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Rapid homogeneous immunoassay of peptides based on bioluminescence resonance energy transfer from firefly luciferase
JOURNAL
OF
BIOSCIENCE AND BIOENGINEERING
Vol. 93, No. 6, 537-542. 2002
Rapid. Homogeneous Immunoassay of Peptides Based on Bioluminescence Resonance Energy Transfer from Firefly Luciferase YUKARI
YAMAKAWA,’
HIROSHI UEDA,‘“” ATSUSHI KITAYAMA,‘$$ NAGAMUNE’
AND TERUYUKI
Department of Chemistry and Biotechnology, Graduate School of Engineering, 7-3-l Hongo, Bunkyo-ku, Tokyo 113-8656, Japan’
University of Tokyo,
Received 8 February ZOOZ/Accepted27 February 2002
A sensitive homogeneous immunoassay is needed in the field of clinical diagnostics. Here we propose a rapid and potentially sensitive homogeneous immunoassay of peptide epitope based on the bioluminescence resonance energy transfer (BRET). A GST-peptide tag fused to the N-terminus of firefly luciferase, and Cy3 or Cy3.5labeled anti-peptide antibody were prepared to detect BRET from the hybrid luciferase to the fluorolabeled antibody. By measuring the spectral change due to BRET, the amount of c-myc peptide was successfully determined in a short period. [Key words: fusion protein, bioluminescent protein, homogeneous immunoassay, peptide epitope]
Conventional heterogeneous immunoassays always require multiple steps including B/F separations, where antigen-antibody complex is separated from free antigen and free antibody. However, there is an increasing need for homogeneous immunoassays without any separation steps especially in the field of clinical diagnostics. Here we devised a new homogeneous immunoassay that takes advantage of a phenomenon occurring in nature, namely, the Fiirster resonance energy transfer between a light-emitting protein (e.g., aequorin, or Renilla luciferase) and an acceptor fluorophore (e.g., GFP) (1, 2). The technique is related to an analytical technique using fluorescence resonance energy transfer (FRET). In this process, one fluorophore (the “donor”) transfers its excited-state energy to another fluorophore (the “acceptor”), which usually emits fluorescence of a different color. FRET efficiency depends on the spectral overlap, the relative orientation, and the distance between the donor and acceptor fluorophores. Generally, FRET occurs when the donor and acceptor are 1O-l 00 8, apart, hence it enables a homogeneous immunoassay by attaching the donor and acceptor fluorophores to the antigen and the antibody (3). As with any fluorescence technique, however, photobleaching and autofluorescence can limit the usefulness of FRET. In addition, it can be complicated by direct excitation of the acceptor fluorophore. Furthermore, FRET may be impractical in tissues that are easily damaged by the excitation light or that are photosensitive. The bioluminescence resonance energy transfer (BRET), which we use for the
new immunoassay,
offers the advantages of FRET but without the consequences of fluorescence excitation. In BRET, the donor fluorophore of the FRET technique is replaced by the activated luciferin bound to luciferase. In the presence of a substrate, bioluminescence from the luciferase excites the acceptor fluorophore through the same Fiirster resonance energy transfer mechanisms described above. Here, a model homogeneous immunoassay system based on BRET from the firefly luciferase to synthetic red fluorochromes was established to assay c-myc peptide as a model antigen (Fig. 1). The method will find a wide range of applications by exchanging the epitope peptide and corresponding antibodies. MATERIALS
AND METHODS
Materials Escherichia coli strain XLl-Blue (hsdR17, supE44, recA1, endAl, gyrA46, thi-1, relA1, lac [F’, proAB, lacl~ZAMl5::TnlO(tet’), Camr]) was used as a host for DNA recombination. E. coli strain TGI (supE, A(hsdM-mcrB)S (r;m;McrB), thi, A(lac-proAB) [F’, traD36, proAB, 1acPZ AM15]) was used for the DNA sequencing and the expression of fusion proteins. Cells were cultured in LB-broth (1% bactotryptone, 0.5% yeast extract, and 0.5% NaCl, pH 7.9, with ampicillin (50 pg/ml) when necessary. The plasmids pGEM-Luc and pGEX5X-3 were purchased from Promega Japan (Tokyo) and Amersham Pharmacia Biotech (Tokyo), respectively. The labeling kits for cyanine dyes were from Amersham Pharmacia Biotech. Restriction endonucleases and modification enzymes were obtained from Takara (Kyoto). The c-myc tag peptide was synthesized by Genosys Biotechnologies (The Woodlands, TX, USA). Luciferin-Na and coenzyme A were from Wako (Osaka). Other reagents unless otherwise indicated were either from Wako or Sigma, of highest grades available. Plasmids construction The c-myc tag-luciferase gene fusion vector pGEX-myc-luc was constructed as follows. The structural gene for Photinus pyralis firefly luciferase was digested from
* Corresponding author. e-mail: [email protected] phone: +81-(0)471-36-3644 fax: +81-(0)471-36-3642 Present address: 5Department of Integrated Biosciences, Graduate School of Frontier Sciences, University of Tokyo, Bldg. FSB-401, 5-l -5 Kashiwano-ha, Kashiwa, Chiba 277-8562, Japan and gSNational Institute for Basic Biology, Okazaki, Aichi 444-8585, Japan. 537
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YAMAKAWA ET AL. (a) No peptide
Cy3-Antibody (b) With peptide
FIG. 1. Principle of the BRET-based homogeneous immunoassay. (a) When peptide-tagged luciferase is mixed with fluorolabeled antibody, BRET induces a red shift in bioluminescence. (b) Addition of peptide inhibits the binding of the tagged luciferase and the labeled antibody, thus BRET between them. the plasmid pGEM-Luc with BumHI and WI, and ligated with pGEX-5X-3 digested with BumHI and s&I, yielding pGEX-luc which expresses GST-luciferase fusion protein. Two complementary oligonucleotides, 5’-GATC CAG GAA CAA AAA CTC ATC TCA GAA GAG GAT CTG AAT TCG CG-3’ and 5’-GATC CG CGA ATT CAG ATC CTC TTC TGA GAT GAG TTT TTG TTC CTG3’, each coding for residues 410 through 419 of human cmyc with the amino acid sequence of NH,-EQKLISEEDL-CO,-, with BamHI-digested DNA compatible sequences at the ends, were synthesized. The oligonucleotides were annealed and inserted into the BumHI site of pGEX-luc in the sense orientation, yielding pGEX-myc-luc, which was expected to express GST-luciferase fusion protein with a c-myc tag sequence between them. Production of myc-tagged luciferase The E. coli TG-1 cells transformed by the plasmid pGEX-myc-luc were cultured in 5 ml of LB medium containing ampicillin (50 &ml) at 37°C with shaking overnight. The culture was used to inoculate 100 ml of LB medium containing ampicillin (50 &ml) at 30°C with vigorous shaking. When the OD,,, reached - 1.O, the fusion protein expression was induced by adding 0.1 mM IPTG and the culture shaken overnight. The cells were harvested by