1 Department of Chemistry and Biotechnology, Graduate School of Engineering, University of Tokyo, Hongo, Bunkyo-ku, Tokyo 113-8656, Japan, 2 Centre for Protein Engineering, MRC Centre, Hills Road, Cambridge CB2 2QH, UK, 3 Department of Biomolecular Engineering, Graduate School of Engineering, Tohoku University, Aoba, Aramaki, Aoba-ku, Sendai 980-8579, Japan, and 4 Roche Diagnostics, Chief Technology Office, 2929 7th Street, Suite 100, Berkeley, CA 94710, USA
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Abstract |
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Keywords: antibody variable region/chimeric protein/fluorescence resonance energy transfer/fluoroimmunoassay/green fluorescent protein
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Introduction |
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GFP, isolated from the jellyfish Aequorea victoria, emits intrinsic intense and stable greenish fluorescence without any cofactors (Morise et al., 1974) and shows less photobleaching than fluorescein (Arai et al., 1998
). Since the advent of gene cloning, GFP has been widely used for monitoring gene expression and/or studying the subcellular localization of cellular proteins in living cells (Wang and Hazelrigg, 1994
; Barthmaier and Fyrberg, 1995
; Baulcombe et al., 1995
; Hu and Cheng, 1995
; Olson et al., 1995
). Also, the stability of GFP and its bright fluorescence underline its potential utility for fluoroimmunoassays in vitro (Aoki et al., 1996
; Arai et al., 1998
). Furthermore, recent developments of many mutant GFPs with enhanced fluorescence and/or altered excitation/emission wavelengths have further enlarged the scope of utilization of these proteins. For example, a fluorescence-enhanced variant EGFP (enhanced green fluorescent protein), having double amino acid substitutions F64L and S65T, fluoresces 35 times more intensely than wild-type GFP when excited at 488 nm (Cormack et al., 1996
). On the other hand, a color variant EBFP (enhanced blue fluorescent protein) containing four amino acid substitutions (F64L, S65T, Y66H and Y145F), is known to have blue-shifted excitation and emission peaks (380 and 440 nm, respectively) (Heim and Tsien, 1996
). While such variants as EGFP and EBFP are ideal fluorolabels for the sensitive multicolor detection of target molecules with fluorescence microscopy, they are also useful for fluorescence resonance energy transfer (FRET) studies.
FRET has been widely used in studies of biomolecular structure and dynamics. With a FRET signal being developed when two labels approach distances in the order of 10100 Å, the technique is suitable for investigating spatial relationships of interesting molecules in biochemistry (Wu and Brand, 1994; Selvin, 1995
). Since the emission spectrum of EBFP overlaps the excitation spectrum of EGFP, the combination of both fluorophores is one of the ideal pairs for FRET studies (Heim and Tsien, 1996
; Mitra et al., 1996
). Until now, the FRET between GFP variants including this has been successfully used as fluorescent indicators for [Ca2+]i (Miyawaki et al., 1997
, 1999
; Romoser et al., 1997
), demonstration of Bcl-2Bax interaction in vivo (Mahajan et al., 1998
) and detection of the myosin heads swing (Suzuki et al., 1998
).
Sandwich immunoassays such as enzyme-linked immunosorbent assay (ELISA) and fluoroimmunoassay (FIA) are widely used techniques to determine antigen concentration. The assay has several merits such as high sensitivity and specificity and low background. However, it also has a weakness that it usually needs a long measurement time owing to several cycles of consecutive binding and washing steps. In addition, there is one basic limitation in the assay that the antigen to be measured requires at least two epitopes to be captured and detected. As a way to circumvent these limitations, recently we described a new immunoassay approach called open sandwich ELISA, a novel ELISA based on the variable interchain interaction of separated VH and VL chains from a single antibody variable region (Ueda et al., 1996; Suzuki et al., 1999
). In short, the assay exploits the reassociation of the generally weak VHVL complex by a bridging antigen. With the use of an immobilized VL and enzyme-tagged VH fragments, one can measure <10 ng/ml antigen in a shorter time period than the conventional sandwich assay from which one incubation/washing cycle can be omitted (Suzuki et al., 1999
). However, as a heterogeneous immunoassay it requires at least one bound and free (B/F) separation, which considerably limits the throughput of the assay. More recently we also described an open sandwich FIA, a homogeneous derivative of the assay which utilized FRET between fluorolabeled VH and VL fragments to detect their association in liquid phase (Ueda et al., 1999
). In the assay, when fluorescein-labeled VH and rhodamine-labeled VL fragments were added to the sample solution, the antigen concentration could be determined almost instantaneously by monitoring the VHVL association with FRET between the two fluorochromes. Although the measurement itself was simple and rapid, the optimization of the fluorolabeling conditions needed several laborious empirical trials (Ueda et al., 1999
).
