Quantification of Eu31 in Quantum-Dye-Labeled Materialsby Ashing and Dissociation Enhancement

Keiko Saito, Reiko T. Lee, and Yuan C. Lee1

Department of Biology, Johns Hopkins University, 3400 North Charles Street, Baltimore, Maryland 21218

Received October 6, 1997

Quantum Dye (QD) is an extremely stable macrocy-clic chelate of Eu31, measurable with a time-resolvedfluorometer even without the aid of enhancement so-lution, albeit at a low sensitivity. However, QD is noteasily quantified by the popular dissociation-enhance-ment methodology (I. Hemmila et al., 1984, Anal. Bio-chem. 137, 335–343), because of difficulty in dissociat-ing Eu31 completely from QD. We found that Eu31 inQD can be accurately measured by simple ashing fol-lowed by the dissociation-enhancement methodology.Validity of the method was tested by determiningEu(NO3)3 with or without ashing and analyzing Eu31

in other chelates known to be amenable to accuratemeasurement by the dissociation enhancement. © 1998

Academic Press

Key Words: Quantum Dye; europium; dissociationenhancement; fluorescence.

Lanthanides, such as Sm31, Tb31, and Eu31, havebeen gaining favor in recent years as fluorescenceprobes in the measurement of biologically active com-pounds such as enzymes, lectins, and receptors (1–5),as well as in histochemical studies (6, 7). One of theadvantages is their high sensitivity, comparable to themethods using radioisotopes (8–11).

The labeling of proteins or amino compounds withEu31 is typically performed using phenylisothiocya-nate derivative of EDTA (9, 11) or diethylenetriamine-pentaacetic acid (DTPA)2 dianhydride (12–14) (Fig. 1).In the dissociation-enhanced lanthanide fluorescent

immunoassay (DELFIA) technique (9, 15), Eu31 is firstdissociated from the original chelate to form a newcomplex with 1-(b-naphthoyl)-3,3,3-trifluoroacetone (b-NTA), which, in the presence of detergents, becomeshighly fluorescent (9). Because the fluorescence of Eu31

has a decay time lasting over 104 times longer thanthat of polystyrene and other plastics (16), Eu31 che-lates can be measured in a plastic microtiter plate bytime-resolved fluorometry (11).

Quantum Dye (QD; Fig. 1), originally developed asa histochemical marker, is a macrocyclic Eu31 che-late (1, 17), which displays low but measurable flu-orescence. We have been using QD to label lectinsand neoglycoproteins to perform quantitative analy-sis in glycobiology successfully (Y. C. Lee et al., un-published results). However, there has not been areliable method for determining the absolute amountof QD. This is because the stability of Eu31 chelationin QD does not permit effective and reliable dissoci-ation enhancement, and the current price of QD isprohibitive for it to be measured accurately byweighing. In this paper, we describe a simple andreliable method for quantification of QD using a com-bination of ashing and dissociation-enhancementtechnology.

MATERIALS AND METHODS

Materials

N1-(p-Isothiocyanatobenzyl)diethylenetriamine-N1,N2,N3,N3-tetraacetic acid (DETATA-ITC) (8, 9), a dis-sociation-enhancement solution [15 mM b-NTA, 50 mMtrioctylphosphine oxide (TOPO), and 0.1% (w/v) TritonX-100 in 0.1 M acetic acid adjusted to pH 3.2 withpotassium hydrogen phthalate] (9), and 96-well micro-plates were obtained from Wallac (Gaithersburg, MD).Quantum Dye (17, 18) was from Research Organics,Inc. (Cleveland, OH). Potassium hydrogen phthalateand Triton X-100 were from Sigma Chemical Co. (St.Louis, MO). Europium nitrate, b-NTA, TOPO, and

1 To whom correspondence should be addressed. Fax: (410) 516-8716. E-mail: yclee@jhu.edu.

2 Abbreviations used: DTPA, diethylenetriaminepentaacetic acid;DELFIA, dissociation-enhanced lanthanide fluorescence immunoassay;b-NTA, 1-(b-naphthoyl)-3,3,3-trifluroacetone; QD, Quantum Dye; DE-TATA-ITC, N1-(p-isothiocyanatobenzyl)diethylenetriaminetetraaceticacid; TOPO, trioctylphosphine oxide; BSA, bovine serum albumin.

0003-2697/98 $25.00 311Copyright © 1998 by Academic PressAll rights of reproduction in any form reserved.

