WO2016178773A1

WO2016178773A1 - System and method for detecting clostridium difficile toxins

Info

Publication number: WO2016178773A1
Application number: PCT/US2016/026071
Authority: WO
Inventors: Ambrose Lin-Yau Cheung; George Lee Newcomb
Original assignee: Saureus, Inc.
Priority date: 2015-04-06
Filing date: 2016-04-05
Publication date: 2016-11-10

Abstract

The invention generally relates to disease detection and medical diagnosis. More particularly, the invention relates to novel systems and methods for detecting Clostridium difficile toxins and related diseases and disorders. In one aspect, the invention generally relates to a method for detecting one or more Clostridium difficile toxins in a subject, comprising contacting a sample from a subject with monoclonal anti-TcdB or anti-TcdA F(ab)2 fragments projecting outwardly; and detecting the presence or absence of TcdB or TcdA by discerning the sample for an agglutination reaction, wherein agglutination indicates the presence of TcdB or TcdA in the sample. In yet another aspect, the invention generally relates to a kit useful for detecting one or more Clostridium difficile toxins, comprising a probe disclosed herein.

Description

SYSTEM AND METHOD FOR DETECTING CLOSTRIDIUM DIFFICILE

TOXINS

Priority Claims and Related Patent Applications

[0001] This application claims the benefit of priority from U.S. Provisional Application Serial No. 62/143,305, filed on April 6, 2015, the entire content of which is incorporated herein by reference in its entirety.

Technical Field of the Invention

[0002] The invention generally relates to disease detection and medical diagnosis. More particularly, the invention relates to novel systems and methods for detecting Clostridium difficile toxins and related diseases and disorders.

Background of the Invention

[0003] Antibiotic-associated diarrhea attributable to C difficile toxins (CD AD) refers to a wide spectrum of diarrheal illnesses caused by the toxins produced by this organism, such as cases of severe colitis with or without the presence of pseudomembranes. CD AD is a common nosocomial problem and is the most frequent cause of diarrhea in hospitalized patients and in nursing homes. (Mylonakis, et al. 2001 Arch. Intern. Med. 161 :525-533.)

[0004] C. difficile has been established to be the cause of pseudomembranous colitis.

(Larson, et al. 1978 Lancet 1 : 1063-1066.) This disease is frequently induced when competing bacteria in the colonic flora are reduced by the administration of oral antibiotics, resulting in C. difficile proliferation and ultimately secretion of exotoxins that cause the diarrheal disease and dreaded complications such as toxic megacolon, colonic rupture and death. (Voth, et al. 2005 Clin. Microbiol. Rev. 18:247-263.) C. difficile secretes two exotoxins, toxin A (TcdA) and toxin B (TcdB). TcdA is enterotoxic while both TcdA and TcdB are cytotoxic. This disease is spread by spores from diarrhea of previously infected patients commonly found in nursing homes and hospital wards. C. difficile spores are very hardy and can remain dormant for a long time. Following ingestion by patients, and occasionally in normal individuals, whose colonic flora are altered by oral antibiotic intake, these spores can germinate, proliferate and secrete toxins, leading to CD AD. (Khanna, et al. 2012 Aliment. Pharmacol. Ther. 35:613-61821; Leffler, et al. 2012 Am. J. Gastroenterol. 107:96-98.)

[0005] The infection rates for C. difficile are reported to be around 10% after 2 weeks of hospitalization but may reach 50% after 4 or more weeks. Whenever a hospitalized patient develops diarrhea after antibiotic exposure, CD AD is frequently suspected, resulting in lengthening of treatment and additional costs of hospitalization. Even with antimicrobial therapy, relapses can occur in -20% of the patients, making CD AD a significant burden to the health care system. (Kyne, et al. 2002 Clin. Infect. Dis. 34:346-353.)

[0006] Although diagnostic assays are available, the diagnosis of CD AD remains

challenging due to difficulty in differentiating disease, which is due to the presence of toxins, from bacteria colonization. First, cytotoxicity assays for TcdA/TcdB toxins which are the gold standards for detection of specific toxins are time-consuming, poorly standardized and requires tissue culture not readily available in many healthcare facilities. (Wilkins, et al. 2003 J. Clin. Microbiol. 41 :531-534.) Second, real time-PCR assays for tcdA, tcdB and binary toxin genes detect cognate mRNA, DNA but not secreted toxins, a major limitation for RT-PCR. Given that over 50%) of hospitalized and nursing home patients are colonized with C. difficile spores, overdiagnosis with RT-PCR is a real problem, resulting in unnecessary treatment, patient isolation and unwarranted decontamination. (Clabots, et al. 1992 J. Infect. Dis. 166:561-567.) Third, EIA (ELISA and lateral flow) for toxin detection are specific, but less sensitive (49-66%>) with clinical samples than the cytotoxicity assay. ( Alcala, et al. 2008 J. Clin. Microbiol. 46:3833- 3835.) Although detection of GDH (a conserved C. difficile antigen) by EIA has largely replaced C. difficile culture in many hospitals, this assay does not confirm the secretion of TcdA/TcdB toxins.

