Enzyme-linked immunoassays using diffusion rate limiting media

Abstract

One aspect of the invention provides a method for detecting a target organism in a sample, the method comprising: a. Optionally immobilizing the target organism on a solid phase; b, incubating the target organism and one or more reporter enzyme detection probes together to form one or more target organism-enzyme detection probe compounds; c, removing any uncombined reporter enzyme detection probe; d. Contacting the one or more target organism-enzyme detection probe complexes with a diffusion rate limiting medium containing one or more substrates, and incubating the one or more target organism-enzyme detection probe complexes with the one or more substrates in the diffusion rate limiting medium, to generate one or more detectable products in the diffusion rate limiting medium; and e. Detecting the one or more detectable products by detecting the visible spots.

Description

TECHNICAL FIELD

[0001] The present invention relates to enzyme-linked immunoassays using diffusion rate limiting media. In particular, the present invention relates to a method for detecting a target organism in a sample. BACKGROUND

[0002] Different research procedures and assays are employed in the fields of medicine, molecular biology, etc. For example, assays are widely used in immunology for determining the rate of activation of cells in response to vaccines, infections, allergens, etc. Assays involving antibodies as a key component are typically referred to as immunoassays, which are the preferred analytical method for the repeated quantitative analysis of biomolecules, such as polypeptide molecules, of biomedical importance. Examples of immunoassays include, but are not limited to, enzyme-linked immunosorbent assay (ELISA), enzyme-linked immunospot (ELISpot) assay, FluoroSpot assay.

[0003] ELISpot is used to quantify cells with a targeted secreted marker. It is limited to secreted markers, only secretions are captured by specific probes. The capture antibodies are bound to a membrane on the platform / plate and thus also bind the secretions to the membrane. The cells are then washed away and the platform is treated with probes and substrate, in the presence of secreted markers, colored spots will form on the membrane at the binding sites. Three main problems can be found in ELISpot. First, the membrane coating on the platform significantly increases the cost of each assay compared to using standard platforms. Second, the requirement to wash away the cells means that surface markers cannot be targeted. Third, this means that the target cells are removed and cannot be harvested for further use.

[0004] ELISA is a quantitative and qualitative immunoassay, usually used to detect biomolecules, such as antigens, antibodies, cytokines, etc., in whole cell populations. Cell-based ELISA can also detect cell surface markers. However, during the ELISA assay, cells are either fixed (a process in which cells are perforated and thus die) or washed away. Quantitative ELISA is limited to relative quantification by colorimetric concentration calibration against a pure reference. Therefore, it cannot distinguish the exact number of cells presenting the cell surface marker or secreted marker of interest. As a population study, a sufficient limit of detection must be reached to give a detectable result. Small amounts of positive results in a large sample can not be detected, giving false negative results.

[0005] Flow cytometry is used to quantify cells with specific cell surface markers in large populations. Recently, flow cytometry has also been used for cell secreted markers, but requires more steps to tag the cell surface marker, which can then capture the secreted marker. The equipment used for the assay is very expensive, and while cells can be sorted after analysis, the process can result in high percentage of cell loss and is unable to isolate individual cells without additional separation techniques.

[0006] Thus, there is a significant unmet need for inexpensive single assays that can be used to analyze cell surface markers, cell secreted markers, or a combination of both, that can isolate cells into single cells and maintain cell viability. Little expensive equipment is needed, and can be performed on any platform format, such as a petri dish, 96 well plate, or even a clear vial. SUMMARY

[0007] One aspect of the present invention provides a method for detecting a target organism in a sample, the method comprising

[0008] a. optionally immobilizing the target organism on a solid phase;

[0009] b. incubating the target organism with one or more reporter enzyme detection probes to form one or more target organism-enzyme detection probe complexes;

[0010] c. removing any unbound reporter enzyme detection probes;

[0011] d. contacting the one or more target organism-enzyme detection probe complexes with a diffusion rate limiting medium containing one or more substrates, and incubating the one or more target organism-enzyme detection probe complexes with the one or more substrates in the diffusion rate limiting medium to generate one or more detectable products; and

[0012] e. detecting the one or more detectable products by detecting visible spots;

[0013] wherein the diffusion rate limiting medium is any suitable medium that reduces the diffusion rate of the one or more detectable products and thereby allows the formation of the visible spots.

[0014] Another aspect of the present invention provides a method for quantifying the amount of a target organism in a sample, the method comprising

[0015] a. detecting the target organism according to the method of the present invention; and

[0016] b. quantifying the amount of the target organism in the sample based on the number of visible spots. BRIEF DESCRIPTION OF DRAWINGS Figure 1

[0017] [ Figure 1 This illustrates the general concept of a method for detecting target organisms in a sample according to the present invention. Figure 2

[0018] [Figure 2] shows [A] an ELISpot for detecting target substances secreted by a target organism; [B] a method according to the invention for detecting target substances secreted by a target organism via platform selective capture; and [C] a method according to the invention for detecting target substances secreted by a target organism via surface marker tagging. Figure 3

[0019] [ Figure 3 The image illustrates a method according to the invention, wherein two or optionally three markers can be used to specifically identify cells with targeting properties that elicit a single positive or negative response, such as the presence or absence of a colorimetric change. Figure 4

[0020] [Figure 4] illustrates a method according to the invention, wherein three biomarkers are used to specifically identify cells having a combination of multiple properties that elicit a variety of possible responses. Combinations of responses can be produced using different colorimetric substrates, fluorescent substrates, or other substrate-to-product reactions, and only a few are depicted here: [A] shows responses from all three biomarkers; [B] shows responses from two of the three possible biomarkers; [C] shows a response from only one of the three possible biomarkers. These are merely examples of possibilities, and other combinations are possible. Figure 5

[0021] [Figure 5] shows, as [ Figure 1 Two other embodiments of the invention described herein depict alternative applications of diffusion rate limiting media and substrates. [A] shows an example application of the diffusion rate limiting media on a sample already treated with a reporter enzyme detection probe. [B] shows an example application in which a platform containing the diffusion rate limiting media can be pre-prepared, and a sample pre-treated with a reporter enzyme detection probe is applied to the surface. Figure 6

[0022] [ Figure 6 The graph depicts the relationship between the number of countable visible spots (y-axis) and the diffusion rates of the substrate and detectable products (x-axis), controlled by the variables listed in this paper. Figure 7

[0023] Figure 7 Representative pictures showing methylene blue diffusion through the following different agar gel compositions: 0.25%, 0.5%, 0.75%, 1% and 1.5% (W / V) agar in water. Each gel composition was prepared 3 times (except 0.5% (n = 2)) and 5 methylene blue diffusion (pixel diffusion distance shown in white arrows) measurements were recorded for each gel. Figure 8 Summary of methylene blue diffusion distances after 1 hour. Figure 8

[0024] Figure 8 Graph showing the relationship of methylene blue diffusion to agar composition. (n=3). Figure 9

[0025] Figure 9 Pictures taken with a macro ccd camera showing: i) ELISpot wells, ii) ELISA wells and iii) a graphical depiction of the different layers in the experiment, where A) and B) columns (read from bottom to top) of wells were coated with (1) rabbit anti-human CD27 primary antibody, (2) CD27+ cells, (3) rabbit anti-human CD27 antibody labeling and (4) HRP-conjugated goat anti-rabbit Fc region secondary antibody. C) column (read from bottom to top) of wells were coated with (1) rabbit monoclonal antibody (mAb) anti-CD27 primary antibody and (2) CD27+ cells and were not further labeled. Pictures Ai) and Aii) were developed with 50:50 (TMB:2xRPMI), Bi) and Ci) were developed with 1:1:2 (4xRPMI:1.2% agarose:TMB) and Bii) and Cii) were developed with 1:1:2 (4xRPMI:1.2% agarose:TMB). Each experiment was performed in triplicate, n=3. Figure 10

[0026] Figure 10 Summary of the number of visible spots (pits) counted when different concentrations of agar were used as diffusion rate limiting media (developing media). Figure 11

[0027] Figure 11 Summary of the number of visible spots (pits) counted when different concentrations of Carbopol 940 were used as diffusion rate limiting media (developing media). Figure 12

[0028] Figure 12 ​​​​​​shows the 100x magnification visible spots (pits) seen through the ELISA well using an upward looking microscope. Figure 13

[0029] [ Figure 13 ] summarizes the results of the inventive method using different diffusion rate limiting media (chromogenic media) in the final phase of the assay. Figure 14

[0030] [ Figure 14 ] shows a graphical depiction of the non-immobilized version of the inventive method. CD27 positive cells were completely coated with rabbit anti-human CD27 antibody followed by HRP conjugated goat anti-rabbit Fc region antibody. Figure 15

[0031] [ Figure 15 ] shows photographs taken with a macro ccd camera of the following: A) a microscope slide containing 10 μL of CD27 labeled cells mixed with 0.028% carboxypon-ELISA specific TMB mix, B) a microscope slide containing 10 μL of CD27 labeled cells mixed with 0.028% carboxypon-ELISpot specific TMB mix, C) a microscope slide containing 10 μL of negative control mixed with 0.028% carboxypon-ELISA specific TMB mix, and D) a microscope slide containing 10 μL of negative control mixed with 0.028% carboxypon-ELISpot specific TMB mix. Figure 16

[0032] [ Figure 16 ] shows photographs taken with an upward looking microscope of the following: A) 10x magnification of multiple visible spots (pits) formed on a microscope slide and B) 100x magnification of visible spots formed at the site of target cells in the vicinity of non-target cells, indicating no product formation by the absence of color. Figure 17

[0033] [ Figure 17 ] shows photographs taken with a macro ccd camera of the following: A) a negative control in which CD27 positive cells were immobilized with rabbit anti-human CD27 antibody and not labeled with SARS-Cov-2 antigens; and B) antigen specific memory B cells that are CD27 positive cells immobilized with rabbit anti-human CD27 antibody and labeled with SARS-Cov-2 spike and nucleocapsid proteins. Each experiment was performed in triplicate, n=3. Figure 18

[0034] [ Figure 18 ] shows a column graph representation of the data in Table 1 from negative controls and SARS-Cov-2 specific memory B cells, expressed as the number of spots per 5 x 10 5 The examples illustrate

[0035] All publications, patent applications, patents, and other references mentioned herein are incorporated by reference in their entirety. The publications and applications discussed herein are provided solely for their disclosure prior to the filing date of the present application. Nothing herein is to be construed as an admission that the present application is not entitled to antedate such publications. Further, the references cited herein are not admitted to be prior art to the application. The materials, methods, and examples are illustrative only and not intended to be limiting.

