determinant; a synthetic peptide; a non-peptide organic determinant of known size; a lectin determinant, a carbohydrate binding module, or other carbohydrate binding determinant; a lipid binding determinant; a metallothionein determinant that binds metals or other metal binding determinants; an immunoreceptor tyrosine-based inhibitory motif (ITIM); an Fc determinant that non-covalently binds to an Fc binding portion of a signal transduction pathway in the biological cell; or a biotin or (strept)avidin determinant that non-covalently binds to a biotin or (strept)avidin binding portion of a signal transduction pathway in the biological cell.

21. 13. The biosensor system of claim 12, wherein the analyte binding component is an affibody, an aptamer, or a soluble receptor, or the analyte binding component comprises an IgG fragment that is a single chain antibody or a single chain diabody.

22. 13. The biosensor system of claim 12, wherein the target analyte is a beneficial enterobacteria, a pathogenic bacteria, a protein biomarker, a small molecule toxin, a metabolite, or a chemical weapon, or a small molecule bound to a polymeric carrier.

23. 1. A biosensor system for detecting a target analyte, comprising: (a) a biological cell of a predetermined type; (b) a signal generation reporter within the biological cell, the signal generation reporter being responsive to a predetermined change occurring within the biological cell; and (c) a signal transduction pathway or activation mechanism associated with the signal generating reporter, the signal transduction pathway or activation mechanism operating to induce a predetermined change in the biological cell; and (d) a universal detection moiety associated with the activation mechanism, the universal detection moiety operative to trigger the activation mechanism; and (e) an analyte binding moiety associated with the universal detection moiety, the analyte binding moiety being specific for both the universal detection moiety and a target analyte; (f) when an analyte-binding component, which also binds a target analyte, binds to the universal detection component, the universal detection component inhibits the activation mechanism, thereby reducing a predetermined change in the biological cell, thereby causing the signal generating reporter to generate an attenuated signal or no signal.

24. 24. The biosensor system of claim 23, wherein the biological cell is a prokaryotic cell, a eukaryotic cell, a yeast cell, an insect cell, a mammalian cell, an animal cell, a plant cell, a non-germ cell, a fixed cell, a drug-treated cell, a chemically treated cell, an osmotically treated cell, an irradiated cell, an artificial cell, a synthetic cell, a follicular dendritic cell, a natural killer cell, a macrophage, a monocyte, a mononuclear phagocyte, a neutrophil, an eosinophil, or a basophil.

25. 24. The biosensor system of claim 23, wherein the signal generating reporter is a dye having fluorescent, ultraviolet, or visible properties; an enzyme adapted to generate a luminescent or fluorescent signal; a fluorescent, charged, or magnetic nanoparticle, nanodot, or quantum dot; a fluorescent protein; or other calcium-sensitive luminescent or fluorescent molecule.

26. 24. The biosensor system of claim 23, wherein the signal transduction pathway or activation mechanism is activated by a change in pH or temperature of the biological cell; a change in electrical or magnetic properties of the biological cell; activation of a G protein-coupled receptor signal transduction pathway in the biological cell; activation of a phosphatidylinositol pathway in the biological cell; activation of a signal transduction pathway in the biological cell that releases diacylglycerol, ceramide, or other lipophilic messenger molecules; activation of a signal transduction pathway in the biological cell that releases or generates nitric oxide, cAMP, cGMP, or other cyclic nucleotides; activation of a signal transduction pathway in the biological cell that releases or generates superoxide, hydrogen peroxide, carbon monoxide, hydrogen sulfide, or other secondary redox signal transduction molecules; or a conformational change in a receptor expressed by the biological cell, which occurs only after the universal detection component binds to a target analyte.

27. The biosensor system of claim 23, wherein the universal detection component comprises an antibody variable diversity joining (VDJ) region, a Fab fragment, or other antibody determinant; a T cell variable joining (VJ), variable diversity joining (VDJ), or other T cell receptor determinant; a synthetic peptide; a non-peptide organic determinant of known size; a lectin determinant, a carbohydrate binding module, or other carbohydrate binding determinant; a lipid binding determinant; a metallothionein determinant that binds metals or other metal binding determinants; an immunoreceptor tyrosine-based inhibitory motif (ITIM); an Fc determinant that non-covalently binds to an Fc binding portion of a signal transduction pathway in the biological cell; or a biotin or (strept)avidin determinant that non-covalently binds to a biotin or (strept)avidin binding portion of a signal transduction pathway in the biological cell.

28. 24. The biosensor system of claim 23, wherein the analyte binding component is an affibody, an aptamer, or a soluble receptor, or the analyte binding component comprises an IgG fragment that is a single chain antibody or a single chain diabody.

29. 24. The biosensor system of claim 23, wherein the target analyte is a beneficial enterobacteria, a pathogenic bacteria, a protein biomarker, a small molecule toxin, a metabolite, or a chemical weapon, or a small molecule bound to a polymeric carrier.

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