Maritza B. Torres, B. S.1
,
Flor de la C. Heres Álvarez, M.D.2,
Aida Rodríguez Hernández, B. S.3,
Luis Sorell Gómez, M.D. Ph.D.4,
María B. Cabalé Vilariño5
ABSTRACT: Lipoprotein (a) [Lp (a)] has been identified as an independent risk factor for atherosclerotic disease (coronary artery disease, cerebral and peripheral vascular disease). Consequently, measurement of serum levels of this lipoprotein has become particularly important in recent years. In this work we describe the results obtained with the use of a visual immunoassay method for measuring Lp (a) levels, the AuBioDOT TM Lp (a). We compared this method with the BioSCREENTM ELISA Lp (a) assay (both developed by Heber Biotec S.A., Cuban Center for Genetic Engineering and Biotechnology). We found that AuBioDOT TM Lp (a) is a fast and simple method for the detection of elevated Lp (a) in serum, blood and plasma, which showed a correlation of r=0.987, a sensibility of 88.8% and a specificity of 100%. In the 100 samples studied, the frequency distribution of Lp (a) showed a right standard deviation, similar to the one reported in other studies. Our study demonstrated the utility of the AuBioDOT TM
Lp (a) assay as a fast and economical method to identify elevated levels of lipoprotein (a).
Lipoprotein (a) [Lp (a)] is similar to low-density
lipoproteins (LDL) in that it contains apolipoprotein B (apo B). However, its structure is characterized by the presence of apolipoprotein (a) [apo (a)], which binds apolipoprotein B-100 (apo B) by means of disulfide
bonds.1-3 The clinical determination of serum levels of Lp (a) has become especially important in recent years after it has been identified as an independent risk factor for
atherosclerotic vascular disease. Elevated levels of serum Lp (a) have been associated with a higher incidence and severity of coronary artery disease, and cerebral and peripheral vascular disease. 2,4,5 Lp (a) has also been reported as a risk factor for recurrence of re-stenosis after coronary angioplasty and aortic-coronary transplants. The degree of re-stenosis
has been found to be directly associated with serum Lp (a) levels.6,7 Some authors have found elevated levels of serum Lp (a) in patients with diabetes mellitus and renal failure.
Nevertheless, the role of Lp (a) in these disorders has not been well defined.8,9 Lp (a) has also been reported to have thrombogenic action due to the structural similarity between apo (a)
and plasminogen (a protein involved in clot lysis) and a probable competition with plasminogen activators. Elevated levels of serum lipoprotein (a) are considered to have an inhibitory effect on fibrinolytic mechanisms.
10,11 The potential atherogenic and thrombogenic effects of Lp (a) have prompted researchers to seek new methods for the quantification of this lipoprotein. Serum and plasma
concentrations of Lp (a) have been measured by radial immunodiffusion,12 electroimmunoassay,13 immunoelectrophoresis assay,13 nephelometry,14 immunoradiometric assay,14-17
and enzyme immunoassay.15-18 Among these, the enzyme-linked immunosorbent assay (ELISA), a highly sensitive and specific method based on the use of monoclonal (mAB) and/or polyclonal antibodies, has
been broadly used in the quantification of Lp (a).16-20 Although these techniques have proved effective for the quantification of Lp (a), their use
requires a lot of time and equipment as well as highly skilled professionals. Moreover, most authors2,21,22 believe that the risk of atherogenesis markedly increases in
patients with Lp (a) levels above 300 mg/L. In view of this, some researchers have suggested the use of survey methods for early detection of Lp (a) levels above 300 mg/L, in a fast and economical way.23,24 Recently, the Cuban Center for Genetic Engineering and Biotechnology (CCGEB) has developed a visual immunoassay method for determining elevated serum, plasma or blood
levels of Lp (a). This immunoassay has been called AuBioDOTTM Lp (a), and brings results in 30 minutes, without the need for specialized personnel or laboratories.(25)
It is evaluated and compared with the BioSCREENTM Lp (a), a commercially available quantitative method.21
Methods
Serum samples were examined from 100 employees of the La Estrella cookie and candy factory in Havana: 64 women and 36 men. Their average age was 42 years, and the group ranged from 22 to 67.
