4
Determination of 25-Hydroxyvitamin D in Serum by HPLC and Immunoassay, Ursula Turpeinen, * Ulla Ho- henthal, and Ulf-Håkan Stenman (Helsinki University Cen- tral Hospital, Laboratory, Haartmaninkatu 2, 00290 Hel- sinki, Finland; * author for correspondence: fax 358-9- 4717-4945, e-mail [email protected]) Vitamin D status is usually assessed by measuring the serum concentration of 25-hydroxyvitamin D [25(OH)D]. Its measurement is important as a clinical indicator of nutritional vitamin D deficiency, which is one of the causes of osteoporosis (1). Vitamin D exists in two forms: cholecalciferol (vitamin D 3 ) and ergocalciferol (vitamin D 2 ). Vitamin D 2 is further metabolized to 25(OH)D 2 . Vitamin D 3 is formed in the skin from its precursor 7-dehydrocholesterol after ultraviolet irradiation or is absorbed from the diet (2). It is further hydroxylated in the liver to 25(OH)D 3 as the first step of its conversion in the kidney to 1,25-dihydroxyvitamin D 3 , which is the biologically active form. 25(OH)D 3 is the main circulating form of vitamin D. Clinically it is important to measure both forms of 25-hydroxyvitamin D to monitor the effect of vitamin D 2 supplementation on total vitamin D status. The first routine methods for measurement of 25(OH)D concentrations in human plasma were based on compet- itive protein binding and used vitamin D-binding protein and a tritium-labeled tracer (3). These methods were replaced by a simpler, rapid RIA (4 ), and a radioiodinated tracer was incorporated into the RIA in 1993 (5 ). This assay principle is the basis of several commercially available methods. Quantitative HPLC assays have been developed based on ultraviolet detection and normal-phase separation (6 ), combined use of normal- and reversed-phase separa- tions (7 ), or reversed-phase separation alone (8 ). Recently, reversed-phase HPLC methods for 25(OH)D 3 in human plasma have been developed with normal-phase prepurifi- cation of the sample (9 ) or liquid extraction only (10 ). Earlier HPLC methods for 25(OH)D 3 in serum were designed mainly for research purposes and were there- fore too complicated for routine use. The present method was designed to be easy to use, sensitive, and rapid with simple sample preparation. Separation and quantification of 25(OH)D 3 from 25(OH)D 2 are achieved with an iso- cratic elution. To 0.5 mL of serum, we added 350 L of methanol–2- propanol (80:20 by volume). The tubes were mixed in a Multitube vortex mixer for 30 s. 25(OH)D was extracted by mixing three times (60 s each time) with 2 mL of hexane. The phases were separated by centrifugation, and the upper organic phase was transferred to a conical tube and dried under nitrogen. The residue was dissolved in 100 L of mobile phase. Calibration curves were con- structed using four concentrations of 25(OH)D 3 (15–120 nmol/L; cat. no. H-4014; Sigma Chemical Co.) and human serum albumin (50 g/L; The Finnish Red Cross). For chromatography we used an Agilent series 1100 HPLC system with a quaternary pump. Separation was performed on a LiChrospher 60 RP select B column (4 250 mm; 5 m bead size; Merck) maintained at 40 °C. The mobile phase was 760 mL/L methanol in water, and the flow rate was 1 mL/min. Detection was at 265 nm, and the injected volume was 50 L. The chromatographic separations obtained with calibrators and human sera are shown in Fig. 1. A patient sample containing 25(OH)D 2 is shown in Fig. 1C. The 25(OH)D 3 and 25(OH)D 2 peaks are completely resolved with retention times of 20.8 –21.1 min and 23.1 min, respectively. The prominent peak at 18.1– 18.4 min is retinol. Our HPLC assay is based on that of Aksnes (8), but we used isocratic rather than gradient elution to separate 25(OH)D 3 from 25(OH)D 2 and retinol. The percentage of methanol in the mobile phase is critical for separation of these analytes. Extraction of the serum samples with hexane before HPLC analysis was simple and fast (30 min), and it gave high and reproducible recoveries of 25(OH)D 3 . Total recoveries of 15, 30, 60 and 120 nmol/L 25(OH)D 3 added to five different sera were 85–105%. The peak areas of the endogenous analyte were subtracted from the supplemented sera before compari- son. 25(OH)D 2 and 25(OH)D 3 (Fig. 1C) can be separately quantified, and there were no interfering peaks, although in some samples an extra peak appeared between that of retinol and 25(OH)D 3 . To clearly separate all of the peaks with the mobile phase used, a column 250 mm in length was necessary. With a 150-mm column, the peaks par- tially overlapped. The sensitivity of the ultraviolet detec- tor is also critical, e.g., the HP 1100 diode array and variable wavelength detectors (Agilent Technologies) pro- vided reliable results, but the HP 1090 did not. Samples containing up to 5 g/L hemoglobin or 100 mol/L bilirubin did not interfere with the quantification of 25(OH)D 3 . Our assay was linear at 15–200 nmol/L. The mean slope, intercept, and correlation coefficient (r) for the calibration curve were 0.222 (95% confidence interval, 0.212– 0.231), 2.2 nmol/L (0.41– 4.0 nmol/L), and 0.9993, respectively. The lower limit of detection, defined as the lowest concentration with a minimum signal-to-noise ratio of at least 3:1, was 3 nmol/L. The limit of quantifi- cation, defined as the lowest concentration with a signal- to-noise ratio of 10:1, was 10 nmol/L. A serum concentra- tion of 30 –37 nmol/L is considered indicative of vitamin D deficiency (11, 12). Our lower limit of quanti- fication, 10 nmol/L, is sufficient to detect subnormal serum concentrations. If 1 mL of serum is used, the lower limit of quantification can be lowered to 5 nmol/L. These limits of quantification are comparable to those reported earlier for other HPLC methods (8, 10). The within-assay and total CVs calculated from 10 –15 replicates of samples containing 21.6 –167 nmol/L are shown in Table 1. The within-assay CVs of the RIA were 3–10%, and the total CVs were 4 –17%, calculated from the two controls of the assay reagent set. The 25-hydroxyvitamin D RIAs were from DiaSorin. The assays were performed according to the manufactur- er’s instructions. 25(OH)D was also measured on the Liaison analyzer (Byk-Sangtec) which uses a competitive chemiluminescence (LIA) format with one incubation (DiaSorin). The antibody used is the same as in the RIA. Clinical Chemistry 49, No. 9, 2003 1521

