Anal. Chem. 1993, 65, 3031-3635
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Ultrasensitive Heterogeneous Immunoassay Using Photothermal Deflection Spectroscopy Chao-Yi Tu, Takehiko Kitamori, and Tsuguo Sawada' Department of Industrial Chemistry, Faculty of Engineering, The University of Tokyo, 7-3-1 Hongo, Bunkyo, Tokyo 113, Japan
Hiroko Kimura and Shigetaka Matsuzawa Department of Forensic Medicine, School of Medicine, Juntendo University, 2-1-1 Hongo, Bunkyo, Tokyo 113, Japan
A novel ultrasensitive, highly selective immunoassay method that uses gold ultrafine particles as the labeling material and laser-induced photothermal beam deflection quantitation is proposed. Ultrasensitivelaser spectrometric analysis of actual biological samples is always obstructed by the background and its fluctuation. However, we could adequately suppress this obstruction by substituting polystyrene microspheres in the antigen-antibody solid-phasereaction instead of the conventionalnitrocellulosemembranebecause the nonspecific adsorption velocity of the admixture proteins was highly restrained and the area of the antigen-antibody reaction was increased. The conditions of the antigen-antibody reaction were optimized using immunoglobulin G (IgG),and the method was shown to be applicable to the determination of trace and weak immunological reactive antigen in a highly dense biological matrix by determining trace IgE in human serum. A significant difference from the background was obtained for 8.4 X IU/mL using a 50-pL sample, corresponding to the absolute quantity of 2.0 pg (10.7 amol), a result that was at least 1 order of magnitude superior to results obtainedby EIAand RIA. INTRODUCTION A sensitive, simple, rapid immunoassay method that could take the place of radioimmunoassay (RIA) and enzyme immunoassay (EIA) would be desirable for medical and biochemical studies. Troublesome labeling materials and chemical reactions are unsuitable for the clinical diagnosis and basic study of tumors, hormonal abnormalities, and infectiousdiseasesthat require analysisof ultratrace biological substances. Therefore, various techniques to achieve ultrasensitive immunoassayhave been applied to antigen-antibody reactions, separation of antigen-antibody reaction products, labels, antibodies,and detection and determination methods.' We have proposed a photothermal immunoassay (PIA) in which antigen-antibody reaction products in turbid solutions were detected and determined ultrasensitively using laserinduced photoacoustic spectroscopy as a photothermal technique.2 We successfully demonstrated that PIA was at least 3 orders of magnitude more sensitive than the conventional
* To whom all correspondence should be addressed.
(1) Gosling, J. P. Clin. Chem. 1990, 36, 1408. (2) Kitamori,T.; Suzuki, K.; Sawada, T.; Cohshi,Y. Anal. Chem. 1987, 59, 2519. 0003-2700/93/0360-3631$04.0010
immunoassay with turbidimetry. However, as we pointed out, further reduction and stabilization of nonspecific (nonimmunological)background reactionsis required for practical application. Although demonstrations of ultratrace determination by laser spectrometry in pure chemicalsystems have shown promise,s background reduction and stabilizationare still serious problems in ultrasensitive laser spectrometry, particularly for immunoassay of ultratrace substances among many admixtures, as in serum. Therefore, in the present study, a heterogeneous immunoassay method in which a process for the separation of immunologicallybound and free particles is included in the procedure was introduced to PIA. The proposed method is a heterogeneous immunoassay that uses gold ultrafine particles as a labeling material. This method has been used as a simple semiquantitative In it, a liquid biological sample such as serum is dropped on a membrane f i i , antigens in the sample are adsorbed on the membrane, and then gold ultrafine particles coated with antibodies are fixed immunologically. The amount of the antigen is semiquantitatively estimated by eye as positive or negative from the red color tone of the stain on the membrane presented by the adsorbed gold ultrafine particles. Details of this method are described in the Experimental Section. This simple method is rather insensitive compared with other immunoblotting te~hniques.