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Anal. Chem. 1991, 63,268-272
applied excitation a t 2w, is minimal.
CONCLUSIONS A new form of mass discrimination in FTMS has been observed and is attributed to interaction of a nonuniform excitation field with ion clouds that are initially asymmetrically distributed in the cell. Trapping frequencies in a single section cell may be observed directly in this way. Evaluation of the effect in a dual-cell instrument during an ion equilibration experiment indicates mass-dependent shifts as high as 60% occur, which can have significant impact on quantitative measurements. A mechanism for damping of the oscillation is unclear, but truncation of the periodic behavior within a few milliseconds is not consistent with ion-ion or ion-neutral collisions; alternately, there appears to be some correlation between the onset of a strong magnetron oscillation and the cessation of the trapping event. The data presented also indicate that the dual-cell ion equilibration pulse sequence will be a useful tool in probing the z axis evolution of the ion cloud during FTMS experiments. LITERATURE CITED Giancaspro. C.; Verdun, F. R . Anal. Chem. 1988, 5 8 , 2097-2099. Giancaspro, C.; Verdun. F. R.; Muller, J. F. Int. J . Mass Spectrom. Ion Processes 1988, 7 2 , 63-71. Hofstadier, S. A,; Laude, D. A., Jr. J. Am. SOC.Mass Spec. 1990, 7 , 35 1-360. Hogan, J. D.; Laude, D. A., Jr. Anal. Chem. 1990, 6 2 , 530-535. Hogan, J. D.: Laude, D. A.. Jr. Unpublished materials. Cody, R. B.; Kinsinger, J. A.; Ghaderi, S.; Amster. I.J.; McLafferty, F. W.; Brown, C. E. Anal. Chim. Acta 1985, 178, 43-66.
(7) (8) (9) (10) (11) (12) (13) (14) (15) (16) (17) (18) (19) (20)
(21) (22) (23) (24)
Wise, M. B. Anal. Chem. 1987, 5 9 , 2289-2293. Kerley. E.; Russell. D. Anal. Chem. 1989. 61, 53-57. Dunbar, R. C. Int. J. Mass Spectrom. Ion Processes 1985. 56, 1-7. Wang, M.; Marshall, A. G. Anal. Chem. 1990, 62, 515-520. Hanson, C. D.; Castro, M. E.: Kerley. E. L.; Russell, D. H. Anal. Chem. 1990, 6 2 , 520-526. Wang, M.; Marshall, A. G. Anal. Chem. 1989, 61, 1288-1293. Morse, P. M.; Feschback, H. Methods of Theoretical Physlcs; McGraw-Hili: New York, 1953; pp 1252-1260. Sharp, T. E.; Eyler, J. R.; Li, E. Int. J. Mass Specfrom. Ion Phys. 1972, 9 , 421-439. Hofstadler, S. A.; Laude, D. A., Jr. Int. J . Mass Specfrom. Ion Processes, in press. Hunter, R. L.; Sherman, M. G.; McIver, R. T., Jr. Int. J. Mass Specfrom. Ion Phys 1903, 50, 47-54. Alleman, M.: Kofel, P.; Kellerhals, Hp.; Wanczek, K. P. Int. J . Mass. Spectrom. Ion Processes 1987, 75, 47-54. Van De Guchte, W. J.; Van Der Hart, W. J. Int. J. Mass Spectrom. Ion Processes 1990, 95, 317-326. Hwng, S. K.; Rempei, D. L.; Gross, M. L. Int. J . Mass Specfrom. Ion Processes 1986, 72, 15-31. Van De Guchte, W. J.; Van Der Hart, W. J. Int J. Mass Spectrom. Ion Processes 1908, 8 2 , 17-32. Delong, S. E.; Mitchell, D. W.; Cherniak, D. J.; Harrison, T. M. Int. J . Mass Spectrom. Ion Processes 1909, 9 1 , 273-282. Alleman, M.: Kellerhals, H. P.; Wanczek, K. P. Chem. Phys. Lett. 1981, 84. 547-551. Chen, L.; Marshall, A. G. Int J. Mass Spectrom. Ion Processes 1987, 79, 115-125. Dunbar, R. C.; Chen, J. H.; Hays, J. D. Int. J. Mass Specfrom. Ion Processes 1984, 5 7 , 39-56.
