IL225380A - Bacterial immunoassay and kit - Google Patents
Bacterial immunoassay and kitInfo
- Publication number
- IL225380A IL225380A IL225380A IL22538013A IL225380A IL 225380 A IL225380 A IL 225380A IL 225380 A IL225380 A IL 225380A IL 22538013 A IL22538013 A IL 22538013A IL 225380 A IL225380 A IL 225380A
- Authority
- IL
- Israel
- Prior art keywords
- group
- udp
- toxin
- antibodies
- small gtpase
- Prior art date
Links
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Classifications
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
- C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
- G01N33/569—Immunoassay; Biospecific binding assay; Materials therefor for microorganisms, e.g. protozoa, bacteria, viruses
- G01N33/56911—Bacteria
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
- G01N33/573—Immunoassay; Biospecific binding assay; Materials therefor for enzymes or isoenzymes
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K2317/00—Immunoglobulins specific features
- C07K2317/20—Immunoglobulins specific features characterized by taxonomic origin
- C07K2317/21—Immunoglobulins specific features characterized by taxonomic origin from primates, e.g. man
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K2317/00—Immunoglobulins specific features
- C07K2317/30—Immunoglobulins specific features characterized by aspects of specificity or valency
- C07K2317/33—Crossreactivity, e.g. for species or epitope, or lack of said crossreactivity
-
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Description
BACTERIAL IMMUNOASSAY AND KIT
BACTERIAL IMMUNOASSAY AND KIT
TECHNOLOGICAL FIELD
This invention pertains to the field of diagnostic methods and kits for detecting Clostridium bacteria. The methods and kits provide rapid, sensitive, and accurate assays for the presence of toxigenic strains of Clostridium bacteria in a biological sample.
REFERENCES
References considered to be relevant as background to the presently disclosed subject matter are listed below:
1. Just, I. et al. (1995) Nature 375:500-503.
2. Genth, H. et al. (2006) FEBS letters 580:3565-3569.
3. Wu, Z.L. et al. (2011) Glycobiology 21 :727-733.
4. Darkoh, C. et al. (2011) J. Clin. Microbiol 49:2933-2941.
. WO 2012/166848
Acknowledgement of the above references herein is not to be inferred as meaning that these are in any way relevant to the patentability of the presently disclosed subject matter.
BACKGROUND
Clostridium difficile is the leading cause of nosocomial enteric infections. The microorganism is a gram-positive, spore-forming anaerobic bacillus that was first linked to disease in 1978, when it was identified as the causative agent of pseudomembranous colitis. It has been associated with gastrointestinal infections ranging in severity from asymptomatic colonization to severe diarrhea, pseudomembranous colitis, toxic megacolon, intestinal perforation, and death. Clostridium difficile infection (CDI) can develop if the normal gastrointestinal flora is disrupted by antibiotic therapy and a person acquires toxin-producing C. difficile, typically via the fecal-oral route. The use
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of antibiotics is the major risk factor for CDI occurrence, and this includes almost every antibiotic. Although found also in the community, most cases, as well as outbreaks of C. £¾/?cz7.?-associated disease (CDAD) occur in health care settings and long-term-care facilities. Among these patients, C. difficile is the most common infectious cause of acute diarrheal illness.
Toxin production by the bacterium is essential for disease to occur. C. difficile' 's primary virulence factors are toxins A (Ted A) and B (TcdB), which are responsible for inflammation, fluid and mucous secretion, and mucosal damage, which lead to diarrhea or colitis. Toxin A attracts neutrophils and monocytes, and toxin B degrades the colonic epithelial cells, both leading to the aforementioned clinical effects. In addition, some strains of C. difficile produce an additional toxin known as binary toxin (CDT). There are higher virulent strains, like the recently identified NAP l/BI/027 that has caused numerous outbreaks of clinically severe disease in North America and Europe. NAP l BI/027 produces 16 times more toxin A and 23 times more toxin B than other strains, possibly due to a deletion in a negative regulatory gene. It should be noted that only toxigenic strains of C. difficile produce clinical disease, but toxin production does not guarantee symptomatic progression.
Most often, CDAD is presented as mild to moderate non-bloody diarrhea. Mild CDAD can quickly progress to moderate or severe disease. Therefore prompt diagnosis and treatment is crucial. Stopping the antibiotic administration is the most important step in the initial treatment of CDAD. In addition, appropriate oral antimicrobial therapy directed specifically against C. difficile is given for treatment of mild to moderate CDAD.
