Anal. Chem. 1997, 69, 2711-2715
Application of Optical Chromatography to Immunoassay Toshiyuki Hatano, Takashi Kaneta, and Totaro Imasaka*
Department of Chemical Science and Technology, Faculty of Engineering, Kyushu University, Hakozaki, Fukuoka 812, Japan
Optical chromatography, a new separation technique involving the use of a radiation force and a medium flow, is used for trace analysis of protein. Two polystyrene beads, coated with antibody (anti-mouse IgG), are combined in the presence of an antigen (mouse IgG). The bound (B) and free (F) beads are readily separated by optical chromatography, and the B/F ratio can be correlated with the concentration of antigen (protein). Nanomolar concentrations of protein can be measured by this technique. The rates of the forward and reverse immunological reactions were independently determined by measuring the time of formation and dissociation, respectively, of the immunobeads. When a particle is irradiated by a laser beam, reflection and refraction occur at the surface of the particle. This, in turn, alters the direction of propagation of the laser beam. For the overall process, the momentum of the light is altered, which induces radiation pressure on the particle. This radiation force can be used for the trapping and manipulation of particles.1,2 In an earlier paper, we reported on a new separation technique using a combination of a radiation force and a medium flow, which we referred to as optical chromatography.3 The radiation force is proportional to the square of the particle diameter, while the force induced by the medium flow is proportional to the diameter of particle. Thus, the particle drifts at a point where the radiation force is identical to the force induced by the medium flow. On the other hand, it is well known that particles, when coated with an antibody, are combined (agglutinated) with one another in the presence of antigen (for example, protein agglutinization), as a result of an immunological reaction.4 The separation of bound (B) and free (F) immunobeads on the basis of their particle size may be performed using optical chromatography, as expected from data presented in the preceding paper.5 This paper reports a comparative study of two approaches for the immunoassay of proteins. The first is a modification of a conventional method that is commonly used for immunoassay. The basic principle of this technique is shown in Figure 1. The solution containing the protein (antigen) is mixed with polystyrene beads (immunobeads) coated with an antibody which are coagulated via an immunological reaction, as shown in Figure 1A. The (1) Ashkin, A. Phys. Rev. Lett. 1970, 24, 156-159. (2) Ashkin, A.; Dziedzic, J. M.; Bjorkholm, J. E.; Chu, S. Opt. Lett. 1986, 11, 288-290. (3) Imasaka, T.; Kawabata, Y.; Kaneta, T.; Ishidzu, Y. Anal. Chem. 1995, 67, 1763-1765. (4) Rosenzweig, Z.; Yeung, E. S. Anal. Chem. 1994, 66, 1771-1776. (5) Kaneta, T.; Ishidzu, Y.; Mishima, N.; Imasaka, T. Anal. Chem. 1997, 69, 2701-2710 (preceding paper in this issue). S0003-2700(97)00081-4 CCC: $14.00
© 1997 American Chemical Society
Figure 1. Concept of immunoassay using optical chromatography. (A) Formation of a pair of beads by agglutination. (B) B/F separation by optical chromatography.
mixture of bound and free beads is introduced into a capillary for separation by optical chromatography, as shown in Figure 1B. The B/F ratio is plotted against the concentration of antigen in order to construct an analytical curve. The second approach is based on on-line monitoring of the immunological reaction. First, the solution containing the immunobeads is introduced into a capillary, and they drift when reaching a position where the radiation force and medium flow are equal. The solution containing antigen is then introduced into the capillary. An antigen molecule combines with two immunobeads, and, as a result, the number of combined beads increases with increasing concentration of antigen. In this approach, it is possible to visualize the immunological reaction. Once two beads are combined, the bound bead begins moving from the equilibrium position for single beads and reaches a new equilibrium position corresponding to a pair of beads. The rates of forward and reverse immunological reactions can be determined by measuring the time periods for formation and dissociation of the bound bead. It is possible, in theory, to determine the rate constant for a single protein molecule by this technique, since it is possible to visualize the chemical reaction of a single molecule of antigen. In this work, we report on and discuss the potential advantages of optical chromatography in applications to immunoassay. EXPERIMENTAL SECTION Apparatus. A block diagram of the experimental apparatus used in this study is shown in Figure 2. An argon ion laser (GLG3200, Nippon Electric Co., Tokyo, Japan; 514.5 nm, 500 mW) is used as a light source for optical chromatography. The laser Analytical Chemistry, Vol. 69, No. 14, July 15, 1997 2711
Figure 3. Photograph of immunobeads separated by optical chromatography. The concentration of mouse IgG is 100 ng/mL. One coagulated bead consisting of two beads is observed in the picture, in addition to four single beads. The laser beam is introduced from the left-hand side, and the solution is flowing from the right-hand side. The distance between the marks is 1 mm. Table 1. Numbers of Coagulated and Single Beads at Different Concentrations of Mouse IgGa no. of particles Figure 2. Experimental apparatus for optical chromatography.