centrifugation at 6OOOxg for 10 min at 4°C washed with 10 ml of ice-cold PBS (10 mM phosphate, 137 mM NaCl, and 13 mM KCl, pH 7.2) and resuspended in 5 ml of PBS. They were frozen at -8O”C, and thawed for lysis at 25°C. Cells were further lysed by a sonicator and then centrifuged at 12,OOOxg for 10 min to remove the cell debris. The supernatant was applied to 1 ml of glutathione Sepharose 4B (Amersham Pharmacia Biotech) and eluted with 7 ml of Glutathione Elution Buffer (10 mM glutathione in 50 mM Tris-HCl, pH 8.0). Fractions containing luciferase activity were pooled and concentrated by ultrafiltration using Amicon Centricon(Millipore Japan, Tokyo). Preparation of the monoclonal antibody The hybridoma 9E10.2 secreting anti-myc MAb 9ElO was obtained from American Type Culture Collection (Rockville, MD, USA). The hybridoma cells were injected into pristane-primed BALB/c mice, and were grown in ascitic fluids. The MAb was purified with a MAb Trap G2 column (Amersham Pharmacia Biotech). The concentration of MAb was determined by BCA protein assay with bovine serum albumin as a standard, and the spectrometry of the protein assuming an EzgOnm(1 mg/ml) of 1.4 (4). Fluorolaheling of antibody One milligram per batch of 9ElO MAb was coupled with the dyes (Cy3.0 or Cy3.5) of the
corresponding Mab labeling kits according to the manufacturer’s instructions. The amount of dye incorporated into a protein (F/P ratio) was estimated by uv/visible absorption spectrophotometry assuming the A,,, of Cy3.0 and A,,, of Cy3.5 to be 150,000 M-l cm-‘, and A,, of Cy5 to be 250,000 M-’ cm-‘. Typical F/P values obtained were 4.556.0. Characterization of the fusion proteins One hundred microliters each of the fusion proteins in PBS (2 @ml) was poured into the wells of a Falcon 3912 microplate (Becton Dickinson, Oxnard, CA, USA), and incubated overnight at 4’C. The wells were then washed three times with PBS-T (PBS with 0.005% Tween20). After the washing, any remaining sites for nonspecific binding were blocked with Block Ace (Dainippon Pharmaceutical, Osaka) at 25°C for 2 h. After three times more washes with PBS-T, various concentrations of anti-c-myc antibody 9ElO were added and incubated at 25°C for 30 min. The wells were then washed again three times, and probed with HRP-labeled anti-mouse IgG (Biosource, Camerillo, CA, USA) for 30 min at 25°C. After vigorous washing, 0.04% o-phenylenediamine-HCl, 0.1% H,O, in 50 mM Na-succinate, pH 5.0 was added to each well and Adg, was determined. Bioluminescent spectral analysis 10 nM GST-c-myc-luciferase, 0.5 uM Cy3- or Cy3.5-labeled 9ElO antibody and human cmyc peptide (408428) (Cosmo-Bio, Tokyo) in a total of 100 ul were mixed and incubated at 0°C for 1 h. The addition of 100 pl of substrate solution (50 mM Tris-HCl, 10 mM MgSO,, 2 mM EDTA, 2 mM luciferin-Na, 10 mM ATP, 600 uM coenzyme A, and 66.6 uM DTT, pH 8.0) activated yellow-green bioluminescence. Luminescence and fluorescence emission spectra were recorded with a F-2000 fluorescence spectrometer (Hitachi, Tokyo) immediately
after the addition of substrate. Scanning was accomplished within 1 min, and luminescence was stable during that period. RESULTS
Production and purification of the fusion protein To test the performance of the system, myc tag (5) was chosen as a model peptide antigen, because an anti-peptide antibody with good affinity was readily available. For the detection of myc peptide with BRET, a fusion protein expression vector, pGEX-myc-luc, was designed to efficiently express the firefly luciferase in E. coli that was N-terminally tethered with myc peptide. As a control, an expression vector for GST-luc fusion protein was also made. The proteins were expressed in E. coli TG-1, and purified to homogeneity using a glutathione affinity column (Fig. 2a). Sufficient amounts of purified proteins (9.6 and 1.6 mg of GST-mycluc and GST-luc, respectively) were obtained from 100 ml cultures. Characterization of the fusion protein To investigate whether GST-myc-luc protein retained the desired properties, binding of anti c-myc antibody 9E 10 (5,6) to the protein was tested. A fixed concentration (2 ug/ml) of the fusion protein was adsorbed in each well of the microplate, and the binding of 9ElO at variable concentrations to the wells was tested by sandwich ELISA. When the signal was plotted against the concentration of 9ElO antibody, a clear increase in binding according to the increasing concentration of the antibody was observed (Fig. 2b). On the other hand, when the same concentration of GST-luc without cmyc tag was used for coating, negligible binding of 9E10 antibody was observed, clearly suggesting tag-specific binding of the antibody. To exclude the possibility that the
BRET-BASED IMMUNOASSAY
VOL. 93,2002
(a)
539
M123456M
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cr myc (ng/ml) FIG. 2. (a) Purification of the fusion proteins. Lanes 1 and 4, Lysate from TG-1 expressing GST-luc (1) or GST-myc-luc (4); Lanes 23 and 5-6, first and second fractions of the glutathione column eluate for GST-luc (2-3) or GST-myc-luc (5-6). M, Molecular marker. Coomassie-stained 10% SDS-polyacrylamide gel is shown. (b) Specific binding of 9ElO antibody to GST-myc-luc protein. The adsorbed fusion proteins (triangle for GST-luc, circle for GST-myc-luc, and cross for PBS only) were added with various concentrations of 9ElO antibody. The amount of bound antibody was probed with peroxidase-labeled anti-mouse IgG.
amounts of adsorbed fusion proteins were different, the bioluminescent activities of the bound proteins were assayed. Little difference in the light emission between the wells adsorbed with GST-myc-luc and GST-luc was observed (data not shown). Accordingly, the specific binding of 9E 10 to the myc-tag in GST-myc-luc was confirmed. In addition, the bioluminescent activity of GST-myc-luc in the wells was not inhibited by anti c-myc antibody added before the bioluminescent reaction, showing that the fusion protein retained its enzymatic activity not only in the free state but also in the 9ElO-bound form (data not shown). Luminescence energy transfer to Cydye-labeled antibody To test whether the luminescence energy transfer, a phenomenon similar to that found in luminous coelenterates (7, S), could occur between a c-myc tagged luciferase and fluorolabeled anti c-myc antibody, at first suitable fluorochromes were investigated. Among a number of red dyes, cyanine dyes Cy3, Cy3.5 and Cy5, each having a larger molar absorption coefficient than traditional rhodamine derivatives, were tested. Figure 3 shows the excitation and emis-
FIG. 3. Normalized spectra of cyanine dyes. Excitation (dotted line) and emission (stitched line) spectra of Cy3 (a), Cy3.5 (b) and Cy5 (c) are compared with the normalized luciferase emission spectrum (thin line).
