Anal. Chem. 1995,67, 1278-1282
Homogeneous Ummunoassay Using - Photothermal Beam Deflection Spectroscopy Hajime Sakashita,t Akihito Tomita,t Yoko Umeda, Hitoshi Natukawa, and Hiroshi Kishioka**t Yokkaichi Research Laboratory, Kyowa Hakko Kogyo Co., Ltd., 2-3 Daikyo-cho, Yokkaichi, Mie 510, Japan
Takehiko Kitamori and Tsuguo Sawada Department of Industrial Chemistry, Faculty of Engineering, The University of Tokyo, 7-3-1 Hongo, Bunkyo-ku, Tokyo 113, Japan
We developed a novel homogeneous immunoassay using photothermal beam deflection (PBD) spectroscopy. Antibody-coated glass beads having a particle sue of 50 pm and antibody-coated colloidal gold ultrahe particles 20 nm in size were used, and an immune complex containing colloidal gold particles on antibody-coatedglass beads was formed by immunological reaction in solution. An excitation beam was directed from below the sample, and a PBD signal was thus generated was measured. The PBD signal was not afhected by nonreacted antibody-coated colloidal gold particles which were suspended in the solution up to the colloidal gold concentration of 0.008%. This result indicates that homogeneous immunoassay is possible. Human a-fetoproteinwas assayed to examine the sensitivity of this method, and a detection limit of 0.25 ng/mL was obtained. This was more sensitive by 1 order of magnitude than the latex agglutination method, the conventional homogeneous rapid assay. Some improvements of reagents and techniques can be expected to enhance the sensitivity further. Immunoassay is useful for analysis of ultratrace biological substances because of its specificity and high sensitivity.’ Radioimmunoassay (RIA) using radioactive labeling materials has recently caused public discussion with respect to disposal of waste matter. Alternatively, enzyme immunoassay and fluoroimmunoassay (FIA) were developed. These techniques are conducted in a heterogeneous system that separates immunologically bound and free labels. This separation requires a washing procedure, which is time-consuming and prevents rapid determination and automation. Therefore, a homogeneous assay that does not separate bound and free labels is desired, but satisfactory sensitivity has not been obtained yet. In recent years, EMIT-type homogeneous immunoa~say,~ fluorescence polarization immuPresent address: Research Laboratory, Kyowa Medex Co., Ltd., 600-1 Minami-ishiki, Nagaizumicho, Sunto-gun, Shimoka 411, Japan. (1) Gosling, J. P. Clin. Chem. 1990, 36, 1408-1427. (2) Monroe, D. Anal. Chem. 1984, 56, 920A-93lA. (3) Voller, A; Bidwell, D.E.; Collins, W. P., Eds.Alternatiue Immunoassay; John Wiley & Sons: New York, 1985; pp 77-86. (4) Maggio, E. T., Ed. Enzyme-Immunoassay; CRC Press: Boca Raton, FL, 1980. (5) Ollerich, M. J. Clin. Chem. Clin.Biochem. 1980, 18, 197-206. (6) Diamandis, E. P.; Christopoulos, T.K. Anal. Chem. 1990,62,1149A-l157A (7) Rubenstein, K. E.: Schnider, R. S.; Ullman, E. F. Biochem. Biophys. Res. Commun. 1972, 47, 846-851.
