Anal. Chem. 1997, 69, 5024-5030
Bioligand Interaction Assay by Flow Injection Absorptiometry Jaromir Ruzicka* and Ari Ivaska†
Department of Chemistry, University of Washington, Box 351700, Seattle, Washington 98195-1700
A novel technique for the study of bioligand interactions based on combining flow injection on renewable surfaces with UV-visible absorptiometry is introduced. The concept is proven by monitoring the binding of various proteins to protein G and the binding of various insulin analogs to a monoclonal anti-insulin antibody, thus providing a comparison with the performance of surface plasmon resonance (SPR)-based techniques. The advantages of the bioligand interaction flow injection absorptiometry approach are speed, low cost, no need for regeneration of solid substrate, and spectral resolution not available with SPR sensing. It is believed that this technique will have an impact on the entire biosensor field since it allows simultaneous monitoring of labeled as well as nonlabeled species in real time over a wide spectral range with high sensitivity. Real-time label-free measurement of bioligand interactions is an important tool of molecular biology, pharmacology, and immunology as it facilitates the study of molecular recognition, receptor characterization, and affinity ranking. Among the instrumental techniques designed for this purpose, surface plasmon resonance (SPR) integrated with a flow injection system (FI) has acquired a dominant position due to its versatility and ability to monitor ligand/ligand interactions without the need for fluorescence or radioisotope labeling. This is why the number of papers dealing with FI-SPR increased rapidly since the pioneering work of Jo¨nsson et al. was been published,1 introducing Pharmacia’s BIAcore as a unique biosensor system.2-7 The method for bioligand interaction study is based on monitoring the rate of formation and dissociation of binding partners, of which the first one is immobilized on a solid substrate, while the second one is brought in transient contact with it by means of a flowing carrier stream. Automation of this process is achieved by means of a flow injection technique1,8,9 that is used
to control the flow of the carrier stream and injection of binding partners and of auxiliary reagents. In this way, timing of biological interactions as they take place on the activated solid substrate monitored by SPR in a microflow cell is well reproduced. The success of the FI-SPR format has prompted research into the use of other surface sensing transducers for bioligand binding. In addition to SPR,10,11 the use of microrefractometry,12-14 thinfilm interferrometry,15,16 ellipsometry,9,17 reflectometric interferrence spectroscopy,14 and quartz microbalance18 has been explored. For various reasons, all these techniques share two significant drawbacks. First, the layer of the coating gel on the surface of which the binding is to take place must be very thin and perfectly uniform; otherwise, the detection limit and reproducibility of measurements will be unsatisfactory. Second, none of these techniques yields spectral information that would allow identification of bioligands. Conventional UV absorptiometry has been the main detection method in chromatography of proteins in both ion exchange and affinity format. Tryphtophan (280 nm), phenylalanine (257 nm), and tyrosine (275 nm) are responsible for the broad band of protein absorption in the UV (260-300 nm), where their cumulative effect contributes to variation between extinction coefficients of individual proteins.19 On the other hand, absorption around 220 nm, due to the peptide bond, is proportional to the molecular weight of the proteinsyielding a cumulative response similar to that of SPR. While absorptiometry in these UV regions shall allow label-free measurement of bioligand interactions, scanning of the UV-visible spectrum will enable selective monitoring of biomolecules with characteristic spectra and the use of numerous stains and labels that will provide an additional means of identification and quantitation of the bound analyte. Turning absorptiometry into a surface sensing technique is accomplished in this work by combining flow injection optosensing20-22 with the jet ring (JR) cell23-25 adapted in this work
Permanent address: Labaoratory of Analytical Chemistry, A° bo Akademi University, FIN-20500 Turku/A° bo, Finland. (1) Jo¨nsson, U.; Fa¨gerstam, L.; Ivarsson, B.; Johnsson, B.; Karlsson, R.; Lundh, K.; Lo ¨fa˚s, S.; Persson,B.; Roos, H.; Ro ¨nnberg. I.; Sjo ¨lander, S.; Stenberg, E.; Sta˚hlberg , R.; Urbaniczky, C.; O ¨ stlin, H.; Malmquist, M. BioTechniques 1991, 11 (5), 620-627. (2) Karlsson, R. Anal. Biochem. 1994, 221, 142-151. (3) Karlsson, R.; Roos H.; Fa¨gerstram, L.; Persson, B. Methods: A Companion to Methods in Enzymology; Academic Press: San Diego, CA, 1994; Vol. 6, pp 99-110. (4) Karlsson, R.; Sta˚hlberg, R. Anal. Biochem. 1995, 228, 274-280. (5) Szabo, A.; Stolz, L.; Granzow, R. Curr. Biol. 1995, 5, 699-705. (6) BIA J. 1995, 2, 5-9 (Pharmacia Biosensor, Uppsala, Sweden). (7) BIA J. 1996, 3, 6-7 (Pharmacia Biosensor, Uppsala, Sweden). (8) Sjo ¨lander, S.; Urbaniczky, C. Anal. Chem. 1991, 63, 2338-2345. (9) Jo¨nsson, U.; Ro¨nnberg, I.; Malmquist, M. Colloid Surf. A 1985, 13, 333339.
