Anal. Chem. 1996, 68, 3551-3554
Chemiluminescence Enzyme Immunoassay Using Bacterial Magnetic Particles Tadashi Matsunaga,* Masashi Kawasaki, Xie Yu, Noriyuki Tsujimura, and Noriyuki Nakamura
Department of Biotechnology, Tokyo University of Agriculture and Technology, Koganei, Tokyo 184, Japan
A novel chemiluminescence enzyme immunoassay using bacterial magnetic particles (BMPs) has been developed for highly sensitive and rapid detection of immunoglobulin G. Antibody was immobilized onto BMPs using the heterobifunctional reagents sulfosuccinimidyl 6-[3′-(2pyridyldithio)propionamido]hexanoate (sulfo-LC-SPDP) and sulfosuccinimidyl 4-(N-maleimidomethyl)cyclohexane-1-carboxylate (sulfo-SMCC). For the highly sensitive immunoassay method using these BMPs, a good relationship was obtained between the luminescence intensity and mouse IgG concentration in the range of 1-105 fg/mL. Furthermore, in order to reduce assay time and to simplify operations, a rapid chemiluminescence enzyme immunoassay method has been developed. The rapid method was completed in 10 min. A linear relationship was obtained between the luminescence and mouse IgG concentration in the range of 10-1000 ng/mL. Radioimmunoassay still plays a major role in medicine and related areas1 due to its sensitivity, but radioactive materials are unwelcome in posttreatment. Therefore, enzyme immunoassay (EIA) has become an important analytical method in clinical diagnostics2 because of its sensitivity, specificity, and general applicability. Improvement of EIA in terms of reducing assay time and simplifying operations is one of the major trends in development of immunoassay technology.3 The use of magnetic particles in immunoassay enables separation of the bound and free analyte by application of a magnetic field. For example, proteins can be attached covalently to solid supports, such as magnetic particles, preventing the desorption of antibody during assay conditions. Because the particles are dispersed evenly throughout the reaction mixture, they allow rapid reaction kinetics without the need for continuous mixing or shaking, provide for the precise addition of antibody, and facilitate ease of use. The magnetic particles serve as both the solid support and the means of separation in the system. Amine-terminated magnetic particles (∼1 µm diameter) developed by Advanced Magnetics, Inc. (Cambridge, MA) are available commercially and have been used for solid phase immunoassay.4-7 The determination of chlorothalonil (2,4,5,6-tetrachloro-1,3-benzenedicarboni(1) Ekins, R.; Chu, F.; Micallef, J. J. Biolumin. Chemilumin. 1989, 4, 59-78. (2) Diamandis, E. P.; Christopoulos, T. K. Clin. Chem. 1991, 37, 625-636. (3) Kricka, L. J.; Phil, D.; Path, F. R. C. J. Clin. Immunoassay 1993, 16, 267271. (4) Lawruk, T. S.; Gueco, A. M.; Jourdan, S. W.; Scutellaro, A. M.; Fleeker, J. R.; Herzog, D. P.; Rubio, F. M. J. Agric. Food Chem. 1995, 43, 1413-1419. (5) Luk, J. M. C.; Lindberg, A. A. J. Immunol. Methods 1991, 137, 1-8. (6) Schlaeppi, J.-M. A.; Kessler, A.; Fory, W. J. Agric. Food Chem. 1994, 42, 1914-1919. (7) Yeung, J. M.; Newsome, W. H. Bull. Environ. Contam. Toxicol. 1995, 54, 444-450. S0003-2700(96)00369-1 CCC: $12.00
