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Evaluation of binding of human serum albumin (HSA) to monoclonal and polyclonal antibody by means of piezoelectric immunosensing technique
S@jRs ACT~JA~ORS ELSEVIER
Sensors
and Actuators
B 42 (1997)
89-94
Evaluation of binding of human serum albumin (HSA) to monoclonal and polyclonal antibody by means of piezoelectric immunosensing technique G. Sakai a,*, T. Saiki a, T. Uda b, N. Miura a, N. Yamazoe a a Department
of Materials b School
Science
and Technology,
of Bioresources, Received
Hiroshima
25 October
Graduate School of Engineering Sciences, K)ushu Unirersity, Fukuoka 816, Japan Prefectural Unirersity, Shol!bara, Hiroshima 727, Japan
1996; received
in revised
form
26 December
Kasuga-shi,
1996
Abstract Binding of human serum albumin (HSA) to its monoclonal and polyclonal antibody was examined by using a quartz crystal immunosensor on which the immobilized monoclonal antibody was exposed to HSA and polyclonal antibody stepwise. The association constants and rate constants of the respective steps of immunoreactions were estimated from the resonant frequency shifts at steady state and the time course of resonant frequency. The binding of HSA to the polyclonal antibody (second step) was shown to be much weaker and slower than that to the monoclonal antibody (first step), probably owing to the steric hindrance and orientation effects of the HSA already bound to the immobilized monoclonal antibody. 0 1997 Elsevier Science S.A. Keyxaords:
Immunoreaction;
Kinetic
analysis;
Quartz
crystal
microbalance;
1. Introduction
Recent advances in the quartz crystal microbalance (QCM) technique have made it possible to detect quite sensitively the mass change on the resonator surface even when the quartz crystal is immersed in liquid media [l-5]. The simplicity, low cost, and ready availability of piezoelectric transducers have encouraged the development of various immunosensors [6121. The change in resonant frequency varies linearly with mass of protein (antigen or antibody) adsorbed on the electrode surface of the quartz even in a liquid media, following Sauerbrey’s equation, just as in the case of gas adsorption [13,14]. Thus, piezoelectric immunosensors
bio-related
can not only detect very small amounts of
substances specifically
but also monitor
* Corresponding author. 0925-4005/97/$17.00 PIISO925-4005(97)00188-3
8 1997 Elsevier
Science
S.A. All rights
reserved.
Sandwich
method
directly immunoreactions between antibodies and antigens at the solid (electrode of quartz crystal)/liquid (diluting media) interface. The QCM technique can be applied for the analysis of the kinetics
of specific bind-
ing of antifluorescyl antibody and Fab to fluorescein lipids in Langmuir-Blodgett films on the quartz crystal [15]. Meanwhile, we have designed a piezoelectric human serum albumin (HSA) sensor immobilized with anti-HSA monoclonal antibody and have found that the HSA sensitivity of the sensor can be much improved by adopting a ‘sandwich’ method in which HSA is combined with monoclonal and polyclonal antibody stepwise [16]. Owing to the high sensitivity and high stability of the frequency response, this ‘sandwich’ method enables one to analyze the nature of both immunoreactions involved in the respective steps. In this paper, we have tried to examine the binding affinities and kinetics of the immunoreactions of HSA with the monoclonal and polyclonal antibody on the surface of the QCM on the basis of its frequency response behavior.
90
G. Sakai
et al. /Sensors
and Actuators
2. Experimental
3 42 (1997)
89-94
10 ppm HSA soin. +
2.1. Reagents
Anti-I-ISA monoclonal antibody (IgG) (molecular weight about 150 000) was obtained from a hybridoma clone by the fusion of mouse myeloma cells of immunized mice. Anti-HSA polyclonal antibody was donated by Shionogi Pharmacia. HSA (Sigma) and bovine serum albumin (BSA, Sigma) were dissolved in a phosphate buffer saline (PBS, pH 7.2) containing 0.1% sodium azide. An acidic buffer solution (PH 3.0) was prepared with 0.2 M disodium hydrophosphate and 0.1 M citric acid. 2.2. Sensing device The quartz resonator used was of AT-cut (diameter 13 mm, MAXTEC) attached with sputtered Au electrodes (diameter 11 mm). Its basic resonant frequency was 6 MHz. Anti-HSA monoclonal antibody was immobilized on top of one of the gold electrodes of the quartz resonator by dropping 10 yl of its PBS solution (500 mg 1 - I), followed by drying in static atmospheric air for 20 min. The resonator was then mounted between silicon rubber sheets inside a flow cell made of PTFE [17], as shown in Fig. 1. Only the antibody-immobilized surface of the resonator was exposed to the flow of sample solution. Each sample (about 1 cm3) was circulated through the cell by a micro-tube pump at a rate of 0.44 cm3 min- ‘. The resonant frequency was monitored with a universal counter (Advantest, TR.5822) at room temperature. 3. Results and discussion
+PBS (pH 7.2)
PBS+ +*cidic soln. (pH 7.2) (pH 3.0) Fig. 2. Typical response transient of the sensor on successive exposure to 10 ppm HSA and 1000 ppm poiyclonal antibody.
