Processing

Please wait...

Settings

Settings

Goto Application

1. WO2020111449 - CELL-BASED METHOD FOR DETERMINING BOTULINUM TOXIN ACTIVITY

Publication Number WO/2020/111449
Publication Date 04.06.2020
International Application No. PCT/KR2019/010467
International Filing Date 19.08.2019
IPC
G01N 33/50 2006.01
GPHYSICS
01MEASURING; TESTING
NINVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
33Investigating or analysing materials by specific methods not covered by groups G01N1/-G01N31/131
48Biological material, e.g. blood, urine; Haemocytometers
50Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
G01N 33/68 2006.01
GPHYSICS
01MEASURING; TESTING
NINVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
33Investigating or analysing materials by specific methods not covered by groups G01N1/-G01N31/131
48Biological material, e.g. blood, urine; Haemocytometers
50Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
68involving proteins, peptides or amino acids
G01N 33/53 2006.01
GPHYSICS
01MEASURING; TESTING
NINVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
33Investigating or analysing materials by specific methods not covered by groups G01N1/-G01N31/131
48Biological material, e.g. blood, urine; Haemocytometers
50Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
53Immunoassay; Biospecific binding assay; Materials therefor
C07K 16/12 2006.01
CCHEMISTRY; METALLURGY
07ORGANIC CHEMISTRY
KPEPTIDES
16Immunoglobulins, e.g. monoclonal or polyclonal antibodies
12against material from bacteria
C12N 5/0793 2010.01
CCHEMISTRY; METALLURGY
12BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
NMICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
5Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
07Animal cells or tissues
071Vertebrate cells or tissues, e.g. human cells or tissues
079Neural cells
0793Neurons
C12Q 1/02 2006.01
CCHEMISTRY; METALLURGY
12BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
QMEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
1Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
02involving viable microorganisms
CPC
C07K 16/1246
CCHEMISTRY; METALLURGY
07ORGANIC CHEMISTRY
KPEPTIDES
16Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
12against material from bacteria
1203from Gram-negative bacteria
1246from Rickettsiales (O)
C07K 16/1282
CCHEMISTRY; METALLURGY
07ORGANIC CHEMISTRY
KPEPTIDES
16Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
12against material from bacteria
1267from Gram-positive bacteria
1282from Clostridium (G)
C07K 16/18
CCHEMISTRY; METALLURGY
07ORGANIC CHEMISTRY
KPEPTIDES
16Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
18against material from animals or humans
C07K 2317/33
CCHEMISTRY; METALLURGY
07ORGANIC CHEMISTRY
KPEPTIDES
2317Immunoglobulins specific features
30characterized by aspects of specificity or valency
33Crossreactivity, e.g. for species or epitope, or lack of said crossreactivity
C07K 2317/34
CCHEMISTRY; METALLURGY
07ORGANIC CHEMISTRY
KPEPTIDES
2317Immunoglobulins specific features
30characterized by aspects of specificity or valency
34Identification of a linear epitope shorter than 20 amino acid residues or of a conformational epitope defined by amino acid residues
C07K 2317/565
CCHEMISTRY; METALLURGY
07ORGANIC CHEMISTRY
KPEPTIDES
2317Immunoglobulins specific features
50characterized by immunoglobulin fragments
56variable (Fv) region, i.e. VH and/or VL
565Complementarity determining region [CDR]
Applicants
  • 휴젤(주) HUGEL INC. [KR]/[KR]
Inventors
  • 이치건 LEE, Chee Gun
  • 엄지현 OUM, Ji Hyun
  • 아제이비제야쿠마 AJAY, Vijayakumar
  • 구이시앙아이 GUI, Xiangai
Agents
  • 이재영 LEE, Jae Young
Priority Data
10-2018-015064029.11.2018KR
10-2018-015099729.11.2018KR
10-2018-015970112.12.2018KR
Publication Language Korean (KO)
Filing Language Korean (KO)
Designated States
Title
(EN) CELL-BASED METHOD FOR DETERMINING BOTULINUM TOXIN ACTIVITY
(FR) PROCÉDÉ À BASE DE CELLULES POUR DÉTERMINER L'ACTIVITÉ DE LA TOXINE BOTULIQUE
(KO) 보툴리눔 독소 활성을 결정하는 세포 기반 방법
Abstract
(EN)
