Enzyme-linked immunosorbent assay for human proapolipoprotein A-I using specific antibodies against synthetic peptide.
Apolipoprotein A-I (apoA-I), the major protein component of human high density lipoprotein, appears intracellularly as an intermediate precursor (proapoA-I) with a hexapeptide extension (Arg-His-Phe-Trp-Gln-Gln) at its amino terminus. To investigate the regulation of processes that regulate plasma apoA-I levels, a sensitive and simple assay for proapoA-I is required. We describe a specific enzyme-linked immunosorbent assay (ELISA) for quantification of proapoA-I using monospecific rabbit antibodies raised against the peptide: Arg-His-Phe-Trp-Gln-Gln-Asp-Glu-Pro. The monospecificity of antibodies to propeptide has been checked and no cross-reaction with mature apoA-I has been found although three first mature apoA-I amino acids (Asp-Glu-Pro) were included in the immunizing peptide. The assay is a non-competitive sandwich ELISA in which polystyrene microtiter plates were used with antibodies to propeptide adsorbed on the wells. After incubation with plasma samples, the bound proapoA-I was revealed by labeled rabbit polyclonal antibodies directed against mature apoA-I. The working range was 10 to 100 ng/ml, recovery of proapoA-I added to plasma was 94.6 to 106.5%, and the intra- and interassay coefficients of variation were 3.8% and 7.9%, respectively. A delipidation step using diisopropylether-n-butanol was necessary to expose antigen sites of proapoA-I in native lipoproteins. Mean level of proapoA-I in normal subjects was 87 +/- 15 micrograms/ml. It represented 7.1% of total apoA-I while in Tangier serum it represented 29%.
Barkia
A
A
SERLIA, Institut Pasteur, Lille, France.
Martin
C
C
Puchois
P
P
Gesquiere
J C
JC
Cachera
C
C
Tartar
A
A
Fruchart
J C
JC
D016632:Apolipoprotein A-I; D001054:Apolipoproteins A / Q000097:blood; D002138:Calibration; D005260:Female; D006801:Humans; D007106:Immune Sera; D007124:Immunoenzyme Techniques; D008297:Male; D010455:Peptides / Q000276:immunology; D011498:Protein Precursors / Q000097:blood; D013631:Tangier Disease / Q000097:blood
D016632:Apolipoprotein A-I; D001054:Apolipoproteins A; D007106:Immune Sera; D010455:Peptides; D011498:Protein Precursors