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Fluorescence immunoassay based on fluorescer microparticles
Colloids and Surfaces B: Biointerfaces 18 (2000) 13 – 17 www.elsevier.nl/locate/colsurfb
Fluorescence immunoassay based on fluorescer microparticles Alexander Kamyshny *, Shlomo Magdassi Casali Institute of Applied Chemistry, The Hebrew Uni6ersity of Jerusalem, 91904 Jerusalem, Israel Received 9 March 1999; accepted 20 August 1999
Abstract A novel fluorescence immunoassay based on specific interaction of an antibody with an antigen preadsorbed onto fluorescer (perylene) microparticles (mean diameter 0.8 – 1.0 mm) is described. The microparticles of perylene are formed by precipitation in IgG solution, and the obtained dispersion is stable in the protein at concentrations higher than 1 mg ml − 1. Dissolving the particles in a suitable solvent leads to fluorescence when exciting by light of a proper wavelength. The dependencies of the fluorescence intensity on the concentrations of antigen, antibody and fluorescer were studied. © 2000 Elsevier Science B.V. All rights reserved. Keywords: Immunoassay; Microparticles; Perylene; IgG; Fluorescence
1. Introduction The routine fluorescence immunoassay is based on monitoring the light emitted from a fluorescent label, which is chemically conjugated with antigen or antibody while excited by light of a suitable wavelength [1,2]. The sensitivity of nonenzymatic fluorescence assay is determined by the number of quanta emitted per analyte molecule. The simplest ways to increase the sensitivity are to increase the molar ratio of fluorescer to analyte (antigen, antibody) and to use a system with high emission quantum yield. Efficient fluorescent conjugates usually have * Corresponding author. Tel.: +972-2-658-4965; fax: + 972-2-652-8250. E-mail address: [email protected] (A. Kamyshny)
molar labelling ratio of the order of 3:1 [1]. Covalent binding of a large number of label molecules usually leads to a decrease in IgG specific binding activity and could decrease the quantum yield owing to the ‘inner filter effect’ (reabsorption of emitted light). There are several patents in which microcapsules with fluorescer [3,4], dye and pigment dispersion [5] were used for amplification of immunoassay sensitivity. Recently a novel chemiluminescence immunoassay, which combines microparticles and microemulsion, was described [6]. It is based on a specific interaction of an antibody with an antigen preadsorbed onto fluorescer microparticles and subsequent dissolving of particles in microemulsion-forming mixture, containing hydrogen peroxide and bis-(2,4,6-trichlorophenol) as reagents of the chemiluminescence reaction.
0927-7765/00/$ - see front matter © 2000 Elsevier Science B.V. All rights reserved. PII: S 0 9 2 7 - 7 7 6 5 ( 9 9 ) 0 0 1 2 3 - X
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A. Kamyshny, S. Magdassi / Colloids and Surfaces B: Biointerfaces 18 (2000) 13–17
This study describes a novel heterogeneous fluorescence immunoassay based on an analogous approach. After specific binding to antibody, the perylene microparticles with bound antigen can be dissolved in a suitable solvent and quantitatively detected by fluorescence monitoring.
