Enzyme-linked immunoabsorbant assay of apolipoprotein AII in plasma, with use of a monoclonal antibody.

1986 ◽  
Vol 32 (6) ◽  
pp. 967-971 ◽  
Author(s):  
E A Stein ◽  
L DiPersio ◽  
A J Pesce ◽  
M Kashyap ◽  
J T Kao ◽  
...  
Keyword(s):  
Monoclonal Antibody ◽  
Solid Phase ◽  
Low Density Lipoprotein ◽  
Density Lipoprotein ◽  
Cross Reactivity ◽  
Lipoprotein Subfractions ◽  
Parallel Displacement ◽  
Standard Curve ◽  
Human Apolipoprotein ◽  
Plasma High Density

Abstract We produced a monoclonal antibody (C2-22) to human apolipoprotein (Apo) AII and describe its use in an enzyme-linked immunoabsorbant assay (ELISA) for Apo AII in human plasma and lipoprotein subfractions. No cross reactivity of the antibody with Apo CI, CII, CIII, E, or ablumin was detected. Apo AI and low- and very-low-density lipoprotein cross reacted by 0.25%, less than 0.2%, and less than 0.3%, respectively. Whole plasma high-density lipoprotein (HDL) and HDL subfractions (HDL2 and HDL3) produced parallel displacement curves. This quantitative ELISA is based on competition between solid-phase-bound Apo AII and free Apo AII. Bound C2-22 is detected by alkaline-phosphatase-labeled second antibody. The standard curve for the assay is linear for plasma diluted 500-fold originally containing 140 to 1140 mg of Apo AII per liter. Delipidation of plasma samples exposed no additional antigenic sites. Within- and between-run CVs were respectively 8.4% and 8.7% at 327 mg/L of Apo AII, and 6.8% and 7.4% at 587 mg/L. Results correlated well with those by a polyvalent-antisera-based RIA procedure: r = 0.916, p less than 0.01, RIA = 0.896 ELISA -19.1 mg/L.

Two new monoclonal antibody-based enzyme-linked assays of apolipoprotein B.

1986 ◽  
Vol 32 (8) ◽  
pp. 1484-1490 ◽  
Author(s):  
S G Young ◽  
R S Smith ◽  
D M Hogle ◽  
L K Curtiss ◽  
J L Witztum
Keyword(s):  
Monoclonal Antibody ◽  
Solid Phase ◽  
Low Density Lipoprotein ◽  
Density Lipoprotein ◽  
Low Density ◽  
Apo B ◽  
Competitive Assay ◽  
Microtiter Plates ◽  
Ldl Particles ◽  
Direct Assay

Abstract We describe two new monoclonal antibody-based, solid-phase immunoenzymometric assays for the quantification of apolipoprotein (apo) B in plasma: a competitive assay and a direct assay. For both, we utilize 96-well microtiter plates and native low-density lipoprotein (LDL) for preparing the standard curve. A single monoclonal antibody, MB24, is used in the competitive assay. The direct assay involves use of two monoclonal antibodies, MB24 and MB47. These two antibodies bind to distinct, unrelated apo B epitopes expressed by all LDL particles, and both antibodies detect apo B in very low-density and intermediate-density lipoproteins as well as LDL. With the two-step competitive assay, which involves use of LDL-coated microtiter plates, the intra- and interassay reproducibility in plasma apo B measurements averaged 6% and 12%, respectively. In the one-step direct assay, which takes less than 2 h for completion, plasma samples and peroxidase-labeled MB24 are incubated simultaneously on MB47-coated microtiter wells. The amount of labeled MB24 bound to lipoproteins trapped by MB47 is proportional to apo B concentration. With the direct assay, intra- and interassay CVs averaged 7% and 12%, respectively. These assays are simple, reproducible, involve convenient incubation intervals, and do not require radioisotopes; thus they can be widely applied in clinical laboratories.

scholarly journals Indirect Sandwich Enzyme-Linked Immunosorbent Assay (ELISA) for Plasma Apolipoprotein E