[0036] In the event of conflict, the document, including definitions, controls. Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this subject matter belongs. As used herein, the following definitions are provided to facilitate understanding of the present application.

[0037] The term “comprise” is used in the inclusive, rather than exclusive, sense of “include.” As used in the specification and in the claims, the phrase “comprises” is to be construed as an open-ended term that means that the listed steps or options will be followed, but additional steps or options can also be employed. The term “including” is used to mean “comprising” and vice versa. The term “consisting” is used to mean “consisting only of” and vice versa. The term “by” can be used to mean “without the use of” or “without the involvement of.” The term “based on” is used to mean “based at least in part on.” The term “based on” is used to mean “based at least in part on.”

[0038] As used in the specification and claims, the singular form “a,” “an” and “the” include plural references unless the context clearly dictates otherwise.

[0039] As used in the specification and claims, the term “and / or” is intended to cover the meanings of “A and B,” “A or B,” “A and B,” “A or B,” “A,” and “B.”

[0040] As used herein, the term “chromophore” refers to a label that causes a color change within the visible spectrum, which can be observed without the aid of instrumental devices.

[0041] ​As used herein, the term "fluorophore" refers to a molecule that has the property of fluorescence. Fluorophores absorb photons and emit photons of lower energy, and visualization requires the aid of a fluorescence instrument device.

[0042] As used herein, the term "chemiluminescence" refers to the emission of light as a result of a chemical reaction that occurs as a substrate is converted to a product. The product is in an excited state and will emit light as it returns to the baseline state. Visualization requires the aid of a dim light environment (e.g., a dark room or dark box).

[0043] As used herein, the term "reporter enzyme detection probe" includes a reporter enzyme component comprising an enzymatic activity coupled to a detection probe component comprising a target binding moiety. The reporter enzyme is optionally a peroxidase, such as horseradish peroxidase, or a phosphatase, such as alkaline phosphatase, although any stable enzyme that can produce an ionizable product can be used, including, for example, lyases, hydrolases, synthases, synthetases, oxidoreductases, dehydrogenases, oxidases, transferases, isomerases, ligases, proteases such as trypsin, proteinases, peroxidases, glucose oxidase, myeloperoxidase, oxidases, monooxygenases, cytochromes, phosphatases (such as alkaline phosphatase), decarboxylases, lipases, caspases, amylases, peptidases, transaminases, and kinases. Additional enzymes can include DNA or RNA polymerases, TAQ, restriction enzymes, Klenow fragment, DNA ligase. The target binding moiety can be a primary target binding moiety (e.g., a biopolymer such as an antibody or antigen) that selectively binds a target substance, or a secondary target binding moiety that selectively binds a primary detection agent. For example, the secondary target binding moiety can comprise a biopolymer such as an antibody or antigen that binds an antibody primary detection agent. Alternatively, the secondary target binding moiety comprises an avidin or streptavidin that selectively binds a biotinylated primary detection agent (e.g., a biotinylated primary detection agent). Other biopolymer target binding moieties that specifically bind to a target substance or primary detection agent are provided in addition to antibodies and antibody fragments, including, for example, antibody mimetics, aptamers, binding polypeptides (such as receptors), and binding polypeptide mimetics, nucleic acids, carbohydrates, and / or lipids. Examples include nucleic acid and / or peptide aptamers, affibodies, and anticalins. For example, according to embodiments, a primary antibody can be linked to an enzyme, antigen, etc., so long as a secondary antibody has specificity for the linked molecule and is attached to a different enzyme than the primary antibody.

[0044] As used herein, the term "primary detection agent" means an agent that selectively binds to a surface ligand of a target organism, a surface marker of a target organism, or a target substance secreted by a target organism (e.g., a secretory marker of a target organism). The primary detection agent is optionally coupled to a detectable label, such as biotin. In addition to antibodies and antibody binding fragments, other primary detection agents that specifically bind to a surface ligand, surface marker, or target substance are provided, including, for example, antibody mimics, binding polypeptides (such as receptors), binding polypeptide mimics, nucleic acid and peptide aptamers, affimers, and anticalins.

[0045] The present disclosure relates to a novel enzyme-linked immunoassay that uses diffusion rate limiting media in the final stage along with substrates to allow accumulation of product concentration around the reaction site and generation of detectable spots from one or more target of interest. Different combinations of capture antibodies (optional), primary antibodies, enzyme probes (e.g., horseradish peroxidase, alkaline phosphatase, etc.), substrates (e.g., chromophores, fluorophores, chemiluminescent substrates, and / or others), and vessels (e.g., 96-well plates, glass slides, etc.) can be adapted into the system to detect cell surface markers and secretory markers, setting the cost to a fraction of conventional methods. Furthermore, depending on the combination used, the sample can remain viable for other experiments, and microscopy work can be performed on transparent vessels (e.g., 96-well plates).

[0046] One aspect of the present invention provides a method for detecting a target organism in a sample, the method comprising:

[0047] a. optionally immobilizing the target organism on a solid phase;

[0048] b. incubating the target organism with one or more reporter enzyme detection probes to form one or more target organism-enzyme detection probe complexes;

[0049] c. removing any unbound reporter enzyme detection probes;

[0050] d. contacting the one or more target organism-enzyme detection probe complexes with a diffusion rate limiting medium containing one or more substrates, and incubating the one or more target organism-enzyme detection probe complexes with the one or more substrates in the diffusion rate limiting medium to generate one or more detectable products in the diffusion rate limiting medium; and

[0051] e. detecting the one or more detectable products by detecting visible spots;

[0052] wherein the diffusion rate limiting medium is any suitable medium that reduces (limits) the diffusion rate of the one or more detectable products and thereby allows formation of the visible spots.

[0053] In some embodiments, step b) comprises incubating the target organism with one or more primary detection agents specific for the target organism prior to incubation with the one or more reporter enzyme detection probes to form the one or more target organism-enzyme detection probe complexes. In further embodiments, the one or more primary detection agents bind to one or more surface ligands of the target organism, one or more surface markers of the target organism, or one or more target substances secreted by the target organism (e.g. a secretory marker of the target organism). In preferred embodiments, the one or more primary detection agents are antibodies or binding fragments thereof, or are antigens. In some embodiments, the one or more primary detection agents comprise biotin conjugated to an antibody or binding fragment thereof specific for the target organism or target substance.

[0054] In some embodiments, the one or more surface markers of the target organism are selected from, but not limited to, the following list:

[0055] ■B cells (target organism): CD27, CD19, CD21, CD20, CD38, CD23, CD138, IgA, IgG, IgM, B cell receptor.

[0056] ■T cells (target organism): CD4, CD8, T cell receptor, CD44, CD25, CD30.

[0057] ■Adult mesenchymal stem cells (target organism): CD10, CD13, CD73, CD105, CD271.

[0058] ■Embryonic stem cells (target organism): CD15, SSEA-3, CD324, CD90, CD117, CD29.

[0059] In some embodiments, the one or more target substances secreted by the target organism are selected from, but not limited to, the following group, which includes:

[0060] ■B cells (target organism): IgA, IgG, IgM, INF-gamma, IL-6, IL-10, IL-13, Lymphotoxin (TNF family cytokine).

[0061] ■T cells (target organism): IL-1, IL-4, IL-5, IL-6, IL9, IL-13, TGFbeta.

[0062] In some embodiments, step b) further comprises inhibiting an endogenous enzyme activity of the target organism (e.g., human cells and E. coli). Inhibiting an endogenous enzyme activity of the target organism is to reduce the likelihood of background false positives (false spots) generated by the endogenous enzyme activity of the target organism. In particular embodiments, if the target organism expresses an endogenous enzyme (e.g., peroxidase) that is the same as or similar (in activity) to the reporter enzyme of the one or more reporter enzyme detection probes, step b) further comprises contacting the target organism with an inhibitor of the endogenous enzyme (e.g., a peroxidase inhibitor). For example, a peroxidase inhibitor is used to reduce background false positives (false spots) generated by endogenous peroxidase activity of some particular target organisms (e.g., human cells and E. coli).

[0063] Figure 1 FIGS. 1 to 5 illustrate different configurations of the methods of the present application. In Figure 1 ] The target organism with a specific marker (depicted as a surface marker in the examples) is labeled with a marker-specific reporter enzyme detection probe. It is mixed to form a suspension within a diffusion rate-limiting medium containing a substrate specific to the reporter enzyme. When the reporter enzyme converts the substrate to a detectable product, a region of higher concentration is generated relative to the rest of the diffusion rate-limiting medium due to the limited diffusion of the detectable product, resulting in a visible spot.

[0064] In some embodiments, the target organism is a cell or a portion thereof, or a microorganism or a portion thereof. The target organism (e.g., a cell or a microorganism) can be alive (viable) or dead. Preferably, if the target organism is not alive, the surface ligand or surface marker of the target organism should be intact.

[0065] In some other embodiments, the detection of the target organism can be carried out by detecting a target substance secreted by the target organism. According to this embodiment, the target substance can serve as a ligand for the reporter enzyme detection probe or the primary detection agent. In some embodiments, the target substance is selected from the group consisting of biopolymers, biomarkers, and proteins secreted by cells or microorganisms. In further embodiments, the target substance is selected from the group consisting of tumor markers, autoantigens, hormones, chemokines, cytokines, cardiac proteins, nucleic acid molecules, lipids, and carbohydrates. Typically, the method for detecting a target substance secreted by a target organism according to the present application can be carried out as follows: the primary detection agent is adhered to the cell surface for the first selection, the target substance capture antibody is then linked to the primary detection agent through a scaffold system (e.g., biotin-streptavidin), the secretion of the target substance is allowed to occur, and the other end of the target substance is then tagged with a reporter enzyme detection probe (see [FIG. 2]). Optionally, the cell secreting the target substance can be immobilized on a solid phase as disclosed below (see [FIG. 3]).Figure 3 ])。

[0066] In some embodiments, the sample is a bodily fluid, a body tissue or any material or composition that can contain target organisms such as cells or microorganisms, preferably the sample is a blood sample.