A false negative outcome may result in a patient being thought to be free of CDI. Such patient may then receive sub-optimal care, and may not be appropriately isolated, thus increasing the risk of transmission of C. difficile. Moreover, as mentioned above, misdiagnosis may delay appropriate treatment, which is crucial to prevent progression to a more severe disease, and by that increasing the risk of life endangering clinical complications. The consequences of false positive results are perhaps less appreciated, when patients may undergo termination of needed broad-range antimicrobial therapy or receive unnecessary antibiotic treatment for CDI. False-positively misdiagnosed patients might also be nursed in wards together with real cases, and consequently might be exposed to increased risk of acquiring true CDI. There is 02213843U-01
therefore a need for sensitive and selective assays to provide early detection of C. difficile infection.
Currently, there are a variety of tests for diagnosis of C. difficile.
(a) CytoToxin assay (CTA). The current gold standard assay for C. difficile is the cell-culture cytotoxic assay. While all C. difficile strains express the common antigen Glutamate Dehydrogenase, production of toxins A and B is restricted to toxigenic strains. Both toxin A and toxin B contribute to the pathogenesis of CD AD. Diagnosis of C. difficile is primarily accomplished by detecting toxins in the stools of individuals with suspected disease. The CTA detects not only the presence of toxins but also their activity. Therefore, detection of toxin-related activity through CTA is considered to be the gold standard in diagnosis of C. difficile. The CTA involves exposing cultured cells to fecal extracts in the absence or presence of an antagonizing anti-toxin antibody. Fecal samples that are toxigenic C. difficile-positive have a cytopathic effect on the cultured cells that have not been treated with antagonizing anti-toxin antibody.
(b) Cytotoxigenic assay. Cytotoxigenic culture has been used as an alternative gold standard method to CTA testing. The Cytotoxigenic assay detects toxin activity as well, however, in this test bacterial culture supernatants are used instead of fecal sample supernatants.
(c) Bacterial culture. C. difficile can also be detected by culturing the organism under anaerobic conditions.
(d) Enzyme immunoassay (EIA) of antigens in stool. EIA kits are currently used for detection of two types of antigens in stool: toxins A and B, and a surface-associated enzyme of C. difficile, Glutamate Dehydrogenase (GDH). Detection of GDH is used usually for screening purposes, as it provides information about the presence of C. difficile in the tested stool specimen. However, it does not distinguish between the possibility of carrying bacterial asymptomatic colonization state, or alternatively an active toxin-producing strain.
(e) Molecular diagnostics in stool. Commercial molecular diagnostic tests are now available and used in hospitals for detection of CDI. An example for these tests is the quantitative real-time polymerase chain reaction (qPCR) test that amplifies the toxin B gene (tcdB gene).
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(f) Colon examination. Conducting a sigmoidoscopy or colonoscopy by the physician in order to confirm CDI.
(g) Imaging tests. Computerized tomography scan, which provides the physician with detailed images of the colon. The scan can show the thickening of the wall of the colon, which is a common symptom in pseudomembranous colitis.
One or more of these tests may be needed to confirm the presence of C. difficile, C. difficile causes disease by secreting toxins A and B. These toxins act as glucosyltransferase enzymes that specifically catalyze the mono-glucosylation of small GTPases (also known as the Ras superfarnily of GTPases). Mono-glucosylation of small GTPases (such as Rho, Rac, and Cdc42) by C. difficile toxins A and B induces changes of actin dynamics and apoptosis. These toxins glucosylate small GTPases at the amino acid threonine located at position 35 or 37 in a domain termed switch J. UDP-glucose selectively serves as a cosubstrate for the monoglucosylation reaction catalysed by toxins A and B (1).
