ANALYST. APRIL 1989, VOL. 114 529 Terbium Chelate Labels for Fluorescence lmmunoassays Jerzy Siepak Department of Instrumental Analysis, A. Mickiewicz University, 60-780 Poznan, Poland Bovine serum albumin was labelled with a terbium complex by means of a reagent prepared from d i et h y I e net r i a m i n e pen ta a cet i c acid o r d i et h y I en et r i a m i n e pe n t a p h 0s p h o n ic a ci d a n d p-a m i n osa I icy I ic acid . The ternary mixed ligand complexes showed strong fluorescence and were stable in very dilute solution (10-10 M). The fluorescence of the conjugated products was compared with regard to their application in the fluoroimmunoassay of bovine serum albumin. Keywords: Fluoroimmunoassa y; terbium chelates; carboxylic and phosphonic complexones; bovine serum albumin In the immunoassay field the use of lanthanides is of growing interest.1 Some lanthanides, especially europium, terbium, erbium. samarium and gadolinium, form highly fluorescent chelates with many different organic ligands."." Lanthanide chelates with complexones, used as reagents in the fluorescent labelling of proteins, are widely applied in histochemistry3 and immunochemistry.5 Complexes of lanthanides with complex- ones have high stability and solubility in water, and the formation ccinstants with lanthanides are > 10'5.&X Bailey et used terbium chelates with diethylenetriaminepentaacetic acid (DTPA) and p-aminosalicylic acid @-AS) and human serum albumin (HSA) for determining nanogram amounts of HSA. We describe here the application of the terbium complexes of DTPA and diethylenetriaminepentaphosphonic acid (DTPP) with p-AS to the simple fluoroimmunoassay of bovine serum albumin (BSA). Experimental Apparatus The corrected fluorescence spectra were recorded using a Perkin-Elmer MPF-3 spectrofluorimeter with a xenon lamp as the radiation source. The excitation wavelength was 328 nm for TbI" and the emission was monitored at 545 nm.All measurements were carried out in 1 X 1 cm square quartz fluorescence cells. Reagents DTPA and DTPP were prepared as described previously.9310 Dimethyl sulphoxide (DMSO), triethylamine and p-AS sodium salt were obtained from Sigma (Poole, Dorset). Terbium chloride was prepared by dissolving the oxide (Tb407. 99.9% ; Fluka) in hydrochloric acid and evaporating the excess of the acid.The concentration of TbC13 in aqueous acidic solution (pH d 3) was measured by complexometric titration with standard EDTA using xylenol orange (Merck) as the indicator.8 Bovine serum albumin (BSA) monomer ( 5 pg ml-I) (Pierce) was used as a standard solution. Coomassie Brilliant Blue G-250 (CBB) (Pierce) was used as a standard solution (1 X 10-3 M) for the spectrophotometric method. Bovine IgG (11.3 mg 1-1) (Bio-Rad Laboratories) was used as a standard solution. Procedure The conditions for the preparation of ternary fluorescence complexes with DTPA and DTPP have been described.9-11 By applying the earlier tested methods, the following ternary complexes, showing strong fluorescence, were obtained: Tb"' - p-AS + DTPA - BSA and Tb"' - p-AS + DTPP - BSA.The properties of these complexes were compared in this work. DTPP or DTPA (0.1 mmol; 57.8 mg) and triethylamine (2 ml) were dissolved in 2 ml of DMSO. p-AS was dried at 110 "C until no further decrease in mass occurred and 0.1 mmol (18 mg) of the anhydrous salt was dissolved in 2 ml of DMSO. The p-AS solution was added dropwise to the stirred DTPP or DTPA solution and the mixture was stirred for 30 min at room temperature. BSA was labelled with the p-AS + DTPP or p-AS + DTPA derivative by adding 0.1 ml of the rapidly stirred suspension to a solution of 67 mg of BSA in 5 ml of 0.1 M phosphate buffer (pH 7) with continuous mixing. The mixture was kept overnight at 4°C and the excess of label was removed by dialysis for 36 h against three 1-1 volumes of 9 g 1-1 sodium chloride solution.For use in a fluorescence immunoassay, 0.4 ml of the labelled BSA preparation was mixed with 0.2 ml of 0.04 M terbium chloride solution and this mixture was diluted 1 + 99 with 0.1 M phosphate buffer (pH 7). The excess of terbium ions was precipitated as phosphate and could be removed by centrifugation at 1500 g for 5 min. Diluted label (0.1 ml) was mixed with 0.1 ml of a 1 + 49 dilution of BSA antibodies. After allowing it to stand for 30 min at room temperature. 