Serum samples were obtained from peripheral venous blood, centrifuged at 4000 x g for 10 minutes after coagulation at room temperature, and kept at –20oC for no longer than one month.
The level of Lp (a) in these samples was quantified by the AuBioDOTTM Lp (a) immunoassay and by the BioSCREENTM Lp (a) micro ELISA assay.
ASSESSMENT OF LP (A) LEVELS BY AUBIODOTTM Lp (a)
In this immunoassay, white opaque polystyrene sheets were used. Anti-apo (a) monoclonal antibodies (mAB) were deposited in each well and used as a non-plasminogen cross-reacting
capture antibodies. These sheets were submerged in a buffer solution of PBS-tween 20 at 0.5% for five minutes. Then, 20 L of pattern serum with a concentration of 300 mg/L of Lp
(a) were added to each well. A control serum with high concentrations of Lp (a) (900 mg/L) was added to one well, and a control serum with low concentrations of Lp (a) (100 mg/L),
was added to another well. Furthermore, 20 L of diluted serum (1 in 100) were added to the remaining wells. Sheets were incubated at room temperature for 10 minutes. After three
washes with PBS-tween 20 at 0.5%. 20 L of another anti-apo (a) mAB, conjugated with colloidal gold, were added to each well. After incubation at room temperature for ten
minutes, and washing, a silver ion-revealing solution was added to amplify the reaction. This solution produces insoluble light or dark metallic brown-colored reactions.
Results were interpreted by simple visual inspection of the reaction areas. The intensity of the color of each reaction varies with the concentration of Lp (a) in the sample. To classify
samples as positives or negatives, the intensity of their color was compared with that of the pattern sample. If the sample's color was equal or darker than that of the pattern sample, the
sample was classified as positive (= 300 mg/L). If the sample's color was lighter than that of the pattern sample, the sample was classified as negative (<300 mg/L).
Each sample was classified independently by three different analysts. The final classification of samples into positive or negative was based on at least two out of three of the analysts obtaining the same results.
The measurements obtained by the three analysts for each sample fell into one of the following four categories: 0, 0.33, 0.66, and 1. 0
: when all three analysts classified the sample as "negative" 0.33: when two of the analysts classified the sample as "negative" 0.66
: when two of the analysts classified the sample as "positive" 1: when all three analysts classified the sample as "positive"
USE OF ELISA FOR THE DETERMINATION OF LP(A) SERUM LEVELS
BioSCREENTM Lp (a) is a sandwich ELISA immunoassay used in the quantitative determination of Lp (a) levels in human serum. This method, uses an anti-apo (a) monoclonal antibody as the capture antibody, and an anti-apo B peroxidase-linked monoclonal antibody, as the detector antibody. For our study, this technique was carried out as proposed by Sorell et al.21
STATISTICAL PROCESSING
The sensitivity (S) and specificity (Sp) of the AuBioDOTTM Lp (a) method was calculated with the following formulas:
S = true positives/true positives + false negatives x 100. The bi-serial correlation test (br) was used to compare a dichotomized qualitative variable (samples classified by AuBioDOT) with a quantitative variable (serum concentrations of Lp
(a) determined by ELISA) aimed at calculating the degree of association between these two methods.26
Sp = true negatives/true negatives + false positives x 100.