[0067] In some embodiments, the target organisms are immobilized by direct binding to the solid phase, optionally by adsorption to the solid phase, or indirectly to the solid phase by a capture molecule that binds the target organisms coupled to the solid phase. The capture molecule coupled to the solid phase binds to the same or different surface ligand or surface marker of the target organisms compared to the one or more reporter enzyme detection probes or the one or more primary detection agents. In preferred embodiments, the capture molecule is an antibody or binding fragment thereof, an antigen or a ligand. For example, the capture molecule is an anti-CD27 antibody. The capture molecule typically immobilizes T cells, NK cells and memory B cells, plasmablast B cells and plasma B cells from a blood sample on the plate. Of all the cell types mentioned above, only memory B cells and plasmablast B cells express the B cell receptor and directly bind to the reporter enzyme detection probes or the primary detection agents. In some embodiments, the capture molecule allows selection (specific targeting) of the target organisms.

[0068] In some embodiments, the solid phase is a reaction vessel, a bead, a platform or a plate. Exemplary reaction vessels can be selected from the group comprising a 96-well plate, a 6-well plate, a petri dish and a tissue suitable tube such as a falcon tube. In other embodiments, the surface of the solid phase is selected from the group comprising metal, gold, stainless steel, plastic, glass, silica, polycarbonate, polyester, PVDF, polystyrene, nitrocellulose and cellulose.

[0069] In some embodiments, the incubation in step b) is performed in solution under conditions to form one or more target organism-enzyme detection probe complexes. The solution for incubation in step b) can be selected from, but not limited to, the group comprising RPMI1640 + 10% fetal bovine serum, DMEM, HEPES, MEM, DMEM F12, IMDM, M199, Ham's F12, Ham's F10, HPLM, fetal bovine serum and human serum. The incubation conditions in step b) are typically 30 minutes to 4 hours, preferably 1 hour to 3 hours; and the temperature can range from 20°C to 40°C; preferably 25°C to 40°C, most preferably 35°C to 40°C.

[0070] The one or more reporter enzyme detection probes bind to one or more surface ligands of the target organism, one or more surface markers of the target organism, one or more target substances secreted by the target organism, or the one or more primary detection agents. In other embodiments, the reporter enzyme detection probe is selected from an antibody or binding fragment thereof, an antigen, a drug, or a peptide, directly or indirectly conjugated to an enzyme or catalyst that can change color upon contact with a substrate.

[0071] In embodiments, the reporter enzyme detection probe comprises a primary target binding moiety (e.g., for direct binding to a target organism or direct binding to a target substance) or a secondary target binding moiety (e.g., for indirect binding to a target organism or indirect binding to a target substance) and a reporter enzyme comprising an enzymatic activity, wherein the target binding moiety is covalently bound to the reporter enzyme.

[0072] In another embodiment, the reporter enzyme is or comprises a lyase, a hydrolase, a synthase, a synthetase, an oxidoreductase, a dehydrogenase, an oxidase, a transferase, an isomerase, a ligase, a protease (such as trypsin, proteinase), a peroxidase, a glucose oxidase, a myeloperoxidase, an oxidase, a monooxygenase, a cytochrome, an alkaline phosphatase, a decarboxylase, a lipase, a caspase, an amylase, a peptidase, a transaminase, and / or a kinase activity. In another embodiment, the reporter enzyme is selected from a DNA or RNA polymerase, TAQ, a restriction enzyme, a Klenow fragment, and a DNA ligase. In a preferred embodiment, the reporter enzyme is or comprises horseradish peroxidase or alkaline phosphatase.

[0073] In some embodiments, the reporter enzyme detection probes are different, i.e., each reporter enzyme detection probe is specific for one specific marker (i.e., one specific surface marker, one specific surface ligand, or one specific secreted marker) (see, e.g., [Figure 4]). In some embodiments, the use of more than one different reporter enzyme detection probe allows for targeting one target organism having different markers (surface markers, surface ligands, and / or secreted markers). In some other embodiments, the use of more than one different reporter enzyme detection probe allows for targeting different target organisms having different markers (surface markers, surface ligands, and / or secreted markers). Thus, according to one aspect, the method of the application allows for detecting one or more target organisms in a sample, the method comprising

[0074] a. optionally immobilizing the one or more target organisms on a solid phase;

[0075] b. incubating the one or more target organisms with one or more reporter enzyme detection probes to form one or more target organism-enzyme detection probe complexes;

[0076] c. removing any unbound reporter enzyme detection probes;

[0077] d. contacting the one or more target organism-enzyme detection probe complexes with a diffusion rate limiting medium containing one or more substrates and incubating the one or more target organism-enzyme detection probe complexes with the one or more substrates in the diffusion rate limiting medium to generate one or more detectable products; and

[0078] e. detecting the one or more detectable products by detecting visible spots;

[0079] wherein the diffusion rate limiting medium is any suitable medium that reduces the diffusion rate of the one or more detectable products and thereby allows the formation of the visible spots.

[0080] In some embodiments, any unbound reporter enzyme detection probe is removed by washing the solid phase.

[0081] According to particular embodiments, when the target organism is not immobilized on a solid phase, any unbound reporter enzyme detection probe is removed by centrifugation. Typically, the wash is performed in a vessel such as a tissue culture grade sterile test tube (e.g., a 15 mL falcon tube). The target organism such as a cell (with higher density) will settle (form a pellet) under the centrifugal force, and the unbound reporter enzyme detection probe will remain in solution. The supernatant (containing the unbound reporter enzyme detection probe) is discarded and fresh media is added to further dilute the unbound reporter enzyme detection probe. The pelleted cells are resuspended, and the process is repeated 3 times. Finally, the labeled and unlabeled target organisms (such as cells) remain in the media. The unbound reporter enzyme detection probe is discarded.

[0082] The substrate is a molecule that is catalyzed by the reporter enzyme to provide a detectable product that is chromogenic, a detectable product that is fluorogenic, or a detectable product that is chemiluminescent. The detectable product provides a visible spot. In some embodiments, the substrate is selected from, but not limited to, the group comprising tetramethylbenzidine (TMB), 5-bromo-4-chloro-3-indolyl-beta-D-galactopyranoside (X-Gal), 5-bromo-4-chloro-3-indolyl phosphate (BCIP), p-nitrophenol (PNPP), 3,3'-diaminobenzidine (DAB), 4-(trifluoromethyl)umbelliferone phosphate, 4-methylumbelliferyl beta-D-galactopyranoside (Mu-Gal), adamantyl 1,2-dioxetane phosphate, ortho-nitrophenyl-beta-D-galactopyranoside (ONPG), nitroblue tetrazolium (NBT), 2,2'-azinobis[3-ethylbenzothiazoline-6-sulfonic acid]-diammonium salt (ABTS), o-phenylenediamine dihydrochloride (OPD), nitroblue tetrazolium chloride (NBT), N-acetyl-Leu-Glu-His-Asp-7-amino-4-trifluoromethylcoumarin, N-acetyl-Leu-Glu-Thr-Asp-7-amino-4-trifluoromethylcoumarin, and 3-oxo-3H-phenoxazin-7-yl-butyrate (resorufin).

[0083] According to the method of the present application, one or more substrates, which can be the same or different, can be used. Typically, when more than one different reporter enzyme detection probe is used, more than one different substrate is used. The markers of the target organisms are targeted so that they can be distinguished. The use of more than one substrate allows for the detection of one target organism with different markers (surface markers, surface ligands, and / or secreted markers) or the detection of different target organisms with different markers (surface markers, surface ligands, and / or secreted markers).

[0084] [Table 1] Non-exhaustive examples of substrates

[0085] The diffusion rate limiting medium (DRLM) is designed to carry the substrate (which ultimately generates a visible spot upon catalysis by the reporter enzyme of the reporter enzyme detection probe); to be porous so as to allow the flow of unconverted substrate and converted detectable product (i.e. before and after catalysis by the reporter enzyme); to slow the diffusion rate of the converted substrate, concentrating the product into a detectable product, thereby allowing the formation of a visible spot around the site of the reporter enzyme probe reaction site; to provide an interface for the reaction between the reporter enzyme (part of the reporter enzyme detection probe, which binds to the target organism) and the substrate. The diffusion rate limiting medium (DRLM) is any suitable medium that limits the diffusion rate by physical properties (e.g. pore size) or by chemical properties (such as charge, hydrophobicity, pH, etc.).

[0086] In contrast to ELISpot and ELISA, where the enzymatically converted product is either absorbed in the membrane of the ELISpot well forming a colored spot (see Figure 2A ] or in ELISA forming a coloration in the whole solution, the method of the present invention uses a diffusion rate limiting medium to slow down the diffusion of the product and trap it around the cell that has been labeled by the reporter enzyme detection probe, allowing the formation of a detectable spot within the diffusion rate limiting medium (DRLM).

[0087] In some embodiments, the diffusion rate limiting medium (DRLM) can also provide an environment for the target organism viability, for example providing osmotic pressure regulation and pH buffering as well as providing nutrients.

[0088] In further embodiments, the diffusion rate limiting medium (DRLM) is non-toxic (for example for non-endpoint applications).

[0089] The diffusion rate limiting medium (DRLM) that reduces the diffusion rate of the detectable product can have different positions relative to the target organism. That is, the target organism can be above the DRLM ([ Figure 5A ], below the DRLM ([ Figure 5B ], or within the DRLM ([ Figure 1 ].

[0090] In some embodiments, the diffusion rate limiting medium is a semi-solid medium, a membrane, or a viscous fluid. According to embodiments, the semi-solid medium is selected from the group consisting of agar, agarose, hydrogel (e.g. Carbopol 940, 2NapFF), gelatin, silicone gel, cellulose derivatives, and SDS-PAGE gel. In another embodiment, the membrane is selected from the group consisting of paper membrane, fiber membrane, cellulose membrane, and plastic membrane. In further embodiments, the viscous fluid is selected from the group consisting of cellulose derivatives (carboxymethyl cellulose, etc.), polysaccharide or monosaccharide gel (syrup, low concentration hydrogel, tapioca gel, guar gum, gellan gum, xanthan gum, acacia gum, hydroxyethyl cellulose, HPC (hydroxypropyl cellulose)), and low concentration hydrogel.

