WO2013006024A1 - Diagnostic d'entamoeba histolytica - Google Patents

Diagnostic d'entamoeba histolytica Download PDF

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Publication number
WO2013006024A1
WO2013006024A1 PCT/MY2011/000172 MY2011000172W WO2013006024A1 WO 2013006024 A1 WO2013006024 A1 WO 2013006024A1 MY 2011000172 W MY2011000172 W MY 2011000172W WO 2013006024 A1 WO2013006024 A1 WO 2013006024A1
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WIPO (PCT)
Prior art keywords
protein
histolytica
family
subject
phosphoglucomutase
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PCT/MY2011/000172
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English (en)
Inventor
Rahmah Binti Noordin
Boon Huat Lim
Zeehaida Mohamed
Nurulhasanah OTHMAN
Weng Kin WONG
Zi Ning TAN
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Universiti Sains Malaysia
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Priority to IN626CHN2014 priority Critical patent/IN2014CN00626A/en
Priority to BR112014000135A priority patent/BR112014000135A2/pt
Priority to MX2014000007A priority patent/MX358619B/es
Publication of WO2013006024A1 publication Critical patent/WO2013006024A1/fr
Priority to ZA2014/00469A priority patent/ZA201400469B/en

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    • 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
    • G01N33/56905—Protozoa
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P33/00—Antiparasitic agents
    • A61P33/02—Antiprotozoals, e.g. for leishmaniasis, trichomoniasis, toxoplasmosis
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
    • Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
    • Y02A50/00—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE in human health protection, e.g. against extreme weather
    • Y02A50/30—Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change

Definitions

  • the present invention relates to methods for diagnosing and/or treating an Entamoeba histolytica (E. histolytica) infection in a subject.
  • E. histolytica Entamoeba histolytica
  • E histolytica is a protozoan parasite that is the causative agent of extraintestinal amoebiasis, which manifests as amoebic liver abscess (ALA).
  • ALA amoebic liver abscess
  • E. histolytica causes high rates of morbidity and mortality in people especially in developing countries. For example, E. histolytica affects an estimated 480 million people annually; about 10 percent of these people develop colitis, ALA, or other symptoms.
  • WW Health Organization World Health Organization (WHO) reported that in 1997, amoebiasis was ranked second for causing the most number of deaths worldwide compared to any other parasitic diseases.
  • E. histolytica infection Diagnosis of E. histolytica infection is often difficult. Amoebic dysentery caused by E. histolytica is easily confused with monocytic erythrophagocytosis and erythrophagocytosis caused by Entamoeba coli. Early diagnostic assays included microscopy and culture.
  • Antibody detection is another method that can be used to detect antibodies of E.histolytica in serum samples.
  • this method may only be useful in non- endemic areas as the currently available commercial tests use a complex mixture of native E. histolytica proteins and often cannot differentiate current and past infection when used in endemic areas (Zengzhu et al., 1999).
  • Kelantan where the level of endemicity is unknown, the interpretations of the commercial test results are often difficult because of the high background levels of seropositivity especially when the antibody titre is low (Zeehaida et al., 2008). Seropositivity can persist for years, thus resulting in a high background due to healthy subjects giving positive results. It is thus impossible to obtain conclusive results in endemic areas using these commercial tests.
  • Another diagnostic method involves detection of a lectin found on the surface of E. histolytica and E. dispar trophozoites. Infection of a cell by Entamoeba involves binding of this lectin to Gal/GalNAc residues on the surface of the target cell.
  • the lectin which has a molecular mass of 260 kDa, is composed of two subunits of 170 kDa and 35 kDa. Diagnostic assays that use monoclonal antibodies raised against purified native 170 kDa antigen were found to have problems with false positives.
  • Monoclonal antibodies against a recombinantly produced form of the 170 kDa subunit and the use of the antibodies for detecting the 170 kDa antigen resulted in the making of a commercially available immunoassay, TechLab Entamoeba histolytica II kit, for E. histolytica detection.
  • a second commercially available immunoassay, Alexon"ProSpecT Entamoeba histolytica Microplate Assay also detects both pathogenic and non- pathogenic Entamoeba species, through use of rabbit polyclonal antisera.
  • the present invention relates to detecting at least one E. histolytica protein, fragment, variant or mutant thereof and/or antibody that specifically binds to the E. histolytica protein, fragment, variant or mutant thereof in a serum sample of a subject.
  • the presence of the protein, fragment, variant or mutant thereof and/or antibody that specifically binds to the protein, fragment, variant or mutant thereof in the serum sample may be indicative of the subject having or as being at risk of having E. histolytica infection.
