Immunoaffinity CE for
Proteomics Studies This 2-D separation technique can complement MS-based proteomics methods.
Norberto A. Guzman Johnson & Johnson Pharmaceutical Research & Development Terry M. Phillips National Institutes of Health
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living organism is a highly evolved system composed of many interwoven molecular networks. These networks primarily involve proteins that enable and control virtually every cellular chemical process. They do this through a very large number of molecular interactions with other proteins, sugars, lipids, nucleic acids, and low-mass molecules, including enzyme–substrate interactions (1–6). A vast amount of information has accumulated on the chemical and physicochemical molecular interactions involved in the specific affinity of an antibody–antigen interaction, as well as for other related affinity associations among a wide range of substances (7–13). The high selectivities and affinities of antibody–antigen interactions make them among the most finely tuned noncovalent bonds exhibited by proteins. Biospecific molecular interactions among a large number of substances have been used extensively for designing affinity chromatographic purifications for a wide range of low-mass molecules and larger biomolecules (8–13). Most of these interactions between a ligand and its corresponding receptor occur when one of the affinity partners is immobilized. Under such conditions, the properties of the interaction(s) differ from those exhibited by a similar system in free solution (14). Therefore, solid-phase purification is a constant challenge for the user, and almost every step of the association and dissociation processes must be optimized and validated for maximum performance (14 –16). Although thousands of substances have been purified using affinity chromatography, the © 2005 AMERICAN CHEMICAL SOCIETY
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teractions in CE (20–24). However, in this article, the primary emphasis will be on immunoaffinity CE (IACE) with the use of an immobilized selective adsorbent (23, 25–29) for applications in proteomics. Properly bound to a solid support, an affinity ligand PNGase F can selectively adsorb a target analyte or a pH 7.5 ligate found in a solution containing a sim24 h at 37 °C ple or complex mixture over a wide concentration range. MercaptoIACE at the microscale differs from the ethylamine Pepsin traditional model of affinity chromatograpH 4.5 30 min at phy at the macroscale in various ways: The 4 h at 37 °C 37 °C F (ab´ )2 fragment Fab´ fragment immobilization of an affinity ligand occurs in a small binding area of a capillary or chanOH (b) nel to capture selective target analyte(s), the A O Si OH preferred placement of the binding area is OH near the inlet of the capillary or channel, and OH O CH2 CH3 the desorption of the bound target anaB O Si O Si O (CH ) NH lyte(s) is immediately followed by separation 2 2 3 of the released analyte(s) within the capillary O CH2 CH3 OH or channel under the influence of an electriO OH O CH2 CH3 O cal field. For the separation, it is possible to C O Si O Si (CH ) NH C CH2 N 2 3 use pressure or a combination of pressure SO3 Na OH O CH2 CH3 and an electrical field. This latter procedure O OH O CH2 CH3 O may enhance selectivity of the analytes and/ D O Si O Si (CH ) NH C CH2 N or the speed of separation. 2 3 S S Fab´ fragment IACE requires several important operaOH O CH2 CH3 tional steps. The sample is introduced into the capillary or channel, and the target anaFIGURE 1. Antibody reactions. lyte(s) are selectively captured by one or (a) Enzymatic processes of deglycosylation and pepsinolysis of IgG to yield F(ab´)2 more affinity ligand(s) bound either to the fragments. PNGase F removes N-linked oligosaccharides and generates deglycosylated IgG. Pepsin cleavage of the deglycosylated IgG yields a F(ab´)2 fragment. Fimatrix that forms the analyte concentrator nally, the dimeric antigen-binding fragment was subjected to a mild reduction of or directly to the inner wall of the capillary the –S–S– group(s), which generated two monomeric Fab´ fragments containing or channel. Washing and cleanup remove the hinge-region cysteine(s). (b) Various chemical reactions used to covalently imexcess sample and nonspecifically bound unmobilize an IgG antibody fragment to a silanol-containing surface (25, 28; adapted with permission from Ref. 30). wanted material. The separation capillary or channel is conditioned. The bound analyte is eluted by means of a small plug of an appropriate desorption buffer and separated by one or more modes of CE. The separated analyte(s) can be detected by UV abapplication that has benefited most is the purification of proteins sorption, fluorescence, laser-induced fluorescence (LIF), chemiand peptides. This article will discuss the most relevant informa- luminescence, electrochemical, radioactive, or MS methods. The tion on the use of immunoaffinity separations coupled with CE affinity ligand bound in the analyte concentrator is regenerated. for the