In this study, we attempted a site-specific and efficient fluorolabeling of antibody variable regions with the GFP variants with the use of an E.coli cytoplasmic expression system. In addition to the construction and functional assay of the chimeric proteins consisting of E.coli thioredoxin (Trx) connected to VH/VL regions of anti-hen egg lysozyme (HEL) antibody HyHEL-10 and EBFP/EGFP domains, respectively, we evaluated their utility in the homogeneous non-competitive open sandwich FIA (Figure 1).
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Materials and methods |
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DNA fragments encoding EGFP and EBFP were prepared from plasmids, pEGFP and pEBFP-N1 (Clontech, CA, USA), by standard PCR using Pfu DNA polymerase (Stratagene, CA, USA) and the primers 5'-CCGCGGCCGCCATGGTGAGCAAGGGCGAGGAGCTG-3' and 5'-CCCTCGAGCTTGTACAGCTCGTCCATGCCGAG-3' (cleavage sites by NotI and XhoI are shown in bold letters). The amplified fragments were digested with NotI and XhoI and cloned into pBluescriptII KS+ (Stratagene). E.coli XL1-Blue (Stratagene) was used as a host strain for transformation. DNA sequencing was performed using a fluorescence DNA sequencer, SQ-5500 (Hitachi, Tokyo, Japan) and Thermosequenase sequencing kit (Amersham, Amersham, UK). For the expression of chimeric proteins, pET TRX Fusion System 32 (Novagen, WI, USA), a fusion expression system with E.coli thioredoxin (trxA), was employed to enhance the solubility of the expressed proteins in E.coli cytoplasm (LaVallie et al., 1993). The NcoIHindIII fragment of pVLPhoA (Suzuki et al., 1999
) encoding HyHEL-10 VL fragment was cloned into the multiple cloning sites of pET32b(+) between NcoI and HindIII to give the plasmid pET32/VL. To make the plasmids pET32/VLEGFP and pET32/VLEBFP, respectively, the amplified fragments for EGFP or EBFP were digested with NotI and XhoI and cloned into pET32/VL between NotI and XhoI sites. pET32/VHEBFP was constructed by substituting the VL fragment of pET32/VLEBFP with the HyHEL-10 VH fragment derived from pVHPhoA (Suzuki et al., 1999
) with the use of NcoI and HindIII (Figure 2
).
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E.coli AD494(DE3)pLysS (Novagen), a strain lacking thioredoxin reductase gene (trxB), was transformed with the plasmids and selected on LB agar plates containing 50 µg/ml ampicillin, 34 µg/ml chloramphenicol and 15 µg/ml kanamycin. For all the cultivations thereafter, LB medium containing 50 µg/ml ampicillin, 34 µg/ml chloramphenicol and 15 µg/ml kanamycin was used. A 100 ml volume of medium was inoculated with 1 ml overnight culture at 30°C of the strains harboring either pET32/VHEBFP or pET32/VLEGFP and cultured at 30°C for 6 h. Then 1 l of medium was inoculated with each strain and cultured at 25°C for 3 h. At OD600 0.5, IPTG was added to a final concentration of 1 mM to induce the expression of the fusion proteins and cells were further cultured for 12 h at 16°C. Harvested cells were resuspended in sonication buffer (50 mM TrisHCl, 50 mM NaCl, 2 mM EDTA, pH 8.0) and disrupted by freezethaws and sonication with a Branson Sonifier with 60 W output, 50% duty cycle per second for 5 min on ice. The two supernatants obtained by centrifugation at 15 kg for 20 min at 4°C were mixed and applied to a 4 ml column of HEL-Sepharose made of CNBr-activated Sepharose 4B (Pharmacia, Uppsala, Sweden) and HEL (Seikagaku, Tokyo, Japan) by standard protocol. The column was washed with 20 volumes of 0.1 M TrisHCl, 0.5 M NaCl, pH 8.5 and then the bound materials were eluted with 5 volumes of 0.1 M Na2HPO4NaOH, pH 12. Each fraction of 1 ml was immediately neutralized with 100 µl of 1 M TrisHCl, pH 6.8. Fractions emitting fluorescence were pooled, dialyzed against phosphate-buffered saline (PBS; 10 mM phosphate, 137 mM NaCl, 13 mM KCl, pH 7.4) and stored at 80°C until use. Sufficient fluorescence and antigen binding activities were retained for at least 1 year when stored at 80°C. The protein concentration was determined by BCA assay kit (Pierce, Rockford, IL, USA) with bovine serum albumin (BSA) as standard. When each TrxVHEBFP or TrxVLEGFP was purified individually, S-protein agarose (Novagen) was used according to the manufacturer's protocol except that 4.5 M MgCl2 was employed for the elution.
