Biosensors & Bioelectronics 6 ( 1991) Sol-505
Fluorescence polarization immunoassay employing immobilized antibody* Makoto
Tsuruoka
TOYOBO Co. Ltd Research Center. Katata. Ohtsu-shi.
Shiga. 520-02. Japan
Eiichi Tamiya 81 lsao Karube Research Center for Advanced Science and Technology, University ofTokyo, Komaba, Meguro-ku. Tokyo, 153, Japan Received 4 May 1990; revised version received 4 January
1991; accepted 4 January
1991)
Abstract: The use of an antibody immobilized on latex or silver colloid in fluorescence polarization immunoassay (FPI) is assessed. In FPI it is possible to detect antigens of high molecular weight because the molecular weight of the antibody is effectively increased. In the assay for rabbit immunoglobulin G a limit of detection lower by two orders of magnitude and an assay range wider by one order of magnitude can be obtained in comparison with conventional FPI. The detection limit is IO-” mol I-’ and the total assay time for one sample is 8 min. This assay combines a low detection limit with a short assay time. Keywords: bound-free separation, fluorescein isothiocyanate, fluorescence polarization, immobilized antibody, immunoassay, immunoglobulin G, latex, molecular weight, silver colloid.
INTRODUCTION Wider availability of rapid and easy immunoassay methods is desired in the clinical or health care
fields. Fluorescence polarization assay has been increasingly used in immunochemistry and biochemistry (Levison et al., 1976; Maeda et al., 1982: Schray & Artz. 1988; Herninget al.. 1990). In immunochemistry, fluorescence polarization immunoassay (FPI) is known as a rapid assay method for detecting antigens of relatively low molecular weight such as haptens and which needs no bound-free separation process (Popelka er al., 1981; Sawada et al., 1984; Fiore et al., 1988). However, in FPI it is difficult to detect antigens of *Paper presented at Biosensors’90. Singapore, 2-4 May 1990 0956~X%3/91/$03.50 Q 1991 Elsevier Science Publishers
Ltd.
high molecular weight because the fluorescence polarization value changes little in association with an antigen-antibody reaction (Haber & Bennett, 1962). The authors have effectively increased the molecular weight of the antibody by immobilizing it on the surface of latex or metal particles. Applying this immobilized antibody to FPI, antigens of high molecular weight such as immunoglobulin G have been measured at a lower detection limit and over a wider assay range than with conventional FPI.
EXPERIMENTAL Materials
Fluorescein isothiocyanate (FITC) was used as a fluorescent label. R.IgG (rabbit immunoglobulin 501
M. Tsuruoka. E. Tamiya, I. Karuhe
G), anti-R.IgG (gamma chain specific IgG made in a goat), and FITC-conjugated R.IgG were obtained from Organon Teknika Corporation. Two types of polystyrene latex (0.077 and 0.22 ,um particle size, both soap free) were obtained from Sekisui Chemical Co., Ltd. Anti-R.IgG (antibody IgG made in a goat) immobilized on silver colloid (Roth, 1982) with a particle diameter of lo-15 nm was obtained from E. Y. Laboratories, Inc. Phosphate buffer (O-07 mol I-‘, pH 7-O with 0.05 wt.% sodium azide) was used to dilute the solutions. All materials and buffer were of reagent grade. The concentrations of materials are mainly given in units of M (mol l-‘), and were simply calculated from the weight/volume concentrations. Apparatus Polarization values were determined using a fluorescent spectrophotometer (Japan Spectroscopic Co., Ltd., model FP-707). A 150 W xenon arc lamp was used as the light source. The light emitted was detected by a photomultiplier tube (PMT). The wavelength filters were diffraction gratings, which were adjusted to a central wavelength of 485 nm with a bandwidth of 5 nm for excitation and a central wavelength of 525 nm with a bandwidth of 10 nm for emission. The polarizing filters are automatically rotatable. The fluorescent sample is placed in a square 10 mm X 10 mm quartz cell that is maintained at 37°C during measurement. Preparation of immobilized antibody Anti-R.IgG (10 mg ml-’ concentration, 0.5 ml volume) was mixed with latex (2 wt.% concentration, 0.5 ml volume) (Kuge & Obana, 1981). The mixture was incubated at 37°C for 3 h with vibration. It was centrifuged at 14 000 X g for 5 min, the supernatant was removed, and the precipitate was then redispersed with 1.0 ml of phosphate buffer.
