Affinity Capillary Electrophoresis for the Assessment of Complex

Aug 23, 2000 - The formation of complexes of human rhinovirus (serotype HRV2 and HRV14) with .... particles of human papillomavirus by capillary elect...
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Anal. Chem. 2000, 72, 4634-4639

Affinity Capillary Electrophoresis for the Assessment of Complex Formation between Viruses and Monoclonal Antibodies Vadim M. Okun,† Bernhard Ronacher,‡ Dieter Blaas,‡ and Ernst Kenndler*,†

Institute of Analytical Chemistry, University of Vienna, Wa¨hringerstrasse 38, A-1090 Vienna, Austria, and Institute of Medical Biochemistry, Vienna Biocenter (VBC), University of Vienna, Dr. Bohr Gasse 9/3, A-1030 Vienna, Austria

The formation of complexes of human rhinovirus (serotype HRV2 and HRV14) with nonaggregating neutralizing monoclonal antibodies was investigated by affinity capillary electrophoresis. The method is based on preincubation of virus with antibody, followed by CE analysis. At low antibody-to-virus ratios, peaks corresponding to the complexes were broad, pointing to the presence of a heterogeneous population of virions with various numbers of antibodies bound; at a high molar ratio between virus and antibody, the peak became narrow again, indicating saturation of the 60 equivalent viral epitopes with the antibodies being attached bivalently. As SDS was used as an additive in the background electrolyte to allow for separation, its influence on complex formation was investigated. Once formed, HRV2-antibody complexes were found to be stable in the presence of the detergent but complex formation in buffer containing SDS was severely impaired. HRV14-antibody complexes were rapidly dissociated by SDS. The method proved to be useful for a rapid assessment of complex formation and might allow for an estimation of the binding stoichiometry. Noncovalent molecular interactions, based on the principle of affinity recognition are of fundamental importance. Among numerous techniques allowing assessment of these reactions both qualitatively and quantitatively, affinity capillary electrophoresis (ACE) has recently gained increasing popularity.1 The method combines ligand-receptor interaction in solution with the separation potential of capillary electrophoresis. For high-affinity systems, the components can be precolumn incubated at various ratios followed by CE analysis, which will then reveal peaks of bound and free reagents, thus enabling quantitation of both. This approach was used in a variety of CE-based immunoassays (for reviews, see, e.g., refs 2 and 3), for the evaluation of binding constants and binding stoichiometries4,5 and characterization of the specific activity of different protein isoforms.6 Another ap* Corresponding author: (e-mail) [email protected]; (phone) +43 1 4277 523 05; (fax) +43 1 4277 9523 . † Institute of Analytical Chemistry. ‡ Institute of Medical Biochemistry. (1) Wehr, T.; Rodriguez-Diaz, R.; Zhu, M. In Capillary Electrophoresis of Proteins; Cazes, J., Ed.; Chromatographic science series 80; Marcel Dekker: New York, 1998. (2) Bao, J. J. J. Chromatogr., B: Biomed. Sci. Appl. 1997, 699, 463-480. (3) Schmalzing, D.; Nashabeh, W. Electrophoresis 1997, 18, 2184-2193.