ANALYTICAL BIOCHEMISTRY 258, 311–314 (1998)ARTICLE NO. AB982612

DTPA and its dianhydride (12–14) were from AldrichChemical Co. (Milwaukee, WI).

Man31–BSA, a conjugate of bovine serum albuminand thiomannoside, was prepared as described (19),and Man9GlcNAc2Asn was obtained from soybeanagglutinin by protease digestion and HPLC fraction-ation (20). The labeling of Man9GlcNAc2Asn withDTPA dianhydride has been reported (14). Labelingof Man31–BSA with QD and labeling of an anti-(C-reactive protein) IgG with DETATA-ITC were ac-cording to the procedure recommended by the man-ufacturers of individual reagents. Structures of theproducts obtained using DETATA-ITC and DTPAdianhydride are shown in Fig. 1.

General Methods

Time-resolved fluorescence of Eu31 was measured ina microtiter plate, with or without the dissociation-enhancement solution (hereafter referred to as en-hancement solution), using a time-resolved fluoromet-ric microplate reader (Victor, Model 1420, Wallac).When the enhancement solution is to be used, a suit-able amount (5–50 ml) of sample solution was dilutedwith the enhancement solution (200 ml) and incubatedfor 15 min at room temperature before the measure-ment. Fluorescence (ex 5 340 nm, em 5 615 nm) wasdetected by means of single photon counting, using axenon flash lamp (1000 Hz) for a total measuring timeof 1 s, with a 50-ms delay time and 250-ms countingtime per flash (9).

Ashing of Eu31 Chelates

The ashing method is basically that used in a well-established organic phosphate determination (21). Asample solution (30 ml) of Eu31 chelate was placed in a13 3 100 mm Pyrex test tube and mixed with 30 ml of10% Mg(NO3)2 in 95% ethanol. The sample was takento dryness and ashed by passing the tube over a strongBunsen burner flame for several seconds. The ashedsample was dissolved by incubating in 1.5 ml of 0.1 Macetic acid at 55°C for 30 min. An appropriate amountof the dissolved sample (20–200 fmol Eu31) was takenfor measurement of Eu31 fluorescence using the disso-ciation-enhancement method. Various amounts of anEu(NO3)3 solution were ashed in the same fashion to beused as standard for quantification of Eu31.

RESULTS

Stability of QD

QD–Man31–BSA (ca. 8 pmol) was incubated with 200ml of DTPA solutions of different concentrations in

FIG. 1. Structures of three Eu31 chelates. (A) Quantum Dye, (B)protein conjugate of DETATA-ITC [N1-(p-isothiocyanatobenzyl)di-ethylenetriaminetetraacetic acid], and (C) diethylenetriaminepenta-acetic acid (DTPA) monoamide, where R can be the structure shownin the enclosure (Man9GlcNAc2Asn).

FIG. 2. Stability of QD. Effect of DTPA on the QD–Man31–BSA. F,No DTPA; Œ, 1 mM DTPA; �, 10 mM DTPA.

312 SAITO, LEE, AND LEE

microtiter wells at room temperature, and the fluores-cence counts were recorded over the course of 5 days(Fig. 2). Even with 10 mM DTPA, the dissociation ofEu31 was extremely slow, dropping only 20% after120 h.

Enhancement of QD Fluorescence

Samples of ca. 1 pmol of QD–Man31–BSA containedin wells of a microtiter plate were treated either with200 ml of water or with the enhancement solution, andthe fluorescent counts were measured over a 5-dayperiod. As soon as the enhancement solution wasadded, the fluorescence increased 270-fold over that inwater. As shown in Fig. 3, the fluorescence subse-quently increased slowly but steadily, but even after100 h, only ca. 50% increase over the initial value wasseen. The fluorescence in water, which was too low tobe plotted in the figure, showed a slight decline. After5 days, fluorescence in the enhancement solution wasca. 480-fold higher than that in water. This value wasstill lower than that obtainable by ashing and dissoci-ation enhancement (see later). In contrast, when DE-TATA and DTPA-based Eu31 chelates were treatedwith the enhancement solution, Eu31 was efficientlyreleased from the chelate within a few minutes.