sion spectra of these dyes. The luminescent spectrum of luciferase is also shown in each panel to compare the spectral overlap of the luminescent and dye excitation spectra. To achieve an efficient BRET, close approximation (~50 A) of the dye and the luciferase active site is crucial. Hence, succinimidyl esters were selected to achieve N-terminal labeling because the antigen binding sites of the antibody exist near the N-termini of its heavy and light chains. As a result, labeled antibodies with an F/P ratio of 4.5 to 6.0 were obtained, and they were tested for the luminescence energy transfer measurement. The bioluminescence of firefly luciferase is triggered by the addition of the substrates luciferin and ATP to the enzyme. The effect of the fluorolabeled antibody on the luminescent color was investigated by adding each antibody to the GST-myc-luc solution (20 nM) in a cuvette, and the luminescence spectra were recorded immediately after substrate addition using a fluorescence spectrometer with no excitation light (Fig. 4). When Cy3-labeled antibody (1.2 PM) was added, a reduction in the light emission at around 550 nm, and increase around 570 nm were observed, suggesting a transfer of energy from the epitopetagged luciferase to the Cy3-labeled anti-epitope antibody. No such spectral change in light emission was observed with GST-luc in the presence of the Cy3-labeled antibody (data not shown). When Cy3.5-labeled antibody (1.0 PM) was added, a similar but distinct spectral change was observed. In this case a clearer peak at 596 nm probably due to BRET was observed, and the intensity at 550 nm was sig-
540
YAMAKAWA ET AL.
J. BIOSCI.BIOENG.,
-
PpL
.. ... .. . . .
CY3
-------
CY3.5
I 550
E c 0.8 - ,, 8 (L ll : ‘g 0.6 3
650
600
700
Wavelength (nm) FIG. 4. Spectral change due to the addition of Cy3- and Cy3.5 labeled antibodies. GST-myc-luc (40nM) alone (black line) or with 2.4 pM Cy3-labeled antibody (dotted line) or 2.0 pM Cy3.5-labeled antibody (stitched line) was incubated in 50 mM Tris-HCI, pH 8.0 for 30 min at 0°C before the bioluminescent reaction.
2
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Peptide FIG. 6. Standard curves of BRET-based immunoassay. Peptide at the indicated concentration was mixed with GST-myc-luc and Cy3 labeled antibody (square), or GST-myc-luc and Cy3.5-labeled antibody (circle) in 50 mM Tris-HCI, pH 8.0, for 1 h at 0°C. Immediately after the addition of substrate, the luminescent spectra were recorded and the luminescence ratio calculated. Each point represents the mean+one SD of three determinations. Curves based on the model were fitted with Kaleida Graph 3.0. Parameters a and b in Eq. 9 are 0.905 and 29.0 for Cy3.0, and 0.23 and 62.7 for Cy3.5, respectively.
0
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o myc (PM) FIG. 5. Antibody concentration dependence of BRET. 20 nM (circle) or 10 nM (square) of GST-myc-luc was incubated with the indicated amount of anti-myc antibody, and luminescent spectra were recorded. The luminescence ratio at specified wavelengths (BRET index) was calculated. GST-luc (cross) was used instead of GST-myc-luc in (a).
nificantly reduced (data not shown). Figure 5 shows the relationship between the labeled MAb concentration and the luminescence ratio. The largest ratio was obtained with the shorter wavelength near the emission maximum of the luciferase (550 nm), and the longer wavelength near that of Cy3 (57Onm) or Cy3.5 (596nm). So BRET indices were taken as the ratios of the light emissions at 5701596nm and 55Onm. In the case of both Cy3 and
Cy3.5 labeling, there was a labeled MAb-dependent increase in BRET index when GST-myc-luc protein was used. However, a very modest increase was observed when GSTluc protein was used (Fig. 5a). We could not detect significant spectral change when CyS-labeled antibody was added. However, the addition of labeled MAb induced significant quenching of the luminescence, implying the existence of BEET (data not shown, see discussion). BRET-based homogeneous immunoassay To use BRET to perform a homogeneous immunoassay, a limited amount of the tagged luciferase (10 nM) and excess Cy3- or Cy3.5-labeled antibody (0.5 PM) were mixed in the presence of various concentrations of c-myc peptide. Excess antibodies were used to obtain an efficient BEET signal. After preincubation, the substrate solution was added and luminescent spectra measured, from which the ratio of the luminescence at two wavelengths was calculated. Then the relationships between the BRET index and c-myc peptide concentration were plotted. For both Cy3 and Cy3.5 labeling, as shown in Fig. 6, a clear peptide concentration-dependent inhibition of BRET was observed. The change was more prominent when Cy3.5-labeled antibody was used, with the BRET index dropping from 0.8 to around 0.3 upon the addition of peptide. The practical measurable concentration range of the peptide was roughly 0.2 to 2.0 PM. A simple model was Comparison with the model built to evaluate the obtained result. Ab: anti c-Myc pep: c-myc peptide Luc: GST-myc-Luc Ag: pep+Luc
bep~Abl=K,bepl[Abl
(1)
[Luc.Ab]=K,[Luc][Ab]
(2)
If we assume K,=K2=K,
then by adding Eqs. 1 and 2, we
BRET-BASED
VOL. 93,2002
obtain the equilibrium
equation.
