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Ligand-based sandwich assay for detection of recombinant expressed protein

IP.com Disclosure Number: IPCOM000249625D
Publication Date: 2017-Mar-08
Document File: 8 page(s) / 414K

Publishing Venue

The IP.com Prior Art Database

Abstract

Disclosed herein are methods and compositions for the detection and quantification of recombinant proteins of interest with the addition of small epitope tags expressed in microbes, animal, plant, etc., using ligand specific sandwich assay. Expression constructs are designed with two or more epitope tags on the N-terminus and/or C-terminus and/or within the body of the gene of interest. The tag-specific antibodies or other protein binding molecules like aptamers, single chain antibodies, etc., are then used within an assay platform(s) designed for protein detection and quantification. It is anticipated that use of the methods and compositions described herein would allow for: detection and quantitation of recombinant proteins without the need for ligands specific to the protein of interest; using of a variety of platforms from traditional ELISA to the more contemporary "no wash" systems (e.g. AlphaLISATM, TR-FRET) for protein detection and quantitation; that are amenable to high throughput analysis through automation.

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“Ligand-based sandwich assay for detection of recombinant expressed protein." Summary Disclosed herein are methods and compositions for the detection and quantification of recombinant proteins of interest with the addition of small epitope tags expressed in microbes, animal, plant, etc., using ligand specific sandwich assay. Expression constructs are designed with two or more epitope tags on the N-terminus and/or C-terminus and/or within the body of the gene of interest. The tag-specific antibodies or other protein binding molecules like aptamers, single chain antibodies, etc., are then used within an assay platform(s) designed for protein detection and quantification. It is anticipated that use of the methods and compositions described herein would allow for: detection and quantitation of recombinant proteins without the need for ligands specific to the protein of interest; using of a variety of platforms from traditional ELISA to the more contemporary “no wash” systems (e.g. AlphaLISATM, TR- FRET) for protein detection and quantitation; that are amenable to high throughput analysis through automation. Rationale Recombinant protein may be expressed in a wide variety of expression systems. Constructs containing the gene of interest can be expressed in viral culture, or in microorganism cultures that include bacterial cultures such as Escherichia coli, or fungal cultures such as Saccharomyces cerevisiae as well as mammalian or other eukaryotic cells. Complex organisms may also be used as expression systems including but not limited to plants such as tobacco, insects, or animals such as mouse. Any protein, characterized or uncharacterized, could be expressed using recombinant techniques. The recombinant protein could be of known or unknown structure or function. Regardless of the organism used for expression, the recombinant protein’s coding gene could originate from any organism, including but not limited to virus, bacteria, fungus, eukaryotic cell line, plant or animal. When compared to the source, the protein’s coding gene may or may not be modified by addition, deletion or substitution of bases. The coding gene may be modified for optimized expression in the organism used for expression. The protein’s coding gene could be exactly as found in another organism or be entirely synthesized, bearing no resemblance to any known protein coding gene. It could be a dimer or a fusion of multiple protein species. It could be a protein of known medicinal value such as, but not limited to human growth factor or insulin. It could be a protein of value to research, such as, but not limited to an antibody, trypsin, pepsin or a CAS protein. It could be an enzyme of known or unknown function. It could be a viral protein or immunizing agent. Recombinant protein may be expressed for many purposes. For example, recombinant proteins can be produced with the intent of purification prior to use in downstream processes. As another example, recombinant protein expres...