isolation, purification, quantification, and characterization of proteins and peptides in a miniaturized environment. Such Antibody basics analyses are more important than ever. The explosive and volu- In general, either polyclonal antibodies (natural antibodies made minous growth of proteomics research, fueled by the completion by immunizing animals) or monoclonal antibodies (antibodies of the DNA sequences of ~100 genomes, is expected to lead to produced in the laboratory by a single clone of cells) are used in the understanding of the expression, function, and regulation of immunoaffinity chromatography and IACE. Both whole bivalent the entire set of proteins encoded by an organism (17–19). (capable of binding two antigens or two haptens) and monovalent (capable of binding one antigen or one hapten) antibody fragments are used. Immunoaffinity CE Antibodies are bifunctional and include both binding and efThe concept of affinity interaction at the microscale level has been described for the study of several reversible molecular in- fector elements. They belong to the group of immunoglobulin (a)
Glycosylated lgG
Deglycosylated lgG
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O
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molecules, which consist of several protein chains. Each chain contains discrete structural domains of ~110 amino acids that are capable of independent folding into their native structure of two stacked sheets twisted into the “immunoglobulin fold” and stabilized by disulfide bonds. There are several types of immunoglobulin, but we will refer primarily to immunoglobulin G (IgG). IgG antibodies are 150-kD glycoproteins with a tetrameric structure consisting of two identical 50-kD glycosylated proteins (“heavy chains”) and two identical 25-kD proteins (“light chains”), which are normally not glycosylated. Each light chain is associated with and covalently linked via a disulfide bridge to the N-terminal region of a heavy chain; the Cterminal halves of the two heavy chains are associated with each other and form the tail of the molecule. The heavy chains are also covalently linked to each other via disulfide bridges in the socalled hinge region. IgG molecules are therefore bilaterally symmetrical structures and usually adopt a Y shape. They have two binding sites for large molecules (antigens) or small molecules (haptens) per intact IgG molecule. Antibodies can be enzymatically modified to produce functional fragments that are suitable for use as immunoaffinity ligands (29, 30). Cleavage of an intact IgG molecule with the enzyme papain will produce two identical antigen-binding fragments called Fab fragments, which are essentially monovalent antibodies. If a similar digestion is performed using pepsin, a single F(ab´)2 fragment is produced (Figure 1a). This fragment is bivalent, like the original IgG, and is capable of binding two antigenic molecules. Pepsin cleavage cuts the IgG molecule below the disulfide bridge that holds the two antigen-binding arms of the antibody together. Chemically reducing the disulfide bridge produces two Fab´ fragments, each with a reactive thiol group at the end furthest from the antigen-binding site, which can be used to covalently immobilize the Fab´ to a solid support (27, 30). Prior to the enzymatic treatment with pepsin, IgG is deglycosylated with the enzyme PNGase F, which removes N-linked oligosaccharides (Figure 1a). The removal of potential steric hindrance from neighboring carbohydrate moieties near the hinge region facilitates the action of pepsin (30, 31). Antibody–antigen interactions form noncovalent complexes when the shape of the antibody’s binding sites conforms to that of the electron cloud of the antigen or hapten. This conformational matching is responsible for the strength of antibody–antigen interactions, producing a “closeness of fit” between the electron-cloud shape of the antigen and the “space fill” of the area within the antibody’s antigen-binding site. This close fit is essential because antibody–antigen interactions are held together by a collection of weak intermolecular forces, namely hydrogen bonding, ionic interactions, van der Waals forces, and hydrophobic interactions (32). Although individually these forces are relatively weak (with the exception of ionic interactions), the sum of
their overall effects produces a strong bond if the antibody and antigen are in sufficiently close contact. Although views differ about the sequence in which these forces interact, there is a logical course of events. The interaction starts with an attraction formed by hydrogen bonding between an antibody and an antigen, followed by electrostatic interaction. The two molecules are now so close that van der Waals forces come into play. In the final step, water molecules are expelled from the space between the antibody and the antigen, and hydrophobic attractive forces are exerted, preventing re-entry of the water. At this stage, the antibody–antigen interaction is complete. Knowledge of these molecular interactions is useful when designing approaches for analyte recovery in IACE.