(10) Daniels, P. B.; Deacon, K. J.; Eddowes, M. J.; Pedley, D. G. Sens. Actuators 1988, 15, 11-18. (11) Liedberg, B.; Lundstro ¨m, I. Sens. Actuators 1993, 11, 63-72. (12) Nellen, Ph. M.; Lukosz, W. J. Sens. Actuators 1990, B1, 592-596. (13) Cush, R.; Cronin, J. M.; Stewart, W. J.; Maule C. H.; Molloy, J.; Goddard, N. J. Biosens. Bioelectron. 1993, 8, 347-353. (14) Piehler, J.; Brecht, A.; Gauglitz, G. Anal. Chem. 1996, 68, 139-143. (15) Gauglitz, G.; Brecht A.; Kraus, G.; Nahm, W. Sens. Actuators 1993, B11, 21-27. (16) Kawaguchi, T.; Shiro, T.; Iwata, K. Thin Solid Films 1990, 191, 369-381. (17) Azzam, R. M. A. Ellipsometry and Polarized Light; North Holland Publishing: Amsterdam, 1987. (18) Ebato, H.; Gentry, Ch. A.; Herron, N. J.; Mueller, W.; Okahata, Y.; Ringsdorf, H.; Suci, P. Anal. Chem. 1994, 66, 1683-1689. (19) CRC Handbook of Biochemistry and Molecular Biology, 3rd ed., Vol. IIsProteins; Fasman, G. D., Ed.; CRC Press: Cleveland, OH, 1976. (20) Ruzicka, J.; Hansen, E. H. H. U.S. Patent 4, 973.561, 1990. (21) Ruzicka, J.; Hansen, E. H. H. Anal. Chim. Acta 1985, 173, 3.
5024 Analytical Chemistry, Vol. 69, No. 24, December 15, 1997
S0003-2700(97)00653-7 CCC: $14.00
†
© 1997 American Chemical Society
Figure 1. (A) Principle of BIA-FIA technique. During the five-step protocol, beads are introduced (1), captured (2), perfused by analyte (3), perfused by carrier solution (4), and discharged (5) when the measurement is completed. Response curves shown in Figures 2-8 have been obtained by collecting data during steps 2-4. (B) Multiwavelength response obtained by binding nonlabeled opiates antiserum (sheep) on protein G Sepharose 4B beads. During the first step (1), absorbance (A1) in the cell filled with carrier stream is low, rising sharply when the JR cell is filled with beads carrying protein G. When the cell is filled (2), the spectrum is zeroed to provide baseline. Injected antiserum perfuses beads during the association phase (3) and is captured on bead surfaces and monitored as its amount increases (A2). Dissociation takes place (4) as the beads are perfused by PBS buffer. (Due to strong bonds between these two bioligands, only a small decrease of signal is observed.) In the last step (5), beads are discarded. The traces show the raw data collected during steps 2-4.
for measurement of absorbance in UV-visible spectroscopy. The configuration of JR cell for absorbance measurement (Figure 1A) allows for the use of transparent beads as the solid surface for bioligand binding and monitoring by fiber-optic-based spectrophotometry. The bioligand interaction assay by flow injection absorptiometry (BIA-FIA) experimental protocol, as introduced in this work, is a five-step procedure where the absorbance spectrum is monitored during steps 2-4: (1) Beads with one of the bioligands immobilized on the surface are introduced into the JR cell. (22) Ruzicka, J.; Hansen, E. H. H. Flow Injection Analysis, 2nd ed.; Wiley: New York, 1988. (23) Ruzicka, J.; Pollema, C. H.; Scudder, K. M. Anal. Chem. 1993, 65, 35663570. (24) Mayer, M.; Ruzicka, J. Anal. Chem. 1996, 68, 3808-3814. (25) Willumsen, B.; Christian, G. D.; Ruzicka, J. Anal. Chem. 1997, 68, 34823489.