2. Materials and methods IgG was isolated from human serum by ammonium sulfate precipitation, purified by caprylic acid and concentrated by additional ammonium sulfate precipitation as described by Harlow and Lane [7]. Goat anti-human IgG, goat anti-rabbit IgG, goat anti-human FITCIgG, bovine serum albumin (BSA), perylene and polypropylene multiwell plates were purchased from Sigma Chemicals Co. Toluene (A.R. grade) was purchased from Y.T. Baker. All other reagents were of analytical grade. Protein concentrations were determined spectrophotometrically (Hitachi double-beam spectrophotometer, model U-2000) using the Bradford method [8]. Perylene concentrations were determined by measuring either the absorbance at 439 nm or the fluorescence intensity (excitation wavelength 350 nm, emission wavelength 470 nm) of its solution in 1,4-dioxane by using calibration curves. Luminescence measurements were carried out with luminescence spectrometer LS 50B (Perkin – Elmer). The reported values are mean values of three measurements. The perylene microparticles were prepared by precipitation from solution in the presence of protein, human IgG (antigen). A perylene solution in 1,4-dioxane (5 mg ml − 1) was added dropwise to the IgG solutions in 0.1 M PBS (phosphate buffered saline), pH 7.4, while stirring, at a volume ratio of 1:4 (the final concentration of perylene in the dispersion was 1 mg ml − 1). The resulting dispersions were shaken at 25°C for 20 h. The dispersion was then centrifuged (microcentrifuge ‘Microliter’, Hettich) at 11 000× g for 15 min, and the amount of bound IgG (mg protein per mg perylene) was calculated from the difference of its concentrations
before and after adsorption and from the known amount of perylene added. The isotherm of IgG binding to perylene microparticles was recently presented in Ref. [6]. The size of the perylene particles in the dispersions was measured with Particle Size Analysis CIS-1 system (Galai Production Ltd., Israel), z potentials were determined using Zetamaster-S (Malvern, UK). The reported values are mean values of three measurements. (the deviation of z potential did not exceed 9 0.2 mV). The immunoassay experiments were conducted as follows, while the first four steps were carried out as in routine ELISA tests [9–11]. 1. Incubation of antibody solutions (goat antihuman IgG in carbonate buffer, pH 9.6) at concentrations ranging from 22 to 2.2 mg ml − 1 in polypropylene plate wells (200 ml per well) at 37°C for 1 h. 2. Washing the plate with PBS (thrice repeated). 3. Blocking the plate wells with 1% BSA in PBS (300 ml per well) at 37°C for 1 h. 4. Washing the plate with PBS containing 0.05% Tween 20 (thrice repeated). 5. Incubation of the perylene dispersion with bound human IgG in the plate wells (200 ml per well) at 37°C for 1 h. Since the dispersion contains a large amount of free IgG molecules, which may compete with IgG adsorbed on the particles for specific binding with antibody, the dispersion was centrifuged prior to the incubations described above. The supernatant was discarded and the precipitate was washed twice by PBS with subsequent centrifugation and finally resuspended in PBS containing 1% BSA and 0.05% Tween 20 (‘stock dispersion’). 6. Washing the plates with PBS containing 0.05% Tween 20 (twice) and then with distilled water (once). 7. Drying the plate. 8. Measuring the fluorescence intensity at 470 nm (excitation at 350 nm) in the plate wells after addition of 200 ml of toluene to each well (excitation and emission slits 5 nm). The scheme of fluorescence immunoassay is presented in Fig. 1.
A. Kamyshny, S. Magdassi / Colloids and Surfaces B: Biointerfaces 18 (2000) 13–17
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3. Results and discussion
Fig. 1. Scheme of proposed fluorescence immunoassay.
The size of the perylene particles precipitated from IgG solutions strongly depends on the bulk protein concentration. The mean size of the perylene particles precipitated in 0.1 M PBS, pH 7.4 in the absence of the protein is 48.5 mm. Precipitation in the presence of IgG results in formation of much smaller particles. These particles, at protein concentrations higher than 0.7 mg ml − 1, have a mean size of 0.8–1.0 mm (by volume distribution). In general, a decrease in the IgG concentration results in a gradual increase in the mean particles size, as presented in Fig. 2. This figure also presents the dependence of the bound amount of IgG calculated as a percentage of a maximal binding corresponding to the plateau value of the binding isotherm [6]. Correlation of these dependencies is obvious: the higher amount of the bound IgG, the smaller perylene particles, which are formed in the protein solution. It was shown previously that the plateau of the isotherm corresponds to a very high saturation value (0.45 mg IgG per 1 mg of perylene), and the isotherm most probably reflects the entrapment of the main
Fig. 2. Perylene particle size and a percentage of the maximal amount of bound IgG (calculated from the binding isotherm presented in Ref. [6]) as functions of the initial bulk IgG concentration (0.1 M PBS, pH 7.4; [perylene]=1 mg ml − 1, 6= 3.9 ml). Error bars correspond to standard deviations ( 9 S.D., n= 3).
