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Flow-injection immunoassays: Present and future
MICROCHEMICAL
JOURNAL
45, 121-128 (1992)
Flow-Injection
Immunoassays:
Present and Future
CY H. POLLEMA,* JAROMIRRUZICKA,*" AKE LERNMARK,? AND GARY D. CHRISTIAN* *Department of Chemistry and fDepartment of Medicine, University of Washington, Seattle, Washington 98195 Received November 15, 1991; accepted November 18, 1991 The recent development of the flow-injection immunoassay (FIIA), beginning with one of the first publications in 1980 by Lim and Miller, has resulted in a growing field which combines the precise and reproducible timing of flow-injection analysis (FIA) with immunoassays to yield assays which are carried out in a nonequilibrium time frame. These often faster assay methods require small volumes of sample and reduced sample handling. The advancement of this field is briefly reviewed in this paper with respect to the general methods and detection schemes applied. A novel method utilizing sequential injection (SI) is also introduced. The method uses immunomagnetic beads to create a renewable reaction column which does not require in-line regeneration. 8 iwz Academic PKSS, Inc.
INTRODUCTION
The application of flow-injection analysis (FIA) to immunology has resulted in a method which offers promise of faster and more reproducible assays. FIA is advantageous in that it is possible to utilize the kinetics of immunochemical binding. Traditional batch type immunoassays are time-consuming, partially because of the equilibrium-based measurements carried out. FIA-based techniques are capable of non-equilibrium-based measurements. The timing attainable with a flow-injection system allows for short precise contact times. Several flowinjection immunoassays (FIIA) developed have exploited these advantages to yield assay times as short as 1 min (1). Two major types of analysis exist, homogeneous and heterogeneous, and several applications of both techniques to flow injection will be discussed as will the various detection schemes utilized. Future prospects involving the application of SI to immunoassays will be presented along with some initial results from a system being developed in our laboratories. FLOW-INJECTION
IMMUNOASSAYS
Homogeneous assays. Each of the two approaches to immunoassays, homogeneous and heterogeneous, has advantages. The typical method for homogeneous assay involves monitoring a change in the physical characteristics of the antigen-antibody complex. One of the earliest papers combining FIA and the immunoassay took advantage of an energy transfer reaction which occurred when fluorescein-labeled albumin was bound to rhodamine-labeled anti-albumin antibodies (2). The result was a loss of fluorescent intensity of the labeled albumin, ’ To whom correspondence should be addressed. 121 0026-265X/92 $1SO Copyright All rights
0 1992 by Academic Press, Inc. of reproduction in any form reserved.
122
POLLEMA
ET AL.
which when contacted with unlabeled albumin would again increase in intensity as the rhodamine-labeled albumin antibodies competitively bound to the unlabeled albumin. The sensitivity of the assay was in the nanomolar region. Several other detection methods exist, including turbidimetric (3, 4), electrochemical (5), ellipsometry (6), and fluorescence (2, 2, 7). The advantage to homogeneous assay is that no separation step is required; however, this also presents the problem of interferences from the sample matrix. One such problem discussed by Miller (8) is the interference which occurs in the emission region for fluorescein-labeled molecules, caused by albumin-bound bilirubin. Albumin, however, can be removed by the use of the dye Cibacron blue conjugated on agarose beads. Miller states that this treatment of the sample can reduce the serum fluorescence at 520 nm by 75%. Heterogeneous assays. Heterogeneous assays are more often applied since a detectable change in signal resulting from the formation of the antigen-antibody complex is not always possible. Heterogeneous assays most often carry out the separation step using an immobilized reactor which binds the species of interest while allowing the rest of the matrix to pass through the reactor. The bound sample is then detected by a