(2) Trapped beads are perfused with the carrier stream, and the baseline for the subsequent absorbance measurements is established. (3) The second bioligand is injected and perfused through the beads. (4) The zone of the second bioligand is washed out by the carrier stream. (5) Spent beads are automatically discarded. The UV-visible spectrum recorded at the beginning of the second step is zeroed to serve as a baseline for continuous monitoring of spectral response during the second and third steps when association and dissociation of binding partners take place (Figure 1B). The thus obtained sensogram is similar to that obtained by BIAcore, with two exceptions: first, the response is in conventional absorbance units (rather than in RU units), and second, it contains spectral information that allows compensation for perturbance due to the refractive index and yields additional Analytical Chemistry, Vol. 69, No. 24, December 15, 1997
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Table 1. Experimental Protocol As Shown in FIAlab Software instrument syringe pump syringe pump syringe pump syringe pump
address
command
Comments: Fill the Syringe internal valve position IN internal set flow rate (µL/s) internal fill syringe internal delay until done
multiport valve syringe pump syringe pump syringe pump
Comments: Flush the Flow Cell internal set valve position internal valve position OUT internal dispense (µL) internal delay until done
multiport valve syringe pump syringe pump syringe pump
Comments: Aspirate the Beads internal set valve position internal set flow rate (µL/s) internal aspirate (µL) internal delay until done
Comments: Load Beads to the JR Cell multiport valve internal set valve position syringe pump internal set flow rate (µL/s) syringe pump internal dispense (µL) syringe pump internal delay until done syringe pump internal set flow rate (µL/s) syringe pump internal dispense (µL)
value
150
1 200
2 50 75
1 25 425 2 25
Comments: Start Spectrum Collection spectrometer reference scan to disk spectrometer start absorbance scan syringe pump internal delay until done multiport valve syringe pump syringe pump syringe pump
Comments: Sample Aspiration internal set valve position internal set flow rate (µL/s) internal aspirate (µL) internal delay until done
Comments: multiport valve syringe pump syringe pump syringe pump spectrometer
Sample Injection and Bead Perfusion internal set valve position internal set flow rate (µL/s) internal dispense (µL) internal delay until done stop scanning
Comments: Disposal of Spent Beads contact closure on syringe pump internal set flow rate (µL/s) syringe pump internal empty syringe syringe pump internal delay until done contact closure off
6 25 25
1 2 300
150
selectivity inherent in multivariate responses. Since the beads are discarded after each measurement and can be preactivated prior to injection, the experimental protocol is simplified. EXPERIMENTAL SECTION Apparatus. The sequential injection system26 modified to handle bead suspension24,25,27 has been described previously. Since bioligand studies require optimized configuration of the flow path to minimize material consumption and to obtain a “square “ zone profile,25 the flow path between the multiposition valve and the JR cell was kept short (15 cm of a 0.5 mm i.d. tube), and the flow rates were kept low (2 µL/s) during the binding and washout steps (Table 1). The JR cell was redesigned for absorbance monitoring (Figure 1A) using quartz fiber-optic cables, so that the light path was 1.5 mm long, with an average beam diameter 100 µm. The probed volume was 0.012 µL, whereas the volume of the cell cavity to be filled with beads was 2.5 µL. The ring gap was mechanically (26) Ruzicka, J. Analyst 1994, 119, 1925-1934. (27) Egorov, O.; Ruzicka, J. Analyst 1996, 120, 1959-1962.
