- Email: [email protected]
Fluorescence labelling with europium chelate of β-diketones and application in time-resolved fluoroimmunoassays (TR-FIA)
ICAL METHODS
ELSEVIER
Journal
of
Immunological Methods 179 (1995) 233-241
Fluorescence labelling with europium chelate of P-diketones and application in time-resolved fluoroimmunoassays (TR-FIA) Yun-Xiang Ci
*, Xiao-Da
Yang, Wen-Bao Chang
Department of Chemistry, Peking University, Be&g
100871, People’s Republic of China
Received 8 February 1994, revised received 15 July 1994,accepted 31 October 1994
Abstract Five P-diketone derivatives were studied for multiple labelling of proteins. The labelled proteins were characterized by absorption and fluorescence measurements. It was found that proteins labelled with chlorosulfonylthenoyltrifluoroacetone (CTTA) were able to form highly fluorescent complexes with Eu3’ which exhibited prolonged fluorescence whereas the Eu3+ complex of hydrolyzed CTTA exhibited almost no fluorescence, and so unreacted ligand gave no background signal in immunoassays even if it was not removed from the labelled reagent. The effect of labelling on the biological activity of albumin and polyclonal antibody was studied and it was also shown that the new probe could be used in time-resolved fluorescence immunoassays. Keywords:
P-Diketone;
Multiple labelling; Time-resolved
1. Introduction Since the development of the time-resolved fluorescence immunoassay, considerable progress has been made in this field. In conventional FIA methods background fluorescence is a major problem, but this can be virtually eliminated by
Abbreviations: CTTA, 5-chlorosulfonyl-2-thenoyltrifluoroacetone; TTA, thenoyltrifluoroacetone; TAABTA, trifluoroacetylaminohenzoyltrifluoroacetone; DMABTA, N,N.dicarboxymethylaminobenzoyltrifluoroacetone; ABTA, aminobenzoyltrifluoroacetone; NTA, naphthoyltrifluoroacetone; EDC, 1-ethyl-3(3-dimethylamino-propyl)carbodiimide: TR-FIA, time-resolved fluoroimmunoassay; MC, macromolecule complex; Eu, europium; TOPO, trioctylphosphine oxide. * Corresponding
author.
0022-1759/95/$09.50 0 1995 Elsevier XSDI 0022..1759(94)00289-4
Science
fluoroimmunoassay
using the fluorescent lanthanide chelates as labels in combination with time-resolved fluorometry. Many authors (Diamandis and Christopoulos, 1990; Hemmilti, 1989; Soini, 1990; Soini and Lovgren, 1987) have reviewed the approaches using europium and other lanthanide chelate labels in time-resolved fluorescence immunoassays and it has been reported that some sensitive assays, such as the assay for cu-fetoprotein, are able to detect approximately 300000 molecules per sample (Diamandis and Christopoulos, 1990). Among several methodologies for time-resolved fluorescence assays, two are generally regarded as very successful. In the Delfia system (Soini and Kojola, 1983; Soini and Lovgren, 1987; Soini, 19901, an ethylenediaminetetraacetic acid (EDTA) derivative is used to bind Eu’+ to the
B.V. All rights reserved
234
Y.-X. Ci et al. /Journal
of Immunological Methods 179 (1995) 233-241
immunoreactants and Eu3+ is released with an enhancement solution for measurement under optimum conditions. The second method, which was popularized by Diamandis and his co-workers (Diamandis et al., 1989a,b; Diamandis and Morton, 1989; Diamandis and Christopoulos, 1990), uses an organic chelating ligand (BCPDA) as the label. Using this method, the amount of ligand is quantified in the presence of excess Eu3+ either directly on the surface of dried microtiter plates or in solution analogously to the Delfia method after the dissociation of the labelled solid phase component (Kropf and Gressner, 1991). The relative merits and limitations of these two methodologies have been discussed by Diamandis (Diamandis, 1988). One advantage of the second method is that the problem of exogenous contamination for Eu3+ in the environment and on skin surfaces can be eliminated. This is especially significant for those assays performed on samples in which fluorescent lanthanide ions are abundant. For this reason, the Diamandis method may be practically superior to the Delfia system. Usually, an Eu 3+ chelating ligand suitable for use in the second method should form relatively stable complexes with Eu3+, be highly fluorescent, have an excitation wavelength preferably close to 337 nm (the emission wavelength of a nitrogen laser), react covalently with the protein of interest but interact minimally with serum components (Diamandis and Morton, 1988). Furthermore the ligand should ideally be cheap and plentiful. Although some ligands meet the first five requirements, few satisfy the last one. p-diketones are generally relatively inexpensive reagents with excellent fluorescence properties when chelated. However, they have proved unsatisfactory as direct fluorescent probes because of the severe problem of quenching and very low thermodynamic stability (Hemmila, 1985). We have speculated that some @-diketones could be used as BCPDA-type probes because in the second method the requirement for thermodynamic stability could be compensated by increasing the concentration of Eu3+ and the incorporation of the probes into protein molecules could yield a ‘bonus’ effect (Diamandis et al., 1989a,b). Recently we described the first success-
ful p-diketone ligand: 5chlorosulfonylthenoyltrifluoroacetone (CTTA) (Ci and Yang, 1992). In this paper, the labelling of protein with five @diketone derivatives is described. Of these CTTA was found to be the only one which was suitable for multiple labelling and CTTA-labelled reagents were tested in the development of a solid-phase immunoassay.
2. Materials and methods 2.1. Instrumentation Fluorescence measurement was performed using a Hitachi M850 and an LS-SOB fluorospectrophotometer. The sample absorption was measured on a Beckman DU-7 spectrophotometer. 2.2. Materials Human serum albumin (HSA) and poly-lysine was purchased from Sigma Chemical Company. Bovine serum albumin (BSA) was obtained from the Beijing Biochemical Plant (China). Polyclonal antibodies (anti-BSA) and antiserum were kindly provided by the Biology Department of Peking University. Sephadex G-25 and G-100 were purchased from the Shanghai Biochemical Plant (China). Trifluoroacetylaminobenzoyltrifluoroacetone (TAABTA), N,N,-dicarboxymethylaminobenzoyltrifluoro-acetone (DMABTA), aminobenzoyl-trifluoroacetone (ABTA), and naphthoyltrifluoro-acetone (NTA) were prepared in our laboratory (Wang, 1993). 96-well microtiter plates for immunoassays were obtained from the Yuhuan Plastic Plant of Zhejiang (China). 2.3. Synthesis of chlorosulfonyl-P-diketone derivatives
The chlorosulfonyl group was introduced into the TTA, TAABTA, and NTA structures through direct chlorosulfonation of the P-diketone: @diketone + 2ClS0,H -
ClSO,-P-diketone
+ HCl
Y-X. Ci et al./Journal of ImmunologicalMethods 179 (1995) 233-241
p-diketone and CISO,H were mixed in the ratio 5 : 1 and stirred whilst heating to 50-60°C. This temperature was maintained until the reaction was complete. The excess ClSO,H was degraded and separated by pouring the reaction mixture into ice cold water. The products were then recrystallized in anhydrous chloroform. The spectra of the products showed that their structures were as follows: ClSO, Lrj
COCH ,COCF,
235
in a small volume of dimethylsulfoxide (DMSO) and reacted for 1 h before the mixture was added to the protein solution. The derivatization reaction was allowed to proceed for 1 h at room temperature. After the derivatization reaction, the labelled proteins were isolated by applying the reaction mixture to a column of Sephadex G-25 (1.0 x 25 cm> which was equilibrated and eluted with 0.9% NaCl solution (1 ml/min). The effluent was monitored by measuring absorbance values at both 280 nm and 350 nm.