  • the present invention relates to a method of diagnosing at least one subject as having or as being at risk of having E. histolytica infection, the method comprising detecting the presence of at least one E.
  • histolytica protein, fragment, variant or mutant thereof in a serum sample from the subject wherein the presence of the protein, fragment, variant or mutant thereof is indicative of the subject having or as being at risk of having E. histolytica infection.
  • the method may be in vitro or in vivo.
  • the protein may be selected from the group consisting of pyruvate phosphate dikinase, phosphoglucomutase, pyruvate:ferredoxin oxidoreductase, phosphoglucomutase/ phosphomannomutase family protein, BspA family leucine rich repeat protein, and Cullin family protein.
  • the protein may have a molecular weight of 42kDa, 70kDa, 79kDa, or 110kDa.
  • the present invention provides serum markers, kits, vaccines, agents, uses and/or methods of treating E. histolytica infection comprising the E. histolytica proteins and/or antibodies thereof.
  • preferred embodiments of the present invention allow an optimal use of these E. histolytica proteins in the method of diagnosing E. histolytica infection and/or related diseases from a subject's serum to take advantage of the specificity, sensitivity and simplicity of the method. This and other related advantages will be apparent to skilled persons from the description below.
  • Figure 1 is a gel photo of a western blot done to show the representative profile of excretory-secretory antigen (ESA) preparation obtained from E.histolytica culture when probed with serum samples from ALA patients and control serum.
  • ESA excretory-secretory antigen
  • Figure 2 is a gel photo of a western blot done to show the representative profile of crude soluble antigen (CSA) preparation obtained from E.histolytica culture when probed with serum samples from ALA patients and control serum.
  • Figure 3 is an MS/MS result for ESA 110 kDa protein.
  • Figure 3A is Mascot search result from Matrix Science database (MSDB) search engine;
  • Figure 3B is peptide summary reports from MSDB search engine;
  • Figure 3C is protein views from MSDB search engine.
  • Figure 4 is an MS-MS identification of CSA 70kDa.
  • Figure 4A is a first possible MS-MS identification of CSA 70kDa;
  • Figure 4B is second possible MS-MS identification of CSA 70kDa;
  • Figure 4C is third possible MS-MS identification of CSA 70kDa.
  • agent refers to a substance that reduces or eliminates expression of any one of the E. histolytica proteins.
  • an agent according to any aspect of the present invention may be capable of reducing the expression of at least pyruvate phosphate dikinase, phosphoglucomutase, pyruvate:ferredoxin oxidoreductase, phosphoglucomutase/ phosphomannomutase family protein, BspA family leucine rich repeat protein, and/or Cullin family protein in a cell.
  • agents include, but are not limited to antisense molecules, antibodies or antibody fragments, proteins or polypeptides as well as small molecules and the like.
  • amebiasis refers to the disease caused by E. histolytica.
  • the term “amebiasis” is used interchangeably with the term “amoebiasis”.
  • the symptoms often are quite mild and can include loose stools, stomach pain, and stomach cramping. Amebic dysentery is a severe form of amebiasis associated with stomach pain, bloody stools, and fever. Rarely, E. histolytica invades the liver and forms an abscess. Even less commonly, it spreads to other parts of the body, such as the lungs or brain.
  • an antibody refers to any immunoglobulin or intact molecule as well as to fragments thereof that bind to a specific epitope.
  • Such antibodies include, but are not limited to polyclonal, monoclonal, chimeric, humanised, single chain, Fab, Fab', F(ab)' fragments and/or F(v) portions of the whole antibody.
  • an antibody that may be capable of specifically binding to at least one E. histolytica protein that may be found in the serum of a subject may be detected for diagnosis of E. histolytica infection and/or related diseases.
  • antibody fragment refers to an incomplete or isolated portion of the full sequence of the antibody which retains the antigen binding function of the parent antibody.
  • antibody fragments include Fab, Fab', F(ab')2, and Fv fragments; diabodies; linear antibodies; single-chain antibody molecules; and multispecific antibodies formed from antibody fragments. Fragments of the antibodies according to any aspect of the present invention are encompassed by the invention so long as they retain the desired affinity of the full-length antibody. In particular, it may be shorter by at least one amino acid.
  • biomarker refers to any diagnostic marker such as a biochemical marker, serological marker, genetic marker, or other clinical or echographic characteristic that can be used to classify a sample from an individual as a sample from a subject with E. histolytica infection.
  • the biomarker may be a serum biomarker, where the marker may be found in the serum, plasma or blood of the individual.
