Cell, method and kit for conducting an assay for neutralizing antibodies
View Patent ↗The present invention provides a cell for use in a one-step cell-based assay for an extracellular ligand (e.g., IFNα) that initiates a ligand-specific signal at the nucleus of the cell and for neutralizing antibodies against the extracellular ligand. The cell-based one-step assay allows both the extracellular ligand concentration and the neutralizing antibody titer to be quantified in a single sample (e.g., serum) without the need for sample dilution and addition of exogenous extracellular ligand.
1. A cell having a cell-surface receptor for a ligand selected from the group consisting of a cytokine and a growth factor in the external environment surrounding the cell, wherein binding of the ligand to the cell surface receptor initiates a specific signal at the nucleus of the cell, said cell comprising:
(a) a first DNA construct having a sequence comprising
(i) a first set of one or more transcription control elements comprising a chimeric promoter, said first set of one or more transcription control elements being inducible by the signal initiated by the binding of said ligand to the cell surface receptor, and
(ii) a portion encoding a first measurable tag, driven by said first set of one or more transcription control elements, which tag can be detected when the first set of one or more transcription control elements is induced by the signal initiated by the binding of the ligand to the cell surface receptor; and
(b) a second DNA construct having a sequence comprising:
(i) a second set of one or more transcription control elements different from said first set; and
(ii) a portion encoding the ligand and a signal peptide that causes secretion of the ligand to the external environment surrounding the cell, said portion being driven by said second set of one or more transcription control elements.
2. The cell of claim 1 , wherein said second set of one or more transcription control elements comprises an inducible transcription control element which is inducible by a first protein that is not the same as the ligand.
3. The cell of claim 2 , wherein said inducible transcription control element is a tetracycline (Tet)-responsive element (TRE) and wherein the cell further comprises an additional DNA construct that constitutively expresses a reverse tetracycline repressor (rTetR) that turns off/represses the action of said inducible TRE in the absence of tetracycline or doxycycline as an inducer in a Tet-On expression system.
4. The cell of claim 2 , wherein said inducible transcription control element is a tetracycline (Tet)-responsive element (TRE) and wherein the cell further comprises an additional DNA construct that constitutively expresses a tetracycline-controlled transactivator (tTA) that binds to said inducible TRE in the absence of tetracycline or doxycycline to activate transcription from TRE as a Tet-Off expression system.
5. The cell of claim 2 , further comprising:
(c) a third DNA construct having a sequence comprising
(i) a third set of one or more transcription control elements different from the first and second set, and being inducible by a second protein that is not the same as the ligand or the first protein; and
(ii) a portion encoding an antagonist for the ligand, driven by said third set of one or more transcription control elements.
6. The cell of claim 5 , wherein the sequence of said third DNA construct further comprises, on a separate cistron from said portion encoding the antagonist for the ligand, a second portion also driven by said third set of one or more transcription control elements and encoding a third measurable tag, which third tag is independently measured in the presence of said first tag.
7. The cell of claim 6 , wherein the sequence of said second DNA construct further comprises, on a separate cistron from said portion encoding the ligand, a second portion also driven by said second set of one or more transcription control elements and encoding a second measurable tag, which second tag is independently measured in the presence of said first tag and said third tag.
8. The cell of claim 1 , wherein the ligand is a cytokine.
9. The cell of claim 1 , wherein the ligand is an interferon.
10. The cell of claim 9 , wherein said first set of one or more transcription control elements comprises an interferon stimulatory response element (ISRE).
11. The cell of claim 1 , wherein the ligand is tumor necrosis factor α (TNFα).
12. The cell of claim 11 , wherein said first set of one or more transcription control elements comprises an NFκB binding site.
13. The cell of claim 1 , wherein the ligand is erythropoietin (EPO).
14. The cell of claim 13 , wherein said first set of one or more transcription control elements comprises a signal transducer and activator of transcription #5 element (STAT5).
15. The cell of claim 1 , which is a mammalian or avian cell.
16. The cell of claim 1 , which is a human cell.
17. The cell of claim 1 , which is a human promonocytic cell.
18. The cell of claim 17 , wherein the human promonocytic cell is a U937 cell.
19. The cell of claim 1 , wherein the sequence of said second DNA construct further comprises, on a separate cistron from said portion encoding the ligand, a second portion also driven by said second set of one or more transcription control elements and encoding a second measurable tag, which second tag is independently measured in the presence of said first tag, and vice versa.
20. The cell of claim 19 , wherein said first or second measurable tag is a luciferase.
21. The cell of claim 20 , wherein said luciferase is Renilla luciferase.
22. The cell of claim 20 , wherein said luciferase is firefly luciferase.
23. The cell of claim 19 , wherein said first and second measurable tags are different luciferases.
24. A kit for determining the level in a sample of a ligand selected from the group consisting of a cytokine and a growth factor in the external environment surrounding the cell, wherein binding of the ligand to the cell surface receptor initiates a ligand-specific signal at the nucleus of a cell, comprising:
a reagent containing a plurality of the cell of claim 1 ; and
either a testing device having a plurality of wells or a container.
25. The kit of claim 24 , wherein said reagent is disposed in the wells of said testing device or in said container.
26. The kit of claim 24 , wherein said reagent is frozen.
27. A method for determining the level in a sample of a ligand selected from the group consisting of a cytokine and a growth factor in the external environment surrounding the cell that initiates a ligand-specific signal at the nucleus of a cell when binding to a cell surface receptor on the cell, or of a neutralizing antibody either against the ligand or against an antagonist of the ligand, comprising:
incubating the cell of claim 1 in a mixture with a sample in which the level of the ligand or the neutralizing antibody is sought to be determined; and
determining the level of the first measurable tag in the mixture relative to the level of the first measurable tag in the absence of the sample to thereby determine the level in the sample of the ligand or neutralizing antibody.