Immobilization on a solid support Although numerous affinity ligands can be immobilized on a solid support, we will describe primarily the immobilization of an antibody or an antibody fragment on a matrix or directly on the inner surface of the capillary or channel. The exquisite specificity and high affinity of the antibody–antigen interaction are responsible for the highly selective adsorption of a target analyte, but the maximum efficiency only occurs if the binding region of the antibody is correctly oriented when the IgG, or IgG fragment, is immobilized on a solid support. Several chemistries have been described for the immobilization of antibodies (32, 33). Although direct adsorption to activated surfaces has been used in affinity chromatography, such approaches are inefficient for IACE. The two most effective procedures are covalent attachment directly to the chemically modified matrix surface and attachment via a secondary molecule. In covalent attachment, many different kinds of reactive molecules, such as succinimide ester, epoxide, and amine end chains, can be attached to modify silica and glass surfaces. The free thiol (or sulfhydryl) groups are perhaps the most effective for orienting antibody fragments, because these chemical groups form convenient attachment points for Fab´ fragments that also contain free thiol groups. Thus, the antibody fragment is immobilized via the formation of a disulfide bridge (30; Figure 1b). The other successful approach is to use a secondary immobilized binder molecule, such as the bacterial coat protein A or G, which has the ability to bind an IgG molecule via its Fc or tail portion, leaving the antigen-binding region of the molecule free. In a similar manner, egg-white protein (avidin) can be easily immobilized to a matrix surface and will strongly bind all types of antibodies once they have been modified by the attachment of the vitamin biotin. The use of hydrazine-activated biotin enables attachment of the biotin molecule to the sugars that are present in the Fc or tail portion of the IgG molecule. When this biotinmodified or biotinylated antibody is reacted with avidin, the two bind irreversibly, orienting the antigen-binding portions of the F E B R U A R Y 1 , 2 0 0 5 / A N A LY T I C A L C H E M I S T R Y
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(a)
(b)
Toxicity
No toxicity 4
Cy A 2 Cy A
1 3 2
3
4
FIGURE 2. Cyclosporin A (Cy A) in tears obtained from corneal-transplant patients during (a) normal and (b) drug-toxicity episodes in the course of their treatments. Total concentrations of immunoreactive cyclosporin A (bar graphs) were obtained by ELISA (arbitrary concentrations), and total concentrations of immunoreactive cyclosporin A and metabolites were obtained by IACE (actual concentrations) using a single immobilized antibody directed against cyclosporin A. ELISA provides quantitative results that are the sum of the intact drug plus the metabolites, although it is possible that a cross-reacted substance is included as part of the result. IACE provides qualitative and quantitative information on the intact drug and the metabolites AM1, AM9, AM1c, and AM4N (1–4, respectively; adapted with permission from Ref. 38.)