1 ml of polyethylene glycol 6000 (200 g 1-1 in 0.1 M phosphate buffer, pH 7) was added to each centrifuge tube and the precipitates were sedimented by centrifugation at 1500 g for 20 min. The supernatants were removed by aspiration and the precipitates resuspended in 2 ml of 0.1 M phosphate buffer (pH 7).The assay tubes were placed directly in the cell compart- ment of a Perkin-Elmer spectrofluorimeter. Results The strong fluorescence of the stable terbium chelates was utilised for the determination of trace amounts of BSA (Fig. 1). It was demonstrated that the spectrofluorimetric method is significantly more sensitive than the spectrophotometric method, and that the sensitivity of the determination of BSA is higher when the DTPP (Table 1) is cross-linked. It was found that the spectrophotometric method permits the deter- mination of only microgram amounts of BSA, whereas the spectrofluorimetric method is applicable to nanogram amounts (Table 1). As shown in Fig. 2, fluoroimmunoassay of BSA showed a good correlation with the spectrophotometric method (CBB).The spectrofluorimetric and spectrophotometric methods showed a linear dependence on BSA concentration within the range 2-1000 ng ml-1. In the regression analysis of determinedANALYST, APRIL 1989, VOL. 114 530 ;; 0.8 z 0.6 - Q) 6 0.4 f a n 6 0.2 0 300 400 500 600 Wavelengthin m Fig. 1. Typical absorption (broken line) and excitation and emission (solid line) spectra of terbium chelates with p - A S + DTPP and BSA (2.1 x 10-8 \I) Table 2. Comparison of fluorescence properties of terndry complexes with phosphonic (DTPP) and carboxylic (DTPA) complexones Values i n parenthews refer t o DTPA Fluorescence Stability of complex intensity. "/" with ti m e/d Concentration of pH 7.0. pH 4.0. complexh DTPP DTPA DTPP DTPP 1.0 x lo-" 29.8 7.0 7 (6) 3 (2) 4.0 x lo-" 60.5 14.2 7 (6) 3 (2) 8.0 x lo-" 91.6 22.1 7 (6) 3 (2) Table 1.Dependence o f the rangc of determination of BSA on pH for the studied complexes Rectilinear Ternary Limit of range of BSA PH complex detection concentration range DTPA . . 0.5ngml-1 10-29.5 ng ml-1 4.0-11.0 DTPP . . 0.1 ngml-1 2-29 ng ml-1 2.5-1 2 .0 CBB . . 0.1pgml-1 0.5-7.5 pg ml-1 5.5-1 1.25 10.5 500 1000 Fig. 2. Calibration graphs for BSA detcrmination by ( u ) fluoroim- munoassay (A. DTPP; B, DTPA) and ( b ) spectrophotometric (CBB) methods versus found BSA concentrations, the slopes were 0.955 (DTPP), 0.940 (DTPA) and 0.830 (CBB). Rectilinear calibration graphs of fluorescence intensity versus BSA concentration were obtained for the ranges 2.&29 ng ml-1 (DTPP) and 1@29 ng ml-1 (DTPA).The relative standard deviation for 10 ng ml-1 of BSA was 3.0% for DTPP and 4.2% for DTPA for 15 measurements. The spectroflu- orimetric assay showed good reproducibility, the coefficients of variation being 0.46% for DTPP and 0.30% for DTPA. From comparisons of the rectilinearity range, BSA determi- nations, detection limits, pH determinations and stabilities of the DTPP and DTPA complexones with time, we concluded that the application of the DTPP complexone provides more favourable analytical results. The differences can be attri- buted to the difference in the stabilities of these complexes (fluorescence intensity, Table 2), and also their acid - base properties and the complex-forming properties of DTPP and DTPA. It was demonstrated that bovine TgG can be determined according to the proposed procedure, allowing fluoro- immunoassay to be performed.Discussion The ternary mixed ligand complexes of Tb11' - p-AS + DTPA (or DTPP) - BSA form highly fluorescent chelates (Fig. 1). The absorption of light by the ligands is followed by efficient energy transfer