RESULTS AND DISCUSSION It is known that the polarization value P used in the fluorescence polarization assay obeys the equation
502
Biosensors & Bioelectronics 6 ( 199 1) 501-505
where r is the lifetime of the excited state of the fluorescent substance, n is the coefficient of viscosity of the solvent, Vis the effective volume of the fluorescent substance (molecular weight times specific volume), R is the gas constant, T is absolute temperature, and P,J is the value of P at 0 K. The definition of the polarization is p _ -- Ill
- 4 III + 11
where III is the fluorescence component parallel to the linearly polarized light used for excitation and 4is the fluorescence component normal to that (Perrin, 1926; Weber, 1953). According to eqn (I), P increases as the molecular weight of a fluorescent-labelled substance increases, on condition that the temperature and viscosity of the solution are constant. FPI is based on measuring the P values corresponding to the apparent molecular weight of a fluorescentlabelled antigen before and after it is combined with the antibody. Therefore, in FPI it is difficult to detect an antigen whose molecular weight is nearly equal to or larger than that of the antibody, because the apparent change in molecular weight associated with antigen-antibody reaction is relatively small. It is expected that the measurement of antigens of relatively high molecular weight would become feasible if the effective molecular weight of the antibody could be increased. The authors increased the effective molecular weight of the antibody by immobilizing the antibody on the surface of latex or metal particles. When the immobilized antibody was used in FPI, antigens of high molecular weight such as immunoglobulin G could be measured. The result was compared with that obtained using conventional FPI. Figure 1 shows the process for obtaining the calibration curve for rabbit IgG in the conventional FPI. Any concentration mentioned below is the final one after mixing every solution for measurement. Firstly, the concentration of FITC-labelled R.IgG was fixed, at which the fluorescent signal was sufficiently higher than the background of the buffer solvent. Secondly, the concentration of anti-R.IgG (gamma chain specific) for FPI was determined from the curve of the polarization of FITC-labelled R.IgG as a function of the antiR.IgG concentration. Finally, anti-R.IgG (0.034 ml volume) was added to R.IgG samples (O-816 ml volume) of various concentrations. FITC-labelled R.IgG (2.55 ml volume) was added to the mixture
Immunoassay using immobilized antibody
Biosensors & Bioelectronics6 (1991) 501-505 (1) Decision on concentration
of FITC-labelled
4 Decision on concentration
R.IgG
of anti-R.IgG
00 Addition of anti-R.IgG to R.IgG samples Incubation at 37°C 1 for 3 min Addition of FITC-labelled R.IgG 4 Plotting of P values 2. 3.4 and 5 min after the addition
Fig. 1. Processfor obtaining the calibration curve of rabbit I#? I, deciding the reagent concentrations for FPI; II, determining the calibration curve of R.&G by FPI.