.
RECEIVED for review August 27,1990. Accepted October 29, 1990. This work is supported by the Welch Foundation and by a grant from the Texas Advanced Technology and Research Program.
Immunoassay Method for the Determination of Immunoglobulin G Using Bacterial Magnetic Particles Noriyuki Nakamura, Kohji Hashimoto, and Tadashi Matsunaga*
Department of Biotechnology, Tokyo University of Agriculture and Technology, Koganei, Tokyo 184, Japan
We have developed a novel immunoassay method using bacterlai magnetic partlcles for the determination of Immunoglobulin G (IgG). Fluorescein Isothbcyanate (FITC) conJugatedant1 IgG-bacterial magnetk partlcles were prepared. The fluorescence quenching caused by agglutination of FITC-ant1 IgG antibody-bacterial magnetic partlcie conjugates was measured by uslng a fluorescence spectrophotometer. The aggregates based on speclfk knmunoreactlon were separated by a gelatin solution. The aggregation of bacterial magnetic partlck conjugates was enhanced by appllcatlon of a magnetic field. The relative fluorescence intensity correlated itnearly with a concentration of IgG in the range 0.5-100 ng/mL.
INTRODUCTION Determination of serum immunoglobulin levels is of value in various clinical fields. A variety of techniques has previously been employed in solid-phase immunoassay, for example, the use of latex particles ( I ) and liposomes (2). Agglutination reactions which use carriers are often used due to their im-
* Corresponding author.
proved handling in the laboratory. In these methods, the solid phase on which the immunological reaction takes place must be separated from the reaction solution by centrifugation before the measurements are carried out. The use of magnetic particles allows separation of the bound and free antibody fractions by application of a magnetic field. This technique also overcomes the problem of mixing during the incubation period (3-7). Magnetotactic bacteria, which orient and swim along geomagnetic fields, are known to produce magnetic particles (8). Purified bacterial magnetic particles (BMPs) are small in size (500-1000 A) and disperse very well because they are covered with a stable lipid membrane (9-11). Large quantities of bioactive substances may be immobilized on BMPs due to the presence of this membrane. Moreover, the location of the immobilized bioactive substance can be controlled with a magnetic field. This paper describes the immobilization of anti-mouse IgG antibody onto BMPs. In addition, a novel solid-phase fluoroimmunoassay using antibody immobilized onto BMPs has been developed for the rapid determination of mouse IgG concentration. EXPERIMENTAL SECTION Materials, FITC-conjugated goat anti-mouse IgG antibody was obtained from Sigma Chemical Co. (St. Louis, MO). The
0003-2700/91/0363-0268$02.50/00 1991 American Chemical Society
ANALYTICAL CHEMISTRY, VOL. 63, NO. 3, FEBRUARY 1, 1991
mouse IgG whole molecule was obtained from Jackson Immunoresearch Laboratories, Inc. (West Grove, PA). Gelatin was obtained from Merck (Darmstadt, West Germany). 142Hydroxyethyl)piperazine-4-2-ethanesulfonicacid (HEPES) and polyoxyethene (20) sorbitan monolaurate (Tween 20) were purchased from Wako Chemical Industries, Ltd. (Osaka, Japan). Other reagents were commerciallyavailable reagents or laboratory grade materials. Distilled water was used in all procedures. Cultivation of Magnetotactic Bacteria. Helical-shaped magnetotactic bacteria were isolated from sludge obtained from ponds in Tokyo and were cultured as previously described (12). This medium contained, per liter, 10 mL of Wolfe's vitamin solution, 5 mL of Wolfe's mineral solution, 2 mL of ferric quinate solution (in 100 mL of water containing 0.27 g of FeC13and 0.19 g of quinic acid), 0.45 mL of 0.1% resazurin, 0.68 g of KH2POF 0.12 g of NaN03, 0.05 g of sodium thioglycolate, 0.37 g of tartaric acid, 0.37 g of succinic acid, and 0.05 g of sodium acetate. The pH of the medium was adjusted to 6.75 with NaOH solution before sterilization. The cells were cultured in 5 L of medium under microaerobic conditions at 25 "C for 7-10 days. Magnetotactic