Immunonephelometry of apolipoprotein A-II in plasma

A quantitative assay based on endpoint immunonephelometry was developed for human apolipoprotein A-II (apoA-II) in plasma or serum. Dilution of plasma samples with a 0.1 mol/L solution of sodium cholate enhanced the quantification. We used either purified apoA-II as the primary standard or plasma as a secondary standard. Results correlated well (r = 0.90) with those by a double-antibody radioimmunoassay for 63 serum samples from both normal and hyperlipemic individuals. The interassay coefficient of variation for the immunonephelometric assay was 7% within a working range between 0.05 and 0.7 microgram of apoA-II per sample (corresponding to a 1500-fold final dilution of serum). No extraction of samples with organic solvent is necessary if the triglyceride concentration is less than 4 g/L.     

Immunonephelometric determination of the apolipoprotein A-II

A fully mechanized immunonephelometric method is described for the rapid and specific determination of apolipoprotein A-II in serum. The method utilizes commercially available sheep antiserum against human apolipoprotein A-II. Nephelometry was performed with the Behring Nephelometer Analyzer (BNA). A single determination can be performed in 12 minutes, requiring 10 microliters sample volume. The measuring range is about 0.08 to 1.25 g/l apolipoprotein A-II. Precision is characterized by intra-assay coefficients of variation of 3.37%, 3.93% and 4.49% for apolipoprotein A-II concentrations of 1.22 g/l, 0.376 g/l and 0.185 g/l, and inter-assay coefficients of variation of 4.27% for an apolipoprotein A-II concentration of 0.404 g/l, respectively. Accuracy of the method is shown by the close correlation of results with those from radial immunodiffusion (r = 0.913, y = 1.091 x -0.033, n = 75).     

Competitive enzyme immunoassay for apolipoprotein A-II

A competitive enzyme immunoassay for apolipoprotein A-II was developed. Microtitre plates were used as a solid phase and coated with anti-apolipoprotein A-II antibodies. Purified apolipoprotein A-II, labelled with horseradish peroxidase was used as competing ligand. The assay was examined with respect to the optimal amounts of specific anti-apolipoprotein A-II antibodies and apolipoprotein A-II-enzyme conjugate. The displacement curves showed a good parallelism between serum and purified apolipoprotein A-II. Delipidation of serum did not affect the content of apolipoprotein A-II. Cross-reactivity with apolipoprotein A-I was minimal. The intra- and inter-assay coefficients of variation in the medium range were 9.0 and 11.8% respectively. The assay might be well-suited for clinical routine.     

Immunonephelometry of apolipoprotein A-II in hyperlipoproteinemic serum

Immunonephelometry of apolipoprotein A-II (apo A-II) in serum requires that factors responsible for nonspecific increases of light scattering be eliminated. Complete delipidation is not necessary for immunonephelometry of apo A-II; in fact, light scattering of immunocomplexes from delipidated HDL is greater than from isolated apo A-II. Therefore, using the isolated peptide as a standard does not suffice for measurements in serum. The concentration of protein influences the development of light scattering and must be considered in standardizing this measurement.     

Automated immunoturbidimetric assay of serum apolipoprotein A-II using the COBAS-BIO™ centrifugal analyser: Influence of hyperlipoproteinemia

A quantitative automated immunoturbidimetric procedure for the analysis of apolipoprotein A-II (Apo A-II) in human serum is described. Dilution of antibody with a 5% solution of PEG 6000 enhanced the quantification. The within- and between-assay coefficients of variation were less than 5%. Results correlated well with those obtained by classic electroimmunodiffusion and immunonephelometry. No extraction of samples with organic solvent was necessary, whatever the triglyceride concentration. Large lipoproteins such as VLDL and immunocomplexes did not affect the method, nor was there interference from icteric or hemolyzed serum or from serum with excessive hyperlipemia. Physiological values of Apo A-II were determined in a normal population. Concentrations were found to be age-dependent, and higher in women than in men. The procedure is very suitable for the rapid, precise, reproducible and inexpensive assay of Apo A-II.     

