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Technical Briefs |
1 Lipoprotein and Atherosclerosis Group, University of Ottawa Heart Institute, Ottawa Civic Hospital, Ottawa, Ontario, K1Y 4E9 Canada
2 The Gladstone Institute of Cardiovascular Disease, Cardiovascular Research Institute, University of California, San Francisco, CA 94141-9100
3 Département des Sciences Biologiques, Université du Québec, Montréal, CP 8888, Québec, H3C 3P8 Canada
aauthor for correspondence: fax 415-285-5632, e-mail rraffai{at}gladstone.ucsf.edu
Numerous methods have been described to determine the apolipoprotein E (apoE) phenotype or genotype of individuals (1)(2). These techniques are relatively time-consuming, and interpretation of the results can be difficult. Here, we report the development of a rapid and specific antibody-based test for the identification of the apoE2 isoform. Previously, we characterized the binding properties of a panel of anti-human apoE monoclonal antibodies (mAbs) (3)(4)(5). The locations of epitopes of selected mAbs from the panel are presented in Fig. 1A
. Although neither mAb 6C5 nor 3H1 showed apoE isoform specificity, mAb 2E8 recognized an epitope that includes the apoE LDL receptor-binding site and resembles the LDL receptor in terms of its fine specificity.
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Several apoE variants that are defective in their ability to mediate binding of lipoproteins to the LDL receptor and are associated with type III hyperlipoproteinemia are poorly recognized by mAb 2E8. These include the relatively common apoE2 (Arg158-Cys) isoform as well as many, but not all, of the rare variants, including apoE2 (Arg136-Ser). Thus, similar to DNA-based phenotyping assays, our immunoassay is designed to detect the most common apoE2 isoforms. When mixtures of purified apoE2 and apoE3 were prepared and tested in a sandwich ELISA format using mAb 6C5 for capture and the 2E8-horseradish peroxidase (HRP) conjugate for the identification, there was selective identification of the apoE3 isoform (Fig. 1D
). Similarly, there was selective identification of apoE2 in mixtures of apoE4 and apoE2 by this test (not shown). We have exploited this isoform specificity to develop an antibody-based test that can identify individuals who are homo- or heterozygous for expression of the apoE2 allele. Compared with existing methodologies, the antibody-based test is inexpensive, rapid, and simple and could easily be incorporated in a clinical laboratory setting.
Blood and DNA samples were obtained from consenting individuals attending the University of Ottawa Heart Institute Lipid Clinic and the San Francisco General Hospital Lipid Clinic. In all cases, the apoE genotype of all individuals was determined by standard PCR amplification followed by digestion with HhaI (data not shown) (2). Initially, the antibody test was developed as a sandwich RIA. In all cases, the concentration of total apoE in donor plasma was determined by a sandwich RIA in which immobilized mAb 6C5 was the capture antibody and 125I-labeled 3H1 was the detection antibody.
For the determination of apoE in plasma, the assay was performed by coating Immunolon II Removawells (Dynatech) with mAb 6C5 overnight at a concentration of 2 mg/L in phosphate-buffered saline (PBS), pH 7.5. Once coated, the wells can be stored for at least 1 week at 4 °C. Before use, the wells were washed with PBS containing 0.25 mL/L Tween 20 (PBS-Tween) and blocked for 1 h with PBS containing 10 g/L bovine serum albumin (PBS-BSA). The wells were then filled and serially diluted with 100 µL of sample plasma previously diluted 1:20 in PBS containing 10 g/L BSA and 0.1 mL/L Tween 20 (PBS-BSA-Tween). After incubation for 1 h at room temperature, the wells were emptied and washed three times with PBS-Tween. The wells were then filled with 100 µL of 125I-labeled 3H1, which corresponded to 100 000 cpm, diluted in PBS-BSA-Tween and were incubated for 1 h at room temperature. The wells were then emptied, washed three times as before, and counted in a gamma counter. The bound radiolabeled antibody counts were plotted as a function of the plasma dilution to quantify plasma apoE concentrations (Fig. 1B
). Determination of the E2 phenotypic isoform in plasma was performed in parallel, using the same format but with 125I-labeled 2E8 as the identification antibody (Fig. 1C
). Ten confirmed unrelated E2 homozygotes and 2 unrelated E2/E3 heterozygotes were unambiguously ascribed the correct phenotype, and 8 individuals were correctly identified as not having inherited an APOE2 allele.
The three antibodies recognized lipid-free and lipid-associated apoE with the same affinity and isoform specificity (5), and the test worked well with both fresh plasma and plasma frozen at -20 °C. The test has been adapted to an ELISA format using 3H1-HRP and 2E8-HRP conjugates as detection antibodies for quantification and isoform identification, respectively (Fig. 1E
; conjugation performed by Bethyl Inc.). The basic experimental method of the ELISA format is identical to the RIA format described above. However, the bound conjugated antibodies were detected by incubating the washed wells with 100 µL of hydrogen peroxide and o-phenylenediamine (Sigma), and the color was allowed to develop for 3–5 min before the reaction was quenched with 100 µL of 2.5 mol/L sulfuric acid. The absorbance of the reaction mixture at 490 nm was determined in a Spectra MAX 250 ELISA reader (Molecular Devices).
Using the ELISA format, we have correctly identified APOE2 inheritance for the 10 apoE2 homozygotes, 10 E2/E3 heterozygotes, and 2 E4/E2 heterozygotes who were tested. All E3/E3, E4/E3, and E4/E4 individuals who were tested were also correctly categorized as having not inherited an APOE2 allele. With the ELISA format, plasma from individuals known to lack an APOE2 allele reached half maximum 2E8 mAb binding at a dilution of 0.03 ± 0.01 times the value obtained for confirmed apoE2 homozygotes, whereas plasma from individuals with one confirmed allele was centered at 0.12 ± 0.05 times this value. The sensitivities of the ELISA and RIA versions of the test were similar (Fig. 1
, C and E).
Development of a complete antibody-based test for the determination of apoE phenotype remains our goal. More specifically, we hope to generate mAbs capable of unambiguously identifying the apoE4 isoform, similar to a previously described mAb (6). Because the inheritance of this allele has been demonstrated to be associated with an increased risk for the development of atherosclerosis and late-onset Alzheimer disease (7), such a tool would be of considerable use to evaluate health risks associated with the expression of this allele.
Acknowledgments
We thank Janet Borthwick and Lito Castro for help in collecting human plasma samples. We also thank Denise Murray and Barbara Westree for help with manuscript preparation, Gary Howard and Stephen Ordway for editorial assistance, and Neile Shea for help with the preparation of the figure. This work was supported by the Heart and Stroke Foundation of Ontario (Operating Grant T3142) and NIH Program Project Grant HL 41633.
References
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3% relative to non-apoE2-containing samples and 10–20% in the equimolar heterozygous form.