bacteria grown to stationary phase (about 2 X l@cells/mL) were centrifuged at 5o00g for 10 min at 4 "C. The collected cells were washed with 10 mM HEPES buffer (pH 7.4). Preparation of BMPs. BMPs were extracted from magnetotactic bacteria by the following methods. (1)Ultrasonication: Magnetotactic bacteria suspended in 20 mL of HEPES buffer (about lo1*cells in total) were disrupted by using an ultrasonic disruptor (Tomy Seiko Co. Ltd., Tokyo, Japan; UR-POOP) operated for 5 min at 0 "C over 10 times. BMPS were collected from the sonicated cell fraction by using a samarium-cobalt (SmCo) magnet (18 x 11 X 14 mm) that produced an inhomogeneous magnetic field (0.4 T on the surface of the magnet and an average gradient of 0.2 T/cm). BMPs collected at the bottom of the tube due to the presence of the magnet, and the supernatant was removed. BMPs were washed with HEPES buffer a t least five times. (2) Lysozyme treatment: Approximately 1O'O cells were incubated with 1 mL of 0.1% lysozyme solution for 1 h at 37 "C before 0.1 mL of 10% SDS solution was added. BMPs were separated from the cellular disrupted fraction as described above. (3) Lysozyme and 5 N NaOH treatment: BMPs were extracted after lysozyme treatment of the cells and were then incubated with 5 N NaOH for 12 h at room temperature. (4) Ultrasonication and organic solvent treatment: BMPs were extracted by ultrasonication. Organic thin films covering the magnetic particles were then extracted with chloroform-methanol (2:1, v/v) for 1 h at room temperature. For electron microscopy, various types of BMPs were placed on the surface of carbon-coated collodion-covered mesh grids and air-dried. Electron microscopy was carried out by using a JEOL JEM 200CX high-resolution transmission electron microscope and a Hitachi (Tokyo, Japan) H-700H transmission electron microscope. Determination of Mean Particle Size a n d Aggregation. BMP concentration was adjusted to 130 pg/mL in water, and the particles were dispersed by using a sonication tub (Tocho, UC-0310 100 W). Particle size was measured with a particle size analyzer (Shimadzu, Kyoto, Japan; SA-CP3: multimode, Accel 240 rpm/min). The dispersive nature was estimated by the time ( t , = 0 . 5 ~ that ~ ) the absorbance of particle suspension was reduced to half of the initial value (ao) caused by aggregation. BMP samples of the following concentrations were prepared 250, 125, and 63 pg/mL. Absorbance (at 660 nm) was measured by using a Hitachi 320A spectrophotometer. Lipid Analysis of Magnetosome Membranes. BMPs were extracted from magnetotactic bacteria by passing cell samples through a French press (1000 kg/cm2) at 4 "C five times. The lipids of the magnetosome membrane that covers the BMP were extracted with chloroform-methanol as described by Kates (13) and dissolved in the same solvent. Fatty acids were hydrolyzed and esterified by methanolic hydrogen chloride. The methyl ester of the fatty acids was extracted with hexane and analyzed by gas chromatography (Hitachi, 163) using a DEGS column. Thin-layer chromatography (TLC) was performed on a silica gel plate (silica gel 60F254,Merck) with chloroform-methanol-water (65:25:4, v/v/v) as the development solvent. Phospholipid, glycolipid, cholinelipid, aminolipid, amidolipid, and carbonyllipid were examined by using Dittmer reagents (14). The colors and Rfvalues
289
were compared with standard samples. Colored spots were removed from the TLC plate and used for the determination of phospholipid concentration. Immobilization of FITC-Conjugated Anti IgG Antibody onto BMPs. BMPS were found to be covered with a lipid membrane containing amino residues as shown in the results section. FITC-conjugated anti IgG antibody was directly immobilized onto the magnetic particles by modifying the lipid membrane with glutaraldehyde. A 5-mg sample of BMPs was dispersed by sonication and incubated with 2.5% glutaraldehyde solution in 1 mL of phosphate-buffered saline (PBS, pH 7.4) for 1h at room temperature. The modified BMPs were washed with PBS, dispersed, and incubated with FITC-conjugated anti IgG antibody