Determination of 25-Hydroxyvitamin D in Serum by HPLC and Immunoassay

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Page 1: Determination of 25-Hydroxyvitamin D in Serum by HPLC and Immunoassay

Determination of 25-Hydroxyvitamin D in Serum byHPLC and Immunoassay, Ursula Turpeinen,* Ulla Ho-henthal, and Ulf-Håkan Stenman (Helsinki University Cen-tral Hospital, Laboratory, Haartmaninkatu 2, 00290 Hel-sinki, Finland; * author for correspondence: fax 358-9-4717-4945, e-mail [email protected])

Vitamin D status is usually assessed by measuring theserum concentration of 25-hydroxyvitamin D [25(OH)D].Its measurement is important as a clinical indicator ofnutritional vitamin D deficiency, which is one of thecauses of osteoporosis (1 ). Vitamin D exists in two forms:cholecalciferol (vitamin D3) and ergocalciferol (vitaminD2). Vitamin D2 is further metabolized to 25(OH)D2.Vitamin D3 is formed in the skin from its precursor7-dehydrocholesterol after ultraviolet irradiation or isabsorbed from the diet (2 ). It is further hydroxylated inthe liver to 25(OH)D3 as the first step of its conversion inthe kidney to 1,25-dihydroxyvitamin D3, which is thebiologically active form. 25(OH)D3 is the main circulatingform of vitamin D. Clinically it is important to measureboth forms of 25-hydroxyvitamin D to monitor the effectof vitamin D2 supplementation on total vitamin D status.