solutions. On contact to 10 mg 1-l HSA in PBS, the resonant frequency shifted downward by about 70 Hz (first step). The 90% response time was about 7 min. On the subsequent introduction of 1000 mg l- l polyclonal antibody solution, the resonant frequency decreased further by about 160 Hz, taking a rather long time of approximately 30 min (second step). Since the resonant frequency returned reversibly to the initial level by treatment with acidic buffer solution (pH 3.0) and PBS, HSA measurements could be repeated many times with the same device [16]. Each step of immunoreaction was subjected to equilibrium as well as kinetic analyses as described below. 3.2. Mnthematicnl derivation of association constants and kinetic constants
The first step response (frequency shift) represents an immunoreaction between free HSA in liquid media (HSA,,,) and the monoclonal antibody immobilized on the sensor surface (AbbmounJ. HSAn,
3.1. Responsetransient
Fig. 2 shows the response transient of the sensor immobilized with anti-HSA monoclonal antibody on successive exposure to HSA and polyclonal antibody Antibody on Au electrode
1000 ppm polyclonal
+ Abr.,,,;?
HSA-Abround
where k,,, and kd,l are kinetic constants of the forward and reverse reactions, respectively, and the superscript m indicates monoclonal antibody. Although the immobilized monoclonal antibody has two binding sites per molecules, it can be assumed to form a one-to-one conjugate with HSA, because of the large molecular size of HSA compared to the size of the monoclonal antibody. The second step response on resonant frequency results from an immunoreaction between free polyclonal antibody and unreacted sites of the HSA already bound by the immobilized monoclonal antibody: Ab’&,, + HSA-Abg:,,d;+;AbP-HSA-AbrO,,,
Acrylic
resin
Fig. 1. Schematic view of cross-section of the flow cell equipped with a piezoelectric immunosensor.
(1)
(2)
where superscript p means polyclonal antibody. According to our results reported earlier [16], this step also produces a one-to-one conjugate (AbP-HSA-AbE&,,) when the (HSA-Ab&J complex is formed adequately. The association constants of Eqs. (1) and (2), Ki and K2, are given by
G. Sakai
Kd. 1
et al. /Sensors
ad
Actuators
B 42 (1997)
91
89-94
stage) to 0, (time t). After rearrangement, ing equation is obtained:
Kd,2
Let us consider the first step. A Langmuir-type adsorption model has already been applied to evaluating the association constant of an immunoreaction [15]. A similar treatment was carried out in our system. It is assumed that the immobilized monoclonal antibody molecules provide ‘adsorption sites’ for free HSA molecules just as in the case of gas adsorption on a solid surface, the fractions of occupied and unoccupied sites being 0, and 0, ( = 1 - QJ, respectively. The fully occupied state (0, = 1) is realized when a sufficiently high concentration of HSA is in contact with Ab&,, at equilibrium. The frequency shift up to this state, denoted 4max, corresponds to the adsorption capacity of the adsorbent. When one obtains a frequency shift (Af) up to time t at a given HSA concentration, 8, and 8, are expressed as follows: (4) The rate (Y) of formation round) is given by
1
t = k.,WSAl + ki.,
ln
k,,WAl
the follow-
k$-=W - (kJHSA1 + ‘b,#, (8)
By using Eqs. (3) and (4), this equation is transformed to k,,, . t = -!- In AB
1 l-AAf
where
B= A = (1 + 1/~1FWY4L,,max and [HSAlAfo,mw This equation indicates that the quantity
of the right-hand side increases linearly with time t, the slope giving the value of k,,,. A similar treatment for the second step can allow the estimation of k,,,.