CITA COCH,COCF,
0 p ClSO,
NHCOCF,
CTAABTA
00 W ClSO,
COCH ,COCF,
CNTA 2.4. Labelling of proteins with P-diketone derivatives and isolation of labelled proteins In order to label the proteins with chlorosulfonyl-@diketones, they were dissolved in 0.1 mol/l carbonate buffer at a concentration of about 2-5 mg/ml and then a freshly prepared solution of P-diketone derivatives in dimethylformide (DMF) was added to the protein solution. The amount of P-diketone derivative varied according to the desired molar ratio of P-diketone/ protein of the products. The reactions were complete after 1 h at room temperature. Proteins were derivatized with p-aminobenzoyltrifluoroacetone and m-l\r,N-dicarboxymethylaminobenzoyltrifluoroacetone using lethyl-3(3-dimethylamino-propylj-carbodiimide (EDC). EDC and the @diketone were dissolved
2.5, Characterization of labelled protein by absorbance and fluorescence The absorption spectra of labelled proteins were obtained at their stock concentration (50100 pg/ml) with a Beckman DU-7 spectrophotometer. The molar ratio (RM) of p-diketone to protein was obtained after dividing the concentration by the molar concentration of the protein. The concentration of p-diketone bound to protein was calculated through the maximum absorbance (at 320-380 nm) and the molar absorption coefficient (E) of free p-diketones was adopted (for each chlorosulfonyl-@diketone, the E was that of the original molecule). The fluorescence spectra were measured with a Hitachi M8.50 using 1 pg/ml of labelled protein, 1 x 10e5 mol/l of Eu3+, and 0.1 mol/l of pH 8.2 Tris buffer. The fluorescence half life values were measured on the PE LS-SOB equipment using the same conditions. The fluorescence properties of labelled proteins were studied by fluorescence measurements of solutions which differed in concentration, pH, buffer, auxiliary ligands, and concentration of Eu3+. Chlorosulfonyl-P-diketones were hydrolyzed by incubating a weighed amount in a 0.1 mol/l carbonate buffer pH 9.5 at room temperature for 1 day. Then the solutions were diluted approximately to cover the range from 10eh to lo-’ mol/l, the solution buffer was 0.1 mol/l Tris-HCl buffer pH 8.2, containing 1 X lop5 mol/l of Eu’+, and the fluorescence was measured with the Hitachi M850.
236
Y-X. Ci et al. /Journal
of Immunological Methods I79 (1995) 233-241
2.6. Preparation and isolation of macromolecular complexes of CTTA-labelled protein
sulfate (SDS), 2 X lop6 mol/l Eu3+, and 1 X lop5 mol/l TOPO.
The macromolecular complexes were prepared by either heating the protein solution (pH 8.2, 10-6-10-5 mol/l of Eu3+, and 100-1000 ng/ml of CTTA-labelled protein) to 50°C for 2 h (the heating method) or adding lop5 mol/l of trioctylphosphine oxide (TOP01 to the solution (the TOP0 method). The macromolecular complexes were separated by applying the solution to a Sephadex G-100 column (1.0 X 20 cm> which was equilibrated and eluted with 0.15 mol/l NaCl solution, and the effluent was monitored by fluorescence measurements at excitation/ emission wavelengths (ex/em> of 350/614 nm.
2.9. Immunoassay procedure The wells were coated overnight at 4°C with 200 ~1 of anti-BSA serum (l/20 diluted with coating buffer). After the wells were blocked, 100 ~1 of BSA standards and 100 ~1 of 4.0 pg/ml CTTA-labelled BSA (RM = 30) were added and incubated at 37°C for 2 h. Finally, 250 ~1 of dissociating solution were added and 1 h later, fluorescence was measured using the LS-SOB instrument under the above conditions.
3. Results and discussion 2.7, Assessment of biological activity of CTTA labelled antibody Rabbit polyclonal anti-BSA antibodies were labelled with C’ITA and purified by the method described above. The wells of a microtiter plate were coated overnight at 4°C with 100 ~1 of a 5 pg/ml BSA solution in coating buffer and then blocked for 2 h at 37°C with 200 ~1 blocking buffer. After blocking, 100 ~1 of a 100 pg/ml solution of labelled antibody (differing in RM value) were added and incubated for 2 h at 37°C. Finally, 250 ~1 of dissociating solution were added and the fluorescence was measured after 1 h in the PE LS-SOB instrument using excitation/ emission wavelengths of 350/614 nm, a delay time of 0.10 ms and a gate time of 1.00 ms. Between each step, the wells were rinsed three times with wash solution. 2.8. Buffer details The coating buffer was 0.05 mol/l carbonate solution, pH 9.5. The dilution buffer was pH 7.4 phosphate buffer saline (PBS, 0.1 mol/l NaCl, and 0.05 mol/l sodium phosphate) containing 0.5% Tween 20. The blocking buffer was the dilution solution but containing in addition 0.1% ovalbumin. The wash solution contained 0.9% NaCl, 0.5% Tween 20. The dissociating solution consisted of 4 mol/l urea, 1% sodium dodecyl
Usually, studies of a new fluorescent label would include the following: (i) molecule design and synthesis of the new compound; (ii) studies on the fluorescence characteristics of free molecules bearing the new label; and (iii) studies of labelling effects and fluorescence characterization of the new probe bound to protein. In Delfia systems, the probe is a combined label system (a non-fluorescent chelate of the Eu3+-EDTA derivative + an enhancement solution containing p-diketone in which highly fluorescent chelate is formed). This is an improvement over the use of direct fluorescent labels but still, nevertheless, conforms to the above plan. Considering the possible enhancing effect of carrier protein, we hypothesized that poor fluorescence of the free probe molecule may not automatically mean poor fluorescence when the probe is conjugated to protein. Accordingly, we first investigated the fluorescence characteristics of labelled protein, and afterwards studied the differences between the free probe and the protein conjugate. Preliminary labelling and characterization studies were carried out using BSA and HSA as model proteins. 3.1. Labelling the protein For protein labelling with chlorosulfonyl-P-diketones, the optimal parameters of protein labelling with BCPDA (Diamandis et al., 1988)
Y-X. Ci et al. /Journal
of Immunological Methods I79 (199s) 233-241
were employed except that organic solvent was used for dissolving the p-diketones. The organic solvents tested included 1,6dioxane, DMSO, and DMF. Of these the solvent DMF was preferred. In all derivatization reactions, the unreacted derivatives were either water soluble (p-ABTA and m-DCMABTA) or could be hydrolyzed into water soluble compounds. Accordingly, both dialysis and gel chromatography on a Sephadex G-25 column could be used successfully for separating the labelled protein from unreacted p-diketones. Using the same molecular ratio of reactants (protein : p-diketone = 1: 1001, the resulting molar ratios of P-diketone to BSA were: 42 (CTTA), 63 (CTABTA), 17 (CMABTA, EDC), 28 (ABTA, EDC), and 18 (CNTA). Apparently the binding of P-diketone to protein through chlorosulfonyl groups is usually more effective (leading to high RM values) and suitable for multiple labelling. In addition, labelling with chlorosulfonyl-P-diketones involves most of the amino groups of the protein if sufficient reagent is present. For instance, the molar ratios of p-diketone : BSA and p-diketone : HSA after maximal labelling were over 60. 3.2. Characteristics of labelled protein Compared with the absorption spectrum of BSA, the spectra of labelled BSA showed two differences. Firstly, absorption intensities below 300 nm were increased. Secondly, a new absorption band resulting from bound /3-diketone appeared with peaks at: 350 nm (BSA-CTTA), 358 nm (BSA-CTAABTA), 320 nm (BSADCMABTA), 320 nm (BSA-ABTA), and 330 nm (BSA-CNTA), respectively. The absorbance at these wavelengths was used to calculate the molar ratios of P-diketone : protein. Compared with the p-diketones incorporated into proteins, the three chlorosulfonyl-P-diketones (CTTA, CTAABTA, and CNTA) almost lost their absorption band at 300-400 nm when hydrolyzed. This result was probably due to the introduction of sulfonyl groups which may introduce an instability in the P-diketone structure in alkaline solution so that part of the P-diketone structure disintegrates during hydrolyzation. Fur-