~J It has also been difficult to apply to quantitative assays because a suitable method of detecting the gold ultrafine particles on the membrane is lacking. Nevertheless, this method showed potential as an ultrasensitive immunoassay when a laser photothermal method was used for detection. Because the gold ultrafine particles possess a strong optical absorption band in the blue region (giving them a red color) owing to the size effect? they are chemically and optically stable against strong radiation and do not obstruct immunologicalactivity. Hence, the particles are a suitable labeling material for laser photothermal spectrometry aimed at immunoassay. The adsorbed labeling material is determined ultrasensitively by a photothermal beam deflection method (or optical beam deflection; OBD). This does not require a measurement cell, which is an (3) Maaujima, T.; Wada, K.; Yoshida, H.; Imai,H. J. Photoacoust. 1983,1, 347. (4) Treado, P. J.; Briggs, L. M.; Morris, M. D. J. Chromatogr. 1990, 511, 341. (5) Diamandis, E. P.; Christopoulos, T. K.; Bean, C. C. J . Imrnunol. Methods 1992, 147, 251. (6) Van Dongen,J. J. M.; Hooijkaas, H.; Comans-Bitter, W. M.; Benne, K.; Van Os,T. M.; De Josselin de Jong, J. J . Immunol. Methods 1986, 80, 1. (7) Kimball, S. R.; Rannels, S. L.; Elenky, M. B.; Jefferson, L. S., J. Immunol. Methods 1988,106, 217. (8) Heimgartner, U.; Kozulic, B.; Mosbach, K. J. Immunol. Methods 1990,132,239. (9) Doremus, R. H. J. Chem. Phys. 1964,40, 2389.
0 1993 Amerlcan Chemlcal Soclety
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ANALYTICAL CHEMISTRY, VOL. 65, NO. 24, DECEMBER 15, 1993
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X';CJ. Flgure 1. Schematic illustratlons to immunoblotting methods using colloidal gold ultrafine particles: (a) a conventional method which uses nitrocellulose membrane as the antigen-antlbody solidphase reaction; (b) a modified method in which polystyrene microspheres are introduced in the solid phase; (c)another modification of the doubie-antibodymethod. The latex mlcrospheres, coated wRh an antigen, were lmmunologlcally bound with antibodytoated colloidal gold. The microspheres were collected on a membrane filter from the unbound components: (d) the conventional method shown in a, and (e) the proposed methods shown in b and c.
advantage. In fact, regarding the sensitivity, we compared the densitometric and OBD methods in the quantitative measurement of dot-ELISA (enzyme linked immunosorbent assay) spots, which is one of the current immunoblotting methods.I0 The dot-ELISA spots on a porous membrane were visualized by the diaminobenzidine technique. We concluded that the OBD method could detect a t least about 1/10 of the quantities of the immunoglobulin E (IgE) and carcinoembryonic antigen (CEA) detected by densitometry. However, it is not sufficient to simply introduce the photothermal deflection technique a t the measurement step of the analytical procedure because of the background problem mentioned above. Although the gold ultrafine particle method is a heterogeneous method and quantitative detection can be carried out by introducing OBD, it is still difficult to apply the method to ultrasensitive laser spectrometry because conventional immunoblotting procedures and conditions are not designed for ultrasensitive measurements. In the present study, we developed a modified gold ultrafine method that uses polystyrene microspheres as a solid substrate and introduces immunoassay techniques to obtain a sufficiently low and stable background level for ultrasensitive measurement. Human IgG was used to optimize the procedure and to compare performance. Furthermore, this method was proved practical for actual biological samples by determining trace IgE in human serum, an immunoglobulin which is closely related to allergic reactions and requires very high selectivity and sensitivity for its assay. (10)Matsuzawa, S.; Kimura, H.; Tu,C.; Kitamori, T.; Sawada, T. J. Immunol. Methods 1993, 161, 59.