1996 ◽  
Vol 33 (2) ◽  
pp. 119-126 ◽  
Author(s):  
Patrick Kee ◽  
Renze Bais ◽  
Stan K Sobecki ◽  
Susan Branford ◽  
Kerry-Anne Rye ◽  
...  
Keyword(s):  
Apolipoprotein E ◽  
Low Density Lipoprotein ◽  
Sandwich Elisa ◽  
Density Lipoprotein ◽  
Gel Permeation Chromatography ◽  
Cross Reactivity ◽  
Enzyme Linked Immunosorbent Assay ◽  
Lipoprotein Subfractions ◽  
Apo E ◽  
Plasma Apolipoprotein

We have developed an indirect sandwich ELISA for measuring plasma apolipoprotein E (apo-E), using commercially available antibodies. A monoclonal anti-apo-E was used as the capture antibody and the captured apo-E detected with polyclonal anti-apo-E antiserum (goat). The detecting antibody was quantitated using horseradish peroxidase-conjugated rabbit immunoglobulin to goat immunoglobulins. There was no detectable cross-reactivity between the three antisera. Interference with the assay by apolipoprotein A-1, bilirubin and haemoglobin was not significant up to 1·7 g/L, 1250 μmol/L and 13·0 g/dL, respectively. The ELISA method showed high correlation with an established immunonephelometric method (ELISA apo-E=104 Immunonephelometric apo-E+16; r2 = 0·954, P < 0·0001, n = 39). The assay has a measuring range between 5 and 560 mg/L. The coefficient of duplicates was 20%, within-run coefficients of variation (CV) ranged from 3·7 to 6·0% and between-run CV from 6·1 to 15·1%. The reference range determined for 168 normotriglyceridaemic subjects was 20 to 130 mg/L. In an analysis of the lipoprotein subfractions isolated by ultracentrifugation as the fraction of density less than 1·25 g/mL and separated by gel permeation chromatography, apo-E was found to be associated with very low-density lipoprotein and large high-density lipoprotein.

Sensitive methods to study human apolipoprotein B metabolism using stable isotope-labeled amino acids

1996 ◽  
Vol 270 (6) ◽  
pp. E1022-E1036 ◽  
Author(s):  
T. Demant ◽  
C. J. Packard ◽  
H. Demmelmair ◽  
P. Stewart ◽  
A. Bedynek ◽  
...  
Keyword(s):  
Stable Isotope ◽  
Apolipoprotein B ◽  
Low Density Lipoprotein ◽  
Density Lipoprotein ◽  
Constant Infusion ◽  
Gas Chromatography Mass Spectrometry ◽  
Low Density ◽  
Lipoprotein Subfractions ◽  
Human Apolipoprotein ◽  
Spectrometry Technique

The objective of the study was to develop a sensitive method using stable isotope-labeled tracers that would permit the determination of apolipoprotein B (apoB) metabolism in very low-density lipoprotein subfractions (VLDL1, Sf 60-400; VLDL2, Sf 20-60), intermediate-density lipoprotein (IDL, Sf 12-20), and low-density lipoprotein (LDL, Sf 0-12). Six normolipidemic subjects were given trideuterated leucine, and its clearance from plasma and appearance in the four apoB-containing lipoprotein fractions were followed by use of a highly sensitive gas chromatography-mass spectrometry technique in which the m + 3-to-m + 2 ion ratio was selectively monitored. This analytic approach permitted the precise measurement of low enrichments in IDL and LDL and extension of the turnover out to 250-300 h. A compartmental model was developed to derive rate constants from the plasma and apoB enrichment curves. The model was uniquely identifiable once parameter dependencies had been introduced to reduce the number of unknowns. Values were obtained for apoB input into all lipoprotein density intervals, together with rates of interconversion and catabolism; these agreed well with results from radioiodinated tracer experiments. An alternative model structure was also explored in which input occurred only into VLDL1. Altering the protocol of tracer administration (bolus vs. primed constant infusion) and dose (over a 10-fold range) had no influence on the results obtained. The analytic and modeling approach described will permit stable isotopes to be used to elucidate key features of apoB metabolism in normal and pathological states.

