Development of Highly Fluorescent Detection Reagents for the

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Development of Highly Fluorescent Detection Reagents for the Construction of Ultrasensitive Immunoassays Qiu-Ping Qin,* Timo Lo 1 vgren, and Kim Pettersson

Department of Biotechnology, University of Turku, Tykisto¨katu 6, FIN-20520 Turku, Finland

We developed two kinds of highly fluorescent streptavidinbased conjugates for use as universal detection reagents in ultrasensitive immunoassays. The direct conjugate was produced by covalently linking streptavidin to poly(Glu: Lys) which was labeled heavily with Eu chelates; the indirect conjugate was made by first conjugating bovine serum albumin (BSA) to poly(Glu:Lys) labeled heavily with Eu chelates and then further linking streptavidin to the conjugate of BSA-poly(Glu:Lys)-Eu chelate. Both direct and indirect conjugates were used to construct a highly sensitive time-resolved fluorometric assay for prostate-specific antigen (PSA). Of two monoclonal antibodies used in the assay, one was coated on the well surface of the microtitration strips, and the other was biotinylated. When 10 µL of sample volume was used, we found that the assay using the indirect conjugate had a detection limit of 0.006 µg/L, which was approximately 5.6-fold more sensitive than the one using Eu chelate directly labeled detection antibody and 6.8-fold more sensitive than the one using Eu chelate-labeled streptavidin. However, the assay that used the direct conjugate was 1.5-fold more sensitive than the one that utilized the indirect conjugate. When 45 µL of sample volume was used, a detection limit of 0.001 µg/L was achieved by using the direct conjugate. This improvement in sensitivity should be equally obtainable for the analytes other than PSA. We further demonstrated that the final immunoassay performance was affected not only by the quality of the streptavidin-based conjugate used but also by the quality of the biotinylated antibody reagent. The universal detection reagents described here are believed to be particularly useful for the construction of ultrasensitive time-resolved fluorometric immunoassays and are potentially applicable in other fields such as immunohistochemistry and nucleic acid detection. A large number of different immunoassays have been developed for routine use in clinical practice as well as in biomedical research. These are either competitive or noncompetitive assays. For a sandwich-type noncompetitive assay, the detection limit obtained by using a radioisotopic label is higher than that obtained using a nonradioisotopic label such as chemiluminescence and * To whom correspondence should be addressed. Tel.: +358 2 333 8095. Fax: +358 2 333 8050. E-mail: [email protected]. 10.1021/ac001351z CCC: $20.00 Published on Web 03/06/2001

© 2001 American Chemical Society

time-resolved fluorescence. This is largely because these nonradioisotopic labels have a much higher specific activity than a radioisotopic label.1,2 In addition to the specific activity of a label, other factors, such as the level of a nonspecific signal and the affinity constant of an antibody, also affect the assay sensitivity.1,3,4 The strategies so far adopted to improve the sensitivity of immunometric assays are basically focused on these three aspects. In the assay system where lanthanide chelate labels are used, the background can be substantially reduced by time-resolved fluorometry. This is because the emitted fluorescence is temporally long-lived and spatially well separated from the excitation wavelength. The signal is specifically detected after a certain delay time, and in this way, the short-lived nonspecific signal is efficiently reduced.5 In certain clinical situations, ultrasensitive immunoassays are highly desirable; for instance, ultrasensitive TSH assay with a detection limit of 0.01 mIU/L is needed to help differentiate euthyroid from clinical and subclinical thyroid dysfunction.6 Ultrasensitive assays for prostate-specific antigen with detection limits of 0.01-0.02 µg/L can be of considerable value for early detection of prostate cancer relapse and estimation of tumordoubling time after radical prostatectomy.7 Usually, the sensitivity of a sandwich-type fluorometric assay can be improved by increasing the sample volume. However, this mechanism carries some disadvantages. Besides the increases in sample consumption in itself, higher sample volumes may slow the assay kinetics, bring about greater matrix effects, and reduce the upper range of an assay. Thus, there is an increasing need to improve the assay sensitivity while still using small sample volumes. One way to achieve this goal is to use multiple labeling of an antibody or covalent conjugation of an antibody with a heavily labeled carrier protein.8 It is already known that about 10 or even more Eu chelates can usually be incorporated into one antibody molecule without having significant effects on the binding affinity (1) Ekins, R. P. In Alternative Immunoassays; Collins, W. P., Ed.; John Wiley & Sons: Chichester, 1985; pp 219-237. (2) Soini, E.; Kojola, H. Clin. Chem. 1983, 29, 65-68. (3) Kricka, L. J. Clin. Chem. 1994, 40, 347-357. (4) Jackson, T. M.; Ekins, R. P. J. Immunol. Methods 1986, 87, 13-20. (5) Lo ¨vgen, T.; Pettersson, K. In Luminescence Immunoassay and Molecular Applications; Van Dyke, K., Van Dyke, R., Eds.; CRC Press: Boca Raton, FL, 1990; pp 233-250. (6) Kaplan, M. M. Clin. Chem. 1999, 45, 1377-1383. (7) Yu, H.; Diamandis, E. P.; Prestigiacomo, A. F.; Stamey, T. A. Clin. Chem. 1995, 41, 430-434. (8) Diamandis, E. P. Clin. Chem. 1991, 37, 1486-1491.

