Site-directed mutagenesis and structure-function analysis of the human apolipoprotein A-I. Relation between lecithin-cholesterol acyltransferase activation and lipid binding.

A number of immunological techniques--radioimmunoassay, enzyme-linked immunosorbent assay (ELISA), electroimmunoassay, radial immunodiffusion, and a variety of immunoprecipitin assays--have been used to quantify apolipoproteins in plasma. This paper outlines their technical details and discusses their major advantages and drawbacks. The most sensitive procedures, RIAs and ELISAS, are best suited to quantifying those apoproteins found in low concentration in plasma. Immunoturbidimetric assays, on the other hand, which are readily automated, are being widely used to quantify apolipoproteins A-I and B. Apolipoprotein quantification is complicated by the interaction of the proteins with lipids, which can often mask their antigenic determinants. This problem may be circumvented by pretreatment of the samples, by selection of appropriate standards, or by the use of polyclonal or monoclonal antibodies that interact with permanently exposed epitopes on the lipoproteins' surfaces. Our proposed methods for measurement of the individual apolipoproteins give consideration to these approaches.

Immunological assays of apolipoproteins in plasma: methods and instrumentation.

Potential involvement of dissociated apoA-I in the ABCA1-dependent cellular lipid release by HDL.

Apolipoprotein A1 (apo A1), the major protein of high-density lipoprotein, is secreted as a proprotein and then cleaved by an uncharacterized metalloproteinase. Here this enzyme is identified as C-terminal procollagen endoproteinase/bone morphogenetic protein-1 (BMP-1). Studies with recombinant BMP-1, BMP-1 antibody, and BMP-1 siRNA establish this proteinase as the major or only apo A1-converting activity secreted by human liver-derived (HepG2) cells and CHO cells stably expressing human apo A1. BMP-1 stimulates the conversion of newly secreted proapo A1 to its phospholipid- (PL-) binding form. In this way it promotes formation of functional HDL and reverse cholesterol transport, while inhibiting filtration and clearance of uncleaved proprotein. Alpha2-macroglobulin, a protease inhibitor secreted as part of the innate immune response, inhibits BMP-1 activity and blocks the maturation of proapo A1. The decrease in circulating apo A1 levels that is characteristic of the response to inflammation and infection may be mediated, at least in part, via BMP-1 by this novel mechanism.

Bone Morphogenetic Protein-1 (BMP-1) Cleaves Human Proapolipoprotein A1 and Regulates Its Activation for Lipid Binding†

Regulation of apoAI processing by procollagen C-proteinase enhancer-2 and bone morphogenetic protein-1

Enzyme-linked immunosorbent assay for human proapolipoprotein A-I using specific antibodies against synthetic peptide.

Chemical approach to synthetic immunogens in clinical chemistry

Apolipoprotein A-I (apoA-I), the major protein com- ponent of human high density lipoprotein, appears intracellularly as an intermediate precursor (proapd-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 mono- specific 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 apd-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 apd-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 f 15 pg/ml. It represented 7.1% of total apoA-I while in Tangier serum it represented 29%. - Barkia, A., C. Martin, P. Puchois, J. C. Gesquiere, C. Cachera, A. Tartar, and J. C. Fruchart. Enzyme-linked immunosorbent assay for human proapolipoprotein A-I using specific antibodies against synthetic peptide. J. Lipid Res. 1988. 29: 77-84.

papers and notes on I methdobgy Enzyme-linked immunosorbent assay for human proapolipoprotein A4 using specific antibodies against synthetic peptide

http://link.springer.com/content/pdf/10.1007/978-94-010-9595-2_161.pdf

Use of synthetic peptides for specific assays of proproteins: Assay of proapolipoprotein A-I

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Enzyme-linked immunosorbent assay for human proapolipoprotein A-I using specific antibodies against synthetic peptide.

Chemical approach to synthetic immunogens in clinical chemistry

papers and notes on I methdobgy Enzyme-linked immunosorbent assay for human proapolipoprotein A4 using specific antibodies against synthetic peptide

Use of synthetic peptides for specific assays of proproteins: Assay of proapolipoprotein A-I