© 1996 American Chemical Society
trile), a broad spectrum fungicide, was carried out using magnetic particles-based enzyme immunoassay with a detection limit of 0.07 ng/mL.4 Magnetic bacteria have been isolated from fresh and marine sediments and are known to produce magnetic particles.8-11 Much research has been carried out regarding the mechanism of production of these magnetic particles and their function as navigational compasses in vivo. The bacterial magnetic particles (BMPs) are small in size (50-100 nm) and disperse very well because they are covered with a stable lipid membrane.12 Enzymes and antibodies have been immobilized on BMPs using both bifunctional reagents and glutaraldehyde and have been found to have higher activities than those immobilized onto artificial magnetic particles.13 On the basis of these properties, BMPs have been applied to fluoroimmunoassay,14-16 mRNA recovery,17 and DNA carriers.18 In this study, to develop highly sensitive immunoassay using antibody-immobilized BMPs, we employed alkaline phosphatase instead of fluorescein isothiocyanate for a label to chemiluminescence EIA. Furthermore, in order to reduce assay time and to simplify operations, we developed a chemiluminescence EIA using antibody-immobilized BMPs for the determination of mouse immunoglobulin G (IgG). EXPERIMENTAL SECTION Materials. Sulfosuccinimidyl 6-[3′-(2-pyridyldithio)propionamido]hexanoate (sulfo-LC-SPDP) and sulfosuccinimidyl 4-(Nmaleimidomethyl)cyclohexane-1-carboxylate (sulfo-SMCC) were purchased from Pierce (Rockford, IL), and Lumi-phos 530, which includes lumigen PPD [4-methoxy-4-(3-phosphonophenyl)spiro(1,2-dioxetane-3,2′-adamantane] disodium salt (3.3 × 10-4 M) as fluorophore, was obtained from Wako Pure Chemical Industries, Ltd. (Osaka, Japan). Bovine serum albumin (BSA) and mouse IgE were purchased from Seikagaku Co. (Tokyo, Japan). Mouse IgG, goat anti-mouse IgG antibody, and alkaline phosphatase(8) Blakemore, R. P. Science 1975, 190, 377-379. (9) Matsunaga, T.; Tadokoro, F.; Nakamura, N. IEEE Trans. Magn. 1990, 26, 1557-1559. (10) Matsunaga, T.; Sakaguchi, T.; Tadokoro, F. Appl. Microbiol. Biotechnol. 1991, 35, 651-655. (11) Sakaguchi, T.; Burgess, J. G.; Matsunaga, T. Nature 1993, 365, 47-49. (12) Balkwill, D. L.; Maratea, D.; Blakemore, R. P. J. Bacteriol. 1980, 141, 13991408. (13) Matsunaga, T.; Kamiya, S. Appl. Microbiol. Biotechnol. 1987, 26, 328-332. (14) Nakamura, N.; Hashimoto, K.; Matsunaga, T. Anal. Chem. 1991, 63, 268272. (15) Nakamura, N.; Burgess, J. G.; Yagiuda, K.; Kudo, S.; Sakaguchi, T.; Matsunaga, T. Anal. Chem. 1993, 65, 2036-2039. (16) Nakamura, N.; Matsunaga, T. Anal. Chim. Acta 1993, 281, 585-589. (17) Sode, K.; Kudo, S.; Sakaguchi, T.; Nakamura, N.; Matsunaga, T. Biotechnol. Tech. 1993, 7, 688-694. (18) Takeyama, H.; Yamazawa, A.; Nakamura, C.; Matsunaga, T. Biotechnol. Tech. 1995, 9, 355-360.