237
thermore, this result suggested that the chelate of chlorosulfonyl-p-diketones hydrolyzed with europium would not be highly fluorescent. From pH 2-10, the fluorescence of labelled proteins was investigated at a concentration about 7 X 10m8 mol/l (in terms of @diketone) in excess Eu3+ (10P6 mol/l). Studies showed that only CTTA-labelled proteins were highly fluorescent while the fluorescence of proteins labelled with other p-diketones was minimal. The reason for this may be the different interactions between P-diketone and protein. 3.3. Fluorescent characterization of CTTA labelled protein The fluorescent spectrum of CTTA-labelled BSA exhibited two excitation peaks: one was at 285 nm and the other at 350 nm; both lead to the characteristic emission of europium at 614 nm. The optimal conditions for BSA-CTTA to form fluorescent chelate were 10-5-10-6 mol/l of Eu3+, pH 4-6 (in buffer containing phosphate) and pH 8-9. Generally, 2 X 10m6 mol/l Eu3+ and pH 8.2 buffer of 0.1 mol/l Tris-HCl or sodium dicarbonate were adopted. The fluorescent lifetime of labelled BSA was determined to be about 300 ps and could be increased to more than 500 kus using the enhancement conditions described later. For labelled BSA, it could be seen from the 285 nm excitation band that the protein molecule could transfer its energy to Eu3+, suggesting a role for the carrier protein. Accordingly, we compared the fluorescence properties of several CTTA-labelled proteins including BSA-C’ITA, HSA-CTTA, OVA-CTTA, IgG-CTTA, TGCTTA, polylysine-CTTA, and labelled BSA with a lysine bridge (BSA-Lys-CTTA). We found that polylysine-CTTA was characterized by low fluorescence and the fluorescence intensity of BSALys-CCTA was reduced to about l/3 of directly labelled BSA of the same concentration in term of CTTA. The other five proteins (labelled BSA, HSA, OVA, TG, and IgG) were highly fluorescent while their excitation spectra under 300 nm differed from protein to protein. As described above, hydrolyzed CTTA showed
238
Y’S-X. Ci et
‘oooo~
B
10
A
0’
d
~
IXd
$
’
’ ’ 8
1111’ ’
’ “‘1
1x10.’ 1x1o-6
Concentration
of
1 xm5
CTTA
Methods 179 (1995) 233-241
the peptide bonds of the protein molecule could take part in coordinations with Eu3+ and cause the increasement of stability. Another assay showed that the addition of lop5 mol/l of EDTA to the labelled TG system did not quench the fluoresence. The dissociation constant was not studied further. However, the enhancement of stability was very striking. A similar fluorescence enhancement (4-6-fold) has been seen in the BCPDA: Eu3+ labelling system (Diamandis et al., 1989a,b), although the fluorescence enhancement effect was more outstanding here (lOOO-lOOOO-fold) together with a similar degree of enhancement of chelate stability. It is evident that carrier protein is the most important factor for the two enhancement effects described above. For certain ligands such as CTTA, the complexes may enter the protein microphase so that the functional groups and/or the peptide bonds of the protein molecule are able to participate in coordination reactions with Eu3+. In the same time the protein phase provides a hydrophobic environment which prevents quenching and dissociation. We also proposed that this was the reason why the other P-diketones were not fluorescent and why polylysineCTTA and BSA-Lys-CTTA exhibited poor fluorescence (because their structures may not be suitable for forming mixed ligand complexes).
A A
1
0.1 n,J
of Immunological
cl
1000
100
al. /Journal
1x1cr4
(mol/L)
Fig. 1. Fluorescence of Eu3+-CTTA complex in 0.1 M Tris-HCl buffer of pH 8.2. CTTA was either hydrolyzed (in lo-’ M of Eu”+ ) or conjugated to HSA (in 4 x 10m6 M of Eu3+). The concentration of CTTA in the HSA-CTTA conjugate was assessed by absorbance as described in the materials and methods section.
little absorption at 300-400 nm and was not fluorescent. The assay (Fig. 1) showed that under pH 8-9 and in the presence of excess Eu3+ hydrolyzed CTTA merely formed a very poorly fluorescent chelate, and it was clear that there was a several thousand fold enhancement between CTTA bound to HSA (p-CTTA) and hydrolyzed CTTA. It has previously been shown that the association constant of the Eu3’ complexes with thenoyltrifluoroacetone (TTA) is of the order of 106-107. Studies of the dissociation constant of CTTA bound to HSA using the equimolar series method (the concentration of Eu3+ was kept 1.0 x lop6 mol/l> demonstrated that the labelled protein tended to form p-CTTA: Eu3+= 1: 1 complexes and that the association constant of _D-CTTA : Eu3 + was at least in the order of lOlo, very different from the constant of Eu3+ : TTA. We proposed that the functional groups or/and
3.4, Fluorescence
enhancement with an auxiliary reagent and the formation of macro-molecular complexes of CTTA labelled protein
Theoretically, Eu3 + coordinated to C’ITAlabelled protein should not be saturated and auxiliary ligands or micelles (or a combination of the two) can be used to improve the fluorescence of labelled proteins. Several reagents have been used for this purpose including phenanthroline, pyridine, TOPO, Triton X-100, and Tween 20. Experimentally, only TOP0 was found to improve the fluorescence but the addition of 1% Triton X-100 eliminated the enhancement effect of TOPO. The optimal concentration of TOP0 in solution was 1 X lop5 mol/l and the fluorescence intensity was increased to about five times the original.
Y-X. Ci et al. /Journal
of Immunological Methods I79 (1995) 233-241
239
chromatography of labelled HSA after different treatments. After enhancement by the two methods the effluent peaks of HSA-CTTA were eluted earlier than the unenhanced protein. This suggested the formation of new molecular complexes (MC).
3
20
0
Effluent
volume
(tube)
Fig. 2. Gel filtration chromatography of differently treated HSA labelled with CTTA using Sephadex G-100. The column was equilibrated and eluted with 0.9% NaCl at a flow rate of about 0.5 ml/min. The volume of each tube was about 2.5 ml. The four elution curves were derived using identical conditions. (a) labelled HSA, (b) labelled HSA enhanced by heating; (c) mixture of (a) and (b) (2: 1, v/v); (d) labelled HSA enhanced by TOPO.