EXPERIMENTAL SECTION Methods and Procedures. Schematic illustrations of modified immunoblotting methods using colloidal gold ultrafine particles are shown in Figure 1 together with the conventional method. In these drawings,only one antibody is shown as bound to one gold ultrafine particle to simplifythe explanation,although several antibody molecules could presumably be bound to one particle based on the results of our preliminary stoichiometric experiment. The polystyrene microspheres, gold ultrafine particles, antibodies, and antigens are not in proportion to their actual sizes. The details of these methods are described in the Results and Discussion; however, in these heterogeneous immunoassays, the solid-phasereaction is a nitrocellulosemembrane or polystyrene latex microspheres, while the labeling material is gold ultrafine particles. The analyte antigen molecules are adsorbed on the reaction solid phases, and then the labeled antibody moleculesare fixed on the solid-phasereaction through antigen-antibody binding. Excess reagent (labeledantigen) and admixtures are removed by washing the solid phase. We introduced polystyrenemicrospheresfor the solid-phasereaction to obtain sufficient reduction of the background level for ultrasensitive detection, although the nitrocellulose membrane was used for the conventionalmethod. The optimized procedure and parameters of reaction conditionsare also given in the Results and Discussion section. Reagents and Materials. The colloidal gold ultrafine particles were prepared by Frens' method." Their size ranged from 20 to 40 nm. The diameter of the uniform polystyrene latex microspheres was 0.9 pm (HD 08-4, Takeda, Japan). Lyophilized and over 98% pure human IgG was purchased from Kokusai Shiyaku (Japan),and IgE standard serum was obtained (11) Frens, G. Nature Phys. Sci. 1973,242, 20.
ANALYTICAL CHEMISTRY. VOL. 65. NO. 24, DECEMBER 15, I993 Excltation beam
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h m Behringwerke (Germany); they were used without further purification. Rabbit anti-human I& was purchased from ICN Immuno-Biological(Israel). Affmity chromatographically purified goat anti-human IgE was obtained from TAG0 and rabbit anti-goat IgG from KPL Inc. A nitrocellulosemembrane (pore size 0.45 em, Schleicher-Schuell, Germany) and polycarbonate membrane (pore size 0.8 em, Costar Corp.) were used. Dot-ELISA for IgG was used, with a minor modification as a referenceanalysis accordingtothernethodpreviouslyreported."' For the assay of human IgG by the proposed PIA method, gold ultrafine particles coated with a rabbit antiserum tu human IgG wereusedasthereageot. WhenPIAwasappliedtoIgEin human serum,the double antibody method was also used. The primary antibody was goat anti-human IgE and the secondaryone rabbit anti-goat I& coated on gold ultrafine particles. The details of the coating conditions me given in the literature.'2 Apparatus. A schematic illustration of the laboratoryconstructed OBD system is shown in Figure 2. The excitation beamwasthe514.5-nmemissionlineofanArlaserwithanoutput of 250 mW, and it was focused on the sample line hy a cylindrical lens. The beam intensity was modulated by a mechanical chopper, and the modulation frequency was set a t 318 H Etu achieve the hest signal-to-noise ratio. The probe beam was a HeNelaser,passingabout 7Oemover themembrane. Deflection oftheprobe beamwasdetected byahifeedgeandaphotodiode. The signal from the photodiode was synchronously amplified while reducing the noise with a lock-in amplifier. It was then recorded by a computer system as data. The irradiation area of the excitation beam on the membrane was scanned by a pulsed stage. Examples of scanned OBD signals on the membrane are shown in Figure 3. The OBD signal corresponds to the distribution of polystyrene microspheres on the membrane that adsorbed gold ultrafiine particles. The scanning peak area was calculated, and the quantity of the gold ultrafiine particles was obtained from the mea. The photothermal beam deflection effect is quite sensitive notonlytoadsorhedmaterialsonasolidsurfacebutalsotosmall changes in offset of vertical probe beam, and this characteristic results in poor reproducibility and incorrect quantitative measurement. Thus, we developed a signal correction method by using phase and the OBD system installed with this correction function was used in the present experiments. The measurements of absorption spectra of materials on a membrane were carried out by a gas-coupled microphone-type pbotoacousticspectrometer (PAS),which was also a laboratoryconstructed system.