noassay? etc. have been proposed as homogeneous immunoassay techniques. The application of these methods, however, is limited to low molecular weight substances. The development of a homogeneous immunoassay that can cover a wide range from low to high molecular weight substances with high sensitivity is therefore demanded. Photothermal spectroscopy is a highly sensitive spectroscopy? and one such methods, photoacoustic spectroscopy (PAS),achieved a detection limit absorption coefficient of cm, times higher than the signal-noise ratio.1° Photothermal beam deflection (PBD) spectroscopy has attracted increasing attention as an improvement on PAS for the assay of solid samples.” It utilizes the “mirage effect”: temperature field gradient occurs in the surrounding medium due to the heat generated from the sample; when a probe beam is passed through the site, deflection is produced, by which the amount of sample is measured. Previously, application of PBD to thin-layer chromatography and other techniques has been reported.12 We recently reported that Fe-oxine complex adsorbed on a single microparticle about 50 ,um in size can be measured more sensitively than that adsorbed to a plate.13J4 This is because the temperature field gradient is amplified by the microparticle curvature. The sensitivity is higher by about 2-3 orders of magnitude than that of the usual absorption microspectrophotometry. As an application of this method to biological determination, the spectral difference between leukemic and normal white blood cells was detected. We have reported an ultrasensitive immunoassay using antibody-coated latex and antibody-coated colloidal gold particles. According to this procedure, polystyrene latex microspheres 0.9 pm in mean size are coated with antibody. The carriers are reacted with antigen, washed, and then reacted with colloidal gold ultrafine particles having a mean sue of 20-40 nm coated with antibody and washed on a commercially available nitrocellulose membrane. After removal of nonreacted material, the sample is dried, and the PBD signal is detected using argon laser as the excitation beam. The sensitivity for detection of IgE reaches 2.0 pg in absolute quantity
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1278 Analytical Chemistry, Vol. 67, No. 7, April 1, 1995
(8) Provost, Y.; Friotti, J. J. Pharm. Clin. 1984,3, 197-213. (9) Harris, T. D.Anal. Chem. 1982, 54, 74L4-750A (10) Kitamon, T.; Fujii, M.; Sawada, T.; Goshi, Y. J. Spectrosc. Soc. Jpn. 1985, 34, 359-365. (11) Boccara, A C.; Fourier, D.; Bodaz, J. J. Appl. Phys. Lett. 1980, 36, 130132. (12) Chen, T.I.; Moms, M. D. Anal. Chem. 1984,56, 19-21. (13) Wu, J.; Kitamori, T.; Sawada, T.J. Appl. Phys. 1991, 69, 7015-7020. (14) Wu, J.; Kitamori, T.; Sawada, T. Anal. Chem. 1991, 63, 217-219. 0003-2700/95/0367-1278$9.00/0 0 1995 American Chemical Society
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Figure 1. Illustration of the immunoassay systems using antibodycoated glass beads and antibody-coated colloidal gold ultrafine particles. (a) Reaction of antibody-coated glass beads with antigen in solution. (b) Reaction of antibody-coated glass beads: antigen complex with antibody-coated colloidal gold in solution. (c) PBD spectroscopy of antibody-coated glass beads: antigen-antibodycoated colloidal gold complex in solution.
(10.7 amol), which is superior by at least 1order of magnitude to the results obtained by RIA and EIA.15 In this paper, we describe a novel homogeneous immunoassay that uses PBD spectroscopy. This technique utilizes the advantages of PBD spectroscopy to measure the substance on microparticles with high sensitivity and to concentrate the substance to be measured on the solid phase. A carrier that has a particle sizeamplifying PBD signal in solution and can concentrate antigen by the coating antibody is used. The principle of this method is illustrated in Figure 1. (a) Glass beads having a particle size of 50 p m are used as the carriers that can amplify PBD signal in solution and concentrate antigen by the coating antibody, and they are reacted with antigen in the cell. (b) Antibody labeled with a (15)
Tu,C. Y.; Kitamon, T.; Sawada, T.Anal. Chem. 1993, 65, 3631-3635.