The formation of stable macromolecular complexes was first reported by Diamandis et al. (1990). These authors obtained a BCPDA-labelled streptavidin-based macromolecular complex (SBMC) following incubation at 50°C for 3 h in the presence of Eu3+. In the present study it was found that CTTA-labelled protein can also form MC and both of the enhancement methods are catalytic. Moreover, the aggregation reaction can be rapidly completed by adding TOP0 to the protein solution. From the work of Diamandis and Christopou10s and our own experiments, it can be concluded that the formation of MC is accompanied by a 2-6-fold fluorescence enhancement when using BCPDA-type probes. The mechanism must, however, be complicated and we postulate that it is probably related to the development of Eu3+ complexes between proteins, which could both prevent fluorescence quenching by water and increase the efficiency of energy transfer (Ci et al., 1989). In general, the complexes were not stable but when the H,O which coordinated with europium was replaced by TOP0 or dehydrogenized by heating, the hydrophobicity between the proteins increased and stable macromolecular complexes were formed. 3.5. CTTA labelling effects on the binding activity
Fluorescence could also be enhanced by heating the solution to 50°C for more than 1 h. This gave a 4-6-fold improvement. However, the two enhancement methods were not additive, following enhancement by one method, the fluorescence of the labelled protein could not be increased by another method. The detection limits of labelled proteins after enhancement were similar and in all cases were about 10-l’ mol/l (in terms of CITA). At the same time the lifetime of the fluorescence increased to 400 ps (heating method) and 520 ps (TOP0 method). Fig. 2 illustrates Sephadex G-100 gel filtration
of protein
It was found that if the labelled BSA was denaturated by heating to 100°C it was no longer fluorescent in excess Eu3+. This result appears to suggest a positive correlation between biological activity and the degree of fluorescence. The effect of labelling with CTTA on the binding activity of HSA antigen was investigated using an immunodiffusion assay. There were no significant differences in the reaction of samples having molar ratios of CTTA : HSA ranging from zero to 60. In assessing the binding activity of the
Y.-X. Ci et al. /Journal
240
of Immunological Methods 179 (1995) 233-241
3.6. Conclusion
-s
_
; ii
Among several P-diketone derivatives synthesized and tested, CTIA was the only one able to form a highly fluorescent chelate with Eu3+ (in excess) when conjugated to protein molecules. A particular characteristic of CITA was that hydrolyzed CTTA was no longer fluorescent. The mechanisms of action of the carrier proteins should be further investigated. Our studies have shown that the new probe is suitable for multiple labelling of proteins which can then be used for developing TR-FIA systems.
70 F-
60 50
\
40
L?
I 303
0
0.5
1
1.5
2
Acknowledgements Concentration
of BSA (~glml)
Fig. 3. Standard curve of BSA in assay using CTTA-labelled BSA.
labelled polyclonal anti-BSA antibody, it was observed that the antibody could tolerate the incorporation of a maximum number of CTTA up to 44 and still retain considerable binding activity. The influence of unreacted CITA on the fluorescence blank of the immunoassay was also tested by either the addition of 10-4-10-5 mol/l hydrolyzed CTTA to the labelled antibody solution or a supplementary incubation of 200 pi/well of hydrolyzed CITA solution (10-4-10-5 mol/l) just before the measurement of dissociation. No differences in fluorescence intensity were observed in comparison with the control experiment and it was concluded that the separation of unreacted CTI’A could be omitted. The competitive immunoassay was performed as described in the materials and methods section. The standard curve obtained is shown in Fig. 3. The calculated limit of detection of BSA in the assay, less than 100 ng/ml, was not very satisfactory. This was because most of the assay conditions were not optimal. In particular the fluorescence dissociation solution was found to be ineffective for CTTA-labelled reagents in later experiments. Nevertheless it will be possible to use CTTA labelled reagents when devising new immunoassays.
We are San Wang tions. This ral Science
grateful to Prof. Suyun Wang and Ms. for kindly providing antibody preparawork was supported by National NatuFoundation of China.
References Chan, M.A., Bellem, A.C. and Diamandis, E.P. (1987) Timeresolved immunofluorometric assay of a-fetoprotein in serum and amniotic fluid with a novel detection system. Clin. Chem. 33, 2000-2003. Christopoulos, T.K. and Diamandis, E.P. (1990) Ultrasensitive time-resolved fluorescence method for cY-fetoprotein. Clin. Chem. 36, 1497-1502. Ci, Y.-X. and Lan, Z.-H. (19881 Fluorescence enhancement of the europium(III)-thenoyltrifluoro-acetone-trioctylphosphine oxide ternary complex by gadolinium(II1) and its application to the determination of europium(II1). Analyst 113, 1453-1457. Ci, Y.-X. and Lan, Z.-H. (1989) Fluorometric determination of samarium and gadolinium by enhancement of fluorescence of samarium-thenoyltrifluoroacetone-l,lO-phenanthroline ternary complex by cadolinium. Anal. Chem. 61, 1069-1072. Ci, Y.-X. and Yang, X.-D. (1992) Labelling of proteins with 5-chlorosulfonyl-2-thenoyltrifluoroacetone (CTTA) and application to immunoassay. Chin. Chem. Lett. 12, 10071010. Diamandis, E.P. (1988) Immunoassays with time-resolved fluorescence spectroscopy: principles and applications [review]. Clin. Biochem. 21, 139-150. Diamandis, E.P. and Christopoulos, T.K. (1990) Europium chelate labels in TR-FIA and DNA hybridization assays [review]. Anal. Chem. 62, 1149A-1157A.
Y-X
Ci et al. /Journal
of Immunological Methods I79 (1995) 233-241
Diamandis, E.P. and Christopoulos, T.K. (1991) The biotin(streptlavidin system: principles and applications in biotechnology [review]. Clin. Chem. 37, 625-636. Diamandis. E.P. and Morton, R.C. (1988) Time-resolved fluorescence using a europium chelate of 4,7-bisfchlorosulfophenyl)-l,l0-phenanthroline-2,9-dicarboxylic acid (BCPDA). Labelling procedures and application in immunoassays. J. Immunol. Methods 112, 43-512. Diamandis, E.P., Morton, R.C., Reichstein, E. and Khosravi, M.J. (1989a) Multiple fluorescence labelling with europium chelators. Application to time-resolved immunoassay. Anal. Chem. 61. 48-53. Diamandis, E.P.. Papanastasiou-Diamandi, A., Lustig, V., Khosravi, M.J. and Tan, A. (1989b) Time-resolved immunofluorometric assay of human pancreatic isomylase in serum with use of two monoclonal antibody. Clin. Chem. 35, 1915-1920. Hemmila, I. (1985) Fluoroimmunoassays and immunofluorometric assays [review]. Clin. Chem. 31, 359-370. Hemmila, I. (1989) Fluorescent labels for use in fluorescence immunoassay [review]. Appl. Fluor. Technol. 1, l-8. Hemmila, I.. Dakubu, S., Mukkala, V.-M., Sitari, H. and Lovgren, T. (1984) Europium as a label in time-resolved immunofluorometric assays. Anal. Biochem. 137,335-343. Kakabakos, SE., Christopoulos, T.K. and Diamandis, E.P. (1992) Multianalyte immunoassay based on spatially distinct fluorescent areas quantified by laser-excited solidphase time-resolved fluorometry. Clin. Chem. 38,338-342. Khosravi, M.J. and Diamandis, E.P. (1987) Immunofluorome-
241
try of choriogonadotropin by time-resolved fluorescence spectroscopy. Clin. Chem. 33, 1994-1999. Khosravi, M.J., Chan, M.A., Bellem, A.C. and Diamandis, E.P. (1988) A sensitive time-resolved immunofluorometric assay of ferritin in serum with monoclonal antibody. Clin. Chim. Acta 175, 267-275. Kropf. J. and Gressner, A.M. (1991) Time-resolved immunofluorometric assays with measurement of a europium chelate in solution: application for sensitive determination of fibronectin. Anal. Biochem. 197, 258-265. Morton, R.C. and Diamandis, E.P. (19901 Streptavidin-based macromolecular complex labelled with a europium chelator suitable for time-resolved fluorescence immunoassay applications. Anal. Chem. 62, 1841-1845. Soini, E. (19901 Biospecific assays with time-resolved fluorescence detection [review]. Trends Anal. Chem. 9, 90-93. Soini, E. and Kojola, H. (1983) Time-resolved fluorometer for lanthanide chelates - A new generation of nonisotopic immunoassays. Clin. Chem. 29, 65-68. Soini, E. and Lovgren, T. (1987) Time-resolved fluorescence of lanthanide probes and applications in biotechnology [review]. CRC Crit. Rev. Anal. Chem. 18, 105-154. Suonpla, M.H. et al. (19921 Europium-labelled streptavidin as a highly sensitive universal label. Indirect time-resolved immunofluorometry of FSH and TSH. J. Immunol. Methods 149, 247-253. Wang, M.-C. (1993) Syntheses of new lanthanide chelating reagents for fluoroimmunoassay. M.D. Thesis, Chem. Dept. Peking Univ.