3.3. Estimation of association constants and maximum frequency shifts Fig. 3 shows the frequency shift at equilibrium (Afo) as a function of concentration of (a) HSA (first step) or (b) polyclonal antibody (second step). The value Afo in
of the conjugates (HSA-Ab-
f(HS.b,-)J,~ 0””dj= ~$-W~V -~~,,~A = k,,WAl
- (k,WW
+ kd,,)“A
(5) where [HSA] is the concentration of free HSA in liquid media. At equilibrium, the rate must be zero and this condition leads to
B = A
k,WAl k,,,[HSA]
K,[HSA] + kd,, =-I + KJHSA]
200 : (4
(6)
The value Af in Eq. (4) is now represented by Afo, the frequency shift up to the equilibrium state (or saturation) at the given HSA concentration. Rearrangement of Eq. (6) gives Afo =
20
4imaxK[HSAI~ or
40
60
80
100
120
Cont. of HSA / ppm
1 + K,[HSA]
WW _ [HSAI I
1
(7) Afo Afo,,,, Afo,maxK1 By plotting [HSA]/A’, versus [HSA], K, (association constant) and AfO,maxcan be obtained. K, for the second step can be estimated similarly. Recently, Ebato et al. [15] and Ebara and Okahata [18] estimated the rate constants of some reactions of proteins from the time course of frequency shift. A similar analysis was applied here to estimate the rate constant (k,,, and ka,2) of the forward reactions (Eqs. (1) and (2)). It is assumed that the amount of HSA,,, in the liquid media is sufficiently large compared with that of the adsorption sites of Ab&,d so that its concentration is kept substantially constant during the reaction. Under this condition, the inverse of reaction rate, Eq. (5), can be integrated with respect to 8, from 0 (initial
400 (b) N z $
300 200 100
Cont. of polyclonal antibody / ppm Fig. 3. Dependence of A& of the sensor on concentration of HSA (a) and polyclonal antibody (b).
92
G. Sakai
et al. /Sensors
and Actuators
3 4.2 (1997)
3.4. Estimation
0'
I 0
1 1.0
05
1.5
[HSA] X lo6
0
0.2
0.4
0.6
023
1.0
1.2
89-94
of rate constmts
By using the values of K and AfO,maxobtained above, the right-hand term of Eq. (9), (l/U) In l/(1 -A Af), can be calculated for a given value of Af: The observed time courses of Af for the first and second steps were thus analyzed based on Eq. (9), as shown in Fig. 5(a) and (b). In these particular examples, the concentration of free HSA (first step) and free polyclonal antibody (second step) were 10 and 1000 mg 1- I, respectively, and the time course for the second step was measured after the first step was saturated by exposure to 100 mg l- ’ HSA solution. The almost linear correlations obtained ensure that the rates of the immunoreactions are essentially controlled by the fraction of unoccupied sites of the immobilized antibody or antigen on the surface of quartz crystal and the concentration of substrate (antigen or antibody) in liquid media, as assumed. The slopes of the correlations give the rate constants, k,,, and ka,2, which were determined to be (2.7 &- 0.1) x lo4 and 95 & 1 M-’ s- l1 respectively. These data indicate that the hrst step immunoreaction proceeds much faster than the second step one.
1.4
[poly] x los Fig. 4. Relationships between [substrate concentration]/Ajl, and [substrate concentration]. (a) HSA; (b) polyclonal antibody. Linear lines depicted in these figures were obtained by the least squares method: R = 0.993 (a); R = 0.959 (b).
the second step was measured after the first step finished by the exposure to the flow of 100 mg 1-j HSA solution. By using these values of A&, [HSA]/Af and [AbP]/A& were plotted, according to Eq. (7), as a function of @ISA] and [AbP] for the first and second steps, respectively, as shown in Fig. 4. The solid linear lines are derived based on the least squares method. From the slopes of these lines, the maximum frequency shifts (A&,,,) for the first and second steps are estimated to be 160 2 10 and 440 + 90 Hz, respectively. From the intercepts, the association constants (K, and K2) are calculated to be (5.4 1 1.4) x lo6 and (1.6 ir 0.8) x 10’ M - ‘, respectively. These results show that the stability of HSA-monoclonal antibody conjugate is about 34fold higher than that of HSA-polyclonal antibody conjugate. The reproducibility of such an analysis was tested by repeating the experiments with other QCM devices. It was found that, when a fixed concentration of monoclonal antibody solution was used for immobilization, AfO,maxvalues were reproduced within fluctuations by 30%. Nevertheless, the fluctuations of K, and K2 values were far lower, ensuring that those values are almost independent of the fluctuations in the amount of immobilized monoclonal antibody.
Time I s 6.0 /
0
100
200
300 400 Time / s
500
600
Fig. 5. Relationships between (l/AB) In I(1 -A Afs) and time i for the first step (a) and the second step (b). (The definition of A and B are given in the text.) Linear lines depicted in these figures were obtained from the fitting by using Eq, (9): R = 0.982 (a); R = 0.989 (b).