[0091] In some embodiments, the detectable product is a chromogenic, chemiluminescent, or fluorescent molecule. In further embodiments, the detectable product can have radiological and / or electrochemical properties.

[0092] Visible spots can be detected manually with a microscope or automatically with a suitable device such as a macro camera. If the visible spots are outside the visible spectrum that can be seen by the human eye, then the visible spots are detected automatically with a suitable device such as a macro camera adapted to this spectrum. In the case of chromogenic data, typically a picture of each well of a 96-well plate can be taken with a device such as a macro camera, and the visible spots can be quantified with software. If chemiluminescence or fluorescence is used, then special filters and lamps for fluorescence and chemiluminescence can be used to detect and quantify the visible spots.

[0093] The size and formation of the visible spots is controlled by the diffusion rate of the substrate and / or detectable product. The diffusion rate is typically influenced by the physical properties of the diffusion rate limiting medium (DRLM), such as porosity; chemical properties, such as charge or pH; the incubation temperature, which can be important for the reporter enzyme, and where the higher the temperature the faster the diffusion rate provided; the incubation duration; the chemical and physical properties of the diffusion rate limiting medium (DRLM) and the substrate (and their interaction); the reporter enzyme kinetics (the speed at which the reporter enzyme converts the substrate and thereby forms the visible spot) and the effective concentration of substrate supplied for the reaction. In preferred embodiments, the detectable product is trapped by a slow diffusion rate and forms a high concentration around the target organism and forms a visible spot.

[0094] The selection of a suitable diffusion rate limiting medium (DRLM) can be made as follows. A literature search and / or experimental testing must be performed to determine the suitability of the diffusion rate limiting medium (DRLM). The following key functions of the diffusion rate limiting medium should be considered:

[0095] 1. The ability to carry the substrate.

[0096] 2. The porosity and / or other chemical and / or physical properties that can influence the diffusion of the substrate to the enzyme reaction site and the diffusion of the detectable product outwards.

[0097] 3. The ability to manipulate the diffusion rate limiting medium properties to influence the product diffusion rate to allow accumulation of concentration around the enzyme to form a detectable visible spot.

[0098] 4. To provide an interface for the enzyme and substrate reaction.

[0099] 5. To be non-inhibitory to the reporter enzyme / substrate reaction.

[0100] 6. For non-endpoint assays, the diffusion rate limiting medium should be non-toxic and provide conditions to maintain the viability of the targeted sample, including pH, osmotic pressure and nutrients.

[0101] After selecting one or more materials, testing of optimal diffusion rate limiting medium composition and concentration should be performed. This should be performed by preparing a series of compositions and concentrations of the one or more materials that make up the diffusion rate limiting medium (in diluent and substrate). Examples of diluents are tissue culture medium and / or phosphate buffered saline (PBS) and / or solvents compatible with the one or more materials, sample type, and substrate. The substrate is specifically selected for the reporter enzyme conjugated on the detection probe. Positive control reporter enzyme detection probes specific for surface markers common to the sample type should be used to evaluate optimal diffusion rate limiting medium according to the following variables: composition of the one or more materials, concentration of the one or more materials, substrate type, substrate concentration, temperature, pH, and time (other variables should also be considered, but can vary by application). The results given from a series of optimization steps should resemble the graphical depiction in Figure 6 ]. The optimal composition and concentration of the one or more materials should generate the maximum number of visible spots as described in region 2 of Figure 6 ].

[0102] [ Figure 6 ] shows a graphical depiction of the number of spots (y-axis) versus the diffusion rate of the substrate and product as controlled by the DRLM properties (from high to low, x-axis). The graph is divided into 3 regions. Region 1 shows a low count of spots where the DRLM properties are not sufficient to limit diffusion of the substrate / product. This would result in a uniform color of the medium with few detectable visible spots, or in some cases random non-specific product precipitation resulting in no count. Region 2 shows optimal DRLM properties and the results should show a medium with little coloration and a high count of visible spots. Region 3 shows a low count of visible spots where the DRLM properties are too limiting of diffusion such that the diffusion of the substrate / detectable product is limited. The results of such a test would be expected to show minimal enzyme activity, thus a decreasing number of visible spots and little to no coloration of the medium.

[0103] The variables that affect substrate and product diffusion are, but not limited to, the physical and chemical properties of the DRLM, changes in the composition and concentration of the DRLM materials, substrate type and concentration, product type and concentration, incubation temperature, and pH. Changes in each variable are expected to generate a shift in the graph as Figure 6The depicted graph. Zone 1 shows typical results in cases where the DRLM does not limit the diffusion rate sufficiently resulting in fast diffusion of the product into the bulk medium. Typical characteristics of such results are that the medium undergoes a color change, a clear countable detectable visible spot is below expectation, and in some cases random precipitation of the product occurs. Zone 2 shows the expected results in cases where the DRLM meets the optimal conditions to form the highest amount of clear countable detectable visible spots. Zone 3 shows scenarios where the DRLM and conditions limit diffusion beyond the optimal value limiting the formation of detectable visible spots. The reduction of detectable visible spots can be due to insufficient substrate availability, product diffusion too slow to remain within the microscopic confines of the reaction point, or other factors such as unexpected enzyme inhibition.

[0104] Considerations for the duration of incubation for visible spot formation must allow for the formation of an optimal number of size / volume detectable visible spots (spots) and cannot be too long to result in diffusion of the visible spots to an undetectable degree. The incubation temperature should also be considered, too low can reduce enzyme activity in a way that limits visible spot formation. Conversely, too high can hinder the enzyme or denature the enzyme such that the reaction cannot occur, also resulting in a reduction of visible spot formation.

[0105] In embodiments, any of the antibodies disclosed herein can be a monoclonal antibody, a polyclonal antibody, a chimeric antibody, and / or a monospecific antibody.

[0106] In some embodiments, the incubation step d) is performed between 20°C and 40°C, preferably between 23°C and 37°C. The incubation time is at least 30 minutes, preferably 30-45 minutes.

[0107] Another aspect of the present application provides a method for quantifying the amount of a target organism in a sample, the method comprising

[0108] ■detecting the target organism according to the method of the application disclosed herein; and

[0109] ■quantifying the amount of the target organism in the sample based on the number of visible spots.

[0110] Another aspect of the present application provides a method for selecting and isolating a target organism in a sample containing different organisms, the method comprising

[0111] ■detecting the target organism according to the method of the application disclosed herein; and

[0112] ■isolating the target organism.

[0113] Once the target organism is detected by the method of the present application, it can be isolated by methods known in the art, such as pipetting.

[0114] The method of the present application for detecting a target organism (or one or more target organisms) in a sample has many advantages over ELISpot. For example, the method of the present application uses a B cell specific surface marker as the target for the capture antibody (capture molecule) to immobilize the cells on the solid phase, then uses the specific antigen binding mechanism of the B cell for the reporter enzyme detection probe. By this procedure, the targeted B cells of interest can be isolated and, since the target is antigen binding rather than a secreted marker, there is no need to wait for cell differentiation and secretion. This results in a reduction of the traditional 7 day B cell assay to 1 day and provides a true B cell count by direct detection rather than indirect detection after secretion or cell differentiation. Another advantage is that the mechanism for obtaining visible spots (i.e. color change) is performed in DRLM rather than on the platform surface as in ELISpot, which limits the use of ELISpot to secreted markers and requires a more expensive membrane coated platform; and unlike ELISA, it can isolate and quantify cells. A further advantage is that the platform used can be transparent, allowing for microscopic studies, and DRLM can be designed to maintain cell visibility. Furthermore, the present application enables multi-marker detection (multiplexing) and cell isolation on a single identical cell group in a single assay. Thus, for example, the method of the present application can replace the following in one go: analysis of cell surface markers in one cell group with FACS, detection of secreted markers in another cell group with ELISpot or ELISA, then recovery of cells after FACS cell sorting for dilution for cloning.

[0115] Those skilled in the art will appreciate that the application described herein is susceptible to variations and modifications other than those specifically described. It is to be understood that the application includes all such variations and modifications which fall within its spirit or essential characteristics. The application also includes all of the steps, features, compositions and compounds referred to or indicated in the specification, individually or collectively, and any and all combinations of any two or more of said steps or features. The disclosure is thus to be considered as illustrative and not restrictive, the scope of the application being indicated by the appended claims, and there are no intentions to limit the application to the exact abstraction shown. The application is further illustrated by the following examples.

[0116] The foregoing description will be more fully understood with reference to the following Examples. Such Examples, are, however, merely illustrative of the method of practicing the application and are not intended to limit the application in any way. Example

[0117] 1 - Concentration of diffusion rate limiting medium that affects diffusion rate (this is example text). This example shows how the present application can be helpful.

[0118] Objectives:

[0119] The goal of this experiment was to demonstrate how the concentration of a diffusion rate limiting medium affects the diffusion rate using agar (a possible diffusion rate limiting medium (DRLM) candidate) and methylene blue (a diffusant) as a model.

[0120] Materials:

[0121] Agar and methylene blue were purchased from Sigma-Aldrich. Isopropyl alcohol (IPA) was purchased from Anaqua. Grade 1 water was generated by Merck Direct-Q® 5 UV. A climate-controlled cabinet from MRC Lab was used to maintain 25 °C and 50% non-condensing humidity. A macro ccd camera for taking images was purchased from sh-renyue, including the calibration software S-eye. Data processing software, Icy software (containing Image J), was downloaded from https: / icy.bioimageanalysis.org / . SDS gel casting apparatus was purchased from Bio-Rad.

