title: Simultaneous detection of three CNS indicator proteins in complex suspensions using a single immuno-PCR protocol.
abstract: The diagnosis of infections and protection against their transmission are aided greatly by determination of indicator proteins. However, protein assays are mostly restricted to single-antigen determinations and are often limited in sensitivity and specificity. Consequently, there is a large demand for high-sensitivity immunoassays for analysis of several antigens in protein suspensions. A novel immuno-polymerase chain reaction (PCR) assay is described for the simultaneous detection of central nervous system (CNS) indicators such as the neuron-specific enolase, the glial fibrillary acid protein, and the cellular prion protein. Coated antigens are immunocomplexed with specific antibodies and a DNA fragment is subsequently amplified by PCR. The PCR product obtained corresponds to the antigen signal. Background signals are a critical factor, primarily when using complex protein suspensions, but we were able to reduce background noise dramatically by including two heating steps, the first for protein denaturation and the second for detachment of immunocomplexed DNA, enabling optimal DNA amplification. Using these methods and depending on the antigen and antibody affinity, a sensitivity enhancement of 2 to 3 orders of magnitude is achieved for CNS indicator detection using the immuno-PCR approach compared with ELISA procedures carried out under identical conditions.
authors.affiliation: Institute of Hygiene, University of Münster and University Hospital Münster, 48149 Münster, Germany. tkuczius@uni-muenster.de
authors.forename: Thorsten
authors.initials: T
authors.lastname: Kuczius
authors.forename: Karsten
authors.initials: K
authors.lastname: Becker
authors.forename: Andreas
authors.initials: A
authors.lastname: Fischer
authors.forename: Wenlan
authors.initials: W
authors.lastname: Zhang
mesh_terms: D000818:Animals; D000906:Antibodies / Q000737:chemistry / Q000276:immunology; D000936:Antigen-Antibody Complex; D002417:Cattle; D002490:Central Nervous System / Q000201:enzymology / Q000378:metabolism*; D004247:DNA / Q000737:chemistry / Q000378:metabolism; D003720:Densitometry; D005904:Glial Fibrillary Acidic Protein / Q000032:analysis* / Q000276:immunology; D010751:Phosphopyruvate Hydratase / Q000032:analysis* / Q000276:immunology; D016133:Polymerase Chain Reaction; D011328:Prions / Q000032:analysis* / Q000235:genetics / Q000276:immunology; D011489:Protein Denaturation; D011994:Recombinant Proteins / Q000032:analysis / Q000235:genetics / Q000378:metabolism; D012756:Sheep; D013535:Suspensions / Q000737:chemistry*
chemical_list: D000906:Antibodies; D000936:Antigen-Antibody Complex; D005904:Glial Fibrillary Acidic Protein; D011328:Prions; D011994:Recombinant Proteins; D013535:Suspensions; D004247:DNA; D010751:Phosphopyruvate Hydratase