Competition between bound and free peptides in an ELISA-based procedure that assays peptides derived from protein digests

BACKGROUND: We describe an ELISA-based method that can be used to identify and quantitate proteins in biological samples. In this method, peptides in solution, derived from proteolytic digests of the sample, compete with substrate-attached synthetic peptides for antibodies, also in solution, generated against the chosen peptides. The peptides used for the ELISA are chosen on the basis of their being (i) products of the proteolytic (e.g. tryptic) digestion of the protein to be identified and (ii) unique to the target protein, as far as one can know from the published sequences. RESULTS: In this paper we describe the competition assay and we define the optimal conditions for the most effective assay. We have performed an analysis of the kinetics of interaction between the four components of the assay: the plastic substratum to which the peptide is bound, the bound peptide itself, the competing added peptide, and the antibody that is specific for the peptide and we compare the results of theoretical simulations to the actual data in some model systems. CONCLUSION: The data suggest that the peptides bind to the plastic substratum in more than one conformation and that, once bound, the peptide displays different affinities for the antibody, depending on how it has bound to the plat

An ELISA-based procedure for assaying proteins in digests of human leukocytes and cell lines, using specifically selected peptides and appropriate antibodies

BACKGROUND: We describe the application of an ELISA-based assay (the Peptidomatrix) that can be used to simultaneously identify and quantitate a number of proteins in biological samples. The biological sample (blood component, biopsy, culture or other) is first lysed to release all the proteins, without any additional separation. The denatured proteins in the sample are then digested in bulk with the desired proteolytic enzyme(s). The peptides in the digest are then assayed by appropriate antibodies, using a competition ELISA protocol. RESULTS: As an example of its use, the present paper applies the Peptidomatrix to the assay of four membrane proteins MDR1 (P-glycoprotein or ABCB1), MRP1 (ABCC1), BCRP/MXR (ABCG2) and the alpha subunit of the Na, K_ATPase (ATP1A1), present in a number of cell lines and in human lymphocytes. We show that we can detect and quantitate these proteins, using a series of peptide-antibody pairs, and that we can differentiate between cell lines or cell preparations that express the target proteins and those that do not. CONCLUSION: We have devised a simple, ELISA-based proteomics assay that enables the quantitation of designated proteins in a cell or tissue sample, and that can be used in any laboratory, with minimal specialized equipment

A role for α-Synuclein in axon growth and its implications in corticostriatal glutamatergic plasticity in Parkinson’s disease

Background α-Synuclein (α-Syn) is a protein implicated in the pathogenesis of Parkinson’s disease (PD). α-Syn has been shown to associate with membranes and bind acidic phospholipids. However, the physiological importance of these associations to the integrity of axons is not fully clear. Methods Biochemical, immunohistochemical and ultrastructural analyses in cultured neurons, transgenic mouse brains, PD and control human brains. Results We analyzed the ultrastructure of cross-sectioned axons localized to white matter tracts (WMTs), within the dorsal striatum of old and symptomatic α-Syn transgenic mouse brains. The analysis indicated a higher density of axons of thinner diameter. Our findings in cultured cortical neurons indicate a role for α-Syn in elongation of the main axon and its collaterals, resulting in enhanced axonal arborization. We show that α-Syn effect to enhance axonal outgrowth is mediated through its activity to regulate membrane levels of the acidic phosphatidylinositol 4,5-bisphosphate (PI4,5P2). Moreover, our findings link α-Syn- enhanced axonal growth with evidence for axonal injury. In relevance to disease mechanisms, we detect in human brains evidence for a higher degree of corticostriatal glutamatergic plasticity within WMTs at early stages of PD. However, at later PD stages, the respective WMTs in the caudate are degenerated with accumulation of Lewy pathology. Conclusions Our results show that through regulating PI4,5P2 levels, α-Syn acts to elongate the main axon and collaterals, resulting in a higher density of axons in the striatal WMTs. Based on these results we suggest a role for α-Syn in compensating mechanisms, involving corticostriatal glutamatergic plasticity, taking place early in PD