Eu31 Quantification in Eu31 Chelates after Ashing

When the fluorescence of various amounts ofEu(NO3)3 was determined with or without ashing, bothseries of samples gave straight lines which overlappedeach other (Fig. 4), indicating quantitative recovery ofEu31 after ashing. When the DETATA-ITC-labeled an-ti(C-reactive protein) IgG and the DTPA-modifiedMan9GlcNAc2Asn were measured by the standard dis-sociation-enhancement method with or without ash-

ing, the fluorescence counts after ashing were 95–100%of the values without ashing (Table 1). These resultsestablished the fact that the ashing per se does notinterfere with the standard dissociation-enhancementprocess.

However, when QD-labeled Man31–BSA was treatedsimilarly, the fluorescence after ashing and dissocia-tion enhancement was approximately 2.4-fold greaterthan that without ashing. The value obtained afterashing probably represents the true Eu31 content, andit is higher than the value obtainable after 5 days ofdirect dissociation enhancement (Fig. 3). Therefore,QD-labeled samples can be quantified by ashing anddissociation enhancement, using Eu(NO3)3 solutionssimilarly treated as standards (Fig. 4).

DISCUSSION

DELFIA (9) is a sensitive and powerful method thatcan compete with radioisotopic methods in quantifica-tion of biochemical and biological reactions. When bio-logical samples are Eu31-labeled with polycarboxylate-

FIG. 3. Enhancement of QD fluorescence. The microtiter well con-tained 1 pmol QD–Man31–BSA and 200 ml of the enhancement solu-tion.

FIG. 4. Standard curves of Eu(NO3)3 by dissociation enhancement.E, Without ashing; F, with ashing. Each point represents an average(61.2%) of triplicate determinations.

TABLE 1

Quantification of Different Eu13 Chelates by DissociationEnhancement with and without Ashing

ChelatesParent

compounds

Ashing a

% 6 SEbWithout With

— Eu(NO3)3 1,038,951 1,057,580 101 6 1.3QDc Man31–BSA 527,550 1,246,260 236 6 7.9DETATAc Anti-CRP IgG 499,904 468,684 94 6 1.8DTPAd Man9GlcNAc2Asn 562,732 588,132 105 6 5.1

a Average fluorescence (cps) of triplicate determination.b (With ashing/without ashing) 3100.c Modified with its isothiocyanate.d Modified with its dianhydride.

313DETERMINATION OF QUANTUM DYE BY ASHING

type chelates, such as EDTA or DTPA derivatives, thequantification can be made quite rapidly by the disso-ciation-enhancement technique. Moreover, the factthat these chelates yielded identical fluorescencecounts with or without ashing confirms the notion thatdirect treatment with the enhancement solution is suf-ficient for the quantification of the absolute amount ofpolycarboxylate-type Eu31 chelates.

Although a different enhancing solution containing4,4,4-trifluoro-1-(2-thiophene)-2,4-butanedione wasproposed for QD (6), it was reported to be less effectivethan the Hemmila enhancing solution mentionedabove (6). Therefore, in this work, we chose to use thelatter enhancing solution. We found, however, thateven with the Hemmila enhancing solution, the en-hancement of Eu31 fluorescence in QD is a very slowprocess that does not reach the maximum value obtain-able by ashing even after 5 days. Therefore to use theenhancement solution to measure the Eu31 in QD-modified samples is inconvenient at best and is inac-curate. The approach we describe in this paper is asimple ashing technique, which oxidatively decom-poses organic material completely and returns Eu31 toa form easily extractable into 0.1 M acetic acid. Thatashing does not affect the measurement of Eu31 in theHemmila enhancing solution containing b-NTA,TOPO, and Triton X-100 (9) was confirmed by theidentical fluorescence counts obtained for Eu(NO3)3with or without enhancement. We found that the com-plete dissolution of ashed samples requires some care,and the dissolution is more efficient in 0.1 M acetic acidthan in water or the enhancement solution. Although aseemingly large (1.5 ml) volume of 0.1 M acetic acid isused to ensure complete dissolution, it is a small priceto pay in view of the high sensitivity of Eu31 fluores-cence. Therefore, the ashing combined with the disso-ciation-enhancement technique is a rapid and accuratemethod for quantification of QD-labeled samples. Al-though the proposed method is aimed at standardiza-tion of a stock QD-labeled sample solution, the ease ofoperation renders itself useful even as a routinemethod.

ACKNOWLEDGMENTS

The authors are grateful to Dr. Nana Kawasaki for the sample ofQD–Man31–BSA. This work was supported by NIH Research GrantDK09970 and by Oriental Yeast Co., Tokyo, Japan.

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