Effect of the number of apolipoprotein(a) kringle 4 domains on immunochemical measurements of lipoprotein(a)

Abstract Lipoprotein(a) [Lp(a)] has been measured in numerous clinical and epidemiological studies by a variety of immunochemical methods. However, little, if any, consideration has been given to the confounding effect of the size heterogeneity of apolipoprotein(a) [apo(a)] on the measurement of Lp(a). We developed three direct-binding enzyme-linked immunosorbent assays (ELISAs) with detecting antibodies of different specificities to evaluate the effect of apo(a) size on Lp(a) measurement. The three assays used the same monoclonal antibody to capture the apo(a)-containing particles and were calibrated (in nanomoles per liter) with a serum containing apo(a) with 21 kringle 4 domains. Using all three ELISAs, we measured Lp(a) in a group of 723 subjects selected to have a single apo(a) band, as determined by a high-resolution phenotyping system. Essentially identical results were obtained by the two methods that measured Lp(a) by use of either a polyclonal antibody against apo B or a monoclonal antibody against apo(a) that does not recognize the kringle 4 type 2 repeats. In contrast, the ELISA using a monoclonal antibody specific for apo(a) kringle 4 type 2 repeats overestimated Lp(a) concentration in samples containing apo(a) with more than 21 kringle 4 domains and underestimated Lp(a) samples containing apo(a) with fewer than 21 kringle 4 domains. Thus, these differences in Lp(a) values varied as a function of apo(a) size. We conclude that antibody specificity and apo(a) size heterogeneity can significantly affect Lp(a) measurements.

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A monoclonal-antibody-based enzyme-linked immunosorbent assay of lipoprotein(a)

Clinical Chemistry ◽

10.1093/clinchem/36.2.192 ◽

1990 ◽

Vol 36 (2) ◽

pp. 192-197 ◽

Cited By ~ 30

Author(s):

W L Wong ◽

D L Eaton ◽

A Berloui ◽

B Fendly ◽

P E Hass

Keyword(s):

Monoclonal Antibody ◽

Immunosorbent Assay ◽

Clinical Laboratory ◽

Low Density Lipoprotein ◽

Density Lipoprotein ◽

Enzyme Linked Immunosorbent Assay ◽

Apo B ◽

Lipoprotein A ◽

Premature Coronary Heart Disease ◽

Large Populations

Abstract Lipoprotein(a) [Lp(a)] is a low-density lipoprotein (LDL)-like lipoprotein particle recently described as a risk factor for premature coronary heart disease, stroke, and atherosclerosis. Structurally, Lp(a) is similar to LDL in that it has comparable lipid composition and contains apolipoprotein B-100 (apo B-100). In addition, Lp(a) contains the glycoprotein apolipoprotein(a) [apo(a)], which is disulfide-linked to apo B-100. The recent awareness of a striking correlation between atherosclerosis and concentrations of Lp(a) in plasma prompted our development of an accurate quantitative assay for plasma Lp(a), a monoclonal-antibody-based enzyme-linked immunosorbent assay for Lp(a) that is shown to be sensitive, precise, and highly specific. The response to several isoforms of Lp(a) is linear, and as many as 80 samples can be quantified on one plate. This easily performed assay is suitable for use in the clinical laboratory and for screening large populations.

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The unique lipoprotein(a): properties and immunochemical measurement

Clinical Chemistry ◽

10.1093/clinchem/36.12.2019 ◽

1990 ◽

Vol 36 (12) ◽

pp. 2019-2026 ◽

Cited By ~ 83

Author(s):

J J Albers ◽

S M Marcovina ◽

M S Lodge

Keyword(s):