[0007] Thus, there is an ongoing unmet need for a fast, simple, sensitive, highly specific and cost-effective test for detecting C. difficile toxins at hospitals and nursing care facilities. Summary of the Invention

[0008] The invention is based in part on the discovery and development of a simple, fast and quantitative diagnostic assay for C. difficile toxins that is relatively inexpensive compared to existing tests and highly efficient as it can be performed by routine personnel.

[0009] The system and method disclosed herein is based on a simple-to-use agglutination platform that can be performed in low-tech settings, without any sophisticated operator skill, and then analyzed and quantified with an imaging-processing algorithm.

[0010] In one aspect, the invention generally relates to a method for detecting one or more Clostridium difficile toxins in a subject. The method includes: contacting a sample from a subject with a plurality of probing beads, wherein each probing bead comprises on its surface from about 4 to ~8 Z-domain protein having IgG binding domains bound thereto Fc portion of IgG with polyclonal or several distinct monoclonal anti-TcdB or anti-TcdA F(ab)₂ fragments projecting outwardly; and detecting the presence or absence of TcdB or TcdA by discerning the sample for an agglutination reaction, wherein agglutination indicates the presence of TcdB or TcdA in the sample.

[0011] In another aspect, the invention generally relates to a probe useful for detecting one or more Clostridium difficile toxins in a subject, the probe comprising a plurality of beads, wherein each bead comprises on its surface from about 4 to ~8 Z-domain protein having IgG binding domains bound thereto Fc portion of IgG with anti-TcdB or anti-TcdA F(ab)₂ fragments projecting outwardly.

[0012] In yet another aspect, the invention generally relates to a kit useful for detecting one or more Clostridium difficile toxins, comprising the probe disclosed herein.

[0013] The detection platform comprises the 4Z protein attached to optimized latex beads for enhanced binding to multiple rabbit anti-TcdA or TcdB MAbs, each with a distinct epitope. Using commercially available rabbit polyclonal antibodies, the assay reaches a level of sensitivity higher than those observed in clinical cases for TcdA (levels of 0.5-1 μg/ml) and TcdB (levels of 0.13-0.2 μg/ml) in stool samples. (Terhes, et al. 2009 J. Clin. Microbiol.

47:3478-3481.) Unlike the lateral flow assay (Alere Inc.), the assay disclosed herein is virtually free of interference from stool filtrates. The sensitivity and specificity of the assay can be increased, while maintaining a faster {e.g., 10 min or shoter) and inexpensive format (about $30 or less), including: (1) use of multiple rabbit MAbs with distinct epitopes; (2) use of multiple human MAbs with distinct epitopes (3) replacing C6 linker with Dendrimer; (4) replacing 4Z with 8Z protein.

[0014] The assay conditions, stability and shelf life can be verified and fine-tuned with CDAD-positive and CDAD-negative clinical samples. The assay can also be assessed with negative samples spiked with known amounts of TcdA or TcdB to evaluate interference from stool particulates. Additionally, quantitate agglutination by image analysis can be achieved. The assay disclosed herein can be designed to enjoy high sensitivity (>95%) and specificity (95%) for clinical samples.

Brief Description of the Drawings

[0015] FIG. 1 schematically illustrates an embodiment of the invention using genetically engineered 4Z protein having IgG binding domains.

[0016] FIG. 2 schematically illustrates an exemplary agglutination reaction between rabbit anti-TcdA or anti-TcdB MAbs on optimized latex beads and C. difficile toxin from stool filtrate of patients.

[0017] FIG. 3 shows an exemplary comparison of IgG binding of 4Z protein and protein A by ELISA.

[0018] FIG. 4 shows exemplary agglutination assays of optimized latex beads with 4Z protein and rabbit anti-TcdA (A) or anti-TcdB antibodies (B).

[0019] FIG. 5 schematically illustrates a prototypic design of a detection system.

[0020] FIG. 6 shows exemplary image analysis of kit prototype agglutinations over a range of target concentrations

[0021] FIG. 7 shows exemplary Image capture and analysis system

Definitions

[0022] As used herein, the term "antibody" is used in the broadest sense and encompasses various antibody structures, including but not limited to monoclonal antibodies, polyclonal antibodies, multispecific antibodies (e.g., bispecific antibodies), and antibody fragments so long as they exhibit the desired antigen-binding activity. [0023] As used herein, the term "antibody fragment" refers to a molecule other than an intact antibody that comprises a portion of an intact antibody that binds the antigen to which the intact antibody binds. Examples of antibody fragments include but are not limited to Fv, Fab, Fab', Fab'-SH, F(ab')₂; diabodies; linear antibodies; single-chain antibody molecules (e.g., scFv); and multispecific antibodies formed from antibody fragments.