for 12 h at 4 "C. Antibody-coupled BMPs were washed with PBS several times to remove excess antibody and kept at 4 "C in PBS. Artificial magnetite particles (lo00 A in diameter) obtained from TDK Corp. (Tokyo, Japan) were washed with PBS and dispersed by sonication. A 5-mg sample of particles was then incubated with 1 mL of (y-aminopropy1)triethoxysilane(yAPTES) for 10 min at room temperature. Magnetite particles coated with 7-APTES were washed with PBS, dispersed, and incubated with 2.5% glutaraldehyde solution for 1 h at room temperature. The modified magnetite particles were incubated with FITC-conjugated anti IgG antibody. FITC-anti IgG antibody-magnetite particles were kept at 4 "C in PBS. The concentration of antibody in the solution was determined by the Lowry method (15) before and after immobilization, and the quantities of antibody immobilized onto BMPs were calculated. Fluoroimmunoassay of IgG by Using FITC-Conjugated Anti IgG Antibody Immobilized onto BMPs. IgG standard samples were prepared by appropriately diluting the IgG whole molecule in PBS. They (10 pL) were diluted into 100 pL of gelatin veronal buffer (GVB; pH 8.3) containing Tween 20 (0.16 ~01%). The BMP suspension (1mL) containing 100 fig of FITC-anti IgG antibody-BMP conjugates and 100 fiL of each diluted standard were mixed and incubated in a test tube (16.5 mm 0.d.) for 15 min at 37 "C. The agglutination reaction was enhanced by applying an inhomogeneous magnetic field (the test tube was placed on a Sm-Co magnet) which increased aggregation of FITC-anti IgG antibody-BMPs when FITC-anti IgG antibody-BMPs reacted with IgG. The strength of the applied magnetic field was adjusted by changing the position of the magnet and measured by using a flux meter (Shimadzu, GK-3). Then the mixtures were added to 2 mL of GVB containing 0.16% Tween 20 and mixed for a few seconds. The fluorescence intensity of FITC-conjugated anti IgG antibody-BMPs decreased because of agglutination. Fluorescence intensity of FITC-labeled magnetic particles was determined by using a fluorescence spectrophotometer (Hitachi, F-1200) with the excitation wavelength set at 490 nm and the emission wavelength at 520 nm using a 10 X 10 mm glass cuvette at 25 "C. The value was estimated after 15 min, when the fluorescence intensity stabilized.
RESULTS C h a r a c t e r i z a t i o n of B M P s Prepared by V a r i o u s Methods. BMPs were isolated from magnetotactic bacteria by various methods (Table I). When BMPs were prepared by ultrasonication, they were well dispersed in water. T h e median diameter was 0.12 pm. The time that the absorbance of BMP suspension was reduced t o half ( t , = 0 . 5 ~was ~ ) 124 min when 63 pg/mL suspension was used. On the other hand, that of artificial magnetite particles (1000 A) was 44 min. When BMPs were isolated by lysozyme treatment, they were aligned and slightly aggregated (median diameter 0.94 rm). Larger aggregates were observed when lysozyme and NaOH treatment was used. Figure l a shows an electron micrograph of BMPs prepared by various procedures. BMPs, which were extracted with chloroform-methanol after ultrasonication, were not covered with a magnetosome membrane (Figure lb). BMPs without magnetosome membranes formed larger aggregates than if this membrane was present. t, = 0.5Uo was 30 min when 63 r g / m L suspension was used. A lipid fraction was extracted from the magnetosome membrane; lipids were hydrolyzed, and the fatty acid content
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Table I. Separation of Bacterial Magnetic Particles from Magnetotactic Bacteria by Various Treatments
treatment
membrane thickness, A
mediana diameter, Ctm
surface area, m2/g
ultrasonication lysozyme lysozyme + NaOH ultrasonication + chloroform-methanol
44 36 35 0
0.12 0.94 1.87 12.52
12.84 3.08 0.98 0.30
250 pg/mL
t, = 0.5ao,minb 125 ~ g / m L
35.0 24.0
63.0 46.0
10.7
14.0
63 %/mL 124.0 102.0 43.0 30.0
a Particle concentration was adjusted to approximately 130 pg/mL (absorbance 100) for particle size analysis. Absorbance (a) of particle suspension was measured at 660 nm.
