ABS1030 Sigma-AldrichAnti-Apolipoprotein D Antibody

Lipoprotein metabolism in brain has not yet been fully elucidated, although there are a few reports concerning lipids in the brain and lipoproteins and apolipoproteins in the cerebrospinal fluid (CSF). To establish normal levels of lipoproteins in human CSF, total cholesterol, phospholipids, and fatty acids as well as apolipoprotein E (apoE) and apoA-I levels were determined in CSF samples from 216 individuals. For particle characterization, lipoproteins from human CSF were isolated by affinity chromatography and analyzed for size, lipid and apolipoprotein composition. Two consecutive immunoaffinity columns with antibodies, first against apoE and subsequently against apoA-I, were used to define four distinct lipoprotein classes. The major lipoprotein fraction consisted of particles of 13;-20 nm containing apoE and apoA-I as well as apoA-IV, apoD, apoH, and apoJ. In the second particle class (13;-18 nm) mainly apoA-I and apoA-II but no apoE was detected. Third, there was a small number of large particles (18;-22 nm) containing no apoA-I but apoE associated with apoA-IV, apoD, and apoJ. In the unbound fraction we detected small particles (10;-12 nm) with low lipid content containing apoA-IV, apoD, apoH, and apoJ. In summary, we established lipid and apolipoprotein levels in CSF in a large group of individuals and described four distinct lipoprotein classes in human CSF, differing in their apolipoprotein pattern, lipid composition, and size. On the basis of our own data and previous findings from other groups, we propose a classification of CSF lipoproteins.

We have previously shown that plasma lipoproteins can be separated by analytical capillary isotachophoresis (ITP) according to their electrophoretic mobility in a defined buffer system. As in lipoprotein electrophoresis, HDL show the highest mobility followed by VLDL, IDL, and LDL. Chylomicrons migrate according to their net-charge between HDL and VLDL, because ITP has negligible molecular sieve effects. Three HDL subfractions were obtained which were designated fast-, intermediate-, and slow-migrating HDL. To further characterize these HDL subfractions, a newly developed free-solution ITP (FS-ITP)-system was used, that allows micro-preparative separation of human lipoproteins directly from whole plasma (Böttcher, A. et al. 1998. Electrophoresis. 19: 1110-1116). The fractions obtained by FS-ITP were analyzed for their lipid and apolipoprotein composition and by two-dimensional nondenaturing polyacrylamide gradient gel electrophoresis (2D-GGE) with subsequent immunoblotting. fHDL are characterized by the highest proportion of esterified cholesterol of all three subfractions and are relatively enriched in LpA-I. Together with iHDL they contain the majority of plasma apoA-I, while sHDL contain the majority of plasma apoA-IV, apoD, apoE, and apoJ. Pre-beta-HDL were found in separate fractions together with triglyceride-rich fractions between sHDL and LDL. In summary, ITP can separate the bulk of HDL into lipoprotein subfractions, which differ in apolipoprotein composition and electrophoretic mobility. While analytical ITP permits rapid separation and quantitation for diagnostic purposes, FS-ITP can be used to obtain these lipoprotein subfractions on a preparative scale for functional analysis. As FS-ITP is much better suited for preparative purposes than gel electrophoresis, it represents an important novel tool for the functional analysis of lipoprotein subclasses.

Apolipoprotein D (apoD), also known as gross cystic disease fluid protein-24 (GCDFP-24), is a minor protein moiety of high-density lipoproteins in human plasma. ApoD is expressed in a subset of breast carcinomas and has been proposed as a tumor marker and prognostic indicator for breast cancer progression. Here we describe a new sensitive time-resolved fluorimetric immunoassay for quantification of human apoD in biological specimens using affinity-purified polyclonal anti-human apoD rabbit antibodies and Eu3+ as a specific probe. Both purified apoD and normal human pool-serum served as reliable primary and secondary standards in the direct sandwich dissociation-enhanced lanthanide fluorescence immunoassay (DELFIA). Plasma apoD concentrations measured by the DELFIA were 99.6 +/- 32 microg/ml. The detection limit of the DELFIA procedure was 0.5 ng/ml after sample dilution of 1/8000. The intra-assay coefficient of variation averaged 3.5%, whereas the inter-assay coefficient of variation averaged 6.9%. The concentration of apoD in breast cyst fluids ranged from 6.82 to 28.37 mg/ml. Based on the low detection limit and the high specificity of the DELFIA procedure, we have applied this technique for the measurement of apoD in breast cancer cell supernatants. In estrogen-receptor positive cells, i.e., T-47D and ZR-75-1 cells, 42.6 +/- 1.4 and 2.7 +/- 0.2 ng apoD/ml supernatant after 4 days in culture without induction of apoD synthesis were measured. A comparison of the direct sandwich DELFIA procedure with an electroimmunoassay commonly used to assay apoD revealed correlation coefficients of 0.986 (serum) and 0.975 (cyst fluids). The present findings indicate that the direct sandwich DELFIA is appropriate for apoD quantification in plasma and breast cyst fluids. Furthermore, the technique should permit studies on the induction of apoD synthesis in the low picomolar range in different carcinoma cells to gain insight into the expression of this atypical apolipoprotein.