Serum apolipoprotein A-II, a biochemical indicator of alcohol abuse

The possible use of high density lipoprotein (HDL) cholesterol, HDL phospholipids, apolipoproteins (APO) A-I and A-II as markers of alcohol abuse was studied in 78 intemperate drinkers. The mean value for each of these parameters was higher in drinkers than in control subjects. The most significant increase was observed in the plasma apo A-II levels (+45%). A composite index of gammaglutamyltranspeptidase (GGTP) and apo A-II was superior to GGTP alone in discriminating drinkers (+14%). Moreover, apo A-II assay is simple to perform.     

Turbidimetric assay of IgG with use of single monoclonal antibodies

We describe a turbidimetric assay for quantifying total immunoglobulin G (IgG) in serum with use of a single monoclonal antibody. The reaction, monitored by a centrifugal analyzer, is technically simple, rapid, and precise. Buffer of low ionic strength and polyethylene glycol are required for formation of detectable antibody-antigen complexes. We measured IgG concentrations in 49 polyclonal sera (Group 1) and 84 sera containing monoclonal IgG (Group 2) in assays in which we used either of two anti-IgG monoclonal antibodies (HG6 or HG8). Results compared well with those obtained with a nephelometric assay involving polyclonal antiserum, except for sera from four persons of Group 2 whose immunoglobulins were not detected by antibody HG6. HG6 bound IgG from these four sera in a solid-phase binding assay. HG6 and HG8 recognize epitopes on the Fab and Fc regions of IgG, respectively, and they do not compete for binding to the whole molecule. However, use of the two monoclonal antibodies combined failed to improve the sensitivity or range of the assay. We conclude that light-scattering assays of IgG can be validly performed with a single monoclonal antibody.     

HDL apolipoprotein A-I and HDL apolipoprotein A-II concentrations in male company employees in Westphalia aged 40 years and older

The major structural components of high density lipoproteins were determined in the sera of 638 male employees aged 40 years and older. It was demonstrated that the HDL apolipoprotein A-I/HDL cholesterol ratio as well as the HDL apolipoprotein A-II/HDL cholesterol ratio are similarly correlated to a cumulative score of established risk factors for atherosclerosis. Most important, however, is the finding that the correlation of these ratios to the risk factor rating of atherosclerosis is found in subgroups with normal or elevated HDL cholesterol values. Furthermore, it is shown that the relative content of apolipoproteins A-I and A-II in individual HDL is partly dependent on the plasma concentration of HDL cholesterol and triglycerides. It is concluded that HDL composition may have an additional predictive significance for the development of atherosclerosis.     

Enzyme-linked immunosorbent assay for apolipoprotein C-I

A non-competitive sandwich enzyme-linked immunosorbent assay for apolipoprotein C-I was developed. Sheep antibody to this apolipoprotein C-I, purified by affinity chromatography, was used for coating the wells of a microtiter plate and as a conjugate with alkaline phosphatase. The linear range of the assay was from 80 ng to 15 ng. It was sensitive down to 5 ng. The intra-assay variation coefficient was 2.8%, and the inter-assay variation coefficient 5.3%. The mean concentration of apolipoprotein C-I was 61 +/- 20 mg/l in healthy normal males, and 65 +/- 19 mg/l in females. Apolipoprotein C-I levels were positively correlated with the total cholesterol concentration in both sexes (p less than 0.002). A significant correlation with triacylglycerol was only observed in males (p less than 0.05). A significant increase of apolipoprotein C-I was observed in type V hyperlipoproteinaemia, and in the only studied case of type III.     

Determination of apolipoprotein A and its constitutive A-I and A-II polypeptides by separate electroimmunoassays.