  • Non-limiting examples of diagnostic markers suitable for use in the present invention are pyruvate phosphate dikinase, phosphoglucomutase, pyruvate:ferredoxin oxidoreductase, phosphoglucomutase/ phosphomannomutase family protein, BspA family leucine rich repeat protein, Cullin family protein and include mRNAs or DNAs of these proteins.
  • the term “comprising” is herein defined to be that where the various components, ingredients, or steps, can be conjointly employed in practicing the present invention. Accordingly, the term “comprising” encompasses the more restrictive terms “consisting essentially of and “consisting of.” With the term “consisting essentially of it is understood that the method according to any aspect of the present invention “substantially” comprises the indicated step of detecting and/or using an E. histolytica protein as "essential" element. Additional steps may be included. Accordingly, a method “consisting essentially of a step of detecting all the E. histolytica proteins disclosed in a serum sample, will be novel in view of a method that uses only one of the E. histolytica proteins disclosed.
  • fragment' refers to an incomplete or isolated portion of the full sequence of an E.histolytica protein.
  • a fragment of pyruvate phosphate dikinase may not comprise the full sequence but comprises the active site(s) that confers the sequence with the characteristics and function of the peptide.
  • it may be shorter by at least one nucleotide or amino acid and may be an immunogenic fragment.
  • a fragment may also refer to a fragment of an antibody according to any aspect of the present invention where the antibody comprises the active site that recognises the corresponding antigen.
  • the fragment may at least be 10 amino acids in length.
  • subject typically refers to humans, but also to other animals including but are not limited to other primates, rodents, canines, felines, equines, ovines, porcines, and the like.
  • the term "method for analysing and/or detecting” refers to any method which makes it possible to measure the level of gene expression. These methods are generally well known to those skilled in the art and may be chosen according to the transcription or translation rates.
  • mutant of an E.histolytica protein refers to one which has at least one amino acid sequence that varies from at least one reference sequence of the protein via substitution, deletion or addition of at least one amino acid, but retains the function of the protein.
  • the terms “specific binding” or “specifically binding” refer to the interaction between a protein or peptide and an agonist, an antibody, or an antagonist. In particular, the binding is between an antigen and an antibody. The interaction is dependent upon the presence of a particular structure of the protein recognized by the binding molecule (i.e., the antigen or epitope). For example, if an antibody is specific for epitope "A", the presence of a polypeptide containing the epitope A, or the presence of free unlabeled A, in a reaction containing free labeled A and the antibody will reduce the amount of labeled A that binds to the antibody.
  • a "variant" of at least one of E.histolytica protein, as used herein, refers to an amino acid sequence that is altered by one or more amino acids.
  • the variant may have "conservative” changes, wherein a substituted amino acid has similar structural or chemical properties (e.g., replacement of leucine with isoleucine). More rarely, a variant may have "nonconservative" changes (e.g., replacement of glycine with tryptophan).
  • Analogous minor variations may also include amino acid deletions or insertions, or both. Guidance in determining which amino acid residues may be substituted, inserted, or deleted without abolishing biological or immunological activity may be found using computer programs well known in the art, for example, DNASTAR software.
  • the E. histolytica proteins, fragment, variant or mutant thereof may be used for the detection of ALA and other forms of extaintestinal amoebiasis by detecting the presence of at least one E. histolytica protein, fragment, variant or mutant thereof in a sample.
  • the sample may be serum, plasma or blood of a subject.
  • These E. histolytica proteins may also be used in antigen detection tests for intestinal amoebiasis and/or vaccines against amoebiasis.
  • the E. histolytica protein may be selected from the group consisting of pyruvate phosphate dikinase, phosphoglucomutase, pyruvate:ferredoxin oxidoreductase, phosphoglucomutase/ phosphomannomutase family protein, BspA family leucine rich repeat protein, and Cullin family protein.
  • BspA family leucine rich repeat protein is a surface family protein encoded by 116 Bsp- A like genes in the genome, and 41 of them associated with transposable elements. It plays a role in attachment and penetration to the host cells and has been shown to be involved in protein-protein interaction. It binds to the extracellular matrix components, fibronectin and fibrinogen.
  • Cullin family protein is one of a scaffold of E3 enzyme, a multisubunit ubiquitin-ligase, which involves in facilitation of ubiquitin from ubiquitin- conjugation enzyme ( E2), to the Lys residue in the substrate molecule, and mainly occur in the eukaryotic cells.