[0024] As used herein, the term "chimeric" antibody refers to an antibody in which a portion of the heavy and/or light chain is derived from a particular source or species, while the remainder of the heavy and/or light chain is derived from a different source or species.

[0025] As used herein, the term "Fc region" herein is used to define a C-terminal region of an immunoglobulin heavy chain that contains at least a portion of the constant region. The term includes native sequence Fc regions and variant Fc regions. In one embodiment, a human IgG heavy chain Fc region extends from Cys226, or from Pro230, to the carboxyl-terminus of the heavy chain. However, the C-terminal lysine (Lys447) of the Fc region may or may not be present. Unless otherwise specified herein, numbering of amino acid residues in the Fc region or constant region is according to the EU numbering system, also called the EU index, as described in Kabat et al., Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service, National Institutes of Health, Bethesda, Md., 1991.

[0026] As used herein, the term "human antibody" is one that possesses an amino acid sequence that corresponds to that of an antibody produced by a human or a human cell or derived from a non-human source that utilizes human antibody repertoires or other human antibody- encoding sequences. This definition of a human antibody specifically excludes a humanized antibody comprising non-human antigen-binding residues.

[0027] As used herein, the term "humanized" antibody refers to a chimeric antibody comprising amino acid residues from non-human HVRs and amino acid residues from human FRs. In certain embodiments, a humanized antibody will comprise substantially all of at least one, and typically two, variable domains, in which all or substantially all of the HVRs (e.g., CDRs) correspond to those of a non-human antibody, and all or substantially all of the FRs correspond to those of a human antibody. A humanized antibody optionally may comprise at least a portion of an antibody constant region derived from a human antibody. A "humanized form" of an antibody, e.g., a non-human antibody, refers to an antibody that has undergone humanization.

[0028] As used herein, the term "monoclonal antibody" refers to an antibody obtained from a population of substantially homogeneous antibodies, i.e., the individual antibodies comprising the population are identical and/or bind the same epitope, except for possible variant antibodies, e.g., containing naturally occurring mutations or arising during production of a monoclonal antibody preparation, such variants generally being present in minor amounts. In contrast to polyclonal antibody preparations, which typically include different antibodies directed against different determinants (epitopes), each monoclonal antibody of a monoclonal antibody preparation is directed against a single determinant on an antigen. Thus, the modifier

"monoclonal" indicates the character of the antibody as being obtained from a substantially homogeneous population of antibodies, and is not to be construed as requiring production of the antibody by any particular method. For example, the monoclonal antibodies to be used in accordance with the present invention may be made by a variety of techniques, including but not limited to the hybridoma method, recombinant DNA methods, phage-display methods, and methods utilizing transgenic animals containing all or part of the human immunoglobulin loci, such methods and other exemplary methods for making monoclonal antibodies being described herein.

[0029] As used herein, the terms "polyclonal antibody" refer to a composition of two or more different antibody molecules that are capable of binding to or reacting with different specific antigenic determinants on the same or on different antigens. In the context of the present invention, the individual antibodies of a polyclonal antibody bind to different antigenic determinants of the HER family. Preferably the individual antibodies of a polyclonal antibody of the invention bind to different epitopes of the HER family, more preferably distinct and substantially non-overlapping epitopes. The variability of a polyclonal antibody is generally thought to be located in the variable regions of the antibody molecules.

[0030] As used herein, the terms "variable region" or "variable domain" refer to the domain of an antibody heavy or light chain that is involved in binding the antibody to antigen. The variable domains of the heavy chain and light chain (VH and VL, respectively) of a native antibody generally have similar structures, with each domain comprising four conserved framework regions (FRs) and three hypervariable regions (HVRs). (See, e.g., Kindt et al. Kuby Immunology, 6th ed., W.H. Freeman and Co., page 91 (2007).) A single VH or VL domain may be sufficient to confer antigen-binding specificity. Furthermore, antibodies that bind a particular antigen may be isolated using a VH or VL domain from an antibody that binds the antigen to screen a library of complementary VL or VH domains, respectively. See, e.g., Portolano et al., J. Immunol. 150:880-887 (1993); Clarkson et al., Nature 352:624-628 (1991).

[0031] As used herein, the term "epitope" refers to a part of a larger molecule (e.g. antigen or antigenic site) having antigenic or immunogenic activity in an animal. An epitope having immunogenic activity is a portion of a larger molecule that elicits an antibody response in an animal. An epitope having antigenic activity is a portion of a larger molecule to which an antibody immunospecifically binds as determined by any method known in the art. Antigenic epitopes are not necessarily immunogenic. An antigen is a substance to which an antibody or antibody fragment immunospecifically binds, e.g. a toxin, virus, bacteria, protein or DNA. An antigen or antigenic site often has more than one epitope, unless it is very small, and is often capable of stimulating an immune response. Epitopes may be linear or conformational. A linear epitope generally consists of about 6 to 10 adjacent amino acids on a protein molecule that are recognized by an antibody. In contrast, a conformational epitope consists of amino acids that are not arranged sequentially, but where an antibody recognizes a particular three-dimensional structure. When a protein molecule folds into a three-dimensional structure, the amino acids forming the epitope are juxtaposed, enabling the antibody to recognize the conformational epitope. In a denatured protein only linear epitopes are recognized. A conformational epitope, by definition, must be on the outside of the folded protein.