Electroimmunoassays ("rocket" electrophoresis) are described for human serum apolipoprotein A and its constitutive A-I and A-II polypeptides. Purified lipoprotein A, A-I, and A-II were used to prepare monospecific antisera and to standardize assays. These specific, rapid (5-8 h), precise (the within-and between-assay coefficients of variations are 5 and 7%, respectively), and accurate (by gravimetry) assays are applicable to measurement of these polypeptides in whole serum and in various density classes of lipoproteins. Comparable results are obtained with intact and delipidized lipoproteins. Results correlated well with those obtained by radial immunodiffusion or radioimmunoassay. However, the present procedure is more rapid than the former and simpler than the latter immunoassay. Concentrations of A-I and A-II in the serum of normal men and women were similar (143 +/- 24 and 146 +/- 78 mg/dl, respectively, for A-I and 78 +/- 17 and 83 +/- 25 mg/dl for A-II). Subjects with type lla, llb, and IV hyperlipoproteinemias had similar concentrations of both polypeptides, while patients with type I disease, lecithin:cholesterol acyltransferase deficiency and LP-A deficiency had lowest concentrations of A-I (0.3-30 mg/dl) and A-II (11-20 mg/dl). The molar ratio of A-I/A-II in the serum and high-density lipoproteins was close to unity.     

Identification of apolipoprotein A-II in cerebrospinal fluid of pediatric brain tumor patients by protein expression profiling

BACKGROUND: Our aim was to detect differences in protein expression profiles of cerebrospinal fluid (CSF) from pediatric patients with and without brain tumors. METHODS: We used surface-enhanced laser desorption/ionization time-of-flight (SELDI-TOF) mass spectrometry and Q10 ProteinChip arrays to compare protein expression profiles of CSF from 32 pediatric brain tumor patients and 70 pediatric control patients. A protein with high discriminatory power was isolated and identified by subsequent anion-exchange and reversed-phase fractionation, gel electrophoresis, and mass spectrometry. The identity of the protein was confirmed by Western blotting and immunohistochemistry. RESULTS: Of the 247 detected protein peak clusters, 123 were differentially expressed between brain tumor and control patients with a false discovery rate of 1%. Double-loop classification analysis gave a mean prediction accuracy of 88% in discriminating brain tumor patients from control patients. From the 123 clusters, a highly overexpressed protein peak cluster in CSF from brain tumor patients was selected for further analysis and identified as apolipoprotein A-II. Apolipoprotein A-II expression in CSF was correlated with the CSF albumin concentration, suggesting that the overexpression of apolipoprotein A-II is related to a disrupted blood-brain barrier. CONCLUSIONS: SELDI-TOF mass spectrometry can be successfully used to find differentially expressed proteins in CSF of pediatric brain tumor and control patients. Apolipoprotein A-II is highly overexpressed in CSF of pediatric brain tumor patients, which most likely is related to a disrupted blood-brain barrier. Ongoing studies are aimed at finding subtype specific proteins in larger groups of pediatric brain tumor patients.     

Gold-labeled nanoparticle-based immunoresonance scattering spectral assay for trace apolipoprotein AI and apolipoprotein B

BACKGROUND: Apolipoprotein AI (ApoAI) and ApoB are risk indicators of cardiovascular disease. We describe the use of immunoresonance scattering to measure the ApoAI and ApoB in serum. METHODS: We used a trisodium citrate method to prepare 9.0-nm gold nanoparticles labeled with goat anti-human ApoAI and ApoB antibodies. The immune reaction between gold-labeled antibodies and antigens took place in Na2HPO4-NaH2PO4 buffer solution (pH 6.4 for ApoAI and pH 6.0 for ApoB) in the presence of 75 g/L polyethylene glycol (PEG). We used a transmission electron microscope to observe the shape of the gold nanoparticles. Results were compared with those obtained by immunoturbidimetric methods. Twenty-five human serum samples were assayed by the immunoresonance scattering assay preset with the data indicated and by an immunoturbidimetric assay. RESULTS: The presence of PEG greatly enhanced the intensity of resonance-scattering peaks at 560 nm. The intensity (DeltaI) was proportional to concentration at 0.00833-0.3333 mg/L ApoAI and 0.00197-0.1972 mg/L ApoB. The detection limits were 2.04 and 0.96 microg/L for ApoAI and ApoB, respectively. The results for human serum samples were in agreement with those obtained with an immunoturbidimetric method. Linear regression analysis revealed a correlation coefficient, slope, and intercept of 0.915, 0.966, and 68.53 mg/L, respectively, for ApoAI and 0.919, 0.996, and 15.46 mg/L for ApoB. CONCLUSION: This method showed high sensitivity and good selectivity for quantitative determination of ApoAI and ApoB in human serum, with satisfactory results.     

