http://rdf.ncbi.nlm.nih.gov/pubchem/patent/ES-2715526-T3
Outgoing Links
| Predicate | Object |
|---|---|
| assignee | http://rdf.ncbi.nlm.nih.gov/pubchem/patentassignee/MD5_5cf9e383d1d59d2d4845b33c43a9e4af |
| classificationCPCAdditional | http://rdf.ncbi.nlm.nih.gov/pubchem/patentcpc/G01N15-1459 |
| classificationCPCInventive | http://rdf.ncbi.nlm.nih.gov/pubchem/patentcpc/G01N33-56972 http://rdf.ncbi.nlm.nih.gov/pubchem/patentcpc/G01N33-5094 |
| classificationIPCInventive | http://rdf.ncbi.nlm.nih.gov/pubchem/patentipc/G01N33-569 http://rdf.ncbi.nlm.nih.gov/pubchem/patentipc/G01N33-50 |
| filingDate | 2015-11-27-04:00^^<http://www.w3.org/2001/XMLSchema#date> |
| grantDate | 2019-06-04-04:00^^<http://www.w3.org/2001/XMLSchema#date> |
| inventor | http://rdf.ncbi.nlm.nih.gov/pubchem/patentinventor/MD5_61719b3433e06640ab9cd65cb58b8e9d http://rdf.ncbi.nlm.nih.gov/pubchem/patentinventor/MD5_62c9a8aef4f57295259381dcc3f5d563 http://rdf.ncbi.nlm.nih.gov/pubchem/patentinventor/MD5_a5b57d710621cd67b8304ae9072048d6 |
| publicationDate | 2019-06-04-04:00^^<http://www.w3.org/2001/XMLSchema#date> |
| publicationNumber | ES-2715526-T3 |
| titleOfInvention | Method for the detection of antigen specific immune cells in extra-blood fluids |
| abstract | Method for enhanced detection of antigen-specific immune cells in extra-blood fluids, consisting of the following steps: - the isolation of vital immune cells from an extra-blood liquid by means of appropriate washing and centrifugation steps, e.g. by the application of physiological serum as a wash buffer and centrifugation buffer at 200 to 500 g, - the determination of the total number of cells, for example, for each stimulation preparation, 105 to 1.5x106 cells are needed, - the previous staining of immune cells isolated with an antibody coupled to a fluorochrome which is directed against the antigens expressed in the cells, eg CD45, or by fluorochromes suitable for staining vital cells, eg, carboxyfluorescein diacetate succinimidyl ester , CFDA-SE, - the removal of unbound antibodies or fluorochromes by washing steps, characterized by - the mixing of the previously stained immune cells with heparinized whole blood of the same person, - stimulation of the samples mixed with the antigen of interest, with or without the addition of anti-CD49d and anti-CD28 costimulatory antibodies, anti-CD49d alone or anti -CD28 alone, - incubation for 30 min at 4 h at 35 to 39 ° C and 3 to 10% CO2, - the addition of a secretion inhibitor, eg brefeldin A, monensin or others, - incubation during 2 to 12 hours at 35 to 39 ° C and 3 to 10% CO2, - red cell lysis and leukocyte fixation, - washing steps for leukocyte isolation, - increased cell membrane permeability by the addition of a substance that increases the permeability of the cell membrane, eg saponin, - incubation with specific antibodies labeled with fluorochrome against activation markers, eg, anti-CD69, anti-IFN-γ, during at least 10 min on ice or at room temperature or 35 ha sta 39 ° C, - new washing steps for the elimination of unbound antibodies, - the analysis of cells in a flow cytometer and the discrimination of immune cells of the extra-blood fluid from those of the blood by means of the signal fluorescent which was used for prior staining, eg, CD45 antibody fluorochrome, CFSE, where the cells reactive against the stimulus are distinguished from non-reactive cells by positivity with respect to the activation markers. |
| priorityDate | 2014-11-29-04:00^^<http://www.w3.org/2001/XMLSchema#date> |
| type | http://data.epo.org/linked-data/def/patent/Publication |
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