WO2017187179A1 - Dosage d'immunodiagnostic - Google Patents

Dosage d'immunodiagnostic Download PDF

Info

Publication number
WO2017187179A1
WO2017187179A1 PCT/GB2017/051181 GB2017051181W WO2017187179A1 WO 2017187179 A1 WO2017187179 A1 WO 2017187179A1 GB 2017051181 W GB2017051181 W GB 2017051181W WO 2017187179 A1 WO2017187179 A1 WO 2017187179A1
Authority
WO
WIPO (PCT)
Prior art keywords
antigen
assay
tvy486
animal
vivax
Prior art date
Application number
PCT/GB2017/051181
Other languages
English (en)
Inventor
Michael Ferguson
Jennifer FLEMING
Lalitha SASTRY
Lauren SULLIVAN
Original Assignee
University Of Dundee
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by University Of Dundee filed Critical University Of Dundee
Publication of WO2017187179A1 publication Critical patent/WO2017187179A1/fr

Links

Classifications

    • 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
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2469/00—Immunoassays for the detection of microorganisms
    • G01N2469/20—Detection of antibodies in sample from host which are directed against antigens from microorganisms

Definitions

  • the present invention relates to assays for detecting whether or not an animal is or has been infected with Trypanosoma vivax.
  • the invention relates to specific antigens for use in the assays of the invention, as well a specific types of assays and assay devices which employ said specific antigens.
  • Trypanosoma vivax is a protozoan parasite of the genus trypansomatidae spread primarily by biting insects. Together with T brucei and T congolense, it is a causative agent of African Animal Trypanosomosis (AAT) in cattle. T vivax causes a severe version of AAT, often characterised by hemorraghic fever as well as the more typical weight loss, fatigue and anaemia .
  • T vivax does not require midgut gestation within the vector it can be transmitted mechanically by body fluid contamination and hematophagous flies. This has allowed the spread of the disease in South America, an area previously free from T vivax. Over eleven million cattle are estimated to be at risk in this region in addition to the 46 million cattle at risk in sub-Saharan Africa.
  • the present invention is based on the development of a low-cost pen-side diagnostic test for T vivax infection in cattle using lateral flow text (LFT) technology.
  • LFT lateral flow text
  • the inventors of the present invention have developed a kit for diagnosis of Trypanosoma vivax infection, which exhibits excellent specificity and sensitivity and is capable of discriminating infection with Trypanosoma vivax and other Trypanosome species, and/or identifying mixed Trypanosoma vivax and other Trypanosome species infection, with ease and economically. Further, the inventors developed a diagnostic kit including purified antigens from Trypanosoma vivax for use in detecting whether or not an animal has been infected with Trypanosoma vivax.
  • a method of detecting whether or not an animal has been infected with Trypanosoma vivax comprising: a) contacting a fluid sample from the animal with an isolated antigen comprising a sequence selected from SEQ ID No: 1 and 2 and antigenic fragments thereof; and
  • isolated is understood to mean that the antigen has been removed or otherwise purified, such that it is substantially free from other Trypanosoma vivax material and/or proteins, or material and/or proteins from a host harbouring nucleic acid which is capable of expressing the antigen.
  • the detection of any complexes being formed between the purified antigen and any antibodies present in the fluid sample is carried out using a detection agent which is coloured and can optionally be detected, when present in sufficient quantities, by the naked eye.
  • a purified antigen comprising a sequence selected from SEQ ID NO: 1 and 2 or antigenic fragment thereof for use in a method of detecting whether or not an animal is and/or has been infected with Trypanosoma vivax.
  • At least a portion of the purified antigen may comprise, consist essentially of, or consist of the sequence identified in SEQ ID No: 1 or 2, or antigenic fragment thereof.
  • the purified antigen may be identical to the identified sequences or antigenic fragment thereof, or may comprise a sequence or portion of sequence which is at least 95%, 98%, 99%, 99.5% identical to the identified sequences or antigenic fragment thereof.
  • the purified antigen may be larger in size and may comprise additional sequence at the 5' and/or 3' ends of the identified sequences.
  • the identified sequences include 3 amino acids at the 5' end which remain following cleavage of a TEV sequence.
  • the sequences of the present invention may exclude these three amino acids.
  • At least a portion of the purified antigen sequence comprises, consists essentially of, or consists of the sequence identified in SEQ ID No: 1 , or variants as described above, for use in a method of detection as described herein.
  • the antigens of the present invention were selected on the basis of (a) being identified by proteomics as belonging to a small group of antigens present in a total detergent lysate of T. vivax parasites that bind specifically and selectively to the immunoglobulin G (IgG) fraction of calves experimentally infected with T.vivax and (b) being similar to invariant surface glycoproteins previously known in the art.