The first routine methods for measurement of 25(OH)Dconcentrations in human plasma were based on compet-itive protein binding and used vitamin D-binding proteinand a tritium-labeled tracer (3 ). These methods werereplaced by a simpler, rapid RIA (4), and a radioiodinatedtracer was incorporated into the RIA in 1993 (5). This assayprinciple is the basis of several commercially availablemethods. Quantitative HPLC assays have been developedbased on ultraviolet detection and normal-phase separation(6), combined use of normal- and reversed-phase separa-tions (7), or reversed-phase separation alone (8). Recently,reversed-phase HPLC methods for 25(OH)D3 in humanplasma have been developed with normal-phase prepurifi-cation of the sample (9) or liquid extraction only (10).

Earlier HPLC methods for 25(OH)D3 in serum weredesigned mainly for research purposes and were there-fore too complicated for routine use. The present methodwas designed to be easy to use, sensitive, and rapid withsimple sample preparation. Separation and quantificationof 25(OH)D3 from 25(OH)D2 are achieved with an iso-cratic elution.

To 0.5 mL of serum, we added 350 �L of methanol–2-propanol (80:20 by volume). The tubes were mixed in aMultitube vortex mixer for 30 s. 25(OH)D was extractedby mixing three times (60 s each time) with 2 mL ofhexane. The phases were separated by centrifugation, andthe upper organic phase was transferred to a conical tubeand dried under nitrogen. The residue was dissolved in100 �L of mobile phase. Calibration curves were con-structed using four concentrations of 25(OH)D3 (15–120nmol/L; cat. no. H-4014; Sigma Chemical Co.) and humanserum albumin (50 g/L; The Finnish Red Cross).

For chromatography we used an Agilent series 1100HPLC system with a quaternary pump. Separation wasperformed on a LiChrospher 60 RP select B column (4 �250 mm; 5 �m bead size; Merck) maintained at 40 °C. The

mobile phase was 760 mL/L methanol in water, and theflow rate was 1 mL/min. Detection was at 265 nm, andthe injected volume was 50 �L. The chromatographicseparations obtained with calibrators and human sera areshown in Fig. 1. A patient sample containing 25(OH)D2 isshown in Fig. 1C. The 25(OH)D3 and 25(OH)D2 peaks arecompletely resolved with retention times of 20.8–21.1 minand 23.1 min, respectively. The prominent peak at 18.1–18.4 min is retinol. Our HPLC assay is based on that ofAksnes (8 ), but we used isocratic rather than gradientelution to separate 25(OH)D3 from 25(OH)D2 and retinol.The percentage of methanol in the mobile phase is criticalfor separation of these analytes. Extraction of the serumsamples with hexane before HPLC analysis was simpleand fast (30 min), and it gave high and reproduciblerecoveries of 25(OH)D3. Total recoveries of 15, 30, 60 and120 nmol/L 25(OH)D3 added to five different sera were85–105%. The peak areas of the endogenous analyte weresubtracted from the supplemented sera before compari-son. 25(OH)D2 and 25(OH)D3 (Fig. 1C) can be separatelyquantified, and there were no interfering peaks, althoughin some samples an extra peak appeared between that ofretinol and 25(OH)D3. To clearly separate all of the peakswith the mobile phase used, a column 250 mm in lengthwas necessary. With a 150-mm column, the peaks par-tially overlapped. The sensitivity of the ultraviolet detec-tor is also critical, e.g., the HP 1100 diode array andvariable wavelength detectors (Agilent Technologies) pro-vided reliable results, but the HP 1090 did not. Samplescontaining up to 5 g/L hemoglobin or 100 �mol/Lbilirubin did not interfere with the quantification of25(OH)D3.