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defined to allow liquid to pass but to retain the beads and was magnetically opened when the beads were to be discarded (Figure 1A, step 5). Prior to injection, the beads were kept in suspension (about 5 mL of 1:20 dilution in PSB of the stock slurrysenough for about 50 measurements) by a stream of air bubbles provided by a peristaltic minipump. The system was assembled from commercial components: microfluidic system FIAlab 3000 (Alitea USA, http://www. flowinjection.com) was operated in sequential injection mode.24-26 The FIAlab 3000 system also comprised a UV-visible fiber-optic scanning spectrophotometer that used a stabilized deuterium lamp (AIS Model UV-2, Analytical Instruments System Inc.) as the light source. A personal computer (Dell, Dimensions XPS Pro200n) was used for data collection, fluid control, and data evaluation under the control of an integrated software package (FIAlab and FIAplot, Alitea USA). This spectrophotometer-software combination made it possible to collect time/absorbance data either of the entire spectrum of interest or at four selected wavelengths. A typical measurement cycle comprised five steps, as depicted in Figure 1 and detailed in Table 1. Materials and Reagents. The beads used were Sepharose 4B gel (size 45-165 µm) recombinant protein G or protein A agarose (Zymed Laboratories Inc, San Francisco, CA). The bead suspension was prepared from the purchased material, which consisted of a 50% slurry, by dilution with PBS buffer 1:20. Water was “nanopure” grade prepared by using the Sybron|Barnstead NANOpure II water purification system (Dubuque, IA). Phosphate-buffered saline (PBS) was made in nanopure water and comprised 0.5 M NaCl, 0.081 M Na2HPO4, and 0.019 M NaH2PO4, pH 7.40. This solution was used as a carrier stream as well as for dilution of all samples used in this work. Monoclonal mouse IgG1 anti-insulin antibodies clone K36aC10, FITC-labled bovine insulin, sheep insulin, porcine insulin, human recombinant proinsulin, and the FITC-labled goat anti-mouse IgG were from Sigma (St. Louis, MO, http://www.sigma.seal.com./safinechem. html). Opiates antiserum (sheep) was from the opiates diagnostic kit, list no. 9673, obtained from Abbott Laboratories (Abbott Park, IL). The bovine serum albumin was from Calbiochem (San Diego, CA). Mouse anti-rat IgG2a was obtained from Pharmingen (San Diego, CA, http://www.pharmingen.com), and the rat IgG2a antibody 10-3.6.4 was from the corresponding hybridoma (ATCC TIB-a2). Immobilization of the insulin antibody to the protein G beads was accomplished by allowing a 2 mL of a suspension consisting of 1850 µL of PBS buffer, 100 µL of bead slurry, and 50 µL of ascites fluid to incubate for 1 h at room temperature during constant rotation. RESULTS AND DISCUSSION In contrast to any SPR format where activation of the biosensing surface must be done individually on each chip after it has been mounted in the flow cell, in BIA-FIA format there are two choices: either preactivate an entire batch of beads, the suspension of which is then used in a number of experiments, or, as in SPR format, use an “empty” biosensing surface and perform activation, washing, and ligand immobilization in the flow cell separately prior to each individual ligand/ligand binding experiment. The disadvantage of the second approach is that it occupies the instrument time and is reagent consuming, but it has the advantage to monitor in situ the immobilization and activation processes as they proceed on the bead surfaces. Use of preac-
Figure 2. (A) Spectra obtained with JR cell filled with protein G Sepharose 4B beads, perfused by PBS buffer. (B) Spectra obtained with the same beads after spectrum A has been zeroed to provide a baseline, and the the were then perfused with goat anti-mouse IgG antibody labeled by FITC.