Measurement of fluorescence spectra and determination of antigen concentration by FRET
The fluorescence spectra of TrxVHEBFP and TrxVLEGFP after purification were measured using an F-2000 fluorescence spectrophotometer (Hitachi, Tokyo, Japan). In the case of antigen concentration determination, 1 µl of a 1000-fold concentration of antigen solution in PBS was added to 1 ml of PBS containing 1 mg/ml BSA (Sigma, St. Louis, MO, USA) and 100 µg/ml TrxVHEBFP/TrxVLEGFP mixture purified by HEL-affinity chromatography. After 5 min incubations, the emission spectra were measured with 380 nm excitation at 4°C. The fluorescence emission intensity ratio I(506 nm)/I(444 nm) was calculated to evaluate FRET.
Determination of antigen concentration using microplate fluororeader
A 10 µl volume of a 10-fold concentration of antigen in PBS and 90 µl of 100 µg/ml TrxVHEBFP/TrxVLEGFP mixture in PBS containing 1 mg/ml BSA or 10% fetal bovine serum (FBS) was applied to each well of Fluoro Nunc Maxisorp white plate strips (Nalge Nunc International, NY, USA) and mixed with a microplate mixer, MPX-96 (Iwaki, Tokyo, Japan). The samples were analyzed with the microplate fluororeader, CytoFluor II (Perseptive Japan, Tokyo, Japan), using bandpass filters of 360/40 nm for excitation, 460/40 nm for EBFP emission and 530/25 nm for EGFP emission. The fluorescence emission ratio I(530 nm)/I(460 nm) was calculated to evaluate FRET.
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Results |
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After several fruitless attempts to express fusion proteins of antibody variable domain and GFP derivatives in E.coli periplasm, we decided to employ a cytoplasmic expression system. As a result of the use of a thioredoxin-fusion expression vector and a trxB E.coli strain AD494(DE3)pLysS as a host, sufficient amounts of soluble and active TrxVHEBFP and TrxVLEGFP could be obtained. Figure 3 shows that the mixture of TrxVHEBFP and TrxVLEGFP could be efficiently purified by one-step purification with HEL-Sepharose, indicating the expression of certain amounts of the correctly folded chimeric proteins with sufficient antigen binding activity when mixed together with the counterparts. As judged from their molecular weights, most of them were full-length products of TrxVHEBFP/TrxVLEGFP (59 kDa), while some (~5%) of them were truncated to VHEBFP/VLEGFP (46 kDa) devoid of Trx domain. However, virtually no VH or VL alone (12 kDa) was detected. From the BCA protein assay and the purity estimated as above, about 6 mg of purified soluble TrxVHEBFP/TrxVLEGFP chimeric protein mixture could be obtained from a total of 2 l of culture.
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The purified TrxVHEBFP and TrxVLEGFP were tested for whether they still retained fluorescent activities. As shown in Figure 4, the individually purified TrxVHEBFP or TrxVLEGFP proteins with the S-tag affinity column had excitation/emission spectra almost indistinguishable from those of parental EBFP (Heim and Tsien, 1996
) or EGFP (Cormack et al., 1996
), respectively. Because the emission maxima of TrxVHEBFP and TrxVLEGFP were 444 and 506 nm, respectively, and their excitation maxima 380 and 487 nm, respectively, these values were used for the subsequent FRET measurements.