Results
Of the 100 serum samples analyzed by using the BioSCREENTM Lp (a) method, 64 were found to contain concentrations of Lp (a) lower than 300 mg/L, while 36 had concentrations
of this lipoprotein above 300 mg/L. Figure 1 shows a histogram of the Lp (a) serum concentration frequency distribution in the samples. The analysis of the samples by using AuBioDOTTM Lp (a) showed that 32 samples were
positive, and 68 negative. When we compared these results with the ones obtained by using ELISA, we found that 4 of the 68 negative samples obtained by AuBioDOTTM Lp (a), were
positive when analyzed by ELISA for they had concentrations of Lp (a) = 302, 314, 331, and 366 mg/L. This showed an 88.8% sensitivity and a 100% specificity for AuBioDOTTM
Lp (a) for the detection of elevated serum levels of Lp (a) (Figure 2) Figure 2
shows the degree of association between these two methods for detecting elevated Lp (a) levels. The correlation obtained was 0.987.
Discussion
The distribution of Lp (a) levels in the serum samples studied displayed a right standard
deviation, in which the highest frequencies were found on the lowest levels. This result is similar to the one obtained in other studies.22
The 88.8% sensitivity obtained (due to two false negative results) could be explained by the genetic polymorphism of apo (a). Depending on its molecular mass, the concentration of Lp (a) can be over- or underestimated
17,20,26 and the influence of the different phenotypes of apo (a) depends on the combination of antibodies used in the study as well as on its specificity. The method of reference [BioSCREENTM
Lp (a)] is a type of ELISA based on a combination of an anti-apo (a) mAB and an anti-apo B mAB. Therefore, it is considered to be less susceptible to individual variations due to the genetic polymorphism of apo (a)21
than AuBioDOTTM Lp (a), in which two anti-apo mAB are used. We must also note that the concentrations of Lp (a) found in the four samples classified as negative by the AuBioDOTTM
Lp (a), but positive by the BioSCREENTM Lp (a) (302, 314, 331, and 366 mg/L) were very close to the cutoff point (300 mg/L).
The use of a common pattern serum to calibrate the systems and the state of conservation of the samples could explain the high correlation found between the two methods compared in our study (r = 0.987).
In short, visual immunoassay, based on the use of monoclonal antibodies is a reliable and easy method for the detection of elevated Lp (a) levels in human serum, blood or plasma,
with which results can be obtained within 30 minutes. We believe that the non-instrumental visual immunoassay system AuBioDOTTM Lp (a), is a
useful alternative for the fast and economical identification of elevated levels of lipoprotein (a), an important independent risk factor for atherosclerotic disease.
References
1. Utermann G. The mysteries of lipoprotein (a). Science 1989;246:904-10.
2. Karmansky I, Gruener N. Structure and possible biological roles of Lp (a). Clin Biochem. 1994;27:151-62.
3. Morrisett JD, Guyton JD, Gaubatz JW, Gotto AM Jr. Lipoprotein (a): structure, metabolism and epidemiology. In Gotto Am ed. Plasma Lipoproteins. Amsterdam: Elsevier Science Pub. BV, Chapter 4, 1987:129-52.
4. Hearn JA, De Macio SJ, Roubin GS, Hammarstrom M, Sgoutas D. Predictive value of lipoprotein (a) and other serum lipoproteins in the angiographic diagnostic of coronary disease Am J Cardiol 1990;66:1176-80.
5. Nishino M, Ito T, Yasuno M, Nago N, Matsuo H, Goto T. Serum Lipoprotein (a) as a risk factor for thoracic atherosclerosis in subjects aged > 40 years. Am J Cardiol 1993;24:965-969.
6. Hoff HF, Beck G J, Skibinski CI, Vega G, Grayburn M, Serum Lp (a) levels as a predictor of vein graf stenosis after coronary artery bypass surgery in patients. Circulation 1988;77:1238-44.
7. Desmarais RL, Sarembuck IJ, Ayers CR, Vernon SM, Powers ER, Gimple LW, Elevated Serum Lipoprotein (a). Is a risk factor for clinical recurrence after coronary balloon angioplasty? Circulation 1995;91:1403-09.