Quantitative determination of human plasma apolipoprotein A-I by laser immunonephelometry.

1981 ◽  
Vol 27 (6) ◽  
pp. 856-859 ◽  
Author(s):  
M Rosseneu ◽  
R Vercaemst ◽  
N Vinaimont ◽  
P Van Tornout ◽  
L O Henderson ◽  
...  
Keyword(s):  
Density Lipoprotein ◽  
Normal Subjects ◽  
Similar Data ◽  
Plasma Samples ◽  
Standard Curve ◽  
Human Apolipoprotein ◽  
Technical Procedure ◽  
Apolipoprotein A ◽  
Plasma High Density

Abstract A technical procedure is described for quantitation of human apolipoprotein A-I (apo A-I) in normal plasma or serum by immunonephelometry. Dilution of the plasma samples with 6 mol/L guanidine chloride ensures maximum exposure of the antigenic sites of the apoprotein and enables optimum quantitation of the apo A-I without requiring extraction with organic solvents. Similar data are obtained by this assay and with radioimmunoassay for normal subjects (1.2--1.5 g/L), and the results obtained on 31 patients are correlated with a coefficient of 0.92. The apo A-I values are correlated with values for plasma high-density lipoprotein cholesterol (r = 0.64). The interassay CV for immunonephelometry is about 7% and the standard curve is linear between 0.1 and 1.0 microgram of apo A-I per sample, corresponding to a 150-fold dilution of serum or plasma. The assay is applicable to plasma samples containing as much as 4 g of triglycerides per liter. At higher concentrations plasma delipidation is required.

scholarly journals Rationale and design of the expanded combination of evolocumab plus empagliflozin in diabetes: EXCEED-BHS3 trial

2020 ◽  
Vol 11 ◽  
pp. 204062232095924
Author(s):  
Ikaro Breder ◽  
Jessica Cunha Breder ◽  
Isabella Bonilha ◽  
Daniel B. Munhoz ◽  
Sheila T. Kimura Medorima ◽  
...  
Keyword(s):  
Endothelial Dysfunction ◽  
Ischemia Reperfusion Injury ◽  
Ischemia Reperfusion ◽  
Low Density Lipoprotein ◽  
Density Lipoprotein ◽  
Open Label ◽  
Lipoprotein Subfractions ◽  
Regular Treatment ◽  
Hdl Function ◽  
Cell Adhesion Molecule 1

Background: Patients with type 2 diabetes mellitus (T2DM) remain at increased cardiovascular residual risk and endothelial dysfunction, even after optimizing metabolic control and treatment by sodium-glucose-2 transporter inhibitors (SGLT2-is). The present study was based on the hypothesis that proprotein convertase subtilisin/kexin 9 inhibitor (PCSK9i) therapy may mitigate endothelial dysfunction in T2DM patients who are on regular treatment by SGLT2-i. Methods: The EXCEED-BHS3 is a prospective, single-center, investigator-blinded, open-label, randomized clinical trial. Participants ( n = 110) will be randomized (1:1) to either empagliflozin 25 mg/day alone or empagliflozin 25 mg/day plus evolocumab 140 mg every 2 weeks in addition to optimal medical care. The primary endpoint was defined as the change in the 1-min flow-mediated dilation (FMD) after 16 weeks of treatment. The secondary endpoint is the FMD change after ischemia/reperfusion injury protocol (reserve FMD) after 16 weeks of treatment. Exploratory outcomes comprise the change in FMD and reserve FMD after 8 weeks of treatment and the change after 16 weeks of treatment in the following parameters: plasma levels of nitric oxide, vascular cell adhesion molecule-1 and isoprostane, high-density lipoprotein (HDL) and low-density lipoprotein subfractions profile, HDL function, blood pressure, body mass index, waist circumference and adipokines. Conclusion: This will be the first study to evaluate the add-on effect of PCSK9i on endothelial function of T2DM patients under regular use of empagliflozin. Trial registration: ClinicalTrials.gov identifier: NCT03932721

scholarly journals Low density lipoprotein and very low density lipoprotein are selectively bound by aggregated C-reactive protein.