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A. Kamyshny, S. Magdassi / Colloids and Surfaces B: Biointerfaces 18 (2000) 13–17
Fig. 3. Dependence of z potential of IgG/perylene microparticles on the amount of bound protein (as a percentage of maximal).
fraction of the protein molecules within the particles, while a smaller fraction of total bound IgG is adsorbed onto the particles’ surface [6]. The presence of IgG on the surface of the perylene microparticles was supported by measuring their z potentials (Fig. 3). Microparticles with high amount of the bound protein (more than 40% of a maximal value) have a z potential of about − 20 mV, while large particles precipitated from aqueous solution without protein have a negative z potential of − 38 mV. Since IgG molecules at the same conditions have z potential of − 4 mV [12], it may be concluded, that the particles’ surface is partially occupied by the IgG molecules, and such an adsorbed protein layer protects the perylene particles against aggregation. The dispersions of the perylene microparticles are stable (no sedimentation) at IgG concentrations higher than 1 mg ml − 1, for at least several days, and can be used as antigen – fluorescer noncovalent conjugates in fluorescence immunoassay. An important feature of these microparticles is the possibility to achieve an extremely high molar fluorescer-to-protein ratio (up to 1400), as was found from measuring the binding isotherm [6].
Thus, one may expect a drastic increase in the immunoassay sensitivity compared to the commercial chemical conjugate, which have a molar fluorescer:antibody ratio of 2–4. In our recent research [6] it was shown that human IgG adsorbed onto perylene microparticles, does not lose the recognition ability relative to goat anti-human IgG in ELISA test. Therefore, the perylene microparticles with bound IgG could be evaluated in a fluorescence immunoassay. Fig. 4 presents the dependence of the fluorescence intensity in the plate wells (after subtraction of the intensity in blank wells, which does not have antibody coating) on the concentration of antibody and perylene. From these results, it is clear that an antibody (goat anti-human IgG) can be detected by the proposed fluorescence immunoassay at a concentration of 2.2 mg ml − 1 (0.44 mg per well) at human IgG concentration of 43 mg ml − 1 (8.6 mg per well) and perylene concentration of 100 mg ml − 1 (20 mg per well). At these conditions the fluorescence intensity in blank wells does not exceed 50% of that in sample wells (the blank fluorescence intensity for samples corresponding to curve 1 in Fig. 4 does not exceed 20%).
A. Kamyshny, S. Magdassi / Colloids and Surfaces B: Biointerfaces 18 (2000) 13–17
In order to verify the specificity of the proposed immunoassay we compared the recognition ability of the human IgG-coated microparticles, while using goat anti-human or goat anti-rabbit IgGs as antibodies. The microplate wells were coated with these antibodies at a concentration of 22 mg ml − 1 and after the standard washing and blocking procedures, were incubated, as described in Section 2, with microparticles containing bound human IgG (concentrations of antibody, human IgG and perylene correspond to the last point of curve 1 in Fig. 4). It was found, that in the case of goat anti-rabbit IgG as antibody the mean value of the fluorescence intensity (after subtraction of the blank intensity) was 87 arbitrary units as compared with a value of 270 arbitrary units for specific goat anti-human IgG, i.e. approx. three times lower (the same order of magnitude as in the case of chemiluminescence immunoassay [6]). These data undoubtedly confirm the specificity of the proposed fluorescence immunoassay.
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In order to compare the proposed immunoassay with the most frequently used assay based on FITC (fluoresceine isothiocyanate)-labelled antibody, the following experiment was performed: microplate wells were coated by human IgG (22 mg ml − 1) and, after the blocking stage, were incubated with goat anti-human FITC-IgG (1/64 dilution, while the specific IgG concentration before dilution is 2 mg ml − 1). It was found that the value of the fluorescence intensity (lex = 492 nm, lem = 518 nm) at about the same conditions, as for the proposed perylenebased assay (curve 4 in Fig. 4), is 9 11, i.e. 25 times lower as compared with the proposed fluorescence immunoassay. It may be concluded that specificity, high emission intensity and simple methodology make the proposed fluorescence immunoassay promising for creation of new diagnostic kits. The subsequent studies will be directed toward improving the sensitivity and monitoring of the proposed immunoassay.
.