G. Sakai
et al. /Sensors
and Actuators
polyclonal antibody HSA molecules immobilized monoclonal antibody Quartz crystal Fig. 6. Schematic drawing of HSA conjugates immobilized sensor surface.
on the
3.5. Stnte of HSA conjugate HSA has several binding sites to form various conjugates with the polyclonal antibody in solution. The HSA already bonded to the immobilized monoclonal antibody on the surface of quartz crystal, however, has been reported to form only a one-to-one conjugate with the polyclonal antibody unless the HSA population on the surface is too low [16]. It seems that the HSA bonded to the monoclonal antibody in the first step is fixed in orientation or configuration rather tightly, leaving no more than one unoccupied site available for the conjugation with the polyclonal antibody. This situation is illustrated schematically in Fig. 6. It is considered that formation of the HSA-polyclonal antibody conjugate (second step) suffers such steric hindrance effects more severely than that of the HSA-monoclonal antibody conjugate (first step). This view is consistent with the present result that Ki is about 34 times as large as K2. In addition, the rate constant /<,,r (first step) was far larger than k,,, (second step), suggesting also the difference in the steric hindrance effect. It should be remarked that even the value of k,,, is lower than the rate constants of the usual immunoreactions in liquid media, which are reportedly in the range lo’-lo8 M- ’ s- *, This suggests that the first step is also hindered sterically or orientationally, while such hindrance becomes more severe for the second step. However, these speculations are to be verified by further investigations.
References
PI PI [31
c41
151
T. Nomura, M. Iijima, Electrolytic determination of nanomolar concentrations of silver in solution with a piezoelectric quartz crystal, Anal. Chim. Acta 131 (1981) 97-102. K.K. Kanazawa, J.G. Gordon II, Frequency of a quartz microbalance in contact with liquid, Anal. Chem. 57 (1985) 17701771. S. Bruckenstein, M. Shay, Experimental aspects of use of the quartz crystal microbalance in solution, Electrochim. Acta 30 (1985) 1295-1300. M. Thompson, C.L. Arthur, G.K. Dhaliwal, Liquid-phase piezoelectric and acoustic transmission studies of interfacial immunochemistry, Anal. Chem. 58 (19%) 1206-1209. E.S. Grabbe, RI?. Buck, O.R. Melroy, Cyclic voltammetry and quartz microbalance electrogravimetry of IgG and anti-IgG reactions on silver, J. Electroanal. Chem. 223 (1987) 67-78.
B 42 (1995)
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93
[61J.E. Roederer, G.J. Bastiaans, Microgravimetric
immunoassay with piezoelectric crystals, Anal. Chem. 55 (1983) 2333-2336. I71 R.C. Ebersole, M.D. Ward, Amplified mass immunosorbent assay with a quartz crystal microbalance, J. Am. Chem. Sot. 110 (1988) 8623-8628. H. Muramatsu, E. Tamiya, I. Karube, Determination of microbes and immunoglobulins using a piezoelectric biosensor, J. Memb. Sci. 41 (1989) 281-290. [91 H. Muramatsu, Y. Watanabe, M. Hikuma, T. Ataka, I. Kubo, E. Tamiya, I. Karube, Piezoelectric crystal biosensor system for detection of Edzerichiu co& Anal. Lett. 22 (1989) 2155-2166. 1101C. Barnes, C. D’Silva, J.P. Jones, T.J. Lewis, A Concanavalin A-coated piezoelectric crystal biosensor, Sensors and Actuators B 3 (1991) 295-304. E. Prusak-Sochaczewski, J.H.T. Luong, A new approach to the development of a reusable piezoelectric crystal biosensor, Anal. Lett. 23 (1990) 401-409. M. Muratsugu, F. Ohta, Y. Miya, T. Hosokawa, S. Kurosawa, 1121 N. Kamo, H. Ikeda, Quartz crystal microbalance for the detection of microgram quantities of human serum albumin: relationship between the frequency change and the mass of protein adsorbed, Anal. Chem. 65 (1993) 2933-2937. [I31 G. Sauerbrey, Venvendung von Schwingquartzen zur Wagung dunner Schichten und zur Mikrowagung, Z. Phys. 155 (1959) 206-222.