Enzyme linked immunosorbent assay for human apolipoprotein C-III

A sandwich enzyme linked immunosorbent assay (ELISA) for apolipoprotein C-III was developed. Anti apolipoprotein C-III immunoglobulins were used both for coating of microtitre plates and for the preparation of an anti apolipoprotein C-III horse-radish peroxidase conjugate. Under optimized assay conditions, the sensitivity lies around 0.3 ng apolipoprotein C-III with a working range of 1 to 6 ng. Standard curves are parallel for purified apolipoprotein C-III, for untreated plasma and for lipoprotein fractions. Delipidation did not affect the content of apolipoprotein C-III in plasma. The assay was evaluated by comparison with an immunonephelometric assay for apolipoprotein C-III, yielding a correlation coefficient of 0.982 (n = 79). The mean intra- and interassay CV for the whole working range of the assay were 3.6% and 4.2% respectively.     

Nephelometric assay of apolipoprotein A-I with a centrifugal analyzer

We developed an automated immunonephelometric assay for quantification of human apolipoprotein A-I (apo A-I) with a fluorescence light-scattering microcentrifugal analyzer. The presence of polyethylene glycol and Tween 20 in the reaction mixture ensures maximum exposure of the antigenic sites of the apoprotein so that immune complex formation occurs more rapidly (reaction is complete within 2 min) and to a greater extent. Lipemia and hemolysis do not interfere with the measurement of apo A-I. The method requires only 10 microL of specimen and is fast and easy to perform. Results vary linearly with apo A-I concentrations to 2.5 g/L. Assay precision (CV) was 3.1% for a specimen with an apo A-I concentration of 1.45 g/L, and the lower limit of detection was 0.15 g/L. Values for a candidate Reference Material agree well with those reported in an international survey (Clin Chem 1985;30:223-8).     

Determination of apolipoprotein B in apolipoprotein CII/CIII-containing lipoproteins by an immunoenzymmetric assay.

A solid phase sandwich immunoenzymmetric assay is described for the determination of apolipoprotein B in apolipoprotein CII- and/or CIII-containing lipoproteins (chylomicron remnants, VLDL, IDL). During a first incubation step the particles containing apolipoproteins CII and/or CIII are bound to antibodies against these components, while the antibodies are immobilised on microtitre plate wells. After washing, anti-apolipoprotein B is added in a second incubation step. Finally peroxidase-labelled anti-sheep IgG reacts with the bound anti-apolipoprotein B, thus allowing the quantification of complexes containing both B and CII/CIII apolipoproteins. The amounts of these complexes were correlated with total cholesterol, triacylglycerols, HDL- and LDL-cholesterol, apolipoproteins AI and B, as well as with age and sex of the subject. A total of 258 individuals was studied, including patients with lipid metabolism disorders, patients with manifest coronary heart disease, and healthy controls. The assay described in this article was compared with radial immunodiffusion after pretreatment of samples. The immunoenzymmetric assay was easier and faster to perform and had a lower detection limit than the classical VLDL apolipoprotein B determination using ultracentrifugation. Furthermore, it could be performed directly on native serum. Most importantly, the study revealed elevated apolipoprotein CII/CIII-B levels in coronary heart disease patients of both sexes compared with normal subjects. Furthermore, in male coronary heart disease patients a negative correlation was found between the concentrations of apolipoprotein CII/CIII-B and HDL-cholesterol. These results suggest a delayed VLDL-HDL exchange of lipids and proteins in these patients which results in an accumulation of atherogenic apolipoprotein B-containing VLDL and IDL.     