  • Such proteins comprise a signal peptide, an extracellular domain, a transmembrane domain and internal domain.
  • the invention is directed in particular to the antigenic properties of the extracellular domain and SEQ ID No: 1 and 2 correspond to extracellular domain sequences of 2 proteins which were identified as having an infection:control intensity response ratio >10.
  • the antigens of the present invention have been shown not to cross-react with serum obtained from animals infected with T. congolense.
  • the antigens of the present invention may be considered as being specific for T. vivax and hence can be used for specifically identifying T. vivax as the causative pathogen for any animal identified as suffering from AAT.
  • the antigens of the present invention may be used alone in any method or device etc according to the present invention, or may be used in conjunction with another antigen or antigens, such as GM6 antigen which is/are intended to simply identify if an animal is infected with any trypanosome species.
  • the methods of the present invention are conventionally known as immunoassays.
  • the present invention is not limited to a particular type of immunoassay and the immunoassay may take a variety of forms.
  • certain embodiments of the present invention include an Enzyme-Linked immunosorbent Assay (EL!SA), dipstick immunoassay, sandwich assay, competitive assay, immunochromatographic assay radioimmunoassay, flow-through immunoassay, etc.
  • the present invention is in the form of a immunochromatographic assay and may be a lateral flow type assay known in the art.
  • Immunochromatographic assays also referred to as a rapid test due to its convenient and rapid features, is based on a principle in which an antibody in a fluid sample reacts with a tracer antigen bound to a coloured particle and then combines with a capture antigen or antibody located on the inner surface of pores of a nitrocellulose membrane to form a coloured band while transferring through the pores by a capillary phenomenon, thereby identifying positivity or negativity with the naked eye.
  • the ICA takes the form of a Lateral flow assay.
  • Lateral flow assays are typically based on the use of a series of capillary beds, such as pieces of porous paper or sintered polymer.
  • Each of these elements has the capacity to transport a fluid (e.g., blood or urine) spontaneously.
  • the first element (the sample pad) acts as a sponge and holds an excess of sample fluid. Once soaked, the fluid migrates to the second element (conjugate pad) in which there is stored a so-called conjugate, a dried format of bio-active particles (see below) in a salt-sugar matrix that contains everything to guarantee an optimized chemical reaction between the antigen and an antibody specifically reactive with the antigen that has been immobilized on the particle's surface.
  • the conjugate pad may further comprise control particles, which are designed simply to confirm to a user that a particular assay has worked appropriately. While the sample fluid dissolves the salt- sugar matrix, it also dissolves the particles and in one combined transport action the sample and conjugate mix while flowing through the porous structure. In this way, any antibodies present in the fluid sample bind to the particles while migrating further through the third capillary bed. This material has one or more areas, or stripes where a third molecule has been immobilized. By the time the sample-conjugate mix reaches these areas/stripes, antibodies have been bound to the particles and the third 'capture' molecule binds the complex. After a while, when more and more fluid has passed the stripes, particles accumulate and the stripe-area changes color.
  • control particles are designed simply to confirm to a user that a particular assay has worked appropriately. While the sample fluid dissolves the salt- sugar matrix, it also dissolves the particles and in one combined transport action the sample and conjugate mix while flowing through the porous structure. In this way, any
  • the control that captures any particle and thereby shows that reaction conditions and technology worked appropriately
  • the second contains a specific capture molecule and only captures those particles onto which a desired antibody molecule has been complexed.
  • the fluid After passing these reaction zones the fluid enters the final porous material, the wick, which simply acts as a waste container.
  • Lateral Flow assay can operate as either competitive or sandwich assays.
  • the present invention further relates to a lateral flow assay device for detecting the antibodies to Trypanosoma vivax present in a fluid test sample