variety of methods, including electrochemical detection (9-Z4), fluorescence (15, Z6), and chemiluminescence (17-20). The drawbacks in heterogeneous assays are that the separation step often complicates the system and following the assay, the bound sample must be removed from the column without affecting the activity of the reaction surface within the column. This regeneration is, at best, time consuming, and may lower the precision of the method due to changes in the reaction surface. The novel method proposed at the end of this review utilizes immunomagnetic beads and a sequential-injection (SI) system to avoid the drawback of in-line regeneration. Detection methods. Since antibody-antigen binding in itself does not produce a detectable signal, several techniques have been proposed to yield useful information from this binding in flow-injection systems. These techniques, some of which are summarized in a timeline in Table 1, typically involve a tag such as an enzyme, radioisotope, or fluorophore to monitor the reaction with a variety of detectors. Electrochemical detection has been applied in several methods (4, 944). Tang et al. monitored the reaction of 2,6-dichloroindophenol (DCIP) by following the production of DCIPH, to obtain detection limits of 0.38 PmolZliter in an assay for phenytion (4). Other enzyme-based electrochemical assays follow the production of hydrogen peroxide or ammonia (9-ZZ). The difficulty associated with electrochemical detection is maintaining a clean electrode surface in the presence of a biological matrix. Proteins are known to strongly adsorb to electrode surfaces, and continuous washing of the electrode surface is required to help prevent fouling. Fluorimetry has also been applied to immunoassays as a method of detection (1, 2, 7, 8, 15, 16). Locascio-Brown et al. used liposomes which can encase up to lo5 water-soluble fluorescent molecules to bind antigens in a sample (16). When the liposomes are passed into a reactor, those which contain bound antigen will not be retained in the column and will flow on to be detected. Turbidity measurements
FLOW-INJECTION
IMMUNOASSAY!3
123
TABLE 1 Timeline of the Development of FIIA Date published
Analyte
Detection principle
1980 1982 1985 1985
Albumin I& I& Fetoprotein Insulin Hydroxyprogesterone kG Lysozyme IgG k&G H,O, I&s Theophylline Insulin Liposomes w W Thyroxin Theophylline Valproic acid Albumin Theophylline Theophylline IS Salmonella Phenytoin
Fluorescence Fluorescence Immunoprecipitation Chemiluminescence
1985 198.5 1985 1987 1987 1988 1988 1989 1989 1989 1989 1990 1990 1990 1990 1991
Ellipsometry
Ref.
(2)
(1) (6) (191
(5)
Amperometric Turbidity Chemiluminescence Amperometric Potentiometric Fluorescence Chemiluminescence Chemiluminescence
(15) cw
(17)
Fluorescence
(7)
Fluorescence Fluorescence Amperometric Potentiometric Amperometric Amperometric
(8) (16) (14) (10) (91 (4)
based on immunoprecipitation provide a simple analytical technique requiring only a spectrophotometer when high sensitivity is not needed (6). Chemiluminescence-based assays have been reported, and may prove to be of future interest in the development of FIIA (17-20). Shellum reported an assay based on the acridinium chemiluminescence reaction (20). Limits of detection were 50 attomol (atto = lo- 18)of mouse IgG with a total analysis time of 18 min. Chemiluminescence is difficult to monitor and quantify due to the rapid decay of the single intensity. Therefore FIA brings an advantage which has been successfully applied (17-20). Vlasenko et al. investigated the mechanism of enhanced chemiluminescence with a FIA system (17). In this study the rate constants for the reactions between the oxidized forms of peroxidase with luminol and p-iodopheno1 as well as several other substrates were also investigated. In addition, homogeneous assays for insulin, antibodies to insulin, and antibodies to the trinitrophenyl group were contemplated. Kinetics. Most FIIA assays proposed so far allow prolonged contact time for binding. These times extend up to 15 min using stop flow techniques, yet very little is known concerning how much contact time can be shortened, indeed the kinetics of binding may be such that only a very short time is needed to bind an
124
POLLEMA
ET AL.
