Intentional Digestion of Target Protein for Detection by Competitive ELISA
Publication Date: 2015-Jan-27
The IP.com Prior Art Database
"Intentional digestion of target protein for detection by competitive ELISA"
Disclosed herein are methods and compositions for the intentional digestion of target proteins (e.g. with trypsin or other proteases) in human, animal, plant, etc., test samples followed by detection/quantification of one or more of the resulting peptides using a competitive ELISA design. In silico analysis may be used to determine the target peptide of interest focusing on antigenicity, uniqueness of sequence, solubility of the resulting peptide and/or other properties or characteristics of the protein. The antibodies or other protein binding molecules like aptamers, single chain antibodies, etc., used in the assays may be produced against one or more target protein-specific peptides to be detected. It is anticipated that use of the methods and compositions described herein would allow for quicker assay development time in at least one or more areas including but not limited to – 1) reducing the buffer component impact for proteins that are difficult to extract or solubilize, 2) producing a single assay that recognizes a conserved peptide across protein families, 3) allowing for unique peptide detection from digestion product regardless of protein conformation, and 4) detection of a specific peptide tag that can be attached to a protein of interest.
A well established method to detect and quantify small molecules is the competitive ELISA (Deshpande, 1996). Digesting the protein of interest into peptides allows for the transition from a sandwich ELISA to a competitive format. In this method the one or more peptides act as the surrogate for the protein of interest and its detection and quantification is representative of the entire protein. This method makes possible the comparison between proteins expressed in different systems, as it eliminates differences in protein conformation/folding that may be system dependent. For example protein expressed in eukaryotic versus prokaryotic systems or monocots versus dicots. The peptide specific binding molecule can be generated (Table 1) and optimized by immunization strategies, by screening or panning phage display, single chain antibody, aptamer, etc. or combinations thereof. In some cases, libraries (phage display, single chain antibody, aptamer, etc) or an optimized kit can be purchased. These or similar libraries and kits may be commercially available and purchased from vendors such as Creative-Biolabs, Lucigen, Dyax, Pierce and BasePair Biotechnologies. Any protein of interest may be used in the competitive ELISA. For example, the protein of interest may be endogenous/native or exogenous/transgenic with respect to the organism and/or sample. The protein may be natural or synthetic. The protein may be created from DNA sequences arising different sources or different proteins so that the resulting protein is a chimeric or fusion protein.