ELSEVIER
Journal
of
Immunological Methods 179 (1995) 233-241
Fluorescence labelling with europium chelate of P-diketones and application in time-resolved fluoroimmunoassays (TR-FIA) Yun-Xiang Ci
*, Xiao-Da
Yang, Wen-Bao Chang
Department of Chemistry, Peking University, Be&g
100871, People’s Republic of China
Received 8 February 1994, revised received 15 July 1994,accepted 31 October 1994
Abstract Five P-diketone derivatives were studied for multiple labelling of proteins. The labelled proteins were characterized by absorption and fluorescence measurements. It was found that proteins labelled with chlorosulfonylthenoyltrifluoroacetone (CTTA) were able to form highly fluorescent complexes with Eu3’ which exhibited prolonged fluorescence whereas the Eu3+ complex of hydrolyzed CTTA exhibited almost no fluorescence, and so unreacted ligand gave no background signal in immunoassays even if it was not removed from the labelled reagent. The effect of labelling on the biological activity of albumin and polyclonal antibody was studied and it was also shown that the new probe could be used in time-resolved fluorescence immunoassays. Keywords:
P-Diketone;
Multiple labelling; Time-resolved
1. Introduction Since the development of the time-resolved fluorescence immunoassay, considerable progress has been made in this field. In conventional FIA methods background fluorescence is a major problem, but this can be virtually eliminated by
Abbreviations: CTTA, 5-chlorosulfonyl-2-thenoyltrifluoroacetone; TTA, thenoyltrifluoroacetone; TAABTA, trifluoroacetylaminohenzoyltrifluoroacetone; DMABTA, N,N.dicarboxymethylaminobenzoyltrifluoroacetone; ABTA, aminobenzoyltrifluoroacetone; NTA, naphthoyltrifluoroacetone; EDC, 1-ethyl-3(3-dimethylamino-propyl)carbodiimide: TR-FIA, time-resolved fluoroimmunoassay; MC, macromolecule complex; Eu, europium; TOPO, trioctylphosphine oxide. * Corresponding
author.
0022-1759/95/$09.50 0 1995 Elsevier XSDI 0022..1759(94)00289-4
Science
fluoroimmunoassay
using the fluorescent lanthanide chelates as labels in combination with time-resolved fluorometry. Many authors (Diamandis and Christopoulos, 1990; Hemmilti, 1989; Soini, 1990; Soini and Lovgren, 1987) have reviewed the approaches using europium and other lanthanide chelate labels in time-resolved fluorescence immunoassays and it has been reported that some sensitive assays, such as the assay for cu-fetoprotein, are able to detect approximately 300000 molecules per sample (Diamandis and Christopoulos, 1990). Among several methodologies for time-resolved fluorescence assays, two are generally regarded as very successful. In the Delfia system (Soini and Kojola, 1983; Soini and Lovgren, 1987; Soini, 19901, an ethylenediaminetetraacetic acid (EDTA) derivative is used to bind Eu’+ to the
B.V. All rights reserved
234
Y.-X. Ci et al. /Journal
of Immunological Methods 179 (1995) 233-241
immunoreactants and Eu3+ is released with an enhancement solution for measurement under optimum conditions. The second method, which was popularized by Diamandis and his co-workers (Diamandis et al., 1989a,b; Diamandis and Morton, 1989; Diamandis and Christopoulos, 1990), uses an organic chelating ligand (BCPDA) as the label. Using this method, the amount of ligand is quantified in the presence of excess Eu3+ either directly on the surface of dried microtiter plates or in solution analogously to the Delfia method after the dissociation of the labelled solid phase component (Kropf and Gressner, 1991). The relative merits and limitations of these two methodologies have been discussed by Diamandis (Diamandis, 1988). One advantage of the second method is that the problem of exogenous contamination for Eu3+ in the environment and on skin surfaces can be eliminated. This is especially significant for those assays performed on samples in which fluorescent lanthanide ions are abundant. For this reason, the Diamandis method may be practically superior to the Delfia system. Usually, an Eu 3+ chelating ligand suitable for use in the second method should form relatively stable complexes with Eu3+, be highly fluorescent, have an excitation wavelength preferably close to 337 nm (the emission wavelength of a nitrogen laser), react covalently with the protein of interest but interact minimally with serum components (Diamandis and Morton, 1988). Furthermore the ligand should ideally be cheap and plentiful. Although some ligands meet the first five requirements, few satisfy the last one. p-diketones are generally relatively inexpensive reagents with excellent fluorescence properties when chelated. However, they have proved unsatisfactory as direct fluorescent probes because of the severe problem of quenching and very low thermodynamic stability (Hemmila, 1985). We have speculated that some @-diketones could be used as BCPDA-type probes because in the second method the requirement for thermodynamic stability could be compensated by increasing the concentration of Eu3+ and the incorporation of the probes into protein molecules could yield a ‘bonus’ effect (Diamandis et al., 1989a,b). Recently we described the first success-
ful p-diketone ligand: 5chlorosulfonylthenoyltrifluoroacetone (CTTA) (Ci and Yang, 1992). In this paper, the labelling of protein with five @diketone derivatives is described. Of these CTTA was found to be the only one which was suitable for multiple labelling and CTTA-labelled reagents were tested in the development of a solid-phase immunoassay.
2. Materials and methods 2.1. Instrumentation Fluorescence measurement was performed using a Hitachi M850 and an LS-SOB fluorospectrophotometer. The sample absorption was measured on a Beckman DU-7 spectrophotometer. 2.2. Materials Human serum albumin (HSA) and poly-lysine was purchased from Sigma Chemical Company. Bovine serum albumin (BSA) was obtained from the Beijing Biochemical Plant (China). Polyclonal antibodies (anti-BSA) and antiserum were kindly provided by the Biology Department of Peking University. Sephadex G-25 and G-100 were purchased from the Shanghai Biochemical Plant (China). Trifluoroacetylaminobenzoyltrifluoroacetone (TAABTA), N,N,-dicarboxymethylaminobenzoyltrifluoro-acetone (DMABTA), aminobenzoyl-trifluoroacetone (ABTA), and naphthoyltrifluoro-acetone (NTA) were prepared in our laboratory (Wang, 1993). 96-well microtiter plates for immunoassays were obtained from the Yuhuan Plastic Plant of Zhejiang (China). 2.3. Synthesis of chlorosulfonyl-P-diketone derivatives
The chlorosulfonyl group was introduced into the TTA, TAABTA, and NTA structures through direct chlorosulfonation of the P-diketone: @diketone + 2ClS0,H -
ClSO,-P-diketone
+ HCl
Y-X. Ci et al./Journal of ImmunologicalMethods 179 (1995) 233-241
p-diketone and CISO,H were mixed in the ratio 5 : 1 and stirred whilst heating to 50-60°C. This temperature was maintained until the reaction was complete. The excess ClSO,H was degraded and separated by pouring the reaction mixture into ice cold water. The products were then recrystallized in anhydrous chloroform. The spectra of the products showed that their structures were as follows: ClSO, Lrj
COCH ,COCF,
235
in a small volume of dimethylsulfoxide (DMSO) and reacted for 1 h before the mixture was added to the protein solution. The derivatization reaction was allowed to proceed for 1 h at room temperature. After the derivatization reaction, the labelled proteins were isolated by applying the reaction mixture to a column of Sephadex G-25 (1.0 x 25 cm> which was equilibrated and eluted with 0.9% NaCl solution (1 ml/min). The effluent was monitored by measuring absorbance values at both 280 nm and 350 nm.