[0122] Methods:

[0123] Prepare 4 mL agar gels with the following compositions: 0.25% (W / V), 0.5% (W / V), 0.75% (W / V), 1% (W / V), and 1.5% (W / V) in grade 1 water and add them to the SDS gel casting apparatus. Add an IPA layer on top of the gel as a leveler and let the gel set for 30 min. Then pour off the IPA and wash thoroughly with water. Then blot the top of the gel by sliding a lint-free paper towel between the slides. Add 200 µL of methylene blue to the top of the gel and place the apparatus in a climate-controlled cabinet at 25 °C, 50% non-condensing humidity for 1 hour. After 1 hour, remove the gel from the climate-controlled cabinet and rinse the remaining methylene blue off with water and blot as done previously. Take images of each gel using a fixed macro camera. Use an ISO certified calibration ruler for pixel-mm calibration and use ImageJ software to calculate the distance of methylene blue diffusion after 1 hour from the number of pixels.

[0124] Results:

[0125] Take all photos using S-eye software in fixed settings and save in jpg format. Use ImageJ to determine the pixel / mm correlation against the calibration ruler. Take five calibration points and plot in pixels vs. length (mm). The R 2 = 1 of the best fit line (trend line) is 1 with the equation: (Y (pixels) + 8.073) / 341.7 = x (length, mm). Then use the equation to calculate the distance traveled by the methylene blue.

[0126] The methylene blue 1 hour diffusion distance was determined by measuring the pixel length from the top of the agar to the methylene blue diffusion front (see Figure 7 ] The methylene blue 1 hour diffusion distance was determined for different agar compositions (0.25% (W / V), 0.5% (W / V), 0.75% (W / V), 1% (W / V), and 1.5% (W / V)). Each agar composition was repeated three times, and five measurements were taken for each repetition. The one hour diffusion distance for each agar composition is plotted in Figure 8 ] The best fit line (trend line) is in agreement with a linear regression model, R 2 = 0.97 (coefficient of determination R 2 > 90% or 0.9 is very strong to complete correlation) with the equation: y (diffusion rate in mm / hour) = -0.954 x (agar composition % (W / V)) + 3.6168. As the agar composition (%) W / V) increases, the methylene blue 1 hour diffusion distance decreases, indicating a slowing of the diffusion rate (mm / hour).

[0127] Conclusion:

[0128] The methylene blue 1 hour diffusion distance decreases proportionally with increasing agar concentration (%) W / V) and vice versa. This provides evidence that as the concentration of the diffusion rate limiting medium increases, the diffusion rate within the diffusion rate limiting medium decreases.

[0129] 2 - Presence and absence of diffusion rate limiting medium

[0130] Objective:

[0131] To demonstrate that only when a suitable diffusion rate limiting medium (DRLM) is present to slow the diffusion rate of the substrate and detectable product at the last step of the assay (independent of the container) does a clearly defined spot (visible spot) form.

[0132] Materials:

[0133] Rabbit anti-human CD27 antibody, HRP-conjugated goat anti-rabbit Fc region antibody and Lymphoprep were purchased from Abeam. Phosphate-buffered saline (PBS), fetal calf serum (FCS), RPMI 1640 with phenol red, ethylenediaminetetraacetic acid (EDTA), sodium bicarbonate (NaHC03) and peroxidase inhibitor were purchased from Thermo Fisher Scientific. ELISpot plates, trypan blue solution, RPMI 1640 without phenol red and agarose were purchased from Merck / Sigma-Aldrich. ELISA plates were purchased from Jet Biofil. 3,3',5,5'-tetramethylbenzidine (TMB) was purchased from Mabtech. Agarose was purchased from Bio Basic.

[0134] A macro-CCD camera for taking images was purchased from Shanghai Yuntai, including the calibration software S-eye. The data processing software, Icy software (containing Image J), was downloaded from https: / / icy.bioimageanalysis.org / .

[0135] Methods:

[0136] ELISA and ELISpot plates were coated with rabbit anti-human CD27 antibody (0.5 pg per well in 50 pL) in phosphate-buffered saline (PBS) at pH 7 at least 12 hours before the experiment. The wells were then washed six times with PBS and blocked with RPMI 1640 containing 10% FCS.

[0137] On the day of the experiment, fresh blood samples were collected from the volunteers. The blood was immediately diluted (50:50) in RPMI 1640 containing 1 mg / mL EDTA. Note: RPMI 1640 for cell culture work was RPMI 1640 with phenol red. Lymphocyte isolation was performed as follows: Take the diluted blood on top of a 10 mL lymphoprep surface, then centrifuge at 800g for 30 min with the brake function off. The lymphocyte layer was extracted using a sterile disposable pipette and fresh RPMI 1640 + 10% FCS + 1 mg / mL EDTA + 0.2% NaHC03 was added to dilute the lymphocytes containing lymphoprep. The lymphocytes were centrifuged at 300g for 10 min. The supernatant was removed and fresh RPMI 1640 + 10% FCS + 1 mg / mL EDTA + 0.2% NaHC03 was added, which was then centrifuged at 300g for 10 min. This washing process was repeated 3 times.

[0138] The lymphocytes were then resuspended in RPMI 1640 + 10% FCS + 1 mg / mL EDTA + 0.2% NaHCO3. 100 μί of the resuspended cells were taken and diluted in 900 μί of RPMI 1640 + 10% FCS + 1 mg / mL EDTA + 0.2% NaHCO3 (1 : 10 dilution). The cells were vortexed, 100 μί of this cell suspension was taken and added to 100 μί of trypan blue solution, which was then vortexed. 20 μί of the trypan blue suspended cells were added to a Neubauer modified counting chamber and the cells were counted.

[0139] 5 x 10 5 were seeded in RPMI 1640 + 10% FCS + 1 mg / mL EDTA + 0.2% NaHCO3 and incubated at 37°C for 2 hours. After 2 hours, the wells were washed six times in warm (37°C) PBS. The positive CD27 cells were further labelled with rabbit anti-human CD27 antibody (0.5 μg per well in 50 μί) prepared in RPMI 1640 + 10% FCS + 1 mg / mL EDTA + 0.2% NaHCO3. The negative control wells were given only RPMI 1640 + 10% FCS + 1 mg / mL EDTA + 0.2% NaHCO3. The plate was then incubated at 37°C for 2 hours. After 2 hours, the wells were washed six times in warm (37°C) PBS.

[0140] The wells were treated with peroxidase inhibitor for 15 min at 4°C, followed by 10 min at room temperature (RT). After peroxidase inhibitor treatment, the wells were washed six times in warm (37°C) PBS. HRP-conjugated goat anti-rabbit Fc region antibody prepared in RPMI 1640 + 10% FCS + 1 mg / mL EDTA + 0.2% NaHCO3 (1 : 1000 dilution) was added to the rabbit anti-human CD27 antibody labelled wells and RPMI 1640 + 10% FCS + 1 mg / mL EDTA + 0.2% NaHCO3 was added to the negative control wells. The plate was incubated at RT for 1 hour. After 1 hour, the wells were washed six times in warm (37°C) PBS.

[0141] Wells without diffusion rate limiting media were prepared by taking the TMB stock solution and mixing with an equal volume of 2x RPMI 1640 without phenol red. 40 μΐ of the TMB / RPMI 1640 mix was added to each well. Note: The RPMI 1640 used in this work stage did not contain phenol red. Wells with semi-solid media were prepared by taking a 1 : 1 :2 ratio of 4x RPMI: 1.2% agar (or agarose) (warmed to 80°C): TMB stock solution to give a 0.3% agar (or agarose) TMB semi-solid media. 40 μΐ of semi-solid TMB media was added to each well. The plate was then incubated at 37°C for 30 min. Photographs were taken using a macro ccd camera.

[0142] Results:

[0143] [ Figure 9 ] Ai) ELISpot and Aii) ELISA plate both platforms without diffusion rate limiting media (such as semi-solid media) produced clumpy precipitates in solution. The cells did not form clearly defined spots and therefore no quantifiable data could be obtained. Note: The TMB solution used in this experiment used ELISpot specific TMB which forms a precipitate.

[0144] [ Figure 9 ] Bi) ELISpot and Bii) ELISA plate both platforms with diffusion rate limiting media produced countable visible spots (pits). By reducing the diffusion rate of the TMB as the reaction is occurring, the cells concentrate the precipitate locally at the site of the reaction and therefore form a colour and visible spot (pit). The 0.3% agar / RPMI / TMB diffusion rate limiting media (chromogenic media) was used for the ELISpot plate (see Figure 9 ] Bi). Whereas the 0.3% agarose / RPMI / TMB diffusion rate limiting media (chromogenic media) was used for the ELISA plate (see Figure 9 ] Bii). Both plates used a low concentration of agar or agarose as the base component of the diffusion rate limiting media (chromogenic media) to form visible spots (pits). This has demonstrated that the method of the present application can be adapted to a variety of vessels and diffusion rate slowing media.

[0145] [ Figure 9Cii) ELISA plate both platforms (negative controls) containing diffusion rate limiting media produced almost no visible spots (pits). These negative controls show that in the absence of rabbit anti-human CD27 antibody and HRP conjugated goat anti-rabbit Fc region antibody labeling almost no visible spots (background noise) are present. More importantly, as seen in the above example, it is the presence of cells with the specific target marker that triggers the formation of blue dye showing clearly distinguishable visible spots (pits).

[0146] Conclusion:

[0147] The results of both ELISpot and ELISA plate platforms are that only in the presence of the appropriate DRLM do detectable and quantifiable spots form. In both cases, if the appropriate DRLM is not used, the experiment will not produce quantifiable data.

[0148] 3 - Visible spot formation and diffusion rate

[0149] Objective:

[0150] To demonstrate that the formation of visible spots is dependent on the diffusion rate.

[0151] Materials:

[0152] Rabbit anti-human CD27 antibody, HRP conjugated goat anti-rabbit Fc region antibody and Lymphoprep were purchased from Abeam. Phosphate buffered saline (PBS), fetal calf serum (FCS), RPMI 1640 with phenol red, ethylenediaminetetraacetic acid (EDTA), sodium bicarbonate (NaHC03) and peroxidase inhibitor were purchased from Thermo Fisher Scientific. ELISpot plates, trypan blue solution, RPMI 1640 without phenol red and agar were purchased from Merck / Sigma Aldrich. ELISA plates were purchased from JET BIO FILTRATION. 3,3',5,5'-tetramethylbenzidine (TMB) was purchased from MABOTEC. Carbopol 940, sodium hydroxide (NaOH) and glycerol were purchased from Acros Organics. Grade 1 water was generated by Merck Direct-Q® 5 UV.

