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.
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.
Malle
E
E
Karl-Franzens University Graz, Institute of Medical Biochemistry, Austria.
Münscher
G
G
Müller
T
T
Vermeer
H
H
Ibovnik
A
A
D000911:Antibodies, Monoclonal / Q000276:immunology; D002846:Chromatography, Affinity; D004591:Electrophoresis, Polyacrylamide Gel; D004797:Enzyme-Linked Immunosorbent Assay / Q000379:methods; D018604:Epitope Mapping / Q000379:methods*; D005063:Europium; D005455:Fluorescent Antibody Technique; D015200:Fluoroimmunoassay; D006801:Humans; D007106:Immune Sera / Q000276:immunology; D015151:Immunoblotting; D007074:Immunoglobulin G / Q000276:immunology; D007525:Isoelectric Focusing; D000685:Serum Amyloid A Protein / Q000276:immunology* / Q000302:isolation & purification
D000911:Antibodies, Monoclonal; D007106:Immune Sera; D007074:Immunoglobulin G; D000685:Serum Amyloid A Protein; D005063:Europium