Analytical methods and arrays for use in the same
AssigneeSenzagen AB
InventorsSven Henrik Johansson, Robin Mikael Gradin
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Abstract
The present invention relates to a method for identifying agents which are capable of inducing respiratory sensitization in a mammal, and arrays and analytical kits for use in such methods.
Claims57
A method for identifying agents capable of inducing respiratory sensitization in a mammal comprising or consisting of the steps of:
- (a) providing a population of dendritic cells or a population of dendritic-like cells;
- (b) exposing the cells provided in step (a) to a test agent;
- and
- (c) measuring in the cells of step (b) the expression of two or more biomarkers selected from the group defined in Table A;
- wherein the expression of the two or more biomarkers measured in step (c) is indicative of the respiratory sensitizing effect of the test agent of step (b).
The method according to any one of the preceding claims wherein step (c) comprises or consists of measuring the expression of all of the biomarkers listed in Table A(i).
The method according to any one of the preceding claims wherein step (c) comprises or consists of measuring the expression of one or more biomarkers selected from the group defined in in Table A(ii), for example, 2, or 3 of the biomarkers listed in Table A(ii).
The method according to any one of the preceding claims wherein step (c) comprises or consists of measuring the expression of all of the biomarkers listed in Table A(ii).
The method according to any one of the preceding claims wherein step (c) comprises or consists of measuring the expression of three or more of the biomarkers selected from the group defined in Table A, for example, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, or 28 of the biomarkers listed in Table A.
The method according to any one of the preceding claims wherein step (c) comprises or consists of measuring the expression of all of the biomarkers listed in Table A.
The method according to any previous claim further comprising:
- d) exposing a separate population of the dendritic cells or dendritic-like cells to one or more negative control agent that is not a respiratory sensitizer in a mammal;
- and
- e) measuring in the cells of step (d) the expression of the two or more biomarkers measured in step (c)
- wherein the test agent is identified as a respiratory sensitizer in the event that the expression of the two or more biomarkers measured in step (e) differs from the expression of the two or more biomarkers measured in step (c).
The method any previous claim further comprising:
- f) exposing a separate population of the dendritic cells or dendritic-like cells to one or more positive control agent that is a respiratory sensitizer in a mammal;
- and
- g) measuring in the cells of step (f) the expression of the two or more biomarkers measured in step (c)
- wherein the test agent is identified as a respiratory sensitizer in the event that the expression of the two or more biomarkers measured in step (f) corresponds to the expression of the two or more biomarkers measured in step (c).
The method according to any one of the preceding claims wherein step (c) comprises measuring the expression of a nucleic acid molecule of one or more of the biomarkers.
The method according to any one of the preceding claims wherein measuring the expression of one or more of the biomarkers in step (c) is performed using one or more binding moieties, each capable of binding selectively to a nucleic acid molecule encoding one of the biomarkers identified in Table A.
The method according to any one of the preceding claims wherein step (c) is performed using an array.
The method according to any one of the preceding claims wherein the method is performed in vitro, in vivo, ex vivo or in silico.
The method according to any one of the preceding claims wherein the population of dendritic cells or population of dendritic-like cells comprises or consists of immortal and/or non-naturally occurring cells.
The method according to any one of the preceding claims wherein the population of dendritic cells or population of dendritic-like cells is a population of dendritic-like cells.
The method according to any one of the preceding claims for identifying agents capable of inducing a respiratory hypersensitivity response.
The method according to any one of the preceding claims wherein the hypersensitivity response is a humoral hypersensitivity response.
The method according to any one of the preceding claims for identifying agents capable of inducing a type I hypersensitivity response in a mammal.
The method according to any one of the preceding claims for identifying agents capable of inducing respiratory allergy.
The method according to any one of the preceding claims wherein the method is indicative of the relative sensitizing potency of the sample to be tested.
The method according to any one of the preceding claims wherein the method comprises one or more of the following steps:
- (i) cultivating dendritic or dendritic-like cells;
- (ii) seeding cells of (i) in one or more well(s), e.g. wells of one or more multi-well assay plates;
- (iii) adding to a one or more well(s) of (ii) the agent(s) to be tested;
- (iv) adding to one or more separate well(s) of (ii) one or more positive control(s);
- (v) adding to one or more separate well(s) of (ii) one or more negative control(s);
- (vi) incubating cells in wells of (iii)-(v), preferably for about 24 hours;
- (vii) isolating purified total RNA from cells of (vi) and, optionally, convert mRNA into cDNA;
- (viii) quantifying expression levels of individual mRNA transcripts from (vii), e.g. using an array, such as an Affymetrix Human Gene 1.0 ST array, and/or a Nanostring code set;
- (ix) exporting and normalizing expression data from (viii);
- (x) isolating data from (ix) originating from biomarkers of the GARD Prediction Signature (i.e. the biomarkers of Table A);
- (xi) applying a prediction model to data from (x), e.g. a frozen SVM model previously established and trained on historical data, e.g. data obtained in Example 1, to predict the respiratory sensitization effect of tested agents(s) and negative/positive control(s).
Use of two or more biomarkers selected from the group defined in Table A for identifying respiratory sensitizing agents, preferably wherein one or more of the biomarkers is selected from the group defined in Table A(i).
Use of two or more binding moieties each with specificity for a biomarker selected from the group defined in Table A for identifying respiratory sensitizing agents, preferably wherein one or more of the binding moieties has specificity for a biomarker selected from the group defined in Table A(i).
A method use, array or kit substantially as described herein.
Dependencies are inferred from the claim text.