incubation at 37°C for 3 min. Anti-R.IgG and FITC-labelled R.IgG were added to yield each final concentration fixed above. Fluorescence polarization values were recorded at 2, 3,4 and 5 min after the addition of FITC-labelled R.IgG. Each mean P value was the average of three P values (triplet) measured by readjusting the polarizing filter for emission; values between 2 and 5 min thereafter were again averaged and the standard deviations were calculated. All P values were indicated as a relative value. Figure 2 shows the dependence of the polarization of FITC-labelled R.IgG solution on the anti-R.IgG concentration. To 2.55 ml of FITClabelled R.IgG was added 0.85 ml of anti-R.&G at each concentration. In constructing the calibration curve of R.IgG, R.IgG and FITC-labelled R.IgG are combined competitively with the antibody; therefore, a low limit of R.IgG detection can be after
obtained as the concentration of FITC-labelled R.IgG is lower. In this experiment, the concentration of FITC-labelled R.IgG (1.1 X 10m9M) was chosen to give a 25:l sample-to-buffer fluorescence ratio, which was high enough to obtain reliable P values. A calibration curve for R.IgG obtained by conventional FPI is shown in Fig. 3. The final concentration of FITC-labelled R.IgG is the same as in the case of Fig. 2. The concentration of antiR.IgG (gamma chain specific) was determined as 5.3 X ‘10-s M, at which the large slope and change in Pvalue on the curve in Fig. 2 were observed. In the assay, the total assay time for one sample is about 8 min including incubation time, which can be estimated from the calibration process (Fig. 1, part II). The assay range is approximately 10-9-10-7 M. The lower detection limit is about lO-8 M. In the case of detecting antigens of high molecular weight such as IgG by conventional FPI, there are problems that a low limit of detection cannot be obtained and that the detectable concentration range is narrow, as can be seen from Fig. 3. In order to avoid these disadvantages, the use of an antibody with an increased effective molecular weight in FPI was investigated. The antibody was immobilized on the surface of polystyrene latex or silver particles to increase its effective molecular weight. Table 1 shows the effective molecular weights of the latex and silver particles used as carriers for the antibody calculated using the simple equation effective molecular
100
I--+-
where d is the particle diameter, p is density of the
/I
- 4-J I___
840
weight = (n/6)d3pN,
100
c
s
960
IgG]
moliL
Fig. 2. Dependence of the polarization of FITC-labelled R.IgG solution on anti-R.IgG concentration. Reagents are in a phosphate bu#er (007 M, pH 7.0). Each value plotted represents the mean of polarization values measured 1. 2 and 3 min afte the addition of anti-R.IgG. Error bars represent one standard deviation.
-1 -_/t--t\
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880
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10-T 10-G IIT5
mol/L
Fig. 3. Calibration curve for rabbit IgG obtained used conventional FPI. Error bars represent one standard deviation.
M. Tsuruoka. E. Tamiya I. Karuhe
Effective
TABLE 1 weights of latex particles
molecular
Latex A Latex B Silver colloid
Biosensors & Bioelectronics 6 ( 1991) 501-505
da (nm)
Material
220 II 15
Polystyrene Polystyrene Silver
and
silver
EJective molecular weight
3.5 X lo9 1.5 x 108 1.1 x 10’
ODiameter of particle, which is treated as a sphere.
Wavelength
Wavelength
(“r-n)
(a)
(nm )
(b)
Wavelength
(“IT
)
Cc)
Fig. 4. Light scattering properties of latex and free I&G: (a) latex A (220 nm particle size): (6) latex B (77 nm particle size): (c)free IgG (anti-R&G) solution. The scattering level
is given in arbitrary units.
material that forms the particle, and N, is the Avogadro number. The effective molecular weight of a silver particle 15 nm in diameter is about 70 times larger than the molecular weight (cu. 1.5 X 105)of free IgG antibody. Accordingly, the antibody-to-antigen molecular weight ratio in FPI using the antibody immobilized on silver colloid is about 70:1, while that in conventional FPI is l:l. 504