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proach is used for low-affinity interactions where one of the interacting components is added to the background electrolyte, enabling the maintenance of equilibrium conditions during separation. Changes in migration time of the analyte (as a function of concentration of the interacting component dissolved in the buffer) can be used to determine binding constants.7-9 So far, analysis of macromolecular interactions has been limited to compounds with molecular weights of up to ∼200 000 (e.g., complexes between a small antigen and an IgG molecule; see, e.g., refs 10-12) and to binding stoichiometries of a maximum of 1:4.4 Recently, we applied CE for the systematic investigation of more complex biomolecules such as human rhinoviruses (HRVs).13-16 HRVs, the main causative agents of common cold infections, are icosahedral particles of ∼30 nm in diameter. They are assembled from 60 copies each of four different viral proteins (VP1-VP4) and an RNA genome resulting in a molecular mass of ∼8 × 106 Da (for a review, see, e.g., ref 17). We demonstrated that these macromolecular complexes could be analyzed by CE, enabling rapid and easy assessment of conformational state and purity of the virions in a given viral preparation. Further, we reported on the separation of native virus from subviral B-particles, which are the end products of virus uncoating and are composed of VP1, VP2, and VP3 only.18 A ternary detergent mixture (0.5% sodium deoxycholate, 0.05% SDS, 0.5% Triton-X100R) added to the background electrolyte was specifically designed to prevent viral (4) Chu, Y. H.; Lees, W. J.; Stassinopoulos, A.; Walsh, C. T. Biochemistry 1994, 33, 10616-10621. (5) Okun, V. M.; Bilitewski, U. Electrophoresis 1996, 17, 1627-1632. (6) Okun, V. M. Electrophoresis 1998, 19, 427-432. (7) Guszczynski, T.; Copeland, T. D. Anal. Biochem. 1998, 260, 212-217. (8) Mammen, M.; Gomez, F. A.; Whitesides, G. M. Anal. Chem. 1995, 67, 3526-3535. (9) Colton, I. J.; Carbeck, J. D.; Rao, J.; Whitesides, G. M. Electrophoresis 1998, 19, 367-382. (10) Schmerr, M. J.; Jenny, A. Electrophoresis 1998, 19, 409-414. (11) Lin, S.; Hsiao, I. Y.; Hsu, S. M. Anal. Biochem. 1997, 254, 9-17. (12) Qian, X. H.; Tomer, K. B. Electrophoresis 1998, 19, 415-419. (13) Schnabel, U.; Groiss, F.; Blaas, D.; Kenndler, E. Anal. Chem. 1996, 68, 4300-4303. (14) Okun, V. M.; Ronacher, B.; Blaas, D.; Kenndler, E. Anal. Chem. 1999, 71, 2028-2032. (15) Okun, V. M.; Blaas, D.; Kenndler, E. Anal. Chem. 1999, 71, 4480-4485. (16) Okun, V. M.; Ronacher, B.; Blaas, D.; Kenndler, E. Anal. Chem. 2000, 72, 2553-2558. (17) Couch, R. B. In Fields Virology; Fields, B. N., Knipe, D. M., Howley, P. M., Eds.; Lippincott-Raven Publishers: Philadelphia, 1996; Vol. 1, pp 713-734. (18) Korant, B. D.; Lonberg Holm, K.; Noble, J.; Stasny, J. T. Virology 1972, 48, 71-86. 10.1021/ac000250y CCC: $19.00