  • a homolog to Cullin protein CDC53 has been found in each parasites apicomplexa (PF08-0094 in Plasmodium falcifarum and 80.m02207 in Toxoplasma gondii. Since these two £. histolytica proteins i.e. Bsp-A family (leucine rich repeat protein) and Cullin family protein are found to circulate in serum samples of ALA patients whose pus samples contain amoebic DNA, they may be useful in the diagnosis of ALA.
  • the E. histolytica protein may have a molecular weight of 42kDa, 70kDa, 79kDa, or 110kDa.
  • the method may be an in vitro method. Since this method is rapid, non-invasive and relatively inexpensive for detecting the presence of, or the absence of at least one E. histolytica protein, it is particularly advantageous.
  • the method may comprise detection of at least one E. histolytica protein in a sample of an individual.
  • the sample may be blood, plasma or serum.
  • the quantitative data associated with the E. histolytica protein markers can be any data that allows generation of a result useful for diagnosis of E. histolytica and related disease, including measurement of DNA or RNA levels associated with the markers but is typically protein expression patterns.
  • Protein levels can be measured via any method known to those of skill in the art that generates a quantitative measurement either individually or via high-throughput methods as part of an expression profile.
  • a serum sample may be applied to a specific binding agent or panel of specific binding agents to determine the presence and quantity of the E. histolytica protein markers.
  • DNA and RNA expression patterns can be evaluated by northern analysis, PCR, RT-PCR, Taq Man analysis, FRET detection, monitoring one or more molecular beacon, hybridization to an oligonucleotide array, hybridization to a cDNA array, hybridization to a polynucleotide array, hybridization to a liquid microarray, hybridization to a microelectric array, cDNA sequencing, clone hybridization, cDNA fragment fingerprinting, serial analysis of gene expression (SAGE), subtractive hybridization, differential display and/or differential screening.
  • SAGE serial analysis of gene expression
  • the method may be an in vivo method.
  • in vivo imaging may be utilized to detect the presence of E. histolytica proteins in the blood.
  • Such methods may utilize, for example, labeled antibodies or ligands specific for such proteins.
  • a detectably-labeled moiety e.g., an antibody, ligand, etc., which is specific for the polypeptide is administered to an individual (e.g., by injection), and labeled cells are located using standard imaging techniques, including, but not limited to, magnetic resonance imaging, computed tomography scanning, and the like.
  • Detection may utilize one, or a cocktail of, imaging reagents.
  • the method according to any aspect of the present invention may comprise the detection of at least 2 or 6 of the E.
  • the method may comprise the detection of at least 3, 4 or 5 of the E. histolytica proteins listed above.
  • Numerous methods for obtaining expression data are known, and any one or more of these techniques, singly or in combination, are suitable for determining expression patterns and profiles in the context of the present disclosure.
  • an in vitro method of diagnosing at least one subject as having or as being at risk of having Entamoeba histolytica (E. histolytica) infection comprising detecting the presence of at least one E. histolytica protein, fragment, variant or mutant thereof selected from the group consisting of pyruvate phosphate dikinase, phosphoglucomutase, pyruvate:ferredoxin oxidoreductase, phosphoglucomutase/ phosphomannomutase family protein, BspA family leucine rich repeat protein, and Cullin family protein, in a sample from the subject, wherein the presence of the protein, fragment, variant or mutant thereof is indicative of the subject having or as being at risk of having E.
  • E. histolytica protein, fragment, variant or mutant thereof selected from the group consisting of pyruvate phosphate dikinase, phosphoglucomutase, pyruvate:ferredoxin oxidoreductase,
  • the sample may be at least one biological fluid which includes but are not limited to cervical-vaginal fluid (CVF), cord blood, neonatal serum, cerebrospinal fluid (CSF), amniotic fluid, serum, plasma, urine, cerebrospinal fluid, breast milk, mucus, saliva, sweat and the like.
  • CVF cervical-vaginal fluid
  • CSF cerebrospinal fluid
  • amniotic fluid serum, plasma, urine, cerebrospinal fluid, breast milk, mucus, saliva, sweat and the like.
  • the sample may be blood, plasma or serum.
  • a method of diagnosing at least one subject as having or as being at risk of having E. histolytica infection comprising detecting the presence of at least one antibody or fragment thereof that binds specifically to an £. histolytica protein, fragment, variant or mutant thereof in a serum sample from the subject, wherein the presence of the antibody or fragment thereof is indicative of the subject having or as being at risk of having E. histolytica infection.
  • Antibody detection may include any method known in the art. Example of methods include but are not limited to ELISAs, dot blot, Western blots, flow-through rapid test, lateral flow rapid tests, biosensor and the like.