adequate amount of sample with increased selectivity. There are few studies which have addressed the initial binding. Jonsson et al. utilized ellipsometry to follow the binding of IgG to a protein (6). The study reported two kinetic runs which were carried our for 60 min, and indicated that saturation of the IgG occurred within 7 min. Initial binding was detected after approximately 1 min. Vlasenko et al. discussed the binding of human IgG as well as T4 (17). Kinetics in the range of &lo min were considered, as was the effect of incubation time on the sensitivity with respect to human IgG concentration. The investigation of initial binding rates during FIIA assays is needed to determine the optimum time and conditions for the assay. Further investigations of these kinetics may serve to shorten the time required for current assay methods and may lead to the restriction of side reactions. SEQUENTIAL-INJECTION
IMMUNOASSAYS
The new system which we are developing has previously been described (22) and is illustrated in Fig. 1A. The sequential-injection ($3) system used allows for a number of solutions (wash, immunomagnetic bead suspensions, and samples) to be selected and sequentially aspirated into the single reaction channel. The system utilizes a single selector valve (rather than injection) and a cam driven sinusoidal flow pump for solution manipulation. The configuration illustrated has the advantage that it is possible to carry out a number of different assays without physical restructuring. The assay procedure, as outlined in Fig. lB, is as follows. (i) Immunomagnetic beads which are coated with antibodies against the analyte are aspirated into the reaction coil (RC) and the electromagnetic field surrounding this coil is activated. This traps the beads within the flowing stream and provides an immobilized reaction surface. Next, the beads are washed to assure no unbound particles remain, and the wash is sent to waste. (ii) The analyte which has been spiked with a labeled competing reagent is then aspirated into the RC and the flow is stopped for a specified contact time. During this time, the analyte and labeled reagent compete for the available sites in the reactor. The more analyte which is present, the more labeled reagent is displaced during competition and remains unbound. (iii) Following the stopped flow, a flow reversal sends the unbound portion of the sample mixture to the detector to determine the amount of unbound labeled reagent present. This yields a signal at the detector which is directly related to the analyte concentration. (iv) Finally, the electromagnetic field is turned off and the reactor is flushed to waste to be regenerated off-line. The system is then ready for another assay. The use of immunomagnetic beads and a magnetic field to form a renewable reaction column within an SI system is novel, and therefore, some initial requirements needed to be met for the technique to be proven useful. First, it is necessary to hold the beads without substantial loss during the analysis in order to obtain quantitative results, and second, the beads must undergo binding within the magnetic field to a sufftcient extent to yield an analytical signal. The behavior of magnetic beads within a flowing stream was investigated with respect to flow rate and magnetic field strength. The voltage supplied to the
FLOW-INJECTION
125
IMMUNOASSAYS
(A)
Waste
Wash
strp
1
step
2
step 3
step .I
FIG. 1. (A) Diagram of sequential injection system, comprising a cam driven sinusoidal flow pump (SFP), a reaction coil (RC) located in an electromagnetic field (M), an eight-port multiposition valve (MPV), and a fluorescence detector (D). (B) Steps of the SIIA magnetic procedure: (1) Aspirate the immunomagnetic beads into the electromagnetic field and turn on the electromagnet. (2) Aspirate the sample spiked with labeled antibody into the reactor coil and stop flow. (3) Following a specified contact time expel1 the unbound sample mixture to the detector. (4) Turn off the electromagnet and remove the column to prepare for another assay.
magnet was varied from 1 to 7 V; above this range the coil of the magnet began to heat. One volt applied to the magnet retained 15% of the beads at a maximum flow rate during the sinusoidal flow cycle of 0.74 ml/min; 7 V retained 61% at the same flow rate. Percentages are based on measurement of the light scatter produced by the beads when passed through the detector, the range (t&100%) is from baseline to the signal produced when no magnetic field is applied and the sample is passed by the detector. Figure 2 shows the results of maintaining a constant DC voltage of 5 V to the magnet and varying the flow rate. It can be seen that at a maximum flow rate of 0.50 ml/min, no beads are lost from the magnetic field. To further
126
POLLEMA
ET AL.
50
40 0 .3
FIG. 2. Influence
0.4
0.5
0.6
0.7
of the flow rate on the retention of immunomagnetic
0.x
beads in the reactor coil.
