Bioconjugate Chem. 2004, 15, 927−930
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Labeling of Steroids on Solid Phase Jari Peuralahti, Katriina Suonpa¨a¨, Kaj Blomberg, Veli-Matti Mukkala, and Jari Hovinen* PerkinElmer Life and Analytical Sciences, Turku Site, P.O. Box 10, FIN-20101 Turku, Finland. Received March 19, 2004; Revised Manuscript Received May 27, 2004
Up to four tetra-tert-butyl-1-[4-aminoacetamido)benzyl]diethylenetriaminetetrakis(acetato) derivatives of Fmoc glutamic acid (1) were attached to two steroids (17R-hydroxyprogesterone-3-Ocarboxymethyloxime 2 and 1,3,5(10)-estratriene-3,16R,17β-triol-6-one-6-O-carboxymethyloxime, 3)) on solid phase using an oligopeptide synthesizer. Upon deprotection and conversion to the corresponding europium(III) chelates, these steroid conjugates were used in DELFIA-based competitive fluoroimmunoassays. The more chelates conjugated to 17-R-hydroxyprogesterone, the more diluted antiserum could be used in an immunoassay for 17-R-hydroxyprogesterone, without any alteration of the measurement range. Hence, 17-R-hydroxyprogesterone tracers with several chelates are useful when a high serum dilution factor is desired i.e., when only a limited quantity of antiserum is available. The result demonstrates the suitability and usefulness of lanthanide(III) chelates as multilabels in bioaffinity assays.
INTRODUCTION
Chart 1
Steroids with additional functional groups are often needed for biological, microbiological and medicinal applications (1). For example, in competitive immunoassays, haptens coupled to a label molecule are used as tracers (2, 3). Most commonly, a spacer arm with a reactive ω-substituent (amino or thiol) is introduced to the steroid structure before the labeling reaction, which is performed in solution between the hapten and an isothiocyanato, haloacetyl, or 3,5-dichloro-2,4,6-triazinyl derivative of the label molecule. Steroids tethered to a carboxylic acid group, in turn, are coupled to labels bearing an amino function using carbodiimide (4). The solution-phase labeling has been used in the preparation of haptens labeled with up to two europium(III) chelates (5). Because in all of these cases the labeling reaction is performed in the presence of an excess of an activated label or an activator, laborious purification procedures cannot be prevented. Especially, when attachment of several label molecules is needed, purification and characterization of the desired biomolecule conjugate may be extremely difficult. The purification problems can be avoided by performing the labeling reaction on solid phase. Hence, most of the impurities can be removed by washings when the biomolecule conjugate is still anchored to the solid support, and after release to the solution, only one chromatographic purification is needed. Fluorescent label molecules attached to monomers for solid-phase synthesis are often organic dyes. Although organic chromophores can be utilized in several applications, these labels and labeled biomolecules suffer from many known drawbacks, such as Raman scattering, low water solubility, and especially concentration quenching. In addition, the sensitivity is compromised by the phosphorescence of the microtitration plates. Instead, the unique properties of lanthanide(III) chelates (6-9) such as long decay-time luminescence make them ideal labels for microtitration plate-based assays. Furthermore, large * To whom correspondence should be addressed. Tel: + 358 2 2678 513, fax: + 358 2 2678 380, e-mail: jari.hovinen@ perkinelmer.com.