[Ag.Ab]=K[Ag][Ab] Dividing
(3)
Eq. 1 by Eq. 2 gives
bep.Abl [Luc.Ab]
[pep1 =[Lucl
[pep1 [PepI,
]pep.Abl+
= [Luc.Ab]+
(4)
[Luc] = [Lucl,
where [pep], and [Luc], are total peptide and luciferase concentrations, respectively. From Eq. 4, [pep.Ab]/[Luc.Ab]
+ 1 =[pep],/[Luc],+
[Ag.Ab]/[Luc.Ab]
=([pep],+[Luc],)/[Luc],
1 (5)
When we take the concentration of antigen-antibody plex as x, transforming Eq. 3 gives
com-
x=[Ag.Ab]=K[Ag][Ab]=K([Ag],-x)([Ab],-x)
Kx2-(~([Agl,+[Abl,)+l)x+K[Agl,[Abl,=0
(6)
x= W&&+ W&J + 1-A
(7)
2K
(D,=(K([&l,+[Abl,)+1F4~L%&L%J From Eqs. 5 and 7,
[Lucl, ‘Luc’Abl=[Luc],+[pep],x = K([Lucl,+[pepl,+[Abl,)+l-~
(8)
2K(l+E)
(~2=W(Wl,+ bepI,+ [AblJ+ 1)’
-4WLucl,+
bepl,)[AblJ
Based on this equation, the BRET index curves (y) were fitted to the following equation
?,,,+b~([Lucl,+x+[Abl~)+l-JzJj
(9)
) 2K(1+& (D;=(K([Luc],+x+[Ab],)+
1>*-4E(Z([Lu~]~+x)[Ab]~)
where a and b are parameters, [Luc],=O.O2 PM, [Ab],= 1.0 PM, and K= 1.25 x 107/M. As shown in Fig. 6, both curves showed relatively good fits with R values of 0.913 and 0.974 for Cy3 and Cy3.5 labeling, respectively. DISCUSSION To our knowledge this is the first demonstration of a BRET immunoassay using firefly luciferase as a luminescence donor. Previous BRET studies have used blue light emitters of Renilla luciferase (9-14) and quite recently, aequorin (15). Firefly luciferase is relatively easy to express in E. coli in large quantity, and a number of stabilized mu-
IMMUNOASSAY
541
tants have also been reported (16-l 8). Considering production costs and ease of use, firefly luciferase seems a far better choice than other enzymes, at least for in vitro diagnostics as presented here. The use of a suitable fluorophore is a key factor if this system is to work properly. Although at first we attempted to label the antibody with rhodamine X succinimide, we could not detect any spectral change upon addition of the labeled antibody. This was probably because its absorption was less than that of cyanine fluorophores, and hence the Fbrster resonance energy transfer distance R, for this luciferin-rhodamine X pair might be shorter than the distance between the luciferase active site and the N-terminus of the antibody H or L chains. Another consideration is the spectral overlap. We obtained a better response as a BRET index on labeling with Cy3.5 than Cy3. A condition for radiationless energy transfer is a significant overlap between the luciferase emission spectrum and the acceptor fluorophore excitation spectrum (J integral). However, the calculated J integral is smaller between the luciferase emission and Cy3.5 excitation than Cy3 excitation spectra (Fig. 3, data not shown). The reason for this apparent discrepancy might be that the emission spectrum of luciferase significantly overlaps with that of Cy3, and considerable background emission at 570nm is observed without BRET. Therefore, to improve sensitivity, in addition to obtaining greater spectral overlap between the luciferase emission and the acceptor excitation spectra, less overlap between the two emission spectra (in other words, larger stokes shift of the fluorophore) should be considered as well. We also attempted to use Cy5 which has a higher maximum absorption and longer emission wavelength (Fig. 3). However, we could not detect any spectral change due to BRET (data not shown). It is possible that the J integral between the luciferase emission and Cy5 extinction was not sufficient to cause a detectable BRET. Another possibility is the selfquenching of Cy5 due to multiple labeling of the 9E 10 antibody. When we compared the fluorescence of the labeled antibody and free fluorochrome at the same maximum absorbance, CyS-labeled antibody showed significant quenching of 75%, while Cy3- and Cy3.5-labeled antibodies showed 5-35%. A high extinction coefficient and greater overlap of excitation and emission spectra of Cy5 might cause the efficient intramolecular FRET, thus leading to such remarkable quenching. Here we showed the feasibility of a homogeneous immunoassay of peptides based on the BRET phenomenon. Though the sensitivity was not remarkable, according to the simulation it can be improved by reducing the concentration of antibody (data not shown). When we reduce the antibody concentration, we may also have to reduce the luciferase concentration to obtain sufficient BRET signal, otherwise, we will have a larger background derived from unbound luciferase. In addition, the use of a higher affinity antibody is preferable to obtain a sufficient binding equilibrium in a limited incubation period. Hence the use of a specialized sensitive luminometer equipped with optimal filter sets and a smaller cuvette will surely enhance the utility of the system. The equipment should not be too expensive because unlike FRET, there is no need for excitation.
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ET AL.
A future possibility is the use of this system in vivo. The natural fluorescent protein DsRed has a similar excitation/emission spectra to Cy3.5, though its maximum extinction coefficient is less than one tenth that of Cy3.5 (22,500 M-l) (19). The possible use of DsRed as a BRET companion with firefly luciferase is now under investigation (Arai et al., in preparation). ACKNOWLEDGMENTS
We are grateful to Tadashi Mikami in SS Pharmaceutical, Tokyo for his generous help in the production of 9E10.2 antibody, and Noriho Kamiya and Ryoichi Arai in University of Tokyo for their helpful comments. This study was supported in part by a Grantin-Aid for Scientific Research on Priority Areas (296-10145107) and Grant-in-Aid for Scientific Researches (B 09450301 and S 13854003) from the Ministry of Education, Science, Sports and Culture of Japan. REFERENCES
1. Hart, R. C., Matthews, J. C., Hori, K., and Cormier, M. J.: Renilla reniformis bioluminescence: luciferase-catalyzed production of nonradiating excited states from luciferin analogues and elucidation of the excited state species involved in energy transfer to Renilla green fluorescent protein. Biochemistry, l&2204-22 10 (1979). 2. Ward, W. W. and Cormier, M. J.: An energy transfer protein in coelenterate bioluminescence. Characterization of the Renilla green-fluorescent protein. J. Biol. Chem., 254, 781788 (1979). 3. Selvin, P. R. and Hearst, J. E.: Luminescence energy transfer using a terbium chelate: improvements on fluorescence energy transfer. Proc. Natl. Acad. Sci. USA, 91, 10024-10028 (1994). 4. Morimoto, K. and Inouye, K.: Single-step purification of F(ab’)2 fragments of mouse monoclonal antibodies (immunoglobulins Gl) by hydrophobic interaction high performance liquid chromatography using TSKgel Phenyl-5PW. J. Biothem. Biophys. Methods, 24, 107-117 (1992). 5. Munro, S. and Pelham, H. R B.: An Hsp70-like protein in the ER: identity with the 78 kd glucose-regulated protein and immunoglobulin heavy chain binding protein. Cell, 46, 291300 (1986). 6. Schiweck, W., Buxbaum, B., Schiitzlein, C., Neiss, H. G., and Skerra, A.: anti-c-myc antibody 9ElO: the VH domain has an extended CDR-H3 and exhibits unusual solubility. FEBS Lett., 414,33-38 (1997). 7. Morise, H., Shimomura, O., Johonson, F. H., and Winant, J.: Intermolecular energy transfer in the bioluminescent sys-
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BIOSCIENCE AND BIOENGINEERING
Vol. 93, No. 6, 537-542. 2002
Rapid. Homogeneous Immunoassay of Peptides Based on Bioluminescence Resonance Energy Transfer from Firefly Luciferase YUKARI
YAMAKAWA,’
HIROSHI UEDA,‘“” ATSUSHI KITAYAMA,‘$$ NAGAMUNE’
AND TERUYUKI
Department of Chemistry and Biotechnology, Graduate School of Engineering, 7-3-l Hongo, Bunkyo-ku, Tokyo 113-8656, Japan’
University of Tokyo,