assure this, in assays, a washing step is included after loading the beads to ensure no unbound beads are present when the sample is introduced. The initial studies indicate that the renewable column can be reproducibly formed within the flowing stream. This is indicated in Fig. 3, which shows peak profiles for a calibration run for OX-19 (a monoclonal antibody against an antigen expressed on rat thymocytes and T lymphocytes) using the proposed system. The three-point calibration with each point carried out in duplicate indicates a linear relationship for concentrations of OX-19 (R = 0.997). This calibration is obtained by first aspirating the beads into the magnetic field and then turning on the electromagnet. The beads are then washed to assure no unbound beads are present, and the wash is sent to waste. Next, the sample is aspirated into the “column” of beads and the flow is stopped. After a defined time the unbound sample is expelled to the detector. The reproducibility of the duplicates and the linear relationship between response and samples of different concentrations indicate that the reaction is uniform and reproducible. The idea of a renewable reaction column was also presented by Kindervater and Schmid (21) for pesticide determination. Fufure trends. Many detection schemes and assays have been developed since the introduction of FIIA in 1980. Advancements in FIA also continue with the addition of double injection (24, 25), and more recently sequential injection (23). Both these techniques offer great promise in applications to immunoassays due to
FLOW-INJECTION
IMMUNOASSAYS
127
FIG. 3. Peak profiles obtained for a calibration plot of 0X-19, at concentrations of antibody of 1,2, and 10 &ml, with each measurement being carried out in duplicate.
the dramatic reduction of volumes required. In double injection, one injects the reagent and sample consecutively in a single line which causes the two zones to merge within the continuously flowing carrier stream. This is superior to dual injection into separate streams and subsequent mixing because of the more reproducible behavior of zone overlap. Dual injection has difficulties in reproducible zone merging due to uneven wear of peristaltic pump tubing. Double injection has corrected for this by using a single channel and single pump. More recent is sequential injection which uses a multiposition valve with one reaction channel. Solution transport can be carried out using either a peristaltic pump or a syringe pump. The syringe pump has the advantage of being more rugged without the problems of changes in peristaltic pump tubing that occur over time as wall thicknesses change and tubing stretches. However, a lower sampling frequency is obtained, since some time is required to fill the syringe. Sequential injection is designed as a more robust method with precision as high as 0.3% RSD (26), and a design which may be adapted to many of the current assay techniques for automation. Most recent papers have begun to show the use of chemiluminescence in immunoassay detection (27-20). This methodology for chemiluminescence band immunoassays may prove to be of future interest because of its added selectivity and high sensitivity. Chemiluminescence measurements are, however, difficult to ex-
128
POLLEMA
ET AL.
ecute reproducibly and effkiently in a batch mode because the rapid decay of emitted light requires high precision of solution manipulations. Therefore, the precision of solution handling offered by SI systems may, in the future, become a preferred technique for chemiluminescence band immunoassays. ACKNOWLEDGMENTS The authors thank Walter Lindberg and Kurt Scudder for their help and the many useful discussions
REFERENCES 1. 2. 3. 4. 5. 6. 7. 8. 9. 10. Il. 12. 13. 14. IS. 16. 17. 18. 19. 20. 21. 22. 23. 24. 25. 26.