Stokes shift and very sharp emission bands enable the simultaneous use of four lanthanides (i.e. Eu, Tb, Dy, and Sm). Thus, the heterogeneous DELFIA technique is widely applied in assays where exceptional sensitivity and robustness is required (6, 7). Quite recently, we reported a solid-phase method for the labeling of oligopeptides (10) and oligonucleotides (11, 12). The approach involves synthesis of oligonucleotide and oligopeptide building blocks, which can be introduced to the biomolecule structure using commercial oligonucleotide and oligopeptide synthesizers by phosphoramidite and Fmoc chemistry, respectively. Upon comple-
10.1021/bc049929p CCC: $27.50 © 2004 American Chemical Society Published on Web 06/25/2004
928 Bioconjugate Chem., Vol. 15, No. 4, 2004
Technical Notes
Scheme 1. Labeling of Steroids on Solid Phase
tion of the chain assembly, the oligomer is deprotected and finally treated with the appropriate lanthanide(III) citrate to give rise to the desired biomolecule conjugate. Here the strategy is exploited in multilabeling of steroids using a peptide synthesizer. Accordingly, several tetra-tert-butyl-1-[4-aminoacetamido)benzyl]diethylenetriaminetetrakis(acetato) derivatives of Fmoc glutamic acid (1; Chart 1) were coupled to steroids tethered to a carboxyl acid function (2, 3). Upon deprotection and conversion to the corresponding europium(III) chelate, these steroid conjugates were used in competitive fluoroimmunoassays based on DELFIA technology. The fluorescence signal was approximately proportional to the number of chelates attached to the steroids: the signal with 17-R-hydroxyprogesterone tethered to two or four europium(III) chelates was two and a half and five times higher, respectively, compared to the monolabeled derivative, 4, demonstrating the suitability of lanthanide(III) chelates to multilabeling. EXPERIMENTAL PROCEDURES
General. The steroid compounds (2 and 3) were purchased from Steraloids (Newport, RI). The nonluminescent block, 1, was synthesized as described previously (10). 17-R-OHP-3-CMO-Eu (4), rabbit anti-17-R-OHP antiserum, and the time-resolved fluorometer VICTOR2V were products of PerkinElmer LAS. Anti-rabbit coated
Figure 1. Reversed-phase HPLC profile (crude reaction mixture) of 17-R-hydroxyprogesterone labeled with four nonluminescent europium(III) chelates synthesized in the aid of block 1 and the steroid analogue 2 detected at 240 nm. The peak at tR 20.06 min is the desired product. For chromatographic conditions, see Experimental Procedures.
microtitration plates, blood spot standards, assay buffer, wash solution, and enhancement solution were from the DELFIA Neonatal 17-R-OH-progesterone kit 1244-039 (PerkinElmer LAS). HPLC analyses were performed on a PerkinElmer LC 2000 instrument using a reversedphase column (LiChrocart 125-3 Purospher RP-18e, 5 µm). The mobile phase used was the following: Buffer A: 0.02 M TEAA (pH 7.5); Buffer B: A in 50% (v/v) acetonitrile. Gradient: from 0 to 1 min 95% A, from 1 to
Technical Notes
Bioconjugate Chem., Vol. 15, No. 4, 2004 929
Table 1. Tracers Synthesized and Their Observed and Calculated Molecular Weights tracera
[M - nH]n-/n calculated
[M - nH]n-/n observed
17-R-OHP-3-CMO-Eu2 (5) 17-R-OHP-3-CMO-Eu4 (6) E3-6-CMO-Eu2 (7)
957.3 (n ) 2) 1143.3 (n ) 3) 944.2 (n ) 2)
957.7 (n ) 2) 1143.5 (n ) 3) 944.2 (n ) 2)
17-R-OHP ) 17-R-hydroxyprogesterone; E3 ) estriol. CMO ) carboxymethyloxime. a
30 min from 95% A to 100% B. Flow rate was 0.6 mL min-1. Electrospray ionization time-of-flight mass spectra (ESI-TOF MS) were recorded on an Applied Biosystems Mariner instrument on negative detection mode. Synthesis of the Steroid Conjugates. The synthesis was performed in 10 µmol scale on an Applied Biosystems 433A instrument using Fmoc chemistry and recommended procedures (O-benzotriazol-1-yl-N,N,N′,N′-tetramethyluronium hexafluorophosphate (HBTU)/1-hydroxybentzotriazole (HOBt) activation; no capping), except the coupling time was increased to 2 h. 35 µmol of 1 was used in all coupling reactions. The last coupling was performed with 15 µmol of 2 or 3. When the chain assembly was completed, the resin was treated with the mixture of crystalline phenol (75 mg), ethanethiol (25 µL), thioanisole (50 µL), water (50 µL), and trifluoroacetic acid (1 mL) for 3 h. The resin was removed by filtration, and the crude steroid conjugate was precipitated with diethyl ether. The precipitate was redissolved in water and treated with aqueous europium(III) citrate (5 equiv/ ligand) Purification was performed on HPLC. The steroid conjugates were characterized on ESI-TOF mass spectrometry. Time-Resolved Fluoroimmunoassays. The 17-Rhydroxyprogesterone derivatives synthesized were tested in a time-resolved fluoroimmunoassay. Standards (3.1 mm disks) punched from blood spots on filter paper were incubated in the wells of anti-rabbit coated microtitration plates with various concentrations of rabbit anti-serum to 17-R-hydroxyprogesterone and 300 pmol/L 17-R-hydroxyprogesterone-Eu tracers in a total volume of 200 µL, for 2 h. Bound and free 17-R-hydroxyprogesteroneEu fractions were separated by washing, and bound Eu was measured, after addition of 200 µL of DELFIA
