WO2013013214A1 - Procédé de détection de micro-organismes - Google Patents

Procédé de détection de micro-organismes Download PDF

Info

Publication number
WO2013013214A1
WO2013013214A1 PCT/US2012/047737 US2012047737W WO2013013214A1 WO 2013013214 A1 WO2013013214 A1 WO 2013013214A1 US 2012047737 W US2012047737 W US 2012047737W WO 2013013214 A1 WO2013013214 A1 WO 2013013214A1
Authority
WO
WIPO (PCT)
Prior art keywords
apparatus system
particle
lanthanide
analyte
europium
Prior art date
Legal status (The legal status 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 status listed.)
Ceased
Application number
PCT/US2012/047737
Other languages
English (en)
Inventor
Jaean Jung
Young Ho Choi
Seung Jae Baek
Hyeonsuk KIM
Tae-Hee Koo
Byeong-gap HWANG
Won-Jung Kim
Eun-jeong JANG
Jingeun RHEE
Heejun Kim
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
MEDISENSOR Inc
Terawave Inc South Korea
Access Bio Inc
Original Assignee
MEDISENSOR Inc
Terawave Inc South Korea
Access Bio 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 MEDISENSOR Inc, Terawave Inc South Korea, Access Bio Inc filed Critical MEDISENSOR Inc
Publication of WO2013013214A1 publication Critical patent/WO2013013214A1/fr
Anticipated expiration legal-status Critical
Ceased legal-status Critical Current

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
    • G01N33/58—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving labelled substances
    • G01N33/582—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving labelled substances with fluorescent label
    • 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
    • G01N33/54366—Apparatus specially adapted for solid-phase testing
    • G01N33/54386—Analytical elements
    • G01N33/54387—Immunochromatographic test strips
    • G01N33/54388—Immunochromatographic test strips based on lateral flow