Figure 4 shows the light scattering properties of latex and free IgG. The wavelength of the incident light is 485 nm, which is the same as that of the excitation beam in the FPI of this work. The light scattering level of latex B (77 nm particle size) at a wavelength of 525 nm (the emission wavelength in FPI) is half of that of latex A (220 nm particle size) (Figs 4(a) and 4(b)). The concentrations (solid component) of both latex A and latex B are the same (5 X 10m4wt.%). The scattering level of latex after immobilizing the antibody was almost the same as that without immobilizing it for both types of latex. The scattering level of free IgG antibody (lo-* M concentration) is nearly equal to the background level of the solvent (Fig. 4(c)). The fluorescence behaviour of the FITC label (lo-’ M concentration) is shown by the broken lines in Fig. 4. As seen from the figure, when latex particles are present in the solution at the concentration used, the scattering level of the latex particles cannot be neglected compared with the fluorescence level of the FITC label. Thus an incorrect fluorescence polarization value is obtained, as is evident from eqn (2). In contrast, the scattering level of antibody immobilized on silver colloid of particle size lo15 nm (10M9~ antibody concentration) was close to the background level of free IgG. It is expected that this occurs because the particle size of the silver colloid is sufficiently smaller than the excitation wavelength (485 nm) that light scattering is very slight, which is not the case for both types of latex. It was therefore decided to employ antibody immobilized on silver colloid in this investigation using FPI. A calibration curve of rabbit IgG obtained using FPI with antibody immobilized on silver colloid is shown in Fig. 5. The curve was obtained by the process outlined in Fig. 1, except that antiRlgG immobilized on silver colloid was substituted for the anti-R.lgG. The final concentration of FITC-labelled R.lgG is I.1 X 10e9 M, which is equal to that in Fig. 3. The concentration of antibody immobilized on silver colloid was determined and fixed at 3.8 X 10m9~ (antibody concentration) in the same way as in conventional FPI. The assay time is 8 min, which is the same as that for conventional FPI. The assay range is approximately lo- “-lO-8 M. The lower detection limit is about lo-” M. It can be concluded that FPI using immobilized antibody has the following advantages over conventional FPI in the measurement of antigens
Immunoassay using immobilized antibody
Biosenson & Bioelectronics 6 ( 199 I ) 501-505
ACKNOWLEDGEMENTS We thank Dr Herning Thierry, Eriberta N. Navera and Yasuhiro Nakano for useful discussion. REFERENCES 92090.0
Lt
0
I
’
16'2 I@ [R WI
KS"0
10-9
Id8
lo-7
mollL
Fig. S. Calibration curvefor rabbit IgC obtained using FPI with antibody immobilized on silver colloid. Ewor bars represent one standard deviation.
of high molecular weight such as IgG. A limit of detection approximately two orders of magnitude lower and an assay range one order wider can be obtained (Figs 3 and 5). In addition, no increase in polarization occurs for high concentrations of antigen (postzone phenomenon) (Fig. 3) which means that the reliability of the assay of antigens at the concentrations near the upper limit of detection would be improved. The low limit of detection and the wide assay range are considered to have been obtained because the effective molecular weight of the antibody immobilized on silver colloidal particles is sufficiently high and because, when antibody immobilized on silver colloid is used, the combination of several antibody molecules with a single molecule of R.IgG antigen (which occurs when using free antibody) can be avoided. The ratio of minimum to maximum polarization values is not so high as would be expected in FPI using immobilized antibody (Fig. 5). The reason is considered to be that the concentration of antibody immobilized on silver colloid was too small compared with that of FITC-labelled R.IgG. The mechanism of rotation of polarizers can be further improved so that more precise values of polarization will be acquired; thus the error in polarization value will be reduced so that the limit of detection obtained in the assay would be even lower. FPI is a very rapid and easy assay method, and by using immobilized antibody in FPI it becomes feasible to detect antigens of high molecular weight. It is expected that such antigens as some peptides or cancer markers, trace tumors, etc. could be detected rapidly using the assay described.
Fiore. M., Mitchell, T., Doan, T., Nelson, R., Winter, G., Grandone, C., Zeng, K., Haraden, R., Smith, J., Harris, K, Leszczynski, J., Berry, D., Safford, S., Barnes, G., Scholnick, A. & Ludington, K. (1988). The Abbott IMx automated benchtop immunochemistry analyzer system. Clin. Chem.. 34, 1726-32.
Haber, E. & Bennett, J. C. (1962). Polarization of fluorescence as a measure of antigen-antibody interaction. Proc. Natl. Acad Sci. USA, 48,1935-42. Heming. T., Kobayashi, S., Tamiya, E. & Karube, I. (1990).Specific liquid DNA hybridization kinetics measured by fluorescence polarization. Anal. Chim. Acta, 244, 207-13.