CITA COCH,COCF,
0 p ClSO,
NHCOCF,
CTAABTA
00 W ClSO,
COCH ,COCF,
CNTA 2.4. Labelling of proteins with P-diketone derivatives and isolation of labelled proteins In order to label the proteins with chlorosulfonyl-@diketones, they were dissolved in 0.1 mol/l carbonate buffer at a concentration of about 2-5 mg/ml and then a freshly prepared solution of P-diketone derivatives in dimethylformide (DMF) was added to the protein solution. The amount of P-diketone derivative varied according to the desired molar ratio of P-diketone/ protein of the products. The reactions were complete after 1 h at room temperature. Proteins were derivatized with p-aminobenzoyltrifluoroacetone and m-l\r,N-dicarboxymethylaminobenzoyltrifluoroacetone using lethyl-3(3-dimethylamino-propylj-carbodiimide (EDC). EDC and the @diketone were dissolved
2.5, Characterization of labelled protein by absorbance and fluorescence The absorption spectra of labelled proteins were obtained at their stock concentration (50100 pg/ml) with a Beckman DU-7 spectrophotometer. The molar ratio (RM) of p-diketone to protein was obtained after dividing the concentration by the molar concentration of the protein. The concentration of p-diketone bound to protein was calculated through the maximum absorbance (at 320-380 nm) and the molar absorption coefficient (E) of free p-diketones was adopted (for each chlorosulfonyl-@diketone, the E was that of the original molecule). The fluorescence spectra were measured with a Hitachi M8.50 using 1 pg/ml of labelled protein, 1 x 10e5 mol/l of Eu3+, and 0.1 mol/l of pH 8.2 Tris buffer. The fluorescence half life values were measured on the PE LS-SOB equipment using the same conditions. The fluorescence properties of labelled proteins were studied by fluorescence measurements of solutions which differed in concentration, pH, buffer, auxiliary ligands, and concentration of Eu3+. Chlorosulfonyl-P-diketones were hydrolyzed by incubating a weighed amount in a 0.1 mol/l carbonate buffer pH 9.5 at room temperature for 1 day. Then the solutions were diluted approximately to cover the range from 10eh to lo-’ mol/l, the solution buffer was 0.1 mol/l Tris-HCl buffer pH 8.2, containing 1 X lop5 mol/l of Eu’+, and the fluorescence was measured with the Hitachi M850.
236
Y-X. Ci et al. /Journal
of Immunological Methods I79 (1995) 233-241
2.6. Preparation and isolation of macromolecular complexes of CTTA-labelled protein
sulfate (SDS), 2 X lop6 mol/l Eu3+, and 1 X lop5 mol/l TOPO.
The macromolecular complexes were prepared by either heating the protein solution (pH 8.2, 10-6-10-5 mol/l of Eu3+, and 100-1000 ng/ml of CTTA-labelled protein) to 50°C for 2 h (the heating method) or adding lop5 mol/l of trioctylphosphine oxide (TOP01 to the solution (the TOP0 method). The macromolecular complexes were separated by applying the solution to a Sephadex G-100 column (1.0 X 20 cm> which was equilibrated and eluted with 0.15 mol/l NaCl solution, and the effluent was monitored by fluorescence measurements at excitation/ emission wavelengths (ex/em> of 350/614 nm.
2.9. Immunoassay procedure The wells were coated overnight at 4°C with 200 ~1 of anti-BSA serum (l/20 diluted with coating buffer). After the wells were blocked, 100 ~1 of BSA standards and 100 ~1 of 4.0 pg/ml CTTA-labelled BSA (RM = 30) were added and incubated at 37°C for 2 h. Finally, 250 ~1 of dissociating solution were added and 1 h later, fluorescence was measured using the LS-SOB instrument under the above conditions.
3. Results and discussion 2.7, Assessment of biological activity of CTTA labelled antibody Rabbit polyclonal anti-BSA antibodies were labelled with C’ITA and purified by the method described above. The wells of a microtiter plate were coated overnight at 4°C with 100 ~1 of a 5 pg/ml BSA solution in coating buffer and then blocked for 2 h at 37°C with 200 ~1 blocking buffer. After blocking, 100 ~1 of a 100 pg/ml solution of labelled antibody (differing in RM value) were added and incubated for 2 h at 37°C. Finally, 250 ~1 of dissociating solution were added and the fluorescence was measured after 1 h in the PE LS-SOB instrument using excitation/ emission wavelengths of 350/614 nm, a delay time of 0.10 ms and a gate time of 1.00 ms. Between each step, the wells were rinsed three times with wash solution. 2.8. Buffer details The coating buffer was 0.05 mol/l carbonate solution, pH 9.5. The dilution buffer was pH 7.4 phosphate buffer saline (PBS, 0.1 mol/l NaCl, and 0.05 mol/l sodium phosphate) containing 0.5% Tween 20. The blocking buffer was the dilution solution but containing in addition 0.1% ovalbumin. The wash solution contained 0.9% NaCl, 0.5% Tween 20. The dissociating solution consisted of 4 mol/l urea, 1% sodium dodecyl
Usually, studies of a new fluorescent label would include the following: (i) molecule design and synthesis of the new compound; (ii) studies on the fluorescence characteristics of free molecules bearing the new label; and (iii) studies of labelling effects and fluorescence characterization of the new probe bound to protein. In Delfia systems, the probe is a combined label system (a non-fluorescent chelate of the Eu3+-EDTA derivative + an enhancement solution containing p-diketone in which highly fluorescent chelate is formed). This is an improvement over the use of direct fluorescent labels but still, nevertheless, conforms to the above plan. Considering the possible enhancing effect of carrier protein, we hypothesized that poor fluorescence of the free probe molecule may not automatically mean poor fluorescence when the probe is conjugated to protein. Accordingly, we first investigated the fluorescence characteristics of labelled protein, and afterwards studied the differences between the free probe and the protein conjugate. Preliminary labelling and characterization studies were carried out using BSA and HSA as model proteins. 3.1. Labelling the protein For protein labelling with chlorosulfonyl-P-diketones, the optimal parameters of protein labelling with BCPDA (Diamandis et al., 1988)
Y-X. Ci et al. /Journal
of Immunological Methods I79 (199s) 233-241
were employed except that organic solvent was used for dissolving the p-diketones. The organic solvents tested included 1,6dioxane, DMSO, and DMF. Of these the solvent DMF was preferred. In all derivatization reactions, the unreacted derivatives were either water soluble (p-ABTA and m-DCMABTA) or could be hydrolyzed into water soluble compounds. Accordingly, both dialysis and gel chromatography on a Sephadex G-25 column could be used successfully for separating the labelled protein from unreacted p-diketones. Using the same molecular ratio of reactants (protein : p-diketone = 1: 1001, the resulting molar ratios of P-diketone to BSA were: 42 (CTTA), 63 (CTABTA), 17 (CMABTA, EDC), 28 (ABTA, EDC), and 18 (CNTA). Apparently the binding of P-diketone to protein through chlorosulfonyl groups is usually more effective (leading to high RM values) and suitable for multiple labelling. In addition, labelling with chlorosulfonyl-P-diketones involves most of the amino groups of the protein if sufficient reagent is present. For instance, the molar ratios of p-diketone : BSA and p-diketone : HSA after maximal labelling were over 60. 3.2. Characteristics of labelled protein Compared with the absorption spectrum of BSA, the spectra of labelled BSA showed two differences. Firstly, absorption intensities below 300 nm were increased. Secondly, a new absorption band resulting from bound /3-diketone appeared with peaks at: 350 nm (BSA-CTTA), 358 nm (BSA-CTAABTA), 320 nm (BSADCMABTA), 320 nm (BSA-ABTA), and 330 nm (BSA-CNTA), respectively. The absorbance at these wavelengths was used to calculate the molar ratios of P-diketone : protein. Compared with the p-diketones incorporated into proteins, the three chlorosulfonyl-P-diketones (CTTA, CTAABTA, and CNTA) almost lost their absorption band at 300-400 nm when hydrolyzed. This result was probably due to the introduction of sulfonyl groups which may introduce an instability in the P-diketone structure in alkaline solution so that part of the P-diketone structure disintegrates during hydrolyzation. Fur-
237