Definitions

  • the present application relates to a method for developing a Rapid Diagnostic Test (RDT) kit through active conjugation of Europium nanoparticles and a highly sensitive method for detecting Chlamydia antigen (Chlamydia Ag) through Time Resolved Fluorescence (TRF) image analysis.
  • RDT Rapid Diagnostic Test
  • TRF Time Resolved Fluorescence
  • Chlamydia may be divided into two species: Chlamydia trachomatis and Chlamydia proctitis.
  • Chlamydia trachomatis includes trachoma, inclusion conjunctivitis, lymphogranuloma venereum conjunctivitis, nonspecific or non-gonorrheal urethritis, and proctitis and is an etiologic agent for diseases in human eyes or genital organs.
  • Chlamydia trachomatis infection spreads throughout the group. It is estimated that non-gonorrheal urethritis occurs over a million cases every year. Since Chlamydia spreads widely, a reliable and highly sensitive test is believed to play an important role in testing existence of organisms.
  • An immunoassay has been used as a test that employs an antibody-antigen immune reaction.
  • Such an immune test mainly uses ELISA or Western blot as a method of combining an optical label with the catalysis of enzyme through the measurement of a signal obtained by labeling a radiation luminescent or fluorescent material on an antibody or an antigen.
  • ELISA or Western blot as a method of combining an optical label with the catalysis of enzyme through the measurement of a signal obtained by labeling a radiation luminescent or fluorescent material on an antibody or an antigen.
  • An antibody or the like which is a target material of an immune sensor, exists in small amount in a biological sample. For this reason, there is a need for highly sensitive signaling technologies that are much superior in detection limit of a sensor to biosensor technologies.
  • an antibody and protein are susceptible to be structurally denaturalized by change in an external environment, an antibody and an antigen are susceptible to be transmuted. Thus, a biometric function is susceptible to be lost.
  • Ultrasensitive immunoassay methods are developed and used in clinical diagnostics to measure extremely low concentrations of specific compounds in highly complex samples. Although the sensitivity, reliability, rapidity, simplicity, and cost of these methods have steadily improved, further improvements are still needed and possible.
  • the interest in new label technologies has especially increased because none of the commonly used direct or enzyme-amplified radioactive, colorimetric, luminescent, or fluorescent reporters fulfills all of the requirements for an ideal label, including specific activity, size, non-toxicity, cost, stability, localization, and detection.
  • Directly detectable labels such as fluorophores suffer from limited sensitivity, and enzyme- amplified or dissociation-enhanced methods lose spatial information.
  • DELFIA dissociation-enhanced lanthanide fluoroimmunoassay
  • the highly fluorescent chelates used in the DELFIA technology can also be used in fluorescent lanthanide(III) chelate nanoparticles because the hydrophobic environment inside the latex protects the fluorescent chelates from environmental effects, such as solvent quenching, and stabilizes the kinetically weak complexes.
  • the adaptation of appropriate chelates for all four lanthanides would enable a nanoparticle-based, quadruple-labeling technology with an extremely low detection limit and a direct, surface readout measurement.
  • Detecting low levels of target marker in a sample using classic fluorochrome is sometimes difficult and prone to errors because specific fluorescence signals tend to be low and are usually mixed with nonspecific signals. Furthermore, autofluorescence produced from specimen can cause interference.
  • the fluorescence half-life of complex chelate of lanthanide elements, e.g., Europium (EU) is as much as six orders of magnitude longer than conventional fluorescent labels. Consequently, the emission from lanthanide chelate can be distinguished from background fluorescence (which has a short decay half-life) by using a time-resolved fluorometer with an appropriate delay, counting, and cycle times.
  • EU Europium
  • This unique dye confers luminescence with a decay time of >500 ⁇ 8, far longer than that of conventional fluorescent probes or autofluorescent samples, typically having decay times of ⁇ 50 ⁇ 8. Thus, time-resolved fluorometry can virtually eliminate autofluorescence.
  • Europium luminescent dyes feature long-wavelength emissions (-610 nm) that are well separated from the excitation peak (-365 nm). This unusually large Stokes shift permits the use of filter combinations that effectively isolate the desired luminescence signal (Harma et al., Europium nanoparticles and time-resolved fluorescence of ultrasensitive detection of prostate-specific antigen. 2001 ;47:561-568).
  • lanthanide ions are dissociated from the chelating structure into the fluorescence enhancement solution.
  • This additional enhancing step is required to provide an environment that effectively eliminates quenching water and contains energy- absorbing chelating compounds to transfer energy further to lanthanide ions.
  • the lanthanide phosphors can be detected directly without any enhancing steps due to the water-protecting crystal structure.
  • the disadvantage of using the lanthanide phosphors is the lack of light- absorbing groups that effectively transfer the absorbed energy to the lanthanide ions. By combining these properties into a latex particle, fluorescent particles with a very high specific activity could be prepared.
