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 Celica D.O.O., LjubljanafiledCriticalCelica D.O.O., Ljubljana
Priority to SI200600207ApriorityCriticalpatent/SI22385B/en
Priority to DE602007011647Tprioritypatent/DE602007011647D1/en
Priority to EP07803258Aprioritypatent/EP2069782B1/en
Priority to PCT/EP2007/059296prioritypatent/WO2008028929A1/en
Priority to AT07803258Tprioritypatent/ATE493650T1/en
Publication of SI22385ApublicationCriticalpatent/SI22385A/en
Publication of SI22385BpublicationCriticalpatent/SI22385B/en
Micro-Organisms Or Cultivation Processes Thereof
(AREA)
Preparation Of Compounds By Using Micro-Organisms
(AREA)
Abstract
The present invention relates to a method for determining the ratio and quality of hybridomas by means of the number of differently labeled lysosomes, usually labeled with green and red fluorescence, where lysosomes originate from eukaryotic parent cells. Due to the property of lysosomes of fusing with one another, also yellow lysosomes formed by the fusion of green and red lysosomes may be found in the population of said green and red lysosomes. The physiological process of lysosomal fusion is one of the events involved in a complex system of expressing tumour and other antigens on the hybridoma surface. Therefore this new method gives, in addition to the data on the amount of hybridomas in a suspension of fused cells, at the same time also indirect information on the immunogenicity of hybridomas, which is of key importance for the effectiveness of a cellular vaccine against cancer in the paticnt's body.
Claims (13)
11Patentni zahtevki 1. Metoda za detekcijo količine in kvalitete hibridomskih celic, ki sestojijo iz evkariontskih celic, s konfokalno mikroskopijo, pri čemer metoda obsega stopnje: (a) barvanje poznih endocitičnih predelkov izvora za celične linije, izvedene iz človeških ali živalskih virov, zlasti kjer so pozni endocitični predelki lizosomi; (b) ločeno barvanje obeh tipov starševskih celic z različnimi fluorescenčnimi barvami; (c) po barvanju fuzijo celic za inkubacijski čas vsaj 2 h pri temperaturi od 25 °C do 35 °C; (d) določanje obsega fuzije poznih endocitičnih predelkov s konfokalno mikroskopijo.A method for detecting the quantity and quality of hybridoma cells consisting of eukaryotic cells by confocal microscopy, the method comprising the steps of: (a) staining late endocytic regions of origin for cell lines derived from human or animal sources, in particular where late endocytic compartments are lysosomes; (b) separate staining of both types of parental cells with different fluorescent colors; (c) after staining, cell fusion for an incubation period of at least 2 hours at a temperature of 25 ° C to 35 ° C; (d) determining the extent of fusion of late endocytic compartments by confocal microscopy.2. Metoda po zahtevku 1, kjer je inkubacijski čas 2-30 h.The method of claim 1, wherein the incubation time is 2-30 h.3. Metoda po katerem koli od zahtevkov 1 ali 2, ki obsega: (a) označevanje poznih endocitičnih predelkov in določanje minimalnega števila poznih endocitičnih predelkov na posamezno starševsko celico s konfokalno mikroskopijo; (b) fuzijo obeh tipov starševskih celic in inkubacijo pri 25-35 °C za 2-30 h; (c) analizo fuzijskih vzorcev za prisotnost hibridomskih celic s konfokalno mikroskopijo; (d) določanje števila hibridomskih celic, ki vsebujejo vsaj minimalno število poznih endocitičnih predelkov, ki izvirajo iz ločenih tipov starševskih celic in ki so ločeno pobarvani z različnimi fluorescenčnimi barvami; (e) določanje količine hibridomskih celic, izražene kot razmerje med številom hibridomskih celic in številom vseh fluorescenčnih celic, detektiranih s konfokalno mikroskopijo.A method according to any one of claims 1 or 2, comprising: (a) labeling the late endocytic compartments and determining the minimum number of late endocytic compartments per parent parent cell by confocal microscopy; (b) fusion