Christian, G. D.; Kelly, T. A. Z’ulanfa 1982, 29, 1109-1112. Lim, C. S.; Miller, J. N. Anal. Chim. Acfa 1980, 114, 183-189. Worsfold, P. J.; Hughes, A.; Mowthorpe, D. J. Analyst 1985, 110, 1303-1305. Hughes, A.; Worsfold, P. J. Anal. Proc. 1985, 22, 16-17. Tang, H. T.; Halsall, H. B.; Heineman, W. R. Clin. C/rem. 1991, 37, 245-248. Jonsson, U.; Ronnberg, I.; Malmqvist, M. Colloids Surfnces 1985, 13, 333-339. Allain, P.; Turcant, A.; Premel-Cable, A. Clin. Chem. 1989, 35, 469-470. Miller, J. N. Anal. Proc. 1989, 26, 317-318. Luong, J. H. T.; Prusak-Sochaczewski, E. Anal. Lett. 1990, 23, 1809-1826. Lee, I. H.; Meyerhoff, M. E. Anal. Chim. Acta 1990,229,47-55. De Alwis, W. U.; Wilson, G. S. Anal. Chem. 1985, 57, 2754-2756. Lee, I. H.; Meyerhoff, M. E. Mikrochim. Acta 1988, 3, 207-221. De Alwis, U.; Wilson, G. S. Anal. Chem. 1987, 59, 2786-2789. Gil, P. E.; Tang, H. T.; Hal&l, H. B.; Heinemann, W. R.; Misiego, A. S. Clin. Chem. 1990,36, 662-665. Locascio-Brown, L.; Plant, A. L.; Durst, R. A. Anal. Chem. 1988, 60, 792-797. Locascio-Brown, L.; Plant, A. L.; Horvath, V.; Durst, R. A. Anal. Chem. 1990,62, 2587-2593. Vlasenko, S. B.; Arefyev, A. A.; Klimov, A. D.; Kim, E. L.; Gorovits, E. L.; Osipov, A. P.; Gavrilova, E. M.; Yegorov, A. M. J. Biolumin. Chemilumin. 1989, 4, 164-176. Hook, K.; Nieman, T. A. Anal. Chem. 1987,59, 869-872. Maeda, M.; Tsuji, A. Anal. Chim. Acta 1985, 167, 241-248. Shehum, C.; Gubitz, G. Anal. Chim. Acta 1989, 227, 97-107. Kindervater, R.; Schmid, R. D. International Workshop on FIA Based on Enzymes or Antibodies, Braunschweig, West Germany, May 14-15, 1990. Pollema, C. H.; Ruzicka, J.; Lemmark, A.; Christian, G. D. Submitted for publication. Ruzicka, J.; Gubeli, T. Anal. Chem. 1991, 63, 1680-1685. Bergamin, F. H.; Krug, F. J.; Zagatto, E. A. G. Anal. Chim. Acta, 1978, 97, 427-431. Bergamin, F. H.; Zagatto, E. A. G.; Krug, F. J.; Reis, B. F. Anal. Chim. Acta 1978, 101, 17-23. Ruzicka, J.; Gubeli, T. Anal. Chem. in press.
JOURNAL
45, 121-128 (1992)
Flow-Injection
Immunoassays:
Present and Future
CY H. POLLEMA,* JAROMIRRUZICKA,*" AKE LERNMARK,? AND GARY D. CHRISTIAN* *Department of Chemistry and fDepartment of Medicine, University of Washington, Seattle, Washington 98195 Received November 15, 1991; accepted November 18, 1991 The recent development of the flow-injection immunoassay (FIIA), beginning with one of the first publications in 1980 by Lim and Miller, has resulted in a growing field which combines the precise and reproducible timing of flow-injection analysis (FIA) with immunoassays to yield assays which are carried out in a nonequilibrium time frame. These often faster assay methods require small volumes of sample and reduced sample handling. The advancement of this field is briefly reviewed in this paper with respect to the general methods and detection schemes applied. A novel method utilizing sequential injection (SI) is also introduced. The method uses immunomagnetic beads to create a renewable reaction column which does not require in-line regeneration. 8 iwz Academic PKSS, Inc.
INTRODUCTION
The application of flow-injection analysis (FIA) to immunology has resulted in a method which offers promise of faster and more reproducible assays. FIA is advantageous in that it is possible to utilize the kinetics of immunochemical binding. Traditional batch type immunoassays are time-consuming, partially because of the equilibrium-based measurements carried out. FIA-based techniques are capable of non-equilibrium-based measurements. The timing attainable with a flow-injection system allows for short precise contact times. Several flowinjection immunoassays (FIIA) developed have exploited these advantages to yield assay times as short as 1 min (1). Two major types of analysis exist, homogeneous and heterogeneous, and several applications of both techniques to flow injection will be discussed as will the various detection schemes utilized. Future prospects involving the application of SI to immunoassays will be presented along with some initial results from a system being developed in our laboratories. FLOW-INJECTION
IMMUNOASSAYS
Homogeneous assays. Each of the two approaches to immunoassays, homogeneous and heterogeneous, has advantages. The typical method for homogeneous assay involves monitoring a change in the physical characteristics of the antigen-antibody complex. One of the earliest papers combining FIA and the immunoassay took advantage of an energy transfer reaction which occurred when fluorescein-labeled albumin was bound to rhodamine-labeled anti-albumin antibodies (2). The result was a loss of fluorescent intensity of the labeled albumin, ’ To whom correspondence should be addressed. 121 0026-265X/92 $1SO Copyright All rights
0 1992 by Academic Press, Inc. of reproduction in any form reserved.