K.A. Davis, T.R. Leary, Continuous liquid-phase piezoelectric biosensor for kinetic immunoassays, Anal. Chem. 61 (1989) 1227-1230. 1151 H. Ebato, CA. Gentry, J.N. Herron, W. Muller, Y. Okahata, H. Ringsdorf, P.A. Suci, Investigation of specific binding of antifluorescyl antibody and Fab to fluorescein lipids in LangmuirBlodgett deposited films using quartz crystal microbalance methodology, Anal. Chem. 66 (1994) 1683-1689. 1161G. Sakai, T. Saiki, T. Uda, N. Miura, N. Yamazoe, Selective and repeatable detection of human serum albumin by using piezoelectric immunosensor, Sensors and Actuators B 24/25 (1995) 134-137. 1171 N. Miura, H. Higobashi, G. Sakai, A. Takeyasu, T. Uda, N. Yamazoe, Piezoelectric crystal immunosensor for sensitive detection methamphetamine (stimulant drug) in human urine, Sensors and Actuators B 13/14 (1993) 188-191. 1181 Y. Ebara, Y. Okahata, A kinetic study of Concanavalin A binding to glycolipid monolayers by using a quartz-crystal microbalance, J. Am. Chem. Sot. 116 (1994) 11209-11212. [I41
Biographies
Go Sn/crri has been a research associate at Kyushu University since 1996. He received a B.Eng. degree in applied chemistry in 1991 and a doctorate in engineering in 1996 from Kyushu University. His current research work is focused on the development of chemical sensors based on semiconductive oxides and piezoelectric quartz crystal. Tuknhiro Soiki received a B.Eng. degree in applied chemistry and an M.Eng. degree in materials science and technology in 1993 and 1995, respectively, from Kyushu University. He currently works at Hitachi.
94
G. Sakai et al. jSensovs and Actmtors B 42 (1997) 89-94
Taim Uda has been a professor at Hiroshima Prefectural University since 1994. He received a B.Eng. degree in 1969 from Yamaguchi University and a doctorate in engineering in 1975 from Kyushu University. His current research concentrates on the basic research of immunological chemistry and its application to biosensors, hTorio Miura has been an associate professor at Kyushu University since 1982. He received a B.Eng. degree in applied chemistry in 1973, an M.Eng. degree in 1975 from Hiroshima University and a doc-
in 1980 from torate in engineering Kyushu University. His current research concentrates on development of new chemical sensors as well as other electrochemical functional devices such as ECD and secondary batteries. Noboru Yamnzoe has been a professor at Kyushu University since 1981. He received a B.Eng. degree in applied chemistry in 1963 and a doctorate in engineering in 1969 from Kyushu University. His current research interests include the development and application of functional inorganic materials.
Sensors
and Actuators
B 42 (1997)
89-94
Evaluation of binding of human serum albumin (HSA) to monoclonal and polyclonal antibody by means of piezoelectric immunosensing technique G. Sakai a,*, T. Saiki a, T. Uda b, N. Miura a, N. Yamazoe a a Department
of Materials b School
Science
and Technology,
of Bioresources, Received
Hiroshima
25 October
Graduate School of Engineering Sciences, K)ushu Unirersity, Fukuoka 816, Japan Prefectural Unirersity, Shol!bara, Hiroshima 727, Japan
1996; received
in revised
form
26 December
Kasuga-shi,
1996
Abstract Binding of human serum albumin (HSA) to its monoclonal and polyclonal antibody was examined by using a quartz crystal immunosensor on which the immobilized monoclonal antibody was exposed to HSA and polyclonal antibody stepwise. The association constants and rate constants of the respective steps of immunoreactions were estimated from the resonant frequency shifts at steady state and the time course of resonant frequency. The binding of HSA to the polyclonal antibody (second step) was shown to be much weaker and slower than that to the monoclonal antibody (first step), probably owing to the steric hindrance and orientation effects of the HSA already bound to the immobilized monoclonal antibody. 0 1997 Elsevier Science S.A. Keyxaords:
Immunoreaction;
Kinetic
analysis;
Quartz
crystal
microbalance;
1. Introduction
Recent advances in the quartz crystal microbalance (QCM) technique have made it possible to detect quite sensitively the mass change on the resonator surface even when the quartz crystal is immersed in liquid media [l-5]. The simplicity, low cost, and ready availability of piezoelectric transducers have encouraged the development of various immunosensors [6121. The change in resonant frequency varies linearly with mass of protein (antigen or antibody) adsorbed on the electrode surface of the quartz even in a liquid media, following Sauerbrey’s equation, just as in the case of gas adsorption [13,14]. Thus, piezoelectric immunosensors
bio-related
can not only detect very small amounts of
substances specifically
but also monitor
* Corresponding author. 0925-4005/97/$17.00 PIISO925-4005(97)00188-3
8 1997 Elsevier
Science
S.A. All rights
reserved.