antibody away from the matrix. The strength of this bond combined with the relative ease of biotinylation have made this one of the most popular approaches to IgG immobilization in any immunoaffinity technique. When these approaches are used, every IgG antibody bound to a matrix is capable of capturing a corresponding antigen or hapten. However, the efficiency of the binding capacity for each IgG antibody differs considerably; this variation is due to many factors. Some of the main factors influencing the binding capacity are the type of chemistry used in binding the IgG antibody to the matrix, the resulting orientation of the IgG antibody molecule with respect to its corresponding antigen or hapten, the degree of affinity of each individual IgG antibody for its corresponding antigen or hapten, and the presence of an optimized environment for binding. There are four commonly used procedures to fabricate an “analyte concentrator–microreactor”, which is an (immuno)sorbent area located near the inlet of the capillary or channel to extract specific analytes and enrich the sample before releasing the bound material for CE separation. One of the most commonly used procedures to link an IgG antibody to an analyte concentrator–microreactor is immobilization of an IgG antibody or fragment to free-floating beads in the restricted space of a fusedsilica capillary, corresponding to the area of the analyte concentrator–microreactor. In a capillary format, this restricted space located near the inlet of the capillary extends 1 mm–10 cm in a capillary with i.d. 75–200 m. The conjugated free-floating beads are retained between two frit structures. A second method is the immobilization of an IgG antibody or fragment to activated beads that are linked covalently to each other and to the inner wall of the capillary. In this particular case, retaining frits are unnecessary. 64 A
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The third approach is the immobilization of an IgG antibody or fragment to an activated sol–gel, monolithic structure, or other polymeric structures; frits are not needed. The last method is the immobilization of an IgG antibody or fragment directly to the inner wall of a previously activated capillary. This procedure does not require beads or frits. Furthermore, the immobilization of the antibody or antibody fragments in fritless structures can occur in a 2–20 cm length of capillary, or up to 40% of the entire length of the capillary. Because the orientation of an immobilized IgG antibody molecule is of critical importance for maximum binding capacity, keep in mind that the active chemical groups involved in the binding of the IgG antibody to the antigen or hapten should not be those that form an integral part of or are located near the binding site of the IgG antibody. The property of the Fab´ antibody fragment that produces a favorable orientation of the molecule on the solid support is the presence of one or more cysteine groups in the hinge region. These groups are located at the opposite end of the fragment from the antigen-binding site and provide stable binding to the support through disulfide bridges, yet they do not interfere with the antigen-binding site.
Recovery of captured analytes In IACE, it can be difficult to recover bound analytes from their capture antibody without using harsh elution conditions. The analyte should be recovered rapidly under gentle, nondenaturing conditions to ensure minimal damage to the capture ligand so that it can be reused many times. Several approaches are used for analyte elution, including changing the pH of the running buffer; adding agents that disrupt the antibody–antigen complex, such as chaotropic ions, which disturb the structure of water; and adding chemicals that alter the internal polarity of the capillary. Lowering the pH of the electrophoresis buffer to 1.5–3.8 is one of the more popular methods of analyte recovery. This change can be achieved by introducing a small plug of acidic buffer into the capillary or channel, thus washing the immobilized antibody–analyte complex in an excess of hydrogen ions (23, 25, 30). Alternatively, the pH of the electrophoretic running buffer may be altered and elution performed on a continuous basis (34). Elution with chaotropic salts, such as thiocyanates, iodide, and chloride, is a way to avoid the denaturing effects often associated with low-pH elution and is a popular approach in immunoaffinity chromatography (35). These salts disrupt the structure of the water molecules around the antibody–analyte complex and interfere with or reduce hydrophobic interactions between the molecules. In a manner similar to that used for pH elution, chaotropic salts are usually applied either as a small plug or as a continuous wash at concentrations of 1.5–8.0 M. Polarity-reducing agents, such as methanol, dioxane, and ethylene glycol, are extremely efficient at reducing antibody–antigen hydrophobic interactive forces and thereby releasing the bound analyte. However, to date, only a few of these agents have been applied to IACE (23, 25, 30, 36).
Applications
800
mAU
400
2 1
0 6
12
18
(b) 800,000
24
30
36
30
36
1 2
TICE
The elution buffer or solution plug may interfere with the identification of an eluted analyte(s). For example, certain plugs may yield a large peak when monitored at lower wavelengths in UV absorption, thereby masking the analyte. However, if the analytes possess intrinsic fluorescence—or are labeled with a chromophore to generate a fluorescent complex—and are monitored by either a noncoherent light source fluorescence or LIF detector, then the plug should not be detectable.