122
POLLEMA
ET AL.
which when contacted with unlabeled albumin would again increase in intensity as the rhodamine-labeled albumin antibodies competitively bound to the unlabeled albumin. The sensitivity of the assay was in the nanomolar region. Several other detection methods exist, including turbidimetric (3, 4), electrochemical (5), ellipsometry (6), and fluorescence (2, 2, 7). The advantage to homogeneous assay is that no separation step is required; however, this also presents the problem of interferences from the sample matrix. One such problem discussed by Miller (8) is the interference which occurs in the emission region for fluorescein-labeled molecules, caused by albumin-bound bilirubin. Albumin, however, can be removed by the use of the dye Cibacron blue conjugated on agarose beads. Miller states that this treatment of the sample can reduce the serum fluorescence at 520 nm by 75%. Heterogeneous assays. Heterogeneous assays are more often applied since a detectable change in signal resulting from the formation of the antigen-antibody complex is not always possible. Heterogeneous assays most often carry out the separation step using an immobilized reactor which binds the species of interest while allowing the rest of the matrix to pass through the reactor. The bound sample is then detected by a variety of methods, including electrochemical detection (9-Z4), fluorescence (15, Z6), and chemiluminescence (17-20). The drawbacks in heterogeneous assays are that the separation step often complicates the system and following the assay, the bound sample must be removed from the column without affecting the activity of the reaction surface within the column. This regeneration is, at best, time consuming, and may lower the precision of the method due to changes in the reaction surface. The novel method proposed at the end of this review utilizes immunomagnetic beads and a sequential-injection (SI) system to avoid the drawback of in-line regeneration. Detection methods. Since antibody-antigen binding in itself does not produce a detectable signal, several techniques have been proposed to yield useful information from this binding in flow-injection systems. These techniques, some of which are summarized in a timeline in Table 1, typically involve a tag such as an enzyme, radioisotope, or fluorophore to monitor the reaction with a variety of detectors. Electrochemical detection has been applied in several methods (4, 944). Tang et al. monitored the reaction of 2,6-dichloroindophenol (DCIP) by following the production of DCIPH, to obtain detection limits of 0.38 PmolZliter in an assay for phenytion (4). Other enzyme-based electrochemical assays follow the production of hydrogen peroxide or ammonia (9-ZZ). The difficulty associated with electrochemical detection is maintaining a clean electrode surface in the presence of a biological matrix. Proteins are known to strongly adsorb to electrode surfaces, and continuous washing of the electrode surface is required to help prevent fouling. Fluorimetry has also been applied to immunoassays as a method of detection (1, 2, 7, 8, 15, 16). Locascio-Brown et al. used liposomes which can encase up to lo5 water-soluble fluorescent molecules to bind antigens in a sample (16). When the liposomes are passed into a reactor, those which contain bound antigen will not be retained in the column and will flow on to be detected. Turbidity measurements
FLOW-INJECTION
IMMUNOASSAY!3
123
TABLE 1 Timeline of the Development of FIIA Date published
Analyte
Detection principle
1980 1982 1985 1985
Albumin I& I& Fetoprotein Insulin Hydroxyprogesterone kG Lysozyme IgG k&G H,O, I&s Theophylline Insulin Liposomes w W Thyroxin Theophylline Valproic acid Albumin Theophylline Theophylline IS Salmonella Phenytoin
Fluorescence Fluorescence Immunoprecipitation Chemiluminescence
1985 198.5 1985 1987 1987 1988 1988 1989 1989 1989 1989 1990 1990 1990 1990 1991
Ellipsometry
Ref.