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To test whether the obtained chimeric proteins could be employed as immunoassay reagents, they were applied to the open sandwich FIA (Figure 1). Increased FRET due to antigen-dependent VH/VL reassociation was expected to be observed. Figure 5
shows the HEL concentration-dependent change in TrxVHEBFP/TrxVLEGFP emission spectra with 380 nm excitation in the presence of 1 mg/ml BSA. As the HEL concentration increased, we could clearly observe an apparent decrease in the fluorescence emission from EBFP and an increase in the emission from EGFP probably due to FRET from EBFP to EGFP. Because the change in spectra was significant and correlated with the antigen concentration, the dose dependences for cognate and non-cognate antigens were assayed. Figure 6
shows the relation between the antigen concentration and a FRET index, the fluorescence emission intensity ratio of I(506 nm)/I(444 nm) after stabilization of the readings. As seen, the fluorescence emission ratio increased significantly in response to the concentration of antigen, HEL, to which HyHEL-10 shows remarkable binding (Smith-Gill et al., 1984a
,b
). The measurable concentration range was roughly from 1 to 100 µg/ml. On the other hand, in the case of human lysozyme (huL) (Sigma), which HyHEL-10 could poorly recognize (Tsumoto et al., 1997
), the emission ratio remained almost unchanged, suggesting no significant hetero-dimerization induction similar to the result with blank samples. In the case of HEL, higher concentrations than 100 µg/ml were difficult to measure under this condition, presumably owing to the saturated antigen binding to the Fv moiety. However, the use of higher concentrations (250 µg/ml) of TrxVHEBFP/TrxVLEGFP mixture allowed the measurement of a higher concentration range (101000 µg/ml) (not shown). In the absence of HEL, the fluorescence emission ratio was observed to increase slightly as the concentration of TrxVHEBFP/TrxVLEGFP mixture increased. However, the increase (0.06 ratio when the concentration was increased from 100 to 400 µg/ml) was so small that we can say that almost all the fusion proteins stay monomeric in this concentration range, permitting the FRET-based homogeneous immunoassay with little influence of spontaneous dimerization (not shown).
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Discussion |
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According to the X-ray structure of HyHEL-10 Fab complexed with HEL (PDB 3HFM), both C-termini of Fv were further from antigen binding surface than N-termini and there seemed no steric hindrance of C-terminally linked EBFP/EGFP and Fv or the antigen. In addition, since the distance between two C-termini of VH and VL domains of the complex is 42 Å, the distance between tethered EBFP/EGFP fluorophores when complexed with HEL might be around or within R0 of EBFP and EGFP of 4043 Å (Tsien, 1998) according to the molecular modeling (not shown). The distance was significantly shorter than the distance where GFP domains were tethered at the Fv N-termini (not shown). Owing to these observations, GFP tagging of Fv was done via the C-termini rather than the N-termini.
As shown in the SDSPAGE after HEL-affinity purification (Figure 3), most of the purified proteins were full-length products of TrxVHEBFP/TrxVLEGFP (59 kDa), whilst the faint bands corresponding to VHEBFP/VLEGFP (46 kDa) devoid of Trx domain were seen. However, virtually no bands for VH/VL (12 kDa) or TrxVH/TrxVL (25 kDa) could be detected as judged from their molecular weights. Although the bands for TrxVHEBFP/TrxVLEGFP or VHEBFP/VLEGFP overlap with each other because of their similar molecular weights, they could be separated after longer electrophoresis (not shown). These data show that almost all VH or VL fragments were efficiently labeled with EBFP or EGFP, respectively, at least immediately after HEL-affinity purification.
The method described here seems to have several advantages over the chemical fluorolabeling method of antibody fragments in the following respects. First, the protein can be labeled site-specifically and efficiently without losing activity. In the case of chemical labeling, site-specific and efficient labeling of the Fv fragment is not always easily achievable. Second, GFP variants show less photobleaching than widely used fluorochromes such as fluorescein, allowing continuous fluorescence measurement (Arai et al., 1998). Third, and more importantly, once the gene for an Fv is obtained, the genetic labeling is relatively easy by employing quick E.coli cultivation. This in turn means that now a number of Fvs can be rapidly screened for their compatibility with open sandwich FIA. Finally, the method has potential use in in vivo antigen quantitation. When TrxVHEBFP and TrxVLEGFP are simultaneously expressed in a cell, we can expect to monitor the intracellular antigen concentration in real time in vivo. This further implies a possibility of randomizing and selecting an Fv which is most suitable for the current assay by monitoring the change in FRET upon antigen expression in the cell. Such an approach has a potential to complement the conventional selection of antibody fragments by several display methodologies (Winter et al., 1994
; Hanes and Plückthun, 1997
; Daugherty et al., 1998
).