Table 11. Fatty Acid Composition of Magnetosome Membrane fatty acid
amt of fatty acid, Pg/mg of magnetosome (%)
0.08 (0.3) 0.24 (0.8) 2.94 (9.2) 13.81 (43.4) 14.72 (46.3) 31.79 (100.0)
c120
C14a C160 C161 C181 TFA" "TFA: total fattv acid.
Diom,
Relative Populat lon
L
0.3 0.4 0.5 0.6
0.8
1 .o 1.5 2.0
3.0 4.0
5.0 6.0 8.0 10.0 15.0
(3)
20.0
30.0 B
- .
Figure 1. (A) Transmission electron micrographof bacterial magnetic particles separated by various procedures: (1) ultrasonication; (2) lysozyme treatment: (3) lysozyme and NaOH treatment; (4) chloroform-methanol (2: 1) after ultrasonication. The bar represents 1 pm. (E) Transmission electron micrograph of bacterial magnetic particles covered with organic thin films (1) and extracted with chloroformmethanol (2:l)after ultrasonication (2). The bar represents 500 A.
was measured. The fatty acid compositions are listed in Table 11. Three saturated fatty acids (Clz0, C14:o,C16:o)and two unsaturated fatty acids (C16:., Cis:.) were identified. Palmitoleic acid and oleic acid accounted for 90% of the total fatty acids. The lipid was developed on silica gel with the solvent chloroform-methanol-water. Three spots confirmed the phospholipids, as detected with Dittmer reagents. One of the phospholipids (R, 0.46) was identified as phosphatidyl ethanolamine by comparison with standards. Phospholipids
Figure 2. Particle size distribution of immunomagnetic particles after the agglutination reaction by applying a magnetic field. IgG concn: (0) 0 ng/mL; (U)100 ng/mL.
comprised 58% of the total lipid, and phosphatidyl ethanolamine accounted for 50% of the total phospholipids present. These results show that a magnetosome membrane containing amino residues covers each BMP. Therefore, proteins may be immobilized onto the membrane after activation using glutaraldehyde. Immobilization of FITC-Conjugated Anti IgG Antibody onto BMPs. FITC-conjugated anti IgG antibody was coupled with BMPs and with artificial magnetite particles. Equal amounts of particles were used. The extent of antibody coupling with BMPs was 263 pg/mg of particles, while that coupling with artificial magnetite particles was 68 pg/mg of particles. BMPs were covered with a stable lipid membrane. 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. Thus the extent of antibody coupling with BMPs was about 4-fold higher than that achieved when artificial magnetite particles were used. Immunoreaction of FITC-Anti IgG Antibody-BMP Conjugates. IgG standard samples were mixed with FITCanti IgG antibody-BMP conjugates in the test tube, and the tubes were incubated a t 37 "C. Then, the fluorescence intensity of the suspension was measured. Since FITC-anti IgG
ANALYTICAL CHEMISTRY, VOL. 63, NO. 3, FEBRUARY 1, 1991
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Table 111. Effect of Gelatin Concentration on Decrease in Fluorescence Intensity of FITC-Conjugated Anti-Mouse IgG Immobilized on Magnetic Particles fluorescence intensity
relative
0 ng/mL gelatin concn, 7‘0
IgC
100 ng/mL IgG
fluorescence intensity, 70
0.1 0.5 1.0 2.0
35 70 310 438
26 40 70 220
74 40 23 50
0
1 IgG
10
concentration
(
100
nghl
)
Flgure 4. Relationship between the relative fluorescence Intensity and IgG concentration. Reaction was carried out at 37 OC for 15 min. A 100-pg sample of FITC-anti IgG antibody-bacterhi magnetic particle conjugates was used. I
0
1
I
15
30
I
I
45
0
15
30
45
60
Incubation time ( mln ) Flgue 3. Effect of the magnetic field on the agglutlnation of FITC-anti IgG antibody-bacterial magnetic particle conjugates (100 pg): (A) no magnetic field; (6)3000 G. IgG concn: (0)0 ng/mL; (0)100 nglmL.