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aggregation and capillary wall adsorption. Most recently, the electrophoretic mobility of four different viral serotypes (HRV2, HRV14, HRV16, HRV49), belonging to either the major or the minor receptor group,19 was determined and the HRV serotypes could be differentiated. This demonstrates that subtle differences at the viral surface are sufficient for their electrophoretic resolution.16 However, affinity interactions of viruses with monoclonal antibodies have not been characterized by CE so far. This is despite the fact that CE analysis can be performed in homogeneous medium without potentially interfering material (solid or gel-like sieving matters, etc.) which is most favorable for bioanalytical applications. In HRVs, antibodies bind to exposed loops connecting the antiparallel β-sheets of the capsid proteins.20 The epitopes have been determined from mutations leading to escape of the virus from neutralization by a panel of monoclonal antibodies (mAbs).21,22 MAbs neutralize viral infectivity by various means such as (i) aggregation, (ii) inhibition of receptor binding by steric hindrance, (iii) inhibition of uncoating by stabilizing the capsid against structural changes as required for RNA release, and most probably (iv) by combinations thereof (for a discussion of neutralization mechanisms, see, e.g., ref 23). Due to the bivalent nature of IgG molecules and the repetitive structure of the viral capsid, antibodies can attach with both arms to the same viral particle over the 2-fold axes of icosahedral symmetry. If the topology of the epitopes or the geometry of the antibody does not allow for bivalent attachment, antibody binding results in aggregation.23 Assessment of the formation of complexes between virus and antibody usually requires sucrose density gradient sedimentation24 or size exclusion chromatography25 to separate complexes from excess antibody. Both methods suffer from the time required for separation as low-affinity complexes might readily dissociate. Moreover, even for analytical purposes, either a substantial amount of material or radiolabeled components are required. All of these restrictions can be avoided by electrophoresis in the capillary format. When affinity interactions including the characterization of complex formation between analytes exhibiting specific affinity for each other are involved, this methodology can be considered as affinity capillary electrophoresis. In this regard, precolumn incubations of the reactants (the present case) as well as in-column interaction between the reactants (with one analyte being dissolved in the background electrolyte) belong to this technique. The main goal of the present study was thus to characterize by ACE the formation of complexes between two different virus serotypes, HRV2 and HRV14, and their relevant (19) Uncapher, C. R.; Dewitt, C. M.; Colonno, R. J. Virology 1991, 180, 814817. (20) Rossmann, M. G.; Arnold, E.; Erickson, J. W.; Frankenberger, E. A.; Griffith, J. P.; Hecht, H. J.; Johnson, J. E.; Kamer, G.; Luo, M.; Mosser, A. G.; Rueckert, R. R.; Sherry, B.; Vriend, G. Nature 1985, 317, 145-153. (21) Sherry, B.; Mosser, A. G.; Colonno, J.; Rueckert, R. R. J. Virol. 1985, 57, 246-257. (22) Appleyard, G.; Russell, S. M.; Clarke, B. E.; Speller, S. A.; Trowbridge, M.; Vadolas, J. J. Gen. Virol. 1990, 71, 1275-1282. (23) Mosser, A. G.; Leippe, D. M.; Rueckert, R. R. In Molecular Aspects of Picornavirus Infection and Detection; Semler, B., Ehrenfeld, E., Eds.; American Society for Microbiology: Washington, DC 20006, 1989; Vol. 1, pp 155-167. (24) Icenogle, J.; Shiwen, H.; Duke, G.; Gilbert, S.; Rueckert, R.; Anderegg, J. Virology 1983, 127, 412-425. (25) Smith, T. J.; Olson, N. H.; Cheng, R. H.; Chase, E. S.; Baker, T. S. Proc. Natl. Acad. Sci. U.S.A. 1993, 90, 7015-7018.