Received 8 February ZOOZ/Accepted27 February 2002
A sensitive homogeneous immunoassay is needed in the field of clinical diagnostics. Here we propose a rapid and potentially sensitive homogeneous immunoassay of peptide epitope based on the bioluminescence resonance energy transfer (BRET). A GST-peptide tag fused to the N-terminus of firefly luciferase, and Cy3 or Cy3.5labeled anti-peptide antibody were prepared to detect BRET from the hybrid luciferase to the fluorolabeled antibody. By measuring the spectral change due to BRET, the amount of c-myc peptide was successfully determined in a short period. [Key words: fusion protein, bioluminescent protein, homogeneous immunoassay, peptide epitope]
Conventional heterogeneous immunoassays always require multiple steps including B/F separations, where antigen-antibody complex is separated from free antigen and free antibody. However, there is an increasing need for homogeneous immunoassays without any separation steps especially in the field of clinical diagnostics. Here we devised a new homogeneous immunoassay that takes advantage of a phenomenon occurring in nature, namely, the Fiirster resonance energy transfer between a light-emitting protein (e.g., aequorin, or Renilla luciferase) and an acceptor fluorophore (e.g., GFP) (1, 2). The technique is related to an analytical technique using fluorescence resonance energy transfer (FRET). In this process, one fluorophore (the “donor”) transfers its excited-state energy to another fluorophore (the “acceptor”), which usually emits fluorescence of a different color. FRET efficiency depends on the spectral overlap, the relative orientation, and the distance between the donor and acceptor fluorophores. Generally, FRET occurs when the donor and acceptor are 1O-l 00 8, apart, hence it enables a homogeneous immunoassay by attaching the donor and acceptor fluorophores to the antigen and the antibody (3). As with any fluorescence technique, however, photobleaching and autofluorescence can limit the usefulness of FRET. In addition, it can be complicated by direct excitation of the acceptor fluorophore. Furthermore, FRET may be impractical in tissues that are easily damaged by the excitation light or that are photosensitive. The bioluminescence resonance energy transfer (BRET), which we use for the
new immunoassay,
offers the advantages of FRET but without the consequences of fluorescence excitation. In BRET, the donor fluorophore of the FRET technique is replaced by the activated luciferin bound to luciferase. In the presence of a substrate, bioluminescence from the luciferase excites the acceptor fluorophore through the same Fiirster resonance energy transfer mechanisms described above. Here, a model homogeneous immunoassay system based on BRET from the firefly luciferase to synthetic red fluorochromes was established to assay c-myc peptide as a model antigen (Fig. 1). The method will find a wide range of applications by exchanging the epitope peptide and corresponding antibodies. MATERIALS
AND METHODS
Materials Escherichia coli strain XLl-Blue (hsdR17, supE44, recA1, endAl, gyrA46, thi-1, relA1, lac [F’, proAB, lacl~ZAMl5::TnlO(tet’), Camr]) was used as a host for DNA recombination. E. coli strain TGI (supE, A(hsdM-mcrB)S (r;m;McrB), thi, A(lac-proAB) [F’, traD36, proAB, 1acPZ AM15]) was used for the DNA sequencing and the expression of fusion proteins. Cells were cultured in LB-broth (1% bactotryptone, 0.5% yeast extract, and 0.5% NaCl, pH 7.9, with ampicillin (50 pg/ml) when necessary. The plasmids pGEM-Luc and pGEX5X-3 were purchased from Promega Japan (Tokyo) and Amersham Pharmacia Biotech (Tokyo), respectively. The labeling kits for cyanine dyes were from Amersham Pharmacia Biotech. Restriction endonucleases and modification enzymes were obtained from Takara (Kyoto). The c-myc tag peptide was synthesized by Genosys Biotechnologies (The Woodlands, TX, USA). Luciferin-Na and coenzyme A were from Wako (Osaka). Other reagents unless otherwise indicated were either from Wako or Sigma, of highest grades available. Plasmids construction The c-myc tag-luciferase gene fusion vector pGEX-myc-luc was constructed as follows. The structural gene for Photinus pyralis firefly luciferase was digested from
* Corresponding author. e-mail: [email protected] phone: +81-(0)471-36-3644 fax: +81-(0)471-36-3642 Present address: 5Department of Integrated Biosciences, Graduate School of Frontier Sciences, University of Tokyo, Bldg. FSB-401, 5-l -5 Kashiwano-ha, Kashiwa, Chiba 277-8562, Japan and gSNational Institute for Basic Biology, Okazaki, Aichi 444-8585, Japan. 537
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YAMAKAWA ET AL. (a) No peptide
Cy3-Antibody (b) With peptide
FIG. 1. Principle of the BRET-based homogeneous immunoassay. (a) When peptide-tagged luciferase is mixed with fluorolabeled antibody, BRET induces a red shift in bioluminescence. (b) Addition of peptide inhibits the binding of the tagged luciferase and the labeled antibody, thus BRET between them. the plasmid pGEM-Luc with BumHI and WI, and ligated with pGEX-5X-3 digested with BumHI and s&I, yielding pGEX-luc which expresses GST-luciferase fusion protein. Two complementary oligonucleotides, 5’-GATC CAG GAA CAA AAA CTC ATC TCA GAA GAG GAT CTG AAT TCG CG-3’ and 5’-GATC CG CGA ATT CAG ATC CTC TTC TGA GAT GAG TTT TTG TTC CTG3’, each coding for residues 410 through 419 of human cmyc with the amino acid sequence of NH,-EQKLISEEDL-CO,-, with BamHI-digested DNA compatible sequences at the ends, were synthesized. The oligonucleotides were annealed and inserted into the BumHI site of pGEX-luc in the sense orientation, yielding pGEX-myc-luc, which was expected to express GST-luciferase fusion protein with a c-myc tag sequence between them. Production of myc-tagged luciferase The E. coli TG-1 cells transformed by the plasmid pGEX-myc-luc were cultured in 5 ml of LB medium containing ampicillin (50 &ml) at 37°C with shaking overnight. The culture was used to inoculate 100 ml of LB medium containing ampicillin (50 &ml) at 30°C with vigorous shaking. When the OD,,, reached - 1.O, the fusion protein expression was induced by adding 0.1 mM IPTG and the culture shaken overnight. The cells were harvested by centrifugation at 6OOOxg for 10 min at 4°C washed with 10 ml of ice-cold PBS (10 mM phosphate, 137 mM NaCl, and 13 mM KCl, pH 7.2) and resuspended in 5 ml of PBS. They were frozen at -8O”C, and thawed for lysis at 25°C. Cells were further lysed by a sonicator and then centrifuged at 12,OOOxg for 10 min to remove the cell debris. The supernatant was applied to 1 ml of glutathione Sepharose 4B (Amersham Pharmacia Biotech) and eluted with 7 ml of Glutathione Elution Buffer (10 mM glutathione in 50 mM Tris-HCl, pH 8.0). Fractions containing luciferase activity were pooled and concentrated by ultrafiltration using Amicon Centricon(Millipore Japan, Tokyo). Preparation of the monoclonal antibody The hybridoma 9E10.2 secreting anti-myc MAb 9ElO was obtained from American Type Culture Collection (Rockville, MD, USA). The hybridoma cells were injected into pristane-primed BALB/c mice, and were grown in ascitic fluids. The MAb was purified with a MAb Trap G2 column (Amersham Pharmacia Biotech). The concentration of MAb was determined by BCA protein assay with bovine serum albumin as a standard, and the spectrometry of the protein assuming an EzgOnm(1 mg/ml) of 1.4 (4). Fluorolaheling of antibody One milligram per batch of 9ElO MAb was coupled with the dyes (Cy3.0 or Cy3.5) of the
