WO2012094427A1 - Procédés de diagnostic - Google Patents

Procédés de diagnostic Download PDF

Info

Publication number
WO2012094427A1
WO2012094427A1 PCT/US2012/020234 US2012020234W WO2012094427A1 WO 2012094427 A1 WO2012094427 A1 WO 2012094427A1 US 2012020234 W US2012020234 W US 2012020234W WO 2012094427 A1 WO2012094427 A1 WO 2012094427A1
Authority
WO
WIPO (PCT)
Prior art keywords
assay
slide
array
cells
fana
Prior art date
Application number
PCT/US2012/020234
Other languages
English (en)
Inventor
Mark Kopnitsky
Original Assignee
Zeus Scientific, Inc.
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 Zeus Scientific, Inc. filed Critical Zeus Scientific, Inc.
Priority to CN201280005699XA priority Critical patent/CN103460043A/zh
Priority to CA2860690A priority patent/CA2860690A1/fr
Priority to AU2012204413A priority patent/AU2012204413A1/en
Priority to EP12732021.6A priority patent/EP2661625A4/fr
Priority to JP2013548490A priority patent/JP2014501932A/ja
Publication of WO2012094427A1 publication Critical patent/WO2012094427A1/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
    • G01N33/543—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals
    • G01N33/54393—Improving reaction conditions or stability, e.g. by coating or irradiation of surface, by reduction of non-specific binding, by promotion of specific binding
    • 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
    • G01N33/5005—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells
    • G01N33/5091—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells for testing the pathological state of an organism
    • 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
    • G01N33/5306—Improving reaction conditions, e.g. reduction of non-specific binding, promotion of specific binding
    • 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
    • G01N33/564—Immunoassay; Biospecific binding assay; Materials therefor for pre-existing immune complex or autoimmune disease, i.e. systemic lupus erythematosus, rheumatoid arthritis, multiple sclerosis, rheumatoid factors or complement components C1-C9
    • 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
    • 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
    • G01N33/574—Immunoassay; Biospecific binding assay; Materials therefor for cancer

