Characterization of a Metal-Specific Monoclonal Antibody - American

Chapter 4

Characterization of a Metal-Specific Monoclonal Antibody 1

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Downloaded by NORTH CAROLINA STATE UNIV on October 6, 2012 | http://pubs.acs.org Publication Date: May 5, 1997 | doi: 10.1021/bk-1997-0657.ch004

D. A. Blake , M . Khosraviani , A. R. Pavlov , and R. C. Blake II 1

Department of Ophthalmology, Tulane University School of Medicine, 1430 Tulane Avenue, New Orleans, LA 70112-2699 Department of Basic Pharmaceutical Sciences, Xavier University of Louisiana, New Orleans, LA 70112-2699 2

A new technique was utilized to characterize the binding specificity of a metal-specific monoclonal antibody, 2A81G5. A KinExA automated immunoassayinstrumentwas employed to measure the concentration of unliganded antibody in a mixture of antibody, antigen, and antibody-antigen complex. Unliganded antibody was captured on a resin containing immobilized antigen and quantified by brief exposure to a fluorescein-labeled anti-species secondary antibody. Instrument response was proportional to the concentration of primary and secondary antibody and was also dependent upon the immobilized antigen concentration, reagent flow rate, and duration of flow. Equilibrium dissociation constants for a Cd(II)ethylenediaminetetraacetic acid complex (Cd-EDTA) and a Cd(II)EDTA-bovine serum albumin conjugate (Cd-EDTA-BSA) were 10 nM and 72 pM, respectively. The instrument could also be used in a competitive immunoassay format to quantify low levels of antigen in solution; the limit of detection for the Cd-EDTA-BSA conjugate was 40 pM or 5 ppt in ionic cadmium

The development of any immunoassay is greatly facilitated by the detailed knowledge of the binding characteristics of the antibodies used in the assay. Immunoassay sensitivity is directly related to the antibody affinity for the antigen and assay selectivity is directly related to the binding specificity of the antibody used in the analysis (7). In addition, the performance of the assay is directly related to the kinetic properties (on and off rates) of the antibody binding reaction. Our laboratory has been developing antibodies to metal-chelate complexes and we have recently demonstrated that these antibodies can be used in competitive immunoassays for metal ions (2). Immunoassays offer significant advantages over more traditional methods of metal ion analysis; they are quick, inexpensive, simple to perform, and sufficiently portable to be used at the site of contamination. In contrast, existing technologies to measure heavy metals require complex

© 1997 American Chemical Society In Immunochemical Technology for Environmental Applications; Aga, D., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1997.

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IMMUNOCHEMICAL TECHNOLOGY FOR ENVIRONMENTAL APPLICATIONS

instrumentation (atomic absorption or inductively coupled plasma emission spectroscopy) in a centralized facility; because of the expense of the iratramentation required for the analysis, samples are often in a queue for long periods of time. As part of our continuing efforts to construct and optimize metal ion immunoassays, we have developed a new technique to rapidly determine the solution equilibrium dissociation constants of antibody-antigen interactions. We have applied these methods in a study of the interactions of the metal-specific monoclonal antibody 2A81G5 with various metal-chelate complexes.