tivated Sepharose 4B beads is more convenient since this material has well-defined properties and is commercially available due to its wide use in affinity chromatography.28 For this reason, Sepharose 4B beads with protein G and protein A bound to their surface were used in the experiments carried out in this work in order to explore the feasibility of the proposed BIA-FIA concept. Initial experiments were designed to fulfill the following conditions: (1) the bead layer must allow transmission of light; (2) the quantity of bound and monitored molecules and their absorbencies must be well reproduced and sufficient to achieve a desirable detection limit, while the bead surfaces must provide specific sites for bioligand interactions; and (3) the monitored surfaces must be in contact with the perfusing carrier stream in order to ensure efficient ligand/ligand exposure during the association phase and the washout during the dissociation step. Optical Properties of the Beads. Light absorption and scattering by beads packed within the monitored and perfused volume is affected by a number of factors, of which three are most important: (1) absorptivity of beads; (2) absorptivity of the protein bound to bead surface; and (3) refractive indexes of the solid and of the surrounding liquid. These factors constitute a background signal that must remain constant during the measurement cycle, while the adsorbtion/desorbtion of an analyte will provide the desired response. Sepharose 4B beads functionalized with protein G and protein A absorb light in the UV-visible (Figure 2A) quite strongly. Yet after the spectrum of the beads was zeroed to provide a baseline, and the bead layer was perfused with a 200 µL injection of 10 µg/mL of goat anti-mouse IgG antibody, a significant increase of absorbance in the UV range was observed (Figure 2B). Since the goat anti-mouse IgG antibody used was conjugated with a fluorescence tag that absorbs at 497 nm, an additional maximum in this region is also observed, confirming the feasibility to monitor both labeled and unlabeled proteins in the BIA-FIA format. Based on this observation, an experimental (28) Affiniy Chromatography. Principles and Methods; Publ. 18-1022-29; Pharmacia: Uppsala, Sweden.
Figure 3. Sequential binding of goat anti-mouse IgG labeled with FITC on protein G Sepharose 4B beads. Four 50 µL injections of 10 µg/mL solution (black bars), each followed by a 200 µL PBS carrier wash (open bar) were carried out to estimate the sensitivity and detection limit of BIA-FIA. The bars may appear to be short and shifted, but this is because they represent the system action and neglect the transport time of the zones to the detector and the zone dispersion. Note that the response levels off due to strong, irreversible binding.
protocol was established, whereby automatic zeroing of the baseline takes place after the beads are introduced into the JR cell. This was achieved automatically by recording the reference scan to disk (step 19, Table 1), resulting in a new absorbance scale (A2), as shown in Figure 1B. Sensitivity and Detection Limit. The first condition for a reliable measurement in BIA-FIA format requires that the cell be charged with a sufficient amount of beads. A too small amount of beads will not fill the detector volume, resulting in loss of response, while a too large amount of beads will result in a decrease of the detection limit, since the binding of the analyte will take place within the uppermost bead layer not probed by the beam. The present configuration of the JR cell requires, as the minimum, 2.5 µL of packed beads. The light scatter by Sepharose 4B beads makes them visible, and the construction of the JR cell, made of Perspex, allows visual control of the beads interrogated by the beam and the initial selection of sufficient quantity of injected bead suspension. During routine runs, a wellreproducible level of the zeroed baseline indicates that the flow cell contains a sufficient amount of beads. Sensitivity of the BIA-FIA format was tested by repeated injection of 50 µL portions of the FITC labeled goat anti-mouse IgG antibody solution (10 µg/mL) into the JR cell charged with a 100 µL injection of the Sepharose 4B protein G bead suspension. The baseline was zeroed at the outset of the run (at 50 s), and four wavelengths were monitored throughout the experiment. The spectra collection was terminated at 500 s, and the beads were discharged (cf. Table 1). The absorbance vs time graphs at four different wavelengths are shown in Figure 3. The recording yielded the following values for sensitivities and detection limits: at 260 nm, 0.133 au/µg (au ) absorbance units), 180 ng; at 280 nm, 0.104 au/µg, 130 ng; at 400 nm, 0.028 au/µg, 250 ng; and at 497 nm, 0.083 au/µg, 80 ng. The detection limit was calculated as a signal 3 times the noise level based on the raw data response in Figure 3. The detection limit of 80 ng corresponds to 0.002 au. While these values were obtained with 100 µL of bead suspension, the sensitivity was 25% lower when the experiment was repeated with 150 µL of bead suspension, because some of Analytical Chemistry, Vol. 69, No. 24, December 15, 1997
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Figure 4. Binding and dissociation of nonlabeled goat anti-mouse IgG antibody to protein G Sepharose 4B beads. A 100 µL injection of 20 µg/mL solution (black bar) was followed by a 100 µL PBS carrier wash (open bar), a 100 µL injection of 0.1 M HCl (shaded bar), and a 200 µL PBS carrier wash (open bar).