In Figure 5, the emission peak at 506 nm without HEL is considered to be due to direct excitation of EGFP rather than the result of FRET. This is because when the individually recorded spectra for TrxVLEGFP and TrxVHEBFP both excited at 380 nm at the same molar concentrations were numerically added, almost the same curve was obtained (not shown). This in turn implies that in the absence of HEL, the FRET from EBFP to EGFP domains is negligible. On the assumption of no FRET without HEL and saturated HEL binding at maximum HEL concentration, the energy transfer efficiency E (E = 1 IDA/ID; IDA = donor intensity in the presence of acceptor; ID = donor intensity in the absence of acceptor) is estimated to be 0.21. This value is more than 0.17, the value obtained from previous open sandwich FIA using chemically fluorolabeled antibody fragments (not shown). While different fluorochrome pairs with different R0 and different labeling targets (N- and C-termini) were employed, one reason for the improvement in efficiency might be site-specific and efficient labeling. Although further improvement in efficiency might be possible with the use of other pairs of GFP mutants with greater R0 (Miyawaki et al., 1997
; Tsien, 1998
Tsien, 1999), the lack of necessity for special filters for the microplate assays is clearly the advantage of the current pair.
We did not remove the thioredoxin domain from the chimeric proteins before FRET assay because we could obtain sufficient responses in the assay. However, if increased sensitivity is required, cleavage of the linker by enterokinase and the removal of the Trx domain might be applied, since we could observe some (23-fold) improvement in sensitivity when the same molar concentrations of Trx-deleted chimeric proteins were used in ELISA detecting HEL-bound chimeric proteins with anti-GFP antiserum (not shown). The reason why such a difference was observed is yet to be clarified. If the interference of Trx with the antigen binding is the reason, C-terminal fusion of Trx domain might be a possible solution to avoid the tedious and costly protease cleavage and purification steps.
If we assume the binding event of TrxVHEBFP/TrxVLEGFP mixture to HEL to be that of antibody Ab to antigen Ag and the fluorescence emission intensity at 506 nm to be a measure of AbAg complex formation, we can derive the approximate association rate constant ka from the intensity curve (Figure 7B). The rate of complex formation AbAg at time t is
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where kd is the dissociation rate constant. Since after time t, [Ab] = [Ab]0 [AbAg], where [Ab]0 is the concentration of Ab at t = 0, the equation can be written as
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On the assumptions that the variation of 506 nm fluorescence intensity of EGFP, F is proportional to the formation of complexes, Fmax is maximum fluorescence intensity when all TrxVHEBFP/TrxVLEGFP form complexes with HEL and HEL concentration, C = 7 µM, which is approximately a 10 times molar excess over TrxVHEBFP/TrxVLEGFP, is constant, the equation becomes
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or
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Thus, a plot of dF/dt versus F gives a slope ks, where ks = kaC kd. From Figure 7C, a plot of dF/dt versus F from 0 to 130 s can roughly fit a linear line (correlation coefficient r2 = 0.905), whose negative slope is ks = 0.014 s1. On the assumption that kd is negligible since kd of Fv obtained with the SPR biosensor Biacore is 2.73x105 ± 1.43x106 s1 at 25°C (Ueda et al., 1996
), ka of TrxVHEBFP/TrxVLEGFP to HEL is calculated to be
. This value is smaller than the value obtained with Biacore previously for Fv (>6.4x103 M1 s1) and for TrxVHEBFP/TrxVLEGFP (4.3 ± 0.2x103 M1 s1) obtained this time at 25°C (not shown). However, considering the difference in temperature, we think the assumptions and calculations roughly appropriate.
Because the immunoassay described here (open sandwich FIA) is homogeneous and non-competitive, operation is easy and quick, taking only a few minutes from mixing the solutions to measuring the fluorescence. Moreover, it can be performed in a microplate format, hence the assay can be robust and suitable for automation. The assay has the limitation that it requires a suitable antibody Fv that has sufficiently weak VHVL interaction without the antigen and is stabilized with the antigen. However, one needs only one clone of antibody to conduct the assay, regardless of the size of the antigen. As such, this homogeneous approach may be the first that works equally well with low molecular weight haptens and high molecular weight antigens. This represents a considerable improvement over fluorescence polarization, cloned enzyme donor immunoassay (CEDIA) (Henderson et al., 1986) and the enzyme-multiplied immunoassay technique (EMIT) (Rubenstein et al., 1972
) technologies that are currently employed in clinical immunoassay-based diagnostics. A selection system for suitable antibodies using a phage display approach has also been proposed (Tsumoto et al., 1997
). If a more general screening method for a suitable Fv is developed, this method may prove to be a convenient and inexpensive alternative to conventional laborious and expensive sandwich immunoassays.
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Notes |
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Acknowledgments |
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Received September 6, 1999; revised January 5, 2000; accepted February 9, 2000.