Kuge, T. & Obana, S. (1981). Application of soap free latex to diagnostics (1); Adsorption equilibrium of human IgG on latex surface. Zgaku no Ayumi, 119, 702-4. Levison. S. A., Dandliker, W. B., Brawn, R. J. & Vanderlaan, W. P. (1976). Fluorescence polarization measurement of the hormone-binding site interaction. Endocrinology 99, 1129-43. Maeda. H., Tsuda, H. & Takeshita, J. (1982). Investigation of factors involved in the uptake velocity of fluorescein diacetate and intracellular fluorescence polarization value. II. Cytotoxicity produced by anticancer agents. Cell. Struct. Funct., 7, 177-82. Pen-in, M. F. (1926). Polarisation de la lumiere de fluorescence. Vie moyenne des molecules dans l’etat excite. J. Phys. Radium, 7, 390-401. Popelka, S. R., Miller, D. M., Holen, J. T. & Kelso, D. M. (1981). Fluorescence polarization immunoassay II. Analyzer for rapid, precise measurement of fluorescence polarization with use of disposable cuvettes. Clin. Chem., 27, 1198-201. Roth, J. (1982).Application of immunocolloids in light microscopy. Preparation of protein A-silver and protein A-gold complexes and their application for localization of single and multiple antigens in paraffin sections. J Histochem. Cytochem., 30, 691-96.
Sawada, M., Yamaguchi,T., Sugimoto,T., Matsuura. S. & Nagatsu. T. (1984). Polarization fluoroimmunoassay of biopterin and neopterin in human urine. Clin. Chim. Acta, 138, 275-82.
Schray, K. J., Artz, P. G. & Hevey, R. C. (1988). Determination of avidin and biotin by fluorescence polarization. Anal. Chem.. 60, 853-5. Weber, G. (1953). Rotational Brownian motion and polarization of the fluorescence of solutions. Adv. Prot. Chem.. 8,415-59.
505
Fluorescence polarization immunoassay employing immobilized antibody* Makoto
Tsuruoka
TOYOBO Co. Ltd Research Center. Katata. Ohtsu-shi.
Shiga. 520-02. Japan
Eiichi Tamiya 81 lsao Karube Research Center for Advanced Science and Technology, University ofTokyo, Komaba, Meguro-ku. Tokyo, 153, Japan Received 4 May 1990; revised version received 4 January
1991; accepted 4 January
1991)
Abstract: The use of an antibody immobilized on latex or silver colloid in fluorescence polarization immunoassay (FPI) is assessed. In FPI it is possible to detect antigens of high molecular weight because the molecular weight of the antibody is effectively increased. In the assay for rabbit immunoglobulin G a limit of detection lower by two orders of magnitude and an assay range wider by one order of magnitude can be obtained in comparison with conventional FPI. The detection limit is IO-” mol I-’ and the total assay time for one sample is 8 min. This assay combines a low detection limit with a short assay time. Keywords: bound-free separation, fluorescein isothiocyanate, fluorescence polarization, immobilized antibody, immunoassay, immunoglobulin G, latex, molecular weight, silver colloid.
INTRODUCTION Wider availability of rapid and easy immunoassay methods is desired in the clinical or health care
fields. Fluorescence polarization assay has been increasingly used in immunochemistry and biochemistry (Levison et al., 1976; Maeda et al., 1982: Schray & Artz. 1988; Herninget al.. 1990). In immunochemistry, fluorescence polarization immunoassay (FPI) is known as a rapid assay method for detecting antigens of relatively low molecular weight such as haptens and which needs no bound-free separation process (Popelka er al., 1981; Sawada et al., 1984; Fiore et al., 1988). However, in FPI it is difficult to detect antigens of *Paper presented at Biosensors’90. Singapore, 2-4 May 1990 0956~X%3/91/$03.50 Q 1991 Elsevier Science Publishers
Ltd.
high molecular weight because the fluorescence polarization value changes little in association with an antigen-antibody reaction (Haber & Bennett, 1962). The authors have effectively increased the molecular weight of the antibody by immobilizing it on the surface of latex or metal particles. Applying this immobilized antibody to FPI, antigens of high molecular weight such as immunoglobulin G have been measured at a lower detection limit and over a wider assay range than with conventional FPI.