Nonradioactive methods for the detection of the toxin glycosyltransferase activity have been described: (i) immunoassay based on two monoclonal antibodies: Mab 102 which recognizes unmodified Racl but not glucosylated Racl, and Mab 23 A8 which recognizes both forms of Racl (2); (ii) a glycosyltransferase assay that takes advantage of a specific phosphatase that is added into the glycosyltransferase reaction to release inorganic phosphate from the leaving group of UDP (from the UDP-glucose cosubstrate). The released phosphate group is then quantitatively detected using colorimetric malachite-based reagents (3); (iii) an assay that uses -nitrophenyl-p-D-glucopyranoside (PNPG) has recently been described (the Cdifftox plate assay) ((4) and WO 2012/166848). The method comprises: a) contacting a sample suspected of containing a toxin-producing strain of C. difficile with a medium comprising an indicator-linked substrate for glucosyltransferase; b) incubating the resulting culture in an anaerobic environment for a time sufficient to allow cleavage of the indicator-substrate link if a C. difficile toxin having glucosyltransferase activity is present; c) detecting cleavage of the indicator-substrate link in the incubated culture from b); and d) determining that a toxin-producing strain of C. difficile is present in the sample based upon detected cleavage in c).
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GENERAL DESCRIPTION
The present invention provides a method for detecting the presence of toxigenic Clostridium bacteria in a bodily sample comprising the following steps:
(a) contacting the sample with a low-molecular-weight GTPase (small GTPase), or functionally-active fragment thereof, and a UDP-hexose molecule;
(b) adding labeled antibodies or antigen-binding fragments thereof specific for binding to hexosylated small GTPase; and
(c) providing conditions whereby the labeled antibodies can produce a signal, the production of the signal indicating the presence of the toxigenic
Clostridium bacteria.
The method of the invention is based on the fact that Clostridium bacteria, and in particular C. difficile, cause disease by secreting toxins that transfer a hexose from
UDP-hexose to mammalian small GTPase proteins such as ho, Racl, etc. In one embodiment of the method of the invention, the activity of toxins A and B of C. difficile in stool samples is determined by coating ELISA plates with recombinant Racl, applying the stool sample, providing UDP-glucose and using recombinant antibodies that recognize specifically the glucosylated Racl but not the non-glucosylated polypeptide, as a direct detection of the toxin activity.
The term "detecting the presence of toxigenic Clostridium bacteria" includes both the qualitative determination of the presence or absence of Clostridium bacteria in the sample, as well as the quantitative determination of the amount of Clostridium toxin present in the sample.
Clostridium bacteria are a known family of bacteria. The method of the invention relates to toxigenic Clostridium bacteria which secrete toxins or cytotoxin molecules. Examples of Clostridium species detected by the method of the invention include Clostridium Difficile (Cd), C. sordellii, C. perfringens and C. novyi.
In one embodiment, the cytotoxin molecule is a member of the large clostridial cytotoxin family. In a further embodiment, the large clostridial cytotoxin is selected from the group consisting of toxins A and B of Cd, lethal and hemorrhagic toxin of C. sordellii, TpeL of C. perfringens and alpha-toxin of C novyi.
In one embodiment, the bodily sample is selected from the group consisting of stool, tissue cultures and culture supernatants, tissue samples and bodily fluids.
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The term "UDP-hexose molecule" is to be understood as referring to the ribonucleoside uridine diphosphate linked to a hexose molecule. In a preferred embodiment, the UDP-hexose molecule is selected from the group consisting of UDP-glucose, UDP-N-acetyl-glucosamine, UDP~galactose, UDP-mannose and UDP-glucuronic acid.
The term "antibody," as used herein, includes, but is not limited to a polypeptide substantially encoded by an immunoglobulin gene or immunoglobulin genes, or fragments thereof which specifically bind and recognize an analyte (antigen). Examples include polyclonal, monoclonal, chimeric, and single chain antibodies, and the like.
The term "antigen-binding fragments thereof " refers to a fragment of an antibody specific for binding to hexosylated small GTPase, wherein the fragment binds to the same antigen or epitope as the antibody from which it is derived. Examples of such fragments include single chain Fv, Fab fragments, F(ab')2 fragments, Fd fragments, Fv fragments, dAb fragments, and isolated complementary determining region (CDR) of sufficient framework to specifically bind.