(a)
600,000
400,000 6
12
18
24
Migration time (min) Several applications of IACE have been described for quantifying peptides and proteins in simple and complex matrixes (23, 25–30, 34, 36–39). FIGURE 3. Separation of two neuropeptides by IACE using an analyte conTypical analytes include IgE, cardiac troponin I, centrator made of glass beads containing immobilized Fab´ fragments. cyclosporin A, neurotensin, angiotensin II, metThe separated peptides are monitored by (a) UV absorption and (b) MS detection. Total enkephalin, cholecystokinin, gonadotropin-rerun time was 38 min. Individual antibodies were directed against neurotensin (1) and angiotensin II (2). A control experiment designed to capture the neuropeptides in a leasing hormone, and various cytokines. Sensitivbuffer (simple matrix) yielded almost identical qualitative data but slightly different ity in IACE has increased at least 100–10,000quantitative data when either C18 or antibody fragments immobilized to glass beads fold, permitting the quantification of analytes were used. However, if the experiment was designed to capture the neuropeptides in from 1 ng/mL with a UV absorbance detector to urine (complex matrix), the qualitative data were very similar, but the quantitative data were several times greater when antibody fragments immobilized to glass beads were 1 pg/mL with a LIF detector. In a few cases, it is used than when C18 immobilized to glass beads was used. (TICE, total ion-current eleceven possible to detect concentrations of femtropherogram; adapted with permission from Ref. 39.) tograms per milliliter (27). Currently, most IACE experiments are performed on instruments equipped with a single capillary or channel. Multicapillary CE instruments have been ly distinguishable. Furthermore, IACE can be coupled to MS to applied primarily to DNA sequencing, which in general presents provide an immediately confirmed result, namely the correct no problem with sensitivity issues because of concentration am- mass of the target antigen or hapten. Neurotensin and angioplification provided by PCR. Some commercial multicapillary tensin II in urine samples were captured directly via their respecCE instruments can be adapted for high-throughput analysis of tive immobilized antibody fragments by means of IACE coupled proteins and peptides, although little information has been re- to a UV detector and a mass spectrometer; their migration times ported in this area (40). To compensate for such low through- and masses were compared with their corresponding standards put, investigators have immobilized multiple capture antibodies (39; Figure 3). in the forepart of the same capillary, enabling selection and capture of several different analytes per single sample introduction High-throughput CE using IACE (37 ). The different eluted analytes are separated by elec- An urgent need exists for a high-throughput CE instrument for trophoresis and detected on-line. A typical application of a CE proteomics research—in particular, for characterizing proteins instrument equipped with a single capillary is shown in Figure 2. and peptides in complex heterogeneous mixtures, such as bioThe Fab´ fragments were immobilized within a portion of the logical fluids, and in homogenates of tissue biopsies and cells. capillary itself (38). The number of genes identified in the Human Genome Project IACE is a 2-D technique (immunocapture and CE separa- appears to be ~25,000–30,000. However, it is estimated that the tion) that has significant advantages when compared with radio- number of polypeptide chains present in the Human Proteome immunoassay, ELISA, and enzyme-multiplied immunoassay Project may well be 50,000 or even 1 million or more. This techniques. Traditional immunoassays are prone to yielding al- estimate does not take into account more than 200 reported cotered data, which result in 2–20% false positives. IACE does not translational and posttranslational modifications, such as glycosylgenerate false positive results because the technology has the ad- ation, hydroxylation, phosphorylation, nitrosylation, methyladitional step of a physical separation after immunocapture or ex- tion, ubiquitination, acetylation, and polypeptide chain folding, traction. For example, certain substances may be closely related which are among the most common (17, 18, 41). structurally to the target analyte(s) and therefore may compete Traditional 2-D slab gel electrophoresis, although extremely for the binding site of the immobilized antibody or antibody useful, has serious limitations when applied to the characterizafragment. However, when it is separated by CE, the cross-react- tion of many proteins and peptides, in particular those found at ing false antigen or hapten will probably have a different migra- low abundance in complex mixtures. Isotope-coded affinity tag tion time than the native antigen or hapten, thus making it easi- technology (18) and multidimensional protein identification F E B R U A R Y 1 , 2 0 0 5 / A N A LY T I C A L C H E M I S T R Y