The present invention relates to a cell and an antibody for determining botulinum toxin activity, and a method for determining activity using same. Currently, in the field of botulinum toxin titer measurement, there is a need to develop a cell-based titer assay (CBPA) to replace the mouse LD50 bioassay (mLD50). The cell and the antibody for the botulinum toxin activity assay of the present invention are a cell and an antibody for CBPA to replace mLD50, and the cell line has a significantly shorter division time compared to a conventional SiMa cell used to determine the botulinum toxin activity and significantly higher sensitivity to botulinum toxin than that of parental cell lines, and therefore is well suited for the detection of toxins as well as for determining the cell-based botulinum toxin activity. Furthermore, the cell line according to the present invention can be stably cultured by attaching to a culture dish coated with poly-d-lysine (PDL), and therefore can be used very usefully as a cell for determining cell-based botulinum toxin activity or detecting toxins. In addition, the antibody is a monoclonal antibody with a markedly high binding affinity and specificity for SNAP25, and overcomes the limitations of conventional CBPA and enables the development of more effective CBPA, and therefore is expected to be actively used in the pharmaceutical and cosmetic fields. The present invention also relates to an optimal CBPA assay using monoclonal antibodies with significantly high binding affinity and specificity for N2-42F cells and SNAP25, which makes it possible to measure titers of botulinum toxin of 0.5 U or less. The CBPA assay using the cells and antibodies of the present invention are expected to be a highly reliable and reproducible cell-based titer assay for botulinum toxin.
(FR)
La présente invention concerne une cellule et un anticorps qui permettent de déterminer l'activité de la toxine botulique, ainsi qu'un procédé de détermination d'activité correspondant. Actuellement, dans le domaine de la mesure de titre de toxine botulique, il est nécessaire de développer un dosage de titre à base de cellules (CBPA) pour remplacer le dosage biologique LD50 de souris (mLD50). La cellule et l'anticorps pour le dosage d'activité de la toxine botulique de la présente invention sont une cellule et un anticorps pour le CBPA destiné à remplacer le mLD50, et la lignée cellulaire présente un temps de division considérablement plus court par comparaison avec une cellule SiMa classique utilisée pour déterminer l'activité de la toxine botulique et une sensibilité considérablement plus élevée à la toxine botulique que celle des lignées cellulaires parentales, et convient donc à la détection de toxines, ainsi qu'à la détermination de l'activité de la toxine botulique à base de cellules. En outre, la lignée cellulaire selon la présente invention peut être cultivée de manière stable par fixation à une boîte de culture revêtue de poly-d-lysine (PDL) et peut donc être utilisée de manière très utile en tant que cellule pour déterminer l'activité de la toxine botulique à base de cellules ou pour détecter des toxines. De plus, l'anticorps est un anticorps monoclonal possédant une affinité et une spécificité de liaison nettement élevées pour SNAP25, et surmonte les limitations du CBPA classique et permet le développement d'un CBPA plus efficace, et devrait donc être activement utilisé dans les domaines pharmaceutique et cosmétique. La présente invention concerne également un dosage CBPA optimal utilisant des anticorps monoclonaux possédant une affinité et une spécificité de liaison considérablement élevées pour les cellules N2-42F et SNAP25, ce qui permet de mesurer des titres de toxine botulique de 0,5 U ou moins. Le dosage CBPA utilisant les cellules et les anticorps de la présente invention devrait être un dosage de titre à base de cellules hautement fiable et reproductible pour la toxine botulique.
(KO)
본 발명은 보툴리눔 독소 활성을 결정하기 위한 세포, 항체, 및 이를 이용한 활성 결정방법에 관한 것이다. 현재 보툴리눔 독소 역가 측정 분야에서는 마우스 LD50 생물검정(mLD50)을 대체하기 위한 세포 기반 역가 분석법(CBPA)의 개발 필요성이 요구되고 있다. 본 발명의 보툴리눔 독소 활성 검정용 세포, 및 항체는 mLD50을 대체하기 위한 CPBA용 세포, 및 항체로서, 상기 세포주는 종래 보툴리눔 독소 활성 결정에 사용되는 SiMa 세포에 비하여 현저하게 짧은 분열 시간을 가지며, 보툴리눔 독소에 대한 민감도가 부모 세포주에 비하여 현저하게 높아, 세포 기반 보툴리눔 독소의 활성 결정뿐만 아니라, 독소의 검출에 매우 적합하다. 나아가, 본 발명에 따른 상기 세포주는 폴리-d-라이신(poly-d-lysine; PDL)으로 코팅된 배양 접시에 부착되어 안정적으로 배양될 수 있으므로, 세포 기반 보툴리눔 독소 활성을 결정 또는 독소를 검출하기 위한 세포로서 매우 유용하게 사용될 수 있다. 또한 상기 항체는 SNAP25에 대한 결합 친화력과 특이성이 현저하게 높은 단일 클론 항체로서, 종래의 CPBA가 가진 한계점을 극복하고, 보다 효과적인 CBPA 개발을 가능하게 하므로, 제약 및 미용 분야에서 활발하게 이용될 것으로 기대된다. 또한 본 발명은 N2-42F 세포, 및 SNAP25에 대한 결합 친화력과 특이성이 현저하게 높은 단일 클론 항체를 이용한 최적의 CBPA 분석법에 관한 것으로, 보툴리눔 독소 0.5 U 이하의 역가를 측정하는 것이 가능하다. 본 발명의 세포 및 항체를 이용한 CBPA 분석법은 신뢰성 및 재현성이 높은 보툴리눔 독소의 세포 기반 역가 분석법이 될 것으로 기대된다.
Latest bibliographic data on file with the International Bureau