  • the E. histolytica proteins may be used either individually or in any combination for detection of specific antibodies (e.g. IgG, IgA) in extraintestinal E. histolytica infections.
  • the proteins may be in native or recombinant forms. Peptides that encompass the antigenic epitopes of these derived from these proteins can also be used.
  • At least one serum/blood or plasma biomarker for diagnosis of an E. histolytica infection in a subject.
  • the biomarker may comprise at least one isolated protein selected from the group consisting of pyruvate phosphate dikinase, phosphoglucomutase, pyruvate:ferredoxin oxidoreductase, phosphoglucomutase/ phosphomannomutase family protein, BspA family leucine rich repeat protein, Cullin family protein, fragment, variant and mutant thereof.
  • the biomarker may comprise a protein, polynucleotide, allele, or transcript of any one of the serum biomarkers mentioned above.
  • the biomarker may be detected in the serum, plasma or blood sample.
  • the presence of any one of the biomarkers may denote the presence of E. histolytica and the quantity of any one of the biomarkers may signify the intensity of the E. histolytica infection.
  • a kit comprising:
  • the kit may be useful for practicing the method of determining whether a subject has E. histolytica infection or whether the subject is susceptible to E. histolytica infection.
  • the E. histolytica protein to be detected may be selected from the group consisting of pyruvate phosphate dikinase, phosphoglucomutase, pyruvate :ferredoxin oxidoreductase, phosphoglucomutase/ phosphomannomutase family protein, BspA family leucine rich repeat protein, and Cullin family protein.
  • the kit may be an assemblage of materials or components, including at least one reagent capable of detecting an E. histolytica protein in a serum, plasma and/or blood sample.
  • the kit may contain a composition including agents according to any aspect of the present invention.
  • kits configured for the purpose of treating E. histolytica infection.
  • the kit is configured particularly for the purpose of treating mammalian subjects.
  • the kit is configured particularly for the purpose of treating human subjects.
  • the kit is configured for veterinary applications, treating subjects such as, but not limited to, farm animals, domestic animals, and laboratory animals.
  • kits Instructions for use may be included in the kit. "Instructions for use” typically include a tangible expression describing the technique to be employed in using the components of the kit to effect a desired outcome, such as to diagnose E. histolytica infection and/or to treat E. histolytica infection.
  • the kit may also contain other useful components, such as, diluents, buffers, pharmaceutically acceptable carriers, syringes, catheters, applicators, pipetting or measuring tools, ban daging materials or other useful paraphernalia as will be readily recognized by those of skill in the art.
  • an agent which specifically binds to at least one E. histolytica protein in serum of at least one subject for treatment of E. histolytica infection.
  • the E. histolytica protein may be is selected from the group consisting of pyruvate phosphate dikinase, phosphoglucomutase, pyruvate:ferredoxin oxidoreductase, phosphoglucomutase/ phosphomannomutase family protein, BspA family leucine rich repeat protein, and Cullin family protein.
  • Agents that modulate the expression of any of the E. histolytica proteins mentioned above may be used in in vivo, ex vivo, and in vitro applications where it may be advantageous to reduce or eliminate the expression or activity of any one of the E. histolytica proteins or a functionally downstream molecule. These agents may find use as drugs for treating E. histolytica infection. These agents may be administered parenterally, topically, orally, or locally for therapeutic treatment. In particular, the compositions may be administered orally or parenterally, i.e., intravenously, intraperitoneally, intradermally, or intramuscularly.
  • the agent may be in a form of a composition that may further comprise a pharmaceutically-acceptable carrier or excipient.
  • aqueous carriers may be used. For example, but not limited to water, buffered water, 0.4% saline, 0.3% glycine, and the like may be used as aqueous carriers.
  • an agent according to any aspect of the present invention that specifically binds to at least one E. histolytica protein in serum of a subject for the preparation of medicament for treatment of E. histolytica infection.
  • a method of treating £. histolytica infection in a subject comprising administering an agent that specifically binds to at least one E. histolytica protein in serum of the subject.
  • the E. histolytica protein may be selected from the group consisting of pyruvate phosphate dikinase, phosphoglucomutase, pyruvate:ferredoxin oxidoreductase, phosphoglucomutase/ phosphomannomutase family protein, BspA family leucine rich repeat protein, and Cullin family protein.
  • ESA excretory-secretory antigen
  • CSA crude soluble antigen
  • HM-1.MSS E. histolytica HM-1.MSS was maintained in TYI-S-33 medium and passaged in Syrian Golden hamster every two weeks in order to maintain its virulence.