(2)
(1) (6) (191
(5)
Amperometric Turbidity Chemiluminescence Amperometric Potentiometric Fluorescence Chemiluminescence Chemiluminescence
(15) cw
(17)
Fluorescence
(7)
Fluorescence Fluorescence Amperometric Potentiometric Amperometric Amperometric
(8) (16) (14) (10) (91 (4)
based on immunoprecipitation provide a simple analytical technique requiring only a spectrophotometer when high sensitivity is not needed (6). Chemiluminescence-based assays have been reported, and may prove to be of future interest in the development of FIIA (17-20). Shellum reported an assay based on the acridinium chemiluminescence reaction (20). Limits of detection were 50 attomol (atto = lo- 18)of mouse IgG with a total analysis time of 18 min. Chemiluminescence is difficult to monitor and quantify due to the rapid decay of the single intensity. Therefore FIA brings an advantage which has been successfully applied (17-20). Vlasenko et al. investigated the mechanism of enhanced chemiluminescence with a FIA system (17). In this study the rate constants for the reactions between the oxidized forms of peroxidase with luminol and p-iodopheno1 as well as several other substrates were also investigated. In addition, homogeneous assays for insulin, antibodies to insulin, and antibodies to the trinitrophenyl group were contemplated. Kinetics. Most FIIA assays proposed so far allow prolonged contact time for binding. These times extend up to 15 min using stop flow techniques, yet very little is known concerning how much contact time can be shortened, indeed the kinetics of binding may be such that only a very short time is needed to bind an
124
POLLEMA
ET AL.
adequate amount of sample with increased selectivity. There are few studies which have addressed the initial binding. Jonsson et al. utilized ellipsometry to follow the binding of IgG to a protein (6). The study reported two kinetic runs which were carried our for 60 min, and indicated that saturation of the IgG occurred within 7 min. Initial binding was detected after approximately 1 min. Vlasenko et al. discussed the binding of human IgG as well as T4 (17). Kinetics in the range of &lo min were considered, as was the effect of incubation time on the sensitivity with respect to human IgG concentration. The investigation of initial binding rates during FIIA assays is needed to determine the optimum time and conditions for the assay. Further investigations of these kinetics may serve to shorten the time required for current assay methods and may lead to the restriction of side reactions. SEQUENTIAL-INJECTION
IMMUNOASSAYS
The new system which we are developing has previously been described (22) and is illustrated in Fig. 1A. The sequential-injection ($3) system used allows for a number of solutions (wash, immunomagnetic bead suspensions, and samples) to be selected and sequentially aspirated into the single reaction channel. The system utilizes a single selector valve (rather than injection) and a cam driven sinusoidal flow pump for solution manipulation. The configuration illustrated has the advantage that it is possible to carry out a number of different assays without physical restructuring. The assay procedure, as outlined in Fig. lB, is as follows. (i) Immunomagnetic beads which are coated with antibodies against the analyte are aspirated into the reaction coil (RC) and the electromagnetic field surrounding this coil is activated. This traps the beads within the flowing stream and provides an immobilized reaction surface. Next, the beads are washed to assure no unbound particles remain, and the wash is sent to waste. (ii) The analyte which has been spiked with a labeled competing reagent is then aspirated into the RC and the flow is stopped for a specified contact time. During this time, the analyte and labeled reagent compete for the available sites in the reactor. The more analyte which is present, the more labeled reagent is displaced during competition and remains unbound. (iii) Following the stopped flow, a flow reversal sends the unbound portion of the sample mixture to the detector to determine the amount of unbound labeled reagent present. This yields a signal at the detector which is directly related to the analyte concentration. (iv) Finally, the electromagnetic field is turned off and the reactor is flushed to waste to be regenerated off-line. The system is then ready for another assay. The use of immunomagnetic beads and a magnetic field to form a renewable reaction column within an SI system is novel, and therefore, some initial requirements needed to be met for the technique to be proven useful. First, it is necessary to hold the beads without substantial loss during the analysis in order to obtain quantitative results, and second, the beads must undergo binding within the magnetic field to a sufftcient extent to yield an analytical signal. The behavior of magnetic beads within a flowing stream was investigated with respect to flow rate and magnetic field strength. The voltage supplied to the
FLOW-INJECTION
125
IMMUNOASSAYS
(A)
Waste
Wash
strp
1
step
2
step 3
step .I
FIG. 1. (A) Diagram of sequential injection system, comprising a cam driven sinusoidal flow pump (SFP), a reaction coil (RC) located in an electromagnetic field (M), an eight-port multiposition valve (MPV), and a fluorescence detector (D). (B) Steps of the SIIA magnetic procedure: (1) Aspirate the immunomagnetic beads into the electromagnetic field and turn on the electromagnet. (2) Aspirate the sample spiked with labeled antibody into the reactor coil and stop flow. (3) Following a specified contact time expel1 the unbound sample mixture to the detector. (4) Turn off the electromagnet and remove the column to prepare for another assay.