EXPERIMENTAL Materials
Fluorescein isothiocyanate (FITC) was used as a fluorescent label. R.IgG (rabbit immunoglobulin 501
M. Tsuruoka. E. Tamiya, I. Karuhe
G), anti-R.IgG (gamma chain specific IgG made in a goat), and FITC-conjugated R.IgG were obtained from Organon Teknika Corporation. Two types of polystyrene latex (0.077 and 0.22 ,um particle size, both soap free) were obtained from Sekisui Chemical Co., Ltd. Anti-R.IgG (antibody IgG made in a goat) immobilized on silver colloid (Roth, 1982) with a particle diameter of lo-15 nm was obtained from E. Y. Laboratories, Inc. Phosphate buffer (O-07 mol I-‘, pH 7-O with 0.05 wt.% sodium azide) was used to dilute the solutions. All materials and buffer were of reagent grade. The concentrations of materials are mainly given in units of M (mol l-‘), and were simply calculated from the weight/volume concentrations. Apparatus Polarization values were determined using a fluorescent spectrophotometer (Japan Spectroscopic Co., Ltd., model FP-707). A 150 W xenon arc lamp was used as the light source. The light emitted was detected by a photomultiplier tube (PMT). The wavelength filters were diffraction gratings, which were adjusted to a central wavelength of 485 nm with a bandwidth of 5 nm for excitation and a central wavelength of 525 nm with a bandwidth of 10 nm for emission. The polarizing filters are automatically rotatable. The fluorescent sample is placed in a square 10 mm X 10 mm quartz cell that is maintained at 37°C during measurement. Preparation of immobilized antibody Anti-R.IgG (10 mg ml-’ concentration, 0.5 ml volume) was mixed with latex (2 wt.% concentration, 0.5 ml volume) (Kuge & Obana, 1981). The mixture was incubated at 37°C for 3 h with vibration. It was centrifuged at 14 000 X g for 5 min, the supernatant was removed, and the precipitate was then redispersed with 1.0 ml of phosphate buffer.
RESULTS AND DISCUSSION It is known that the polarization value P used in the fluorescence polarization assay obeys the equation
502
Biosensors & Bioelectronics 6 ( 199 1) 501-505
where r is the lifetime of the excited state of the fluorescent substance, n is the coefficient of viscosity of the solvent, Vis the effective volume of the fluorescent substance (molecular weight times specific volume), R is the gas constant, T is absolute temperature, and P,J is the value of P at 0 K. The definition of the polarization is p _ -- Ill
- 4 III + 11
where III is the fluorescence component parallel to the linearly polarized light used for excitation and 4is the fluorescence component normal to that (Perrin, 1926; Weber, 1953). According to eqn (I), P increases as the molecular weight of a fluorescent-labelled substance increases, on condition that the temperature and viscosity of the solution are constant. FPI is based on measuring the P values corresponding to the apparent molecular weight of a fluorescentlabelled antigen before and after it is combined with the antibody. Therefore, in FPI it is difficult to detect an antigen whose molecular weight is nearly equal to or larger than that of the antibody, because the apparent change in molecular weight associated with antigen-antibody reaction is relatively small. It is expected that the measurement of antigens of relatively high molecular weight would become feasible if the effective molecular weight of the antibody could be increased. The authors increased the effective molecular weight of the antibody by immobilizing the antibody on the surface of latex or metal particles. When the immobilized antibody was used in FPI, antigens of high molecular weight such as immunoglobulin G could be measured. The result was compared with that obtained using conventional FPI. Figure 1 shows the process for obtaining the calibration curve for rabbit IgG in the conventional FPI. Any concentration mentioned below is the final one after mixing every solution for measurement. Firstly, the concentration of FITC-labelled R.IgG was fixed, at which the fluorescent signal was sufficiently higher than the background of the buffer solvent. Secondly, the concentration of anti-R.IgG (gamma chain specific) for FPI was determined from the curve of the polarization of FITC-labelled R.IgG as a function of the antiR.IgG concentration. Finally, anti-R.IgG (0.034 ml volume) was added to R.IgG samples (O-816 ml volume) of various concentrations. FITC-labelled R.IgG (2.55 ml volume) was added to the mixture
Immunoassay using immobilized antibody
Biosensors & Bioelectronics6 (1991) 501-505 (1) Decision on concentration
of FITC-labelled
4 Decision on concentration
R.IgG
of anti-R.IgG
00 Addition of anti-R.IgG to R.IgG samples Incubation at 37°C 1 for 3 min Addition of FITC-labelled R.IgG 4 Plotting of P values 2. 3.4 and 5 min after the addition
Fig. 1. Processfor obtaining the calibration curve of rabbit I#? I, deciding the reagent concentrations for FPI; II, determining the calibration curve of R.&G by FPI.