Materials and Methods Bovine serum albumin (BSA) (fatty acid ultrafree) was a product of Boehringer Mannheim (Indianapolis, IN). Atomic absorption standard metals were purchased from Perkm-Elmer (Norwalk, CT). These standard metals were handled and disposed of according to the directions in the Materials Safety Data Sheets provided by Perkm-Elmer. Fluorescein isothiocyanate conjugate of affinity purified goatantimouse IgG was a product of Jackson ImmunoResearch Laboratories (West Grove, PA). Polyrr^ylmethacrylate beads (140-170 mesh) were obtained from Bangs Laboratories (Carmel, IN). Reagents for tissue culture and ELISA were obtained as previously described (2). Glassware was mixed-acid washed (3) and plasticware was soaked overnight in 3 N H Q and rinsed liberally with nanopure water before use. Preparation of protein-chelate conjugates. Protein-chelate complexes were prepared as previously described (2). Extent of substitution of free lysine groups was 55% for the BSA-thiourddo-L-benzyl-eth^ acid-cadmium conjugate (Cd-EDTA-BSA) and 30% for the BSA-thioureido-L-benzylethylenediarranetetraacetic acid-mercury conjugate (Hg-EDTA-BSA). Purification of monoclonal antibody. Isolation and characterization of the 2A81G5 monoclonal antibody will be described elsewhere ( D A Blake etal.,J. Biol. Chem., in press). The concentration of IgG in the purified product (405 pg/ml) and the light chain specificity of the antibody (IgGl) was determined by sandwich ELISA (4). Determination of equilibrium dissociation constants. Equilibrium measurements of the interactions of chelated metals with the 2A81G5 monoclonal antibody were performed with a KinExA (Sapidyne Instruments, Inc., Idaho City, ID) automated immunoassay system Polymethylmethacrylate beads (98 ± 8 pm) were coated with antigen by suspending 200 mg (dry weight) of beads in 1.0 ml of phosphate buffered saline (PBS, 137 mM NaCl, 3 mM KC1,10 mM sodium phosphate buffer, pH 7.4) containing 5-100 pg/ml Cd-EDTA-BSA. For some experiments, the beads were incubated with 100 pg/ml of Hg-EDTA-BSA. After 1.0 h of gentle agitation at 37° C, the beads were centrifuged and the supernatant solution was decanted. Any non-specific protein binding sites remaining on the beads were blocked by the subsequent incubation of the beads with 1.0 ml of 10% (w/v) goat serum in PBS containing 0.03% NaN for an additional h at 37° C. The blocked beads were stable at 4° C in the blocking solution for up to 4 weeks. 3

In Immunochemical Technology for Environmental Applications; Aga, D., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1997.


4. BLAKE ET AL.

Characterization of a Metal-Specific Monoclonal Antibody

A bead pack approximately 4 mm high was created in the observation cell of the instrument by drawing 675 pi of a suspension of the blocked beads in PBS (6.7 mg beads/ml) into a capillary cell (i.d. = 1.6 mm) above a rrricroporous screen at a flow rate of 1.5 ml/min and washing the retained beads with sufficient PBS buffer (1.0 ml) to remove the excess goat serum The beads were then gently disrupted with a brief backflush of PBS (50 pi at 300 pl/rrrin), followed by a 20 s settling period to create a uniform and reproducible pack. For measurement of equilibrium dissociation constants, the 2A81G5 monoclonal antibody was mixed with varying concentrations of metal ion in PBS supplemented with 5.0 mM EDTA and 1 mg/ml BSA. Once equilibrium was achieved (less than 2 min in all cases), an aliquot of the reaction mixture was drawn past the beads, followed by 166 pi of the PBS-EDTA-BSA buffer to wash out unbound primary antibody and excess metal chelate. One ml of fluorescein-labeled goat anti-mouse antibody (1.0 pg/ml in PBS-BSA) was then drawn past the beads. All solution transfers up to this point were accomplished with a flow rate of 500 pl/min. Unbound labeled secondary antibody was subsequently removed by drawing 3.0 ml of PBS-EDTA-BSA through the bead pack over a period of 2 min. Data acquisition (initiated at the introduction of primary antibody to the beads) and instrument control were accomplished via a Toshiba Satellite 486/33 notebook computer interfaced to the KinExA using software provided by Sapidyne. Dissociation constants were obtained from nonlinear regression analyses of the equilibrium binding data using a one-site homogeneous binding model. The contribution of each datum to the analysis was weighted by a factor derived from the application of information theory to protein binding reactions (5). Results The KinExA immunoassay instrument is a computer controlled flow spectrofluorimeter designed to achieve the rapid separation and quantification of free, unbound antibody present in reaction mixtures of antibody, antigen, and antibody-antigen complexes. The operating principles of the instrument are illustrated schematically in Fig. 1. Uniform particles were pre-coated with CdEDTA-BSA and deposited in a packed bed above a rrricroporous screenfittedin a capillary flow/observation cell. A small volume of the reaction mixture, typically 0.5 ml, is then percolated through the packed bed of microbeads under negative pressure. Antibodies in the mixture with unoccupied binding sites are available to bind to the immobilized antigen coated on the solid phase. Exposure of the antibody-soluble antigen complex to the immobilized antigen is sufficiently brief (typically 240 ms) to insure that negligible dissociation of the soluble complex occurs during the time of exposure to the beads. The soluble reagents are removed from the beads by an immediate buffer wash. Quantification of the primary antibody thus captured is achieved by the brief exposure of the particles to a fluorescein labeled goat anti-mouse secondary antibody, followed by measurement of the resulting fluorescencefromthe particles after the removal of excess unbound secondary antibody.


