Enzyme-linked immunosorbent assay for human plasma apolipoprotein B.

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.

Apolipoprotein (apo) A-IV has been proposed to play a role in reverse cholesterol transport. ApoA-IV-containing lipoprotein particles (A-IVLp) were isolated from human plasma and interstitial fluid and characterized by immunoaffinity chromatography. Two major A-IVLp subpopulations, lipoprotein particles containing apoA-IV with apoA-I (LpA-I:A-IV) and lipoprotein particles containing apoA-IV without apoA-I (LpA-IV), were identified. The larger subpopulation of A-IVLp is the LpA-IV that represents 70% (protein mass) of the initial particles. Only 5.8% of apoA-IV was recovered in the retained fraction after affinity chromatography with an anti-apoA-I immunosorbent. ApoA-I, apoA-II, apoA-IV, apoB, apoC-III, apoD, apoE, apoH, lecithin: cholesterol acyltransferase (LCAT), cholesteryl ester transfer (CET) protein, proline-rich protein, and a protein of Mr 59,000 were detected in the A-IVLp. These particles contain more than 20% triglycerides (lipid mass). ApoA-IV-containing particles that were isolated from plasma are heterogeneous in size, consisting of two major populations with Stokes' diameters of 10.3 nm and 9.3 nm. Both subpopulations of A-IVLp contain LCAT and CET activities and promote cholesterol efflux from cholesterol-preloaded adipose cells. These data support the hypothesis that A-IVLp particles may be involved in reverse cholesterol transport.

Characterization of apoA-IV-containing lipoprotein particles isolated from human plasma and interstitial fluid.

Plasma-levels of major lipids (cholesterol, triglyceride, high-density-lipoprotein cholesterol), two major apolipoproteins (apo-B and apo-A1), and two ratios (total-cholesterol/apo-B and apo-A1/apo-B) were studied in 218 survivors of myocardial infarction and 160 controls. Apolipoproteins were as good as lipids as discriminators between the populations under the age of 50 and better in the sixth to eighth decades.. Furthermore, values of total-cholesterol/apo-B and apo-A1/apo-B obtained from controls and normolipaemic survivors of myocardial infarction gave a bimodal distribution. The protein moiety of lipoproteins is a better discriminator than lipids between atherosclerotic subjects and controls.

Are Apolipoproteins Better Discriminators than Lipids for Atherosclerosis

Anion-exchange fast protein liquid chromatographic characterization and purification of apolipoproteins A-I, A-II, C-I, C-II, C-III0, C-III1, C-III2 and E from human plasma

We used a noncompetitive enzyme-linked immunoassay to measure apolipoprotein B (apo-B) concentration in human plasma. Goat anti-lipoprotein B immunoglobulins were adsorbed to the surface of polystyrene balls. After washing, this solid-phase antibody was incubated with antigen (plasma from normal or hyperlipoproteinemic fasting subjects), washed, and then incubated with peroxidase-labeled goat anti-lipoprotein B IgG. After a last washing, we measured the bound label, which provided a direct measurement of the antigen. Under optimized assay conditions, the minimum detectable concentration was 50 ng per assay. The assay may be used to measure apo-B in different lipoprotein fractions (low- or very-low-density) and yields values that compared favorably with those obtained by electroimmunoassay (r = 0.86). The assay offers several advantages over existing techniques: sensitivity, specificity, simplicity, avoidance of radioisotopes, and potential for use with monoclonal antibodies.

Noncompetitive enzyme-linked immunoassay for apolipoprotein B in serum.

We describe an isoelectric-focusing method for rapidly separating and quantifying apolipoprotein (apo) C-II and C-III subspecies of triglyceride-rich lipoproteins. Concentrated very-low-density lipoproteins (VLDL) or delipidated apo VLDL are focused on dry acrylamide plates, after their rehydration with ampholytes and urea. Apo C-II and apo C-III in VLDL are resolved into four major bands--C-II (pI 5.01), C-III0 (pI 5.10), C-III1 (pl 4.92), and C-III2 (pI 4.84)--at the same pI values as for purified apo C. This precise technique can be used without delipidating VLDL. The relative percentage of C apoproteins found in VLDL from plasma of normal subjects agreed with previously published data. The ratio of apo C-II to apo C-III decreased in patients with chronic renal failure or with coronary artery disease.

Rapid assessment of the distribution of apolipoproteins C-II and C-III subspecies in triglyceride-rich lipoproteins by isoelectric focusing.

We used a noncompetitiveenzyme-linkedimmunoassayto measure apolipoproteinB (apo-B) concentrationin human plasma. Goat anti-lipoproteinB immunoglobulins were adsorbedto the surfaceof polystyreneballs.Afterwashing,this solid-phase antibody was incubated with antigen (plasma from normal or hyperlipoproteinemic fasting subjects), washed, and then incubatedwith peroxidase-labeledgoat anti-lipoprotein B lgG. Aftera lastwashing,we measuredthe bound label, which provided a direct measurementof the antigen. Under optimized assay conditions,the minimum detectable concentrationwas 50 ng per assay. The assay may be used to measure apo-B in different lipoprotein fractions (low- or very-low-density)and yields values that compared favorably with those obtained by electroimmunoassay(r = 0.86). The assay offersseveraladvantagesover existingtechniques:sensitivity,specificity,simplicity,avoidance of radioisotopes,and potentialfor use with monoclonal antibodies.

NoncompetitiveEnzyme-LinkedImmunoassayfor ApolipoproteinB in Serum

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Noncompetitive enzyme-linked immunoassay for apolipoprotein B in serum.

Rapid assessment of the distribution of apolipoproteins C-II and C-III subspecies in triglyceride-rich lipoproteins by isoelectric focusing.

NoncompetitiveEnzyme-LinkedImmunoassayfor ApolipoproteinB in Serum