antibody-BMP conjugates aggregated due to the immunoreaction, the relative fluorescence of the suspension decreased. Figure 2 shows the particle size distribution of FITC-anti IgG antibody-BMP conjugates after 45 min of immunoreaction. The mean particle size was 3 pm in the absence of antigen. When 100 ng/mL IgG were added to FITC-anti IgG antibody-BMP suspension, the mean particle size was 10 pm. In order to separate the aggregates resulting from a specific immunoreaction from those resulting from a nonspecific aggregation, gelatin was used. Table I11 shows the differences between the fluorescence intensity decrease based on specific and nonspecific aggregations. T o clarify the differences between the fluorescence intensity decrease based on specific aggregation and nonspecific aggregation, the effect of gelatin concentration in GVB was investigated. The gelatin concentration of both samples containing either no IgG or 100 ng/mL IgG was varied. A low gelatin concentration (0.1%) in GVB and a high gelatin concentration (2%) did not clarify the differences due to rapid or slow sedimentation of the immunomagnetic particles as a result of the gelatin viscosity. The difference between the relative fluorescence intensity decrease based on specific and nonspecific aggregation was maximum when 1% gelatin was used. Figure 3 shows the magnetic enhancement of immunoreaction of FITC-anti IgG antibody-BMPs. The relative fluorescence intensity decreased to 39% in the presence of 100 ng/mL IgG when no magnetic field was applied. On the other hand, the relative fluorescence intensity decreased to 15% when a magnetic field was applied. The relative fluorescence decreased with increasing magnetic field. The relative fluorescence intensity decreased to 67%, 5270, and 20% in inhomogeneous magnetic fields (magnetic field gradients) of 400 G (200 G/cm), 2000 G (2000 G/cm), and 3000 G (3300 G/cm), respectively (unpublished data). A homogeneous magnetic field of 2800 G also enhanced the agglutination rate of FITC-anti IgG antibody-BMPs, and the intensity decreased to 18% (unpublished data). A magnetic field makes particles align, and particles are easy to aggregate. As a result, the agglutination of FITC-anti IgG antibodyBMPs was enhanced and the relative fluorescence intensity decreased by applying a magnetic field during incubation. The use of FITC-anti IgG antibody-BMPs for immunochemical
analysis allows easier handling of the separation and agglutination steps. Determination of IgG by FITC-Anti IgG AntibodyBMP Conjugates. IgG concentration was measured by using FITC-conjugated anti-mouse IgGBMPs. Figure 4 shows the relationship between the relative fluorescence intensity and IgG concentration. The relative fluorescence intensity decreased with increasing mouse IgG concentration. A linear relationship was obtained between the relative fluorescence intensity and IgG concentration in the range 0.5-100 ng/mL. The minimum detectable concentration of mouse IgG was 0.5 ng/mL. Fluorescence intensity was reproducible with an average relative error of 5% when a sample containing 10 ng/mL IgG was measured five times. Fluoroimmunoassay for determining mouse IgG concentration using BMPs may be carried out across a wider concentration range having a linear response than is possible when artificial magnetite particles are used. When artificial magnetic particles were used for the measurement, a linear relationship was obtained in the range 5.0-30 ng/mL. The present system is more sensitive and more able to extend to high concentrations. This is because a larger amount of antibody was immobilized onto the BMPs, which were also more dispersed than artificial magnetite particles of a similar size. The sensor described above was applied to the determination of IgG in serum. Serum was directly diluted 106-fold with GVB. The IgG concentration determined by this method was 500 mg/L, whereas that by the latex immunoassay (16) was 600 mg/L. There was a 15% difference between them. Relatively, good agreement was obtained between the values obtained by both methods. Serum may contain several substances which affect aggregation. There was no interference in the measurement for fluorescence intensity, because samples were diluted 106-fold with GVB. Therefore, other materials in serum did not interfere with the measurements.