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Figure 1. Schematic of KinExA Automated Immunoassay Instrument.



4. BLAKE ET AL.


Fig. IA shows representative examples of the time courses for the fluorescence signal when different concentrations of a primary antibody were analyzed in the absence of the soluble antigen. The primary antibody was 2A81G5, a mouse monoclonal raised in response to a Cd-EDTA hapten covalently coupled to keyhole limpet hemocyanin (D.A. Blake et.al, J. Biol Chem, in press). The immobilized ligand was Cd-EDTA-BSA. Data accumulation was initiated when the beads were first exposed to the unlabeled primary antibody. The fluorescence signalfromzero to 90 s thus corresponded to the background signal generated while the primary antibody was exposed to and subsequently washed out of the packed microbead column. The beads were then exposed to a solution of fluorescein labeled goat anti-mouse antibodies (90 to 190 s); excess unbound labeled secondary antibody was then removedfromthe beads with a buffer wash (190 to 300 s). In the absence of the primary antibody (curve a) the fluorescence response approximated a square wave corresponding to the fluorescence of the labeled secondary antibody during its transient passage past the beads in the observation cell. The signal failed to return to that of the background (0.8% higher), indicating a slight nonspecific binding of the fluorescein labeled antibody to the beads. When different concentrations of the primary antibody were analyzed (curves b through d) the fluorescence response from 90 to 190 s reflected the sum of two contributions: the fluorescence of the unbound labeled antibody in the interstitial regions among the beads; and that of the labeled secondary antibody that had bound to primary antibody captured by the immobilized antigen on the beads. Binding of the secondary antibody was an ongoing process that produced a positive slope in this portion of the curve. When the excess unbound label was removed from the beads, the signal that remained was the sum of both nonspecifically and specifically bound antispecies antibody. The instrument response as a function of the concentration of primary antibody is shown in Fig. IB. The standard instrument response was taken as the integrated fluorescence signal over the last 90 s of the experimental trace (the 210 to 300 s interval for curves such as those in Fig. 2A). The signal in Fig. 25 was approximately linear with the concentration of primary antibody up to 0.2 pg/ml. Beyond that concentration deviations from linearity became increasingly apparent. The inset of Fig. 2B shows the instrument response to concentrations of primary antibody up to 2.5 pg/ml. The data were fit to a rectangular hyperbola with a maximum of 360 volt-seconds and a half-maximal response at 2.1 pg/ml. This dependence, which represented the binding curve of primary antibody to the immobilized antigen on the beads, indicated that only a small fraction of the population of immobilized antigen was involved in antibody binding in the approximately linear portion of the binding curve at low antibody concentrations. A primary antibody concentration of 0.1 pg/ml was chosen for standard assay conditions to insure that the instrument response was well within the linear portion of the curve. The signal as a function of the concentration of fluorescein-labeled secondary antibody was also determined. The instrument response was approximately linear with the concentration of secondary antibody up to 2.0 pg/ml (data not shown). The slope of a plot of instrurnent response versus the