the binding took place in the top layer of beads that was not probed by the light beam. Use of 75 µL of bead suspension yielded an erratic response, while a 50 µL bead injection yielded no response to injected analyte. Repeatability of the BIA-FIA concept was tested by injecting 50 µL of a 1:20 dilution of the stock solution of opiates antiserum (sheep) in the flow cell loaded with a 100 µL injection of the protein G Sepharose 4B bead suspension. Five injections gave the repeatability of 5% at 280 nm. Surface-Ligand Exposure and Perfusion Efficiency. One of the principal concerns in the methodology of solution/surfacebased techniques concerns kinetics and efficiency of the solution/ surface analyte transfer. In chromatography, the efficiency is achieved by providing large surface/solution area through the use of particulate and porous material, while in the instruments used in biosensor technology, such as BIAcore, the sensing surface is fabricated on a microchip that is docked into a thin-layer flowthrough cell. Since the BIA-FIA format exploits chromatographic material packed into a microcolumn, the efficiency of perfusion of the monitored surfaces was tested in chromatographic mode by an adsorbtion/desorption experiment of nonlabeled goat antimouse IgG antibody with protein G Sepharose 4B (Figure 4). After injecting 100 µL of goat anti-mouse antibody (20 µg/mL), followed by a 100 µL wash with the carrier stream, a response plateau is reached, confirming that the analyte is strongly bound to protein G. Next, 100 µL injection of 0.1 M HCl was followed by a 300 µL wash with the carrier stream. As one would expect, the response, monitored at four wavelengths, confirms that the strongly bound antibody dissociates during the passage of acid zone through the bead layer, leaving about 20% of undissociated antibody on the protein G. It is noteworthy that the trend of all four curves, obtained at different wavelengths, is the same and that nonlabeled analyte was monitored in this experiment. The sensitivity of the measurement in the UV at 260 and 280 nm is the highest, and signal at 217 nm suffers from low light throughput by fiber optics, while the response in the visible range (497 nm) is small. Sequential Binding of Nonlabeled and Labeled Bioligands. Sequential binding on the same sensor surface is used in the BIAcore system to estimate the rate of adsorption and desorption of different bioligands on the same binding partner.1-7 Using protein G Sepharose 4B beads, three 50 µL injections of 5028 Analytical Chemistry, Vol. 69, No. 24, December 15, 1997
Figure 5. Sequential binding of nonlabeled and labeled bioligands on protein G Sepharose 4B beads, 50 µL injections: opiates antiserum (sheep) (1:20 dilution of the stock solution), FITC-labeled goat anti-mouse IgG antibody (10 µg/mL), and FITC-labeled bovine insulin (50 µg/mL). Black bars indicate injections and open bars wash with carrier PBS. Note the difference in the magnitude of response at 497 nm for labeled and nonlabeled proteins.
opiates antiserum (sheep) (1:20 dilution of the stock solution), FITC-labeled goat anti-mouse IgG antibody (10 µg/mL), and FITCbovine insulin (57 µg/mL) were made sequentially, each injection followed by a 300 µL wash with the carrier stream. The response curves shown in Figure 5 confirm that the first two bioligands bind at a similar rate and remain bound to protein G also during the wash by PBS buffer. Bovine insulin, on the other hand, does not bind to protein G and is washed out from the flow cell. At 260 and 280 nm, both nonlabeled and labeled analytes provide a well-defined response. FITC-labeled goat anti-mouse IgG antibody (second injection) and FITC-labeled bovine insulin (third injection) develop a well-defined response also at 497 nm, while at 400 nm all three analytes exhibit only a minimum signal. Bioligand Interactions. Monitoring of adsorbtion/desorption of ligands on protein G Sepharose 4B is a less demanding task than sequential monitoring of the binding of an antigen/antibody complex. The reason is that, by the time the antibody is to be monitored, the background signal already comprises a contribution from protein G as well as from the antigen, making SPRbased measurement more difficult. Since bioligands of this type may not be available in a quantity sufficient for activation of larger amounts of beads in a batch mode, we have decided to carry out the entire binding experiment in the flow cell. Nonlabeled mouse anti-rat IgG2a (A) and a rat IgG2a antibody (B) were used as a model system to demonstrate capture of the first ligand on protein G Sepharose 4B beads in the flow cell, followed by antibody/antigen interaction as the second step. The abosrbance vs time curves of this experiment are shown in Figure 6. A 50 µL injection of A (50 µg/mL in PBS) followed by a 300 µL wash with PBS carrier, yielded the first step, indicating rapid binding and slow dissociation of the thus-formed complex with protein G on the Sepharose 4B surface. The next 50 µL injection of B (30 µg/mL in PBS), followed by a 300 µL wash with PBS carrier, yielded the second step, indicating rapid binding of rat antigen to anti-rat antibody. A subsequent control run (not shown), where injection of A was omitted and the same amount of B as in the previous experiment was injected, yielded a binding curve of B on protein G Sepharose 4B that was only 20% as high as the second step on Figure 6, confirming that the response curve
Figure 6. Sequential binding of nonlabeled antibody to nonlabeled antigen using proteine G Sepharose 4B beads as the solid phase. Here, 50 µL of bioligands was injected (black bar), followed by a 300 µL PBS wash (open bar). Anti-rat IgG2a (50 µg/mL) was injected first, followed by a PBS wash, and then rat IgG2a antibody (30 µg/mL) was injected. Subsequent blank run with only rat IgG2a antibody allowed correction for nonspecific adsorption of this antibody on protein G and confirmed that 80% of the ligand bound in the experiment shown here was due to specific antibody/antigen interaction.