corresponding Mab labeling kits according to the manufacturer’s instructions. The amount of dye incorporated into a protein (F/P ratio) was estimated by uv/visible absorption spectrophotometry assuming the A,,, of Cy3.0 and A,,, of Cy3.5 to be 150,000 M-l cm-‘, and A,, of Cy5 to be 250,000 M-’ cm-‘. Typical F/P values obtained were 4.556.0. Characterization of the fusion proteins One hundred microliters each of the fusion proteins in PBS (2 @ml) was poured into the wells of a Falcon 3912 microplate (Becton Dickinson, Oxnard, CA, USA), and incubated overnight at 4’C. The wells were then washed three times with PBS-T (PBS with 0.005% Tween20). After the washing, any remaining sites for nonspecific binding were blocked with Block Ace (Dainippon Pharmaceutical, Osaka) at 25°C for 2 h. After three times more washes with PBS-T, various concentrations of anti-c-myc antibody 9ElO were added and incubated at 25°C for 30 min. The wells were then washed again three times, and probed with HRP-labeled anti-mouse IgG (Biosource, Camerillo, CA, USA) for 30 min at 25°C. After vigorous washing, 0.04% o-phenylenediamine-HCl, 0.1% H,O, in 50 mM Na-succinate, pH 5.0 was added to each well and Adg, was determined. Bioluminescent spectral analysis 10 nM GST-c-myc-luciferase, 0.5 uM Cy3- or Cy3.5-labeled 9ElO antibody and human cmyc peptide (408428) (Cosmo-Bio, Tokyo) in a total of 100 ul were mixed and incubated at 0°C for 1 h. The addition of 100 pl of substrate solution (50 mM Tris-HCl, 10 mM MgSO,, 2 mM EDTA, 2 mM luciferin-Na, 10 mM ATP, 600 uM coenzyme A, and 66.6 uM DTT, pH 8.0) activated yellow-green bioluminescence. Luminescence and fluorescence emission spectra were recorded with a F-2000 fluorescence spectrometer (Hitachi, Tokyo) immediately
after the addition of substrate. Scanning was accomplished within 1 min, and luminescence was stable during that period. RESULTS
Production and purification of the fusion protein To test the performance of the system, myc tag (5) was chosen as a model peptide antigen, because an anti-peptide antibody with good affinity was readily available. For the detection of myc peptide with BRET, a fusion protein expression vector, pGEX-myc-luc, was designed to efficiently express the firefly luciferase in E. coli that was N-terminally tethered with myc peptide. As a control, an expression vector for GST-luc fusion protein was also made. The proteins were expressed in E. coli TG-1, and purified to homogeneity using a glutathione affinity column (Fig. 2a). Sufficient amounts of purified proteins (9.6 and 1.6 mg of GST-mycluc and GST-luc, respectively) were obtained from 100 ml cultures. Characterization of the fusion protein To investigate whether GST-myc-luc protein retained the desired properties, binding of anti c-myc antibody 9E 10 (5,6) to the protein was tested. A fixed concentration (2 ug/ml) of the fusion protein was adsorbed in each well of the microplate, and the binding of 9ElO at variable concentrations to the wells was tested by sandwich ELISA. When the signal was plotted against the concentration of 9ElO antibody, a clear increase in binding according to the increasing concentration of the antibody was observed (Fig. 2b). On the other hand, when the same concentration of GST-luc without cmyc tag was used for coating, negligible binding of 9E10 antibody was observed, clearly suggesting tag-specific binding of the antibody. To exclude the possibility that the
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VOL. 93,2002
(a)
539
M123456M
94k 67k 43k
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Wavelength (nm) 0
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0.1
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* . . .. i
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cr myc (ng/ml) FIG. 2. (a) Purification of the fusion proteins. Lanes 1 and 4, Lysate from TG-1 expressing GST-luc (1) or GST-myc-luc (4); Lanes 23 and 5-6, first and second fractions of the glutathione column eluate for GST-luc (2-3) or GST-myc-luc (5-6). M, Molecular marker. Coomassie-stained 10% SDS-polyacrylamide gel is shown. (b) Specific binding of 9ElO antibody to GST-myc-luc protein. The adsorbed fusion proteins (triangle for GST-luc, circle for GST-myc-luc, and cross for PBS only) were added with various concentrations of 9ElO antibody. The amount of bound antibody was probed with peroxidase-labeled anti-mouse IgG.
amounts of adsorbed fusion proteins were different, the bioluminescent activities of the bound proteins were assayed. Little difference in the light emission between the wells adsorbed with GST-myc-luc and GST-luc was observed (data not shown). Accordingly, the specific binding of 9E 10 to the myc-tag in GST-myc-luc was confirmed. In addition, the bioluminescent activity of GST-myc-luc in the wells was not inhibited by anti c-myc antibody added before the bioluminescent reaction, showing that the fusion protein retained its enzymatic activity not only in the free state but also in the 9ElO-bound form (data not shown). Luminescence energy transfer to Cydye-labeled antibody To test whether the luminescence energy transfer, a phenomenon similar to that found in luminous coelenterates (7, S), could occur between a c-myc tagged luciferase and fluorolabeled anti c-myc antibody, at first suitable fluorochromes were investigated. Among a number of red dyes, cyanine dyes Cy3, Cy3.5 and Cy5, each having a larger molar absorption coefficient than traditional rhodamine derivatives, were tested. Figure 3 shows the excitation and emis-
FIG. 3. Normalized spectra of cyanine dyes. Excitation (dotted line) and emission (stitched line) spectra of Cy3 (a), Cy3.5 (b) and Cy5 (c) are compared with the normalized luciferase emission spectrum (thin line).
sion spectra of these dyes. The luminescent spectrum of luciferase is also shown in each panel to compare the spectral overlap of the luminescent and dye excitation spectra. To achieve an efficient BRET, close approximation (~50 A) of the dye and the luciferase active site is crucial. Hence, succinimidyl esters were selected to achieve N-terminal labeling because the antigen binding sites of the antibody exist near the N-termini of its heavy and light chains. As a result, labeled antibodies with an F/P ratio of 4.5 to 6.0 were obtained, and they were tested for the luminescence energy transfer measurement. The bioluminescence of firefly luciferase is triggered by the addition of the substrates luciferin and ATP to the enzyme. The effect of the fluorolabeled antibody on the luminescent color was investigated by adding each antibody to the GST-myc-luc solution (20 nM) in a cuvette, and the luminescence spectra were recorded immediately after substrate addition using a fluorescence spectrometer with no excitation light (Fig. 4). When Cy3-labeled antibody (1.2 PM) was added, a reduction in the light emission at around 550 nm, and increase around 570 nm were observed, suggesting a transfer of energy from the epitopetagged luciferase to the Cy3-labeled anti-epitope antibody. No such spectral change in light emission was observed with GST-luc in the presence of the Cy3-labeled antibody (data not shown). When Cy3.5-labeled antibody (1.0 PM) was added, a similar but distinct spectral change was observed. In this case a clearer peak at 596 nm probably due to BRET was observed, and the intensity at 550 nm was sig-
540
YAMAKAWA ET AL.
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-
PpL
.. ... .. . . .
CY3
-------
CY3.5
I 550
E c 0.8 - ,, 8 (L ll : ‘g 0.6 3
650
600
700
Wavelength (nm) FIG. 4. Spectral change due to the addition of Cy3- and Cy3.5 labeled antibodies. GST-myc-luc (40nM) alone (black line) or with 2.4 pM Cy3-labeled antibody (dotted line) or 2.0 pM Cy3.5-labeled antibody (stitched line) was incubated in 50 mM Tris-HCI, pH 8.0 for 30 min at 0°C before the bioluminescent reaction.