Definitions

  • the present invention relates to the field of diagnostic assay systems, and in particular is useful for diagnosing and assessing autoimmune disorders, and more specifically the invention relates to novel methods for performing auto-antibody diagnostic assays.
  • Antibodies are proteins produced by the body in response to invading or infectious materials. They constitute one of the many means the body has to protect itself from disease. In normal circumstances, the body can recognize "foreign” from “self tissues, and therefore only generates antibodies to those materials that are “foreign” to the body.
  • autoimmune disorders develops when the body begins to produce antibodies to its own tissues.
  • the symptoms associated with the onset of an autoimmune disorder can be varied. Also, any of the early symptoms can mimic those of many other diseases.
  • ANA anti -nuclear antibody
  • the ANA test is a good "first round" screening test to see if somebody has antibody to self tissues.
  • FANA fluorescent anti -nuclear antibody assay
  • HEp-2 FANA test is a continuous, human epithelial cell line that grows rapidly and readily attaches to solid surfaces such as tissue culture flasks and glass slides.
  • the HEp-2 cell also has a large, well defined nucleus.
  • the HEp-2 FANA test is made by growing HEp-2 cells on glass slides and fixing the cells permanently on the surface of the slide. The assay is performed by allowing a serum sample from a patient to react to the cells. If anti-nuclear antibodies are present, they will bind to the cells.
  • the slides are washed and the antibody is labeled with anti -human Immunoglobulin (Ig) labeled with a fluorescent tag.
  • Ig Immunoglobulin
  • Auto-antigens used in the ANA test, represent the majority, but not all, of known auto- antigens expressed on the HEp-2 cell. Some auto-antigens found on the HEp-2 cell are not present in the multiplex suspension. Consequently, the multiplex bead method, such as the AtheNA Multi-Lyte ANA Test System (commercially available from Zeus Scientific, Inc., Bridgewater, NJ), contains a bead mix that is built on the Luminex xMAP® platform
  • the aforementioned AtheNA Multi-Lyte ANA Test System provides substantially equivalent results, and there are those that feel that the ANA multiplex arrays are adequate and there are those that feel that only the HEp2 cell is sufficient in screening for ANA. However, it is generally agreed that following an acceptable screening method, providing the specific data from a multiplex array would be of extreme value.
  • the present invention has been found to achieve substantially similar goals and results without the necessity of using multiplex beads or instrumentation as described therein, by employing, such as by printing, an array of antigen spots in proximity to cells that are fixed on a glass slide or other solid phase, and therefore the present invention is unexpectedly advantageous in that it does not require the involvement of multiplex bead arrays or instrumentation, such as the Luninex instrument referred to in said application.
  • Figure 1 shows examples of positive ANA reactions; specific patterns of reactivity are shown using HEp-2 FANA (performed using a conventional slide method).
  • Figure 2 shows a dual-spotted slide array for cells utilizing the methods of the present invention.
  • Figure 3 shows a slide and flexible grid used to form wells for performing the methods of the present invention.
  • Figure 4 shows a formation of wells on a slide useful for performing the methods of the invention, so that each well contains two areas; one with cells and one with a defined antigen array.
  • the ANA screening test is widely recognized as a good "first round" screening test to see if a patient has antibody to self tissues, the currently most popular being the fluorescent anti-nuclear antibody assay (FANA), and the currently most popular of the FANA tests being the HEp-2 FANA.
  • the assay is performed by allowing the patient serum to react to the cells. If anti-nuclear antibodies are present, they will bind to the cells. The patient antibody is then tagged with anti -human Ig labeled with a fluorescent moiety. When viewed using a properly equipped microscope, specific patterns of reactivity are observed (if present) and some have been highly associated with specific autoimmune disorders.
  • the present invention provides novel methods for the substantially simultaneous analysis of autoantigens in a single sample from a subject by combining the known FANA slide tests and multiplex arrays into a single assay, with minimal modifications to each.
  • the HEp-2 FANA test comprises a slide test using HEp-2 cells that have been allowed to grow in TC media on the surface of glass slides. The slides are then rinsed and the cells fixed with conventional organic solvents, thus
  • This assay can be performed, for example, in the small wells of a microscope slide.
  • a multiplex array platform is any platform that enables one to test for multiple analytes simultaneously. Common array systems involve spotting target molecules in precise locations on a solid support such as glass or plastic. A popular example of a widely used multiplex array platform is the system developed and commercially available based on the Luminex® xMAP® Technology (Luminex Corporation, Austin, TX). Other arrays are known to be fabricated with spots, wells, posts, beads, cantilevers, wires, electrodes or fiber optics (see Clinical Chemistry; 56:12 (2010)).
  • Reporter Molecule as used herein means the reporter molecule is the fluorescent, visual or chemiluminescent tag that is bound to the detection molecule(s) in the assay.
  • the detection molecule may be goat anti-human IgG (for example) that is labeled with phycoerythrin (for example).
  • Cellular substrate as used herein means any cellular material or tissue substrate that is generally adhered to a solid support (glass or plastic microscope slide) and then used as part of a biochemical assay for analysis of biomolecules such as antibody or antigens.
  • the combination assay as provided by the present invention therefore can be performed without the use of, instrumention such as for example, a conventional Luminex xMAP instrument, which is essentially a flow cytometer that has been modified for use of the Luminex polystyrene microspheres in a multiplex bead assay.
  • instrumention such as for example, a conventional Luminex xMAP instrument, which is essentially a flow cytometer that has been modified for use of the Luminex polystyrene microspheres in a multiplex bead assay.
  • the present invention is therefore greatly advantageous over know methods in the art for performing similar assays, in the aspect of simplicity and lack of need for expensive instrumentation, among other advantages which will be apparent to those skilled in the art.
  • the array in the case of the present invention comprises a series of purified antigens that have been spotted onto a glass slide in proximity to the cellular substrate containing cells that are of interest to analyze.
  • the array can be as simple or as complex as desired, depending on the nature of the assay in question. For the ANA example, it is presently preferred that a minimum of 10 highly purified auto-antigens, and preferably a maximum of 20 to 25 highly purified auto-antigens, be used.
  • the practice of the methods of the present invention typically begins with a glass slide.
  • a glass slide with two "areas" per "well” (see Figure 2 of the drawings) is used.
  • the cells of interest are en applied, thereby creating a cellular substrate and fixed into the circular wells in the image shown in Figure 2.
  • the purified antigens are spotted in proximity to the substrate as an array in the square wells shown in Figure 2.
  • This slide is thereafter packaged and provided to the user of the assay system, for further analysis of patient specimens in accordance with the user's needs.
  • the slide may be modified as needed by the user so that wells are formed for each patient specimen.
  • the wells can be configured, for example, so that each well contains one circle (HEp2 cells) and one square (purified antigen array).
  • Figures 3 and 4 of the drawings depict the formation of these wells.
  • This slide can then be used to evaluate patient specimens (for example, serum, urine, plasma, etc.) for reactivity to the combination of the cells and the antigen array, substantially all simultaneously, using suitable reporter molecules well known to those skilled in the art, and thereby providing a novel way to collect the combination of reactivity to naturally occurring biomolecules within fixed cells or tissues, as well as an array of highly specific, highly purified biomolecules.
  • the HEp-2 FANA test comprises a slide test using HEp-2 cells that have been allowed to grow in TC media on the surface of the glass slide. They are rinsed and fixed with some organic solvents, which permeabalizes the membrane and keeps them adhered to the glass slide.
  • This assay has conventionally been performed in tiny wells of a microscope slide.
  • the array in this case according to the invention is a series of purified antigens that have been spotted onto a glass slide for reactivity to the combination of the cells and the antigen array all substantially simultaneously.
  • the present invention could be applied to a myriad of other examples including autoimmune, infectious disease, cancer diagnostics, cytological studies, to name only a few. Other examples will be apparent to those skilled in the art. It will also be appreciated that in addition to analysis of serum samples as described above, the methods and teachings of the present invention can be applied to analysis of any biological fluid that may be obtained from a subject; for example, blood, urine, cerebral spinal fluid and plasma, as well as other biological fluids as are well known to those skilled in the art, can all be suitable for obtaining samples upon which to perform the methods of analysis in accordance with the present invention.