References
Fig. 4. Dependence of fluorescence intensity (470 nm) on antibody (goat anti-human IgG) concentration in wells (coating stage) at different concentrations of antigen (human IgG) and perylene after subtraction of the intensity in blank wells. Curve 1, [human IgG] =0.43 mg ml − 1, [perylene] =1 mg ml − 1, blank intensity 64 9 2; curves 2, 3 and 4, concentrations of human IgG and perylene decrease two, four and ten times in consecutive order, blank intensities 53 9 2, 529 1 and 489 1, respectively. Error bars correspond to standard deviations ( 9S.D., n = 3).
[1] G.D. Johnson, in: D. Catty (Ed.), Antibodies. A Practical Approach, vol. II, IRL, Oxford, 1989, p. 179. [2] T.K. Christopoulos, E.P. Diamandis, in: E.P. Diamandis, E.K. Christopoulos (Eds.), Immunoassay, Academic Press, San Diego, CA, 1996, p. 309. [3] R.M. Mandle, Y.N. Wong, US Patent 4,372,745 (1981). [4] K. Imai, Y. Namura, US Patent 4,916,080 (1986). [5] T.C.J. Gribnau, F. Roeles, J.H.W. Leuvering, US Patent 4,373,932 (1981). [6] A. Kamyshny, S. Magdassi, Colloids Surf. B11 (1998) 249. [7] E. Harlow, D. Lane, Antibodies. A Laboratory Manual, Cold Spring Harbor Laboratories, New York, 1988, p. 283. [8] M. Bradford, Anal. Biochem. 72 (1976) 248. [9] E. Harlow, D. Lane, Antibodies. A Laboratory Manual, Cold Spring Harbor Laboratories, New York, 1988, p. 553. [10] D. Catty, C. Raykundalla, in: D. Catty (Ed.), Antibodies. A Practical Approach, vol. II, IRL, Oxford, 1989, p. 97. [11] A.M. Campbell, Monoclonal antibody technology, in: R.H. Burden, P.M. van Knippenberg (Eds.), Laboratory Techniques in Biochemistry and Molecular Biology, vol. 13, Elsevier, Amsterdam, 1984, p. 33. [12] A. Kamyshny, S. Magdassi, Colloids Surf. B9 (1997) 147.
Fluorescence immunoassay based on fluorescer microparticles Alexander Kamyshny *, Shlomo Magdassi Casali Institute of Applied Chemistry, The Hebrew Uni6ersity of Jerusalem, 91904 Jerusalem, Israel Received 9 March 1999; accepted 20 August 1999
Abstract A novel fluorescence immunoassay based on specific interaction of an antibody with an antigen preadsorbed onto fluorescer (perylene) microparticles (mean diameter 0.8 – 1.0 mm) is described. The microparticles of perylene are formed by precipitation in IgG solution, and the obtained dispersion is stable in the protein at concentrations higher than 1 mg ml − 1. Dissolving the particles in a suitable solvent leads to fluorescence when exciting by light of a proper wavelength. The dependencies of the fluorescence intensity on the concentrations of antigen, antibody and fluorescer were studied. © 2000 Elsevier Science B.V. All rights reserved. Keywords: Immunoassay; Microparticles; Perylene; IgG; Fluorescence
1. Introduction The routine fluorescence immunoassay is based on monitoring the light emitted from a fluorescent label, which is chemically conjugated with antigen or antibody while excited by light of a suitable wavelength [1,2]. The sensitivity of nonenzymatic fluorescence assay is determined by the number of quanta emitted per analyte molecule. The simplest ways to increase the sensitivity are to increase the molar ratio of fluorescer to analyte (antigen, antibody) and to use a system with high emission quantum yield. Efficient fluorescent conjugates usually have * Corresponding author. Tel.: +972-2-658-4965; fax: + 972-2-652-8250. E-mail address: [email protected] (A. Kamyshny)
molar labelling ratio of the order of 3:1 [1]. Covalent binding of a large number of label molecules usually leads to a decrease in IgG specific binding activity and could decrease the quantum yield owing to the ‘inner filter effect’ (reabsorption of emitted light). There are several patents in which microcapsules with fluorescer [3,4], dye and pigment dispersion [5] were used for amplification of immunoassay sensitivity. Recently a novel chemiluminescence immunoassay, which combines microparticles and microemulsion, was described [6]. It is based on a specific interaction of an antibody with an antigen preadsorbed onto fluorescer microparticles and subsequent dissolving of particles in microemulsion-forming mixture, containing hydrogen peroxide and bis-(2,4,6-trichlorophenol) as reagents of the chemiluminescence reaction.