[0153] A macro ccd camera for taking images was purchased from Shanghai Ruiyue, including the calibration software S-eye. Data processing software was downloaded from https: / / icy.bioimageanalysis.org / , Icy software (containing Image J).

[0154] Methods:

[0155] 0.056% Carbopol 940 was prepared by taking 75 mg of Carbopol 940 into 100 mL of room temperature pre-boiled ultrapure water. The wetting process was allowed to proceed overnight with maximum speed mixing using a magnetic stirrer. The volume of 0.1 M NaOH added to the mixture until pH 7.0 was reached was titrated and recorded (33 mL of 0.1 M NaOH was added to the mixture until pH 7.0 was reached). 75 mg in a volume of 133 mL equals 0.056% (W / V) Carbopol 940. A 1 : 1 ratio of TMB was added to each concentration before use.

[0156] 4% (W / V) agar was added to water and autoclaved. The agar was then kept at 80°C until required. The appropriate amount of water and a 1 : 1 ratio of TMB was added to each concentration before use.

[0157] At least 12 hours before the experiment, ELISA plates were coated with rabbit anti-human CD27 antibody (0.5 pg per well in 50 pL) formulated in phosphate buffered saline (PBS) at pH 7. The wells were then washed six times with PBS and blocked with RPMI 1640 containing 10% FCS.

[0158] On the day of the experiment, fresh blood samples were collected from the volunteers. The blood was immediately diluted (50:50) in RPMI 1640 containing 1 mg / mL EDTA. Note: RPMI 1640 used for cell culture work was phenol red containing RPMI 1640. Lymphocyte isolation was performed as follows: Take the diluted blood and add to the top of a 10 mL lymphoprep, then centrifuge at 800g for 30 min with the brake function turned off. The lymphocyte layer was extracted using a sterile disposable pipette and fresh RPMI 1640 + 10% FCS + 1 mg / mL EDTA + 0.2% NaHC03 was added to dilute the lymphocytes containing the lymphoprep. The lymphocytes were then centrifuged at 300g for 10 min. The supernatant was removed and fresh RPMI 1640 + 10% FCS + 1 mg / mL EDTA + 0.2% NaHC03 was added, which was then centrifuged at 300g for 10 min. This washing process was repeated 3 times.

[0159] The lymphocytes were then resuspended in RPMI 1640 + 10% FCS + 1 mg / mL EDTA + 0.2% NaHCO3. 100 μί of the resuspended cells were taken and diluted in 900 μί of RPMI 1640 + 10% FCS + 1 mg / mL EDTA + 0.2% NaHCO3 (1 : 10 dilution). The cells were vortexed, 100 μί of this cell suspension was taken and added to 100 μί of trypan blue solution, which was then vortexed. 20 μί of the trypan blue suspended cells were added to a Neubauer improved counting chamber and the cells were counted.

[0160] 5 x 10 5 were seeded in RPMI 1640 + 10% FCS + 1 mg / mL EDTA + 0.2% NaHCO3 and incubated at 37°C for 2 hours. After 2 hours, the wells were washed six times in warm (37°C) PBS. The positive CD27 cells were further labeled with rabbit anti-human CD27 antibody (0.5 μg per well in 50 μί) prepared in RPMI 1640 + 10% FCS + 1 mg / mL EDTA + 0.2% NaHCO3. Control 1 wells were given only RPMI 1640 + 10% FCS + 1 mg / mL EDTA + 0.2% NaHCO3. The plate was then incubated at 37°C for 2 hours. After 2 hours, the wells were washed six times in warm (37°C) PBS.

[0161] The wells were treated with peroxidase inhibitor for 15 min at 4°C, followed by 10 min at room temperature (RT). After peroxidase inhibitor treatment, the wells were washed six times in warm (37°C) PBS. HRP-conjugated goat anti-rabbit Fc region antibody prepared in RPMI 1640 + 10% FCS + 1 mg / mL EDTA + 0.2% NaHCO3 (1 : 1000 dilution) was added to the sample wells. RPMI 1640 + 10% FCS + 1 mg / mL EDTA + 0.2% NaHCO3 was added to the control 1 wells. The plate was incubated at RT for 1 hour. After 1 hour, the wells were washed six times in warm (37°C) PBS.

[0162] A series of diffusion rate limiting media (DRLM) were prepared as well as PBS as a control 2 (instead of DRLM): 0.3%, 1%, 2% (W / V) agar; 0.028% and 0.014% of Carbopol 940; 50% glycerol, all containing 50% TMB. 40 μΐ of each diffusion rate limiting medium (chromogenic medium) was added to the appropriate well and allowed to develop at 37 °C for 30 min.

[0163] Results:

[0164] The different DRLM tested are listed in Figure 13 . Very few background visible spots (dots) were seen in control 1, where no reporter enzyme probe was added. Note that 9 to 20 spots were considered as background positive or false positive, possibly due to endogenous peroxidase activity. In control 2, a large amount of TMB precipitate mass was observed, but almost no clearly defined visible spots were formed, so no counting was possible. In the case of agar and carbopol, a wide range of visible spot (dot) counts could be observed between the different concentrations used. The average visible spot (dot) counts observed for 2%, 1% and 0.3% W / V agar preparations were 972 ± 451, 1132 ± 219 and 4897 ± 263, respectively. At the same time, the average visible spot (dot) counts observed for 0.028% and 0.014% W / V carbopol preparations were 3903 ± 213 and 6553 ± 300, respectively. The 50% glycerol sample showed a very low number of visible spots (dots) ranging from 20 to 28 dots, which was not significantly different from control 1, due to the enzyme stabilizing properties of glycerol. (n = 3).

[0165] Control 1, which was not coupled with the enzyme probe, had an average of 14 visible spots (dots) caused by endogenous peroxides known to be expressed in B cells [1] . Peroxide inhibitors could not completely inhibit all endogenous peroxide activity of the cells, however, the number of visible spots (dots) in control 1 was still significantly lower than the number of visible spots (dots) in wells incubated with the enzyme probe. Control 2, which contained PBS with 50% TMB, could not be counted accurately due to the excessive large TMB precipitate mass. In addition, the whole solution turned blue / purple. This is because there was no DRLM in the substrate mixture to slow down the substrate / detectable product diffusion rate. The % (W / V) of agar and carbopol greatly influenced the visible spot (dot) count, with high % (W / V) agar (1-2% W / V) (see Figure 10 ] and carbopol (0.028% W / V) (see Figure 11The lower the concentration of the diffusion rate limiting medium, the higher the count of visible spots. Conversely, the higher the concentration of the diffusion rate limiting medium, the lower the count of visible spots. The slower the detectable product diffusion rate allows the concentration around the cell to accumulate to a visible degree. However, if the diffusion rate is slowed too much, the formation of visible spots is inhibited, most likely because the unintended substrate diffusion rate limitation results in insufficient substrate at the reaction site, and the product diffuses too slowly to reach a visible size. In Example 1 above, it was shown that a higher concentration of the diffusion rate limiting medium gave a slower diffusion rate of the methylene blue. It is important to note that the count of visible spots for the 50% glycerol-TMB experiment was similar to the negative control, with very few visible and countable spots. Glycerol is known to be an enzyme stabilizer, resulting in a glycerol-induced conformational change, which is responsible for enzyme stability (in enzyme storage) [2] and inhibition (possibly due to hydrogen bond network disruption) [3] Glycerol as an example of an incompatible medium, it is a viscous liquid that can slow diffusion but is incompatible for enzymatic reactions.

[0166] Microscopic studies using an epifluorescence microscope on ELISA wells (see Figure 12 ) at 100x magnification showed that the visible spots had a dark blue core with a light blue / green halo radiating from the core. This indicates that each spot was generated from one cell. Note: The size of the visible spots was slightly larger than a typical lymphocyte, one spot equals one cell.

[0167] Conclusion

[0168] Visible spots can be formed in DRLM such as semi-solid (agar) or viscous liquid (carbopol) as long as the medium is compatible with the experiment. Incompatible medium (50% glycerol-TMB) proved that visible spots could not be formed due to incompatibility with the enzyme reaction. The composition of the semi-solid or viscous liquid (W / V) greatly affected the number of visible spots formed, due to the diffusion of the chromogenic substrate to the enzyme site and the diffusion of the detectable product from the enzyme site.

[0169] Using the ELISA plate platform allowed for epifluorescence microscopy work (unlike the ELISpot platform with a solid membrane at the bottom). At 100x magnification, the visible spots appeared to have a dark core with a light blue / green halo representing the concentration of the blue chromogenic product, where the light blue / green halo was the leading edge of the product diffusion from the enzyme site. At 100x magnification, the spots were larger than a typical lymphocyte, where one spot represented one cell. References

[0170] 1. Okada, S.S. et al. (2016) ‘Myeloperoxidase in human peripheral blood lymphocytes: Production and subcellular localization’, Cellular Immunology, 300, pp. 18-25. doi:10.1016 / j.cellimm.2015.11.003.

[0171] 2. Ramm, I. et al. (2021) ‘The Impact of Glycerol on an Affibody Conformation and Its Correlation to Chemical Degradation’, Pharmaceutics, 13(11), pp. 1853-1866. https: / doi.org / 10.3390 / pharmaceutics13111853

[0172] 3. Meneses, L. et al (2023) ‘Improving the activity of horseradish peroxidase in betaine-based natural deep eutectic systems’, RSC Sustainability, 1, pp. 886-897. https: / doi.org / 10.1039 / d2su00127f

[0173] 4 - Assay in non-immobilized target organisms (cells)

[0174] Objectives:

[0175] To demonstrate that the method of the invention is also applicable to non-immobilized target organisms (cells).