of both types of parental cells and incubation at 25-35 ° C for 2-30 hours; (c) analysis of fusion samples for the presence of hybridoma cells by confocal microscopy; (d) determination of the number of hybridoma cells containing at least a minimum number of late endocytic compartments originating from separate parental cell types and stained separately with different fluorescent dyes; (e) determination of the amount of hybridoma cells, expressed as the ratio of the number of hybridoma cells to the number of all fluorescent cells detected by confocal microscopy.4. Metoda po katerem koli od zahtevkov 1-3, kjer zaporedno izvedemo naslednje stopnje: - označevanje subcelulamih organelov, zlasti poznih endocitičnih predelkov, ki so vključeni v antigen predstavitveno pot; - kvantifikacija števila poznih endocitičnih predelkov v posamezni celici s konfokalno miksroskopijo; - določanje dobitka hibridomskih celic z detekcijo fluorescenčno označenih, npr. rdečih in zelenih, poznih endocitičnih predelkov, ki izvirajo iz obeh tipov starševskih celic; - določanje obsega zlitih poznih endocitičnih predelkov v hibridomski celici s kvantitativno konfokalno mikroskopijo in analizo slike.A method according to any one of claims 1-3, wherein the following steps are carried out sequentially: - labeling the subcellular organelles, in particular the late endocytic compartments involved in the antigen presentation pathway; - quantification of the number of late endocytic compartments in a single cell by confocal microscopy; - determination of hybridoma cell yield by detection of fluorescently labeled, e.g. red and green, late endocytic compartments originating from both types of parental cells; - determination of the extent of fused late endocytic compartments in a hybridoma cell by quantitative confocal microscopy and image analysis.5. Metoda po katerem koli od zahtevkov 1-4, kjer zaporedno izvedemo naslednje stopnje: a) določanje minimalnega števila označenih poznih endocitičnih predelkov na posamezno starševsko celico; 3 b) zajemanje konfokalnih slik zlitih in kontrolnih vzorcev, kjer je bil postopek fuzije izpuščen; c) zajemanje zaporedja konfokalnih slik, da se optično odreže celica in dobi tridimenzionalne slike, npr. z-sklade, za vsako detektirano hibridomsko celico; d) določanje števila nezlitih poznih endocitičnih predelkov, ki izvirajo iz vsakega tipa starševskih celic in določanje števila zlitih poznih endocitičnih predelkov; e) določanje števila hibridomskih celic v vsakem fuzijskem vzorcu na osnovi minimalnega števila poznih endocitičnih predelkov, označenih z dvema različnima barvama, npr. rdeče in zeleno, ki izvirajo iz vsakega tipa starševskih celic, in mimimalnega števila zlitih poznih endocitičnih predelkov, npr. rumenih.The method of any one of claims 1-4, wherein the following steps are performed sequentially: a) determining the minimum number of labeled late endocytic compartments per parent parent cell; 3 b) capturing confocal images of fused and control samples where the fusion process has been omitted; c) capturing a sequence of confocal images to optically cut a cell and obtain three-dimensional images, e.g. z-stacks, for each hybridoma cell detected; d) determining the number of fused late endocytic compartments originating from each type of parental cell and determining the number of fused late endocytic compartments; e) determining the number of hybridoma cells in each fusion sample based on the minimum number of late endocytic compartments labeled with two different colors, e.g. red and green originating from each type of parental cell, and a minimum number of fused late endocytic compartments, e.g. yellowish.6. Metoda po zahtevku 5, kjer v stopnji a) zajamemo laserske konfokalne z-sklade slik za vsako naključno izbrano s fluorescenčnimi molekulami naloženo neelektrofuzionirano celico.The method of claim 5, wherein in step a), laser confocal z-stacks