Sandwich
method
directly immunoreactions between antibodies and antigens at the solid (electrode of quartz crystal)/liquid (diluting media) interface. The QCM technique can be applied for the analysis of the kinetics
of specific bind-
ing of antifluorescyl antibody and Fab to fluorescein lipids in Langmuir-Blodgett films on the quartz crystal [15]. Meanwhile, we have designed a piezoelectric human serum albumin (HSA) sensor immobilized with anti-HSA monoclonal antibody and have found that the HSA sensitivity of the sensor can be much improved by adopting a ‘sandwich’ method in which HSA is combined with monoclonal and polyclonal antibody stepwise [16]. Owing to the high sensitivity and high stability of the frequency response, this ‘sandwich’ method enables one to analyze the nature of both immunoreactions involved in the respective steps. In this paper, we have tried to examine the binding affinities and kinetics of the immunoreactions of HSA with the monoclonal and polyclonal antibody on the surface of the QCM on the basis of its frequency response behavior.
90
G. Sakai
et al. /Sensors
and Actuators
2. Experimental
3 42 (1997)
89-94
10 ppm HSA soin. +
2.1. Reagents
Anti-I-ISA monoclonal antibody (IgG) (molecular weight about 150 000) was obtained from a hybridoma clone by the fusion of mouse myeloma cells of immunized mice. Anti-HSA polyclonal antibody was donated by Shionogi Pharmacia. HSA (Sigma) and bovine serum albumin (BSA, Sigma) were dissolved in a phosphate buffer saline (PBS, pH 7.2) containing 0.1% sodium azide. An acidic buffer solution (PH 3.0) was prepared with 0.2 M disodium hydrophosphate and 0.1 M citric acid. 2.2. Sensing device The quartz resonator used was of AT-cut (diameter 13 mm, MAXTEC) attached with sputtered Au electrodes (diameter 11 mm). Its basic resonant frequency was 6 MHz. Anti-HSA monoclonal antibody was immobilized on top of one of the gold electrodes of the quartz resonator by dropping 10 yl of its PBS solution (500 mg 1 - I), followed by drying in static atmospheric air for 20 min. The resonator was then mounted between silicon rubber sheets inside a flow cell made of PTFE [17], as shown in Fig. 1. Only the antibody-immobilized surface of the resonator was exposed to the flow of sample solution. Each sample (about 1 cm3) was circulated through the cell by a micro-tube pump at a rate of 0.44 cm3 min- ‘. The resonant frequency was monitored with a universal counter (Advantest, TR.5822) at room temperature. 3. Results and discussion
+PBS (pH 7.2)
PBS+ +*cidic soln. (pH 7.2) (pH 3.0) Fig. 2. Typical response transient of the sensor on successive exposure to 10 ppm HSA and 1000 ppm poiyclonal antibody.
solutions. On contact to 10 mg 1-l HSA in PBS, the resonant frequency shifted downward by about 70 Hz (first step). The 90% response time was about 7 min. On the subsequent introduction of 1000 mg l- l polyclonal antibody solution, the resonant frequency decreased further by about 160 Hz, taking a rather long time of approximately 30 min (second step). Since the resonant frequency returned reversibly to the initial level by treatment with acidic buffer solution (pH 3.0) and PBS, HSA measurements could be repeated many times with the same device [16]. Each step of immunoreaction was subjected to equilibrium as well as kinetic analyses as described below. 3.2. Mnthematicnl derivation of association constants and kinetic constants
The first step response (frequency shift) represents an immunoreaction between free HSA in liquid media (HSA,,,) and the monoclonal antibody immobilized on the sensor surface (AbbmounJ. HSAn,
3.1. Responsetransient
Fig. 2 shows the response transient of the sensor immobilized with anti-HSA monoclonal antibody on successive exposure to HSA and polyclonal antibody Antibody on Au electrode
1000 ppm polyclonal
+ Abr.,,,;?
HSA-Abround
where k,,, and kd,l are kinetic constants of the forward and reverse reactions, respectively, and the superscript m indicates monoclonal antibody. Although the immobilized monoclonal antibody has two binding sites per molecules, it can be assumed to form a one-to-one conjugate with HSA, because of the large molecular size of HSA compared to the size of the monoclonal antibody. The second step response on resonant frequency results from an immunoreaction between free polyclonal antibody and unreacted sites of the HSA already bound by the immobilized monoclonal antibody: Ab’&,, + HSA-Abg:,,d;+;AbP-HSA-AbrO,,,
Acrylic
resin
Fig. 1. Schematic view of cross-section of the flow cell equipped with a piezoelectric immunosensor.
