Mapping of antigenic sites in human neuron-specific enolase by expression subcloning.
Human serum neuron-specific enolase (NSE) is a marker of neurons and of small-cell carcinoma of the lung; improved immunoassays of NSE remain an important goal. Here, we used overlapping complementary DNA (cDNA) clones for reconstruction to express full-length recombinant NSE, and also to express a set of cloned subfragments through the prokaryotic expression vectors pUEX and pUBEX. Subfragments expressed as fusion proteins were used to characterize immunogenic and antigenic regions and epitopes and, expressed as affinity matrices, to derive purified, fractionated polyclonal antibodies. NSE epitope data can be visualized with yeast enolase-1 crystal structure coordinates: The two protein sequences align almost perfectly and are 61% identical. This approach demonstrates the complementarity of cDNA expression with techniques of polyclonal antiserum and monoclonal antibody production and with chemical peptide synthesis in the refinement of immunodiagnostic reagents.
Quinn
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Department of Biochemistry, College of Medicine, University of South Florida, Tampa 33612-4799.
Reeves
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Day
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D000595:Amino Acid Sequence; D000941:Antigens / Q000032:analysis*; D001483:Base Sequence; D003001:Cloning, Molecular; D018076:DNA, Complementary / Q000235:genetics; D006801:Humans; D008969:Molecular Sequence Data; D008970:Molecular Weight; D010446:Peptide Fragments / Q000235:genetics; D010751:Phosphopyruvate Hydratase / Q000737:chemistry / Q000235:genetics / Q000276:immunology*; D011993:Recombinant Fusion Proteins; D011994:Recombinant Proteins / Q000276:immunology; D015183:Restriction Mapping; D017421:Sequence Analysis
D000941:Antigens; D018076:DNA, Complementary; D010446:Peptide Fragments; D011993:Recombinant Fusion Proteins; D011994:Recombinant Proteins; D010751:Phosphopyruvate Hydratase