Analytical Chemistry, Vol. 68, No. 20, October 15, 1996 3551
conjugated anti-mouse IgG antibody (ALP-Ab) were purchased from Cosmo Bio (Tokyo, Japan). Other reagents were of analytical-reagent or laboratory grade. Deionized, distilled water was used in all procedures. Preparation of Bacterial Magnetic Particles (BMPs). BMPs were isolated from the magnetic bacterium Magnetospirillum sp. AMB-1 by the following method. Wet cells (∼1.4 mg) suspended in 10 mL of water were disrupted by three passes through a French pressure cell at 1300 kg/cm2 (Ohtake Works Co. Ltd., Tokyo, Japan). BMPs were collected from the disrupted cell fraction by using a neodymium-boron (Nd-B) magnet (10 mm × 10 mm × 6 mm) that produced an inhomogeneous magnetic field (0.37 T on the surface of the magnet). BMPs were collected at the bottom of the tube due to the presence of the magnet, and the supernatant was removed. The collected BMPs were washed with 10 mM phosphate-buffered saline (PBS, pH 7.4) using ultrasonic cleaner CA 4481 (Kaijo Denki Co. Ltd., Tokyo, Japan) at least three times and kept at 4 °C in PBS containing 0.01% sodium azide before use. Immobilization of Antibody onto BMPs. For the immobilization of antibody onto BMPs, we modified the method reported by Hashida et al.19 At first, sulfo-SMCC (0.06 mg) was added to 1 mL of anti-mouse IgG antibody solution (1 mg/mL) and incubated for 2 h at room temperature. After incubation, the sample was purified using a NAP-10 column (Pharmacia, Uppsala, Sweden), eluting with PBS according to the manufacturer’s instructions. On the other hand, 1.2 mg of sulfo-LC-SPDP was added to 2 mL of BMPs suspension (1 mg/mL). The suspension was then dispersed by sonication and incubated for 2 h at room temperature. After the incubation, the sulfo-LC-SPDP-modified BMPs were separated magnetically from reaction mixture using a Nd-B magnet and washed three times with 1.0 mL of PBS. The sulfo-LC-SPDP-modified BMPs were dispersed in 2 mL of 20 mM dithiothreitol in phosphate buffer containing 100 mM NaCl and incubated for 1 h at room temperature. After being washed three times, the modified BMPs were incubated with the sulfoSMCC-modified anti-mouse IgG solution for 12 h at 4 °C. Antimouse IgG antibody-immobilized BMPs (Ab-BMPs) were washed with PBS three times to remove excess antibody. Highly Sensitive Chemiluminescence EIA of Mouse IgG Using Ab-BMPs and ALP-Ab. Mouse IgG solution (500 µL) was mixed with Ab-BMPs (50 µg) in the test tube and incubated for 1 h at room temperature. Antigen-antibody complex was collected with a Nd-B magnet and washed three times with PBS. BMPs were dispersed by sonication in 200 µL of alkaline phosphatase-conjugated antibody solution (60 ng/mL) and incubated for 1 h at room temperature. After incubation, antigenantibody complex was collected with a Nd-B magnet and washed five times by PBS to remove excess alkaline phosphataseconjugated antibody. Lumi-phos 530 (300 µL) was then added to antigen-antibody complex and dispersed by sonication. After 30 min of incubation at 37 °C, the luminescence intensity was measured using a BLR-301 luminescence reader (Aloka Co., Ltd., Tokyo, Japan). Rapid Chemiluminescence EIA of Mouse IgG. A rapid and simple chemiluminescence EIA procedure was developed as follows. Ab-BMPs (100 µg) and 10 µL of ALP-Ab (100 µg/mL) was added to 500 µL of mouse IgG solution. The mixture was (19) Hashida, S.; Imagawa, M.; Inoue, S.; Ruan, K.-H.; Ishikawa, E. J. Appl. Biochem. 1984, 6, 56-63.
3552 Analytical Chemistry, Vol. 68, No. 20, October 15, 1996
Figure 1. Relationship between luminescence intensity and the amount of magnetic particles. Mouse IgG solution (500 µL: O, 0 pg/ mL; 2, 1 pg/mL) was mixed with Ab-BMPs and incubated for 1 h at room temperature. Antigen-antibody complex was collected with a Nd-B magnet and washed three times with PBS. BMPs were collected and dispersed by sonication in 200 µL of alkaline phosphatase-conjugated antibody solution (60 ng/mL) and incubated for 1 h at room temperature. Lumi-phos 530 (300 µL) was then added to antigen-antibody complex and incubated for 30 min at 37 °C, and luminescence intensity was measured.