beam is focused by a lens with a focal length of 5 cm into a fused silica capillary (GL Science Inc., Tokyo, Japan; 200 µm i.d., 375 µm o.d.) whose polymer coating was removed by heating over a flame that portion which was inserted into a quartz cell. The lengths of the capillaries for inlet and outlet of the solution are 40 cm. The ends of the capillaries are immersed into reservoirs containing buffer solutions. The sample is introduced by gravity using a siphon method. The sample flow rate was adjusted to 100 µm s-1 by changing the levels of the buffer solution. The motion of the particles is observed by a video camera (chargecoupled-device, CCD) equipped with a video tape recorder. The experimental apparatus and the operation conditions are described in detail in the preceding paper.5 Sample. A suspended solution, containing 1.25% of anti-mouse IgG-coated latex beads (2.3 × 1010 particles/mL, 1.0-µm sphere), was supplied from Polyscience, Inc. (Warrington, PA). This solution was stored at 4 °C. A solution which was 20 mM in sodium phosphate (Kishida Chemical Co., Tokyo, Japan) and contained 7.6 × 107 particles/mL of antibody-coated immunobeads was used as a reagent solution. The pH of the solution was adjusted to 7.4 with sodium hydroxide. A sample solution of mouse IgG (1.0 mg/mL, pH 7.8) was purchased from Vector Laboratories, Inc. (Burlingame, CA). Procedure. Three-milliliter aliquots of the suspension of antibody-coated beads were treated for 15 min in an ultrasonic agitator, in order to avoid possible coagulation of the beads. Different amounts of mouse IgG were added to these aliquots to give concentrations of mouse IgG corresponding to 330, 230, 170, 100, 30, 20, and 3 ng/mL. The solution was gently stirred at room temperature for 2 h and was introduced into a capillary. The laser beam was then focused by counter-propagation for the separation of the beads by optical chromatography. For the case of realtime monitoring of the immunological reaction, antibody-coated beads were first introduced into the capillary, and the antigen solution was then allowed to flow into the capillary by gravity. RESULTS AND DISCUSSION Immunoassay of Protein. To determine a blank for these immunological assays, a sample solution containing no mouse IgG was used as a sample. Only single particles were observed, and no coagulated beads were found. Alternatively, a sample solution containing 100 ng/mL mouse IgG was added to the suspension of antibody-coated beads. The result is shown in Figure 3. A 2712 Analytical Chemistry, Vol. 69, No. 14, July 15, 1997
concn (ng/mL)
coagulated (B)
single (F)
B/F
330 230 170 100 30 20 3
5 3 1 1 1 none none
1 1 2 4 4 many many
5 3 0.5 0.25 0.25 0 0
reaction probability 91 86 50 33 33
a The reaction probability is derived by calculating the ratio of the number of spheres in the coagulated particles to that of the total number of spheres. Formation of coagulated particles containing more than three spheres is neglected in this study.
coagulated bead is clearly observed, in addition to four single beads. The coagulated bead is separated 500 µm upstream from the position of the single beads. Similar studies were carried out by changing the concentration of mouse IgG. The results are summarized in Table 1. Apparently, the B/F ratio, and hence the reaction probability, increase with increasing concentrations of mouse IgG. This permits the concentration of protein to be determined on the basis of B/F separation, using optical chromatography. The disadvantage of optical chromatography, as used herein, is poor precision, resulting from the small number of particles involved in the measurement. This disadvantage can be solved by increasing the laser power at the expense of the cost of the laser and also by decreasing the flow rate of the medium at the expense of the sample measurement time, since such a high-power laser and a low flow rate allow expansion of the beam radius at the position of particle drift in the solution.5 Another problem might be a lack of sensitivity, which is essentially determined by the incompleteness of the immunological reaction at low concentration levels. This problem also occurs in conventional methods for immunoassay based on coagulation of the immunobeads. Real-Time Monitor of Immunological Reaction. As described in the previous section, the immunological reaction is reversible; as a result, this limits the sensitivity of immunoassay. If it were possible to visualize the forward and reverse immunological reactions independently on a real-time scale, the concentration of protein could be determined at considerably lower concentration levels. This appears to be feasible by optical chromatography; single antibody-coated particles can be first introduced into a capillary and drift at the point where the radiation force is identical to the force induced by the medium flow, and
Figure 4. Series of photographs for immunobeads separated by optical chromatography. (A) At 0 s, single polystyrene beads drift at an equilibrated position; (B) 30 s, two beads are combined and begin moving to a new position, while a single bead is pulled by a combined bead; (C) 90 s, a pulled single bead is returning to the original position; and (D) 120 s, a combined bead is almost completely separated at a new equilibrated position. The distance between the single combined beads and the coagulated one is about 1 mm. The lens magnification is 100×. The other conditions are the same as in Figure 3.