  • said lateral flow assay device comprising a porous membrane in liquid communication with a conjugate pad and a wicking pad: said conjugate pad located upstream from a detection zone, said conjugate pad having antigen-conjugates comprising an isolated antigen comprising a sequence selected from SEQ ID No: 1 and 2 or antigenic fragment thereof immobilised on a nanopartic!e and; said detection zone having an immobilized first capture reagent, said first capture reagent being configured to bind to at least a portion of any antibody-antigen conjugates formed, so as to generate a detection signal; and a control zone located downstream from said detection zone, wherein a second capture reagent is immobilized within said control zone, said second capture reagent being configured to bind control antigen-conjugate or antibody-conjugate complexes to generate a second detection signal.
  • control takes the form of nanoparticies which have been modified to have an antibody conjugated to the surface of the nanoparficie. These nanoparticies may be captured by antibodies which are capable of binding the antibody which is conjugated to the surface of the nanopartide.
  • the nanoparticies may comprise a chicken IgY antibody conjugated to the surface of the nanoparticies and this may be captured by, for example, a goat, or other mammal anti-chicken IgY antibody.
  • an antigen- antibody complex is detected by a colour particle coupling method, in which examples of coloured particles include a colloidal gold or silver particle, colored glass, or plastic (e.g., polystyrene, polypropylene, latex, etc.) beads.
  • the particles may be gold-nanoparticles and the signal is a visible colour signal which is observed through accumulation of the gold nanoparticies at the sites of the respective capture agents
  • the methods of the present invention may be used to detect any antibodies which are capable of binding to the purified antigen(s) in any sample which is suspected of, or is capable of, harbouring such antibodies.
  • samples for use in the methods of the invention are ex vivo samples taken from the body of an animal to be tested. Such samples may be tested either on site or taken remotely and transferred to a testing facility for assay.
  • the sample is taken from a mammal, such as a pig or a wild or domesticated ruminant (e.g. cattle, buffalo, sheep, goat, camel and deer).
  • a mammal such as a pig or a wild or domesticated ruminant (e.g. cattle, buffalo, sheep, goat, camel and deer).
  • suitable samples include vesicular epithelium, vesicular fluid, blood samples, probang samples (collection of fluid from the throat), cardiac muscle (such as whole heart from young pigs or lambs), semen, saliva and milk.
  • the sample is a sample of blood or serum.
  • the antigens of the present invention may be purified by any known method.
  • the antigens may be extracted from Trypanosoma vivax obtained from an infected animal, or grown in vitro.
  • the antigens may be obtained by recombinant means e.g. from a culture of cells which are transformed to express the antigen.
  • the sequences of the nucleic acid molecules encoding the antigen can be obtained through cloning techniques known in the art.
  • the nucleotide sequences can be amplified from Trypanosoma vivax nucleic acid using PCR and related techniques known to the skilled addressee, or synthesised de novo and cloned into a suitable expression vector. If appropriate the nucleic acid can be modified to facilitate its expression in a particular host organism, by altering one or more codons of the nucleotide sequence.
  • the sequence may also be modified to include a cleavable 5' or 3' tag sequence, such as a His tag sequence known in the art, which is designed to facilitate purification of the antigen.
  • the sequence may be modified to include a tobacco etch virus (TEV) protease cleavage site between an N-terminal tag sequence, such as a hexahistidine affinity tag sequence.
  • TSV tobacco etch virus
  • the expression vector which harbours the nucleotide sequences capable of expressing the antigen of the present invention is then used to transform an appropriate host cell for the expression and production of the polypeptide of the invention.
  • Such methods of expressing proteins in recombinant cells lines are widely known in the art (for example, see Sambrook & Russell, 2000, Molecular Cloning, A Laboratory Manual, Third Edition, Cold Spring Harbor, New York).
  • variants of such antigens may be used.
  • a variant we include a polypeptide comprising the amino acid sequence of the naturally occurring antigen, wherein there have been amino acid insertions, deletions or substitutions, either conservative or non-conservative, such that the changes do not substantially reduce the ability of the variant to bind to said antibodies, compared to the binding ability of the naturally occurring antigen.
  • the variant may have increased affinity for said antibodies compared to that of the naturally occurring 'parent' antigen.
  • the variant may have increased stability, e.g. tolerance to proteases, pH and/or thermal stability.
  • Such variant antigens may be made using methods of protein engineering and site-directed mutagenesis commonly known in the art (for example, see Sambrook & Russell, 2000, supra).
  • the present invention also provides vectors (such as expression vectors), host cells (such as a bacterial (e.g. E.coli) host cells) for expressing the antigens of the invention and methods for making such antigens comprising culturing a host cell or vector according to the invention.