(1)
(2)
where superscript p means polyclonal antibody. According to our results reported earlier [16], this step also produces a one-to-one conjugate (AbP-HSA-AbE&,,) when the (HSA-Ab&J complex is formed adequately. The association constants of Eqs. (1) and (2), Ki and K2, are given by
G. Sakai
Kd. 1
et al. /Sensors
ad
Actuators
B 42 (1997)
91
89-94
stage) to 0, (time t). After rearrangement, ing equation is obtained:
Kd,2
Let us consider the first step. A Langmuir-type adsorption model has already been applied to evaluating the association constant of an immunoreaction [15]. A similar treatment was carried out in our system. It is assumed that the immobilized monoclonal antibody molecules provide ‘adsorption sites’ for free HSA molecules just as in the case of gas adsorption on a solid surface, the fractions of occupied and unoccupied sites being 0, and 0, ( = 1 - QJ, respectively. The fully occupied state (0, = 1) is realized when a sufficiently high concentration of HSA is in contact with Ab&,, at equilibrium. The frequency shift up to this state, denoted 4max, corresponds to the adsorption capacity of the adsorbent. When one obtains a frequency shift (Af) up to time t at a given HSA concentration, 8, and 8, are expressed as follows: (4) The rate (Y) of formation round) is given by
1
t = k.,WSAl + ki.,
ln
k,,WAl
the follow-
k$-=W - (kJHSA1 + ‘b,#, (8)
By using Eqs. (3) and (4), this equation is transformed to k,,, . t = -!- In AB
1 l-AAf
where
B= A = (1 + 1/~1FWY4L,,max and [HSAlAfo,mw This equation indicates that the quantity
of the right-hand side increases linearly with time t, the slope giving the value of k,,,. A similar treatment for the second step can allow the estimation of k,,,.
3.3. Estimation of association constants and maximum frequency shifts Fig. 3 shows the frequency shift at equilibrium (Afo) as a function of concentration of (a) HSA (first step) or (b) polyclonal antibody (second step). The value Afo in
of the conjugates (HSA-Ab-
f(HS.b,-)J,~ 0””dj= ~$-W~V -~~,,~A = k,,WAl
- (k,WW
+ kd,,)“A
(5) where [HSA] is the concentration of free HSA in liquid media. At equilibrium, the rate must be zero and this condition leads to
B = A
k,WAl k,,,[HSA]
K,[HSA] + kd,, =-I + KJHSA]
200 : (4
(6)
The value Af in Eq. (4) is now represented by Afo, the frequency shift up to the equilibrium state (or saturation) at the given HSA concentration. Rearrangement of Eq. (6) gives Afo =
20
4imaxK[HSAI~ or
40
60
80
100
120
Cont. of HSA / ppm
1 + K,[HSA]
WW _ [HSAI I
1
(7) Afo Afo,,,, Afo,maxK1 By plotting [HSA]/A’, versus [HSA], K, (association constant) and AfO,maxcan be obtained. K, for the second step can be estimated similarly. Recently, Ebato et al. [15] and Ebara and Okahata [18] estimated the rate constants of some reactions of proteins from the time course of frequency shift. A similar analysis was applied here to estimate the rate constant (k,,, and ka,2) of the forward reactions (Eqs. (1) and (2)). It is assumed that the amount of HSA,,, in the liquid media is sufficiently large compared with that of the adsorption sites of Ab&,d so that its concentration is kept substantially constant during the reaction. Under this condition, the inverse of reaction rate, Eq. (5), can be integrated with respect to 8, from 0 (initial
400 (b) N z $
300 200 100
Cont. of polyclonal antibody / ppm Fig. 3. Dependence of A& of the sensor on concentration of HSA (a) and polyclonal antibody (b).
92
G. Sakai
et al. /Sensors
and Actuators
3 4.2 (1997)
3.4. Estimation
0'
I 0
1 1.0
05
1.5
[HSA] X lo6
0
0.2
0.4
0.6
023
1.0
1.2
89-94
of rate constmts
By using the values of K and AfO,maxobtained above, the right-hand term of Eq. (9), (l/U) In l/(1 -A Af), can be calculated for a given value of Af: The observed time courses of Af for the first and second steps were thus analyzed based on Eq. (9), as shown in Fig. 5(a) and (b). In these particular examples, the concentration of free HSA (first step) and free polyclonal antibody (second step) were 10 and 1000 mg 1- I, respectively, and the time course for the second step was measured after the first step was saturated by exposure to 100 mg l- ’ HSA solution. The almost linear correlations obtained ensure that the rates of the immunoreactions are essentially controlled by the fraction of unoccupied sites of the immobilized antibody or antigen on the surface of quartz crystal and the concentration of substrate (antigen or antibody) in liquid media, as assumed. The slopes of the correlations give the rate constants, k,,, and ka,2, which were determined to be (2.7 &- 0.1) x lo4 and 95 & 1 M-’ s- l1 respectively. These data indicate that the hrst step immunoreaction proceeds much faster than the second step one.