  • Mass culture of E. histolytica trophozoites were harvested at log phase and pooled into one tube.
  • the trophozoites suspension was subjected to centrifugation, the supernatant was discarded and the pelleted trophozoites were washed three times (by centifugation) with RPMI supplemented with 0.1 % cysteine and 0.02 % ascorbic acid (RPMI-C&A).
  • RPMI-C&A RPMI supplemented with 0.1 % cysteine and 0.02 % ascorbic acid
  • the pelleted trophozoites were resuspended with RPMI-C&A and counted using Trypan blue exclusion method.
  • the cells were then seeded into culture tubes at cell density of 0.5 x 10 6 cells per mL of RPMI-C&A with 80 % filled medium.
  • the tubes were then incubated at 36 °C for 6 hours. Subsequently, the culture tubes were chilled on ice and subjected to centrifugation at 20 000 x g for 2 min at 25 °C. The supernatant was collected and subjected to centrifugation at 5,000 x g for 10 min at 4 °C. Then the supernatant was syringe-filtered through 0.22 pm membrane, and 0.5 M Iodoacetamide added to a final concentration of 1 mM. The supernatant containing ESA was concentrated 1000X using U-tube concentrator with molecular weight cut-off of 10 kDa, and centrifuged at 8000 x g, 16 °C.
  • Protein concentration was determined Bradford protein assay reagent. Complete lysis-M buffer containing 0X protease inhibitor (Roche, USA) was added until a final concentration of 1X protease inhibitor was achieved. The supernatant was again subjected to centrifugation at 10000 x g for 5 min at 4 °C to pellet down the precipitate. Protein concentration of the supernatant was again determined, then stored at - 80 °C.
  • ESA was separated via SDS-PAGE using Bio-Rad protein systems i.e. Mini Protean III Electrophoresis Cell and Protean ® II xi Cell according to Laemmli (1976) protocols with modifications.
  • Bio-Rad protein systems i.e. Mini Protean III Electrophoresis Cell and Protean ® II xi Cell according to Laemmli (1976) protocols with modifications.
  • crude ESA and CSA samples each were mixed with 2X Laemmli sample buffer and boiled for 5 min.
  • protein samples were electrophoreticaliy separated via 6% SDS-PAGE gel at constant current of 25 mA per gel for about 1 h.
  • NCP 0.45 ⁇ nitrocellulose membrane
  • NCP strips were incubated with monoclonal mouse anti-human IgG conjugated with horseradish peroxides (HRP) at dilution of 1 :6000 for another 1 hr.
  • HRP horseradish peroxides
  • Western blot substrates i.e. enhanced chemiluminescence (ECL) blotting reagent (Roche diagnostics, Germany), and/or Tetramethylbenzidine (TMB) substrate for membrane (Sigma, USA) were used to develop the signal and captured using film or image analyzer. Antigenic bands which showed good sensitivity and specificity were identified.
  • ECL enhanced chemiluminescence
  • TMB Tetramethylbenzidine
  • 2-DE was performed.
  • the OFF-GEL apparatus (Agilent Technologies) and 12-well IPG gel strip of pH 3-10 was used according to the manufacturer's instructions.
  • the IPG gel strip was rehydrated in the assembled device with 40 ⁇ of rehydration buffer per well for 15 min prior to sample loading.
  • An amount of 1.5 mg of the CSA and 2 mg ESA were mixed with OFFGEL sample buffer to a final volume of 2 ml, respectively. Subsequently, 150 ⁇ sample volume was loaded into each well.
  • sample was focused with a maximum current of 50 mA, with a maximum power of 200 mW and typical voltages ranging from 400 to 4000 V until 50 kVh was reached for approximately 15 hrs for CSA and 26 hours for ESA.
  • samples from OFFGEL fractions were mixed with sample buffer (4:1 ratio) containing 28.6% SDS and 4.76% 2-mercaptoethanol, then separated using a 12% Sodium Dodecyl Sulphate- Polyacrylamide Gel Electrophoresis (SDS-PAGE) in running buffer (25 mM Tris base, 192 mM Glycine, 0.1 % SDS, pH 8.3).
  • a sample volume of 23 pL per well was evenly loaded, and electrophoresis was ran at room temperature with a constant current of 100 V for 100 minutes. Western blot was then performed as above to confirm the location of the identified bands.
  • the antigenic proteins from the above 2-DE procedure were excised and sent to the Proteomic Laboratory Service Center, Australia for mass-spectrometry analysis (MALDI-TOF-TOF), and using Amoeba DB database. The identity of each protein was obtained, and the corresponding nucleotide sequences were then deduced.