Our assay was linear at 15–200 nmol/L. The meanslope, intercept, and correlation coefficient (r) for thecalibration curve were 0.222 (95% confidence interval,0.212–0.231), 2.2 nmol/L (0.41–4.0 nmol/L), and 0.9993,respectively. The lower limit of detection, defined as thelowest concentration with a minimum signal-to-noiseratio of at least 3:1, was 3 nmol/L. The limit of quantifi-cation, defined as the lowest concentration with a signal-to-noise ratio of 10:1, was 10 nmol/L. A serum concentra-tion of �30–37 nmol/L is considered indicative ofvitamin D deficiency (11, 12). Our lower limit of quanti-fication, 10 nmol/L, is sufficient to detect subnormalserum concentrations. If 1 mL of serum is used, the lowerlimit of quantification can be lowered to 5 nmol/L. Theselimits of quantification are comparable to those reportedearlier for other HPLC methods (8, 10).

The within-assay and total CVs calculated from 10–15replicates of samples containing 21.6–167 nmol/L areshown in Table 1. The within-assay CVs of the RIA were3–10%, and the total CVs were 4–17%, calculated from thetwo controls of the assay reagent set.

The 25-hydroxyvitamin D RIAs were from DiaSorin.The assays were performed according to the manufactur-er’s instructions. 25(OH)D was also measured on theLiaison analyzer (Byk-Sangtec) which uses a competitivechemiluminescence (LIA) format with one incubation(DiaSorin). The antibody used is the same as in the RIA.

Clinical Chemistry 49, No. 9, 2003 1521

Page 2: Determination of 25-Hydroxyvitamin D in Serum by HPLC and Immunoassay

The regression lines between each pair of assay methodswere calculated by the Deming method (13, 14). Thismethod allows for variation in both the x and y axes at thesame time. The correlation by the Deming method was:RIA � 1.02(HPLC) � 1.02 nmol/L (r � 0.829; n � 301).The overall correlation was fairly acceptable, and theslope was close to unity; several samples, however, dis-

played very large differences. This is in agreement with aprevious study in which the correlation was 0.86 withonly 25 samples (15 ). The correlation between the HPLC(x) and the LIA method (y) was: LIA � 1.05(HPLC) � 4.84nmol/L (r � 0.735; n � 203), and that between Liaison andRIA was: Liaison � 1.03(RIA) � 3.78 nmol/L (r � 0.595;n � 203). The correlation between HPLC and the Liaison

Fig. 1. Chromatograms of a calibrator in human albumin containing60 nmol/L 25(OH)D3 (A), a patient sample containing 72 nmol/L25(OH)D3 (B), and a patient sample containing both 25(OH)D3 and25(OH)D2 (C).

1522 Technical Briefs

Page 3: Determination of 25-Hydroxyvitamin D in Serum by HPLC and Immunoassay

assay was poor, and that between RIA and LIA stillworse. The poor correlation between RIA and LIA issurprising because both assays use the same antibody.This may be explained by the use of a separate precipita-tion/extraction step before the immunoassay in the RIA.

We estimated the costs (in US dollars) of the tests basedon 1000, 2000, 3000, and 4000 samples/year. The instru-ment costs for the HPLC method were calculated on thebasis of a leasing fee of 30% of the price of the automatedHPLC instrument (US $38 000). The cost for the HPLCcolumn was US $360. The cost for the DiaSorin RIAreagents (US $383) was calculated on the basis of optimalusage, i.e., with 100 tubes, 6 calibrators, 42 samples, and 2controls analyzed in duplicate. Labor costs were esti-mated according to our local expenses. In our setting, thecosts of the HPLC method (US $10 per sample) wereclearly lower than that of the RIA (US $16–18 per sample),irrespective of the number of samples analyzed per year.We did not calculate the costs for the automated methodbecause they would have been competitive only if theanalyzer was used mainly for other purposes.

The finding (1 ) of lowered hip fracture incidence andhigher circulating concentrations of 25(OH)D has greatlyincreased the use of vitamin D assays. The use of vitaminD supplementation makes reliable measurement of25(OH)D important. Results of an international compari-son of serum 25-hydroxyvitamin D measurements, how-ever, show that 25(OH)D values from different laborato-ries are often not comparable, with interlaboratorydifferences in assays for serum 25(OH)D being up to 38%(16 ). More accurate assays are therefore needed.