The term "antibodies specific for binding to hexosylated small GTPase" is to be understood as referring to an antibody that may also bind non hexosylated small GTPase. However, its affinity to the hexosylated form is much higher. The term "specific for" when referring to an antibody or other binding moiety refers to a binding reaction which is determinative of the presence of the target analyte in the presence of a heterogeneous population of proteins and other biologies. Thus, under designated assay conditions, the specified binding moieties bind preferentially to a particular target analyte and do not bind in a significant amount to other components present in a test sample. Specific binding to a target analyte under such conditions may require a binding moiety that is selected for its specificity for a particular target analyte. A variety of immunoassay formats may be used to select antibodies specifically immunoreactive with a particular protein. For example, solid-phase ELISA immunoassays are routinely used to select monoclonal antibodies specifically immunoreactive with an analyte. See Harlow and Lane (1988) Antibodies, A Laboratory Manual, Cold Spring Harbor Publications, New York, for a description of immunoassay formats and conditions that can be used to determine specific immunoreactivity. The term "much higher" will typically refer to a specific affinity or selective reaction that will be at least twice signal to background (i.e. affinity to the hexosylated small GTPase as compared to affinity to 02213843U-01
the non-hexosylated small GTPase), and more typically more than 3, 4, 5, 6, 7, 8, 9 or
times the background.
The antibody or fragment thereof are labeled. In one embodiment, the antibodies are labeled by attaching to them a detectable label. In a further embodiment, the detectable label is a moiety that is detectable by methods selected from the group consisting of spectroscopic, photochemical, biochemical, immunochemical (such as immune-PCR), electrical, optical and chemical methods.
Examples of detectable labels include biotin for staining with labeled streptavidin conjugate, fluorescent dyes (fluorescein, Texas red, rhodamine, green fluorescent protein), radiolabels (H3, I125, S35, C14, P32), enzymes (horseradish peroxidase [HRP], alkaline phosphatase), and colorimetric labels such as colloidal gold or colored glass or plastic (e.g., polystyrene, polypropylene, latex) beads.
The terms "low-molecular-weight GTPase" or "small GTPase", refer to molecules having masses of 21 to 30 kD, that are monomeric guanine nucleotide binding proteins related to the a subunit of heterotrimeric G proteins. In one embodiment, the small GTPase is selected from the group consisting of Rho, Racl, Ras,
Rab, Arf, Ran, Rheb, Rad, Rit, Cdc42, and Rap. Also included within the scope of these terms are genetically modified small GTPase molecules and small GTPase fragments
(also named "functionally-active fragment thereof') which fulfill the following requirements:
1. Capable of being glucosylated by active toxin only, i.e. containing a region that undergoes glucosylation by a Clostridium toxin;
2. Capable of being recognized by an antibody specific for the glucosylated small GTPase; and
3. Optionally capable of being immobilized on a solid surface.
In one embodiment, the small GTPase is immobilized on a solid phase surface. There are various methods of immobilization as is well known to the skilled man of the art (see for example Nakanishi, ., et ah, (2008), Recent Advances in Controlled Immobilization of Proteins onto the Surface of the Solid Substrate and Its Possible Application to Proteomics, Current Proteomics, 5, 161-175). In one embodiment, the solid phase surface is selected from the group consisting of a microtiter plate (such as a MaxiSorp® plate [followed by a blocking step] or a Ni coated plate that specifically binds His tagged proteins, i.e. recombinant proteins produced in-house or commercially 02213843M-01
available, for example from Cytoskeleton catalogue number RCOl-A), an antibody coated plate (e.g. anti-His or anti-small GTPase), beads (such as Ni coated beads, or anti-His and anti-small GTPase antibody coated beads) and magnetic particles. In a preferred embodiment, the immobilization is performed after mixing the reaction components. However, various alternative combination/conditions are contemplated e.g. conditions that allow immobilization prior to mixing.
Another aspect of the invention is an antibody or antigen-binding fragment thereof specific for binding to glucosylated small GTPase.
A further aspect of the invention is a kit for detecting the presence of toxigenic Clostridium bacteria in a bodily sample comprising:
(a) a small GTPase, or functionally-active fragment thereof;
(b) a UDP-hexose molecule; and
(c) labeled antibodies or antigen-binding fragments thereof specific for binding to hexosylated small GTPase.
The kit will preferably include positive and negative controls.