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of the antibody.5,8 Furthermore, multiple fluorescence labeling with fluorescent europium chelates is free from fluorescence quenching.9 However, there are instances where an antibody may lose part or all of its immunoreactivity when the degree of label incorporation increases.8,10 Furthermore, the nonspecific binding of a heavily labeled antibody may also increase considerably. Other approaches to increase assay sensitivity involve the use of indirect detection reagents with high specific activities. This approach obviates harsh derivatizations of a detection antibody used in an assay and often involves the use of the biotinstreptavidin system.11 Streptavidin, found in the bacterium Streptomyces avidinii, has the ability to specifically bind to the vitamin biotin with a very high affinity (Ka ≈ 1015 M-1).12 Moreover, streptavidin is a very stable molecule, and, in many cases, its biotin-binding activities can be maintained even after extensive derivatizations. Streptavidin has been used for conjugation to horseradish peroxidase13 and alkaline phosphatase.14 The use of a streptavidin-alkaline phosphatase conjugate, together with the use of a diflunisal phosphate substrate and a terbium chelate, has been reported to achieve an assay with improved sensitivity.15,16 Streptavidin has also been used to conjugate with Eu chelates.17 However, the sensitivities of time-resolved immunofluorometric assays achieved by the use of a streptavidin-Eu chelate conjugate are fairly comparable to those obtained by the use of the Eu-labeled detection antibodies.18 A very different method to create a streptavidin-based detection reagent with high specific activity relates to the use of a high-molecular-weight carrier protein. Bovine thyroglobulin (TG, Mr ) 660 kDa), as reported by Diamandiss’ group in Canada, was first highly labeled with the europium chelate of 4,7-bis(chlorosulfophenyl)-1,10-phenanthroline-2,9-dicarboxylic acid (BCPDA). About 175 BCPDA labels were introduced into one molecule of TG.19,20 This heavily labeled TG was then covalently linked to streptavidin. The use of the resultant streptavidin reagent allowed development of a few highly sensitive time-resolved fluoroimmunoassays.21,22 Recently, the same group reported the development of a new streptavidin-based fluorescent conjugate utilizing polyvinylamine (PVA) as a carrier and claimed that a better sensitivity for PSA assay was achieved when using the new conjugate as compared to the old TG-based conjugate.23 (9) Diamandis, E. P.; Morton, R. C.; Reichstein, E.; Khosravi, M. J. Anal. Chem. 1989, 61, 48-53. (10) Hemminki, A.; Hoffren, A. M.; Takkinen, K.; Vehniainen, M.; Makinen, M. L.; Pettersson, K.; Teleman, O.; Soderlund, H.; Teeri, T. T. Protein Eng. 1995, 8, 185-191. (11) Diamandis, E. P.; Christopoulos, T. K. Clin. Chem. 1991, 37, 625-636. (12) Green, M. N. Biochem. J. 1966, 101, 777-780. (13) Bai, G.; Fujiwara, K.; Tanimori, H.; Kitagawa, T. Biol. Pharm. Bull. 1997, 20, 1224-1228. (14) Thurmond, L. M.; Reese, M. J.; Donaldson, R. J.; Orban, B. S. J. Pharm. Biomed. Anal. 1998, 16, 1317-1328. (15) Papanastasiou-Diamandi, A.; Christopoulos, T. K.; Diamandis, E. P. Clin. Chem. 1992, 38, 545-548. (16) Yu, H.; Diamandis, E. P. Clin. Chem. 1993, 39, 2108-2114. (17) Rossler, A. Clin. Chim. Acta 1998, 270, 101-114. (18) Suonpaa, M.; Markela, E.; Stahlberg, T.; Hemmila, I. J. Immunol. Methods 1992, 149, 247-253. (19) Diamandis, E. P.; Morton, R. C.; Reichstein, E.; Khosravi, M. J. Anal. Chem. 1989, 61, 48-53. (20) Morton, R. C.; Diamandis, E. P. Anal. Chem. 1990, 62, 1841-1845. (21) Christopoulos, T. K.; Lianidou, E. S.; Diamandis, E. P. Clin. Chem. 1990, 36, 1497-1502. (22) Reichstein, E.; Morton, R. C.; Diamandis, E. P. Clin. Biochem. 1989, 22, 23-29.