  • Sensitivity Number of ALA-patients' sera reactive with the band
  • E. histolytica proteins were found in the patients' sera and were identified by MS- MS analysis as BspA family leucine rich repeat protein and Cullin family protein.
  • the serum proteomics approach using serum samples from infected humans and hamsters showed a protein band of ⁇ 42kDa to be antigenic and specific, and this protein was as identified as Bsp-A family, leucine rich repeat protein (LRR) of E. histolytica.
  • LRR leucine rich repeat protein
  • Cullin family protein was identified from an antigenic band of ⁇ 79kDa.
  • the ALA patients' sera and the corresponding pus from the liver aspirates were collected from Hospital Universiti Sains Malaysia (HUSM), Kubang Kerian, Kelantan, Malaysia.
  • the Ridascreen, ELISA (Rbiopharm, Germany) was performed to confirm the presence of anti-E.histolytica antibodies in the serum and the real-time polymerase chain reaction was utilized to detect E. histolytica DNA in the pus aspirates.
  • Nine normal serum samples which tested negative for anti-E.histolytica antibodies via ELISA were also collected and used as the negative controls.
  • pooled serum sample from 30 experimentally infected hamsters with ALA was also used as primary antibody in some Western blots. Serum depletion
  • the serum depletion was performed with pooled sera from 15 ALA patients, three individual serum samples from ALA patients, and pooled normal sera from 9 individuals.
  • Two types of high abundant serum proteins were removed (albumin and IgG) using the antibody-based ProteoSeek Albumin and IgG removal kit (Thermo Scientific, USA) according to manufacturer's protocols. Subsequently concentration procedure was carried out using 2 ml Viva spin, MWCO 3000 (Sortorius, USA) and centrifuged for 60 min, 3, 000 X g at 4°C until the total volume was ⁇ 400 ⁇ .
  • the OFF-GEL apparatus (Agilent Technologies) and 12-well IPG gel strip of pH 4-7 was used according to the manufacturer's instructions.
  • the IPG gel strip was rehydrated in the assembled device with 40 ⁇ of rehydration buffer per well for 5 min prior to sample loading.
  • samples from OFFGEL fractions were mixed with sample buffer (4:1 ratio) containing 28.6% SDS and 4.76% 2-mercaptoethanol, then electrophoresed using a 10% Sodium Dodecyl Sulphate-Polyacrylamide Gel Electrophoresis (SDS- PAGE) in running buffer containing 0.3% tris, 1.44% glycine and 0.1% SDS with a pH of 8.3.
  • SDS- PAGE 10% Sodium Dodecyl Sulphate-Polyacrylamide Gel Electrophoresis
  • Protein bands on the SDS-PAGE was electrophoretically transferred onto a nitrocellulose membrane (NCP), 0.45 ⁇ by using a using semi dry transblot (Bio Rad - USA) at a constant current of 12 A for 30 min.
  • the membranes was blocked for 1 hr at room temperature with 5% skimmed milk, and then washed three times with 0.05% Tween 20 in TBS for 30 min. Then the membranes were incubated with two categories of ALA sera; from hamster and human. For set I of the experiment, the blocked membrane was incubated with ALA hamster sera at 1:50 dilution overnight, 4°C.
  • blotted normal sera OFFGEL fractionates were incubated with ALA hamster sera at the same dilution.
  • the membrane was incubated with 0.25 pg/ml IgG purified from ALA patients' sera, overnight, 4°C.
  • Two kinds of control sera at : 00 dilution were used as the primary antibodies i.e. pooled sera from pyogenic liver abscess (PLA) patients and normal sera.
  • Immunodetection was performed with monoclonal mouse anti-hamster IgG and mouse anti-human IgG conjugated with horseradish peroxides (HRP) for set I and set II experiments respectively, each at a dilution of 1:2000 for 1hr.
  • Substrate development was performed using enhanced chemiluminescence blotting reagent (Roche diagnostics, Germany).
  • Selected proteins bands were manually excised from the Colloidal blue-stained gels, transferred into microfuge tubes, and destained with a destaining buffer which consisted of 20% methanol and 5% acetic acid for 15 min. After removing the buffer, the gel bands were sent to Academia Sinica, Taiwan for in-gel digestion preparation and mass spectrometry analysis (MS-MS) using AmoebaDB and human database. Bioinformatic analysis was used to elucidate the amino acid sequences of the proteins. The corresponding nucleotide sequences were then deduced.