Figure 5. Series of photographs for immunobeads separated by optical chromatography. (A) At 0 s, a combined bead is located 1 mm away from the equilibrated position for single beads; (B) 10 s, the combined bead dissociates to two beads, and these begin moving to the position at which single beads are equilibrated; (C) 20 s; (D) 30 s, the separated beads return almost completely to the original position. The lens magnification is 150×. Due to poor spatial resolution of the video system, the dissociated beads are seen as a single point in the picture.
then protein, introduced from the outside, reacts with two polystyrene beads, which then move from the original position to a new position of equilibrium. The sample solution can be moved back and forth to complete the chemical reaction, if necessary, by changing the level of the reservoir when using the siphon method or by changing the polarity of the electrode when using an electroosmotic flow method. The binding event can be visually observed by monitoring the motion of the coagulated bead toward its new position of equilibrium. Dissociation of the coagulated bead can also be confirmed by monitoring the motion of the combined bead, after separation, toward the original position. In this study, we performed a preliminary experiment to demonstrate the basic concepts of this approach. A series of photographs are printed from a video recorder and are shown in Figure 4. Initially, single polystyrene beads are drifting in the equilibrated region. A pair of beads are coagulated in the presence of antigen and begin moving to a new equilibrated position. This indicates that the immunological reaction has taken place and that the chemical species Ab-Ag-Ab is formed. This phenomenon appears more frequently when the concentration of antigen is increased. The coagulated bead is held at the equilibrated position and begins to move back to the original position again, as shown in Figure 5. This behavior was reproducible and was observed in all the experiments performed. The most likely explanation for this is that it is due to a dissociation of the chemical bond formed in the immunological reaction. The dissociated beads return nearly to the original position within 1 min. The time for combination of the immunobeads is plotted
against the concentration of mouse IgG in Figure 6A. The time period decreases with increasing concentration of mouse IgG. Thus, the concentration of protein can be determined from this calibration curve. On the other hand, the time period for dissociation of the combined immunobead was found to be independent of the concentration of antigen contained in the sample solution. Mechanism. The scheme for the immunological reaction can be expressed as
bead-Ab + Ag a bead-Ab-Ag
(1)
When the rate constants for forward and reverse reactions are assumed to be k1 and k2, respectively, the equilibrium constant, K, can be expressed as
K)
k1 [bead-Ab-Ag] ) k2 [bead-Ab][Ag]
(2)
Therefore, the concentration of bead-Ab-Ag is proportional to the concentration of Ag when the latter is sufficiently lower than the concentration of bead-Ab. Two beads are combined with antigen by the following immunological reaction:
bead-Ab-Ag + Ab-bead a bead-Ab-Ag-Ab-bead
(3)
The rate for the forward reaction, r3, can be written as Analytical Chemistry, Vol. 69, No. 14, July 15, 1997
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r3 ) k3[bead-Ab-Ag][bead-Ab] ) k3K[Ag][bead-Ab]2
(4)
where k3 is the rate constant. The concentration of the immunobead is constant in this experiment; nine beads are typically trapped. Thus, the reaction rate is proportional to the concentration of antigen. The time period for combining two immunobeads can be expressed as
T1 ) 1/r3
(5)
The rate for a reverse reaction, r4, can be written as
r4 ) k4[bead-Ab-Ag-Ab-bead]
(6)
T2 ) 1/r4
(7)
where k4 and T2 are the rate constant and the time period for dissociation of a combined immunobead, respectively. Analysis. The collision frequency of the antigen molecule and the immunobead can be calculated from their concentrations and the flow rate of the sample solution (30 µm/s). When the molecular weight of mouse IgG is assumed to be 105, the number of collisions per second is calculated to be 140. On the other hand, the particles are spread by the collisions among immunobeads and tend to move to the center of the equilibrated position. The moving speed of the particle in the congested region can be calculated from eq 18 in the preceding paper.5 The number of collisions between immunobeads is estimated to 5.7 s-1 when the number of beads is 9. Of course, this number increases with increasing number of immunobeads trapped. It is noted that the number of antibody molecules on the immunobeads is estimated to be 8.0 × 104, assuming a molecular weight of 106, while that of antigen molecules is 1.6 × 103, even at 1 µg/mL in the above congested region. Thus, the number of antigen is much less than that of antibody, which is consistent with the assumption used in deriving eq 1. As recognized from Figure 6A, the time needed to combine two immunobeads is 6-64 min, depending on the concentration of antigen. Thus, many collisions (680, 64 times) take place before coagulation of the immunobeads. The antigen molecules collide with immunobeads (5.1-8.5) × 105 times, on average, before a productive immunological reaction, resulting in the formation of a pair of beads. This value is independent of the concentration of antigen within the experimental error. Thus, there are sufficient collisions prior to an immunological reaction. These results are consistent with the assumptions used in deriving eqs 1 and 3, and eqs 2 and 4 appear to be valid for the present conditions. Figure 6B indicates the relationship between the concentration of antigen and the reciprocal value of the time period required for combining the immunobeads. A straight line is obtained, which suggests that eqs 4 and 5 are valid. On the other hand, the time period for dissociation of the combined immunobead was 10 ( 3 min, and this value was independent of the concentration of antigen used in the sample solution. It should be noted that the combined bead is completely separated from the single beads. In other words, no single beads exist in the region of the combined beads. Therefore, no equilibrium exists between the single and combined beads. The dissociation rate, 2714 Analytical Chemistry, Vol. 69, No. 14, July 15, 1997
Figure 6. (A) Relationship between concentration of mouse IgG and reaction time. (B) Data plotted against reciprocal of reaction time. The solid line is drawn by fitting the data to a straight line.