thermore, this result suggested that the chelate of chlorosulfonyl-p-diketones hydrolyzed with europium would not be highly fluorescent. From pH 2-10, the fluorescence of labelled proteins was investigated at a concentration about 7 X 10m8 mol/l (in terms of @diketone) in excess Eu3+ (10P6 mol/l). Studies showed that only CTTA-labelled proteins were highly fluorescent while the fluorescence of proteins labelled with other p-diketones was minimal. The reason for this may be the different interactions between P-diketone and protein. 3.3. Fluorescent characterization of CTTA labelled protein The fluorescent spectrum of CTTA-labelled BSA exhibited two excitation peaks: one was at 285 nm and the other at 350 nm; both lead to the characteristic emission of europium at 614 nm. The optimal conditions for BSA-CTTA to form fluorescent chelate were 10-5-10-6 mol/l of Eu3+, pH 4-6 (in buffer containing phosphate) and pH 8-9. Generally, 2 X 10m6 mol/l Eu3+ and pH 8.2 buffer of 0.1 mol/l Tris-HCl or sodium dicarbonate were adopted. The fluorescent lifetime of labelled BSA was determined to be about 300 ps and could be increased to more than 500 kus using the enhancement conditions described later. For labelled BSA, it could be seen from the 285 nm excitation band that the protein molecule could transfer its energy to Eu3+, suggesting a role for the carrier protein. Accordingly, we compared the fluorescence properties of several CTTA-labelled proteins including BSA-C’ITA, HSA-CTTA, OVA-CTTA, IgG-CTTA, TGCTTA, polylysine-CTTA, and labelled BSA with a lysine bridge (BSA-Lys-CTTA). We found that polylysine-CTTA was characterized by low fluorescence and the fluorescence intensity of BSALys-CCTA was reduced to about l/3 of directly labelled BSA of the same concentration in term of CTTA. The other five proteins (labelled BSA, HSA, OVA, TG, and IgG) were highly fluorescent while their excitation spectra under 300 nm differed from protein to protein. As described above, hydrolyzed CTTA showed
238
Y’S-X. Ci et
‘oooo~
B
10
A
0’
d
~
IXd
$
’
’ ’ 8
1111’ ’
’ “‘1
1x10.’ 1x1o-6
Concentration
of
1 xm5
CTTA
Methods 179 (1995) 233-241
the peptide bonds of the protein molecule could take part in coordinations with Eu3+ and cause the increasement of stability. Another assay showed that the addition of lop5 mol/l of EDTA to the labelled TG system did not quench the fluoresence. The dissociation constant was not studied further. However, the enhancement of stability was very striking. A similar fluorescence enhancement (4-6-fold) has been seen in the BCPDA: Eu3+ labelling system (Diamandis et al., 1989a,b), although the fluorescence enhancement effect was more outstanding here (lOOO-lOOOO-fold) together with a similar degree of enhancement of chelate stability. It is evident that carrier protein is the most important factor for the two enhancement effects described above. For certain ligands such as CTTA, the complexes may enter the protein microphase so that the functional groups and/or the peptide bonds of the protein molecule are able to participate in coordination reactions with Eu3+. In the same time the protein phase provides a hydrophobic environment which prevents quenching and dissociation. We also proposed that this was the reason why the other P-diketones were not fluorescent and why polylysineCTTA and BSA-Lys-CTTA exhibited poor fluorescence (because their structures may not be suitable for forming mixed ligand complexes).
A A
1
0.1 n,J
of Immunological
cl
1000
100
al. /Journal
1x1cr4
(mol/L)
Fig. 1. Fluorescence of Eu3+-CTTA complex in 0.1 M Tris-HCl buffer of pH 8.2. CTTA was either hydrolyzed (in lo-’ M of Eu”+ ) or conjugated to HSA (in 4 x 10m6 M of Eu3+). The concentration of CTTA in the HSA-CTTA conjugate was assessed by absorbance as described in the materials and methods section.
little absorption at 300-400 nm and was not fluorescent. The assay (Fig. 1) showed that under pH 8-9 and in the presence of excess Eu3+ hydrolyzed CTTA merely formed a very poorly fluorescent chelate, and it was clear that there was a several thousand fold enhancement between CTTA bound to HSA (p-CTTA) and hydrolyzed CTTA. It has previously been shown that the association constant of the Eu3’ complexes with thenoyltrifluoroacetone (TTA) is of the order of 106-107. Studies of the dissociation constant of CTTA bound to HSA using the equimolar series method (the concentration of Eu3+ was kept 1.0 x lop6 mol/l> demonstrated that the labelled protein tended to form p-CTTA: Eu3+= 1: 1 complexes and that the association constant of _D-CTTA : Eu3 + was at least in the order of lOlo, very different from the constant of Eu3+ : TTA. We proposed that the functional groups or/and
3.4, Fluorescence
enhancement with an auxiliary reagent and the formation of macro-molecular complexes of CTTA labelled protein
Theoretically, Eu3 + coordinated to C’ITAlabelled protein should not be saturated and auxiliary ligands or micelles (or a combination of the two) can be used to improve the fluorescence of labelled proteins. Several reagents have been used for this purpose including phenanthroline, pyridine, TOPO, Triton X-100, and Tween 20. Experimentally, only TOP0 was found to improve the fluorescence but the addition of 1% Triton X-100 eliminated the enhancement effect of TOPO. The optimal concentration of TOP0 in solution was 1 X lop5 mol/l and the fluorescence intensity was increased to about five times the original.
Y-X. Ci et al. /Journal
of Immunological Methods I79 (1995) 233-241
239
chromatography of labelled HSA after different treatments. After enhancement by the two methods the effluent peaks of HSA-CTTA were eluted earlier than the unenhanced protein. This suggested the formation of new molecular complexes (MC).
3
20
0
Effluent
volume
(tube)
Fig. 2. Gel filtration chromatography of differently treated HSA labelled with CTTA using Sephadex G-100. The column was equilibrated and eluted with 0.9% NaCl at a flow rate of about 0.5 ml/min. The volume of each tube was about 2.5 ml. The four elution curves were derived using identical conditions. (a) labelled HSA, (b) labelled HSA enhanced by heating; (c) mixture of (a) and (b) (2: 1, v/v); (d) labelled HSA enhanced by TOPO.
The formation of stable macromolecular complexes was first reported by Diamandis et al. (1990). These authors obtained a BCPDA-labelled streptavidin-based macromolecular complex (SBMC) following incubation at 50°C for 3 h in the presence of Eu3+. In the present study it was found that CTTA-labelled protein can also form MC and both of the enhancement methods are catalytic. Moreover, the aggregation reaction can be rapidly completed by adding TOP0 to the protein solution. From the work of Diamandis and Christopou10s and our own experiments, it can be concluded that the formation of MC is accompanied by a 2-6-fold fluorescence enhancement when using BCPDA-type probes. The mechanism must, however, be complicated and we postulate that it is probably related to the development of Eu3+ complexes between proteins, which could both prevent fluorescence quenching by water and increase the efficiency of energy transfer (Ci et al., 1989). In general, the complexes were not stable but when the H,O which coordinated with europium was replaced by TOP0 or dehydrogenized by heating, the hydrophobicity between the proteins increased and stable macromolecular complexes were formed. 3.5. CTTA labelling effects on the binding activity
Fluorescence could also be enhanced by heating the solution to 50°C for more than 1 h. This gave a 4-6-fold improvement. However, the two enhancement methods were not additive, following enhancement by one method, the fluorescence of the labelled protein could not be increased by another method. The detection limits of labelled proteins after enhancement were similar and in all cases were about 10-l’ mol/l (in terms of CITA). At the same time the lifetime of the fluorescence increased to 400 ps (heating method) and 520 ps (TOP0 method). Fig. 2 illustrates Sephadex G-100 gel filtration
of protein
It was found that if the labelled BSA was denaturated by heating to 100°C it was no longer fluorescent in excess Eu3+. This result appears to suggest a positive correlation between biological activity and the degree of fluorescence. The effect of labelling with CTTA on the binding activity of HSA antigen was investigated using an immunodiffusion assay. There were no significant differences in the reaction of samples having molar ratios of CTTA : HSA ranging from zero to 60. In assessing the binding activity of the
Y.-X. Ci et al. /Journal
240
of Immunological Methods 179 (1995) 233-241
3.6. Conclusion
-s
_
; ii
Among several P-diketone derivatives synthesized and tested, CTIA was the only one able to form a highly fluorescent chelate with Eu3+ (in excess) when conjugated to protein molecules. A particular characteristic of CITA was that hydrolyzed CTTA was no longer fluorescent. The mechanisms of action of the carrier proteins should be further investigated. Our studies have shown that the new probe is suitable for multiple labelling of proteins which can then be used for developing TR-FIA systems.