monoclonal antibodies. For such systems, the stoichiometry of binding largely exceeds that of molecules previously analyzed with a similar method, due to the multivalent nature of the viruses. MATERIALS AND METHODS Apparatus. The CE system used was an automated HP3D capillary electrophoresis system (Hewlett-Packard, Waldbronn, Germany), equipped with an uncoated fused-silica capillary (Composite Metal Service Ltd., 51.5 cm effective length, 60.0 cm total length, 50 µm i.d.) packed in a standard HP cassette and thermostated at 20 °C during all experiments. Injection was performed at 50 mbar pressure for 9 s. Between all runs, the capillary was conditioned by aspirating 100 mmol/L NaOH, water, and background electrolyte (BGE) for 2 min each by applying ∼950 mbar pressure. The detector signals were recorded at 205 nm. In specified cases, fast spectral scanning mode was used. Data were collected and analyzed using the Hewlett-Packard Chemstation software. Positive polarity mode (detector placed at the cathodic side of the capillary) with 25 kV was used throughout the experiments. Reagents. Human rhinovirus serotypes 2 and 14, as originally obtained from the American Type Culture Collection (ATCC), were produced and purified from infected cell pellets as described previously.16,26 Where purified virus was employed, the concentration was determined spectrophotometrically using an extinction coefficient of 77 at 260 nm (A260) for a 1% w/v solution.27 A contaminant of unknown nature was constantly revealed by electropherograms; its absorbance at 260 nm was taken into account when calculating the viral concentrations. Monoclonal antibody 8F5, directed against a linear antigenic determinant of VP2,28 was purified from hybridoma tissue culture supernatants by standard procedures as described in ref 29. Antibody concentration was determined by assuming an A280 of 13.5 for a 1% solution.30 Ascites fluid containing mAb 17-IA directed against neutralizing immunogen NIm-IA of HRV1421,31 was kindly given to us by Anne Mosser, University Wisconsin, Madison, WI. All other chemicals were of analytical grade and were purchased from E. Merck (Darmstadt, Germany). Reagent solutions were prepared in deionized water. Samples were dissolved in a buffer solution corresponding to half-diluted BGE without SDS added. The sample buffer was supplemented with o-phthalic acid (20 µg/mL) as an internal standard (IS). The BGE was 100 mmol/L boric acid containing 10 mmol/L sodium dodecyl sulfate (SDS); it was adjusted to pH 8.3 with 1 mol/L NaOH. In the sample buffer, SDS was omitted. Buffers were filtered through a 0.45-µm cellulose nitrate membrane before use. All solutions used as BGE were centrifuged for 2 min in a tabletop centrifuge at 5000g prior to CE analysis. (26) Skern, T.; Sommergruber, W.; Blaas, D.; Pieler, C.; Kuechler, E. Virology 1984, 136, 125-132. (27) Rueckert, R. R. In Comprehensive Virology; Fraenkel-Conrad, H., Wagner, R. R., Eds.; Plenum Press: New York, 1976; Vol. 6, pp 131-213. (28) Skern, T.; Neubauer, C.; Frasel, L.; Gruendler, P.; Sommergruber, W.; Zorn, W.; Kuechler, E.; Blaas, D. J. Gen. Virol. 1987, 68, 315-323. (29) Tormo, J.; Fita, I.; Kanzler, O.; Blaas, D. J. Biol. Chem. 1990, 265, 1679916800. (30) Kirschenbaum, D. M. Anal. Biochem. 1973, 55, 166-192. (31) Smith, T. J.; Olson, N. H.; Cheng, R. H.; Liu, H.; Chase, E. S.; Lee, W. M.; Leippe, D. M.; Mosser, A. G.; Rueckert, R. R.; Baker, T. S. J. Virol. 1993, 67, 1148-1158.

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Procedures. (1) Determination of Virus Concentration by CE. In principle, the virus concentration can be easily determined spectrophotometrically by assuming 9.4 × 1012 virions per 1 A260.27 However, despite extensive purification, we constantly observed the presence of variable amounts of a contaminant with absorbance at 260 nm in the virus preparations upon CE analysis.14-16 It did not stain with Coomassie Brilliant Blue when the viral preparation was analyzed by polyacrylamide SDS gel electrophoresis under reducing conditions. It also remained unchanged upon heating to 56 °C and treating with RNAse.14 We therefore believe that it is neither protein nor RNA and does not represent viral material. The contribution of this contaminant (judged from the ratio of the peak heights) to the total absorbance at 260 nm ranged from 32 to 39%, depending on the preparation used. A260 was thus measured in a standard spectrophotometer and corrected for the contribution of the contaminant. This value was then used for the calculation of the effective concentration of pure virus and a linear regression line relying virus peak heights as seen in CE analysis with corrected virus concentration was drawn (results not shown). (2) Affinity CE. For the evaluation of the immunoaffinity interaction between HRV2 and mAb 8F5, 10 µL of virus (0.24 mg/ mL; 0.030 × 10-6 M) and 10 µL of antibody (from 0.018 to 0.92 mg/mL; 0.12 × 10-6-6.13 × 10-6 M), both in sample buffer, were mixed in a microvial. After incubation in sample buffer without SDS at room temperature, CE separation was carried out. In the case of mAb 17-IA, ascites fluid was used and the concentration of the antibody was not determined because of the contaminating proteins present. Evaluation of the affinity interaction between this antibody and HRV14 was thus done by mixing 10 µL of 0.24 mg/mL (30 × 10-9 M) virus with 10 µL of dilutions of the ascites fluids of between 1:250 and 1:10 in sample buffer. The stability of the affinity complex was studied by programming the CE instrument to switch off the voltage, for example, after 4 min of separation time. Separation was then continued after the time periods indicated in the figures. Rhinoviruses are unstable at low pH;27 all solutions containing virus were thus acidified with acetic acid to inactivate virus prior to disposal. Experiments were done according to the safety regulations for work with infectious agents. RESULTS AND DISCUSSION Affinity Complex Formation between HRV2 and mAb 8F5. (1) CE Analysis. For the characterization of an affinity interaction by CE, the components must be sufficiently resolved or the migration behavior of at least one of them must be altered upon complex formation. In addition, the complex must be stable during the time required to reach the detector. To assess whether this was the case for our particular virus-antibody system, a constant amount of virus was mixed with increasing amounts of mAb 8F5, incubated, and analyzed by CE (Figure 1). Relying on the usually high affinities between antibodies and antigens (confirmed by cryoelectron microscopy for the case under consideration; see ref 32), none of them was added to the BGE as commonly done for low-affinity systems. As can be deduced from the top and bottom traces in Figure 1, HRV2 is indeed well separated from mAb 8F5, permitting quantitation of either of the components. (32) Hewat, E. A.; Blaas, D. EMBO J. 1996, 15, 1515-1523.