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Figure 2. Dependence of the instrument response on antibody concentration. A, kinetic traces of fluorescence as a function of time for 0 (a), 0.06 (b\ 0.12 (c) and 0.18 (d) pg/ml of primary antibody. B, linear dependence of the instrument response at low concentrations of primary antibody. Inset, hyperbolic dependence of the iristrument response on higher concentrations of primary antibody.



4. BLAKE ET AL.

Characterization of a Metal-Specific Monoclonal Antibody 55

concentration of secondary antibody was proportional to the concentration of primary antibody employed in the assay; similarly, changing the concentration of secondary antibody changed the slope of a plot of instrument response versus the concentration of primary antibody (data not shown). A secondary antibody concentration of 1.0 pg/ml was chosen as the standard assay condition to insure adequate signal-to-noise characteristics without the excessive consumption of fluorescein labeled protein reagent. Instrument response was also a function of immobilized ligand concentration, reagent flowrate,and duration of flow. The dependence of the signal on the amount of immobilized antigen is shown in Fig. 3A. Set amounts of the beads were exposed to different concentrations of the Cd-EDTA-BSA coating reagent and subsequently incorporated into the standard assay format. The data in Fig. 3A were fit to a rectangular hyperbola that represented the binding curve for the protein-hapten conjugate to the hydrophobic surface of the beads (half-maximal effect, 3.4 pg/ml). A coating solution of 100 pg of protein-hapten conjugate per ml was chosen as the standard operating condition to insure that an excess of immobilized ligand was present on the beads. The dependence of the instrument response on the flowrateof the primary antibody is shown in Fig. 3B. The higher the flowrate,the lower was the signal. The data in Fig. 3B were fit to a single exponential function of flow rate, with an amplitude of 21.8 volt-seconds and a constant in the exponential term of -1.4 min/ml. The value of the amplitude represented the total instrument response expected if the flowratewere zero, where the amount of primary antibody bound to the immobilized antigen had achieved equilibrium for that set of reagents and assay conditions. The fact that lower instrument responses were obtained at higher flow rates indicated that only a percentage of the primary antibody with unoccupied binding sites was removed from solution by binding to the immobilized antigen. The shape of the exponential curve (i.e., the value of the constant term in the argument of the exponent) was independent of the concentration of primary antibody over a 10-fold range (data not shown). These observations indicated that the kinetics of antibody binding to the beads were driven by the effective concentration of the immobilized antigen, the binding partner in excess. Similar data and conclusions were obtained from the dependence of the instrument response on the flowrateof the secondary antibody (data not shown). Flowratesof 0.5 ml/min for both the primary and secondary antibodies were chosen as the standard assay condition as a compromise between assay sensitivity and expediency. Finally, experiments were performed to demonstrate that the instrument response was dependent upon the total number of antibody molecules in the solution drawn past the beads. When antibody solution was diluted while concomitantly decreasing the flowratesuch that the number of antibody molecules in contact with the beads remained constant, identical experimental traces were obtained (data not shown). For high affinity antibodies, this feature allows the investigator to measure binding constants at very low concentrations of primary antibody. When soluble antigen was mixed with the primary antibody, the instrument response of the KinExA became a means of quantifying unliganded antibody. The primary data resembled those in Fig. 2A\ transformation of these signals into the


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Figure 3. Instrument response as a function of immobilized ligand concentration and reagent flow rate. A, individual lots of PMMA beads were exposed Cd-EDTA-BSA at concentrations as shown on the abscissa. B, only the flow rate of the primary antibody past the beads was varied; the total volume of antibody remained constant at 0.5 ml.