  • the polymer shell efficiently removes fluorescence-quenching water from the vicinity of the chelate by producing a hydrophobic environment. Extremely sensitive assays can be carried out using such particle labels.
  • nanosized polymer labels contain 30,000-2,000,000 Europium molecules entrapped by ⁇ -diketones, which have one of the highest quantum yields of the known lanthanide chelators (Harma et al., Europium nanoparticles and time-resolved fluorescence of ultrasensitive detection of prostate-specific antigen. 2001 ;47:561-568). This encapsulation has no negative effect on the fluorescence efficiency. For a 100 nm size Europium particle, the fluorescence yield is equivalent to about 3,000 molecules of fluorescein. Phycobiliprotein B-PE (perhaps the most fluorescent substance known) has a fluorescence yield equivalent to about 30 fluorescein molecules.
  • a rapid diagnostic test (RDT) kit is designed such that the amount of chemical coloring is determined according to the amount of an analysis target in a sample and the chemical coloring is observed by naked eyes. For this reason, the RDT kit encounters a limitation in finding out an objectified value. Accordingly, there is a need to develop an apparatus for overcoming the limitation and enhancing sensitivity and accuracy.
  • Fluorescence is generally the result of a three-stage process.
  • energy is supplied by an external source, such as an incandescent lamp or a laser, and absorbed by the fluorescent compound, creating an excited electronic singlet state.
  • the excited state exists for a finite time during which the fluorescent compound undergoes conformational changes and is also subject to a multitude of possible interactions with its molecular environment. During this time, the energy of the excited state is partially dissipated, yielding a relaxed state from which fluorescence emission originates.
  • the third stage is the fluorescence emission stage wherein energy is emitted, returning the fluorescent compound to its ground state. The emitted energy is lower than its excitation energy (light or laser) and thus of a longer wavelength. This shift or difference in energy or wavelength allows the emission energy to be detected and isolated from the excitation energy.
  • Time- resolved fluorescence involves exciting the fluorescent label with a short pulse of light, then waiting a certain time (e.g., between approximately 100 to 200 microseconds) after excitation before measuring the remaining long-lived fluorescent signal. In this manner, any short-lived fluorescent background signals and scattered excitation radiation are eliminated.
  • RDT rapid diagnostic test
  • sensitivity is significantly improved by immunoassay where an antibody-antigen reaction is induced through specific binding to an antibody of Europium that a luminescent or fluorescent reporter. Further, more objectified, highly sensitive, and accurate analysis can be accomplished through development of a TFR apparatus.
  • the present invention provides an immunoassay diagnostic device capable of qualitatively and quantitatively determining whether there is Chlamydia infection in a biological sample and the degree of the Chlamydia infection through quantification thereof.
  • the present invention also provides a highly sensitive assay system, including but not limited to time-resolved fluorescent dye used in a lateral flow assay format, which ensures improved sensitivity while maintaining the beneficial aspects of lateral flow assay.
  • the highly sensitive assay system is a chromatographic test system using Europium particles. The assay system is characterized in rapidity, convenience, high sensitivity, and specificity.
  • Figure 1 shows scheme for preparing Europium encapsulated nanoparticle bound to antibody.
  • Figure 2 shows an outline for immunochromatographic assay.
  • Figure 3 shows details of lateral flow immunochromatographic assay
  • Figure 4 shows details of time-resolved fluorescence assay.
  • Figure 5 shows details of time-resolved fluorescence assay.
  • Figure 6 shows details of time-resolved fluorescence assay system.
  • Figure 7 shows details of time-resolved fluorescence assay system instrumentation.
  • Figure 8 shows details of time-resolved fluorescence assay system lens
  • an analyte may be without limitation an antibody and an antigen.
  • a specific binding partner may be without limitation an antibody, an antigen, biotin, streptavidin or avidin.
  • the present invention is directed to a method for determining the presence of at least one type of an analyte in a sample comprising applying an amount of the sample to the apparatus described above. If at least one type of analyte is present in the sample, the sample migrates to a wicking membrane where a chemical reaction occurs. The presence of a signal indicates that the analyte is present in the sample, and the signal is a Europium combination.
  • the Europium particles may be Europium encapsulated carboxylate modified polystyrene particles. These particles are known as fluorescent nanomaterials with a superior fluorescent strength.
  • the polystyrene particles are advantageous in nontoxicity, wide surface area, strong adsorption force, and antibody compatibility.
  • polystyrene particles polystyrene is modified to enhance efficiency for binding to an antibody, and encapsulated is Europium which is a lanthanide element that can be observed with fluorescence. These particles excite at UV light (maximum absorbance wavelength: 333 nm) and emit at 613nm. These particles have a very long lifetime of approximately 0.5 milliseconds, which is about 10,000 to 100,000 times the lifetime of most fluorophores. Since their long lifetime allows time- resolved fluorescence to be applicable, light interference is reduced. Thus, these particles may be used in an ultrasensitive specific system.