dispersed by sonication and incubated for 5 min at room temperature. After incubation, antigen-antibody complex was separated magnetically from the reaction mixture using a Nd-B magnet and washed five times with 0.5 mL of PBS. Lumi-phos 530 (500 µL) was then added to the antigen-antibody complex and dispersed by sonication. Luminescence intensity was measured at 37 °C using a BLR-301 luminescence reader, and the integrated values for 5 min were evaluated as luminescence. RESULTS AND DISCUSSION Preparation of BMPs and Immobilization of Anti-IgG Antibody on BMPs. When BMPs were prepared by the French press and ultrasonication method, they were well dispersed in buffer. The size of 70 wt % of BMPs ranged from 50 to 120 nm (mean size 100 nm), and the other 30 wt % was aggregated in the size range of 250-700 nm. Modified antibody was immobilized on BMPs activated with SPDP. The extent of antibody coupling with BMPs was 54 µg/mg of particles. Highly Sensitive Chemiluminescence EIA Using Ab-BMP and ALP-Ab. Optimum Assay Conditions. Figure 1 shows the relationship between the luminescence intensity and the amount of magnetic particles. The luminescence intensity increases with increasing amount of Ab-BMPs in the range from 10 to 30 µg. It was suggested that, when more than 30 µg of antibody-conjugated BMPs was employed, IgG was sufficiently bound to Ab-BMPs. But, when more than 70 µg of antibody-conjugated BMPs was used, the luminescence intensity decreased. Optimum amounts of Ab-BMPs were 30-70 µg. It was suggested that, when the amount of BMPs was high, BMPs aggregated in the buffer and blocked luminescence themselves. Optimization of buffer pH for assay was also carried out. Maximum luminescence intensity occurred at pH 7.5. Buffer pH affected BMP dispersion and antibody reactivity. In this case, these results suggested that BMPs dispersion and antibody reactivity were optimized in neutral pH.
Figure 2. Correlation between luminescence intensity and mouse IgG concentration using ALP-Ab and Ab-BMP. The experiments were performed under various mouse IgG concentration and under the same conditions, except for the amount of BMPs (50 µg).
Determination of Mouse IgG Concentration. As noted above, the conditions for sensitive chemiluminescence EIA were optimized, and then the measurement of IgG concentration was carried out. A good relationship was obtained between the luminescence intensity and IgG concentration in the range of 1-105 fg/mL (Figure 2). The maximum detectable concentration of IgG was 105 fg/mL. This is because the immunological reaction of anti-IgG antibody was saturated. The minimum detectable concentration of IgG was 1 fg/mL (6.7 zmol; 4000 molecules as calculated from Avogadro’s number). For this reason, antibody was specifically immobilized onto the BMPs, which were also dispersed. We have reported the development of fluoroimmunoassay using Ab-BMP.14 Mouse IgG concentration could be detected in the range of 0.5-100 ng/mL. In this paper, the minimum detectable concentration of mouse IgG by sensitive chemiluminescence EIA was 5 × 108 times higher than that of fluoroimmunoassay using Ab-BMP.14 Rapid Chemiluminescence EIA of Mouse IgG. Immunoreaction of ALP-Ab, Mouse IgG, and Ab-BMPs. To reduce the antigen-antibody reaction time, the time course of antigenantibody reaction of ALP-Ab, mouse IgG, and Ab-BMPs was examined. Then, 500 µL portions of mouse IgG standard samples (1000 ng/mL) were mixed with ALP-Ab and Ab-BMPs in the test tubes, and the tubes were incubated at room temperature. After washing five times, the luminescence was measured. Figure 3 shows the time course of the antigen-antibody reaction of ALPAb, mouse IgG, and Ab-BMPs. The luminescence indicates the amount of antigen-antibody complex formed. The increase in luminescence shows that the immunoreaction continues for more than 20 min. The luminescence in the presence of 1000 ng/mL mouse IgG increases linearly when the immunoreaction time was less than 5 min. Although extending the immunoreaction time makes this assay sensitive, the immunoreaction time was fixed at 5 min in the following experiments. Optimum Assay Conditions. The amount of Ab-BMP was found to be a significant factor for optimal conditions. The amount of Ab-BMP influenced the antibody amount on the surface of the BMPs and blocked the luminescence during measurement. Therefore, the correlation between the amount of Ab-BMP and the luminescence on determination of mouse IgG concentration was examined, and the results were shown in Figure 4. When the Ab-BMP was 25-100 µg, the luminescence increased with
Figure 3. Time course of luminescence based on antigen-antibody reaction. Ab-BMPs (100 µg) and ALP-Ab (10 µL, 100 µg/mL) were added to 500 µL of mouse IgG solution: O, 0 ng/mL; b, 1000 ng/ mL. The mixture was dispersed by sonication and incubated for 5 min at room temperature. After incubation, antigen-antibody complex was separated magnetically using a Nd-B magnet. Lumi-phos 530 (500 µL) was then added to the antigen-antibody complex. Luminescence intensity was measured at 37 °C using a luminescence reader. The integrated values in each time were evaluated as luminescence.