[0032] As used herein, the terms "protein" or "polypeptide" refer to any chain of amino acids, regardless of length or post-translational modification. Proteins can exist as monomers or multimers, comprising two or more assembled polypeptide chains, fragments of proteins, polypeptides, oligopeptides, or peptides.

Detailed Description of the Invention

[0033] The invention provides a novel simple, fast and quantitative diagnostic assay for C. difficile toxins that is inexpensive compared to existing tests and highly efficient as it can be performed by routine personnel. The system and method of the invention can be performed without the need for any sophisticated operation skill and then analyzed and quantified with an imaging-processing algorithm.

[0034] The agglutination platform relies on over-expression of a four IgG-binding Z domain for attachment to optimized latex beads (with respect to size, charge density and length of linker between beads and the Z domain protein). Several high affinity rabbit or human monoclonal antibodies (MAb) (for specificity) with differing epitopes for TcdA or TcdB are anchored on the surface of optimized latex beads to increase sensitivity (FIG. 1). The Fc portion of the IgG binds to the Z-domain, projecting every F(ab)₂ fragment outward for more accessible binding to Ted toxins. This contrasts with the regular latex agglutination assay where Fc and F(ab)₂ are anchored equally to the surface of the beads. Because of this unique agglutination setup, there is less interference from stool particulates in binding between the antibody and Ted toxins to yield an agglutination reaction (as opposed to lateral flow where fluid channels can be blocked by stool materials, leading to reduced sensitivity) (FIG. 2). Rabbit or human MAbs are chosen based on better affinity of the Fc fragment of the rabbit or human IgG to the Z-domain (prelim, data) and that the rabbit or human F(ab)₂ may bind antigen better than the mouse counterpart.

[0035] The assay of the invention is more sensitive and selective than traditional latex bead agglutination reactions due to reduced interference from stool particulates and also quantitative due to optical imaging processing. The surface charge density of activated carboxyl groups on latex beads to tether a six carbon linker (to reduce steric constraint) have been optimized for attachment of the 4Z-domain protein (herein called 4Z protein) (FIG. 1). The modified 4Z protein was ~2-log better than Protein A in attachment of rabbit and mouse IgG (FIG. 3). 4Z protein binds Fc of IgG to enable projection of all F(ab)₂ outward for antigen binding. (Brigido, et al. 1991 J. Basic Microbiol 31 :331 '-345; Eliasson, et al. 1989 J. Immunol. 142:575-581.) Rabbit and human IgG binds 4Z protein better than murine IgG (unpublished data). F(ab)₂ of rabbit MAbs can bind antigen more avidly than murine MAbs. (Rocha, et al. 2008 Pathol. Res. Pract. 204:655-662.) Assay sensitivity is augmented by deploying multiple high affinity MAbs against different epitopes of TcdA or TcdB. Digital imaging processing of the agglutination reaction renders the results less subjective and more quantitative and amenable to automation in the future. [0036] Protein A (Spa) is a 50-64 kDa protein that binds the Fc fragment of most mammalian IgGs with high affinity. Spa has a conserved C-terminus and a variable N-terminal IgG binding repeat, thus accounting for divergent IgG binding capacity among S. aureus strains. (Navarre, et al. 1999 Microbiol. Mol. Biol. Rev. 63 : 174-229.) The IgG binding domain of Spa can be genetically engineered to enhance IgG binding on optimized latex beads to increase sensitivity.

[0037] The agglutination platform disclosed herein is based on tethering of engineered 4Z protein to optimized latex beads, which provide the scaffold for enhanced IgG binding. 4Z has been expressed in E. coli and purified successfully on a nickel column from cell lysates. If needed, sensitivity can be increased by the number of IgG binding domains by cloning DNA encoding 8Z domain in pET14b vector, similar to the cloning strategy for 4Z. Replacement of the C6-linker with PANAM dendrimer may lead to increase in the binding of 4Z and its ensuing effect on agglutination. Based on the binding of rabbit or human IgG (vs. murine IgG) to 4Z and that F(ab)₂ of rabbit or human IgG can bind antigen more avidly than murine MAbs, high affinity rabbit and human MAbs for TcdA and TcdB can be raised with non-competing epitopes for C. difficile toxins to improve agglutination.

[0038] The invention also provide an image analysis system has that efficiently quantifies the agglutination reaction in a manner that removes subjective (visual) analysis imparted by a lab technician. A number of image capture devices may be employed to facilitate the analysis.