DISCUSSION Recently, much attention has been focused on the use of ultrafine magnetic particles in various fields, for example, with high-density recording materials (I 7), gas sensors (I8),and drug carriers (19,20) and in immunoassay procedures (21). For the production of synthetic ultrafine particles, the atmosphere, pressure, temperature, and pH must be carefully controlled. Magnetotactic bacteria synthesize magnetic particles under mild conditions. It has been reported that BMPs are covered with a lipid membrane containing proteins (22). In fact, we found that BMP was covered with 98% lipids and 2% other compounds including proteins. Lipids consisted
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of 58% phospholipids and 42% other lipids. This lipid membrane makes BMPs negatively charged. On the other hand, artificial magnetite particles and BMPs without a lipid membrane form large aggregates and are not suitable for use in fluorescence immunoassay. Various kinds of BMPs have been prepared by using ultrasonication and lysozyme. U1trasonication treatment makes BMPs disperse individually. Aligned particles can be obtained by lysozyme treatment. NaOH treatment and methanol-chloroform extraction make BMPs aggregate. The technique of radioimmunoassay has been developed for the analysis of many biologically important substances. However, it requires significant maintenance and treatment of isotopes. Enzyme immunoassay has been also employed. For example, Hybritech has developed a simple and rapid ICON system. Although these separation immunoassay methods have high sensitivity, they are not convenient in some case. There are numerous immunoassays without a separation step that depend on changes in light scattering, absorption, or fluorescence to monitor aggregation. The particle-counting immunoassay, including the latex immunoassay, is based on the principle that the number of free particles decreases during the aggregation reaction (23). Latex immunoassay based on turbidimetry or light scattering is more sensitive (minimum detectable concentration, approximately 1 ng/mL). Mitsubishi Kasei has an automated analyzer based on latex immunoassay that gives very rapid results. Assays dependent on magnetic separation have been developed (3-7). An example of an immunoassay method using magnetic particles that has been commercialized is Magic Lite by Ciba Corning. Also, Du Pont has published information on a rapid separation assay using magnetic chromium dioxide particles. Magnetic affinity immunoassay has also been used for the quantification of hemoglobins S, C, and F, but the use of isotopes was required (21). A variety of immunoassay techniques employing fluorescence detection has been described (24). The fluorescence quenching immunoassay method has been devised in a homogeneous system (25). However, the degree of quenching is sometimes too small to provide a satisfactory signal response. Magnetizable solid-phase fluoroimmunoassay methods have also been devised for detecting antigen-antibody interactions in solution without separating the bound and free components ( 4 ) . The assay described here is based on the agglutination of BMPs covalently coated with FITC-conjugated anti IgG antibody and IgG present in the sample. The decrease of fluorescence intensity of immunomagnetic particles was caused by the agglutination. The aggregation of FITC-anti IgG antibody-BMP conjugates was enhanced during the agglutination reaction by applying a magnetic field. This resulted
in a shortening of the required incubation time. The reaction was completed within 15 min while it takes 30 min for the Ciba Corning and Du Pont methods using magnetic particles. Furthermore, the high sensitivity of this method for IgG determination is achieved by combining an agglutination reaction with fluoroimmunoassay, also nonradioactive and without the separation step. This method is preferable over previously reported magnetic particle based assays because of 4-10 times higher sensitivities (minimum detectable concentrations: 0.5 ng/mL for this method and 5 and 2 ng/mL for Ciba Corning and Du Pont methods, respectively).