then, is simply proportional to the concentration (or number) of the coagulated bead; the number of coagulated beads is unity in all the experiment in this study. Thus, the dissociation rate depends only on the rate constant, k4. If two beads are combined with multiple sites, the reaction rate, and hence the time period for dissociation, may be dependent on the antigen concentration and show a distribution. For example, if 10 sites are used for combining a pair of beads on average, the bead combined with a single site may dissociate more easily, giving a shorter dissociation time. Thus, the dissociation time would depend on the concentration of antigen. Experimentally, neither distribution of the dissociation time nor dependence of antigen concentration was observed within the experimental error. This strongly suggests that the immunobeads are combined at a single site via a single chemical bond. The characteristics of a single-strand chain of DNA have been investigated in detail using an optical trapping technique, in which two latex beads, combined with a DNA molecule, are stretched by moving one of them using a trapping force.6,7 However, characteristics of a single bond for a reversible reaction has, to the best of our knowledge, not been investigated until now, even by a laser trapping technique. The present work, therefore, may be the first demonstration of an investigation of a single reversible bond. Using this approach, it is possible to separate forward and reverse chemical reactions, since no reaction products (coagulated particles in this study) exist in the measurement of the forward reaction, and no unreacted reagents (single particles) exist in the measurement of the reverse reaction. Thus, there is no equilibrium between them. It should be noted that the dissociated beads were combined and began moving to the (6) Cluzel, P.; Lebrun, A.; Heller, C.; Lavery, R.; Viovy, J. L.; Chatenay, D.; Caron, F. Science 1996, 271, 792-794. (7) Smith, S. B.; Cui, Y.; Bustamante, C. Science 1996, 271, 795-798.
equilibrium position for a combined bead again after the picture shown in Figure 5D was taken, although the other beads remained unchanged. The same phenomenon is sometimes observed in other experiments. This implies that a single antigen molecule with a capacity to bind a pair of beads exists on this immunobead; many antigen molecules are probably attached on the immunobeads, but these might be located inside or on concave parts of the bead and might not be effectively utilized for combining two immunobeads. If this is true, the reaction probability might be greatly improved by extending the chemical bond between the polystyrene bead and the antibody molecule. Further studies are, of course, necessary to verify this. However, it might be possible, in theory, to detect a single protein molecule at very low concentrations by the present approach, since a single coagulated particle can easily be separated and measured by optical chromatography. CONCLUSION A newly developed analytical method, optical chromatography, is applied to immunoassay of protein. Analogous to the conventional method, antibody-coated beads are combined in the presence of antigen, which is readily separated by optical chromatography for measurement of the bound/free ratio. The concentration range measured is in the nanomolar or nanograms per milliliter (8) Mishima, N.; Kaneta, T.; Imasaka, T. Unpublished result (1996).
range. Real-time monitoring of the immunological reaction is also demonstrated by causing the immunobeads to drift, by applications of a radiation force and a medium flow, in the presence of protein. The combining of the beads is visually observed, strongly suggesting that an immunological reaction between single antigen and antibody molecules occurs. This approach is expected to be used in the detection of single molecules of protein at low concentration levels. As partly demonstrated in this study, optical chromatography has a variety of unique applications. In our recent preliminary work, it was applied to a microorganism and is used for determination of its maximum power with no calibration being required.8 Thus, this new technique, optical chromatography, has potential to be used not only in immunoassay but also in a wide variety of scientific fields, including biology. ACKNOWLEDGMENT This research is supported by Grants-in-Aid for Scientific Research from the Ministry of Education of Japan and by Takeda Science Foundation.
Received for review January 23, 1997. Accepted March 28, 1997.X AC970081Q X
Abstract published in Advance ACS Abstracts, June 15, 1997.
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