2
1.2
0
2 z
0
53
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I
0.6 ‘1 0
0.1
1
10
~1myc (IJW o.8
-
8:: 0
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0.2
// 0
0.01
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0.01
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100
Peptide FIG. 6. Standard curves of BRET-based immunoassay. Peptide at the indicated concentration was mixed with GST-myc-luc and Cy3 labeled antibody (square), or GST-myc-luc and Cy3.5-labeled antibody (circle) in 50 mM Tris-HCI, pH 8.0, for 1 h at 0°C. Immediately after the addition of substrate, the luminescent spectra were recorded and the luminescence ratio calculated. Each point represents the mean+one SD of three determinations. Curves based on the model were fitted with Kaleida Graph 3.0. Parameters a and b in Eq. 9 are 0.905 and 29.0 for Cy3.0, and 0.23 and 62.7 for Cy3.5, respectively.
0
1
4
0
. . . .. . .I
. . . . **..I
0.1
1
. . * .*_
10
o myc (PM) FIG. 5. Antibody concentration dependence of BRET. 20 nM (circle) or 10 nM (square) of GST-myc-luc was incubated with the indicated amount of anti-myc antibody, and luminescent spectra were recorded. The luminescence ratio at specified wavelengths (BRET index) was calculated. GST-luc (cross) was used instead of GST-myc-luc in (a).
nificantly reduced (data not shown). Figure 5 shows the relationship between the labeled MAb concentration and the luminescence ratio. The largest ratio was obtained with the shorter wavelength near the emission maximum of the luciferase (550 nm), and the longer wavelength near that of Cy3 (57Onm) or Cy3.5 (596nm). So BRET indices were taken as the ratios of the light emissions at 5701596nm and 55Onm. In the case of both Cy3 and
Cy3.5 labeling, there was a labeled MAb-dependent increase in BRET index when GST-myc-luc protein was used. However, a very modest increase was observed when GSTluc protein was used (Fig. 5a). We could not detect significant spectral change when CyS-labeled antibody was added. However, the addition of labeled MAb induced significant quenching of the luminescence, implying the existence of BEET (data not shown, see discussion). BRET-based homogeneous immunoassay To use BRET to perform a homogeneous immunoassay, a limited amount of the tagged luciferase (10 nM) and excess Cy3- or Cy3.5-labeled antibody (0.5 PM) were mixed in the presence of various concentrations of c-myc peptide. Excess antibodies were used to obtain an efficient BEET signal. After preincubation, the substrate solution was added and luminescent spectra measured, from which the ratio of the luminescence at two wavelengths was calculated. Then the relationships between the BRET index and c-myc peptide concentration were plotted. For both Cy3 and Cy3.5 labeling, as shown in Fig. 6, a clear peptide concentration-dependent inhibition of BRET was observed. The change was more prominent when Cy3.5-labeled antibody was used, with the BRET index dropping from 0.8 to around 0.3 upon the addition of peptide. The practical measurable concentration range of the peptide was roughly 0.2 to 2.0 PM. A simple model was Comparison with the model built to evaluate the obtained result. Ab: anti c-Myc pep: c-myc peptide Luc: GST-myc-Luc Ag: pep+Luc
bep~Abl=K,bepl[Abl
(1)
[Luc.Ab]=K,[Luc][Ab]
(2)
If we assume K,=K2=K,
then by adding Eqs. 1 and 2, we
BRET-BASED
VOL. 93,2002
obtain the equilibrium
equation.
[Ag.Ab]=K[Ag][Ab] Dividing
(3)
Eq. 1 by Eq. 2 gives
bep.Abl [Luc.Ab]
[pep1 =[Lucl
[pep1 [PepI,
]pep.Abl+
= [Luc.Ab]+
(4)
[Luc] = [Lucl,
where [pep], and [Luc], are total peptide and luciferase concentrations, respectively. From Eq. 4, [pep.Ab]/[Luc.Ab]
+ 1 =[pep],/[Luc],+
[Ag.Ab]/[Luc.Ab]
=([pep],+[Luc],)/[Luc],
1 (5)
When we take the concentration of antigen-antibody plex as x, transforming Eq. 3 gives
com-
x=[Ag.Ab]=K[Ag][Ab]=K([Ag],-x)([Ab],-x)
Kx2-(~([Agl,+[Abl,)+l)x+K[Agl,[Abl,=0
(6)
x= W&&+ W&J + 1-A
(7)
2K
(D,=(K([&l,+[Abl,)+1F4~L%&L%J From Eqs. 5 and 7,
[Lucl, ‘Luc’Abl=[Luc],+[pep],x = K([Lucl,+[pepl,+[Abl,)+l-~
(8)
2K(l+E)
(~2=W(Wl,+ bepI,+ [AblJ+ 1)’
-4WLucl,+
bepl,)[AblJ
Based on this equation, the BRET index curves (y) were fitted to the following equation
?,,,+b~([Lucl,+x+[Abl~)+l-JzJj
(9)
) 2K(1+& (D;=(K([Luc],+x+[Ab],)+
1>*-4E(Z([Lu~]~+x)[Ab]~)
where a and b are parameters, [Luc],=O.O2 PM, [Ab],= 1.0 PM, and K= 1.25 x 107/M. As shown in Fig. 6, both curves showed relatively good fits with R values of 0.913 and 0.974 for Cy3 and Cy3.5 labeling, respectively. DISCUSSION To our knowledge this is the first demonstration of a BRET immunoassay using firefly luciferase as a luminescence donor. Previous BRET studies have used blue light emitters of Renilla luciferase (9-14) and quite recently, aequorin (15). Firefly luciferase is relatively easy to express in E. coli in large quantity, and a number of stabilized mu-
IMMUNOASSAY
541
tants have also been reported (16-l 8). Considering production costs and ease of use, firefly luciferase seems a far better choice than other enzymes, at least for in vitro diagnostics as presented here. The use of a suitable fluorophore is a key factor if this system is to work properly. Although at first we attempted to label the antibody with rhodamine X succinimide, we could not detect any spectral change upon addition of the labeled antibody. This was probably because its absorption was less than that of cyanine fluorophores, and hence the Fbrster resonance energy transfer distance R, for this luciferin-rhodamine X pair might be shorter than the distance between the luciferase active site and the N-terminus of the antibody H or L chains. Another consideration is the spectral overlap. We obtained a better response as a BRET index on labeling with Cy3.5 than Cy3. A condition for radiationless energy transfer is a significant overlap between the luciferase emission spectrum and the acceptor fluorophore excitation spectrum (J integral). However, the calculated J integral is smaller between the luciferase emission and Cy3.5 excitation than Cy3 excitation spectra (Fig. 3, data not shown). The reason for this apparent discrepancy might be that the emission spectrum of luciferase significantly overlaps with that of Cy3, and considerable background emission at 570nm is observed without BRET. Therefore, to improve sensitivity, in addition to obtaining greater spectral overlap between the luciferase emission and the acceptor excitation spectra, less overlap between the two emission spectra (in other words, larger stokes shift of the fluorophore) should be considered as well. We also attempted to use Cy5 which has a higher maximum absorption and longer emission wavelength (Fig. 3). However, we could not detect any spectral change due to BRET (data not shown). It is possible that the J integral between the luciferase emission and Cy5 extinction was not sufficient to cause a detectable BRET. Another possibility is the selfquenching of Cy5 due to multiple labeling of the 9E 10 antibody. When we compared the fluorescence of the labeled antibody and free fluorochrome at the same maximum absorbance, CyS-labeled antibody showed significant quenching of 75%, while Cy3- and Cy3.5-labeled antibodies showed 5-35%. A high extinction coefficient and greater overlap of excitation and emission spectra of Cy5 might cause the efficient intramolecular FRET, thus leading to such remarkable quenching. Here we showed the feasibility of a homogeneous immunoassay of peptides based on the BRET phenomenon. Though the sensitivity was not remarkable, according to the simulation it can be improved by reducing the concentration of antibody (data not shown). When we reduce the antibody concentration, we may also have to reduce the luciferase concentration to obtain sufficient BRET signal, otherwise, we will have a larger background derived from unbound luciferase. In addition, the use of a higher affinity antibody is preferable to obtain a sufficient binding equilibrium in a limited incubation period. Hence the use of a specialized sensitive luminometer equipped with optimal filter sets and a smaller cuvette will surely enhance the utility of the system. The equipment should not be too expensive because unlike FRET, there is no need for excitation.