material that absorbs the excitation beam and generates heat is reacted with antigen to form an immune complex of antibodycoated particle-antigen-labeled antibody. The abovementioned colloidal gold ultrafine particle is used as the material that absorbs the excitation beam. (c) When the sample is irradiated by the excitation beam, heat is generated by antibody-coated colloidal gold particles that exist in the form of immune complex at the bottom of the sample cell and by free antibody-coated colloidal gold particles that are suspended in solution. Though the free antibody-coated colloidal gold particles exist in solution in greater quantities than the colloidal gold particles forming part of the immune complex, no temperature field gradient from a fixed direction is produced with a small deflection of probe beam, because the free particles are suspended in solution. On the other hand, in spite of a very small quantity, the colloidal gold particles forming the immune complex produce a very large PBD signal because they exist on the carriers that amplify the signal and because they are fixed on the surface of the carriers, causing spatially heterogenous absorption. As a result, the PBD signal from the antibody-coated colloidal gold particles forming the immune complex is greater than that from the nonreacted material. Indeed, this phenomenon makes it possible to detect the target antigen without separating bound and free labels. However, when sample is irradiated by the excitation beam from above, as shown in Figure IC, the greater part of the beam is absorbed by colloidal gold particles in solution. Irradiating the sample from below may remove the effect of the absorption. In this study, we measured human a-fetoprotein by this method. EXPERIMENTAL SECTION Apparatus. A schematic illustration of the experimental setup is shown in Figure 2. The excitation beam was an argon laser (NEC Model GLG 3260) with an emission line of 514 nm, and its output power was 150 mW. The beam intensity was modulated by a mechanical chopper (NF Model 5584). The modulation frequency was set at 20 Hz to achieve the best signal-to-noise ratio. The modulated excitation beam was focused in a line shape with a cylindrical lens to be fitted to the size of sample cell. The sample was irradiated by the excitation beam in 2 directions: from above and from below. The measurement cell used was a four-face transparent cell (3 mm x 3 mm x 45 mm) for fluorometry. The probe beam was a He-Ne laser Uapan Laser Model JLH-pT11) with an emission line of 633 nm, and it was focused just above the sample by using a convex lens with a focal length of 100 mm. The deflection of the probe beam was detected by using the knife edge and a photodiode detection system (Hamamatsu). The signal from the photodiode was synchronously amplified with a two-phase lock-in amplilier (NF Model LI-575). In-phase and outof-phase signal outputs were sent to an A/D converter (Adtek Model R48&AD/2) and processed on a personal computer (NEC Model PC-9801DA). The sample was automatically scanned with a stepping motor-driven translation stage Cokyo Optoelectronics Model XYZ-1OOATA) in the x-direction. The scanning peak area was calculated according to a method described elsewhere.16 Reagents and Materials. Colloidal gold ultrafine particles 20-40 nm in size were prepared according to the method described by Fren et all7 The size of glass beads was 50 pm. (16) Harada, M.; Obata, S.; Kitamori,T.;Sawada, T.Anal. Chem. 1993,65,21812183. (17) Frens, G. Nature Phys. Sci. 1973, 241,20.
Analytical Chemistry, Vol. 67,No. 7,April 1, 1995
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Glass beads was purchased from Toushinrikou (lapan), and quartz glass beads were obtained from Nihon Gaishi gapan). (3-( (2-Aminoethyl) amino) propyl) trimethoxysilane was purchased from Shinetsu Chemistry, Japan, and glutaraldehyde from Wako Pure Chemicals, Japan. The sheep anti-human a-fetoprotein (AFP) antibody used was obtained by immunizing sheep with purified AFP antigen and subjecting the obtained antibody to aflinity purification. The AFP antigen for immunological reaction was AFP Japanese Reference Standard manufactured by the Japan National Institute of Health. Preparationof Antibody-CoatedGlass Beads. Quartz glass beads (mean particle size, 50 pm, Nihon Gaishi) and were 5 g of glass beads (mean particle size, 50 pm, Toushinrikou, Japan) were heated at 200 "C for 3 h. After cooling, the beads were placed in 12.5 mL of 36 mM (5.