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Figure 1. Formation of HRV2-mAb 8F5 complexes as a function of the amount of antibody added as analyzed by CE. A final concentration of 15 nmol/L HRV2 was incubated with the final concentrations of mAb 8F5 indicated in a volume of 20 µL for 60 min at room temperature followed by CE analysis. IS, internal standard; BGE, borate buffer (100 mmol/L borate/boric acid, pH 8.3) with SDS (10 mmol/L ) 0.26%) as detergent. Conditions: untreated fusedsilica capillary, 60.0 (51.5) cm length, 50 µm i.d.; voltage, +25 kV; T, 20 °C.

When increasing amounts of mAb 8F5 were added to a constant amount of HRV2, the virus peak decreased and was gradually shifted toward longer migration times, becoming increasingly broader and indicating heterogeneity. This might be taken to indicate that various numbers of equivalent epitopes were occupied by the antibodies. Upon further increasing the amount of antibody, the peak became sharper again with a concomitant appearance of a peak of free antibody. This is indicative for saturation of the virus at excess antibody concentration with the appearance of a homogeneous population of complexes. The sharp and symmetrical peak (Figure 1, bottom trace) is seemingly the result of the fully saturated complex. All traces of free HRV2 had disappeared and the peak corresponding to excess 8F5 emerged. This peak is obtained also after direct injection from the mAB 8F5 preparation (not shown). Spectral analysis of the peak anticipated as the virus-antibody complex revealed a significant increase of the ratio of the signals at 205 and 260 nm as compared to the spectrum of virus alone. Absorbance at 205 nm is less specific whereas absorbance at 260 nm indicates the presence of nucleic acid. For pure HRV2, the ratio of A205/A260 was found to be ∼6 (as determined for the virus peak in Figure 1, top tracing) whereas it had increased to a value of ∼11 in the peak corresponding to the complex (as determined from Figure 1, bottom tracing). This clearly supports the assumption of an increase in protein content with respect to RNA in the complex. As expected, there is still an absorption maximum at 260 nm indicating the presence of RNA (tracing not shown).

Figure 2. Total electrophoretic mobility of the HRV2-mAb 8F5 complexes as a function of the final concentration of mAb 8F5 added. Data were taken from the runs depicted in Figure 1.