Landscapes

  • Health & Medical Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Immunology (AREA)
  • Engineering & Computer Science (AREA)
  • Chemical & Material Sciences (AREA)
  • Hematology (AREA)
  • Biomedical Technology (AREA)
  • Urology & Nephrology (AREA)
  • Molecular Biology (AREA)
  • Medicinal Chemistry (AREA)
  • Pathology (AREA)
  • Cell Biology (AREA)
  • Biotechnology (AREA)
  • Food Science & Technology (AREA)
  • Microbiology (AREA)
  • Physics & Mathematics (AREA)
  • Analytical Chemistry (AREA)
  • Biochemistry (AREA)
  • General Health & Medical Sciences (AREA)
  • General Physics & Mathematics (AREA)
  • Chemical Kinetics & Catalysis (AREA)
  • Tropical Medicine & Parasitology (AREA)
  • Rehabilitation Therapy (AREA)
  • Rheumatology (AREA)
  • Physiology (AREA)
  • Virology (AREA)
  • Hospice & Palliative Care (AREA)
  • Oncology (AREA)
  • Investigating Or Analysing Biological Materials (AREA)

Abstract

La présente invention concerne un nouveau procédé combinant les propriétés associées à un test sur lame FANA largement répandu à celles d'un dosage multiplex sur puce largement répandu, pour obtenir un seul dosage réalisé sensiblement simultanément, des modifications minimales étant apportées à chaque test. Ceci procure aux médecins et aux autres utilisateurs du procédé des avantages jusqu'alors inconnus, tels que facilité d'utilisation et amélioration de la vitesse du test, qui sont utiles par exemple dans le diagnostic et l'évaluation de maladies auto-immunes.
PCT/US2012/020234 2011-01-05 2012-01-04 Procédés de diagnostic WO2012094427A1 (fr)

Priority Applications (5)

Application Number Priority Date Filing Date Title
CN201280005699XA CN103460043A (zh) 2011-01-05 2012-01-04 诊断方法
CA2860690A CA2860690A1 (fr) 2011-01-05 2012-01-04 Procedes de diagnostic
AU2012204413A AU2012204413A1 (en) 2011-01-05 2012-01-04 Diagnostic methods
EP12732021.6A EP2661625A4 (fr) 2011-01-05 2012-01-04 Procédés de diagnostic
JP2013548490A JP2014501932A (ja) 2011-01-05 2012-01-04 診断方法