To determine nutritional vitamin D status, it is importantthat the method used measures circulating 25(OH)D2 and25(OH)D3 equally to provide total circulating 25(OH)D. Theprimary antibody in the DiaSorin RIA is claimed to recog-nize both forms of vitamin D equally, although the calibra-tion curves are constructed with 25(OH)D3 (15). That makesit possible to monitor the effect of supplemented vitamin D2on total vitamin D status. Of the many protein-bindingmethods developed for the determination of vitamin Dstatus during the past 30 years, 125I-based RIAs have becomemost widely used methods for determining circulating25(OH)D. HPLC methods (6, 7) that use ultraviolet detectionare much less common, apparently because of their labori-ous prepurification steps and the use of normal-phase chro-matography. Recently, relatively simple and sensitive meth-ods based on reversed-phase chromatography have beendeveloped (8–10).

In Finland, vitamin D deficiency is quite common

during winter time, and recognition of low values isimportant (12 ). On the basis of the results obtained in thepresent study, we have decided to switch to the HPLCmethod. Ideally, a mass spectrometric method should beused to validate the HPLC method, but in earlier studiesa similar HPLC method was shown to correlate stronglywith isotope-dilution mass spectrometry (17 ). The goodrecovery and precision also indicate that the HPLC assayprovides more correct results than the immunologicmethods. In our setting, the lower cost also favors use ofthe HPLC method. The automated method is potentiallymore economical if the analyzer is used mainly for otherassays, but further development of this assay is necessaryto make it a viable alternative. We could not explain thereasons for the poor correlation with the immunoassays,but based on results from earlier studies, immunoassaysare affected by nonspecific interference (17 ).

In conclusion, our HPLC method with ultraviolet de-tection enables reliable quantification of 25(OH)D3 and, ifpresent, 25(OH)D2. The short and relatively simple sam-ple preparation and ease of use make it useful for routinedeterminations.

References1. Chapuy MC, Arlot ME, DuBoeuf F, Brun J, Crouzet B, Arnaud S, et al. Vitamin

D3 and calcium to prevent hip fractures in elderly women. N Engl J Med1992;327:1637–42.

2. Holick MF. The cutaneous photosynthesis of previtamin D3: a uniquephotoendocrine system. J Invest Dermatol 1981;76:51–8.

3. Haddad JG, Chyu KJ. Competitive protein-binding radioassay for 25-hydroxy-cholecalciferol. J Clin Endocrinol 1971;33:992–5.

4. Hollis BW, Napoli JL. Improved radioimmunoassay for vitamin D and its usein assessing vitamin D status. Clin Chem 1985;31:1815–9.

5. Hollis BW, Kamerud JQ, Selvaag SR, Lorenz JD, Napoli JL. Determination ofvitamin D status by radioimmunoassay with an 125I-labeled tracer. ClinChem 1993;39:529–33.

6. Gilbertson TJ, Stryd RP. High-performance liquid chromatographic assay for25-hydroxyvitamin D3 in serum. Clin Chem 1977;23:1700–4.

7. Jones G. Assay of vitamins D2 and D3, and 25-hydroxyvitamins D2 and D3 inhuman plasma by high-performance liquid chromatography. Clin Chem1978;24:287–98.

8. Aksnes L. Simultaneous determination of retinol, �-tocopherol, and 25-hydroxyvitamin D in human serum by high-performance liquid chromatogra-phy. J Pediatr Gastroenterol Nutr 1994;18:339–43.

9. Shimada K, Mitamura K, Kitama N, Kawasaki M. Determination of 25-hydroxyvitamin D3 in human plasma by reversed-phase high-performanceliquid chromatography with ultraviolet detection. J Chromatogr 1997;689:409–14.

10. Alvarez JC, De Mazancourt P. Rapid and sensitive high-performance liquidchromatographic method for simultaneous determination of retinol, �-tocopherol, 25-hydroxyvitamin D3 and 25-hydroxyvitamin D2 in humanplasma with photodiode-array ultraviolet detection. J Chromatogr 2001;755:129–35.