  • a sample may be a biological sample.
  • the sample may be without limitation blood, serum, plasma, urine, saliva, sweat, and liquid media processed from a biological or environmental sample.
  • An apparatus used in the method may be without limitation a lateral flow assay format apparatus, and the analyte specifically reacts to Chlamydia.
  • the present invention is also directed to a kit comprising a compartment that contains the apparatus described above, and instructions for using the apparatus as described above.
  • Time-resolved fluorometry technology is a system based on Europium embedded micro particles conjugated with antigens and antibodies. This technology may be used in Chlamydia markers that require highly sensitive tools for their detection.
  • Europium may be used as fluorescent labels to form labeled reagents by adsorbing or covalently binding other proteinaceous compounds such as antibodies or antigens to the surface of the polymeric latex beads.
  • Antibodies may be adsorbed to particles modified to polystyrene (PS) or carboxyl group by hydrophobic bond or ionic bond. Further, the antibodies may be adsorbed to the particles by bonding amide to an amino group of the antibodies through activation of the carboxyl group of the particles with water-soluble carbodiimide l-ethyl-3-(3- dimethylaminopropyl) carbodiimide (ED AC).
  • ED AC water-soluble carbodiimide l-ethyl-3-(3- dimethylaminopropyl) carbodiimide
  • This is a kind of covalent bond which is stronger than hydrophobic bond or ionic bond.
  • a Europium- antibody conjugate may be maintained at a more stable state. A Europium-antibody conjugate is completed based on the foregoing, and high sensitivity is reproduced by comparison and examination of the Europium- antibody conjugate.
  • polymeric particle refers to a spherical or near-spherical polymer particle at various sizes. Preferably, the size is about 0.05-0.5 ⁇ in diameter. However, it is understood that the invention is not limited to the use of any particular type of polymeric particle. In its broadest sense, any substance or particle that can encapsulate the fluorescent dye is encompassed by the present invention.
  • tag refers to a substance that is labeled to the specific binding reagent, which is labeled with time-resolved fluorescent dye.
  • the tag specifically reacts to specific binder immobilized on solid phase.
  • the tag may be biotin when the specific binder immobilized on solid phase is avidin or streptavidin.
  • the inventive test may be performed on-site by a lay person with minimal training while rapid results are obtained after adding one or two drops of sample to a disposable test device or card or strip.
  • the assay is not limited to an antigen/antibody complex.
  • the present invention adopts a lateral-flow immunochromatography method for a rapid chromatography test which is capable of diagnosing Chlamydia trachomatis infection.
  • a lateral flow test kit roughly comprises a membrane and a pad.
  • the membrane and the pad are various in material, dimension, and pore size and were selected according to diagnosis field and kit formation.
  • the membrane and the pad are being tested considering compatibility with reagents and chemicals used in kit formation, such as a Chlamydia sample, a Chlamydia antibody, a Europium conjugate, and a fixed material, for diagnosis of Chlamydia. Each of the most compatible materials was processed.
  • a wicking pad was attached on a top surface of the membrane to fix materials which constitute a control line capable of capturing antibodies and a test line capable of capturing Chlamydia trachomatis and induce and control a flow of fluid.
  • a pad for fixing a nanoparticle-antibody conjugate, i.e., dye was attached to a bottom surface of the membrane, which allowed the dye to migrate onto the membrane by binding to antigens.
  • a target antigen reacts to a labeled specific antibody contained in the dye pad when liquid is absorbed/flows along a card.
  • the materials flow into a film fixed to at least one distinct site of an unlabeled antibody group.
  • An antigen-labeled antibody complex is maintained and captured to form a predetermined line for reading.
  • the predetermined line may be detected by a TRF-mounted reader or any suitable detector.
  • time-resolved fluorometer refers to a tool for measuring time dependence of fluorescence intensity after a short excitation pulse which can also be made as a function of emission wavelength.
  • a wavelength of a light source is 365 nm and power consumption is about 2.5 W.
  • a CCD has 2048 x 2048 pixels each having a size of 7.4um x 7.4um and 12 bits of resolution. In order to increase sensitivity, 4 x 4 pixels are merged into a single pixel before being used. Accordingly, the final resolution uses 512 x 512 pixels.
  • the CCD is turned on after 263 um have passed since an LED was turned off. Each pixel has 12 bits of resolution, but uses only 8 bits of resolution for the purpose of convenience.
  • a silicon detector Si-Detector
  • a 12-bit analog-digital converter (12BIT ADC)
  • 340 nm that is an optimal wavelength to fluorescence is used in the silicon detector and an optical system design is optimized to limit a light-taken region of a source-sample-detector to 1mm x 1mm, it will be estimated that a signal of 6 dB or higher is obtained as compared to conventional methods.
  • Optical loss was measured at each wavelength band in the ultraviolet-visible-infrared (UV-Vis-IR) region of a manufactured optical fiber.
  • a result of the measurement was that light transmissivity of the optical fiber was about 89%.
  • TRF time-resolved fluorescence