0927-7765/00/$ - see front matter © 2000 Elsevier Science B.V. All rights reserved. PII: S 0 9 2 7 - 7 7 6 5 ( 9 9 ) 0 0 1 2 3 - X
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A. Kamyshny, S. Magdassi / Colloids and Surfaces B: Biointerfaces 18 (2000) 13–17
This study describes a novel heterogeneous fluorescence immunoassay based on an analogous approach. After specific binding to antibody, the perylene microparticles with bound antigen can be dissolved in a suitable solvent and quantitatively detected by fluorescence monitoring.
2. Materials and methods IgG was isolated from human serum by ammonium sulfate precipitation, purified by caprylic acid and concentrated by additional ammonium sulfate precipitation as described by Harlow and Lane [7]. Goat anti-human IgG, goat anti-rabbit IgG, goat anti-human FITCIgG, bovine serum albumin (BSA), perylene and polypropylene multiwell plates were purchased from Sigma Chemicals Co. Toluene (A.R. grade) was purchased from Y.T. Baker. All other reagents were of analytical grade. Protein concentrations were determined spectrophotometrically (Hitachi double-beam spectrophotometer, model U-2000) using the Bradford method [8]. Perylene concentrations were determined by measuring either the absorbance at 439 nm or the fluorescence intensity (excitation wavelength 350 nm, emission wavelength 470 nm) of its solution in 1,4-dioxane by using calibration curves. Luminescence measurements were carried out with luminescence spectrometer LS 50B (Perkin – Elmer). The reported values are mean values of three measurements. The perylene microparticles were prepared by precipitation from solution in the presence of protein, human IgG (antigen). A perylene solution in 1,4-dioxane (5 mg ml − 1) was added dropwise to the IgG solutions in 0.1 M PBS (phosphate buffered saline), pH 7.4, while stirring, at a volume ratio of 1:4 (the final concentration of perylene in the dispersion was 1 mg ml − 1). The resulting dispersions were shaken at 25°C for 20 h. The dispersion was then centrifuged (microcentrifuge ‘Microliter’, Hettich) at 11 000× g for 15 min, and the amount of bound IgG (mg protein per mg perylene) was calculated from the difference of its concentrations
before and after adsorption and from the known amount of perylene added. The isotherm of IgG binding to perylene microparticles was recently presented in Ref. [6]. The size of the perylene particles in the dispersions was measured with Particle Size Analysis CIS-1 system (Galai Production Ltd., Israel), z potentials were determined using Zetamaster-S (Malvern, UK). The reported values are mean values of three measurements. (the deviation of z potential did not exceed 9 0.2 mV). The immunoassay experiments were conducted as follows, while the first four steps were carried out as in routine ELISA tests [9–11]. 1. Incubation of antibody solutions (goat antihuman IgG in carbonate buffer, pH 9.6) at concentrations ranging from 22 to 2.2 mg ml − 1 in polypropylene plate wells (200 ml per well) at 37°C for 1 h. 2. Washing the plate with PBS (thrice repeated). 3. Blocking the plate wells with 1% BSA in PBS (300 ml per well) at 37°C for 1 h. 4. Washing the plate with PBS containing 0.05% Tween 20 (thrice repeated). 5. Incubation of the perylene dispersion with bound human IgG in the plate wells (200 ml per well) at 37°C for 1 h. Since the dispersion contains a large amount of free IgG molecules, which may compete with IgG adsorbed on the particles for specific binding with antibody, the dispersion was centrifuged prior to the incubations described above. The supernatant was discarded and the precipitate was washed twice by PBS with subsequent centrifugation and finally resuspended in PBS containing 1% BSA and 0.05% Tween 20 (‘stock dispersion’). 6. Washing the plates with PBS containing 0.05% Tween 20 (twice) and then with distilled water (once). 7. Drying the plate. 8. Measuring the fluorescence intensity at 470 nm (excitation at 350 nm) in the plate wells after addition of 200 ml of toluene to each well (excitation and emission slits 5 nm). The scheme of fluorescence immunoassay is presented in Fig. 1.