[0039] Thus, in one aspect, the invention generally relates to a method for detecting one or more Clostridium difficile toxins in a subject. The method includes: contacting a sample from a subject with a plurality of probing beads, wherein each probing bead comprises on its surface from about 4 to about 8 (e.g., 4, 6, 8) Z-domain protein having IgG binding domains bound thereto Fc portion of IgG with anti-TcdB or anti-TcdA F(ab)₂ fragments projecting outwardly; and detecting the presence or absence of TcdB or TcdA by discerning the sample for an agglutination reaction, wherein agglutination indicates the presence of TcdB or TcdA in the sample.

[0040] In certain preferred embodiments of the method, the sample is a mammal stool sample {e.g., human stool or human stool filtrate samples). In certain preferred embodiments, the sample is a mammal tissue or body fluid {e.g., human tissue or body fluid). In certain embodiments, the test sample is obtained by diluting the originally obtained stool, tissue or bodily fluid samples.

[0041] In certain embodiments, the IgG are murine pAbs. In certain preferred embodiments of the method, the IgG are murine MAbs. In certain embodiments, the IgG are rabbit pAbs. In certain preferred embodiments of the method, the IgG are rabbit MAbs. In certain embodiments, the IgG are human pAbs. In certain preferred embodiments of the method, the IgG are human MAbs.

[0042] In certain preferred embodiments of the method, the IgG comprises anti-TcdB F(ab)₂. In certain preferred embodiments of the method, the IgG comprises anti-TcdA F(ab)₂.

[0043] In certain preferred embodiments of the method, discerning the sample for an agglutination reaction is performed automatically via optical image acquisition and processing. In certain preferred embodiments of the method, discerning the sample for an agglutination reaction is completed in about 10 minutes (e.g., 10 min., 8 min., 5 min., 4 min., 3 min., 2 min. 1 min.) or less.

[0044] In certain embodiments, the method is characterized by a LOD of 0.03 μg/mL (e.g., 0.025 μg/mL, 0.020 μg/mL, 0.015 μg/mL, 0.010 μg/mL) or lower for TcdB. In certain preferred embodiments, the method is characterized by a LOD of at least 0.020 μg/mL (e.g., 0.025 μg/mL, 0.020 μg/mL, 0.015 μg/mL, 0.010 μg/mL) or lower for TcdA. In certain preferred embodiments, the method is characterized by a LOD of 0.016 μg/mL or lower for TcdB. In certain preferred embodiments, the method is characterized by a LOD of 0.016 μg/mL or lower for TcdA.

[0045] In another aspect, the invention generally relates to a probe useful for detecting one or more Clostridium difficile toxins in a subject, the probe comprising a plurality of beads, wherein each bead comprises on its surface from about 4 to about 8 (e.g., 4, 6, 8) Z-domain protein having IgG binding domains bound thereto Fc portion of IgG with anti-TcdB or anti-TcdA F(ab)₂ fragments projecting outwardly.

[0046] In certain embodiments of the probe, the IgG are murine pAbs. In certain preferred embodiments of the probe, the IgG are murine MAbs. In certain embodiments, the IgG are rabbit pAbs. In certain preferred embodiments of the probe, the IgG are rabbit MAbs. In certain embodiments, the IgG are human pAbs. In certain preferred embodiments of the method, the IgG are human MAbs. [0047] In certain preferred embodiments of the probe, the IgG comprises anti-TcdB F(ab)₂.

In certain preferred embodiments of the probe, the IgG comprises anti-TcdA F(ab)₂.

[0048] In certain preferred embodiments of the probe, the beads are latex beads.

[0049] In yet another aspect, the invention generally relates to a kit useful for detecting one or more Clostridium difficile toxins, comprising the probe disclosed herein. In certain

embodiments, the kit further includes a set of control beads not bound with antibodies. In certain embodiments, the kit further includes a reaction medium and a mixing tool.

Examples

Optimization of the latex beads for the agglutination platform

[0050] Using ovalbumin (Ova) and affinity purified rabbit polyclonal anti-Ova antibodies, a variety of conditions were tested to optimize the latex beads for agglutination: (1) size of bead (0.2, 1 and 5 μπι); (2) attachment chemistry on the beads (amine, carboxyl and aldehyde groups); (3) charge density of reactive groups; and (4) length of linkers (C2 - CIO) for attaching latex beads to the IgG-binding protein. It was found that 1 μπι beads with medium density carboxyl group (CML) and C6 linkers produced agglutination at the lowest concentrations of Ova.