8. Haffner S. Lipoprotein (a) and diabetes. Diabetes Care 1993;16:835-40.
9. Irish AB, Simons LA, Saudie E, Hayes JM, Simons J, Lipoprotein (a) levels in chronic renal disease states, dialysis and transplantation. Aust N Z J Med 1992;22:243-48.
10. Hajjar KA, Gavish D, Breslow JL, Nachman RL. Lipoprotein (a) modulates endothelial cell surface fibrinolysis: Potential role in atherosclerosis. Nature 1989;339:302-35.
11. Leerink CB, Gimpel JA, Kortlandt W, Bouma BN, Van Rijn HJM. Kinetic analysis of Lp (a) inhibition of plasminogen activation by tissue plasminogen activator in vitro. Fibrinolysis 1991;5:233-38.
12. Albers JJ, Hazzard WR. Immunochemical quantification of human plasma Lp (a) lipoprotein. Lipids 1974;9:15-27.
13. Marz W, Grob W. Quantification of human serum lipoprotein Lp (a): Zone immunoelectrophoresis assay, a new sensitive method as compared to electroimmunoassay. Cin Chim Acta 1983; 134:265-79.
14. Albers JJ, Adolphson JL, Hazzard WR. Radioimmunoassay of human plasma Lp (a) lipoprotein. J Lipid Res 1993;18:331-38.
15. Marz W, Siekmeier R, Gross E, Gross W. Determination of lipoprotein (a): enzyme immunoassay and immunoradiometric assay compared. Clin Chim Acta 1993;214:153-63.
16. Marz W, Siekmeier R, Grob W, Kostner M, Determination of lipoprotein (a): Evaluation of three methods. Eur J Clin Cem Clin Bioch 1993;31:295-301.
17. Labeur C, Rosseneu M, Henderson O. International Lp (a) standardization. Chem Phys, Lip 1994;67/68:265-70.
18. Wong W, Eaton D, Berlovi A, Frendly B, Hass PA. A monoclonal antibody-based enzyme-linked immunosorbent assay with monoclonal antibodies. Clin Chem 1990;36:192-97.
19. Labeur C, Michlels. Bury J, Usher D, Tosseneu M. Lipoprotein (a) quantified by an enzyme-linked immunosorbent assay with monoclonal antibodies. Clin Chem 1989;35:1380-4.
20. Teddie-Peetrs W, Butman B, Jones G, Venetta TH, Ma Comber P, Ransom J. Quantitation of lipoprotein (a) particles containing various apolipoprotein (a) isoforms by a monoclonal anti-apo (a) capture antibody and a polyclonal anti-apolipoprotein B detection antibody sandwich enzyme immunoassay. Clin Chem 1993;39:1382-9.
21. Sorell L, Rojas G, Rodríguez M, Ramos C, Torres L, Torres MB. A sandwich ELISA based on anti-apo (a) and anti-apo B monoclonal antibodies for lipoprotein (a) measurement. Clin Chim Acta 1995;236:59-70.
22. Nago N, Kayaba K, Hiraoka J. Lipoprotein (a) levels in the Japanese population: influence of age and sex and relation to atherosclerotic risk factors. Am J Epidemiol 1995;141:815-21.
23. Lippi G, Ruzzenente O, Facchinetti R, Guidi G. Semi-quantitative latex method for lipoprotein (a) assay Clin Chem Acta 1994;229:147-51.
24. Mc Namara JR, Campos H, Adolphson JL, Albers JJ. Screening for lipoprotein (a) elevations in plasma and assessment of size heterogeneity using gradient gel electrophoresis. J Lipid Res 1989;30:747-55.
25. Sorell L, Rojas G. A simple visual immunoassay for the rapid detection of high Lp (a) blood levels. Clin Chim Acta 1997;260:65-71.
26. Ostle B. Estadística aplicada Ciencia y Técnica Publishers. Havana. Cuba. 1981:251-61.
This article originally appeared in Spanish in the Revista Cubana de Cardiología y Cirugía Cardiovascular journal, Vol. 24, No. 1, (pp. 29-35), 1998.