of images are captured for each randomly selected non-electrofusion cell loaded with fluorescent molecules.7. Metoda po zahtevku 6, kjer preštejemo število označenih poznih endocitičnih predelkov na celico pod pogojem, da se vsak pozni endocitični predelek lahko pojavi v predhodno določenem številu sosednjih optičnih ravnin, ugotovimo število poznih endocitičnih predelkov za vsako celično populacijo ter ugotovimo minimalno število poznih endocitičnih predelkov na celico.The method of claim 6, wherein counting the number of labeled late endocytic compartments per cell provided that each late endocytic compartment can occur in a predetermined number of adjacent optical planes, determining the number of late endocytic compartments for each cell population and determining the minimum number of late endocytic compartments compartments per cell.8. Metoda po zahtevku 5, kjer v stopnji b) po fuzijskem postopku vzorce inkubiramo pri 25-35 °C za 2-30 h in nato analiziramo s konfokalnimi slikami za prisotnost hibridomskih celic, konfokalne slike zajamemo z lasersko konfokalno mikroskopijo, zlasti, kjer vzbudimo zeleno fluorescenco z argonskim laserjem in rdečo fluorescenco s helij/neonskim laserjem.The method according to claim 5, wherein in step b) after the fusion process the samples are incubated at 25-35 ° C for 2-30 h and then analyzed with confocal images for the presence of hybridoma cells, the confocal images are captured by laser confocal microscopy, in particular where excite green fluorescence with an argon laser and red fluorescence with a helium / neon laser.9. Metoda po zahtevku 5, kjer v stopnji c) fuzijske vzorce analiziramo za prisotnost hibridomskih celic in zajamemo optične ravnine z-skladov s konfokalno mikroskopijo za vsako detektirano hibridomsko celico.The method of claim 5, wherein in step c) the fusion samples are analyzed for the presence of hybridoma cells and the optical planes of the z-stacks are captured by confocal microscopy for each hybridoma cell detected.10. Metoda po zahtevku 5, kjer v stopnji d) ocenimo število zelenih, rdečih in rumenih poznih endocitičnih predelkov z uporabo računalnika.The method of claim 5, wherein in step d), the number of green, red and yellow late endocytic compartments is estimated using a computer.11. Metoda po zahtevku 10, kjer so hibridomi definirani kot celice, ki vsebujejo vsaj minimalno število rdečih, zelenih ali rumenih fluorescenčnih poznih endocitičnih predelkov ali pare drugih fluorescenčnih markerjev in je število zlitih poznih endocitičnih predelkov indikativno za nivo imunogenosti hibridoma.The method of claim 10, wherein the hybridomas are defined as cells containing at least a minimal number of red, green or yellow fluorescent late endocytic compartments or pairs of other fluorescent markers and the number of fused late endocytic compartments is indicative of the level of immunogenicity of the hybridoma.12. Metoda po zahtevku 5, kjer stopnja e) vključuje štetje hibridomov v fuzijskem vzorcu in izrazitev nivoja hibridomskih celic v fuzijskem vzorcu kot razmerje med številom hibridomskih celic in številom fluorescenčno označenih celic v fuzijskem vzorcu.The method of claim 5, wherein step e) comprises counting the hybridomas in the fusion sample and expressing the level of hybridoma cells in the fusion sample as the ratio of the number of hybridoma cells to the number of fluorescently labeled cells in the fusion sample.13. Metoda po katerem koli od zahtevkov 1-12, kjer se hibridomske celice tvorijo z zlivanjem med tumorskimi celicami in evkariontskimi celicami, npr. dendritičnimi celicami.The method of any one of claims 1-12, wherein the hybridoma cells are formed by fusion between tumor cells and eukaryotic cells, e.g. dendritic cells.
SI200600207A2006-09-052006-09-05Method for determination of proportion and quality of hybridoms and application of hybridoms of appropriate quality
SI22385B
(en)