To determine whether a completely saturated complex was obtained under the conditions used, the electrophoretic mobility (measured at the peak apex) versus the amount of mAb (at a constant virus concentration) was plotted. The mobility was calculated from the migration time in the usual way, taking the EOF and the internal standard as references. The reproducibility of the mobility was in the tenth of a percent range, expressed by the relative standard deviation for n ) 6. As seen in Figure 2, the mobility of the complex decreased with increasing amounts of mAb added and attained a plateau at seemingly full saturation. It is noticeable, however, that the curve reached a plateau only when mAb/HRV2 molar ratios largely exceed the theoretical ratio of 30:1,32 which corresponds to an mAb concentration of 0.45 × 10-6 M. This may be due to the presence of inactive antibody or low binding affinity and is topic of a current investigation. In contrast to simple systems (e.g., refs 8 and 33) where a migration shift is caused by the association of a low molecular weight ligand carrying defined numbers of negative charges, the change in migration behavior of the virus upon attachment of antibodies is more complicated. It appears to result from a combination of various factors such as changes in surface charge, conformation, molecular mass, and hydrodynamic properties. It should be noted that a shift of the isoelectric point of a rhinovirus from neutral to acidic was noted upon attachment of neutralizing antibodies; this was interpreted as a conformational change of the virion induced from strain exerted by the antibody upon binding bivalently. However, this idea was dismissed as Fab fragments were able to induce similar changes; it is now currently thought that masking of positive charge by the antibodies is the main reason for the changes in isoelectric point.34 Note that the contaminant in the viral sample (migration time 3.75 min) was not affected upon addition of antibody. This again (33) Lin, S.; Hsu, S. M. Electrophoresis 1997, 18, 2042-2046. (34) Colonno, R. J.; Callahan, P. L.; Leippe, D. M.; Rueckert, R. R.; Tomassini, J. E. J. Virol. 1989, 63, 36-42.

Figure 3. Complex formation as a function of the incubation time. Components were mixed as in Figure 1, bottom tracing, and analyzed by CE immediately after incubation at room temperature for the times indicated. Other conditions are as in Figure 1.

suggests that it cannot be a viral material (see Experimental Section, Procedures). (2) Time Course of Complex Formation. To determine whether incubation for 1 h at room temperature, as used previously for the preparation of complexes for cryoelectron microscopy analysis,32 was sufficient to attain equilibrium, mixtures (prepared as described in the legend to Figure 1, bottom trace) were analyzed by CE immediately after incubation of 10, 40, and 60 min, respectively (Figure 3). Although a peak corresponding to the complex was observed in each case, it was rather broad after a 10-min incubation (upper trace) but successfully sharpened and concomitantly shifted toward longer migration times upon incubation for 40 and 60 min, respectively (middle and lower tracings). This might indicate that initially heterogeneous complexes are formed, which become more homogeneous with increasing saturation of the virus particles with antibody molecules. Since peak shape and migration time only marginally change from 40 to 60 min, it can be assumed that the equilibrium is reached upon 60 min and no more antibodies can attach upon further extension of the incubation time. (3) Stability of the Complex between HRV2 and mAb 8F5 in the Presence of SDS. Repeated attempts at separating HRVs in the absence of detergent have failed due to aggregation and protein adsorption to the capillary wall. Therefore, previous work had been carried out with SDS, deoxycholate, or a combination of detergents (modified RIPA buffer) present in the background electrolyte. BGE, which contained SDS only, was found to provide the best resolution for analysis of native virus particles, and therefore, it was also used in the present study. Although SDS is considered to be a denaturing agent in biochemistry (it is worth mentioning, that it denatures proteins only upon boiling), several (35) Steinmann, L.; Thormann, W. Electrophoresis 1996, 17, 1348-1356.