  • MS-MS mass spectrometry analysis
  • the potential antigenic protein band with approximate molecular weight 42 kDa from OFFGEL fraction no. 4 was detected in the Western blot using two categories of ALA sera i.e. pooled infected hamster and human sera. BspA family, leucine rich repeat protein was identified from this band by MS-MS analysis. Meanwhile, from an antigenic band of ⁇ 79kD probed with individual infected human serum samples, Cullin family protein was identified in the two out of three samples (Tables 3 & 4).

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Abstract

L'invention concerne un procédé in vitro de diagnostic et/ou de traitement d'au moins un sujet ayant ou présentant un risque d'avoir une infection par Entamoeba histolytica (E. histolytica), le procédé consistant à détecter la présence d'au moins une protéine E. histolytica, d'un fragment, d'un variant ou d'un mutant de celle-ci dans un échantillon de sérum provenant du sujet, la présence de la protéine, d'un fragment, d'un variant ou d'un mutant de celle-ci étant indicative du fait que le sujet est atteint d'une infection par E. histolytica ou présente un risque d'en être atteint.
PCT/MY2011/000172 2011-07-07 2011-07-20 Diagnostic d'entamoeba histolytica WO2013006024A1 (fr)

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MX2014000007A MX358619B (es) 2011-07-07 2011-07-20 Diagnóstico de entamoeba histolytica.
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Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP0455620A2 (fr) * 1990-04-30 1991-11-06 Washington University Protéine immunogène et clone d'ADNc
US5459042A (en) * 1992-10-20 1995-10-17 Flores De Castaneda; Maria D. S. Procedure to preserve antigens of Entamoeba histolytica without enzymatic inhibitors

Patent Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP0455620A2 (fr) * 1990-04-30 1991-11-06 Washington University Protéine immunogène et clone d'ADNc
US5459042A (en) * 1992-10-20 1995-10-17 Flores De Castaneda; Maria D. S. Procedure to preserve antigens of Entamoeba histolytica without enzymatic inhibitors

Non-Patent Citations (8)

* Cited by examiner, † Cited by third party
Title
ABD-ALLA, M ET AL.: "Differentiation of Pathogenic Entamoeba histolytica Infections from Nonpathogenic Infections by Detection of Galactose-Inhibitable Adherence Protein Antigen in Sera and Feces", JOURNAL OF CLINICAL MICROBIOLOGY, vol. 31, no. 11, 1993, pages 2845 - 2850 *
INTRARAPUK, A ET AL.: "Comparison Between R-Phycocyanin-Labeled and R- Phycoerythrin-Labeled Monoclonal Antibody (Mab) Probes for the Detection of Entamoeba histolytica Trophozoites", SOUTHEAST ASIAN J TROP MED PUBLIC HEALTH, vol. 32, no. 2, 2001, pages 165 - 171 *
ORTNER, S ET AL.: "Molecular Analysis of Two Hexokinase Isoenzymes from Entamoeba histolytica", MOLECULAR AND BIOCHEMICAL PARASITOLOGY, vol. 73, 1995, pages 189 - 198 *
SANUKI, J-I ET AL.: "Purification and Identification of Major Soluble 40-kDa Antigenic Protein from Entamoeba histolytica: its Application for Serodiagnosis of Asymptomatic Amebiasis", PARASITOLOGY INTERNATIONAL, vol. 50, 2001, pages 73 - 80 *
STANLEY, S ET AL.: "Longitudinal Study of the Antibody Response to Recombinant Entamoeba histolytica Antigens in Patients with Amebic Liver Abscess.", AM. J. TROP. MED. HYG, vol. 58, no. 4, 1998, pages 414 - 416 *
STEPHEN, P ET AL.: "Molecular Modeling on Pyruvate Phosphate Dikinase of Entamoeba histolytica and In Silico Virtual Screening for Novel Inhibitors", J COMPUT AIDED MOL DES, vol. 22, 2008, pages 647 - 660 *
TANYUKSEL, M ET AL.: "Laboratory Diagnosis of Amebiasis", CLINICAL MICROBIOLOGY REVIEWS, vol. 16, no. 4, 2003, pages 713 - 729 *
THAMMAPALERD, N ET AL.: "Pyruvate: Ferredoxin Oxidoreductase from Entamoeba histolytica Recognized by a Monoclonal Antibody", SOUTHEAST ASIAN J TROP MED PUBLIC HEALTH, vol. 27, no. 1, 1996, pages 63 - 70 *

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ZA201400469B (en) 2015-08-26
MX2014000007A (es) 2015-02-12
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BR112014000135A2 (pt) 2018-06-19
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