3033
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for photothermal spectrometry were investigated. The photoacoustic spectra of a gold thin fhand gold ultrafiie particles collected on a nitroeellulose membrane are shown in Figure 4. An absorption peak appears around 617 nm in the PA spectrum of the gold ultratine particles. This absorption band is considered to be a spatially localized plasmon band caused by the size effect of metal ultraijie particles;" hence, it did not appear in the bulk (thin film) gold. Owing to this absorption band, the gold ultrafine particles are red colored. This PA spectrum should be the most reliable absorption spectrum for the gold ultratine particles among spectra measured by various spectroscopic techniques such as turhidimetric and diffuse reflectance s p e c t r o m e t r i e ~because ~ ~ ~ PAS is hardly affected by light scattering. The roughly estimated molar absorptivity a t the absorption peak was in the order of 1W M c n - 2 , and it proved RESULTS A N D DISCUSSION the strong optical absorption of the particle. Hence, gold ultrafhe particles can be detected with high sensitivity by Before biological sampleg were measured, the characterOBD using the 514.5-nm line of the Ar laser as the excitation istieaofthegoldultr~meparticlegusedasalabelingmaterial beam. Therefore, this lasing line waa used for the following (12) Chaloaborty, U. R.;Black, N.;Brooks, H. G.. Jr.; Campbell, C.; measurements. Furthermore, the gold ultrafiie particles have Gluek,K.;Harmon,F.;Hollenbeek,L.;Lawler,S.;Lwison,S.;Maehnal, no serious effects on the immunological properties of antiD. Ann. Bid. Clin. (Poria) 1990,48,403. bodies because their coating on the gold u l t r a h e particles (13) Harsda, M.;Obsta, S.; Kitamori, T.;Sam& T.A d . Chem. 1993,65,2181. is based on nonchemical binding. In addition to their optical
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ANALYTICAL CHEMISTRY, VOL. 65, NO. 24, DECEMBER 15, 1993
and chemical stability, these characteristics of gold ultrafine particles prove that they are a suitable labeling material for laser excitation OBD measurement. In the conventional method (Figure la), a liquid biological samplesuch as serum is dropped on a nitrocellulosemembrane film. The antigen in the sample is adsorbed on and into the nitrocellulose membrane, which acts as a kind of filter. The sample stain on the film is reacted with a 0.1% ’ casein solution to form a casein protective layer on the sample spot to prevent nonspecific adsorption of the gold ultrafiie particles. After the casein protective layer is formed,the membrane is reacted with the suspensionof antibody-coatedcolloidal gold ultrafiie particles, and the gold ultrafhe particles are adsorbed on and into the membrane immunologically. Excess gold ultrafine particles and admixtures are removed by washing the membrane. Ordinarily, the quantity of adsorbed gold ultrafiie particles, which is in proportion to the antigen concentration, is estimated by eye from the shade of the stain. In our preliminary experiment, IgG could be determined quantitatively by OBD measurement of the gold ultrafine particle stains on the membrane. However, more effective background reduction is required to apply this method to an ultratrace and immunologically weak reactive antigen in actual biological samples. One of the main factors affecting background is nonspecific adsorption of the gold ultrafine particles on the membrane substrate during immersion in colloidal solution, as shown in Figure Id. While this background level is negligibly low for conventional measurements, it is a serious problem for ultrasensitive photothermal spectrometric measurements. Therefore, we proposed a modified method using latex microspheres for the solid substrate of the antigen-antibody reaction instead of the nitrocellulosemembrane, as shown in Figure l b and c. In our modification, the liquid sample is poured into a turbid solution of polystyrene latex microspheres, and the antigen is adsorbed on the latex microspheres (Figure lb). After casein blocking of the latex microspheres, the antibody-coated gold ultrafine particles are immunologi d l y fiied on the latex microspheres. The latex microspheres, colored by the adsorbed gold ultrafine particles, are collected on a membrane fiiter while excess particles and admixtures are washed away, as shown in Figure le. The gold ultrafine particles