  • vectors such as expression vectors
  • host cells such as a bacterial (e.g. E.coli) host cells
  • methods for making such antigens comprising culturing a host cell or vector according to the invention.
  • Figure 1 shows a bar graph of ELISA data of recombinant proteins TvY486_0045500 and TvY486_0019690 against pooled T. vivax sera from animals pre-infection (day- 7) and post-infection (day+28).
  • Figure 2. shows examples of the TvY486_0045500 prototype lateral flow tests developed with control and infection sera, as indicated. Top band control line, bottom band TvY486_0045500 diagnostic line.
  • the Mozambique samples (20 sera) were from 2 calves and consisted of 20 7 vivax post-infection sera.
  • the ClinVet samples (72 sera) were from 31 calves and consisted of 27 pre-infection sera and 32 7 vivax post-infection sera and 13 7 congolense post-infection sera.
  • Strains used to infect cattle were: In Mozambique, Y486 and IL700. In Burkina Faso, Sokoroni 19, Napie22, Komborodougou and Gondo Bengaly. At ClinVet , ILRAD560.
  • Sera were collected in Burkina Faso from four calves before and 28 days after experimental infection with 7 vivax. Aliquots (250 ⁇ ) of the pre- and post-infection sera were pooled and IgG fractions were purified on protein-G Sepharose, as previously described [5, 7]. Purified IgG was coupled to CNBr-activated Sepharose 4B (GE Healthcare) at 4mg IgG per milliliter of packed gel, according to the manufacturer's instructions.
  • mice Three BALB/c mice were injected with one stabilate of 7. vivax ILRAD V34. After five days, infected mouse blood was harvested with citrate anticoagulant, adjusted to 5x10 4 parasites per ml with phosphate-buffered saline (PBS) and aliquots of 0.2 ml were injected into the peritoneal cavity of 45 NMR1 mice. The mouse blood was harvested after 7 days and the parasites were purified by centrifugation, to yield a buffy coat enriched in trypanosomes, followed by DE52 ion exchange chromatography to remove white blood cells and residual erythrocytes, as described in [5, 7].
  • PBS phosphate-buffered saline
  • the purified trypanosomes were dissolved to 1 x 10 9 cells.
  • the lysate was incubated for 30 min on ice and then centrifuged at 100,000 g for i h at 4°C.
  • the trypanosome proteins were eluted with 500 ⁇ of 50 mM sodium citrate, pH 2.8, 1 % nOG into tubes containing 100 ⁇ of 1 M Tris pH 8.5 for neutralization.
  • the eluates were further concentrated to 270 ⁇ using a centrifugal concentrator (Millipore, 0.5 ml capacity with 10 kDa MW cut off membrane).
  • the concentrates containing the trypanosome proteins were then transferred to low binding Eppendorf tubes and the proteins precipitated by adding 1 ml ice-cold ethanol and incubation for 24 h at -20°C.
  • the proteins eluted from the post-infection IgG and pre-infection IgG columns were dissolved in SDS sample buffer, reduced with DTT and run on a precast 4-12% Bis-Tris gradient SDS-PAGE (Invitrogen) using the MES running system.
  • the gel was stained with colloidal Coomassie blue and equivalent regions of the infection and control lanes were cut out, reduced and alkylated with iodoacetamide and digested in-gel with trypsin.
  • the tryptic peptides were analysed by LC-MS/MS on a Thermo Orbitrap Velos system and MaxQuant 1.4 software was used to match peptides to the predicted trypanosome protein databases (GeneDB).
  • TvY486_l 106220 9.71 60.63 ribosome binding ubiquitin-conjugating
  • TvY486_0603490 8.25 150.23 kinase/hydrolase
  • TvY486_0019690 A DNA construct encoding residues 42-363 of TvY486_0019690 was amplified from T. vivax (strain ILRAD V34) genomic DNA using the forward and reverse primers 5' -CATATGGAGAATGAGATTGCTCGGG-3' and 5'-
  • TvY486_0045500 which is very similar in sequence to TvY486_0019690, could not be selectively amplified and a construct encoding residues 40-363 was instead synthesised by GenScript and optimised to avoid rare codon combinations in E. coli, unfavourable mRNA structures for protein expression and cis elements.
  • the gene was obtained in a pUC vector with restriction sites (Nde1 and BamH1) in place for downstream cloning.
  • TvY486_0045500 construct was achieved with E. coli BL21- CodonPlus (DE3) RIPL cells (Stratagene) in autoinduction medium [10] containing 50 ⁇ g ml_ "1 ampicillin and 12 ⁇ g ml_ "1 chloramphenicol.
  • TvY486_0019690 was expressed in BL21 (DE3) Gold cells (Stratagene) in autoinduction medium containing containing 50 ⁇ g ml_ "1 ampicillian. Cells were cultured for 24 h at 22°C before harvesting by centrifugation (3,500 x g, 30 min, 4 °C) the bacterial pellet was resuspended in buffer A (50 mM Tris-HCI, pH 7.5, 250 mM NaCI) containing an EDTA-free protease inhibitor cocktail (Roche).
  • buffer A 50 mM Tris-HCI, pH 7.5, 250 mM NaCI
  • protease, uncleaved protein and affinity tag contaminants were removed with a further subtractive IMAC step.
  • Final purification was achieved by size exclusion chromatography (Superdex 200 26/60) eluted with buffer A.
  • proteins were dialysed into PBS and adjusted to at least 1 mg.ml "1 using 10 kDa cut-off centrifugal concentrators. All proteins were >95% pure, as judged by sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS PAGE) and Coomassie blue staining.