Figure 4. Correlation between amount of Ab-BMP and luminescence on determination of mouse IgG concentration: O, 0 ng/mL; b, 1000 ng/mL. The experiments were performed under the same conditions, except for the amount of BMPs. The luminescence was expressed as the integrated values of luminescence intensity for 5 min.
increasing amount of Ab-BMP in the presence of 1000 ng/mL mouse IgG. This was caused by increasing the amount of antigen-antibody complex formed. On the other hand, the luminescence decreased with increasing amounts of Ab-BMP in the range 100-250 µg. This was caused by BMPs blocking luminescence during measurement. The luminescence in the absence of mouse IgG varied little with increasing amounts of Ab-BMP. This indicates that the most effective amount of AbBMP for this assay is 100 µg. ALP-Ab concentration also affects the luminescence based on ALP-Ab bound to IgG-Ab-BMP conjugates. The luminescence increased with increasing ALP-Ab concentration until 100 and 300 µg/mL when 1000 and 10 ng/mL IgG was employed, respectively. Therefore, 100 µg/mL ALP-Ab was used for further experiments. Determination of Mouse IgG Concentration. Mouse IgG concentration was measured by rapid and simple chemiluminescence EIA using Ab-BMP and ALP-Ab. Figure 5 shows the relationship Analytical Chemistry, Vol. 68, No. 20, October 15, 1996
3553
Table 1. Specificity of the Determination of Mouse IgG protein
relative luminescencea
none mouse IgG BSA mouse IgE IgG + BSA + IgE
0.11 ( 0.02 1.00 ( 0.04 0.16 ( 0.03 0.18 ( 0.03 1.06 ( 0.04
a Luminescence obtained from mouse IgG was regarded as 1.00. Concentrations of each protein and Ab-BMP were adjusted to 1000 ng/mL and 100 µg/mL, respectively.
Figure 5. Correlation between luminescence intensity and mouse IgG concentration using Ab-BMP and ALP-Ab. A immunoreaction was carried out for 5 min. Concentration of Ab-BMPs was adjusted to 100 µg/mL.
between the luminescence and mouse IgG concentration. The luminescence increased with increasing mouse IgG concentration. A linear relationship was obtained between the luminescence and mouse IgG concentration in the range 10-1000 ng/mL. The minimum detectable concentration of mouse IgG was 10 ng/mL. Luminescence was reproducible, with coefficients of variation of 7.7% and 8.2%, when samples containing 1000 and 10 ng/mL IgG were measured eight times, respectively. BMP-synthesized Magnetospirillum sp. AMB-1 was covered with 98% lipids and 2% other compounds, including proteins. Lipids consisted of 58% phospholipids and 42% other lipids. This lipid membrane makes BMPs negatively charged. There was slight aggregation in each particle as a result of its own magnetic properties. BMPs were superior in dispersion to artificial magnetite particles of the same size in aqueous solution.14 Table 1 shows the specificity of the determination of mouse IgG in this assay. The concentration of each protein was adjusted
3554 Analytical Chemistry, Vol. 68, No. 20, October 15, 1996
to 1 µg/mL. Except for mouse IgG, the luminescence scarcely increased. The luminescence increased only in the presence of mouse IgG. Luminescence was observed attributable to nonspecific binding between BMP and alkaline phosphatase-conjugated anti-mouse IgG in the experiments in the case of BSA and IgE. These results suggested that the increase of the luminescence was caused by an antigen-antibody-specific reaction. This method demonstrates the advantages of BMPs for the rapid and simple chemiluminescence EIA. The assay described allows results to be obtained in about 10 min, whereas the fluoroimmunoassay using Ab-BMP14 allows results to be obtained in about 15 min. Studies have shown the EIA using Ab-BMPs to be accurate compared with the EIA using antibody-immobilized artificial magnetite particles, and that the specific determination of mouse IgG was possible in the presence of other proteins. Furthermore, this method may be generally applicable to the determination of any suitable antigen.
Received for review April 16, 1996. Accepted August 1, 1996.X AC9603690 X
Abstract published in Advance ACS Abstracts, September 1, 1996.