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We report here the development of two highly fluorescent streptavidin-based detection reagents that can be used for ultrasensitive time-resolved fluorometric assays. A polyamino acid polymer that consists of glutamic acids and lysines and has a mean apparent molecular mass of 100 kDa was used as a carrier molecule. The polymer was highly labeled with fluorescent Eu chelates (∼110 Eu chelates per polymer) before it was covalently linked to streptavidin either in a direct manner or mediated by BSA. The performance of these detection reagents was evaluated with a model assay for PSA and compared with those of Eu chelate-labeled detection antibody and Eu chelate-labeled sreptavidin. EXPERIMENTAL SECTION Materials. ITC-TEKES Eu fluorescent chelate of 4-[2-(4isothiocyanatophenyl)ethynyl]-2,6,-bis{[N,N-bis(carboxymethyl)amino]methyl}pyridine and biotin isothiocyanate (BITC) were obtained from Perkin-Elmer Life Sciences, Wallac Oy, Finland. N-Hydroxysulfosuccinimide (sulfo-NHS) and 2-mercaptoethanol were purchased from Fluka, Germany. 1-Ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDC) and sulfosuccinimidyl(4-iodoacetyl)aminobenzoate (sulfo-SIAB) were obtained from Pierce Chemical Co., Rockford, IL. Bovine serum albumin (BSA) was supplied by Intergen, Purchase, NY. Streptavidin was from Biospa, Italy. Poly(D-Glu,D-Lys; 6:4) (MW 20 000-50 000), 2-(Nmorpholino)ethane sulfonic acid (MES), and dithiothreitol (DTT) were obtained from Sigma Chemical Co., St. Louis, MO. Two IgG1-type monoclonal antibodies Mab5A10 and Mab117 were produced in our own laboratory. Low-fluorescence 12-well Maxisorp microtitration strips (UV-quenched) were purchased from Nunc, Denmark. All other chemicals used were of analytical grade. Instrumentation. The 1420 multilabel counter (Victor) for time-resolved surface measurement of solid-phase fluorescence, the 1230 Arcus fluorometer for time-resolved measurement of DELFIA enhanced fluorescence in tube format, and DELFIA Platewash were obtained from Perkin Elmer Life Sciences. The iEMS incubater/shaker from Labsystems Oy, Finland, was used for plate incubation. The FPLC system, Superdex 200 HR 10/30 (a prepacked column), and Sephacryl S-200 were from Pharmacia Biotech, Sweden. Methods. Coating of Microtitration Wells. The microtitration strips were coated for 2 h at 35 °C with 50 µL/well of 0.2 M phosphate buffer at pH 7.8, containing 0.375 µg of the Mab117. The strips were washed twice with DELFIA wash solution (Wallac Oy, Finland) and blocked for 1 h at room temperature with 100 µL/well of 50 mM Tris-HCl at pH 7.2, containing 0.1% BSA, 0.1% Germall II, and 3% Trehalose. After blocking, the wells were aspirated and left to dry in a laminar hood for 1 h. The coated strips were then stored at 4 °C in a sealed package with desiccant until use. Biotinylation of Monoclonal Anti-PSA Antibody. Biotinylation of Mab5A10 was performed in 50 mM sodium carbonate buffer at pH 9.6, containing a 40-fold, or 80-fold, or 120-fold molar excess of BITC dissolved in dimethylformamide. This mixture was incubated for 3 h at room temperature. The biotinylated antibody was separated from excess reagent by passing the mixture first through a NAP-5 column (Pharmacia Biotech) and then through (23) Scorilas, A.; Diamandis, E. P. Clin. Biochem. 2000, 33, 345-350.

a NAP-10 column (Pharmacia Biotech) with 50 mM Tris-HCl at pH 7.8, containing 0.15 M NaCl as the eluent. BSA was added as a stabilizer to the purified conjugates at a concentration of 0.1%. The Proportion of the Unbiotinylated Antibody. In the biotinylated Mab5A10 preparation, the proportion of the unbiotinylated antibody was determined as follows: To prewashed streptavidincoated strips (Wallac Oy, Finland) was added 50 ng/well of biotinylated Mab5A10 in 20 µL of the DELFIA assay buffer (Wallac Oy, Finland). The strips were incubated for 1 h at room temperature. After that, 25 µL of reaction solution from each well, together with 200 µL of assay buffer, was transferred to the well of the anti-mouse IgG-coated strips (Wallac Oy, Finland), parallel to the wells containing a series of diluted biotinylated Mab5A10 as calibrators. The strips were incubated for 2 h at room temperature and then washed six times. To each well was added 100 ng of Eu-labeled anti-mouse IgG (Wallac Oy, Finland) in 200 µL of the assay buffer. The strips were incubated at room temperature for 2 h with continuous shaking. After the wells were washed six times, 200 µL of enhancement solution (Wallac Oy, Finland) was dispensed into each well. The strips were shaken slowly for 5 min before the fluorescent signals were measured with the 1420 multilabel counter (Victor) using the normal measurement mode. The concentrations of the unbiotinylated Mab5A10 from the transferred wells were determined from the calibration curve that was constructed from the data obtained from the calibrator wells. Subsequently, the proportion of the unbiotinylated Mab5A10 in the biotinylated Mab5A10 preparation was calculated. Fractionation of the Polyamino Acid Copolymer. The fractionation of poly(Glu:Lys) was performed on a 1.5- × 96-cm column of Sephacryl S-200 (Pharmacia Biotech). Twenty-five milligrams of poly(Glu:Lys) dissolved in 1.5 mL of 50 mM carbonate buffer at pH 9.6 was loaded onto the column. Elution buffer was 0.1 M phosphate-buffered saline at pH 7.4. The column was operated at room temperature with a flow rate of 12 mL/h, and fractions of 1.5 mL were collected. For molecular mass estimation, the column was calibrated with F(ab)2 and Fab, the products obtained by papain (Sigma Chemical Co.) digestion of Mab117.24 The fractions corresponding to F(ab)2 were pooled and concentrated by using a Centricon-30 (Amicon). The concentration of poly(Glu:Lys) was determined according to the absorbance value at 214 nm. The fractionated poly(Glu:Lys) has an apparent molecular mass of about 100 kDa and contains approximately 272 amino groups and 409 carboxyl groups per molecule. Labeling of Mab5A10, Streptavidin, and the Fractionated Poly(Glu:Lys). For Mab5A10, labeling was performed at 4 °C for 20 h with a 100-fold molar excess of the ITC-TEKES Eu chelate in 50 mM carbonate buffer at pH 9.6. The labeling of streptavidin was carried out at room temperature for 20 h with a 100-fold molar excess of the Eu chelate in the reaction buffer. For the fractionated polyamino acid polymer with an apparent molecular mass of ∼100 kDa, the labeling reaction was conducted at 37 °C for 36 h with a 3.8-fold molar excess of the Eu chelate over the free amino groups on the polyamino acid. The labeled antibody, streptavidin, and polyamino acid were separated from excess free chelate and possible aggregates by gel filtration on a Superdex 200HR 10/30 column (1.6 × 60 cm), equilibrated, and eluted with 50 mM Tris(24) Parham P. J. Immunol. 1983, 131, 2895-2902.