11. Tannenbaum C, Clark J, Schwartzman K, Wallenstein S, Lapinski R, Meier D,et al. Yield of laboratory testing to identify secondary contributors toosteoporosis in otherwise healthy women. J Clin Endocrinol Metab 2002;87:4431–7.

12. Kauppinen-Makelin R, Tahtela R, Loyttyniemi E, Karkkainen J, Valimaki M. Ahigh prevalence of hypovitaminosis D in Finnish medical in- and outpatients.J Intern Med 2001;249:559–63.

13. Cornbleet PJ, Gochman N. Incorrect least-squares regression coefficients inmethod-comparison analysis. Clin Chem 1979;25:432–8.

14. Linnet K. Evaluation of regression procedures for method comparisonstudies. Clin Chem 1993;39:424–32.

15. Hollis BW. Comparison of commercially available 125I-based RIA methodsfor the determination of circulating 25-hydroxyvitamin D. Clin Chem 2000;46:1657–61.

16. Lips P, Chapuy MC, Dawson-Hughes B, Pols HAP, Holick MF. An internationalcomparison of serum 25-hydroxyvitamin D measurements. Osteoporosis Int1999;9:394–7.

Table 1. Within-run and total precision for 25(OH)D3

in serum.Within-run Total

Mean, nmol/L 21.6 38.7 63.8 138 16.4 47.5 167SD, nmol/L 1.2 2.2 3.4 5.1 1.2 3.0 9.5CV, % 5.6 5.7 5.3 3.7 7.3 6.3 5.7n 14 13 10 15 12 15 15

Clinical Chemistry 49, No. 9, 2003 1523

Page 4: Determination of 25-Hydroxyvitamin D in Serum by HPLC and Immunoassay

17. Lindback B, Berlin T, Bjorkhem I. Three commercial kits and one liquid-chromatographic method evaluated for determining 25-hydroxyvitamin D3 inserum. Clin Chem 1987;33:1226–7.

Antibody Phenotyping Test for the Human Apolipopro-tein E2 Isoform, Robert L. Raffaı,2* Ruth McPherson,1 KarlH. Weisgraber,2 Thomas L. Innerarity,2 Eric Rassart,3 ThomasP. Bersot,2 and Ross W. Milne1 (1 Lipoprotein and Athero-sclerosis Group, University of Ottawa Heart Institute,Ottawa Civic Hospital, Ottawa, Ontario, K1Y 4E9 Canada;2 The Gladstone Institute of Cardiovascular Disease, Car-diovascular Research Institute, University of California,San Francisco, CA 94141-9100; 3 Departement des SciencesBiologiques, Universite du Quebec, Montreal, CP 8888,Quebec, H3C 3P8 Canada; * author for correspondence:fax 415-285-5632, e-mail [email protected])

Numerous methods have been described to determine theapolipoprotein E (apoE) phenotype or genotype of indi-viduals (1, 2). These techniques are relatively time-con-suming, and interpretation of the results can be difficult.Here, we report the development of a rapid and specificantibody-based test for the identification of the apoE2isoform. Previously, we characterized the binding prop-erties of a panel of anti-human apoE monoclonal antibod-ies (mAbs) (3–5). The locations of epitopes of selectedmAbs from the panel are presented in Fig. 1A. Althoughneither mAb 6C5 nor 3H1 showed apoE isoform specific-ity, mAb 2E8 recognized an epitope that includes theapoE LDL receptor-binding site and resembles the LDLreceptor in terms of its fine specificity.