  • Enzyme linked immunosorbent assay ELISA
  • White polystyrene Costar untreated 96 well plates were coated with 50 ⁇ per well of target protein at a concentration of 2 ⁇ g ml_ "1 in plating buffer (0.05 M NaHCOs , pH 9.6). Then blocked with 200 ⁇ of PBS containing 5% bovine serum albumin (BSA) and 0.1 % Tween-20 to block non-specific sites overnight at 4°C. Calf sera were diluted 1 :2500 in PBS containing 5% BSA, 0.1 % Tween- 20 and transferred in triplicate by a liquid handling device (Bio-Tek, Precision) to the ELISA plates and incubated for 1 h at room temperature.
  • plating buffer 0.05 M NaHCOs , pH 9.6
  • BSA bovine serum albumin
  • Tween-20 0.1 % Tween-20
  • chemiluminescent super signal Femto substrate (Pierce) diluted 1 :5 i.e. , 0.5 ml solution A, 0.5 ml solution B with 4 ml PBS was applied to the wells at 50 ⁇ per well and plates were read using an Envision plate reader within 5 minutes of addition of the substrate.
  • TvY486_0045500 residues 40-363 were provided to BBI-Solutions (Dundee, UK) an immunoassay development and manufacturing company that has completed more than 250 lateral flow projects over the last 25 years, with manufacturing sites in Europe, USA and South Africa, and 2400 prototype LFT devices were manufactured.
  • Triplicate aliquots of 5 ⁇ of calf sera diluted with 15 ⁇ of PBS were added to the LFTs followed by an 80 ⁇ of chase-buffer (PBS containing 0.05% Tween 20). Tests were discarded if upper control line was not clearly visible. After 30 min, scoring of the test bands was performed by visual comparison of freshly completed tests with a scoring card [12] and the consensus score from three devices for each serum was recorded. After reading, the nitrocellulose test strips were taken out their cases for photography.
  • T. vivax Selection of candidate diagnostic antigens for T. vivax An immunoprecipitation experiment was carried out to identify candidate diagnostic antigens for T. vivax. Pooled pre-infection (day -7) and post-infection (day +28) calf sera from four animals from Burkina Faso were used to generate IgG antibody columns. Detergent lysate of bloodstream form T. vivax cells was generated from parasites recovered from mice infected with T. vivax strain ILRAD V34.
  • Identical amounts of parasite detergent lysate were mixed with the pre-infection and post-infection Sepharose-lgG beads and proteins bound to the washed beads were eluted with a buffer containing high salt and low pH to break antibody-antigen interactions.
  • the eluted samples from the pre-infection and post-infection columns were concentrated and subjected to SDS-PAGE gels for antigen separation.
  • the pre-infection and post-infection eluates were run on separate SDS-PAGE gels to reduce potential antigen cross-contamination.
  • the Coomassie blue stained gel lanes were cut into ten segments and each subjected to in-gel reduction and alkylation and trypsin digestion.
  • the peptides from each gel slice were separately analysed by LC-MS/MS and the data concatenated for the pre-infection and post-infection eluate samples, respectively. These concatenated data sets were used to search the predicted protein database for T. vivax (Y486) using MaxQuant 1.4.
  • TvY486_0045500 and TvY486_0019690 Two of these stood out as possible immunodiagnostic antigens, TvY486_0045500 and TvY486_0019690. These are two closely related proteins sharing 91 % and 80% amino acid sequence identity and similarity, respectively. These proteins have a typical ISG domain structure, consisting of an N-terminal signal peptide, an ISG domain, a transmembrane domain and a small intracellular domain [14]. We chose to investigate these antigens because ISGs have previously proved to be good diagnostics antigens for T. brucei, T. congolonse and T. evansi [4-6].
  • TvY486_0045500 amino acid residues 40-363
  • TvY486_0019690 amino acid residues 42-363
  • TvY486_0045500 and TvY486_0019690 coated ELISA plates were tested in triplicate with 1 13 randomised cattle sera provided by GALVmed. After data collection, the sera codes were broken and the data are collated. Sera were classified as being either from uninfected animals or from animals that had been exposed to experimental T. vivax infection. Based on this classification, there were 69 infection (positive) and 44 control (negative) sera.
  • T. vivax negative sera were from calves experimentally infected with T. congolense. None of these 13 sera gave a positive reaction with the T. vivax ISGs, either by ELISA or LFT, suggesting that the T. vivax ISGs do not routinely cross-react with T. congolense infection sera. This is in contrast to, for example, the GM6 antigen (also recently developed into an LFT) that cross-reacts with the sera from cattle infected with multiple trypanosome species [15, 16]. Both of these properties are useful, the latter with respect to making a pan-specific cattle AAT diagnostic and the former as a component of a pathogen-identifying diagnostic.
  • Trypanosoma vivax GM6 antigen a candidate antigen for diagnosis of African animal trypanosomosis in cattle.
  • PloS one 8 e78565.