HCl at pH 7.8, containing 0.15 M NaCl. The flow rate was 15 mL/ h, and fractions of 0.5 mL were collected. The fractions containing labeled antibody, streptavidin, or polyamino acid were pooled and concentrated to 0.5 mL by using a Centricon-30 (Amicon). For Mab5A10 and streptavidin, the protein concentration was measured by absorbance at 280 nm. For the polyamino acid, we assumed that all the labeled polyamino acids were completely recovered. Determined against an Eu calibration solution (Wallac Oy, Finland), the labeling degrees for Mab5A10, streptavidin, and polyamino acid were 5, 5, and 109, respectively. Direct Conjugation of Streptavidin to Eu-Labeled Poly(Glu:Lys) through EDC and Sulfo-NHS. Step 1. Activation of Carboxyl Groups of Poly(Glu:Lys). After a buffer change on the NAP-5 column, 35 or 140 µg of Eu-labeled poly(Glu:Lys) was dissolved in 1 mL of 0.1 M MES at pH 6.0, containing 0.15 M NaCl. To each of these solutions were added EDC in a final concentration of 6 mM and 18 mM Sulfo-NHS. This was followed by a 15-min incubation at room temperature in the dark. To quench excess EDC, 2-mercaptoethanol was then added to each of the reaction solutions to a final concentration of 60 mM. The mixtures were allowed to stay at room temperature for 20 min in the dark. Step 2. Conjugation of the Activated Poly(Glu:Lys) to Streptavidin. Each of these solutions containing derivatized poly(Glu: Lys) was mixed in a glass vial with 500 µg of streptavidin dissolved in 1 mL of 0.1 M MES at pH 6.0, containing 0.15 M NaCl. The conjugation reaction was allowed to proceed for 2 h at room temperature in the dark. To quench any unreacted NHS present on the poly(Glu:Lys) molecules, 100 µL of a 1 M glycine (Prolabo, France) solution was added to each of these reaction mixtures. The crude conjugate products were concentrated to 500 µL by using a Centricon-30 and stored at 4 °C for further purification. Indirect Conjugation of Streptavidin to Eu-Labeled Poly(Glu:Lys) Mediated by BSA. Step 1. Activation of Carboxyl Groups of Poly(Glu:Lys). This was performed through EDC and Sulfo-NHS as described above. Step 2. Conjugation of Activated Poly(Glu:Lys) to BSA. Each of the solutions containing derivatized poly(Glu:Lys) was mixed in a glass vial with 1 mg of BSA dissolved in 0.5 mL of 0.1 M Mes at pH 6.0, containing 0.15 M NaCl. The conjugation reaction was left to proceed for 2 h at room temperature in the dark. To quench any unreacted NHS present on the poly(Glu:Lys) molecules and to restore free sulfhydryls on BSA molecules, 100 µL of a 1 M glycine (Prolabo) solution and 10 mg of DTT were added to each of these reaction mixtures. After 2 h of incubation at room temperature, the poly(Glu:Lys)-BSA conjugate from each reaction mixture was separated from excess quenching and reducing reagents by gel filtration on PD-10 columns (Pharmacia Biotech) that were equilibrated and eluted with a 50 mM borate buffer at pH 8.3. As a result, the volume of the conjugate from each mixture was 3.5 mL after separation. Step 3. Activation of Streptavidin with Sulfo-SIAB. Two glass vials of streptavidin solution were prepared, each of which contained 500 µg of streptavidin dissolved in 0.5 mL of a 50 mM borate buffer at pH 8.3. To each streptavidin solution was added 1 mg of Sulfo-SIAB, followed by a 2-h incubation at room temperature in the dark. Subsequently, the excess Sulfo-SIAB molecules were removed by passing the reaction mixture through a NAP-5 column eluted with a 50 mM borate buffer at pH 8.3. At Analytical Chemistry, Vol. 73, No. 7, April 1, 2001

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this stage, 1 mL of activated streptavidin solution was obtained. Fourth, the activated streptavidin was conjugated to the poly(Glu: Lys)-BSA conjugate. This was done by mixing 1 mL of activated streptavidin solution with 3.5 mL of the reduced poly(Glu:Lys)BSA conjugate in a glass vial. The incubation was allowed to proceed for 3 h at room temperature and overnight at 4 °C in the dark. After that, cysteine (Sigma Chemical Co.) was added to a final concentration of 5 mM and was left to react for 15 min at room temperature. Finally, the crude conjugate products were concentrated to 500 µl using a Centricon 30 and stored at 4 °C for further purification. Purification of Crude Direct and Indirect Conjugates. This was performed by gel filtration on a 1.6 × 60 cm column of Superdex 200 by using 50 mM Tris-HCl at pH 7.8 containing 0.15 M NaCl as the equilibration and elution buffer. The column was operated at room temperature with a flow rate of 15 mL/h, and fractions of 0.29 mL were collected. The fractions containing streptavidinbased labeled conjugates were revealed by a PSA assay (see below) and pooled, and then supplemented with BSA to a final concentration of 0.1% before being stored at 4 °C. PSA Assay Procedures. (1) Use of Directly Labeled Mab5A10. After the wells of Mab117-coated strips were washed twice, 10 µL of the PSA calibrators from the Prostatus PSA kit (Wallac Oy, Finland) and 200 ng of Eu-labeled Mab5A10 in 20 µL of the DELFIA assay buffer (Wallac Oy, Finland) were added. The strips were incubated for 20 min at 36 °C with continuous shaking. The wells were then washed six times with the DELFIA wash solution and dried for 5 min. The fluorescencent signals were measured with the 1420 multilabel counter (Victor) using the powerflash surface measurement mode. (2) Use of Biotinylated Mab5A10. After the wells of Mab117coated strips were washed twice, 10 µL of the PSA calibrators and 200 ng of biotinylated Mab5A10 in 20 µL of the DELFIA assay buffer were added. The strips were incubated for 20 min at 36 °C with continuous shaking. The wells were then washed twice with the DELFIA wash solution. To each well was added 30 µL of either directly labeled streptavidin solution (200 ng) or the streptavidinbased conjugate diluted with the DELFIA assay buffer supplemented with skimmed milk powder and Tween 40 to a final concentration of 0.005% and 0.02%, respectively. The strips were further incubated for 20 min at 36 °C with constant shaking. After being washed six times, the wells were dried, and the fluorescence was measured in the same way as described above. The detection limit was defined as the concentration of PSA giving a signal corresponding to that for the zero standard + 2SD. (3) Assay Optimized for Maximum Sensitivity. After the wells of Mab117-coated strips were washed twice, 45 µL of the PSA calibrators and 50 ng of biotinylated Mab5A10 in 5 µL of the DELFIA assay buffer were added. The strips were incubated for 60 min at 36 °C with continuous shaking. The wells were then washed twice with the DELFIA wash solution. To each well was added 30 µL of a streptavidin-Eu-polyaa conjugate (which was a newly made direct conjugate) diluted 20-fold with 0.1 M TrisHCl, pH 7.4, containing 6% BSA. Afterward, the strips were incubated for 40 min at 36 °C with constant shaking. After being washed six times, the wells were dried, and the fluorescence was measured. 1524