70 F-
60 50
\
40
L?
I 303
0
0.5
1
1.5
2
Acknowledgements Concentration
of BSA (~glml)
Fig. 3. Standard curve of BSA in assay using CTTA-labelled BSA.
labelled polyclonal anti-BSA antibody, it was observed that the antibody could tolerate the incorporation of a maximum number of CTTA up to 44 and still retain considerable binding activity. The influence of unreacted CITA on the fluorescence blank of the immunoassay was also tested by either the addition of 10-4-10-5 mol/l hydrolyzed CTTA to the labelled antibody solution or a supplementary incubation of 200 pi/well of hydrolyzed CITA solution (10-4-10-5 mol/l) just before the measurement of dissociation. No differences in fluorescence intensity were observed in comparison with the control experiment and it was concluded that the separation of unreacted CTI’A could be omitted. The competitive immunoassay was performed as described in the materials and methods section. The standard curve obtained is shown in Fig. 3. The calculated limit of detection of BSA in the assay, less than 100 ng/ml, was not very satisfactory. This was because most of the assay conditions were not optimal. In particular the fluorescence dissociation solution was found to be ineffective for CTTA-labelled reagents in later experiments. Nevertheless it will be possible to use CTTA labelled reagents when devising new immunoassays.
We are San Wang tions. This ral Science
grateful to Prof. Suyun Wang and Ms. for kindly providing antibody preparawork was supported by National NatuFoundation of China.
References Chan, M.A., Bellem, A.C. and Diamandis, E.P. (1987) Timeresolved immunofluorometric assay of a-fetoprotein in serum and amniotic fluid with a novel detection system. Clin. Chem. 33, 2000-2003. Christopoulos, T.K. and Diamandis, E.P. (1990) Ultrasensitive time-resolved fluorescence method for cY-fetoprotein. Clin. Chem. 36, 1497-1502. Ci, Y.-X. and Lan, Z.-H. (19881 Fluorescence enhancement of the europium(III)-thenoyltrifluoro-acetone-trioctylphosphine oxide ternary complex by gadolinium(II1) and its application to the determination of europium(II1). Analyst 113, 1453-1457. Ci, Y.-X. and Lan, Z.-H. (1989) Fluorometric determination of samarium and gadolinium by enhancement of fluorescence of samarium-thenoyltrifluoroacetone-l,lO-phenanthroline ternary complex by cadolinium. Anal. Chem. 61, 1069-1072. Ci, Y.-X. and Yang, X.-D. (1992) Labelling of proteins with 5-chlorosulfonyl-2-thenoyltrifluoroacetone (CTTA) and application to immunoassay. Chin. Chem. Lett. 12, 10071010. Diamandis, E.P. (1988) Immunoassays with time-resolved fluorescence spectroscopy: principles and applications [review]. Clin. Biochem. 21, 139-150. Diamandis, E.P. and Christopoulos, T.K. (1990) Europium chelate labels in TR-FIA and DNA hybridization assays [review]. Anal. Chem. 62, 1149A-1157A.
Y-X
Ci et al. /Journal
of Immunological Methods I79 (1995) 233-241
Diamandis, E.P. and Christopoulos, T.K. (1991) The biotin(streptlavidin system: principles and applications in biotechnology [review]. Clin. Chem. 37, 625-636. Diamandis. E.P. and Morton, R.C. (1988) Time-resolved fluorescence using a europium chelate of 4,7-bisfchlorosulfophenyl)-l,l0-phenanthroline-2,9-dicarboxylic acid (BCPDA). Labelling procedures and application in immunoassays. J. Immunol. Methods 112, 43-512. Diamandis, E.P., Morton, R.C., Reichstein, E. and Khosravi, M.J. (1989a) Multiple fluorescence labelling with europium chelators. Application to time-resolved immunoassay. Anal. Chem. 61. 48-53. Diamandis, E.P.. Papanastasiou-Diamandi, A., Lustig, V., Khosravi, M.J. and Tan, A. (1989b) Time-resolved immunofluorometric assay of human pancreatic isomylase in serum with use of two monoclonal antibody. Clin. Chem. 35, 1915-1920. Hemmila, I. (1985) Fluoroimmunoassays and immunofluorometric assays [review]. Clin. Chem. 31, 359-370. Hemmila, I. (1989) Fluorescent labels for use in fluorescence immunoassay [review]. Appl. Fluor. Technol. 1, l-8. Hemmila, I.. Dakubu, S., Mukkala, V.-M., Sitari, H. and Lovgren, T. (1984) Europium as a label in time-resolved immunofluorometric assays. Anal. Biochem. 137,335-343. Kakabakos, SE., Christopoulos, T.K. and Diamandis, E.P. (1992) Multianalyte immunoassay based on spatially distinct fluorescent areas quantified by laser-excited solidphase time-resolved fluorometry. Clin. Chem. 38,338-342. Khosravi, M.J. and Diamandis, E.P. (1987) Immunofluorome-
241
try of choriogonadotropin by time-resolved fluorescence spectroscopy. Clin. Chem. 33, 1994-1999. Khosravi, M.J., Chan, M.A., Bellem, A.C. and Diamandis, E.P. (1988) A sensitive time-resolved immunofluorometric assay of ferritin in serum with monoclonal antibody. Clin. Chim. Acta 175, 267-275. Kropf. J. and Gressner, A.M. (1991) Time-resolved immunofluorometric assays with measurement of a europium chelate in solution: application for sensitive determination of fibronectin. Anal. Biochem. 197, 258-265. Morton, R.C. and Diamandis, E.P. (19901 Streptavidin-based macromolecular complex labelled with a europium chelator suitable for time-resolved fluorescence immunoassay applications. Anal. Chem. 62, 1841-1845. Soini, E. (19901 Biospecific assays with time-resolved fluorescence detection [review]. Trends Anal. Chem. 9, 90-93. Soini, E. and Kojola, H. (1983) Time-resolved fluorometer for lanthanide chelates - A new generation of nonisotopic immunoassays. Clin. Chem. 29, 65-68. Soini, E. and Lovgren, T. (1987) Time-resolved fluorescence of lanthanide probes and applications in biotechnology [review]. CRC Crit. Rev. Anal. Chem. 18, 105-154. Suonpla, M.H. et al. (19921 Europium-labelled streptavidin as a highly sensitive universal label. Indirect time-resolved immunofluorometry of FSH and TSH. J. Immunol. Methods 149, 247-253. Wang, M.-C. (1993) Syntheses of new lanthanide chelating reagents for fluoroimmunoassay. M.D. Thesis, Chem. Dept. Peking Univ.