shown comprises both specific and nonspecific binding. As expected, the response of nonlabeled analytes was most pronounced at 260 and 280 nm, while at 442 and 497 nm the response was much smaller since, in contrast to Figure 5, no chromophore was conjugated to the binding partner. Antibody/Antigen Ranking. Monitoring of sequential binding of different antigens on antibody immobilized on a solid substrate is a challenging task, especially when the antigen is a small molecule.4 Since insulin falls into the category (6 kDa) of low molecular weigh, it was selected by us as a model system to explore the suitability of the BIA-FIA format for small molecule detection. Human recombinant proinsulin (5 µg/mL), porcine insulin (12 µg/mL), sheep insulin (100 µg/mL), and bovine insulin (57 µg/ mL) were selected, of which only the last one was FITC labeled. Four sequential 100 µL injections of each compound, each injection followed by a 400 µL wash with PBS carrier, were performed. The protein G Sepharose 4B beads used in this experiment were preactivated in a batch mode so that their surface contained monoclonal antibody MAb K36aC10 against human insulin, designed to capture insulin analogs. The response curves shown in Figure 7 indicate that the human proinsulin (a) binds but dissociates rapidly, porcine insulin (b) binds rapidly while about 50% of it remains bound, the sheep insulin (c) also binds and about 30% of it remains bound, while FITC-labeled bovine insulin (d) binds and dissociates rapidly so that only about 20% of it remains bound. One might be tempted to make more quantitative observations if only one response curve were available, but comparison of all three responses reveals a problem related to binding process. Since the response at 497 nm has no drift and yields a reliable response for FITC-labeled bovine insulin, it may be speculated that measurements in the UV are affected during the wash period. Considering the very low absorbance values monitored in this experiment (full-scale 0.05 au on background of about 2 au (cf. Figure 2A)) and the good stability of the absorbance/time response curve at 497 nm, the reason for drift at 260 and 280 nm must be sought in the changes of amount of
Figure 7. Sequential binding of nonlabeled and labeled insulin analogs onto protein G Sepharose 4B furnished with monoclonal antinsulin antibody. Human proinsulin (5 µg/mL) (a), porcine insulin (12 µg/mL) (b), sheep insulin (160 µg/mL) (c), and FITC-conjugated bovine insulin (57 µg/mL) (d) were injected in 100 µL portions, each followed by a 400 µL wash with PBS carrier stream. Points of injection are marked 1-4. Note the pronounced response at 497 nm following injection 4, due to the chromophore label. Drifts from baseline at 260 and 280 nm are discussed in the text and in Figure 8.