RESULTS AND DISCUSSION Eu and other lanthanide chelates have widely been used as labels in many time-resolved fluorometric assays.5,25 These lanthanide chelates are either fluorescent or nonfluorescent. In the nonfluorescent lanthanide chelate-based assays, the lanthanide ion is finally dissociated from the immunocomplex on the solid phase into an acidic aqueous solution where a highly fluorescent lanthanide chelate is formed.26 In the fluorescent lanthanide chelate-based assays, the fluorescent lanthanide chelate is immobilized, via the labeled detector antibody, to the solid phase during the final measurement phase.27 The Eu chelate used in this study is a fluorescent one; therefore, the fluorescence was measured directly on the solid phase. The performance of this fluorescent chelate in the immunoassays has previously been shown to be similar to that of the nonfluorescent chelates using a dissociative enhancement procedure.28 Polyamino acids have been studied as enzyme inhibitors,29 drug delivery devices,30 catalysts in organic synthesis,31 etc. To the best of our knowledge, this is the first report in which a polyamino acid polymer was used as a carrier heavily labeled with fluorescent Eu chelates, which was then conjugated to streptavidin to form a universal detection reagent to be used in time-resolved immunoassays. The original poly(Glu:Lys) prepared by the baseinitiated polymerization of the corresponding N-carboxyanhydride was a product of random copolymerization, and its averaged molecular mass ranged from 20 to 50 kDa, as determined by viscosity measurements. This product consists of a mixture of polymeric amino acids differing in the degree of polymerization. The low-molecular-weight polyamino acids are less useful because they have a limited number of amino groups that can be labeled with Eu chelate. To remove the low-molecular-weight polyamino acids, molecular sieve chromatography based on a 1.5- × 96-cm column of Sephacryl S-200 was utilized. As shown in Figure 1A, the poly(Glu:Lys)s were eluted as a rather broad and continuous peak (the line of closed circles). Polyamino acids eluted at fractions 47-52 have an apparent molecular mass of about 100 kDa, since F(ab)2 fragments are eluted from the column at the same position, as shown in Figure 1B. These fractions were pooled and concentrated for subsequent labeling experiment. A total of 4 mg of poly(Glu:Lys) with a molecular mass of about 100 kDa was obtained from 25 mg of the original polyamino acid product. About 110 Eu chelates were introduced into one molecule of the fractionated polyamino acids. More than half of the estimated 274 amino groups remain unlabeled per polyamino acid molecule, provided that the apparent molecular mass based on the gel filtration results correctly represents the true one. This result is interesting since the labeling conditions applied to the polyamino (25) Dickson, E. F.; Pollak, A.; Diamandis, E. P. Pharmacol. Ther. 1995, 66, 207-235. (26) Hemmila, I.; Dakubu, S.; Mukkala, V. M.; Siitari, H.; Lovgren, T. Anal. Biochem. 1984, 137, 335-343. (27) Evangelista, R. A.; Pollak, A.; Allore, B.; Templeton, E. F.; Morton, R. C.; Diamandis, E. P. Clin. Biochem. 1988, 21, 173-178. (28) Lo ¨vgren, T.; Merio, L.; Mitrunen, K.; Makinen, M. L.; Makela, M.; Blomberg, K.; Palenius, T.; Pettersson, K. Clin. Chem. 1996, 42, 1196-1201. (29) Uritani, M.; Nakano, K.; Aoki, Y.; Shimada, H.; Arisawa, M. J. Biochem. (Tokyo) 1994, 115, 820-824. (30) Caponetti, G.; Hrkach, J. S.; Kriwet, B.; Poh, M.; Lotan, N.; Colombo, P.; Langer, R. J. Pharm. Sci. 1999, 88, 136-141. (31) Porter, M. J.; Roberts, S. M.; Skidmore, J. Bioorg. Med. Chem. 1999, 7, 2145-2156.