BRIEF DESCRIPTION OF THE DRAWINGS
In order to better understand the subject matter that is disclosed herein and to exemplify how it may be carried out in practice, embodiments will now be described, by way of non-limiting example only, with reference to the accompanying drawings, in which:
Fig. 1 is a schematic drawing illustrating one embodiment of an assay according to the invention;
Fig. 2 shows the amino acid sequence of human Racl (NCBI Reference sequence NP-008839.2) (SEQ. ID. NO:l). The glucosylation region is underlined. Thr35 and Ser71 are emphasized; Thr35 is the target of glucosylation by C. difficile toxins and Ser is an amino acid in switch II domain that is required for specificity of Closrtidium toxins (see Jank et al., 2006, The Journal of Biological Chemistry, Vol 281, No. 28, pp. 19527-19535. The paper mentions Ser of RhoA which corresponds to Ser of Racl(/Cdc42));
Fig 3 is a bar graph showing relative recognition of glucosylated Racl and unglucosylated Racl peptides by various recombinant antibodies {by measuring color production at A450);
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Fig. 4 is a bar graph showing relative recognition of Racl protein by various recombinant antibodies before and after interaction with toxin B;
Figs. 5 to 7 are bar graphs showing the specific recognition of glucosyltransferase activity of C. difficile toxin A and/or toxin B in one embodiment of the method of the invention;
Figs. 8 and 9 are bar graphs reflecting toxin mediated glucosyltansferase activity in the presence of various assay component combinations using Ni or MaxiSorp® microtitre plates, respectively; and
Figs. 10 and 11 show the influence of an anti-toxin B antibody on a cytotoxin assay and on one embodiment of an assay according to the invention, respectively.
DETAILED DESCRIPTION OF EMBODIMENTS
In the following examples, one embodiment of the method of the invention is described. The general scheme of this embodiment of the method is shown in Fig. 1. C. difficile toxin A or B mediate a glucosyltransferse reaction in which glucose is transferred from UDP-glucose to members of the Rho family of small GTPases e.g. Racl. The method comprises the following steps: (1) The GTPase substrate Racl having a Thr residue is incubated with (2) a C. difficile toxin-containing sample in the presence of UDP-glucose and reaction buffer, and subsequently immobilized on an ELISA plate. (3) HRP-labeled recombinant antibodies specifically detect Racl which is monoglucosylated on residue Thr35, and produce a color signal from a 3,3 ',5,5'-Tetramethylbenzidine (TMB) substrate. The color signal is quantified by measuring the absorbance at 450 nm against a reference wavelength of 620 nm.
Example 1 - Selection of recombinant antibodies that specifically detect the glucosylated form of Racl.
Specific antibodies against the glucosylated form of Racl (Fig. 2) were selected and produced from a phage-display library containing 15xl09 human antibody clones. Two antigen peptides corresponding to Racl 's glucosylation region - amino acids 30-39 (GEYIPTVFND) (SEQ. ID. NO;2) - were synthesized, one of which contained a glucosylated Thr residue, while the other contained a non-glucosylated Thr residue. These peptides were used to select recombinant antibodies that possess a much higher affinity for the glucosylated form of Racl as compared to the non-glucosylated form. 02213843U-01
Antibodies were finally conjugated to HRP (using Innova HRP conjugation system cat# 701-0000) and tested in an Enzyme-Linked Immunosorbent Assay (ELISA) in which the substrate Racl peptides (glucosylated and non glucosylated) were immobilized on a MaxiSorp® microtiter plate. TMB was used as the HRP substrate and the reaction was stopped with 0.16M sulfuric acid (Stop solution). A450/620nm is the optical density (OD) or absorbance of a solution as measured spectroscopically at a wavelength of 450nm and corrected for background absorbance at 620nm. The color intensity is directly proportional to the amount of antigen detected in the sample.
A total of 12 antibodies (Hul - Hul2) recognized the Racl peptide in its glucosylated form yet showed no or low recognition to its non glucosylated form (Fig. 3).
Example 2 - Antibody specificity
The ability of these antibodies to specifically bind to toxin B mediated glucosylated Racl protein was tested in a similar ELISA assay as described above. Samples containing either buffer or native toxin B (Native Antigen CDB-TNL-50) were incubated with His tagged Racl protein (His-Racl) and UDP-glucose in a reaction buffer containing 50mM HEPES, pH 7.5, 50 mM KCL 1 m MnCl2, and MgCI2. His-Racl was next immobilized on a Ni microtiter plate and HRP-conjugated recombinant antibodies (Hul - Hul 2) were added.
Two recombinant antibodies, Hu3 and Hu7, showed significant increased recognition to Racl protein specifically after interaction with toxin B (Fig. 4). This indicates that these antibodies primarily detect the toxin B mediated glucosylated form of Racl and could be used in an in vitro ELISA based detection assay according to the invention.