Rabbit IgG binds more avidly to 4Z protein than Spa

[0051] To increase binding of IgG onto latex beads {i.e., increased sensitivity), Spa was covalently attached to the C-6 linker using 0.3% glutaraldehyde followed by the addition of IgG. It was found that rabbit IgG binds more avidly to Spa than murine IgGl, IgG2a, IgG2b and IgG3 in an ELISA (verified by FITC-labeled rabbit vs. mouse IgG to Spa). Also, the DNA encoding the minimum consensus IgG domain of Spa, called the Z-domain, as cloned. (Braisted, et al. 1996 Proc. Natl. Acad. Sci. U.S.A. 93 :5688-5692; Tashiro, et al. 1997 J. Mol. Biol. 272:573- 590.). Using overlapping PCR, DNA fragments encoding 4 adjoining Z-domains (4Z protein) in pET14b were created, over-expressed with IPTG and purified 4Z protein using a nickel column to yield ~4 mg of protein. Upon comparing the binding of rabbit IgG to 4Z vs. Spa, it was found that the 4Z protein displayed superior binding, with a two-log increase in binding capacity over Spa (FIG. 3). Rabbit IgG binding to the 4Z protein was five times better than mouse IgG. Based on these studies, binding of high affinity rabbit or human anti-TcdA or anti-TcdB IgG to 4Z protein on latex beads can increase the sensitivity of our assay. Purified TcdA and TcdB

[0052] TcdA and TcdB are large proteins secreted by C. difficile. C-terminal fragments of TcdA and TcdB, which lack the toxic activity, have been purified. These fragments have been shown to be immunogenic can be used to raise rabbit MAbs. ( Rupnik, et al. 2005 Microbiology 151 : 199-208; Sauerborn, et al. 1994 Bakteriol. 24 Suppl, 510-511; von Eichel-Streiber, et al. 1992 Mol. Gen. Genet. 233 :260-268.)

Pilot agglutination assays with 4Z-coated latex beads containing rabbit anti-TcdA or anti-TcdB IgG

[0053] Assays using the agglutination platform of optimized latex bead containing 4Z protein and 0.156 mg/mL (optimized by titration) of rabbit polyclonal anti-TcdA or anti-TcdB antibodies (Abnova Inc.) showed LOD at 0.069 μg/mL and 0.034 μg/mL for purified TcdA and TcdB toxins, respectively. LOD is defined as the lowest concentration of TcdA or TcdB added to control stool samples that exhibited agglutination with TcdA or TcdB antibodies. Next conducted were assays with four known clinical samples without any patient identifier. After diluting the stool samples 1 : 10 with PBS + 0.1% azide, the clarified supernatants (via pelleting) were filtered through a 0.45 μπι filter to remove any bacteria. The assay was positive with two known positives and negative with two known negatives (FIG. 4), implying specificity. After spiking the stool filtrate of a CD AD negative clinical sample with dilutions of purified TcdA or TcdB, a LOD of 0.016 μg/mL for both TcdA and TcdB was observed (Table 1). These results are comparable to LOD of purified toxin in PBS, implying a relative lack of interference from stool particulates in our assay.

Table 1. LOD for TcdA and TcdB with Current Assay and Alere Assay*

* - LOD for TcdA and TcdB with current assay and Alere lateral flow assay using a negative diarrheal sample spiked with a range of TcdA or TcdB toxin. For Alere at LOD, the band is very difficult to discern whereas the results for the current assay are still clear at LOD

GLCM analysis of agglutination images

[0054] Analysis of optical images of the agglutination platform with dilutions of TcdB toxin and negative controls with gray-level co-occurrence matrix (GLCM) revealed a number of potential strategies to enumerate the transition from positive (++) to negative (-) samples.

Spatial frequency analysis of the images showed clear peaks in the positive samples at 0.1 to 0.05 inverse pixels, indicating clusters of white 10-20 pixels wide. Using a step size of 10 in the GLCM analysis, the data revealed statistically significant trends in most parameters from (++) to (-) samples. The strongest indicators are angular second moment and contrast. Contrast between these sized pixel regions ranges from 220±20 for (++) down to 65±30 for (-). The second moment varies from 2.6±0.5 for (++) up to 6.7±0.6 for (-), indicating histogram of intensity values can be used to separate positive from negative samples.

[0055] An image analysis process has been developed that quantifies the agglutination reaction in a manner that removes subjective (visual) analysis imparted by a lab technician (FIG. 6). Based on an exemplary configuration, "contrast" and "peak density with a threshold" have shown a linear correlation between signal and toxin concentration in the agglutination reaction (FIG. 6). A number of image capture devices may be employed to facilitate the analysis, including an iPhone 6, an SLR camera fitted with a macro lens, a trifocal microscope affixed with a digital image recorder, and a simple point and shoot camera, all of which can be adapted to provide image data to the image analysis process. An exemplary fully operational prototype of both the agglutination reagent kit as well as the image capture and analysis system is shown in FIG. 7

Current assay is superior to the lateral flow assay

[0056] Using negative clinical samples spiked with TcdA/TcdB or PBS control, the current assay was compared to the lateral flow assay from Alere Inc. As seen in Table 1, the current assay is more sensitive with the spiked clinical sample than the Alere kit even though the Alere kit has a LOD claim of 0.63 and 1.25 ng/mL with purified TcdA and TcdB, respectively. This finding indicates that the Alere EIA is subject to significant interference from stool particulates, thus explaining their reported lack of sensitivity (50-70%) compared to the gold standard.