1982 ◽  
Vol 156 (1) ◽  
pp. 230-242 ◽  
Author(s):  
F C de Beer ◽  
A K Soutar ◽  
M L Baltz ◽  
I M Trayner ◽  
A Feinstein ◽  
...  
Keyword(s):  
Acute Phase ◽  
Solid Phase ◽  
Low Density Lipoprotein ◽  
Density Lipoprotein ◽  
Very Low Density Lipoprotein ◽  
Low Density ◽  
C Reactive Protein ◽  
Calcium Dependent ◽  
Reactive Protein

C-reactive protein (CRP), the classical acute-phase protein, can bind phospholipids by virtue of its specific, calcium-dependent reactivity with phosphorylcholine residues. However, analysis of acute-phase serum by gel filtration and by density gradient ultracentrifugation showed that the CRP was in a free, uncomplexed form, despite the coexistent presence of the various classes of serum lipoproteins, all of which contain phospholipids. In contrast, when isolated CRP was aggregated by immobilization at a sufficient density on a solid phase and then exposed to normal human serum, it selectively bound low density lipoprotein (LDL) and traces of very low density lipoprotein. The reaction was calcium dependent and reversible by free phosphorylcholine but not by heparin. LDL isolated from normal plasma was also bound by aggregated CRP. CRP reacts in vitro with a wide variety of different ligands both of extrinsic and of autogenous origin, e.g., microbial products and damaged cell membranes, respectively. If CRP aggregated in vivo by complexing with these ligands than acquires the capacity to selectively bind LDL, the phenomenon may have significant implications for the function of CRP and for the metabolism, clearance, and deposition of LDL.

scholarly journals Increased midlife triglycerides predict brain β-amyloid and tau pathology 20 years later

Neurology ◽  
2017 ◽  
Vol 90 (1) ◽  
pp. e73-e81 ◽  
Author(s):  
Katarina Nägga ◽  
Anna-Märta Gustavsson ◽  
Erik Stomrud ◽  
Daniel Lindqvist ◽  
Danielle van Westen ◽  
...  
Keyword(s):  
Risk Factors ◽  
Low Density Lipoprotein ◽  
Density Lipoprotein ◽  
Healthy Population ◽  
Lipid Levels ◽  
Tau Pathology ◽  
Lipoprotein Subfractions ◽  
Cognitively Normal ◽  
Normal Individuals ◽  
Β Amyloid

ObjectiveTo evaluate the effect of midlife lipid levels on Alzheimer brain pathology 20 years later in cognitively normal elderly individuals.MethodsThis is a longitudinal cohort study of 318 cognitively normal individuals with data on fasting lipid levels at midlife (mean age 54 years). Presence of β-amyloid (Aβ) and tau pathologies 20 years later (mean age 73 years) were detected by quantifying Alzheimer disease (AD) biomarkers in CSF. In a subset (n = 134), Aβ (18F-flutemetamol) PET was also performed.ResultsCSF Aβ42 and Aβ PET revealed Aβ pathology in approximately 20% of the cognitively healthy population and CSF Aβ42/phosphorylated tau (p-tau) ratio indicated both Aβ and tau pathology in 16%. Higher levels of triglycerides in midlife were independently associated with abnormal CSF Aβ42 (odds ratio [OR] 1.34, 95% confidence interval [CI] 1.03–1.75, p = 0.029) and abnormal Aβ42/p-tau ratio (OR 1.46, 95% CI 1.10–1.93; p = 0.009) adjusting for age, sex, APOE ε4, education, and multiple vascular risk factors. Triglycerides were also associated with abnormal Aβ PET in multivariable regression models, but the association was attenuated in the fully adjusted model. Increased levels of medium and large low-density lipoprotein subfractions were significantly associated with abnormal Aβ PET and large high-density lipoprotein particles were associated with decreased risk of abnormal Aβ PET.ConclusionsIncreased levels of triglycerides at midlife predict brain Aβ and tau pathology 20 years later in cognitively healthy individuals. Certain lipoprotein subfractions may also be risk factors for Aβ pathology. These findings further support an involvement of lipids in the very early stages of AD development.

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