Figure 8. Blank run at identical conditions as given for the experiment shown in Figure 7, but with injection of bovine serum albumin, which does not bind onto protein G Sepharose 4B beads. The upward baseline drift at 260 nm was identified as an impurity being gradually preconcentrated from 1600 µL of the carrier stream that perfused ∼3 µL of beads held in the cell. Note the low and similar absorbency values in Figures 7 and 8.
bound/desorbed species on the beads and not in the instability of the light source or of the detector. The experiment, however, is successful in showing that BIA-FIA can, in the present format, reliably detect binding of a small, nonlabeled antigen such as insulin. Blank Run and Nonspecific Adsorption. Upward drift of baseline close to the detection limit (found to be 180 ng of protein monitored at 260 nm or 80 ng of protein monitored at 497 nm) compromises the performance of the BIA-FIA at this level and was, therefore, investigated by injecting bovine serum albumin (120 µg/mL) in four sequential injections (100 µL each, followed by a 400 µL carrier PBS wash) while the signal was recorded at four different wavelengths (Figure 8). Again, at 260 nm, an upward drift was observed. Since bovine serum albumin is known not to bind to protein G and the shape of the response curve at 260 nm confirms that the absorbance increases during the wash period and not during the injection period, the species adsorbed and monitored was identified as an impurity in the PBS buffer used. Indeed, an experiment using doubly distilled water from a quartz apparatus rather than “nanopure” water yielded curves with Analytical Chemistry, Vol. 69, No. 24, December 15, 1997
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no upward drift at all four wavelengths. Finally, since no downward drift was observed at 280 nm in this blank run when different beads were used than those used in the experiment in Figure 7, it can be concluded that the downward drift observed in the insulin experiment was due to leakage of some protein from the pretreated beads. CONCLUSION The advantages of using microamounts of beads as a renewable layer for (bio)chemical sensing have been established in several papers dealing with reflectance,27 fluorescence,23,25,26 and electrochemical modes of detection,24 as well as in a novel type of titrations,29 yet in this work the use of UV-visible absorbance measurement is applied to an assay which, so far, was thought to be possible only with the use of surface sensing techniques, such as SPR. It is shown here that, by changing the paradigm, the following advantages are gained: (1) Well-characterized beads can be obtained from several different sources, inexpensively and with a wide variety of functional groups. (2) The bead properties are well known from the literature on affinity chromatography, and the behavior of proteins adsorbed on bead surfaces can be predicted from easily accessible data. Therefore, results of binding experiments can be confirmed by chromatography using identical material. (3) Multiwavelength capability of the BIA-FIA technique allows comparison of response curves obtained at different wavelengths, making sensograms more reliable and informative. (4) Changes of the refractive index resulting from injecting a solution with a different salt content than that of the carrier stream are small compared to those found in SPR sensing (see Figures 3 and 4) and can be eliminated by taking the ratio of absorbencies at two different wavelengths. While SPR will respond to any species that would increase the mass adsorbed on the surface, the spectral resolution of the BIA(29) Holman, D. A.; Christian, G. D.; Ruzicka, J. Anal. Chem. 1997, 69, 17631765.
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Analytical Chemistry, Vol. 69, No. 24, December 15, 1997
FIA format makes it possible to distinguish between species of interest and interferences, and the shape of the response curve makes it possible to identify its source: the injected sample or the carrier stream (Figures 7 and 8). While BIA-FIA is shown to be well suited for monitoring of unlabeled species, analytes tagged by a suitable chromophore can also be identified and monitored (Figures 3, 5, and 7). Indeed, it is likely that additional identification of analytes can also be performed on beads retained in the JR cell, by injecting a suitable reactive chromophore. The sensitivity and detection limit of BIA-FIA are somewhat lower than those possible with SPR techniques, but there is room for further improvement. This is because, while the SPR technique probes only a single surface, the beam in the JR cell interacts with a large number of beads, thus being exposed to multiple surfaces. We believe that BIA-FIA will find applications well beyond the study of protein interactions and their assays and will branch into areas of cell biology and drug discovery with use of a variety of semitransparent beads to capture various analytes of interest. ACKNOWLEDGMENT The authors thank Kristina Peterson, Bodil Willumsen, and Craig Beeson for lessons in protein chemistry, valuable comments, and supply of test materials and Garth Klein for providing his expertise in software design and computer technology. To Zymogenetics (Seattle, WA) and Novo Nordisk (Copenhagen, Denmark), we are indebted for material and financial support. The scholarship to A.I. from The Academy of Finland is gratefully acknowledged.
Received for review June 23, 1997. Accepted September 24, 1997.X AC9706537 X
Abstract published in Advance ACS Abstracts, November 15, 1997.