((2-aminoethyl)amino)propyl)trimethoxysilane solution and stirred at room temperature for 1 h. After the reaction, the reaction mixture was washed with distilled water and heated at 110 "C for 1.5 h. After the mixture cooled, 3 mL of 2.5%glutaraldehyde was added, and the mixture was stirred for 1 h at room temperature. After the mixture was washed with distilled water and phosphate-buffered saline (PBS, pH 7.2), sheep anti-human AFP antibody (diluted with PBS, 2 mg/mL, 2 mL) was added and allowed to stand for 1h at room temperature. After reaction, the mixture was washed with PBS and stored in PBS. Preparation of Antibody-CoatedColloidal Gold Particles. The antibody used was sheep anti-human AFP antibody. Colloidal gold solution (20 mL, 0.01%) was adjusted to pH 9.0 with 0.1 N NaOH. To this solution was added sheep anti-human AFP antibody (0.1 mg/mL, 1.2 mL), and the mixture was stirred for 15 min at room temperature. After reaction, 20 mL of 1%bovine serum albumin @SA)-PBS was added, and the mixture was gently stirred for 15 min at room temperature. After reaction, the mixture was centrifuged (11 000 rpm, 1 h, 4 "C) to separate 1280 Analytical Chemistry, Vol. 67,No. 7,April 7, 7995
antibody-coated colloidal gold particles and the supernatant. Twenty milliliters of 1%BSA-PBS was added to scatter antibodycoated colloidal gold particles, and the solution was centrifuged. After this procedure was repeated twice, 6.7 mL of 1%BSA-PBS was added to disperse the particles. The solution was adjusted to the h a 1 concentration of 0.03%and preserved. Preparation of Antibody-CoatedColloidal Gold ParticleAntigen-Antibody-Coated Glass Beads (Immune Complex). To a test tube containing 5 mg of antibody-coated glass beads was added 500 pL of AFP antigen diluted with 1%BSA-PBS (0, 2, 5, and 10 ng/mL), and the mixture was stirred at room temperature for 2 h. After reaction, the mixture was washed with physiological saline three times, and antibody-coated colloidal gold solution (0.03%, 100 pL) was added and stirred at room temperature for 2 h. After reaction, the mixture was washed with physiological saline three times, and finally PBS was added and the solution stored. The prepared immune complex was used as the sample for the preliminary experiment of homogeneous immunoassay. Methods and Procedure. (1) Optimization of AntibodyCoated Colloidal Gold Concentration. To fix the antibodycoated colloidal gold concentration to be used in homogeneous assay, PBD from antibody-coated glass beads alone precipitated in antibodycoated colloidal gold solution in the measurement cell was measured. The quantity of antibodycoated glass beads was 1mg, the concentrations of antibody-coated colloidal gold solution were 0, 0.002,0.004,0.008,0.02, and 0.03%. The excitation beam was directed toward the sample from above and from below. (2) Assay of Immune Complex in Antibody-Coated Colloidal Gold Solution (Preliminary Experiment). As the preliminary experiment for homogeneous immunoassay, PBD spectroscopy was performed for antibody-coated colloidal goldantigen-antibody-coated particle (immune complex) precipitated
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in antibodycoated colloidal gold solution in the measurement cell. The immune complex prepared at AFP antigen concentrations of 0,2,5, and 10 ng/mL was used. The concentrations of antibodycoated colloidal gold solution were 0, 0.002, 0.004, and 0.008%. (3) Immunoassay by Homogeneous System. As a homogeneous system, in a time course test protocol, AFP antigen (0, 10, or 1000 ng/mL, 100 yL) was added to a test tube containing 1mg of anti-AFP antibodycoated quartz glass beads and reacted at room temperature for 24 h. The system was transferred to the measurement cell, to which 100pL of antibodycoated colloidal gold solution (0.01%) was added. PBD signal was measured 0, 10, 30, 60, and 120 min after the addition of the colloidal gold solution to investigate the time course. The sample was irradiated by the excitation beam from bottom. As a next test, 100 pL of AFP antigen solution at each concentration was added to test tubes containing 1 mg of anti-AFP antibodycoated quartz glass beads, and the solution was stirred for 30 min. Then, 100pL of antibodycoated colloidal gold (0.01%) was added, and the solution stirred for 30 min and transferred to the measurement cell to measure PBD signal. RESULTS AND DISCUSSION Figure 3 shows the PBD signal from antibody-coated glass beads alone which were precipitated in antibody-coated colloidal gold solution when excitation beam was directed from above and from below the sample. As shown in the figure, when the sample was irradiated from above, PBD signal was sharply decreased with the increase in colloidal gold concentration. This decrease resulted from the absorption of the excitation beam by colloidal gold particles. The roughly estimated molar absorptivity at the absorp tion peak was on the order of lo4 M cm-2,18,19proving the strong optical absorption of the particles. PBD signal was hardly detected at 0.008%or higher colloidal gold concentrations. On the other hand, when the sample was irradiated from below, PBD signal (18) Van Dongen, J. J. M.; Hooijkaas, H.; Comans-Bitter, W. M.; Benne, IC;Van Os, T. M.; De Josselin de Jong, J. 1.Immunol. Methods 1985, 80, 1. (19) Doremus, R H. 1.Chem. Phys. 1964,40, 2389.