incubation at 37°C for 3 min. Anti-R.IgG and FITC-labelled R.IgG were added to yield each final concentration fixed above. Fluorescence polarization values were recorded at 2, 3,4 and 5 min after the addition of FITC-labelled R.IgG. Each mean P value was the average of three P values (triplet) measured by readjusting the polarizing filter for emission; values between 2 and 5 min thereafter were again averaged and the standard deviations were calculated. All P values were indicated as a relative value. Figure 2 shows the dependence of the polarization of FITC-labelled R.IgG solution on the anti-R.IgG concentration. To 2.55 ml of FITClabelled R.IgG was added 0.85 ml of anti-R.&G at each concentration. In constructing the calibration curve of R.IgG, R.IgG and FITC-labelled R.IgG are combined competitively with the antibody; therefore, a low limit of R.IgG detection can be after
obtained as the concentration of FITC-labelled R.IgG is lower. In this experiment, the concentration of FITC-labelled R.IgG (1.1 X 10m9M) was chosen to give a 25:l sample-to-buffer fluorescence ratio, which was high enough to obtain reliable P values. A calibration curve for R.IgG obtained by conventional FPI is shown in Fig. 3. The final concentration of FITC-labelled R.IgG is the same as in the case of Fig. 2. The concentration of antiR.IgG (gamma chain specific) was determined as 5.3 X ‘10-s M, at which the large slope and change in Pvalue on the curve in Fig. 2 were observed. In the assay, the total assay time for one sample is about 8 min including incubation time, which can be estimated from the calibration process (Fig. 1, part II). The assay range is approximately 10-9-10-7 M. The lower detection limit is about lO-8 M. In the case of detecting antigens of high molecular weight such as IgG by conventional FPI, there are problems that a low limit of detection cannot be obtained and that the detectable concentration range is narrow, as can be seen from Fig. 3. In order to avoid these disadvantages, the use of an antibody with an increased effective molecular weight in FPI was investigated. The antibody was immobilized on the surface of polystyrene latex or silver particles to increase its effective molecular weight. Table 1 shows the effective molecular weights of the latex and silver particles used as carriers for the antibody calculated using the simple equation effective molecular
100
I--+-
where d is the particle diameter, p is density of the
/I
- 4-J I___
840
weight = (n/6)d3pN,
100
c
s
960
IgG]
moliL
Fig. 2. Dependence of the polarization of FITC-labelled R.IgG solution on anti-R.IgG concentration. Reagents are in a phosphate bu#er (007 M, pH 7.0). Each value plotted represents the mean of polarization values measured 1. 2 and 3 min afte the addition of anti-R.IgG. Error bars represent one standard deviation.
-1 -_/t--t\
c
5 920 -
a”
880
[AW
ztf:
C”
-
8f+o L0
US" 10'0 lo-9 l@
[R WI
10-T 10-G IIT5
mol/L
Fig. 3. Calibration curve for rabbit IgG obtained used conventional FPI. Error bars represent one standard deviation.
M. Tsuruoka. E. Tamiya I. Karuhe
Effective
TABLE 1 weights of latex particles
molecular
Latex A Latex B Silver colloid
Biosensors & Bioelectronics 6 ( 1991) 501-505
da (nm)
Material
220 II 15
Polystyrene Polystyrene Silver
and
silver
EJective molecular weight
3.5 X lo9 1.5 x 108 1.1 x 10’
ODiameter of particle, which is treated as a sphere.