A. Kamyshny, S. Magdassi / Colloids and Surfaces B: Biointerfaces 18 (2000) 13–17
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3. Results and discussion
Fig. 1. Scheme of proposed fluorescence immunoassay.
The size of the perylene particles precipitated from IgG solutions strongly depends on the bulk protein concentration. The mean size of the perylene particles precipitated in 0.1 M PBS, pH 7.4 in the absence of the protein is 48.5 mm. Precipitation in the presence of IgG results in formation of much smaller particles. These particles, at protein concentrations higher than 0.7 mg ml − 1, have a mean size of 0.8–1.0 mm (by volume distribution). In general, a decrease in the IgG concentration results in a gradual increase in the mean particles size, as presented in Fig. 2. This figure also presents the dependence of the bound amount of IgG calculated as a percentage of a maximal binding corresponding to the plateau value of the binding isotherm [6]. Correlation of these dependencies is obvious: the higher amount of the bound IgG, the smaller perylene particles, which are formed in the protein solution. It was shown previously that the plateau of the isotherm corresponds to a very high saturation value (0.45 mg IgG per 1 mg of perylene), and the isotherm most probably reflects the entrapment of the main
Fig. 2. Perylene particle size and a percentage of the maximal amount of bound IgG (calculated from the binding isotherm presented in Ref. [6]) as functions of the initial bulk IgG concentration (0.1 M PBS, pH 7.4; [perylene]=1 mg ml − 1, 6= 3.9 ml). Error bars correspond to standard deviations ( 9 S.D., n= 3).
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A. Kamyshny, S. Magdassi / Colloids and Surfaces B: Biointerfaces 18 (2000) 13–17
Fig. 3. Dependence of z potential of IgG/perylene microparticles on the amount of bound protein (as a percentage of maximal).
fraction of the protein molecules within the particles, while a smaller fraction of total bound IgG is adsorbed onto the particles’ surface [6]. The presence of IgG on the surface of the perylene microparticles was supported by measuring their z potentials (Fig. 3). Microparticles with high amount of the bound protein (more than 40% of a maximal value) have a z potential of about − 20 mV, while large particles precipitated from aqueous solution without protein have a negative z potential of − 38 mV. Since IgG molecules at the same conditions have z potential of − 4 mV [12], it may be concluded, that the particles’ surface is partially occupied by the IgG molecules, and such an adsorbed protein layer protects the perylene particles against aggregation. The dispersions of the perylene microparticles are stable (no sedimentation) at IgG concentrations higher than 1 mg ml − 1, for at least several days, and can be used as antigen – fluorescer noncovalent conjugates in fluorescence immunoassay. An important feature of these microparticles is the possibility to achieve an extremely high molar fluorescer-to-protein ratio (up to 1400), as was found from measuring the binding isotherm [6].
Thus, one may expect a drastic increase in the immunoassay sensitivity compared to the commercial chemical conjugate, which have a molar fluorescer:antibody ratio of 2–4. In our recent research [6] it was shown that human IgG adsorbed onto perylene microparticles, does not lose the recognition ability relative to goat anti-human IgG in ELISA test. Therefore, the perylene microparticles with bound IgG could be evaluated in a fluorescence immunoassay. Fig. 4 presents the dependence of the fluorescence intensity in the plate wells (after subtraction of the intensity in blank wells, which does not have antibody coating) on the concentration of antibody and perylene. From these results, it is clear that an antibody (goat anti-human IgG) can be detected by the proposed fluorescence immunoassay at a concentration of 2.2 mg ml − 1 (0.44 mg per well) at human IgG concentration of 43 mg ml − 1 (8.6 mg per well) and perylene concentration of 100 mg ml − 1 (20 mg per well). At these conditions the fluorescence intensity in blank wells does not exceed 50% of that in sample wells (the blank fluorescence intensity for samples corresponding to curve 1 in Fig. 4 does not exceed 20%).