LITERATURE CITED (1) Pandall, K. S. J. Immunol. Methods 1979, 2 6 , 229-244. (2) Yu, B. S.; Choi, Y. K.; Chung, H. Blotechnd. Appl. Biochem. 1987, 9 , 209-216. (3) Pourfarzaneh, M.; Nargessi, R. D. Clin. Chim. Acta 1981, 7 7 7 , 61-63. (4) Nargessi, R. D.; Pourfarzaneh, M.; Landon, J. Clin. Chim. Acta 1981, 7 7 7 , 65-68. (5) Nye, L.; Forrest, G. C.; Greenwood, H.; Gardner, J. S.; Jay, R.; Roberts, J. R.; Landon, J. Clin. Chim. Acta 1978, 69. 387-396. (6)Hersh, L. S.; Yaverbaum, S. Clin. Chim. Acta 1975, 6 3 , 69-75. (7) Guesdon, J.-L.; Avrameas, S. Immunochemistry 1977, 14, 443-447. (8) Blakemore, R. P. Science 1975. 790, 377-379. ( 9 ) Balkwill, D. L.; Maratea, D.; Blakemore, R. P. J. Becteriol. 1980, 747. 1399- 1408. (10) Matsunaga, T.; Kamiya, S. Appl. Microbiol. Biotechnol. 1987, 26, 328-332. (11) Matsunaga, T.; Kamiya, S. Blomsgnetism 7987; Atsuml, K., Kotani, M., Ueno, S., Katila. T.. Williamson, S. J., Eds.; Tokyo Denki University Press: Tokyo, 1988; pp 410-413. (12) Blakemore, R. P.; Maratea, D.; Wolfe, R. S. J. Bacterial. 1979, 740, 720-729. (13) Laboratory Techniques in Biochemisby and Molecular BioJogy; Kates, M., Ed.; Elsevier Science Publishers: Amsterdam, 1972; pp 269-610. (14) Advances in ApplW Mlc#bbbgy; Shaw, N., Ed.: Academic Press: New York, 1974; pp 63-108. (15) Lowry, 0. H.; Rosebrough, N. J.; Farr, A. L.; Randall, R. J. J. B M . Chem. 1951, 793, 265-275. (16) Jolley. M. E.; Wang, J. C. H.; Ekenberg, S. J.; Zuelke, M. S.; Kelso, D. M. J. Immunol. Methods 1984, 6 7 , 21-35. (17) Kiyama, M.; Takada, T.; Nagai, N.; Horikoshl, K. Ext. Abst. Int. Conf. Ferrltes, 4th 1984, 226. (18) Nakatani, Y.; Sakal, M.; Matsuoka, M. proceedingsof the International Meeting on ChemicalSensors; Kodansha: Tokyo, 1983; pp 147-152. (19) Kataoka, N.; Misaki, A. Agric. B b l . Chem. 1983, 47, 2349-2355. (20) Papisov, M. I.; Torchilin, V. P. Int. J. Pharm. 1987, 40, 207-214. (21) Moscoso, H.; Kiefer, C. R.: Kutlar, A.; Garver, F. A. Clin. Chem. 1988, 34, 902-905. (22) Gorby, Y. A.; Beveridge, T. J.; Blakemore, R. P. J. Bacterial. 1988, 770, 834-641. (23) Masson, P. L.; Cambiaso, C. L.; ColletCassart. D.; Magnusson, C. G. M.; Richards, C. B.; Sindlc, C.J.M. I n Methods in Enzymobgy; Langone, J. J., van Vunakis, H., Eds.; Academic Press: New York, 1981; VOI. 74, pp 106-139. (24) Ullman, E. F. Tokai J. Exp. Clin. Med. 1979, 4 , 7-32 (supplement). (25) Shaw, E. J.; Watson, R. A. A.; Landon, J.; Smith, D. S. J. Clln. Pathd. 1977. 30, 526-531.
RECEIVED for review December 8,1989. Accepted October 29, 1990. This work was partially supported by Grant-in-Aid for Special Project Research No. 02205035 from the Ministry of Education, Science, and Culture.