Example 3 - One embodiment of the assay of the invention
In this example, the indicated mixtures of His-Racl, UDP-glucose, and either toxin A or toxin B were incubated in the presence of reaction buffer. Recombinant His-Racl was immobilized after incubation with toxin and UDP-glucose on either a Ni (cat# 436024) or MaxiSorp® microtiter plate from Nunc, and its glucosylation levels were determined using an HRP conjugated Hu3 antibody.
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MATERIALS AND METHODS
Samples that contained different levels of C. difficile toxin A or B (NativeAntigen, cat # CDA-TNL-50 and CDB-TNL-50), were added to indicated mixtures of His tagged Racl protein (His-Racl), UDP-glucose and a reaction buffer comprised of 50mM HEPES, pH 7.5, 50 mM KC1, 1 mM MnCI2, and MgCl2 The reaction mixtures were incubated at 37°C for 30 minutes and then transferred to a Ni or MaxiSorp® microtiter plate for 1 hour to immobilize the His-Racl protein. After immobilization, the plates were washed five times with Phosphate Buffered Saline with Tween (PBST). Finally, an HRP conjugated recombinant Hu3 antibody (prepared as previously described) that specifically detect the giucosylated form of Racl (see Example 1 and 2) was added for 1 hour to detect the level of toxin mediated glucosylation of His-Racl protein. This was followed by another cycle of PBST washing (repeated five times, as previously described), addition of TMB and stop solution. The optical density (OD) was determined as previously described in Example 1 at wavelengths of 450 and 620nm.
RESULTS
The results are presented in Figs. 5 - 9, and show that the in vitro reaction and ELISA detection settings of the assay allow specific detection of small amounts of active toxin.
The recombinant Hu3 antibody was able to detect toxin mediated giucosylated Racl protein in a dose dependent manner (Fig. 5). As little as 1 ng toxin B showed significantly higher signals of about 5 fold compared to the control lacking toxin B. The toxigenic activity of C. difficile is derived from the secretion and activity of both toxin A and B that function as glucosyltransferases to glucosylate small GTPases.
Indeed, the toxigenic activity of toxin A was also detected in the method of the invention, as shown in Fig. 6. Similar to toxin B, toxin A affected signal level of giucosylated Racl in a dose dependent manner. The signal detected after addition of 5ng toxin was significantly increased (in comparison to the control), as well as after adding 50 ng toxin. Notably, the signals obtained with toxin B were higher than those obtained with toxin A, indicating that glucosyltransferase activity of toxin B is higher
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than that of toxin A. This is consistent with the results of cytotoxin assays in tissue culture in which toxin B was found to be more potent that toxin A (1).
Because the toxins are enzymes, their denaturation is expected to negatively affect their activity. To demonstrate that the method of the invention reflects toxin B activity, the production level of glucosylated Racl in the assay using a denatured toxin was determined.
Samples containing 10 ng of native or denatured (by heating to 96°C for 3 minutes) toxin B were added to the reaction mixture and the level of glucosylated Racl was determined. The high signal levels that appear after addition of the native toxin did not appear when the denatured toxin was added (Fig. 7), indicating that the signals are directly related to the toxin's enzymatic activity.
Example 4 - determining assay parameters
Because UDP-glucose selectively serves as the glucose donor in the GTPase glucosylation reaction catalyzed by C. difficile toxins, its requirement in the method of the invention was tested. The glucosylation levels of Racl was determined under the reaction and ELISA conditions of the method of the invention as described above and in mixtures that lacked either His-Racl, toxin B, or UDP-glucose.
As shown in Fig. 8, UDP-glucose was essential for the reactions and Racl glucosylation was detected only when toxin B, His Rac-1 and UDP-glucsoe were present. Similar results were shown in a parallel ELISA setting in which His-Racl was immobilized on a MaxiSorp® plate (Fig. 9), instead of the Ni coated plate, indicating that the immobilization platform does not affect the assay outcome. Taken together, these findings show that the results obtained by the method of the invention reflect toxin mediated glucose transfer (glucosyltransferase) of glucose from its glucose donor, UDP-glucose, to the small GTPase molecule.