[0176] Materials:

[0177] Rabbit anti-human CD27 antibody, HRP conjugated goat anti-rabbit Fc region antibody and Lymphoprep were purchased from Abeam. Phosphate buffered saline (PBS), fetal calf serum (FCS), RPMI 1640 with phenol red, ethylenediaminetetraacetic acid (EDTA), sodium bicarbonate (NaHC03) and peroxidase inhibitor were purchased from Thermo Fisher Scientific. Trypan blue solution, RPMI 1640 without phenol red and agarose were purchased from Merck / Sigma Aldrich. ELISA plates were purchased from Jelte Biofilter. 3,3',5,5'-tetramethylbenzidine (TMB) was purchased from MacCrt BioTec. Agarose was purchased from Bio Basic.

[0178] A macro ccd camera for taking images was purchased from Shanghai Rongyue, including the calibration software S-eye. The data processing software, Icy software (including Image J), was downloaded from https: / / icy.bioimageanalysis.org / .

[0179] Methods:

[0180] On the day of the experiment, fresh blood samples were collected from the volunteers. The blood was immediately diluted (50:50) in RPMI 1640 containing 1 mg / mL EDTA. Note: RPMI 1640 for cell culture work was RPMI 1640 with phenol red. Lymphocyte isolation was performed as follows: Take the diluted blood on top of a 10 mL lymphoprep, then centrifuge at 800g for 30 min with the brake function turned off. The lymphocyte layer was extracted using a sterile disposable pipette and fresh RPMI 1640 + 10% FCS + 1 mg / mL EDTA + 0.2% NaHC03 was added to dilute the lymphocytes containing lymphoprep. The lymphocytes were centrifuged at 300g for 10 min. The supernatant was removed and fresh RPMI 1640 + 10% FCS + 1 mg / mL EDTA + 0.2% NaHC03 was added, which was then centrifuged at 300g for 10 min. This washing process was repeated 3 times.

[0181] The lymphocytes were then resuspended in RPMI 1640 + 10% FCS + 1 mg / mL EDTA + 0.2% NaHCO3. 100 pL of the resuspended cells were taken and diluted in 900 pL RPMI 1640 + 10% FCS + 1 mg / mL EDTA + 0.2% NaHCO3 (1 :10 dilution). The cells were vortexed, 100 pL of this cell suspension was taken and added to 100 uL trypan blue solution, which was then vortexed. 20 pL of the trypan blue suspended cells were added to a Neubauer improved counting chamber and the cells were counted.

[0182] Positive CD27 cells were labeled with rabbit anti-human CD27 antibody (0.5 pg / 5x10 5 The labeled cells and negative control cells were then incubated for 2 hours at 37°C. After 2 hours, the cells were pelleted by centrifugation at 300g for 10 min, followed by a wash with warm (37°C) PBS and re-pelleting. This cell wash process was repeated 3 times.

[0183] The cells were treated with peroxidase inhibitor for 15 min at 4°C, followed by 10 min at room temperature (RT). After peroxidase inhibitor treatment, the cells were washed by centrifugation at 300g for 10 min, followed by a wash with warm (37°C) PBS and re-pelleting. This cell wash process was repeated 3 times.

[0184] HRP-conjugated goat anti-rabbit Fc region antibody prepared in RPMI 1640 + 10% FCS + 1 mg / mL EDTA + 0.2% NaHCO3 diluted 1 : 1000 was added to the rabbit anti-human CD27 antibody labeled cells, and RPMI 1640 + 10% FCS + 1 mg / mL EDTA + 0.2% NaHCO3 was added to the negative control wells. The cells were incubated for 1 hour at RT. After 1 hour, the cells were washed by centrifugation at 300g for 10 min, followed by a wash with warm (37°C) PBS and re-pelleting. This cell wash process was repeated 3 times.

[0185] 0.028% (W / V) Carbopol-TMB was prepared by mixing 0.058% (W / V) Carbopol with TMB at a 1:1 ratio. The TMB solution was tested for specificity to both ELISA and ELISpot assays. The 0.028% (W / V) Carbopol-TMB mixture was added directly to the cell pellet, vortexed and added directly to a microscope slide (10 μΐ^). The microscope slide was developed at 37 °C for 30 min. A macro ccd camera was used to take the photograph.

[0186] Results:

[0187] As illustrated in [ Figure 14 ] non-immobilized CD27 labeled cells show that the cells are completely labeled with rabbit anti-human CD27 primary antibody followed by HRP conjugated goat anti-rabbit Fc region secondary antibody. When mixed with [ Figure 15 ] A) ELISA specific TMB and B) ELISpot specific TMB solution, spots were formed in both cases. Negative control (non-immobilized unlabeled cells) Figure 15 ] C) ELISA specific TMB and D) ELISpot specific TMB solution showed very few visible spots (background).

[0188] Regarding the microscope slide [ Figure 16 ] A) microscopic study, multiple visible spots (dots) were visible at 10x magnification. More importantly, at 100x magnification ( Figure 16 B), the visible spots (dots) had a dark blue core, with a light blue / green halo around the visible spots (dots) that was a diffusion front. Additionally, when compared to nearby non-target cells, the dots covered a volume larger than the size of one cell.

[0189] Conclusion

[0190] The method of the present invention provides visible spots of non-immobilized cells that have been labeled with rabbit anti-human CD27 primary antibody followed by HRP-conjugated goat anti-rabbit Fc region antibody. Each visible spot represents one labeled cell produced by the enzymatic reaction of a chromogen to a visible chromophore, in this example horseradish peroxidase (HRP) is the enzyme and 3,3',5,5'-tetramethylbenzidine (TMB) substrate is the chromogen. The diffuse front of chromophore (3,3',5,5'-tetramethylbenzidine diamine) at lower concentration than the reaction site (or core) appears as a light blue / green halo. The core of the reaction site (surface labeling of the cell) appears as a near black to dark blue. Due to the diffusion effect of the chromophore radiating outward from the core of the reaction site, the visible spot is larger than the labeled cell. Direct comparison of CD27 labeled spots to CD27 negative cells shows that the size of the visible spot is much larger compared to CD27 negative cells. This demonstrates that the diffusion rate of the diffusion rate limiting medium is essential in forming a larger, more easily detected visible spot and that the color formed is not simply due to cell staining.

[0191] 5 - Specific antigen targeting memory B cell assay: SARS-CoV-2 spike and nucleocapsid proteins.

[0192] Objectives:

[0193] To detect antigen-specific memory B cells in peripheral blood mononuclear cells (PBMC) against SARS-Cov-2 spike and nucleocapsid proteins as models.

[0194] Materials:

[0195] Rabbit anti-human CD27 antibody and Lymphoprep were purchased from Abeam. Phosphate-buffered saline (PBS), fetal calf serum (FCS), RPMI 1640 with phenol red, ethylenediaminetetraacetic acid (EDTA), sodium bicarbonate (NaHC03), and peroxidase inhibitor were purchased from Thermo Fisher Scientific. ELISpot plates, trypan blue solution, RPMI 1640 without phenol red, and agar were purchased from Merck / Sigma-Aldrich. 3,3',5,5'-tetramethylbenzidine (TMB) was purchased from MacCruz Biotech. Biotinylated SARS-Cov-2 spike protein (from Wuhan-Hu-1 isolate) and biotinylated nucleocapsid protein (2019-nCov WHU02 isolate) were purchased from Acro Biosystems.

[0196] A macro-CCD camera for taking images was purchased from Shanghai Yuntai Company, including the calibration software S-eye. The data processing software, Icy software (containing Image J), was downloaded from https: / icy.bioimageanalysis.org / .

[0197] Method:

[0198] At least 12 hours before the experiment, the ELISpot plates were coated with rabbit anti-human CD27 antibody (0.5 pg per well / 50 pL) prepared in PBS at pH 7. The wells were then washed six times with PBS and blocked with RPMI 1640 containing 10% FCS.

[0199] On the day of the experiment, fresh blood samples were collected from the volunteers. The blood was immediately diluted (50:50) in RPMI 1640 containing 1 mg / mL EDTA. Note: RPMI 1640 used for cell culture work was RPMI 1640 containing phenol red. Lymphocyte isolation was performed as follows: Take the diluted blood and add it on top of a 10 mL lymphoprep surface, then centrifuge at 800g for 30 min with the brake function turned off. Use a sterile disposable pipette to extract the lymphocyte layer and add fresh RPMI 1640 + 10% FCS + 1 mg / mL EDTA + 0.2% NaHC03 to dilute the lymphocytes containing lymphoprep. Centrifuge the lymphocytes at 300g for 10 min. Remove the supernatant and add fresh RPMI 1640 + 10% FCS + 1 mg / mL EDTA + 0.2% NaHC03, then centrifuge it at 300g for 10 min. Repeat this washing process 3 times.

[0200] The lymphocytes were then resuspended in RPMI 1640 + 10% FCS + 1 mg / mL EDTA + 0.2% NaHC03. Take 100 pL of the resuspended cells and dilute them in 900 pL RPMI 1640 + 10% FCS + 1 mg / mL EDTA + 0.2% NaHC03 (1 : 10 dilution). Vortex the cells, take 100 pL of this cell suspension and add it to 100 pL of trypan blue solution, then vortex it. Add 20 pL of the trypan blue suspended cells to a Neubauer modified counting chamber and count the cells.

[0201] Take 5 x 10 5Cells / confluent were seeded in RPMI 1640 + 10% FCS + 1 mg / mL EDTA + 0.2% NaHCO3 and incubated at 37°C for 2 hours. After 2 hours, the wells were washed six times in warm (37°C) PBS. Positive CD27 cells were further labeled with biotinylated SARS-Cov-2 S protein and biotinylated nucleocapsid protein (100 ng / 50 µL of each antigen per well) prepared in RPMI 1640 + 10% FCS + 1 mg / mL EDTA + 0.2% NaHCO3. Negative control wells were given only RPMI 1640 + 10% FCS + 1 mg / mL EDTA + 0.2% NaHCO3. The plates were then incubated at 37°C for 2 hours. After 2 hours, the wells were washed six times in warm (37°C) PBS.