magnet was varied from 1 to 7 V; above this range the coil of the magnet began to heat. One volt applied to the magnet retained 15% of the beads at a maximum flow rate during the sinusoidal flow cycle of 0.74 ml/min; 7 V retained 61% at the same flow rate. Percentages are based on measurement of the light scatter produced by the beads when passed through the detector, the range (t&100%) is from baseline to the signal produced when no magnetic field is applied and the sample is passed by the detector. Figure 2 shows the results of maintaining a constant DC voltage of 5 V to the magnet and varying the flow rate. It can be seen that at a maximum flow rate of 0.50 ml/min, no beads are lost from the magnetic field. To further
126
POLLEMA
ET AL.
50
40 0 .3
FIG. 2. Influence
0.4
0.5
0.6
0.7
of the flow rate on the retention of immunomagnetic
0.x
beads in the reactor coil.
assure this, in assays, a washing step is included after loading the beads to ensure no unbound beads are present when the sample is introduced. The initial studies indicate that the renewable column can be reproducibly formed within the flowing stream. This is indicated in Fig. 3, which shows peak profiles for a calibration run for OX-19 (a monoclonal antibody against an antigen expressed on rat thymocytes and T lymphocytes) using the proposed system. The three-point calibration with each point carried out in duplicate indicates a linear relationship for concentrations of OX-19 (R = 0.997). This calibration is obtained by first aspirating the beads into the magnetic field and then turning on the electromagnet. The beads are then washed to assure no unbound beads are present, and the wash is sent to waste. Next, the sample is aspirated into the “column” of beads and the flow is stopped. After a defined time the unbound sample is expelled to the detector. The reproducibility of the duplicates and the linear relationship between response and samples of different concentrations indicate that the reaction is uniform and reproducible. The idea of a renewable reaction column was also presented by Kindervater and Schmid (21) for pesticide determination. Fufure trends. Many detection schemes and assays have been developed since the introduction of FIIA in 1980. Advancements in FIA also continue with the addition of double injection (24, 25), and more recently sequential injection (23). Both these techniques offer great promise in applications to immunoassays due to
FLOW-INJECTION
IMMUNOASSAYS
127
FIG. 3. Peak profiles obtained for a calibration plot of 0X-19, at concentrations of antibody of 1,2, and 10 &ml, with each measurement being carried out in duplicate.
the dramatic reduction of volumes required. In double injection, one injects the reagent and sample consecutively in a single line which causes the two zones to merge within the continuously flowing carrier stream. This is superior to dual injection into separate streams and subsequent mixing because of the more reproducible behavior of zone overlap. Dual injection has difficulties in reproducible zone merging due to uneven wear of peristaltic pump tubing. Double injection has corrected for this by using a single channel and single pump. More recent is sequential injection which uses a multiposition valve with one reaction channel. Solution transport can be carried out using either a peristaltic pump or a syringe pump. The syringe pump has the advantage of being more rugged without the problems of changes in peristaltic pump tubing that occur over time as wall thicknesses change and tubing stretches. However, a lower sampling frequency is obtained, since some time is required to fill the syringe. Sequential injection is designed as a more robust method with precision as high as 0.3% RSD (26), and a design which may be adapted to many of the current assay techniques for automation. Most recent papers have begun to show the use of chemiluminescence in immunoassay detection (27-20). This methodology for chemiluminescence band immunoassays may prove to be of future interest because of its added selectivity and high sensitivity. Chemiluminescence measurements are, however, difficult to ex-
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ecute reproducibly and effkiently in a batch mode because the rapid decay of emitted light requires high precision of solution manipulations. Therefore, the precision of solution handling offered by SI systems may, in the future, become a preferred technique for chemiluminescence band immunoassays. ACKNOWLEDGMENTS The authors thank Walter Lindberg and Kurt Scudder for their help and the many useful discussions
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