Landscapes

  • Health & Medical Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Immunology (AREA)
  • Engineering & Computer Science (AREA)
  • Hematology (AREA)
  • Chemical & Material Sciences (AREA)
  • Urology & Nephrology (AREA)
  • Biomedical Technology (AREA)
  • Molecular Biology (AREA)
  • Microbiology (AREA)
  • Physics & Mathematics (AREA)
  • Biotechnology (AREA)
  • Virology (AREA)
  • Tropical Medicine & Parasitology (AREA)
  • Food Science & Technology (AREA)
  • Medicinal Chemistry (AREA)
  • Cell Biology (AREA)
  • Analytical Chemistry (AREA)
  • Biochemistry (AREA)
  • General Health & Medical Sciences (AREA)
  • General Physics & Mathematics (AREA)
  • Pathology (AREA)
  • Peptides Or Proteins (AREA)

Abstract

La présente invention concerne des dosages permettant de détecter si un animal est, ou a été, ou non infecté par Trypanosoma vivax . En outre, l'invention concerne des antigènes spécifiques pour leur utilisation dans les dosages de l'invention, ainsi que des types spécifiques de dosages et des dispositifs de dosage qui utilisent lesdits antigènes spécifiques.
PCT/GB2017/051181 2016-04-29 2017-04-27 Dosage d'immunodiagnostic WO2017187179A1 (fr)

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
GBGB1607560.8A GB201607560D0 (en) 2016-04-29 2016-04-29 Immunodiagnostic assay
GB1607560.8 2016-04-29

Publications (1)

Publication Number Publication Date
WO2017187179A1 true WO2017187179A1 (fr) 2017-11-02

Family

ID=56234176

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/GB2017/051181 WO2017187179A1 (fr) 2016-04-29 2017-04-27 Dosage d'immunodiagnostic

Country Status (2)

Country Link
GB (1) GB201607560D0 (fr)
WO (1) WO2017187179A1 (fr)

Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2020144465A1 (fr) * 2019-01-07 2020-07-16 Genome Research Limited Nouveau vaccin trypanosomique

Non-Patent Citations (3)