Rapid Development ◽

Structural Complexity ◽

Structural Domains ◽

Lipoprotein A ◽

Apolipoprotein A ◽

High Amino Acid ◽

Multiple Copies ◽

High Concentrations ◽

Size Heterogeneity ◽

Genetically Determined

Abstract Lipoprotein (a) [Lp(a)] represents a class of lipoprotein particles defined by the presence of apolipoprotein(a), a unique glycoprotein linked by a disulfide bond to apolipoprotein B-100 to form a single macromolecule. Apolipoprotein(a) is formed by three different structural domains having high amino acid sequence homology with plasminogen. One of the domains, called kringle 4, is present in multiple copies, the number of which varies and is genetically determined. This accounts for the size heterogeneity of apolipoprotein(a) and thus of Lp(a). Because high concentrations of Lp(a) are associated with atherosclerotic cardiovascular and cerebrovascular disease and may inhibit fibrinolysis, interest in measuring Lp(a) has increased considerably, leading to a rapid development of commercially available immunoassays for the measurement of Lp(a) in human plasma. However, the immunochemical measurement of Lp(a) has several peculiar problems in addition to those encountered by the measurements of other apolipoproteins. The major problems that need to be carefully evaluated are (a) the structural complexity and heterogeneity of Lp(a), (b) the homology of apolipoprotein(a) with plasminogen, (c) the lack of standardization of the methods, and (d) the lack of a common means of expressing the Lp(a) values.

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Lipoprotein(a) quantified by an enzyme-linked immunosorbent assay with monoclonal antibodies.

Clinical Chemistry ◽

10.1093/clinchem/35.7.1380 ◽

1989 ◽

Vol 35 (7) ◽

pp. 1380-1384 ◽

Cited By ~ 45

Author(s):

C Labeur ◽

G Michiels ◽

J Bury ◽

D C Usher ◽

M Rosseneu

Keyword(s):

Monoclonal Antibody ◽

Immunosorbent Assay ◽

Plasma Cholesterol ◽

Sample Pretreatment ◽

Hdl Cholesterol ◽

Normal Subjects ◽

Enzyme Linked Immunosorbent Assay ◽

Apo B ◽

Lipoprotein A ◽

Screening Programs

Abstract This new, sensitive, specific "sandwich"-type enzyme-linked immunosorbent assay (ELISA) for quantifying lipoprotein(a) [Lp(a)] in human serum and in ultracentrifugal lipoprotein fractions is based on use of a monoclonal antibody raised against apolipoprotein(a) as coating protein and a polyclonal antibody, raised against either apo B or against Lp(a) and conjugated with peroxidase, for detection of bound Lp(a). Mean intra- and interassay CVs for assay of 16 samples were 3.0% and 5.6%, respectively. Sample pretreatment with urea did not enhance Lp(a) immunoreactivity, and treatment with nonionic detergents decreased binding to the monoclonal antibody. Results correlated well (r = 0.99, n = 38) with those by radial immunodiffusion (RID). The ELISA assay, however, detects amounts corresponding to Lp(a) contents of 10 to 1000 mg/L in plasma samples diluted 1000-fold, compared with 100-500 mg/L for RID. For 92 normolipidemic subjects, the mean Lp(a) concentration was 120 (SD 130) mg/L. In patients undergoing coronary angiography, Lp(a) concentrations increased with the severity of the disease but were not correlated with either HDL cholesterol, triglycerides, apo A-I, or apo B, and only weakly with plasma cholesterol and apo A-II. These two correlations were even weaker in normal subjects, and only the correlation with total cholesterol was valid. Lp(a), measured at birth and at seven days and six months, steadily increased with age. This assay is well suited for measuring Lp(a) in plasma and in lipoprotein fractions and also for screening programs evaluating this significant genetic risk factor for the development of atherosclerosis.

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Lipoprotein(a), apolipoprotein(a) polymorphism and coronary artery disease severity in type 2 diabetic Tunisian population

Biomedical Genetics and Genomics ◽

10.15761/bgg.1000115 ◽

2016 ◽

Vol 1 (3) ◽

Author(s):

Abid Kaouthar ◽

Mili Donia ◽

Trabelsi Imen ◽

Msolli Mohamed Amine ◽

Nouira Semir ◽

...

Keyword(s):

Coronary Artery Disease ◽

Coronary Artery ◽

Disease Severity ◽

Tunisian Population ◽

Lipoprotein A ◽

Coronary Artery Disease Severity ◽

Apolipoprotein A ◽

Artery Disease ◽

Type 2 Diabetic

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Structural and functional polymorphism of lipoprotein(a): biological and clinical implications

Clinical Chemistry ◽

10.1093/clinchem/41.1.170 ◽

1995 ◽

Vol 41 (1) ◽

pp. 170-172 ◽

Cited By ~ 13

Author(s):

A M Scanu

Keyword(s):