Figure 1. (A) Fractionation of poly(Glu:Lys) by chromatography on a Sephacryl S-200 column (1.5 × 96 cm). The vertical dotted lines denote the fractions that were collected and pooled. (B) Separation of F(ab)2 from Fab by chromatography on a Sephacryl S-200 column (1.5 × 96 cm). The same running conditions were used in both cases, as described in the Methods section.

acids are rather stringent as compared to those used for routine labeling of antibodies. The relatively low labeling degree could be due to the molecular structure of the poly(Glu:Lys). Since the polyamino acid contains a high number of carboxyl groups and -amino groups, the electrostatic interactions between negatively charged carboxyl groups and positively charged amino groups may keep some amino groups from being exposed to the labeling chelates. Figure 2 shows the protocols for the direct or indirect conjugation of labeled polyamino acids to streptavidin molecules. In the direct procedures, carboxyl groups are first activated by EDC and then form a rather stable active intermediate with SulfoNHS. After the excess EDC molecules are quenched by 2-mercaptoethanol, streptavidin is added, and its amino group reacts with an activated carboxyl group from the polyamino acid to form a stable amide linkage. Finally, all the unreacted activated carboxylate groups are quenched by glycine. In the indirect approach, activated carboxyl groups from the labeled polyamino acids react with amines from BSA to produce a conjugate of Eupolyamino acid-BSA. Subsequently, primary amine groups from the streptavidin molecule react with the NHS ester groups of SulfoSIAB to make an iodoacetyl-activated streptavidin. After excess Sulfo-SIAB is removed, the iodoacetyl-activated streptavidin is reacted with the sulfhydryl-containing conjugate of Eu-polyamino acid-BSA, resulting in a new conjugate of Eu-polyamino acidBSA-streptavidin by forming stable thioether linkages.

It is possible that the activated carboxyl groups of the labeled polyamino acids may react with the free amines if they are accessible on the same or a neighboring polyamino acid, leading to the formation of intramolecular or intermolecular amide linkages (so-called self-polymerization). However, this should not affect the subsequent conjugations and the use of the final conjugate, since the multilabeled status of polyamino acid remains unchanged even though the polymerized molecule is bigger. The self-polymerization is probably not extensive, because all easily accessible amino groups have been already utilized in the previous labeling experiment, where reaction conditions are more stringent than those used here. Further increases in the labeling degree of polyamino acid should favor elimination of self-polymerization. Purification of the crude conjugates is very important, as it removes unconjugated free streptavidins, BSA, and BSA-streptavidin conjugate from the original products. As shown in Figure 3, free streptavidin and BSA are easily separated from the fluorescent conjugates by chromatography on the Superdex 200 column. Most of the BSA-streptavidin conjugates can also be separated from the indirect conjugates, but the column does not separate well any unconjugated Eu-labeled polyamino acids from the fluorescent conjugates. However, further separation of unconjugated Eulabeled polyamino acids from the fluorescent conjugates did not seem necessary because low background signals were obtained with the use of these purified conjugates (see Tables 1 and 2). The presence of free BSA is likely to consume some activated amino groups from streptavidin molecules, but this should not significantly affect the conjugation results since streptavidin was present in an excess amount. The conjugation between BSA and streptavidin can be avoided if the extra gel filtration is applied to the crude conjugate products of Eu-polyamino acid-BSA. The direct conjugates produced a 1.6-fold higher specific signal than the indirect conjugates. Moreover, the indirect conjugates obtained in the presence of two different amounts of Eu-labeled polyamino acids gave relatively similar specific counts with respect to the PSA concentrations. This observation applies also for the two direct conjugates. However, the background signal obtained by the use of direct conjugates was twice as high as that achieved by the use of indirect conjugates (as shown in Table 1). These results, together with the information from Figure 3, clearly demonstrate that the amount of streptavidin present in the conjugation reactions was more than enough. This probably results in conjugates dominant with a few amide linkages between the labeled polyamino acid molecules and the streptavidin molecules or with a few thioether linkages between the Eupolyamino acid-BSA and the streptavidin molecules. However, since one molecule of streptavidin contains 13 -amines,32 500 µg of streptavidin should have 0.11 µmol of amines. The number of amines introduced into the conjugation reaction is thus theoretically lower than that of carboxyls present in 140 µg (0.57 µmol) and 35 µg (0.14 µmol) of polyamino acids. The results indicate that polyamino acid may exist as a superhelix structure that is tightly held by strong electrostatic interactions. Thus, it should be possible to further increase the specific activity of the fluorescent conjugates by either reducing the amount of streptavidin present in the labeling reaction for the direct conjugates or (32) Argarana, C. E.; Kuntz, I. D.; Birken, S.; Axel, R.; Cantor, C. R. Nucleic Acids Res. 1986, 14, 1871-1882.


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Figure 2. Schematic representation of the conjugation reaction between streptavidin and Eu-labeled poly(Glu:Lys) (A) and the conjugation reaction with the use of streptavidin, BSA, and Eu-labeled poly(Glu:Lys) (B). Eu-PA stands for Eu-labeled polyamino acid, and StrA stands for streptavidin.

reducing the amount of BSA present in the labeling reaction while still maintaining the streptavidin concentration unchanged for the indirect conjugates. Standard curves obtained with the use of the direct conjugate and the indirect conjugate are shown in Figure 4. Both standard curves are linear over the concentration range. The between-well variability (coefficient of variation, CV) at PSA concentrations from 0.095 µg/L and onward was 2-8% for both types of conjugates. The concentration CV at a PSA concentration of 0.019 µg/L was 12.8% when the indirect conjugate was used, which was similar to that observed when the direct conjugate was used (13.2%). As can be seen in Table 1, the specific fluorescence generated by the indirect conjugate was 4 times higher than that produced by Eu-labeled streptavidin or 8 times higher than that achieved by Eu-labeled detection antibody. Furthermore, no increase in background was found with the use of the indirect conjugate, whereas the direct conjugate gave a 2-fold higher signal. Consequently, the assay using the indirect conjugate had a detection 1526