Wavelength
Wavelength
(“r-n)
(a)
(nm )
(b)
Wavelength
(“IT
)
Cc)
Fig. 4. Light scattering properties of latex and free I&G: (a) latex A (220 nm particle size): (6) latex B (77 nm particle size): (c)free IgG (anti-R&G) solution. The scattering level
is given in arbitrary units.
material that forms the particle, and N, is the Avogadro number. The effective molecular weight of a silver particle 15 nm in diameter is about 70 times larger than the molecular weight (cu. 1.5 X 105)of free IgG antibody. Accordingly, the antibody-to-antigen molecular weight ratio in FPI using the antibody immobilized on silver colloid is about 70:1, while that in conventional FPI is l:l. 504
Figure 4 shows the light scattering properties of latex and free IgG. The wavelength of the incident light is 485 nm, which is the same as that of the excitation beam in the FPI of this work. The light scattering level of latex B (77 nm particle size) at a wavelength of 525 nm (the emission wavelength in FPI) is half of that of latex A (220 nm particle size) (Figs 4(a) and 4(b)). The concentrations (solid component) of both latex A and latex B are the same (5 X 10m4wt.%). The scattering level of latex after immobilizing the antibody was almost the same as that without immobilizing it for both types of latex. The scattering level of free IgG antibody (lo-* M concentration) is nearly equal to the background level of the solvent (Fig. 4(c)). The fluorescence behaviour of the FITC label (lo-’ M concentration) is shown by the broken lines in Fig. 4. As seen from the figure, when latex particles are present in the solution at the concentration used, the scattering level of the latex particles cannot be neglected compared with the fluorescence level of the FITC label. Thus an incorrect fluorescence polarization value is obtained, as is evident from eqn (2). In contrast, the scattering level of antibody immobilized on silver colloid of particle size lo15 nm (10M9~ antibody concentration) was close to the background level of free IgG. It is expected that this occurs because the particle size of the silver colloid is sufficiently smaller than the excitation wavelength (485 nm) that light scattering is very slight, which is not the case for both types of latex. It was therefore decided to employ antibody immobilized on silver colloid in this investigation using FPI. A calibration curve of rabbit IgG obtained using FPI with antibody immobilized on silver colloid is shown in Fig. 5. The curve was obtained by the process outlined in Fig. 1, except that antiRlgG immobilized on silver colloid was substituted for the anti-R.lgG. The final concentration of FITC-labelled R.lgG is I.1 X 10e9 M, which is equal to that in Fig. 3. The concentration of antibody immobilized on silver colloid was determined and fixed at 3.8 X 10m9~ (antibody concentration) in the same way as in conventional FPI. The assay time is 8 min, which is the same as that for conventional FPI. The assay range is approximately lo- “-lO-8 M. The lower detection limit is about lo-” M. It can be concluded that FPI using immobilized antibody has the following advantages over conventional FPI in the measurement of antigens
Immunoassay using immobilized antibody
Biosenson & Bioelectronics 6 ( 199 I ) 501-505
ACKNOWLEDGEMENTS We thank Dr Herning Thierry, Eriberta N. Navera and Yasuhiro Nakano for useful discussion. REFERENCES 92090.0
Lt
0
I
’
16'2 I@ [R WI
KS"0
10-9
Id8
lo-7
mollL
Fig. S. Calibration curvefor rabbit IgC obtained using FPI with antibody immobilized on silver colloid. Ewor bars represent one standard deviation.
of high molecular weight such as IgG. A limit of detection approximately two orders of magnitude lower and an assay range one order wider can be obtained (Figs 3 and 5). In addition, no increase in polarization occurs for high concentrations of antigen (postzone phenomenon) (Fig. 3) which means that the reliability of the assay of antigens at the concentrations near the upper limit of detection would be improved. The low limit of detection and the wide assay range are considered to have been obtained because the effective molecular weight of the antibody immobilized on silver colloidal particles is sufficiently high and because, when antibody immobilized on silver colloid is used, the combination of several antibody molecules with a single molecule of R.IgG antigen (which occurs when using free antibody) can be avoided. The ratio of minimum to maximum polarization values is not so high as would be expected in FPI using immobilized antibody (Fig. 5). The reason is considered to be that the concentration of antibody immobilized on silver colloid was too small compared with that of FITC-labelled R.IgG. The mechanism of rotation of polarizers can be further improved so that more precise values of polarization will be acquired; thus the error in polarization value will be reduced so that the limit of detection obtained in the assay would be even lower. FPI is a very rapid and easy assay method, and by using immobilized antibody in FPI it becomes feasible to detect antigens of high molecular weight. It is expected that such antigens as some peptides or cancer markers, trace tumors, etc. could be detected rapidly using the assay described.
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