Example 5 - The anti-toxin antibodies that specifically inhibit toxin B in the cytotoxin assay also inhibit the method of the invention
Toxin B antagonizing antibodies are typically used in the cytotoxin assay to demonstrate the specificity of the assay. Similarly such antibodies were added to the assay of the present invention in order to demonstrate its specificity.
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Claims (23)
1. A method for detecting the presence of toxigenic Clostridium bacteria in a bodily sample comprising the following steps: (a) contacting the sample with a low-molecular-weight GTPase (small GTPase), or functionally-active fragment thereof, and a UDP-hexose molecule; (b) adding labeled antibodies or antigen-binding fragments thereof specific for binding to hexosylated small GTPase; and (c) providing conditions whereby the labeled antibodies can produce a signal, the production of the signal indicating the presence of the toxigenic Clostridium bacteria in the sample.
2. The method of claim 1 wherein the Clostridium bacteria is selected from the group consisting of Clostridium Difficile (Cd), C. sordeliii C perfringens and C novyi.
3. The method of claim 1 wherein the Clostridium bacteria is toxigenic due to the production of a cytotoxin molecule.
4. The method of claim 3 wherein the cytotoxin molecule is a member of the large clostridial cytotoxin family.
5. The method of claim 4 wherein the large clostridial cytotoxin is selected from the group consisting of toxins A and B of Cd, lethal and hemorrhagic toxin of C. sordeliii, TpeL of C. perfringens and alpha-toxin of C novyi.
6. The method of claim 1 wherein the small GTPase or functionally-active fragment thereof is immobilized on a solid phase surface.
7. The method of claim 6 wherein the solid phase surface is selected from the group consisting of a microtiter plate, a MaxiSorp® plate, a Ni coated plate, an antibody coated plate, beads and magnetic particles.
8. The method of claim 7 wherein the Ni coated plate specifically binds an His tagged small GTPase or functionally-active fragment thereof.
9. The method of claim 7 wherein the beads are selected from the group consisting of Ni coated beads, anti-His coated beads and anti-small GTPase antibody coated beads.
10. The method of claim 1 wherein the UDP-hexose molecule is selected from the group consisting of UDP-glucose, UDP-N-acetyl-glucosamine, UDP-galactose, UDP-mannose and UDP-glucuro ic acid. 02213843U-01
11. The method of claim 10 wherein the UDP-hexose molecule is UDP-glucose and the small GTPase is glucosylated by the sample.
12. The method of claim 1 wherein the small GTPase is selected from the group consisting of Rho, Racl, Ras, Rab, Arf, Ran, Rheb, Rad, Rit, Cdc42, and Rap.
13. The method of claim 1 wherein the bodily sample is selected from the group consisting of stool, tissue cultures and culture supematants, tissue samples and bodily fluids.
14. The method of claim 1 wherein the antibodies are labeled by attaching a detectable label to them.
15. The method of claim 14 wherein the detectable label is a moiety that is detectable by a method selected from the group consisting of spectroscopic, photochemical, biochemical, immunochemical, electrical, optical and chemical methods.
16. The method of claim 15 wherein the detectable label moiety is selected from the group consisting of biotin for staining with labeled streptavidin conjugate, a fluorescent dye, a radiolabel, an enzyme and a colorimetric labels.
17. The method of claim 16 wherein the fluorescent dye is selected from the group consisting of fluorescein, Texas red, rhodamine and green fluorescent protein.
18. The method of claim 16 wherein the radiolabel is selected from the group consisting of H3, 1125, S35, C14 and P32.
19. The method of claim 16 wherein the enzyme is selected from the group consisting of horseradish peroxidase and alkaline phosphatase.
20. The method of claim 16 wherein the colorimetric label is selected from the group consisting of colloidal gold, colored glass, and plastic beads
21. The method of any one of claims 1-20 wherein the antibodies are selected from the group consisting of polyclonal antibodies, monoclonal antibodies, chimeric antibodies and recombinant antibodies.
22. The method of claim 21 wherein the recombinant antibodies are selected from the group consisting of single chain Fv, Fab fragments, F(ab')2 fragments, Fd fragments, Fv fragments, dAb fragments, and isolated complementary determining region (CDR) of sufficient framework to specifically bind the small GTPase antigen.
23. An antibody or antigen-binding fragment thereof specific for binding to glucosylated small GTPase. 02213843U-01
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| WO2014147628A1 (en) | 2014-09-25 |
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