limit of 0.006 µg/L, making it 5.6-fold more sensitive than the one using the Eu-labeled detection antibody (detection limit, 0.034 µg/ L) and about 6.8-fold more sensitive than the assay using Eustreptavidin (detection limit, 0.041 µg/L). The assay using the direct conjugate had a detection limit of 0.004 µg/L, which was 1.5-fold more sensitive than the one using the indirect conjugate. Noticeably, though Eu-labeled streptavidin gave a higher specific signal, it did not produce an assay that was more sensitive than the one produced by using Eu-labeled antibody. This is because the Eu-labeled streptavidin generated more background fluorescence at the same time. The levels of the nonspecific binding of the labeled reagent and its variation critically affect the detection limit finally obtained for an assay.4 This explains why the assay using Eu-streptavidin was less sensitive than the one using a Eu detection antibody. By increasing the sampling volume from 10 to 45 µL and the incubation times of the first and second steps, we could achieve a lowest detection limit of 1 ng/L of PSA. As shown in Figure 5,

Figure 3. Purification of the crude conjugates by FPLC on a 1.6× 60-cm column of Superdex 200. (A) The elution profile of the direct conjugation products. The conjugate and the Eu-labeled polyamino acid were coeluted from the column, which formed peak I, while the elution of the unconjugated streptavidin formed peak II, and excess 2-mercaptoethanol, cysteine, and inactivated cross-linker together formed peak III. (B) The elution profile of the indirect conjugation products. Peak I was formed by sequential elution of the streptavidinBSA-Eu-polyamino acid conjugate, the BSA-Eu-polyamino acid conjugate, the unconjugated Eu-labeled polyamino acid, and the BSA-streptavidin conjugate, while peaks II, III, and IV were formed by the elution of free BSA, free streptavidin, and cysteine, respectively.

the functional sensitivity achieved in this system was about 0.004 µg/L of PSA. The assay sensitivity can be further improved by using higher sample volumes; however, the resultant assay then requires a longer incubation time to compensate for the slower kinetics. In the current assay system, we coated each well with 50 µL of antibody solution. This volume would completely cover the bottom surface as well as part of the side surface of the well. The capture antibodies in this coated area are reactive with PSA molecules if they are exposed to these antibodies. The captured PSA molecules further bind to biotinylated antibodies. After removal of the free biotinylated antibodies by wash, the bound biotinylated antibodies will react with the streptavidin-based fluorescent conjugate. However, the Victor counter used in this study measures only a small surface area around the center of the bottom of the microtitration well, and consequently, only a fraction of the whole fluorescence of the well is measured. There has been a report on the use of a U-bottom microtitration well with a small coated surface area around the center of the bottom in an assay for AFP.33 In this well, all the assay reactions were confined to the coated area no matter what volumes of sample

were used. The fluorescence signal measured from the U-bottom well was more than 5-fold that obtained from a coated flat-bottom well. Streptavidin has frequently served as a carrier for different labels, due to its extremely high affinity for biotin, multiple binding sites, and high chemical stability. The Eu chelate-labeled streptavidin has been used in immunoassays as well as in DNA hybridization assays and in immunocytochemical studies.10,34 It was previously observed that the level of Eu chelate labels has a negligible effect on streptavidin affinity.18 In this study, both direct and indirect conjugates were found to retain the ability to bind to the biotinylated antibody. However, this did not necessarily mean that the binding affinity of the streptavidin-based conjugates was the same as that of the free streptavidins. As a rule, labeled streptavidin cannot be used in the one-step sandwich-type immunoassays. This is also true for the streptavidinbased conjugates. The use of the conjugates requires the detection antibody to be biotinylated. Consequently, the final assay performance will be dependent on the quality of the conjugates as well as on the quality of the biotinylated antibodies. There are two factors that can affect the quality of a biotinylated antibody: one is the proportion of the unbiotinylated antibody, and the other is the degree of biotinylation. As shown in Table 2, the biotinylated antibody reagent that does not contain any unbiotinylated antibody produced a maximal binding signal. In contrast, the biotinylated antibody reagent containing 1.6% unbiotinylated antibody produced a quite low binding signal. In addition, the degree of biotinylation also exercises an effect on the binding signal. This is clearly shown in Table 2, where a biotinylated antibody made by the use of an 80-fold molar excess of BITC produced a binding signal about 1.5-fold higher than that achieved with a biotinylated antibody prepared with the use of a 40-fold molar excess of BITC. However, no further increase in the binding signal was observed with a biotinylated antibody that was prepared using a 120-fold molar excess of BITC, suggesting that these two biotinylated antibodies (made with a 80-fold or a 120-fold molar excess of BITC) most likely have the same degree of biotinylation. In principle, a large BITC excess may negatively affect the immunoreactivity of the antibody,35 although in using monoclonal antibody 5A10 this did not seem to be the case. There have been reports on the development of streptavidinbased fluorescent conjugates based on the use of either bovine thyroglobulin or polyvinylamine as the carrier and BCPDA as the Eu chelator. The use of these conjugates has enabled a few highly sensitive immunoassays to be developed.21,22 However, to compare our conjugates with those conjugates solely on the basis of the published data is rather inaccurate, due to the differences in the Eu chelates, the detection sensitivities of the measuring fluorometers, and the assay systems, especially the sampling volume and the antibodies used. The correct way to make such a comparison is to use different streptavidin-based conjugates in the same assay system with the fluorescence measured by the same detector, as we did for the direct conjugate and the indirect conjugate. (33) Christopoulos, T. K.; Lianidou, E. S.; Diamandis, E. P. Clin. Chem. 1990, 36, 1497-1502. (34) Bjartell, A.; Laine, S.; Pettersson, K.; Nilsson, E.; Lovgren, T.; Lilja, H. Histochem. J. 1999, 31, 45-52. (35) Qin, Q. P.; Christiansen, M.; Lovgren, T.; Norgaard-Pedersen, B.; Pettersson, K. J. Immunol. Methods 1997, 205, 169-175.