from the excited singlet state through the triplet state to the resonance levels of the terbium ion. The terbium ion emits energy as a narrow-band line-type emission. The fluorescence of the chelate is characterised by broad excitation in the absorption region of the ligand (328 nm) with a large Stokes shift (>215 nm) and emission lines typical of terbium. Typical excitation and emission spectra of terbium chelates are shown in Fig. 1. The 488- and 545-nm bands are the strongest and are due to the SD4 -+ 7F6 and sD4 -+ 7Fs transitions, which are also hypersensitive.The hypersensitive band observed for TbI" arises as a result of vibronic coupling connected with the influence of the ligand field on the 4f orbital of the Tb"1 ion. The oscillator strengths of certain 4f + 4f transitions in lanthanide(II1) complexes exhibit an espe- cially strong sensitivity to the structural details and chemical nature of the ligand environment. A terbium cation with a high coordination number is capable of binding strongly p-AS + DTPP (or DTPA) with BSA. Hence the high stability of the chelate complex is confirmed by intense fluorescence in dilute solutions. The ternary mixed ligand complexes, TbIII - complexones - BSA, are strongly fluorescent and stable in very dilute solution (10-10 M)? The sensitivities of the two DTPP and DTPA complexones were compared in the determination of BSA, BSA monomer being used as the reference material.The differences in the sensitivities of the two complexones should be attributed to the different acid - base properties. The dissociation constants for DTPA are pK1 = 1.95. pK2 = 2.55, pK3 = 4.23, pK4 = 8.56 and pK5 = 10.45,7,11.13 and those for DTPP are pK1 = 1.05, pK2 = 1.90, pK8 = 8.15, pK9 = 10.10 and pKlo = 12.04.7.11.13 These dissociation constants indicate a much wider range of the acid - base properties for DTPP than for DTPA (ApKlo =: 11, ApKs = 8.5). This provides more favourable conditions for the complexation reaction in a wider pH range. The stability constants of TbIII complexes with DTPP, DTPA, BSA and p-AS are log f3 = 21.90, 19.90, 9.40 and 11.40, respec- tively.7.11.1-i As follows from the stability constants of com- plexes with the Tb"' cation, the Tb - DTPP system has the greatest strength.Further increases in the stability of the fluorescent ternary complexes may be possible by using polyaminocarboxylic, phosphonic, phosphinic and arsonic ligands. This procedure for the determination of proteins may be applied to many similar compounds. Work on such com- pounds and their lanthanide complexes is in progress. P K ~ = 2.80, P K ~ 1 4.45, PKS = 5.50, pK6 = 6.38, pK7 = 7.77, References 1. 2. 3. Soini, E., and Kojola, 13.. Clin. Chem., 1983. 29. 65. Hemmila, I . , Clirz. Chem., 1985, 31, 359. Petersson. K., Siitari, H.. Hemmila, I., Soini, E.. Liivgren, T.. Hanninen. V., Tanner, P.. and Stenman, U . , Clin. Chem., 1983, 29, 60. Chen, F. F., and Scott, C . H., Anal. Lett., 1985, 18, 393. Ferrara, A. I . . Meroni. G., and Bacigalupo, M., Fre.senius Z. And. Chem., 1985. 322, 509. 4. 5 .ANALYST, APRIL 1989, VOL. 114 53 1 6. 7. 8. 9. 10. Moeller, T., Martin, D. T., Thompson, L. L., Ferrus. R., Feistel, G. R.. and Randall. W. J . , Chem. Rev., 1965, 65. 1. Smith. R . M., and Martell, A . E., “Critical Stability Con- stants,” Volume 5 , Plenum Press, New York, 1982. Welcher, F. J . , “The Analytical Uses o f Ethylenediarninetetra- acetic Acid,” Van Nostrand, New York. 1958. Bailey, M. P., Rocks, B. F., and Riley, C., AnalyJt, 1984, 109, 1449. Siepak, J . , Bull. Soc. Amis. Sci. Lett. Poznari. Ser. D, 1988,26, 5 . 11. Siepak, J . , “Properties and Applications of New Organophos- phorus Complexones in Analytical Chemistry.” A. Mickiewicz University, Poznan, 1987. Siepak, J . , paper presented at Euroanalysis VI. Paris, 1987. Kabachnik, M. I., Dyatlova, N. M., Medved, T. Ya, Belgugin, Yu. F., andsidorenko. W. W., Dokl. Akad. NaukSSSR, 1967, 175, 351. Paper 8101 624K Received April 25th, 1988 Accepted September 27th, 1988 12. 13.