Landscapes

  • Health & Medical Sciences (AREA)
  • Immunology (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Engineering & Computer Science (AREA)
  • Molecular Biology (AREA)
  • Biomedical Technology (AREA)
  • Chemical & Material Sciences (AREA)
  • Hematology (AREA)
  • Urology & Nephrology (AREA)
  • Biotechnology (AREA)
  • Microbiology (AREA)
  • Cell Biology (AREA)
  • Food Science & Technology (AREA)
  • Medicinal Chemistry (AREA)
  • Physics & Mathematics (AREA)
  • Analytical Chemistry (AREA)
  • Biochemistry (AREA)
  • General Health & Medical Sciences (AREA)
  • General Physics & Mathematics (AREA)
  • Pathology (AREA)
  • Investigating, Analyzing Materials By Fluorescence Or Luminescence (AREA)
  • Investigating Or Analysing Materials By The Use Of Chemical Reactions (AREA)

Abstract

La présente invention concerne un procédé de dosage de Chlamydia, selon lequel des nanoparticules d'europium et un anticorps anti-Chlamydia sont conjugués au moyen d'une liaison amide pour déclencher une réaction immunitaire contre un antigène de Chlamydia. Ainsi, le conjugué réagit de manière sensible à l'antigène de Chlamydia pour détecter une quantité même infime de l'antigène de Chlamydia. Un appareil à fluorescence en temps résolu (TRF) fait appel à la TRF pour détecter un signal produit par l'europium. Étant donné que l'europium a une fréquence d'émission relativement longue, on obtient une réaction plus sensible et précise, en termes de détection de la fluorescence ou des effets de fond, qu'avec une courte longueur d'onde. Un objet de la présente invention concerne un lecteur de fluorescence ayant une structure simple, offrant une conception peu coûteuse.
PCT/US2012/047737 2011-07-20 2012-07-20 Procédé de détection de micro-organismes Ceased WO2013013214A1 (fr)

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
US201161510019P 2011-07-20 2011-07-20
US61/510,019 2011-07-20

Publications (1)

Publication Number Publication Date
WO2013013214A1 true WO2013013214A1 (fr) 2013-01-24

Family

ID=47558521

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/US2012/047737 Ceased WO2013013214A1 (fr) 2011-07-20 2012-07-20 Procédé de détection de micro-organismes

Country Status (1)

Country Link
WO (1) WO2013013214A1 (fr)

Cited By (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN105445449A (zh) * 2015-04-04 2016-03-30 吉林双正医疗科技有限公司 唾液尿酸快速半定量检测装置及其制备方法
WO2020016308A1 (fr) 2018-07-18 2020-01-23 Ecole Polytechnique Test a diffusion capillaire mettant en œuvre des nanoparticules inorganiques photoluminescentes
GB2609419A (en) * 2021-07-29 2023-02-08 Ams Int Ag Lateral flow test

Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20030175812A1 (en) * 2000-03-20 2003-09-18 Abs (Analytical Biological Services, Inc.) Method for detecting an analyte by fluorescence
US20040219690A1 (en) * 2003-05-02 2004-11-04 Choi Young Ho Chromatographic assay system
US20080199851A1 (en) * 2006-02-21 2008-08-21 Richard Laswell Egan Methods and compositions for analyte detection

Patent Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20030175812A1 (en) * 2000-03-20 2003-09-18 Abs (Analytical Biological Services, Inc.) Method for detecting an analyte by fluorescence
US20040219690A1 (en) * 2003-05-02 2004-11-04 Choi Young Ho Chromatographic assay system
US20080199851A1 (en) * 2006-02-21 2008-08-21 Richard Laswell Egan Methods and compositions for analyte detection

Cited By (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN105445449A (zh) * 2015-04-04 2016-03-30 吉林双正医疗科技有限公司 唾液尿酸快速半定量检测装置及其制备方法
CN105548534A (zh) * 2015-04-04 2016-05-04 吉林双正医疗科技有限公司 心肌标志物的联合检测装置及其制备方法
WO2020016308A1 (fr) 2018-07-18 2020-01-23 Ecole Polytechnique Test a diffusion capillaire mettant en œuvre des nanoparticules inorganiques photoluminescentes
FR3084165A1 (fr) 2018-07-18 2020-01-24 Ecole Polytechnique Test a diffusion capillaire mettant en œuvre des nanoparticules inorganiques photoluminescentes
GB2609419A (en) * 2021-07-29 2023-02-08 Ams Int Ag Lateral flow test

Similar Documents

Publication Publication Date Title
US9594078B2 (en) Chromatographic assay system
CN106872420B (zh) 一种时间分辨荧光定量检测尿微量白蛋白的试剂盒及方法
US20140170674A1 (en) Membraine-Based Assay Devices Utilizing Time-Resolved Up-Converting Luminescence
US8470608B2 (en) Combined visual/fluorescence analyte detection test
WO2016191507A1 (fr) Utilisation de la fluorescence pour la détermination rapide et facile de la s-adénosylméthionine, de la s-adénosylhomocystéine et de l'homocystéine
CA2750531A1 (fr) Appareil et procedes de detection d'une inflammation a l'aide de points quantiques
CN104246500A (zh) 宽范围发光免疫分析
JP4274944B2 (ja) ダイナミックレンジが拡張された粒子利用リガンドアッセイ
US12416627B2 (en) Immunoassay with enhanced sensitivity
JP2005510706A5 (fr)
WO2022236120A9 (fr) Diagnostic de point d'intervention en temps réel et méthode d'utilisation associée
RU2379691C1 (ru) Способ многоаналитного иммуноанализа с использованием микрочастиц
KR101521565B1 (ko) 생체 분자 분석 키트 및 분석 방법
CA2305543A1 (fr) Systeme de dosage capillaire
KR102220357B1 (ko) 면역진단 키트 및 이를 이용한 면역진단 방법
EP3308167A1 (fr) Utilisation de la fluorescence pour la détermination rapide et facile de la s-adénosylméthionine, de la s-adénosylhomocystéine et de l'homocystéine
CN114923886A (zh) 一种基于光栅与磁阵列式的单分子荧光检测方法
CN119199102A (zh) 一种基于均相光激化学发光免疫分析技术的胰蛋白酶原-2检测方法及试剂盒
JP2025510706A (ja) 分析物を検出するための方法
CN117529663A (zh) 用于使用标记固定和放大策略对单分子进行数字免疫传感的方法

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: 12815049

Country of ref document: EP

Kind code of ref document: A1

NENP Non-entry into the national phase

Ref country code: DE

122 Ep: pct application non-entry in european phase

Ref document number: 12815049

Country of ref document: EP

Kind code of ref document: A1