4. BLAKE ET AL.


fraction of occupied binding sites as a function of the concentration offreeantigen produced binding curves as shown in Fig. 4. Fig. 4A shows a binding curve for Cd-EDTA to antibody 2A81G5. The different symbols displayed in the figure correspond to different immobilized antigens employed in the assays. The 2A81G5 antibody has the ability to recognize both Cd-EDTA-BSA and Hg-EDTA-BSA conjugates ( D A Blake et al., J. Biol. Chem, in press) and substitution of HgEDTA-BSA for Cd-EDTA-BSA on the solid phase had no apparent influence on the outcome of the binding experiments. A single binding curve with an apparent equilibrium dissociation constant of 10 nM was drawn through all of the data points. The concentration of 2A81G5 employed in the binding experiment, 1.3 nM in binding sites, was well below the value of the dissociation constant; thus no corrections in the concentration of free ligand were required. When the Cd-EDTA moiety was covalently coupled to the S-amino group of a lysine residue in BSA by the same benzyl-thioureido group as that employed in the immunogen, binding of the antigen to the antibody became much tighter. The equilibrium binding curve for soluble Cd-EDTA coupled to BSA and 2A81G5 is shown in Fig. 45. These binding experiments were conducted by lowering the standard primary antibody concentration 10-fold to 0.01 pg'rnl, while increasing the standard volume of analyte 10-fold to 5.0 ml. The curve drawn through the data points was that for a homogeneous binding interaction with a dissociation constant of 72 pM, a value some 150-fold lower than that for the Cd-EDTA interaction. It was evident that the binding site of 2A81G5 recognized features of the lysylthioureido-benzyl portion of the antigen. Since the concentration of the antibody's binding sites (133 pM) and the value of the dissociation constant were of the same magnitude, the concentration of free antigen on the abscissa of Fig. 45 was calculated as the difference between the concentration of the total antigen minus that bound to the antibody according to the following equation: [Cd-EDTA on BSA]^ = [Cd-EDTA on BSA] - [Antibody] x fraction of occupied binding sites total

The KinExA could also be exploited to devise competitive immunoassays to quantify low levels of the antigen in solution. An apparent binding curve for CdEDTA-BSA that resembled a stoichiometric titration could be generated by raising the concentration of 2A81G5 to 0.1 pg/ml. Fig. 5 shows the dependence of the instrument response on the concentration of soluble Cd-EDTA-BSA at an antibody concentration of 0.1 pg/ml. The linear portion of the curve comprised a standard curve for Cd-EDTA-BSA up to about 400 p M Given that the reproducibility of individual data points in the KinExA was around 5%, a conservative estimate for the limit of detection for Cd-EDTA-BSA using the standard curve in Fig. 5 was approximately 40 pM or 5 parts per trillion in ionic cadmium concentration. Discussion Unlike other automated immunoassay mstrurnents where the interaction to be quantified is that between a soluble and an immobilized binding partner (6-8) the


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Figure 4. Equilibrium binding curves for Cd-EDTA and Cd-EDTA-BSA. A, Concentration of the unliganded antibody in the presence of various concentrations of Cd-EDTA were measured using immobilized Cd-EDTA-BSA (O) or Hg-EDTA-BSA (•) to capture unliganded antibody. B, Binding curve for soluble Cd-EDTA-BSA.

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Figure 5. Effect of soluble Cd-EDTA-BSA on the instrument response in the presence of 0.1 pg/ml of 2A81G5. The concentrations on the abscissa are those of the total Cd(II), not those of the multiply-labeled BSA.