(Alcala, et al. 2008 J. Clin. Microbiol. 46:3833-3835.)

Methods

Assessing the use of dendrimer to augment sensitivity of the assay

[0057] Polyamido amine (PAMAM) dendrimers are a class of tree-like macromolecules composed of repetitive branching monomers radiating from a central core. (Lee, et al. 2005 Nat. Biotechnol. 23 : 1517-1526.) The synthesis employs repetitive steps, with each step doubling the reactive groups and increasing the valency {e.g., fourth generation (G4) polymer will have 64 vs. 4 reactive groups in GO). Based on data from Speroni et al who showed that PAMAM

dendrimers can lead to three-fold increase antigen binding to cognate antibodies, it can be explored to replace the C6-linker with dendrimers to improve agglutination. (Speroni, et al. 2010 Anal. Bioanal. Chem. 397:3035-3042.) G4 PAMAM dendrimer which has a globular structure (to reduce steric hindrance) will be used in excess (to ensure free amino groups available for the second reaction) to link with the carboxyl groups on CML latex beads using EDC. The free amino groups on G4 will then be attached to the unique cysteine residue on the 4Z protein using sulfo-SIAB. The binding efficiency of 4Z to C6-linker vs. the G4 dendrimer will be determined by an ELISA with alkaline phosphatase-conjugated rabbit IgG and subsequent substrate development. If the binding is inadequate as compared to C6 linker, we will consider testing dendrimers beyond G4.

Production of MAbs to TcdA and TcdB for binding to 4Z protein on optimized latex beads

[0058] Rabbit MAbs to TcdA and TcdB can be produced using purified TcdA and TcdB. If antigen toxicity is encountered, C-terminal fragments of TcdA and TcdB can be used that have shown to bind cognate rabbit anti-TcdA or anti-TcdB antibodies to promote agglutination. To verify the binding site of rabbit MAbs, peptide mapping of TcdA and TcdB with individual MAbs can be performed. (Fishetti, et al. 1985 J. Exp. Med. 161 : 11384-1401; Jones, et al. 1988 J. Exp. Med. 167: 1114-1123; Jones, et al. 1985 J. Exp. Med. 161 :623-628.). [0059] Human monoclonal antibodies were produced in Medarex mice which are transgenic for human immunoglobulin genes as described by Babcock et al (Babcock et al. Infect. Immun. 74.6339-6347).

[0060] The rabbit or human MAbs can be attached to 4Z (or 8Z) protein immobilized on the optimized latex bead scaffold. To optimize MAb binding, a dose response curve may be constructed of varying amounts of MAbs with a fixed number of beads. Notably, the number of beads can also be increased to augment sensitivity.

Increasing sensitivity and optimization of agglutination

[0061] The sensitivity of agglutination may be impacted by: (1) increasing the Z-domain to augment IgG binding sites; (2) replacing C6 linker with PANAM dendrimer to enhance 4Z (8Z) binding; (3) increasing the multiplicity of MAbs with different epitopes; and (4) expanding the volume of agglutination platform. The platform control will be beads without 4Z or MAbs. Negative stool samples and spiked samples may be used as respective negative and positive sample controls. A preferred LOD to be achieved in clinical samples below the two EIA kits and better than the reported range of -0.5-1 and 0.13-0.2 μg/mL for TcdA and TcdB in stool samples, respectively (36). Unlike the lateral flow kit, the current assay seems to be virtually free of inhibition from stool filtrates.

[0062] The sensitivity can be enhanced by using magnetic latex beads coated with 4Z and attached MAbs to enable separation and buffer change with a magnet.

[0063] To minimize the subjectivity of the readout, agglutination can be quantified using an image analysis approach. In brief, contrast and 2^nd moment as key discriminators of (+) vs. (-) test can be used initially. Then, a training data set can be used to develop cluster of values.

These data can be employed to train a nearest neighbor analysis algorithm (kNN algorithm), which can be used to prospectively identify (+) vs. (-) samples.

[0064] Stability and shelf life of the agglutination platform, stored at 4°C with 0.1% azide, can be assessed by tracking biweekly agglutination assays. The effect of protein

degradation/denaturation, if observed, can be evaluated, which can be minimized with a protease inhibitor cocktail and changes in storage buffer. Other preservatives {e.g., thiomersal) or varying concentrations of azide can be tried to prolong storage. The preferred shelf-life is at least 6 months. The suitability of a dropper (or other delivery vehicles) to deliver 15 μΙ_, of agglutination platform is also evaluated. A dropper delivering 7.5 μΙ_, of samples (purified toxin, processed stool samples or spikes samples) will be mixed with a 15 [iL of agglutination platform to promote agglutination (FIG. 5 for kit).