0
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AFP concentration (ng I ml) Figure 4. PBD signal from antibody-coated glass beads. Antigenantibody-coated colloidal gold particles in antibody-coated colloidal gold solution when sample was irradiated by the excitation beam from above. Colloidal gold concentration: 0% (0),0.002% (A), 0.004% (O), and 0.008% (0).
kept at a fixed value up to the colloidal gold concentration of 0.008%, but tended to increase at higher concentrations. This phenomenon indicated the augmentation in blank value with the increase in colloidal gold concentration. The reason why the signal from the excitation beam from below was higher might be that the signal from the glass beads was also included. The signal from the quartz glass beads should be lower than that of conventional glass beads because the quartz beads have no impurities. These results suggest that colloidal gold concentrations not exceeding 0.008%are favorable. Figure 4 shows PBD signal from immune complex precipitated in antibody-coated colloidal gold solution at less than 0.008%when the sample was irradiated from above. As shown in the figure, PBD signal was markedly decreased as the colloidal gold concentration increased. At 0.004% or higher concentrations, AFP was not detectable due to the absorption of excitation beam by colloidal gold particles. Figure 5 shows PBD signal when the sample was irradiated from below, toward the immune complex precipitated in antibodycoated colloidal gold solution. As shown in the figure, the elevation in colloidal gold concentration was not associated with a decrease in PBD signal. Because of irradiation from below, the excitation beam did not pass through the colloidal gold solution before reaching the sample, which prevented a large absorption of the beam. Figure 6 shows the time course change in PBD signal in the immunological reaction at AFP concentrations of 0, 10, and 100 ng/mL in colloidal gold solution. This indicates the binding rates of anti-AFP antibodies to AFP. PBD signal was not increased at the AFP concentration of 0 ng/mL. For the AFP concentration of 10 ng/mL, binding rates from PBD signal were constant during with the passage of time, but when an AFP concentration of 100 ng/mL is used, binding rates after the initial constant decrease as binding approaches a steady state level. From these results, the measurement by a homogeneous system was verified. In Figure 7, the determination curve of AFP in homogeneous system is shown. It shows a nonlinear curve resulting from the nature of the immunological reaction. It will Analytical Chemistty, Vol. 67,No. 7,April 1, 1995
1281
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Table I. Reproducibility of AFP Assays (within Run, n = 4)
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result was more sensitive by 1 order of magnitude than the latex agglutination method, the conventional homogeneous rapid assay, and it is expected that improvement of reagents and techniques may further enhance the sensitivity.
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Time (min) Figure 6. Time course of PBD signal in immunological reaction when the sample was irradiated by the excitation beam from below. AFP concentration (nglmL): 0 (0),10 (A), and 100 (0).
show a sigmoidal curve when a higher concentration of antigen is used. At 1h of reaction, the PBD signal for 0.25 ng/mL antigen was clearly distinguishable from the blank levels. It is difticult to calculate the exact lower limit of detection from the sigmoidal determination curve; however, with twice the fluctuation of the blank level, half of those values (0.125 ng/mL) can be detected as signscant differences. The reproducibility of four assays at 0.25 ng/mL had a coefficient of variation of 7.8% (Table l), an adequate level compared with other ultratrace immunoassays. This
1282 Analytical Chemistry, Vol. 67,No. 7,April 1, 1995
CONCLUSION In this paper, we proposed a novel homogeneous immunoassay using photothermal beam deflection spectroscopy: antibodycoated glass beads having a particle size of 50 pm and antibodycoated colloidal gold particles 20 nm in size were used, and an immune complex containing colloidal gold particles on antibodycoated glass beads was formed by immunological reaction. This sample was irradiated by an excitation laser beam from below, and PBD signal thus generated was measured. PBD signal was not affected by free antibody-coated colloidal gold particles suspended in solution up to the colloidal gold concentration of 0.008%. It is therefore revealed that homogeneous immunoassay is possible. The sensitivitywas investigated by measuring human AFP, and a detection limit of 0.25 ng/mL was obtained. This result was more sensitive by 1 order of magnitude than the latex agglutination method, the conventional homogeneous rapid assay. Some improvement of reagents and techniques can be expected to enhance the sensitivity further. Received for review August 26, 1994. Accepted January 24, 1995.@ AC940852F a Abstract published
in Advance ACS Abstracts, February 15, 1995.