A. Kamyshny, S. Magdassi / Colloids and Surfaces B: Biointerfaces 18 (2000) 13–17
In order to verify the specificity of the proposed immunoassay we compared the recognition ability of the human IgG-coated microparticles, while using goat anti-human or goat anti-rabbit IgGs as antibodies. The microplate wells were coated with these antibodies at a concentration of 22 mg ml − 1 and after the standard washing and blocking procedures, were incubated, as described in Section 2, with microparticles containing bound human IgG (concentrations of antibody, human IgG and perylene correspond to the last point of curve 1 in Fig. 4). It was found, that in the case of goat anti-rabbit IgG as antibody the mean value of the fluorescence intensity (after subtraction of the blank intensity) was 87 arbitrary units as compared with a value of 270 arbitrary units for specific goat anti-human IgG, i.e. approx. three times lower (the same order of magnitude as in the case of chemiluminescence immunoassay [6]). These data undoubtedly confirm the specificity of the proposed fluorescence immunoassay.
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In order to compare the proposed immunoassay with the most frequently used assay based on FITC (fluoresceine isothiocyanate)-labelled antibody, the following experiment was performed: microplate wells were coated by human IgG (22 mg ml − 1) and, after the blocking stage, were incubated with goat anti-human FITC-IgG (1/64 dilution, while the specific IgG concentration before dilution is 2 mg ml − 1). It was found that the value of the fluorescence intensity (lex = 492 nm, lem = 518 nm) at about the same conditions, as for the proposed perylenebased assay (curve 4 in Fig. 4), is 9 11, i.e. 25 times lower as compared with the proposed fluorescence immunoassay. It may be concluded that specificity, high emission intensity and simple methodology make the proposed fluorescence immunoassay promising for creation of new diagnostic kits. The subsequent studies will be directed toward improving the sensitivity and monitoring of the proposed immunoassay.
.
References
Fig. 4. Dependence of fluorescence intensity (470 nm) on antibody (goat anti-human IgG) concentration in wells (coating stage) at different concentrations of antigen (human IgG) and perylene after subtraction of the intensity in blank wells. Curve 1, [human IgG] =0.43 mg ml − 1, [perylene] =1 mg ml − 1, blank intensity 64 9 2; curves 2, 3 and 4, concentrations of human IgG and perylene decrease two, four and ten times in consecutive order, blank intensities 53 9 2, 529 1 and 489 1, respectively. Error bars correspond to standard deviations ( 9S.D., n = 3).
[1] G.D. Johnson, in: D. Catty (Ed.), Antibodies. A Practical Approach, vol. II, IRL, Oxford, 1989, p. 179. [2] T.K. Christopoulos, E.P. Diamandis, in: E.P. Diamandis, E.K. Christopoulos (Eds.), Immunoassay, Academic Press, San Diego, CA, 1996, p. 309. [3] R.M. Mandle, Y.N. Wong, US Patent 4,372,745 (1981). [4] K. Imai, Y. Namura, US Patent 4,916,080 (1986). [5] T.C.J. Gribnau, F. Roeles, J.H.W. Leuvering, US Patent 4,373,932 (1981). [6] A. Kamyshny, S. Magdassi, Colloids Surf. B11 (1998) 249. [7] E. Harlow, D. Lane, Antibodies. A Laboratory Manual, Cold Spring Harbor Laboratories, New York, 1988, p. 283. [8] M. Bradford, Anal. Biochem. 72 (1976) 248. [9] E. Harlow, D. Lane, Antibodies. A Laboratory Manual, Cold Spring Harbor Laboratories, New York, 1988, p. 553. [10] D. Catty, C. Raykundalla, in: D. Catty (Ed.), Antibodies. A Practical Approach, vol. II, IRL, Oxford, 1989, p. 97. [11] A.M. Campbell, Monoclonal antibody technology, in: R.H. Burden, P.M. van Knippenberg (Eds.), Laboratory Techniques in Biochemistry and Molecular Biology, vol. 13, Elsevier, Amsterdam, 1984, p. 33. [12] A. Kamyshny, S. Magdassi, Colloids Surf. B9 (1997) 147.