[0065] The preferred embodiments are characterized by: (1) delivering the agglutination platform or stool sample in a fine dropper corresponding to 15 and 7.5 [iL, respectively; (2) the kind of container for diluting and minimally processing the stool sample (FIG. 5); (3) assessing the volume of agglutination platform and test samples for agglutination with appropriate controls (FIG. 4); (4) evaluating the effect of stool filtrates on the agglutination reaction; (5) monitoring quality control, variability and reproducibility of the agglutination platform; (6) buffer for the reaction; (7) comparing the sensitivity and specificity of our agglutination with Meridian and Alere EIA as comparators, using blinded positive and negative stool samples (25 each).

[0066] In this specification and the appended claims, the singular forms "a," "an," and "the" include plural reference, unless the context clearly dictates otherwise.

[0067] Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art. Although any methods and materials similar or equivalent to those described herein can also be used in the practice or testing of the present disclosure, the preferred methods and materials are now described.

Methods recited herein may be carried out in any order that is logically possible, in addition to a particular order disclosed.

Incorporation by Reference

[0068] References and citations to other documents, such as patents, patent applications, patent publications, journals, books, papers, web contents, have been made in this disclosure. All such documents are hereby incorporated herein by reference in their entirety for all purposes. Any material, or portion thereof, that is said to be incorporated by reference herein, but which conflicts with existing definitions, statements, or other disclosure material explicitly set forth herein is only incorporated to the extent that no conflict arises between that incorporated material and the present disclosure material. In the event of a conflict, the conflict is to be resolved in favor of the present disclosure as the preferred disclosure. Equivalents

[0069] The representative examples disclosed herein are intended to help illustrate the invention, and are not intended to, nor should they be construed to, limit the scope of the invention. Indeed, various modifications of the invention and many further embodiments thereof, in addition to those shown and described herein, will become apparent to those skilled in the art from the full contents of this document, including the examples which follow and the references to the scientific and patent literature cited herein. The following examples contain important additional information, exemplification and guidance that can be adapted to the practice of this invention in its various embodiments and equivalents thereof.

Claims

What is claimed is:

1. A method of detecting one or more Clostridium difficile toxins in a subject, the method comprising:

contacting a sample from a subject with a plurality of probing beads, wherein each probing bead comprises on its surface from about 4 to about 8 Z-domain protein having IgG binding domains bound thereto Fc portion of IgG with anti-TcdB or anti- TcdA F(ab)₂ fragments projecting outwardly; and

detecting the presence or absence of TcdB or TcdA by discerning the sample for an agglutination reaction, wherein agglutination indicates the presence of TcdB or TcdA in the sample.

2. The method of claim 1, wherein the sample is a human stool sample.

3. The method of claim 1 or 2, wherein the sample is a diluted human stool filtrate.

4. The method of claim 1, wherein the sample is a human tissue or body fluid.

5. The method of any of claims 1-4, wherein the IgG are human pAbs.

6. The method of any of claims 1-4, wherein the IgG are human MAbs.

7. The method of any of claims 1-4, wherein the IgG are rabbit pAbs.

8. The method of any of claims 1-4, wherein the IgG are rabbit MAbs.

9. The method of any of claims 1-4, wherein the IgG are murine pAbs.

10. The method of any of claims 1-4, wherein the IgG are murine MAbs.

11. The method of any of claims 5-10, wherein the IgG comprises anti-TcdB F(ab)₂.

13. The method of any of claims 5-10, wherein the IgG comprises anti-TcdA F(ab)₂.

14. The method of any of claims 1-13, wherein discerning the sample for an agglutination reaction is performed automatically via optical image acquisition and processing.

15. The method of any of claims 1-14, wherein discerning the sample for an agglutination reaction is completed in about 10 minutes or less.

16. The method of any of claims 1-15, characterized by a LOD of at least 0.016 μ§/ ιί for TcdB.

17. The method of any of claims 1-16, characterized by a LOD of at least 0.016 μ§/ ιί for TcdA.

18. A probe for detecting one or more Clostridium difficile toxins, the probe comprising a plurality of beads, wherein each bead comprises on its surface from about 4 to about 8 Z- domain protein having IgG binding domains bound thereto Fc portion of IgG with anti- TcdB or anti-TcdA F(ab)₂ fragments projecting outwardly.

19. The probe of claim 18, wherein the IgG are human pAbs.

20. The probe of claim 18, wherein the IgG are human MAbs.

21. The probe of claim 18, wherein the IgG are rabbit pAbs.

22. The probe of claim 18, wherein the IgG are rabbit MAbs.

23. The probe of claim 18, wherein the IgG are murine pAbs.

24. The probe of claim 18, wherein the IgG are murine MAbs.

25. The probe of any of claims 18-24, wherein the IgG comprises anti-TcdB F(ab)₂.

26. The probe of any of claims 18-24, wherein the IgG comprises anti-TcdA F(ab)₂.

27. The probe of any of claims 18-26, wherein the beads are latex beads.

28. A kit useful for detecting one or more Clostridium difficile toxins, comprising the probe according to any of claims 18-27.

29. The kit of claim 28, further comprising a set of control beads without antibodies.

30. The kit of claim 28 or 29, further comprising a reaction medium and a mixing tool.