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A future possibility is the use of this system in vivo. The natural fluorescent protein DsRed has a similar excitation/emission spectra to Cy3.5, though its maximum extinction coefficient is less than one tenth that of Cy3.5 (22,500 M-l) (19). The possible use of DsRed as a BRET companion with firefly luciferase is now under investigation (Arai et al., in preparation). ACKNOWLEDGMENTS
We are grateful to Tadashi Mikami in SS Pharmaceutical, Tokyo for his generous help in the production of 9E10.2 antibody, and Noriho Kamiya and Ryoichi Arai in University of Tokyo for their helpful comments. This study was supported in part by a Grantin-Aid for Scientific Research on Priority Areas (296-10145107) and Grant-in-Aid for Scientific Researches (B 09450301 and S 13854003) from the Ministry of Education, Science, Sports and Culture of Japan. REFERENCES
1. Hart, R. C., Matthews, J. C., Hori, K., and Cormier, M. J.: Renilla reniformis bioluminescence: luciferase-catalyzed production of nonradiating excited states from luciferin analogues and elucidation of the excited state species involved in energy transfer to Renilla green fluorescent protein. Biochemistry, l&2204-22 10 (1979). 2. Ward, W. W. and Cormier, M. J.: An energy transfer protein in coelenterate bioluminescence. Characterization of the Renilla green-fluorescent protein. J. Biol. Chem., 254, 781788 (1979). 3. Selvin, P. R. and Hearst, J. E.: Luminescence energy transfer using a terbium chelate: improvements on fluorescence energy transfer. Proc. Natl. Acad. Sci. USA, 91, 10024-10028 (1994). 4. Morimoto, K. and Inouye, K.: Single-step purification of F(ab’)2 fragments of mouse monoclonal antibodies (immunoglobulins Gl) by hydrophobic interaction high performance liquid chromatography using TSKgel Phenyl-5PW. J. Biothem. Biophys. Methods, 24, 107-117 (1992). 5. Munro, S. and Pelham, H. R B.: An Hsp70-like protein in the ER: identity with the 78 kd glucose-regulated protein and immunoglobulin heavy chain binding protein. Cell, 46, 291300 (1986). 6. Schiweck, W., Buxbaum, B., Schiitzlein, C., Neiss, H. G., and Skerra, A.: anti-c-myc antibody 9ElO: the VH domain has an extended CDR-H3 and exhibits unusual solubility. FEBS Lett., 414,33-38 (1997). 7. Morise, H., Shimomura, O., Johonson, F. H., and Winant, J.: Intermolecular energy transfer in the bioluminescent sys-
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tern ofdequorea. Biochemistry, 13,26562662 (1974). 8. Ward, W. W. and Cormier, M. J.: In vitro energy transfer in ReniZZabioluminescence. J. Physic. Chem., 80, 2289-2291 (1976). 9. Angers, S., Salabpour, A., Joly, E., Hilairet, S., Chelsky, D., Dennis, M., and Bouvier, M.: Detection of beta 2-adrenergic receptor dimerization in living cells using bioluminescence resonance energy transfer (BRET). Proc. Natl. Acad. Sci. USA, 97,3684-3689 (2000). 10. Arai, R., Nakagawa, H., Tsumoto, K., Mahoney, W., Kumagai, I., Ueda, H., and Nagamune, T.: Demonstration of a homogeneous noncompetitive immunoassay based on bioluminescence resonance energy transfer. Anal. Biochem., 289,77-81 (2001). 11. Boute, N., Pernet, K., and Issad, T.: Monitoring the activation state of the insulin receptor using bioluminescence resonance energy transfer. Mol. Pharmacol., 60,640-645 (2001). 12. Cheng, Z. J. and Miller, L. J.: Agonist-dependent dissociation of oligomeric complexes of G protein-coupled cholecystokinin receptors demonstrated in living cells using bioluminescence resonance energy transfer. J. Biol. Chem., 276, 48040-48047 (2001). 13. Kroeger, K. M., Hanyaloglu, A. C., Seeber, R. M., Miles, L. E., and Eidne, K. A.: Constitutive and agonist-dependent homo-oligomerization of the thyrotropin-releasing hormone receptor. Detection in living cells using bioluminescence resonance energy transfer. J. Biol. Chem., 276, 1273612743 (2001). 14. Xu, Y., Piston, D. W., and Johnson, C. H.: A bioluminescence resonance energy transfer (BRET) system: application to interacting circadian clock proteins. Proc. Natl. Acad. Sci. USA, 96, 151-156 (1999). 15. Adamczyk, M., Moore, J.A., and Shreder, K.: Quenching of biotinylated aequorin bioluminescence by dye-labeled avidin conjugates: application to homogeneous bioluminescence resonance energy transfer assays. Org. Lett., 3, 17971800 (2001). 16. Kajiyama, N. and Nakano, E.: Thermostabilization of tirefly luciferase by a single amino acid substitution at position 217. Biochemistry, 32, 13795-13799 (1993). 17. Kajiyama, N. and Nakano, E.: Enhancement of thermostability of firefly luciferase from Luciola lateralis by a single amino acid substitution. Biosci. Biotechnol. Biochem., 58, 1170-1171(1994). 18. White, P. J., Squirrell, D. J., Amaud, P., Lowe, C. R., and Murray, J. A.: Improved thermostability of the North American firefly luciferase: saturation mutagenesis at position 354. Biochem. J., 319,343-350 (1996). 19. Mab, M. V., Fradkov, A. F., Labas, Y. A., Savitsky, A. P., Zaraisky, A. G., Markelov, M. L., and Lukyanov, S. A.: Fluorescent proteins from nonbioluminescent Anthozoa species. Nat. Biotechnol., 17,969-973 (1999).