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Table 1. Comparison of the Background Signals, Specific Signals, and Detection Limits Obtained by the Use of Both Conjugates, Eu-Labeled Streptavidin, and Eu-Labeled Detection Antibodya

background signal 0.095 µg/L PSA 0.19 µg/L PSA 9.5 µg/L PSA detection limit

direct conjugate

indirect conjugate

1376 ( 59 (5) 2462 ( 151 (5) 1016 ( 38 (5) 34 × 104 ( 3 × 104 (5) 0.004 µg/L

737 ( 58 (5) 2041 ( 81 (5) 658 ( 4 (2) 23 × 104 ( 7 × 103 (5) 0.006 µg/L

Eu-streptavidin

Eu-Mab5A10

1235 ( 95 (8)

892 ( 52 (2)

6.6 × 104 ( 4 × 103 (5) 0.041 µg/L

3.7 × 104 ( 1 × 103 (2) 0.034 µg/L

aThe counts of a signal are expressed as mean ( SD (replicates). The background signal has been subtracted from all the specific signals. The direct conjugate and the indirect conjugate were made using 140 µg of Eu-labeled polyamino acid. A 25-fold dilution was used for both the direct and the indirect conjugates.

Table 2. Effects of the Biotinylation Degree and the Level of Unbiotinylated Antibody on the Specific and Nonspecific Signalsa molar excess of BITC in biotinylated Mab5A10

% of unbiotinylated Mab5A10 background signal 0.095 µg/L PSA 95 µg/L PSA

40-fold

80-fold

120-fold

50-fold

0.2 669 ( 42 1818 ( 56 19 × 105 ( 2 × 104

0.0 679 ( 27 2416 ( 140 25 × 105 ( 7 × 104

0.4 849 ( 64 2404 ( 49 25 × 105 ( 13 × 104

1.6 1026 ( 31 1392 ( 242 15 × 105 ( 14 × 104

aThe counts of a signal are expressed as mean ( SD. The background signal has been subtracted from all the specific signals. Each dosage was performed in four replicates with the indirect conjugate that was made by using 140 µg of Eu-labeled polyamino acid. The conjugate was diluted 25-fold with assay buffer.

Figure 4. Standard curves (continuous lines) and precision profiles (discontinuous lines) for the PSA assay with the use of the direct conjugate (closed circles) or the indirect conjugate (open circles). The fluorescence signal of the zero standard has been subtracted from all the measurements. Each point was performed in five replicates. Sample volume was 10 µL.

The conjugates we described here are stable at least for 1 month. Further investigation on stability will be done. In addition to the nature of their high fluorescence, the conjugates should have all the advantages typical for time-resolved fluorometric assays. Thus, they are particularly useful in the situation where ultrasensitive detection of an analyte is required. For instance, the fluorescent conjugates may be applicable in the assay for human glandular kallikrein (hK2), which is usually present in the serum samples with a concentration about 1-2 orders of magnitude lower than that of PSA.36 Here the PSA assay developed with (36) Piironen, T.; Lovgren, J.; Karp, M.; Eerola, R.; Lundwall, A.; Dowell, B.; Lovgren, T.; Lilja, H.; Pettersson, K. Clin. Chem. 1996, 42, 1034-1041.

1528 Analytical Chemistry, Vol. 73, No. 7, April 1, 2001

Figure 5. Standard curve (continuous line) and precision profile (dotted line) for the PSA assay with the use of the direct conjugate. The fluorescence signal of the zero standard has been subtracted from all the measurements. Each point was performed in five replicates. Sample volume was 45 µL.

the use of fluorescent conjugates is among the most sensitive ones and should be adequate for use for monitoring of prostate cancer patients for early relapse after radical prostatectomy.37,38 Since there is still room to improve the labeling degree of the polymer and the conjugation parameters, we believe that there is a great potential to develop better conjugates, enabling further improved specific activity. CONCLUSIONS We report methods for the development of highly fluorescent universal detection reagents based on the use of streptavidin and (37) Vassilikos, E. J.; Yu, H.; Trachtenberg, J.; Nam, R. K.; Narod, S. A.; Bromberg, I. L.; Diamandis, E. P. Clin. Biochem. 2000, 33, 115-123. (38) Yu, H.; Diamandis, E. P.; Wong, P. Y.; Nam, R.; Trachtenberg, J. J. Urol. 1997, 157, 913-918.

a Eu chelate-labeled polyamino acid (Glu:Lys). When 10 µL of sampling volume was used, detection limits of 0.004-0.006 µg/L for PSA assays achieved by using these detection reagents compared favorably to that obtained by using either directly Eulabeled streptavidin or Eu-labeled detection antibody. This improvement in sensitivity should be universally applicable for analytes other than PSA. Besides, we have demonstrated that the final immunoassay performance is affected not only by the quality of a streptavidin-based detection reagent but also by the quality of a biotinylated antibody reagent. We believe that the universal detection reagents described here are particularly useful for the construction of ultrasensitive time-resolved fluorometric immu-

noassays and are potentially applicable in other fields, such as immunohistochemistry and nucleic acid detection. ACKNOWLEDGMENT This study was financially supported by a grant (Project 43025) from the Finnish Academy. We thank Antti Valanne and Pirjo Laaksonen for their excellent technical assistance, and Va¨limaa Lasse and Soukka Tero for helpful discussions. Received for review November 20, 2000. Accepted January 23, 2001. AC001351Z


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Development of Highly Fluorescent Detection Reagents for the

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