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Figure 4. Stability of the complex between HRV2 and mAb 8F5 in the presence of SDS. (A) A mixture of HRV2 and mAb 8F5 as in Figure 1, bottom trace, was incubated for 1 h at room temperature; SDS was then added to a final concentration of 10 mmol/L and incubation was continued for an additional hour, followed by CE. (B) Incubation of the components was as in (A), but SDS (10 mmol/L) was present in the reaction mixture from the beginning. CE conditions as in Figure 1.

reports have shown that even bioaffinity reactions can be performed in the presence of SDS.5,35 However, as SDS is clearly not physiologic, an evaluation of potential influences of SDS on the affinity reaction is required. For the affinity reaction described above, virus was incubated with antibodies in BGE without SDS added. It is obvious that during migration through the capillary the analytes are leaving their initial sample zone, and as a consequence, they are in contact with SDS in the separation buffer. To assess the influence of SDS on complex formation and dissociation, three sets of experiments were thus carried out. In the first setup, the affinity complex was formed in (SDS-free) sample buffer as above, and SDS was then added to a final concentration corresponding to that present in the BGE (10 mmol/L). Incubation was continued for 1 h and the sample was analyzed by CE. As seen in Figure 4A, no dissociation of the affinity complex was noted as no free virus was detectable and no distortion of the peak of the complex or any overlapping took place. This indicates that the complex of HRV2 and mAb 8F5 is stable in the presence of SDS. In a second experiment, SDS was present in the reaction mixture from the beginning of the incubation (Figure 4B). The appearance of peaks corresponding to unbound HRV2 together with a small peak of the complex indicates that association of the components is severely impaired but not completely prevented in the presence of the detergent; even a large excess of mAb cannot drive the reaction toward the complex. Finally, the stability of the complex in the absence of the free components was assessed under conditions closely matching those occurring during electrophoresis. Virus was incubated with an excess of 8F5 (as in Figure 1, bottom tracing) and electrophoresis was conducted for 4 min, a time required for resolution of the affinity complex from excess antibody (as estimated from the results shown in Figure 1). Migration was then halted by switching off the voltage for 10 and 15 min, respectively, as indicated by arrows in Figure 5, whereupon electrophoresis was resumed. The time range was chosen because of its being within the migration time of the components in the electrophero4638 Analytical Chemistry, Vol. 72, No. 19, October 1, 2000

Figure 5. Effect of SDS on the stability of the affinity complex of HRV2 and mAb 8F5 assayed in the capillary. Affinity complexes prepared as in Figure 1, bottom trace (in the absence of SDS), were injected into the capillary and electrophoresis was halted after 4 min (see arrows) and resumed after 10 (upper tracing) or 15 min (lower tracing).

grams obtained before. The absence of peak distortion or overlapping allows concluding that under the conditions chosen no dissociation of the HRV2-mAb 8F5 complex takes place in the capillary even in the absence of free antibody and free virus. Taken together, these results indicate that complexes between 8F5 and HRV2 are stable in the presence of SDS but their formation is inhibited in the presence of this detergent. Analysis of the amino acids involved in the interaction between a synthetic peptide derived from VP2 of HRV2 and 8F5 36 and a model of the complex between virus and antibody37 indicates that the interacting surfaces are most probably protected from access of SDS in the complex. It is possible that a strong negative charge imparted to the antigen-binding site and/or to the epitope by the sulfonic acid prevents complex formation by electrostatic repulsion. Affinity Complex of HRV14 and mAb 17-IA. (1) CE Analysis. Aiming to extend our findings to other viral serotypes, affinity complex formation between HRV14 and the neutralizing mAb 17-IA was investigated by CE. Similarly to 8F5, mAb 17-IA binds bivalently over the 2-fold axes of icosahedral symmetry of its cognate serotype HRV14 although the symmetry-related epitopes of neutralizing immunogen-IA are much farther apart than site B in HRV2, which is bound by 8F5.25,32 The interaction of 17-IA with HRV14 has been extensively characterized by cryoelectron microscopy and X-ray techniques.38 For CE analysis, the complex was formed by incubation under conditions identical to that used for HRV2. From Figure 6 it can be seen that antibodyvirus complexes are clearly resolved from virus. In accordance with the results obtained for HRV2, increasing amounts of mAb 17-IA led to an increase in occupancy of the virus with antibody with a concomitant shift of the complex peak toward longer migration times. In addition, the peak corresponding to the complex is again clearly broader than the virus peak (compare Figure 6 upper and bottom tracings). As mentioned before, peak (36) Tormo, J.; Blaas, D.; Parry, N. R.; Rowlands, D.; Stuart, D.; Fita, I. EMBO J. 1994, 13, 2247-2256. (37) Tormo, J.; Centeno, N. B.; Fontana, E.; Bubendorfer, T.; Fita, I.; Blaas, D. Proteins: Struct. Funct. Genet. 1995, 23, 491-501. (38) Smith, T. J.; Chase, E. S.; Schmidt, T. J.; Olson, N. H.; Baker, T. S. Nature 1996, 383, 350-354.