[0202] The wells were treated with peroxidase inhibitor for 15 min at 4°C followed by 10 min at room temperature (RT). After peroxidase inhibitor treatment, the wells were washed six times in warm (37°C) PBS. The HRP-conjugated streptavidin (1 mg / mL) stock solution was diluted (1:1000) in RPMI 1640 + 10% FCS + 1 mg / mL EDTA + 0.2% NaHCO3, added to the test wells, and RPMI 1640 + 10% FCS + 1 mg / mL EDTA + 0.2% NaHCO3 was added to the negative control wells. The plates were incubated at 37°C for 45 min. After 45 min, the wells were washed six times in warm (37°C) PBS.

[0203] Diffusion rate limiting medium (DRLM) consisting of agar, ELISpot specific TMB, and 4x RPMI without phenol red was prepared as follows: Take a 1:1:2 ratio of 4x RPMI: 1.2% agar (warmed to 80°C): stock TMB solution to get a 0.3% agar / RPMI-TMB semi-solid medium. Add 40 µL of semi-solid TMB medium to each well. The plates were then incubated at 37°C for 30 min. Photographs were taken using a macro ccd camera.

[0204] Results:

[0205] CD27 positive cells were immobilized on the experimental wells with rabbit anti-human CD27 antibody. These immobilized CD27 positive cells from peripheral blood samples included T cells [1][3] , natural killer cells [2][3] , memory B cells, and plasma cells, but did not include naive B cells [4]Negative control cells were not labeled with SARS-Cov-2 spike protein and nucleocapsid protein (see Figure 17 A). While SARS-Cov-2 specific memory B cells were labeled with biotinylated SARS-Cov-2 spike protein and nucleocapsid protein (see Figure 17 B). Out of all immobilized CD27+ cells, only cells expressing B cell antigen receptors (BCR) could directly bind to the antigen of interest without the need for MHC molecules [5][6] . Plasma B cells lack surface immunoglobulin expression [7] This makes memory B cells the only cells that are immobilized to the plate surface expressing BCRs that can be tagged with the antigen-enzyme probe and generate visible spots (dots). Obviously, there are some background visible spots (dots) present in the negative control test, however there are significantly more visible spots (dots) present in the test well and this is confirmed by the visible spot (dot) count shown in Table 2 and the column chart of the data in Figure 18 .

[0206] [Table 2] summarizes the visible spot (dot) count of each experimental well shown in Figure 17 [Fig. 2] and includes the mean value and standard deviation of three replicates (n=3).

[0207] Conclusion

[0208] In this study, a direct method for the detection of antigen-specific memory B cells in peripheral blood using the method of the present invention was provided. The method was able to determine the frequency of SARS-Cov-2 antigen-specific memory B cells against SARS-Cov-2 spike protein and nucleocapsid protein in whole peripheral blood mononuclear cells (PBMC) (antigen-specific memory B cell dot count mean (29) - negative control mean (7) = 22 / 5 x 10 5 x 100 = 0.0044%). This is in line with literature work: depending on the vaccination status and the size / number of epitopes present on the antigen, limited dilution analysis results on the frequency of antibody secreting cell precursors are between 0.05% - 0.005% [8] . References

[0209] 1. Hintzen, R Q et al. “Regulation of CD27 expression on subsets of mature T-lymphocytes.” Journal of immunology (Baltimore, Md. : 1950) vol. 151, 5 (1993): 2426-35.

[0210] 2. Silva, Anabel et al. “Application of CD27 as a marker for distinguishing human NK cell subsets.” International immunology vol. 20,4 (2008): 625-30. doi:10.1093 / intimm / dxn022.

[0211] 3. Turaj AH, Hussain K, Cox KL, Rose-Zerilli MJJ, Testa J, Dahal LN, Chan HTC, James S, Field VL, Carter MJ, Kim HJ, West JJ, Thomas LJ, He LZ, Keler T, Johnson PWM, Al-Shamkhani A, Thirdborough SM, Beers SA, Cragg MS, Glennie MJ, Lim SH. Antibody Tumor Targeting Is Enhanced by CD27 Agonists through Myeloid Recruitment. Cancer Cell. 2017 Dec 11;32(6):777-791.e6. doi:10.1016 / j.ccell.2017.11.001. Epub 2017 Nov 30. PMID: 29198913; PMCID:PMC5734932.

[0212] 4. Levesque, Marc C, and E William St Clair. “B cell-directed therapies for autoimmune disease and correlates of disease response and relapse.” The Journal of allergy and clinical immunology vol. 121,1 (2008): 13-21; quiz 22-3. doi:10.1016 / j.jaci.2007.11.030

[0213] 5. Alberts B, Johnson A, Lewis J, et al. Molecular Biology of the Cell. 4th edition. New York: Garland Science; 2002. T Cells and MHC Proteins.

[0214] 6. Paul S, Lal G. The Molecular Mechanism of Natural Killer Cells Function and Its Importance in Cancer Immunotherapy. Front Immunol. 2017 Sep 13;8:1124. doi: 10.3389 / fimmu.2017.01124. PMID: 28955340; PMCID: PMC5601256.

[0215] 7. Ribatti, D. (2017). The discovery of plasma cells: An historical note. Immunology Letters, 188, pp.64-67. doi:https: / doi.org / 10.1016 / j.imlet.2017.06.006.

[0216] 8. Smith MJ, Packard TA, O'Neill SK, Hinman RM, Rihanek M, Gottlieb PA, Cambier JC. Detection and Enrichment of Rare Antigen-specific B Cells for Analysis of Phenotype and Function. J Vis Exp. 2017 Feb 16;(120):55382. doi:10.3791 / 55382. PMID: 28287549; PMCID: PMC5409333.

Claims

1. A method for detecting a target organism in a sample, the method comprising: a. Optionally, the target organism is fixed onto a solid phase; b. Incubate the target organism with one or more reporter enzyme detection probes to form one or more target organism-enzyme detection probe complexes; c. Remove any unbound reporter enzyme detection probes; d. Contacting the one or more target organism-enzyme detection probe complexes with a diffusion rate limiting medium containing one or more substrates, and incubating the one or more target organism-enzyme detection probe complexes and the one or more substrates in the diffusion rate limiting medium to generate one or more detectable products; and e. Detecting one or more detectable products by detecting visible spots; The diffusion rate limiting medium is any suitable medium that reduces the diffusion rate of the one or more detectable products and thereby allows the formation of the visible spots.

2. The method of claim 1, wherein step b) comprises incubating the target organism with one or more primary detection agents specific to the target organism before incubating it with the one or more reporter enzyme detection probes to form the one or more target organism-enzyme detection probe complex.

3. The method of claim 2, wherein the one or more primary detection agents bind to one or more surface ligands of the target organism, one or more surface markers of the target organism, or one or more target substances secreted by the target organism, and wherein the primary detection agent is an antibody or its binding fragment, or an antigen.

4. The method of any one of claims 1 to 3, wherein if the target organism expresses an endogenous enzyme that is the same as or similar to the reporter enzyme of the one or more reporter enzyme detection probes, then step b) further comprises contacting the target organism with an inhibitor of the endogenous enzyme.

5. The method of any one of claims 1 to 4, wherein the target organism is a cell or a portion thereof, or a microorganism or a portion thereof.

6. The method of any one of claims 1 to 5, wherein the solid phase is a reaction vessel, bead, or plate, and wherein the surface of the solid phase is selected from metals, gold, stainless steel, plastics, glass, silica, polycarbonate, polyester, PVDF, polystyrene, nitrocellulose, and cellulose.

7. The method of any one of claims 1 to 6, wherein the target organism is immobilized by directly binding to the solid phase, or indirectly immobilized to the solid phase by a capture molecule coupled to the solid phase in which the target organism is bound.

8. The method of claim 7, wherein the capture molecule is an antibody, an antigen, or a ligand.

9. The method of any one of claims 1 to 8, wherein the diffusion rate limiting medium is a semi-solid medium, a film, or a viscous fluid.

10. The method of claim 9, wherein the semi-solid medium is selected from the group consisting of agar, agarose, hydrogel, gelatin, silicone gel, cellulose derivative and SDS-PAGE gel.

11. The method of claim 9, wherein the membrane is selected from the group consisting of paper membranes, fiber membranes, cellulose membranes, and plastic membranes.

12. The method of claim 9, wherein the viscous fluid is selected from the group consisting of cellulose derivatives, polysaccharide or monosaccharide gels (syrups, low-concentration hydrogels, cassava gels, guar gum, xanthan gum, gum arabic, hydroxyethyl cellulose, HPC (hydroxypropyl cellulose)) and low-concentration hydrogels.

13. The method of any one of claims 1 to 12, wherein the one or more reporter enzyme detection probes bind to one or more surface ligands of the target organism, one or more surface markers of the target organism, one or more target substances secreted by the target organism, or the one or more primary detection agents, and wherein the reporter enzyme detection probes are selected from antibodies or their binding fragments, antigens, drugs, or peptides, which are directly or indirectly conjugated to an enzyme or catalyst that can change color upon contact with a substrate.

14. The method of any one of claims 1 to 13, wherein the substrate is selected from the group consisting of tetramethylbenzidine (TMB), 5-bromo-4-chloro-3-indolyl-β-D-galactopyranoside (X-Gal), 5-bromo-4-chloro-3-indolyl phosphate (BCIP), p-nitrophenol (PNPP), 3,3'-diaminobenzidine (DAB), 4-(trifluoromethyl)umbelliferyl ketone phosphate, 4-methylumbelliferyl ketone β-D-galactopyranoside (Mu-Gal), adamantyl 1,2-dioxane phosphate, o-Nitrophenyl-β-D-galactopyranoside (ONPG), Nitroblue tetrazolium (NBT), 2,2'-bis[3-ethylbenzothiazoline-6-sulfonic acid]-diammonium salt (ABTS), o-phenylenediamine dihydrochloride (OPD), Nitroblue tetrazolium chloride (NBT), N-acetyl-Leu-Glu-His-Asp-7-amino-4-trifluoromethylcoumarin, N-acetyl-Leu-Glu-Thr-Asp-7-amino-4-trifluoromethylcoumarin, and 3-oxo-3H-phenoxazine-7-yl-butyrate (butyrate halogen).

15. A method for quantifying the amount of a target organism in a sample, the method comprising: a. Detecting the target organism according to the method of any one of claims 1 to 14; and b. Quantify the amount of the target organism in the sample based on the number of visible spots.

Images