4. BLAKE ETAL.


immobilized reagent in the KinExA is merely a tool used to separate and quantify the concentration offree,uncomplexed reagent in solution reaction mixtures. When measuring binding interactions in the KinExA, the principal conditions that must be met are: i) binding of the immobilized and the corresponding soluble component must be mutually exclusive and ii) binding to the immobilized component must be sufficiently tight to permit an iristrument response with acceptable signal-to-noise characteristics. Once these conditions are met, the quantitative binding information extractedfromthe data are those of the binding interaction in homogeneous solution between soluble partners. In those alternative cases where one of the principal binding partners is immobilized (6-8), one must always be concerned about the effects of mass transport limitations and surface properties (charge, hydrophobicity, stearic hindrance, etc.) on the apparent strength of the measured interaction (9). The KinExA can be exploited to quantify any protein-ligand interaction, as long as one of the binding partners can be immobilized in such a way that it still binds to the other, and a means can be devised to convert immobilized complexes quantitatively into fluorescent signals that remain proportional to the concentration of the complex. This laboratory has used the KinExA to determine equilibrium binding constants between 2A81G5 and each of 16 different metal-EDTA complexes, as well as 4 different Cd-chelator combinations (D.A. Blake et al., J. Biol. Chem., in press). The values of individual dissociation constants obtained in this study ranged over 7 orders of magnitudefrom10~ to 10" M. The instrument was also exploited to quantify the values of individual rate constants for the binding of antigens to 2A81G5 (data not shown). Instead of exposing an equilibrium mixture of the binding partners to the immobilized antigen on the beads, a solution of the monoclonal antibody was injected into and mixed with a stream of chelated metal ions that reacted for just 11 s before the mixture encountered the packed beads. When used as a platform for conventional immunoassay, the KinExA exhibited a limit of detection for Cd-EDTA-BSA equivalent to that for the corresponding competition ELISA (D.A. Blake, unpublished data). It should be noted that no effort was made to adjust the assay parameters in Fig. 5 to optimize the value of the detection limit. Thus, a combination of greater analyte volume, slower flow rates, and a greater concentration of secondary antibody could conceivably lower the observed limit of detection some 50-fold to the sub-ppt level. Of course, since the operating format of the KinExA only permits the analysis of one sample at a time, the KinExA is not likely to replace standard ELISA formats and instrumentation when large numbers of samples must be analyzed on a routine basis. However, because of its unique ability to provide information about antibody-antigen interactions in solution, the KinExA could be a useful adjunct in any analytical irnmimochemistry laboratory where research and development of new assays is underway. 11

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Acknowledgments This work was supported by a grant to D.A. Blake from the Environmental Protection Agency (R 824029) and by a grantfromthe Department of Defense (93DNA-2) to the Tulane-Xavier Center for Bioenvironmental Research.


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Literature Cited 1. Gosling, J.P. Clinical Chemistry. 1990, 36, 1408-1427 2. Chakrabarti, P.; Hatcher, F.M.; Blake, R.C., II; Ladd, P.A.; Blake, D.A. Anal. Biochem. 1994, 217, 70-75. 3. Thiers, R.C. Meth. Biochem. Anal. 1957, 5, 273-335. 4. Harlow, E.; Lane, D. Antibodies: A Laboratory Manual; Cold Spring Harbor Laboratory, Cold Spring Harbor, N Y , 1988; pp. 553-612. 5. Weber, G. in Molecular Biophysics; Pullman, B., and Weissbluth, M., Eds; Academic Press, New York, N Y , 1965; pp 369-696. 6. Friguet, B.; Chaffotte, A.F.; Djavadi-Ohaniance, L ; Goldberg, M.E. J. Immunol. Methods. 1985, 77, 305-319. 7. Dill, K . ; Lin, M.; Poteras, C.; Fraser, C.; Hafeman, D.G.; Owick, J.C.; Olson, J.D. Anal. Biochem. 1994, 217, 128-138. 8. Malmqvist, M. Nature. 1993, 361, 186-187. 9. Kemeny, D . M . ; Challacombe, S.J., Eds.; ELISA and Other Solid Phase Irnmunoassays: Theoretical and Practical Aspects, John Wiley and Sons: New York, N Y , 1988.


Characterization of a Metal-Specific Monoclonal Antibody - American

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