Applications Claiming Priority (4)

Application Number Priority Date Filing Date Title
US201161429892P 2011-01-05 2011-01-05
US61/429,892 2011-01-05
US201161524630P 2011-08-17 2011-08-17
US61/524,630 2011-08-17

Publications (1)

Publication Number Publication Date
WO2012094427A1 true WO2012094427A1 (fr) 2012-07-12

Family

ID=46457702

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/US2012/020234 WO2012094427A1 (fr) 2011-01-05 2012-01-04 Procédés de diagnostic

Country Status (6)

Country Link
EP (1) EP2661625A4 (fr)
JP (1) JP2014501932A (fr)
CN (1) CN103460043A (fr)
AU (1) AU2012204413A1 (fr)
CA (1) CA2860690A1 (fr)
WO (1) WO2012094427A1 (fr)

Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP3591406A1 (fr) 2018-07-06 2020-01-08 Euroimmun Medizinische Labordiagnostika AG Dispositif et procédé de détection des anticorps
EP3591403A1 (fr) 2018-07-06 2020-01-08 Euroimmun Medizinische Labordiagnostika AG Procédé de détection automatisée d'anticorps dans un échantillon biologique liquide à l'aide d'une puce à antigène et puce à antigène correspondante

Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US6939720B2 (en) * 1995-10-11 2005-09-06 Luminex Corporation Multiplexed analysis of clinical specimens apparatus and method
US20080254482A1 (en) * 2006-11-22 2008-10-16 Invitrogen Corporation Autoimmune disease biomarkers
WO2010030624A1 (fr) * 2008-09-09 2010-03-18 Zeus Scientific, Inc. Procédés de diagnostic et d'évaluation de troubles auto-immuns
US20100285986A1 (en) * 2007-11-02 2010-11-11 Friedrich Menges Single-step multiplex immunoassay

Family Cites Families (10)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JPS60183559A (ja) * 1984-02-29 1985-09-19 Otsuka Pharmaceut Co Ltd 肝細胞膜抗原に対する自己抗体の測定方法
US5518881A (en) * 1993-11-02 1996-05-21 Flinders Medical Centre Transfected cell lines expressing autoantigens and their use in immunoassays for the detection of autoimmune disease
CN1101936C (zh) * 2000-08-14 2003-02-19 李永哲 一种抗核抗体检测试剂及其制备方法
US7189516B2 (en) * 2001-08-17 2007-03-13 Luminex Corporation Method for characterizing autoimmune disorders
US20030104439A1 (en) * 2001-11-30 2003-06-05 Finch Rosalynde J. Methods of identifying cellular target molecules
CN2653510Y (zh) * 2003-09-25 2004-11-03 长沙福滋堂生物技术开发有限公司 抗核抗体检测细胞薄片
CN101017168B (zh) * 2007-02-15 2012-08-29 广州万孚生物技术股份有限公司 荧光胶乳定量层析试纸条及其制备方法
CA2702421C (fr) * 2007-11-13 2013-03-26 Medipan Gmbh Procede de determination du titre final et d'evaluation de celui-ci au moyen d'un test d'immunofluorescence indirect
US8630690B2 (en) * 2009-05-05 2014-01-14 Electric Power Research Institute, Inc. Thermal contraction compensation for superconducting and cryo-resistive cables
EP2362222B1 (fr) * 2010-02-22 2013-06-26 Medipan GmbH Procédé et dispositif pour la détection simultanée d'anticorps liés à des substrats synthétiques et cellulaires et/ou de tissus

Patent Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US6939720B2 (en) * 1995-10-11 2005-09-06 Luminex Corporation Multiplexed analysis of clinical specimens apparatus and method
US20080254482A1 (en) * 2006-11-22 2008-10-16 Invitrogen Corporation Autoimmune disease biomarkers
US20100285986A1 (en) * 2007-11-02 2010-11-11 Friedrich Menges Single-step multiplex immunoassay
WO2010030624A1 (fr) * 2008-09-09 2010-03-18 Zeus Scientific, Inc. Procédés de diagnostic et d'évaluation de troubles auto-immuns