adsorbed on the latex microspheres are determined by OBD measurements. The idea of introducing latex microspheres for the solid phase of the antigen-antibody reaction instead of the nitrocellulosemembrane was based on the following reasons: To inhibit rapid, nonspecific adsorption of the various protein admixtures in actual biological samples, the solid-phase material was changed to polystyrene because the reaction velocity of protein adsorption on polystyrene is slower than that of nitrocellulose. The form of the solid phase was changed from the membrane to microparticlesto increase the reaction area and coating ability of the antigens or antibodies. High specific gravity (1.40) polystyrene microspheres were chosen for effective B/F separation by low speed centrifugation. By combiningall these effects,we expected sufficientbackground reduction and stabilization for OBD measurements. We evaluated the effect of introducing polystyrene microparticles for background reduction using IgG standard solution in phosphate-buffered saline. The evaluation was carried out using an antigen coating on the solid phase, as shown in Figure l a and b. The optimized parameters and conditions of assay were as follows: 50 p L of the IgG solution was added to 100 p L of 1% (w/w) polystyrene microsphere turbid solution; the optimal size of the polystyrene microspheres was 0.9 pm; IgG was adsorbed on the particles by overnight incubation a t 4 OC;the free surface of the particles
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was saturated by adding 100 pL of 0.1% casein solution followed by standing overnight; 100 p L of the rabbit antiIgGcoated gold ultrafine particle reagent was added to the IgG-adsorbed polystyrene microspheres after removal of excess antigen and other proteins; the antigen-antibody reaction was carried out overnight a t 4 “C;the polystyrene microspheres which adsorbed gold ultrafine particles by immunological binding were collected on a polycarbonate membrane (pore size 0.8 pm) without separation of bound and free gold particles. The same procedure and conditions were applied to the conventionalmethod wing a nitrocellulaee membrane as the solid phase. The factor of reaction time, which is the baeis for judging rapidity, was not considered in this step because the effect of introducing latex microspheres was evaluated in comparisonwith the conventionalmembrane method. According to the conventional immunoblotting procedure,6”however, the reaction time a t each step is 10-15 min to avoid nonspecific adsorption because the adsorption velwity of proteins on nitrocellulose is much faster than on polystyrene. The sample preparation time of the modified procedure for OBD measurement can be shortened a t room temperature by further improvement of the procedure. The results obtained using these two proceduresare plotted in Figure 6. Results obtained by the conventionaldot-ELISA method are also plotted. The dotted base stains of ELISA were also measured by OBD. Compared with the nitrocellulose membrane method, the signal change in the latex microaphere method is about 50 times larger; neverthela, the background level is stabilized at the control level and its rate of increase is restricted to about 1order of magnitude less thanthe improvementin signal magnitude. These results show that using polystyrene microspheres for the antigenantibody solid-phase reaction helps control the adsorption velocity to reduce nonspecificprotein adsorption and increase the reaction area. Furthermore, the results show that both the rate of signal change and limit of detection are improved by about 1order of magnitude in comparison with dot-ELISA measurement. The estimated limit of discrimination from the change in background level is about 0.2 ng/mL, which corresponds to 10pg (67 amol) of the absolute amount of I&, although it is difficult to defiie standard deviation for the sigmoidal determination curves that always appear in immunoassay. Next, we used the double-antibodymethod shown in Figure ICto obtain higher sensitivity in applying the assay to IgE in human serum. The first antibody forms immunological bond on the antigen-coatedlatex microspheres,and the second antibody-coated gold ultrafine particles are immunologically adsorbed on the latex microspheres. The IgE concentration
ANALYTICAL CHEMISTRY, VOL. 65, NO. 24, DECEMBER 15, 1993
