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Malle, E; Münscher, G; Müller, T; Vermeer, H; Ibovnik, A.
Quantification and mapping of antigenic determinants of serum amyloid A (SAA) protein utilizing sequence-specific immunoglobulins and Eu3+ as a specific probe for time-resolved fluorometric immunoassay.
J Immunol Methods. 1995; 182(1):131-144 Doi: 10.1016%2F0022-1759%2895%2900035-9
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Leading authors Med Uni Graz: Malle Ernst
Co-authors Med Uni Graz: Müller Thomas
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Abstract:: Serum amyloid A (SAA) protein, the most prominent amongst acute-phase proteins, is the specific precursor protein of secondary reactive amyloidosis. The fact that SAA once released into the circulation as a 'free' protein rapidly associates with lipoproteins of the high-density range indicates a specific role in lipoprotein metabolism. In this study a new sensitive assay for quantification of human SAA protein in biological specimens using affinity-purified polyclonal antibodies and Eu3+ as a specific probe for time-resolved fluorometric immunoassay is presented. Both purified SAA and SAA-rich high-density lipoprotein particles served as reliable standards in the indirect and the direct sandwich dissociation-enhanced lanthanide fluorescence immunoassay (DELFIA). The detection limit of the DELFIA technique presented was 4-10 ng after sample dilution of 1/2500. The intra-assay coefficient of variation averaged 4.3% whereas the inter-assay coefficient of variation averaged 6.2%. Comparison with the nephelometric assay, a widely and commonly used assay for SAA quantification in plasma, revealed correlation coefficients of 0.9428. In addition to polyclonal anti-human SAA antibodies sequence-specific antibodies raised against synthetic peptides corresponding to region; 1-17, 14-30, 27-44, 40-63, 59-72, 68-84, 79-94, and 89-104 of the human SAA amino acid sequence were studied. Sequence-specific antibodies raised against epitopes 27-44, 59-72, 68-84, and 89-104 recognize human SAA protein in the DELFIA assay whereas antibodies raised against epitopes 1-17, 14-30, 40-63 and 79-94 failed to recognize the corresponding epitopes. Results obtained from these studies indicate that the N-terminal domain (1-30) as well as epitopes 40-63 and 79-94 of human SAA are apparently masked by the environment of the lipoprotein particle. From our studies it is proposed that the epitopes 31-39, 64-78, and 95-104 may be responsible for the interaction of SAA-rich high density lipoprotein particles with peripheral cells.
Find related publications in this database (using NLM MeSH Indexing): Antibodies, Monoclonal - immunology; Chromatography, Affinity - immunology; Electrophoresis, Polyacrylamide Gel - immunology; Enzyme-Linked Immunosorbent Assay - methods; Epitope Mapping - methods; Europium - methods; Fluorescent Antibody Technique - methods; Fluoroimmunoassay - methods; Humans - methods; Immune Sera - immunology; Immunoblotting - immunology; Immunoglobulin G - immunology; Isoelectric Focusing - immunology; Serum Amyloid A Protein - immunology
Find related publications in this database (Keywords): Serum Amyloid a Protein; Amyloidosis; Acute-Phase Reaction; High Density Lipoprotein; Apolipoprotein