Low Density Lipoprotein ◽

Density Lipoprotein ◽

The Other ◽

Clinical Implications ◽

Low Density ◽

Functional Polymorphism ◽

Lipoprotein A ◽

Apolipoprotein A ◽

Binding Function

Abstract Lipoprotein(a) [Lp(a)], a variant of low-density lipoprotein, is heterogeneous in density because of variability in the content and composition of its core lipids and size polymorphism of its specific glycoprotein component, apolipoprotein(a) [apo(a)]. In some individuals, density polymorphism may also derive from the fact that Lp(a) contains 2 mol of apo(a) per mole of apoB100, contrary to the more common 1:1 molar stoichiometry. Moreover, the size of apo(a) is polymorphic because of variations in the number of kringle 4 type 2 repeats. Another type of apo(a) polymorphism is related to sequence mutations at the kringle level. Two mutations can occur in kringle 4 type 10: one, Trp72-->Arg, is affiliated with an Lp(a) that is lysine-binding defective; the other, Met66-->Thr, with a normal lysine-binding function. Thus, Lp(a) is structurally and functionally polymorphic, a notion that must be considered in assessing the cardiovascular pathogenicity of this lipoprotein variant and in immunoquantification assays.

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Assessment of Asymptomatic Coronary Artery Disease in Apparently Uncomplicated Type 2 Diabetic Patients: A role for lipoprotein(a) and apolipoprotein(a) polymorphism

Diabetes Care ◽

10.2337/diacare.25.8.1418 ◽

2002 ◽

Vol 25 (8) ◽

pp. 1418-1424 ◽

Cited By ~ 33

Author(s):

C. Gazzaruso ◽

A. Garzaniti ◽

S. Giordanetti ◽

C. Falcone ◽

E. De Amici ◽

...

Keyword(s):

Coronary Artery Disease ◽

Coronary Artery ◽

Diabetic Patients ◽

Type 2 Diabetic Patients ◽

Lipoprotein A ◽

Apolipoprotein A ◽

Artery Disease ◽

Asymptomatic Coronary Artery Disease ◽

Type 2 Diabetic

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Isolation and partial characterization of apolipoprotein (a) from human lipoprotein (a)

Biochemistry and Cell Biology ◽

10.1139/o86-133 ◽

1986 ◽

Vol 64 (10) ◽

pp. 999-1009 ◽

Cited By ~ 64

Author(s):

L. J. Seman ◽

W. C. Breckenridge

Keyword(s):

Amino Acid ◽

Gel Filtration ◽

Density Lipoprotein ◽

Compositional Analysis ◽

Apparent Molecular Weight ◽

Supernatant Fraction ◽

Apo B ◽

Lipoprotein A ◽

Apolipoprotein A ◽

Protein Pellet

A procedure was developed for the dissociation of apolipoprotein (a) (apo (a)) from pure human lipoprotein (a) (Lp(a)) prepared by density gradient ultracentrifugation and gel filtration. Lp(a) was ultracentrifuged through a layer of saline which was adjusted to a density of 1.182 g/mL and contained 30 mM dithiothreitol (50 mM) and phenylmethylsulfonyl fluoride (1.25 mM). Following centrifugation, the lipid and apolipoprotein B (apo B) were recovered as a lipoprotein (Lp(a) B) in the supernatant fraction, while the apo (a) was recovered as a lipid-poor protein pellet. An investigation of the supernatant lipoprotein by electron microscopy and compositional analysis revealed that it was similar in size and composition to low density lipoprotein (LDL) isolated from the same density range and contained apo B100 with an amino acid and carbohydrate composition which was similar to apo B from LDL. Estimates of the apparent molecular weight of the apo (a) varied amongst individuals but was always greater than apo B100 [Formula: see text]. The amino acid composition of apo (a), which was very distinct from apo B, was characterized by a higher content of serine, threonine, proline, and tyrosine, but lower amounts of isoleucine, phenylalanine, and lysine when compared with apo B of Lp(a) or LDL. The apo (a) contained a much higher proportion of carbohydrate, in particular N-acetylgalactosamine, galactose, and N-acetylneuraminic acid (which were three- to six-fold higher) than the apo B of Lp(a). It is concluded that apo (a) is distinct from other apolipoproteins owing to its low avidity for lipid and the nature of the interaction with apo B. Lp(a) consists of an LDL-like particle with a carbohydrate-rich apo (a) attached to the surface of apo B.

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