Figure 7. Stability of the complex between HRV14 and mAb 17-IA in the presence of SDS. A mixture of virus and ascites fluid as in Figure 6, bottom trace, was incubated for 1 h at room temperature, SDS was then added to a final concentration of 10 mmol/L and incubation was continued for 6 min or for 22 min, followed by CE.

Figure 6. Complex formation between HRV14 and mAb 17-IA. HRV14 (30 nmol/L) was incubated with the antibody containing ascites fluid (dilution factors are indicated) for 1 h at room temperature in a final volume of 20 µL followed by CE analysis. Other conditions are as in Figure 1.

broadening is seemingly caused by the formation of different heterogeneous forms of the antibody-virus complex. The peak of the affinity complex, shifted to 4.7 min in Figure 6 (bottom tracing), does not represent the fully saturated virus, as became clear from experiments with higher amounts of antibodies added (not shown). However, use of ascites fluid instead of purified antibody in this experiment made a detailed analysis not possible as many peaks stemming from contaminating proteins with migration times between 5.0 and 6.5 min were seen at higher antibody concentration. (2) Stability of the Complex between HRV14 and mAb 17IA in the Presence of SDS. Experiments similar to those described above for the evaluation of the stability of the HRV28F5 complex in SDS were also carried out with HRV14 and mAb 17-IA. In contrast to HRV2 and mAb 8F5, no complex formation was seen when the incubation of the components was carried out in the presence of SDS (data not shown). Furthermore, addition of SDS to the preformed complex of HRV14 and mAb 17-IA caused extensive dissociation already after 6 min of incubation as seen from CE analysis (Figure 7). This short incubation time led to a serious broadening of the peak of the affinity complex (marked with an arrow), seemingly caused by decomposition. After 22 min of incubation, HRV14 reappeared as a sharp peak, indicating that at least a part of the complex had been completely dissociated

(compare upper tracings of Figure 7 and Figure 6). From these experiments, it can be concluded that SDS severely inhibited binding of the antibody to HRV14 although dissociation of the preformed complex in the presence of SDS is slow enough to allow for its detection under the particular conditions used. CONCLUSIONS For two human rhinovirus serotypes (HRV2 and HRV14), the migration behavior becomes modified upon binding of specific monoclonal antibodies; this allowed identifying affinity complexes with various occupancies of the viruses with antibodies and their separation from unbound reagents. The investigation of the stability of the complex in the presence of SDS, as used for CE separation, revealed pronounced differences between the two systems. Complex formation is described by a “classical” binding isotherm with a plateau indicating complete saturation of virus by specific antibodies when the latter are in excess. Our results for the first time demonstrate the utility of ACE for affinity analysis of extremely large macromolecules and pave the way for their application in rapid screening for potential virus neutralizing capsid binders. ACKNOWLEDGMENT This work was supported by the Austrian Science Foundation (Project P13504-CHE). We thank I. Goesler for preparation and purification of HRV2 and Anne Mosser for the kind gift of ascites fluid containing mAb 17-IA. Received for review February 29, 2000. Accepted June 8, 2000. AC000250Y

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