US 12,359,195 B2 · Secreted reporter-peptides for optimizing cell-based assays for analysis on immuno-assay platforms

Abstract

The present invention relates to small secreted reporter-peptides 15 to 150 amino-acids in length comprising a response element, activated by one or more transcription factors induced by the pharmacology active substance to be analyzed following its interaction with a specific intracellular or cell surface molecule, functionally linked to a response element, a TATA box, a signal peptide, anchor and detection sequences to which antibodies can be raised, and a poy-A tail. The anchor sequence may differ from one peptide to another or may be common to multiple secreted reporter-peptides such that all the peptides can be analyzed simultaneously by ELISA using a detection antibody labelled with for example HRP, or by the use of a commonly available immuno-detection platform such MSD, Gyros, AlphaLisa, or Biacore. The detection sequence is unique to each secreted reporter-peptide and may be labelled with a Sulfo-Tag that permits detection of the peptide on the MSD platform, or Alexa that permits detection on the Gyros platform, or digoxigenin that permits detection of on the PerkinElmer AlphaLISA platform, or left unlabeled for detection by SPR on a Biacore platform. The present invention provides i.a. a substantial improvement of cell-based assays for analysis using automated immune-detection platforms and allows simultaneous analysis of multiple analytes, multiple sampling from a single cell culture, and obviates the necessity to lyse cells and remove cell debris by centrifugation prior to analysis on an automated immuno-detection platform. The present invention also provides a means of increasing the dynamic range, sensitivity and reducing the cost of cell-based assays and can be applied to existing engineered cell lines, such as those containing a reporter-gene such as a luciferase reporter-gene obviating the necessity to extensively re-engineer cell lines containing multiple molecular constructs.

Claims13

Dependent claims

A polynucleotide comprising the following i) to iv) in the direction of 5′ to 3′:

i) a response element that is capable of binding one or more transcription factors and is a 5-fold tandem repeat of gal4 upstream activation sequence (UAS),
ii) a TATA box,
iii) a sequence that encodes a reporter peptide and comprises any one of the sequences set forth in SEQ ID NOS.:
1-3, and
iv) a polyadenylation sequence, and
wherein the transcription factors in i) are chimeric transcription factors comprising the trans-activation domain of Elk-1 fused to a synthetic DNA binding domain capable of binding to the gal4 UAS cis-acting regulatory sequence of the secreted reporter peptide, and
wherein the reporter peptide in iii) comprises a signal peptide, an anchor sequence and a detection sequence that are different from each other, and
wherein the anchor sequence and the detection sequence are selected from c-MYC, V5 protein, or VSV-G protein, and
wherein antibodies bind to the detection sequence and the anchor sequence.

6 more elements

Dependencies are inferred from the claim text.

Secreted reporter-peptides for optimizing cell-based assays for analysis on immuno-assay platforms

Life of this patent

Secreted reporter-peptides for optimizing cell-based assays for analysis on immuno-assay platforms

Life of this patent

Abstract

Claims13