* Cited by examiner, † Cited by third party
Title
A. P. JACKSON ET AL: "Antigenic diversity is generated by distinct evolutionary mechanisms in African trypanosome species", PROCEEDINGS NATIONAL ACADEMY OF SCIENCES PNAS, vol. 109, no. 9, 13 February 2012 (2012-02-13), US, pages 3416 - 3421, XP055398575, ISSN: 0027-8424, DOI: 10.1073/pnas.1117313109 *
DATABASE UniProt [online] 19 October 2011 (2011-10-19), "SubName: Full=Putative uncharacterized protein {ECO:0000313|EMBL:CCD19278.1};", XP002772955, retrieved from EBI accession no. UNIPROT:F9WP05 Database accession no. F9WP05 *
DATABASE UniProt [online] 19 October 2011 (2011-10-19), "SubName: Full=Putative uncharacterized protein {ECO:0000313|EMBL:CCD21631.1};", XP002772954, retrieved from EBI accession no. UNIPROT:F9WVM3 Database accession no. F9WVM3 *

Cited By (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2020144465A1 (fr) * 2019-01-07 2020-07-16 Genome Research Limited Nouveau vaccin trypanosomique
GB2594637A (en) * 2019-01-07 2021-11-03 Genome Res Ltd Novel trypanosomal vaccine
GB2594637B (en) * 2019-01-07 2023-08-02 Genome Res Ltd Novel trypanosomal vaccine

Also Published As

Publication number Publication date
GB201607560D0 (en) 2016-06-15

Similar Documents

Publication Publication Date Title
JP4554218B2 (ja) トリコモナスを検出するための方法および装置
US10948487B2 (en) Peptides, devices, and methods for the detection of Ehrlichia antibodies
Boulangé et al. Development of a rapid antibody test for point-of-care diagnosis of animal African trypanosomosis
Fleming et al. Proteomic identification of immunodiagnostic antigens for Trypanosoma vivax infections in cattle and generation of a proof-of-concept lateral flow test diagnostic device
KR102559678B1 (ko) 아나플라즈마 항체의 검출을 위한 펩티드, 장치 및 방법
US7824875B2 (en) Recombinant antigens for the detection of Coxiella burnetii
KR101753580B1 (ko) 브루셀라 캐니스 항원의 제조 방법
CN113024641B (zh) 新型冠状病毒的重组s蛋白及其制备方法和应用
Reyna-Bello et al. Evaluation of an enzyme-linked immunosorbent assay using recombinant major surface protein 5 for serological diagnosis of bovine anaplasmosis in Venezuela
Ren et al. A brief review of diagnosis of small ruminants brucellosis
Jirapattharasate et al. Development and evaluation of recombinant GRA8 protein for the serodiagnosis of Toxoplasma gondii infection in goats
WO2017187179A1 (fr) Dosage d'immunodiagnostic
CA1256371A (fr) Separation rapide de complexes immuns pour dirofilaria immitis
US20090017556A1 (en) Methods and complexes involving immunoglobulin molecule
AU2012278386B2 (en) A diagnostic kit for the detection of early acute leptospirosis
Hassan et al. Comparison of two antigens for diagnosis of trichinellosis in pigs
Suhr-Jessen et al. Microscopical, biochemical, and immunological studies of the immune defense system of the horseshoe crab, Limulus polyphemus
US20110039281A1 (en) Method of immunological analysis for detection of antibodies against human gstt1 (anti-hgstt1)
CN113784975A (zh) 嵌合蛋白、其生产方法和用途，以及核酸分子、表达盒、表达载体、宿主细胞、用于诊断利什曼病的组合物、用于诊断利什曼病的试剂盒和体外诊断利什曼病的方法
CN114034860A (zh) 一种结核分枝杆菌mtb39a蛋白抗体间接elisa检测方法及其试剂盒
Araújo et al. Development and assessment of a latex agglutination test based on recombinant MSP5 to detect antibodies against...
Ho-Yen Other tests in bacteriology
JP2002017397A (ja) ノルウオークウイルスの免疫学的分析方法、感染症のスクリーニング方法及びスクリーニング用キット
BR102016005202A2 (pt) Proteína multiepítopo recombinante e seu processo de obtenção para uso no diagnóstico de esquistossomose
CA2610995A1 (fr) Dispositif comprenant un facteur proteique, facteur proteique recombine et sequence nucleotidique codant pour ce facteur proteique

Legal Events

Date Code Title Description
NENP Non-entry into the national phase

Ref country code: DE

121 Ep: the epo has been informed by wipo that ep was designated in this application

Ref document number: 17721440

Country of ref document: EP

Kind code of ref document: A1

122 Ep: pct application non-entry in european phase

Ref document number: 17721440

Country of ref document: EP

Kind code of ref document: A1