Several apoE variants that are defective in their abilityto mediate binding of lipoproteins to the LDL receptorand are associated with type III hyperlipoproteinemia arepoorly recognized by mAb 2E8. These include the rela-tively common apoE2 (Arg158-Cys) isoform as well asmany, but not all, of the rare variants, including apoE2(Arg136-Ser). Thus, similar to DNA-based phenotypingassays, our immunoassay is designed to detect the mostcommon apoE2 isoforms. When mixtures of purifiedapoE2 and apoE3 were prepared and tested in a sandwichELISA format using mAb 6C5 for capture and the 2E8-horseradish peroxidase (HRP) conjugate for the identifi-cation, there was selective identification of the apoE3isoform (Fig. 1D). Similarly, there was selective identifi-cation of apoE2 in mixtures of apoE4 and apoE2 by thistest (not shown). We have exploited this isoform specific-ity to develop an antibody-based test that can identifyindividuals who are homo- or heterozygous for expres-sion of the apoE2 allele. Compared with existing method-ologies, the antibody-based test is inexpensive, rapid, andsimple and could easily be incorporated in a clinicallaboratory setting.

Blood and DNA samples were obtained from consent-ing individuals attending the University of Ottawa HeartInstitute Lipid Clinic and the San Francisco GeneralHospital Lipid Clinic. In all cases, the apoE genotype of all

individuals was determined by standard PCR amplifica-tion followed by digestion with HhaI (data not shown) (2 ).Initially, the antibody test was developed as a sandwichRIA. In all cases, the concentration of total apoE in donorplasma was determined by a sandwich RIA in whichimmobilized mAb 6C5 was the capture antibody and125I-labeled 3H1 was the detection antibody.

For the determination of apoE in plasma, the assay wasperformed by coating Immunolon II Removawells (Dyna-tech) with mAb 6C5 overnight at a concentration of 2mg/L in phosphate-buffered saline (PBS), pH 7.5. Oncecoated, the wells can be stored for at least 1 week at 4 °C.Before use, the wells were washed with PBS containing0.25 mL/L Tween 20 (PBS-Tween) and blocked for 1 hwith PBS containing 10 g/L bovine serum albumin (PBS-BSA). The wells were then filled and serially diluted with100 �L of sample plasma previously diluted 1:20 in PBScontaining 10 g/L BSA and 0.1 mL/L Tween 20 (PBS-BSA-Tween). After incubation for 1 h at room tempera-ture, the wells were emptied and washed three times withPBS-Tween. The wells were then filled with 100 �L of125I-labeled 3H1, which corresponded to 100 000 cpm,diluted in PBS-BSA-Tween and were incubated for 1 h atroom temperature. The wells were then emptied, washedthree times as before, and counted in a gamma counter.The bound radiolabeled antibody counts were plotted asa function of the plasma dilution to quantify plasma apoEconcentrations (Fig. 1B). Determination of the E2 pheno-typic isoform in plasma was performed in parallel, usingthe same format but with 125I-labeled 2E8 as the identifi-cation antibody (Fig. 1C). Ten confirmed unrelated E2homozygotes and 2 unrelated E2/E3 heterozygotes wereunambiguously ascribed the correct phenotype, and 8individuals were correctly identified as not having inher-ited an APOE2 allele.

The three antibodies recognized lipid-free and lipid-associated apoE with the same affinity and isoform spec-ificity (5 ), and the test worked well with both freshplasma and plasma frozen at �20 °C. The test has beenadapted to an ELISA format using 3H1-HRP and 2E8-HRP conjugates as detection antibodies for quantificationand isoform identification, respectively (Fig. 1E; conjuga-tion performed by Bethyl Inc.). The basic experimentalmethod of the ELISA format is identical to the RIA formatdescribed above. However, the bound conjugated anti-bodies were detected by incubating the washed wellswith 100 �L of hydrogen peroxide and o-phenylenedi-amine (Sigma), and the color was allowed to develop for3–5 min before the reaction was quenched with 100 �L of2.5 mol/L sulfuric acid. The absorbance of the reactionmixture at 490 nm was determined in a Spectra MAX 250ELISA reader (Molecular Devices).

Using the ELISA format, we have correctly identifiedAPOE2 inheritance for the 10 apoE2 homozygotes, 10E2/E3 heterozygotes, and 2 E4/E2 heterozygotes whowere tested. All E3/E3, E4/E3, and E4/E4 individualswho were tested were also correctly categorized as havingnot inherited an APOE2 allele. With the ELISA format,plasma from individuals known to lack an APOE2 allele

1524 Technical Briefs