1.4
[poly] x los Fig. 4. Relationships between [substrate concentration]/Ajl, and [substrate concentration]. (a) HSA; (b) polyclonal antibody. Linear lines depicted in these figures were obtained by the least squares method: R = 0.993 (a); R = 0.959 (b).
the second step was measured after the first step finished by the exposure to the flow of 100 mg 1-j HSA solution. By using these values of A&, [HSA]/Af and [AbP]/A& were plotted, according to Eq. (7), as a function of @ISA] and [AbP] for the first and second steps, respectively, as shown in Fig. 4. The solid linear lines are derived based on the least squares method. From the slopes of these lines, the maximum frequency shifts (A&,,,) for the first and second steps are estimated to be 160 2 10 and 440 + 90 Hz, respectively. From the intercepts, the association constants (K, and K2) are calculated to be (5.4 1 1.4) x lo6 and (1.6 ir 0.8) x 10’ M - ‘, respectively. These results show that the stability of HSA-monoclonal antibody conjugate is about 34fold higher than that of HSA-polyclonal antibody conjugate. The reproducibility of such an analysis was tested by repeating the experiments with other QCM devices. It was found that, when a fixed concentration of monoclonal antibody solution was used for immobilization, AfO,maxvalues were reproduced within fluctuations by 30%. Nevertheless, the fluctuations of K, and K2 values were far lower, ensuring that those values are almost independent of the fluctuations in the amount of immobilized monoclonal antibody.
Time I s 6.0 /
0
100
200
300 400 Time / s
500
600
Fig. 5. Relationships between (l/AB) In I(1 -A Afs) and time i for the first step (a) and the second step (b). (The definition of A and B are given in the text.) Linear lines depicted in these figures were obtained from the fitting by using Eq, (9): R = 0.982 (a); R = 0.989 (b).
G. Sakai
et al. /Sensors
and Actuators
polyclonal antibody HSA molecules immobilized monoclonal antibody Quartz crystal Fig. 6. Schematic drawing of HSA conjugates immobilized sensor surface.
on the
3.5. Stnte of HSA conjugate HSA has several binding sites to form various conjugates with the polyclonal antibody in solution. The HSA already bonded to the immobilized monoclonal antibody on the surface of quartz crystal, however, has been reported to form only a one-to-one conjugate with the polyclonal antibody unless the HSA population on the surface is too low [16]. It seems that the HSA bonded to the monoclonal antibody in the first step is fixed in orientation or configuration rather tightly, leaving no more than one unoccupied site available for the conjugation with the polyclonal antibody. This situation is illustrated schematically in Fig. 6. It is considered that formation of the HSA-polyclonal antibody conjugate (second step) suffers such steric hindrance effects more severely than that of the HSA-monoclonal antibody conjugate (first step). This view is consistent with the present result that Ki is about 34 times as large as K2. In addition, the rate constant /<,,r (first step) was far larger than k,,, (second step), suggesting also the difference in the steric hindrance effect. It should be remarked that even the value of k,,, is lower than the rate constants of the usual immunoreactions in liquid media, which are reportedly in the range lo’-lo8 M- ’ s- *, This suggests that the first step is also hindered sterically or orientationally, while such hindrance becomes more severe for the second step. However, these speculations are to be verified by further investigations.
References
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Biographies
Go Sn/crri has been a research associate at Kyushu University since 1996. He received a B.Eng. degree in applied chemistry in 1991 and a doctorate in engineering in 1996 from Kyushu University. His current research work is focused on the development of chemical sensors based on semiconductive oxides and piezoelectric quartz crystal. Tuknhiro Soiki received a B.Eng. degree in applied chemistry and an M.Eng. degree in materials science and technology in 1993 and 1995, respectively, from Kyushu University. He currently works at Hitachi.
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G. Sakai et al. jSensovs and Actmtors B 42 (1997) 89-94
Taim Uda has been a professor at Hiroshima Prefectural University since 1994. He received a B.Eng. degree in 1969 from Yamaguchi University and a doctorate in engineering in 1975 from Kyushu University. His current research concentrates on the basic research of immunological chemistry and its application to biosensors, hTorio Miura has been an associate professor at Kyushu University since 1982. He received a B.Eng. degree in applied chemistry in 1973, an M.Eng. degree in 1975 from Hiroshima University and a doc-
in 1980 from torate in engineering Kyushu University. His current research concentrates on development of new chemical sensors as well as other electrochemical functional devices such as ECD and secondary batteries. Noboru Yamnzoe has been a professor at Kyushu University since 1981. He received a B.Eng. degree in applied chemistry in 1963 and a doctorate in engineering in 1969 from Kyushu University. His current research interests include the development and application of functional inorganic materials.