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Table I. Reproducibility of IgE Assays by the Double Antibody PIA (within Run, n = 4) concn (IU/mL) mean (arb units) SD CV (To) 840 84
8.4 0.84 0.084 0.0084 O.OOO84
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in human serum is known to be very low compared with other classes of immunoglobulins. The quantitation of IgE is an important clinicaltest because this immunoglobulin is closely related to allergic reactions. The IgE in serum has been determined by radio immunodiffusion,RIA, and ELISA,lC16 but a simpler, more rapid, sensitive new method would be desirable. A series of standard human serum samples with a known quantity of IgE (Second International Standard, 830IU/mL) were used as a standard solution. For this assay, microtiter plates and ELISA washer (Dynatech) were employed. Like the previous IgG assay, the first immunological binding to form latex microsphere-IgE-anti-IgE complexes was completed after standing overnight a t 4 O C . Following casein quenching of the surface of the microspheres, 100 p L of the colloidal gold ultrafine particle reagent was added (4 "C, overnight) after washing in a centrifuge. The latex microspheres were collected on a polycarbonate membrane filter for OBD measurement of the gold ultrafine particles adsorbed on them. The results obtained are plotted in Figure 6, and they show a good sigmoidal curve. The OBD signal for 8.4 X 109 IU/mL was clearly distinguishable from the background and control levels. The absolute amount of IgE in this sample was 4.2 X 1V IU. It is difficult to convert international units of IgE to an exact weight or other molar unit. However, considering the reported immunological reactivityl4 and our preliminary fluorometric immunoassay measurement, the correspondingabsolute amount and molar amount were roughly estimated as 2.0 pg and 10.7 amol, respectively. It is also difficult to calculate the exact lower (14)Bazaral, M. B.;Hamburger, R. N. J.Allergy Clin. Zmmunol.1972, 49, 189. (15)Ceeka, M.; Lundkviet, U. Immunochemistry 1972,9,1021. (16)Hong, C.S.;Stadler, B. M.; Walti, M.; De Weck, A. L. J. Zmmunol. Methods 1986,64,195. (17)Labroue, H.; Avrameas, S. J. Immunol. Methods 1987,103,S. (18)Kemeny, D.M.;Richards,D.; Johanneeon,A.J.Zmmunol.Methods 1989,120,251.
8.30 8.20 4.36 2.75 1.33 1.10 1.00 1.00
1.343 1.057 0.321 0.282 0.044
0.043 0.039 0.057
16.18 12.89 7.36 10.26 3.30 3.91 3.49 5.70
limit of detection from the sigmoidal determination curve; however, with twice the fluctuation of the background level, half of those values (1.0 pg and 5.3 amol) can be detected as significant differences. The reproducibility of four time assays at 8.4 X 109 IU/mL had a coefficient of variation of 3.9% (Table I), an adequate level compared with other ultratrace immunoassays. The estimated lower limit of detection is the same or about 1 order of magnitude superior to those of EIA and RIA, and a t least 2 orders of magnitude superior to that of dot-ELISA.lb18 Furthermore, the introduction of further techniques to improve sensitivity and selectivity,such as the avidin-biotin double antibody method, should provide results a t least 1 order of magnitude superior to the reported ones. The proposed PIA method can determine antigens and antibodies in the ultratrace region in which conventional EIA has yielded results of zero or undetectable, and PIA is expected to provide another research tool for immunology and biochemistry as well as to enable the diagnosis of various diseases.
CONCLUSION In this study, we improved the colloidal gold immunoblotting method, which has been wed as a quasiquantitative tool for rather high concentration levels, to an ultrasensitive and quantitative immunoassay method. The improvement was mainly due to the design of the reaction system including the solid-phase reaction and labeling material as well to the high sensitivity and quantitative measurements provided by the laser-induced OBD method. In general, it will become more and more important to redesign and optimize conventional analytical procedures for ultrasensitive spectrometry of actual biological samples that have various kinds and large amounts of admixtures because the lower limit of detection of the equipment or the detection method itself has little relevance for these samples. RECEIVED for review May 10, 1993. Accepted September
21, 1993.' @
Abstract published in Adoance ACS Abstracts, November 1,1993.