enhancement solution, into which Eu dissociates and in which it forms highly fluorescent complexes. The fluorescence was measured in a 1420 Victor2V multilabel counter. RESULT AND DISCUSSION
Tracer Synthesis. The present approach for solidphase steroid derivatization is outlined in Scheme 1. Accordingly, two or four building blocks 1 were coupled to an amide resin in 10 µmol scale using Fmoc chemistry. A prolonged coupling time (2 h instead of 30 min) but otherwise standard conditions (3.5 equiv of 1; HBTU/ HOBt as an activator) were used. After the desired number of blocks was introduced, a steroid analogue tethered to a carboxylic acid function (1.5 equiv of 2 or 3) was coupled using similar conditions. When the reaction was completed, the steroid conjugate was released from the resin and deprotected followed by precipitation from diethyl ether. Treatment of the deblocked conjugate with europium(III) citrate converted it to the corresponding lanthanide(III) chelate. The labeled steroids were purified on reversed-phase HPLC and characterized on ESI-TOF mass spectrometry. A typical HPLC profile (crude reaction mixture) is shown in Figure 1 as an illustrative example. In all of the cases, the observed molecular weights were in accordance with the proposed structures (Table 1). The DELFIA Assay. In the DELFIA assay set up for 17-R-hydroxyprogesterone, it is clearly seen that the signal goes up more than what is anticipated from the number of chelates attached to the steroid structure and that an increasing number of chelates makes the analytical sensitivity of the assay somewhat worse (Table 2). The shift of the standard curve to the right as well as the 5-fold increase in signal level when the number of Eu-chelates rises from 1 to 4 indicate that the antiserum has a higher affinity to tracers with more Eu-chelates. However, in 17-R-hydroxyprogesterone assays with these three tracers (4-6) the same sensitivity was observed when 17-R-hydroxyprogesterone antiserum and tracer dilutions were chosen to give the same signal in all three assays (Table 3). Thus, the more chelates conjugated to 17-R-hydroxyprogesterone, the more diluted antiserum could be used. Hence, 17-R-hydroxyprogesterone tracers
Table 2. Dose-Response Values and Analytical Sensitivity with the Three 17-r-Hydroxyprogesterone Tracers (4-6) Tested in an Immunoassays for 17-r-OH-progesterone at Constant Tracer and Anti-Serum Concentrations tracer
tracer (pmol/L)
antiserum (dilution factor)
B0a (fluorescence counts)
4 5 6
300 300 300
60000 60000 60000
27500 70000 146500
standard curve (nmol/L) at B/B0b ) 0.20 B/B0 ) 0.50 B/B0 ) 0.80 36 95 160
6.4 12 20
1.4 2.5 3.5
analytical sensitivityc (nmol/L) 0.6 1.0 1.34
a B was the fluorescence signal from tracer bound to antiserum at zero 17-R-OH-progesterone dose. The background signal, measured 0 from wells containing all reagents except tracer, was between 500 and 1000 in all assays. b B was the fluorescence signal from tracer with 17-R-OH-progesterone present and B/B0 is their ratio. c The analytical sensitivity was defined as the dose calculated from a response 3 SD below the B0 signal (n ) 4).
Table 3. Dose-Response Values and Analytical Sensitivity with the Three 17-r-Hydroxyprogesterone Tracers Tested in an Immunoassays for 17-r-OH-Progesterone at Constant Signal Level tracer
tracer (pmol/l)
antiserum dilution factor
B0a (fluorescence counts)
4 5 6
300 300 150
60000 120000 240000
20252 25428 24114
standard curve (nmol/L) at B/B0b ) 0.20 B/B0 ) 0.50 B/B0 ) 0.80 42 65 62
7.5 9.9 8.6
2.2 2.5 2.3
analytical sensitivityc (nmol/L) 0.9 1.0 1.0
a B was the fluorescence signal from tracer bound to antiserum at zero 17-R-OH-progesterone dose. The background signal, measured 0 from wells containing all reagents except tracer, was between 500 and 1000 in all assays. b B was the fluorescence signal from tracer with 17-R-OH-progesterone present and B/B0 is their ratio. c The analytical sensitivity was defined as the dose calculated from a response 3 SD below the B0 signal (n ) 4).
