Identification and characterization of human eosinophil cationic protein by an epitope-specific antibody.
The eosinophil cationic protein (ECP) is a basic secretion protein involved in the immune response system. ECP levels in biological fluids are an indicator of eosinophil-specific activation and degranulation and are currently used for the clinical monitoring and diagnosis of inflammatory disorders. A polyclonal epitope-specific antibody has been obtained by immunizing rabbits with a conjugated synthetic peptide. A sequence corresponding to a large exposed loop in the human ECP three-dimensional structure (D115-Y122) was selected as a putative antigenic epitope. The antibody was purified on an affinity column using recombinant ECP (rECP) as antigen. The antibody (D112-P123 Ab) specifically recognizes rECP and its native glycosylated and nonglycosylated forms in plasma, granulocytes, and sputum. The antibody detects as little as 1 ng of rECP, can be used both in reducing and nonreducing conditions, and does not cross-react with the highly homologous eosinophil-derived neurotoxin or other proteins of the pancreatic ribonuclease superfamily.
Boix
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Departament de Bioquímica i Biologia Molecular, Facultat de Ciències, Universitat Autònoma de Barcelona, 08193-Bellaterra, Spain.
Carreras
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Nikolovski
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Cuchillo
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CM
Nogués
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MV
D000595:Amino Acid Sequence; D000818:Animals; D000911:Antibodies, Monoclonal / Q000276:immunology*; D000918:Antibody Specificity; D001249:Asthma / Q000378:metabolism; D001798:Blood Proteins / Q000032:analysis* / Q000737:chemistry / Q000276:immunology; D015153:Blotting, Western; D003429:Cross Reactions; D004797:Enzyme-Linked Immunosorbent Assay; D047091:Eosinophil Granule Proteins; D004804:Eosinophils / Q000737:chemistry; D000939:Epitopes / Q000276:immunology; D006031:Glycosylation; D006098:Granulocytes / Q000737:chemistry; D006801:Humans; D008958:Models, Molecular; D008969:Molecular Sequence Data; D010949:Plasma / Q000737:chemistry; D011487:Protein Conformation; D011489:Protein Denaturation; D011499:Protein Processing, Post-Translational; D011817:Rabbits; D012260:Ribonucleases; D016415:Sequence Alignment; D017386:Sequence Homology, Amino Acid; D013183:Sputum / Q000737:chemistry
D000911:Antibodies, Monoclonal; D001798:Blood Proteins; D047091:Eosinophil Granule Proteins; D000939:Epitopes; D012260:Ribonucleases