Human apolipoprotein C-II quantitation by sandwich enzyme-linked immunosorbent assay

A specific, sensitive and accurate, non competitive enzyme-linked immunosorbent assay was developed for the quantitation of human apolipoprotein C-II. Using apolipoprotein C-II and apolipoprotein C-III immunosorbent columns, monospecific anti-apolipoprotein C-II antibodies were prepared for coating and for the preparation of a peroxidase-antibody conjugate. The assay is sensitive down to 0.25 ng apolipoprotein C-II per assay and precise, with mean intra- and inter-assay coefficients of variation of 3.1% and 7.9% respectively. The apolipoprotein C-II concentrations in normolipaemic and hyperlipaemic plasma were not affected by delipidation, and increased only slightly after treatment with detergents or urea. The mean plasma apolipoprotein C-II concentration in a group of 30 normolipaemic subjects, was 33.1 +/- 7.5 mg/l. All hypertriglyceridaemic subjects had significantly elevated apolipoprotein C-II plasma concentrations, which were most pronounced in Fredrickson type III and type V patients. The apolipoprotein C-II profiles, obtained by column fractionation of 6 normolipaemic and 11 hypertriglyceridaemic plasmas, demonstrated a shift of apolipoprotein C-II towards the triglyceride-rich lipoproteins in hypertriglyceridaemic subjects.     

Differential turbidimetric assay for subpopulations of lipoproteins containing apolipoprotein A-I

A differential immunoturbidimetric procedure for the quantitation of apolipoprotein A-I associated with lipoproteins LpA (containing both apolipoprotein A-I and apolipoprotein A-II) and with lipoproteins LpA-I (containing apolipoprotein A-I but no apolipoprotein A-II) is presented. Lipoproteins containing apolipoprotein A-II are precipitated with an anti-apolipoprotein A-II antibody. The resulting immunoprecipitate is sedimented and LpA-IA-I is measured in the supernate. Whereas LpA-IA-I concentrations differed significantly between normolipidaemic men and women (0.75 and 1.00 g/l, respectively), there was virtually no sex related difference in LpAA-I (0.83 and 0.88 g/l, respectively). LpA-IA-I was predominantly correlated with HDL2-cholesterol (rs = 0.630), whereas LpAA-I was statistically associated with HDL3 (rs = 0.417).     

Elevated high density lipoprotein cholesterol levels correlate with decreased apolipoprotein A-I and A-II fractional catabolic rate in women.

High levels of HDL-cholesterol (HDL-C) protect against coronary heart disease susceptibility, but the metabolic mechanisms underlying elevated HDL-C levels are poorly understood. We now report the turnover of isologous radioiodinated HDL apolipoproteins, apo A-I and apo A-II, in 15 female subjects on a metabolic diet with HDL-C levels ranging from 51 to 122 mg/dl. The metabolic parameters, fractional catabolic rate (FCR) and absolute synthetic rate (SR), were determined for apo A-I and apo A-II in all subjects. There was an inverse correlation between plasma HDL-C and the FCR of apo A-I and apo A-II (r = -0.75, P less than 0.001, and r = -0.54, P = 0.036, respectively), but no correlation with the SR of either apo A-I or apo A-II (r = 0.09, and r = -0.16, respectively, both P = NS). Apo A-I levels correlated inversely with apo A-I FCR (r = -0.64, P = 0.01) but not with apo A-I SR (r = 0.30, P = NS). In contrast, plasma levels of apo A-II did not correlate with apo A-II FCR (r = -0.38, P = 0.16), but did correlate with apo A-II SR (r = 0.65, P = 0.009). Further analysis showed that apo A-I and apo A-II FCR were inversely correlated with the HDL-C/apo A-I + A-II ratio (r = -0.69 and -0.61, P = 0.005 and 0.015, respectively). These data suggest that: (a) low HDL apolipoprotein FCR is the predominant metabolic mechanism of elevated HDL-C levels; (b) apo A-I FCR is the primary factor in controlling plasma apo A-I levels, but apo A-II SR is the primary factor controlling plasma apo A-II levels; (c) low HDL apolipoprotein FCR is associated with a lipid-rich HDL fraction. These findings elucidate aspects of HDL metabolism which contribute to high HDL-C levels and which may constitute mechanisms for protection against coronary heart disease.