Non-Patent Citations (1)

* Cited by examiner, † Cited by third party
Title
See also references of EP2661625A4 *

Cited By (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP3591406A1 (fr) 2018-07-06 2020-01-08 Euroimmun Medizinische Labordiagnostika AG Dispositif et procédé de détection des anticorps
EP3591403A1 (fr) 2018-07-06 2020-01-08 Euroimmun Medizinische Labordiagnostika AG Procédé de détection automatisée d'anticorps dans un échantillon biologique liquide à l'aide d'une puce à antigène et puce à antigène correspondante
EP3591401A1 (fr) 2018-07-06 2020-01-08 Euroimmun Medizinische Labordiagnostika AG Procédé de détection automatisée d'anticorps dans un échantillon biologique liquide à l'aide d'une puce à antigène et puce à antigène correspondante
EP3591402A1 (fr) 2018-07-06 2020-01-08 Euroimmun Medizinische Labordiagnostika AG Dispositif et procédé de détection des anticorps
WO2020007597A1 (fr) 2018-07-06 2020-01-09 Euroimmun Medizinische Labordiagnostika Ag Procédé de détection automatisée d'anticorps dans un échantillon biologique liquide en utilisant une puce d'antigène et puce d'antigène correspondante

Also Published As

Publication number Publication date
EP2661625A1 (fr) 2013-11-13
AU2012204413A1 (en) 2013-08-15
EP2661625A4 (fr) 2014-10-29
JP2014501932A (ja) 2014-01-23
CN103460043A (zh) 2013-12-18
CA2860690A1 (fr) 2012-07-12

Similar Documents

Publication Publication Date Title
Hartmann et al. Protein microarrays for diagnostic assays
EP1322960B1 (fr) Test de jeu ordonne de microechantillons d'allergenes
US20080058224A1 (en) Substrate chemistry for protein immobilization on a rigid support
JP2013520664A (ja) 合成基材及び細胞基材に結合した抗体の同時検出による疾病診断のための方法及びシステム
WO2004094458A2 (fr) Jeux ordonnes de peptides-complexes a histocompatibilite majeure (mhc)
JP2010500537A (ja) 非特異的結合を軽減するための変異抗原を含む改良型免疫アッセイ
EP2661625A1 (fr) Procédés de diagnostic
EP3262422B1 (fr) Procédé et système de détection d'anticorps
Bacarese-Hamilton et al. Allergen microarrays
US20120040850A1 (en) Methods for Diagnosis and Assessment of Autoimmune Disorders
US20130217015A1 (en) Hmga2 as a biomarker for diagnosis and prognosis of ovarian cancer
CA2939789C (fr) Detection de globules rouges
CA2938935C (fr) Epreuve de compatibilite croisee pour echantillons de sang
KR20140067104A (ko) 교정제 및 교정 방법
Sohn et al. ANA testing
EP3283888B1 (fr) Procédé de diagnostic in vitro de l'allergie et dispositif associé
Saxena et al. Immunotechnology in Disease Diagnosis
WO2008114152A2 (fr) Nouveau capteur de diagnostic pour la détection rapide et reproductible du domaine de la protéine ro52
JP2007010625A (ja) アシアロｇｍ１発現細胞検出試薬、これを用いた細胞検出方法および細胞分類方法並びに老化測定方法
US20150011419A1 (en) Systems and methods for characterizing lupus erythematosus

Legal Events

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

Ref document number: 12732021

Country of ref document: EP

Kind code of ref document: A1

ENP Entry into the national phase

Ref document number: 2013548490

Country of ref document: JP

Kind code of ref document: A

NENP Non-entry into the national phase

Ref country code: DE

WWE Wipo information: entry into national phase

Ref document number: 2012732021

Country of ref document: EP

ENP Entry into the national phase

Ref document number: 2012204413

Country of ref document: AU

Date of ref document: 20120104

Kind code of ref document: A

ENP Entry into the national phase

Ref document number: 2860690

Country of ref document: CA