930 Bioconjugate Chem., Vol. 15, No. 4, 2004
with several chelates are useful when a high serum dilution factor is desired i.e., when only limited quantities of antibody is available. This is usually the case in immunoassay applications for steroids of which many are widely used in clinical routine laboratories. Thus, when required, considerable amounts of antiserum can be saved and the lifetime of an assay prolonged by attachment of several chelates to the tracer. By contrast, if the labeling were performed using organic chromophores such as fluorescein or cyanine dyes, these dyes in close proximity will partially quench each other and further dilution of antiserum would be impossible. In summary, a straightforward method for labeling of steroids on solid phase is demonstrated. Although an increasing number of biomolecule conjugates are nowadays synthesized entirely on solid phase (13), according to our knowledge, no method for solid phase labeling of steroids have been reported, excluding solid-phase synthesis of steroid-oligonucleotide conjugates. Although only labeling of carboxymethyloxime derivatives of estriol and 17-R-hydroxyprogesterone with several lanthanide(III) chelates is demonstrated, in all likelihood, the method is applicable to other haptens tethered to a carboxylic acid group as well. LITERATURE CITED (1) Pratt, J. J. (1978) Steroid immunoassay in clinical chemistry. Clin. Chem. 24, 1869-1890. (2) Mikola, H., Sundell, A.-C., and Ha¨nninen, E. (1993) Labeling of estradiol and testosterone alkyloxime derivatives with a europium chelate for time-resolved fluoroimmunoassay. Steroids 58, 330-334. (3) Meltola, N., Jauria, P., Saviranta, P., and Mikola, H. (1999) Synthesis of novel europium-labeled estradiol derivatives for
Technical Notes time-resolved fluoroimmunoassays. Bioconjugate Chem. 10, 325-331. (4) Takalo, H., Mukkala, V.-M., Mikola, H., Liitti, P., and Hemmila¨, I. (1994) Synthesis of europium(III) chelates suitable for labeling of bioactive molecules. Bioconjugate Chem. 5, 278-282. (5) Mikola, H., and Hedlo¨f, E. (1997) Synthesis of europiumlabeled digoxin derivatives and their use in time-resolved fluoroimmunoassay. Steroids 59, 472-478. (6) Hemmila¨, I. (1991) Applications of Fluorescence in Immunoassays, in Chemical Analysis: A Series of Monographs on Analytical Chemistry and Its Applications, Vol. 117, John Wiley & Sons, Inc., New York. (7) Hemmila¨, I., Ståhlberg, T., and Mottram, P., Eds. (1994) Bioanalytical Applications of Labelling Technologies, Chapter 9, Wallac Oy, Turku. (8) Hemmila¨, I., Dakubu, S., Mukkala, V.-M., Siitari, H., and Lo¨vgren, T. (1984) Europium as a label in time-resolved immunofluorometric assays. Anal. Biochem. 137, 335-343. (9) Hemmila¨, I., and Mukkala, V.-M. (2001) Time-resolution in fluorometry technologies, labels and applications. Crit. Rev. Clin. Lab. Sci. 38, 441-519. (10) Peuralahti, J., Hakala, H., Mukkala, V.-M., Hurskainen, P., Mulari, O., and Hovinen, J. (2002) Introduction of lanthanide(III) chelates to oligopeptides on solid phase. Bioconjugate Chem. 13, 870-875. (11) Hovinen, J., and Hakala, H. (2001) Versatile strategy for oligonucleotide derivatization. Introduction of lanthanide(III) chelates to oligonucleotides. Org. Lett. 3, 2473-2476. (12) Hakala, H., Ollikka, P., Degerholm, J., and Hovinen, J. (2002) Oligonucleotide conjugates based on acyclonucleosides and their use in DNA hybridization assays. Tetrahedron 58, 8771-8777. (13) Virta, P., Katajisto, J., Niittyma¨ki, T., and Lo¨nnberg, H. (2003) Solid-supported synthesis of oligomeric bioconjugates. Tetrahedron 59, 5137-5174.
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