Secreted Reporter-Peptides for Optimizing Cell-Based Assays for Analysis on Immuno-Assay Platforms

    公开(公告)号:US20220315919A1

    公开(公告)日:2022-10-06

    申请号:US16758694

    申请日:2018-10-25

    摘要: 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.

    DNA-RNA hybrid particles and manufacturing method thereof

    公开(公告)号:US10253317B2

    公开(公告)日:2019-04-09

    申请号:US15321748

    申请日:2015-06-30

    摘要: Disclosed are particles which are introduced into target cells and suppress the expression of specific genes, and a method of manufacturing such particles. More particularly, the present invention relates to DNA-RNA hybrid particles that comprise a DNA strand and an RNA strand that binds to the DNA strand through partial complementary base pairing, in which the DNA strand comprises an aptamer sequence that is able to bind to a target protein produced in a target cell, and the RNA strand comprises an siRNA sequence that binds to a target RNA in the target cell to suppress protein expression from the target RNA. Such hybrid particles are capable of effectively delivering an siRNA therapeutic agent into target cells for the treatment of disease, and have resistance against digestion by in vivo nucleases, DNase and RNase, owing to complementary binding formed between DNA and RNA strands. Also, the present invention relates to a method of manufacturing such DNA-RNA hybrid particles.