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ing their life. A larger preconcentration device, located near the inlet of the transport tubing, is used to clean highly complex sample matrixes. The excess Detector sample and any unwanted material are directed to waste. This Analyte concentrator–microreactor preconcentrator can use nonselective or selective immobilized ligands and can be disposable. The separation fused-silica capilw laries are merged into a single o fl c ti o Electroosm exit capillary for sequential online and/or off-line detection. Single or multiple detectors can be used, including a mass spectrometer (25, 43). This multiple concentration– Sample Waste Buffer separation system (Figure 4) exhibits reproducible migration times and peak areas over many FIGURE 4. Schematic of a prototype multidimensional CE instrument (43; adapted with permisanalyses for every analyte studsion from Ref. 25). ied. It has the potential to perform all functions automatically, technology (better known as MudPIT; 42) have proven to be enabling the trapping, enriching, and eluting of analytes in any unbiased and exquisitely sensitive methods for the automated cell, organelle, tissue, or biological fluid (25). The high-throughcharacterization of a large fraction of the proteome. However, put analytical instrument will capture, enrich, clean, separate, and because of the complexity involved in protein characterization, characterize target analyte(s) with significant speed, high sensiIACE may well be used as a complementary technique and play tivity, and possibly lower cost when made commercially available. an important role in the concurrent proteomic analysis of both In addition, the immobilization of the antibodies or antibody fragments directly on the inner surface of the capillary wall withbiological fluids and tissue proteins. Two different models of a simple solid-phase microextraction out beads, frits, or polymeric matrixes may permit the capture device have been described for use in IACE. The device is de- and characterization of circulating cells that may be present in a signed in a four-part cruciform configuration or in a staggered biological fluid. In addition to the antibodies that can be immobilized on a arrangement. It includes a large-bore tube to transport samples and washing buffers and a small-bore fused-silica capillary to sep- solid support matrix, numerous affinity ligands, including enarate the analytes (25, 30, 43). Another goal is to include the mi- zymes, can also be immobilized. For example, it may be possible croextraction device in the design of a multidimensional CE in- to use the multidimensional CE instrument to capture a protein (using one analyte concentrator containing an immobilized antistrument. Figure 4 is a diagram of a high-throughput IACE instrument body directed against that protein) and to cleave the released capable of performing hundreds of assays per day. It contains protein to generate peptides (using a second analyte concentrathree concentrator–microreactor devices (shown in the enlarged tor containing immobilized trypsin or any other proteolytic enview) located near the inlets of each of three fused-silica capillar- zyme) for further separation and characterization. Alternatively, ies. Each device, designed with a cruciform configuration, has it is possible to cleave the protein into peptides first (first analyte four entrance–exit ports connected to each other via PEEK concentrator) and to capture and tag the peptides with a chrotransport tubes for sample introduction and buffer washes. A se- mophore thereafter (second analyte concentrator) (44). The ries of miniaturized valves (shown as green circles with ) are entire procedure for capturing specific proteins (analyte concenplaced at strategic locations in the instrument to control the di- trator) and digesting them (microreactor) on-line can be autorection of the liquid, enabling the interaction of each analyte mated; this makes the multidimensional CE instrument a powerwith the corresponding immobilized antibody or antibody frag- ful tool for microproteomics studies. ment to a solid support matrix. The valves are designed to maintain a closed environment in the concentrator–microreactor and Future trends to permit optimal conditions of time and temperature for maxi- Over the years, the bottleneck in protein analysis has been idenmum binding of individual antibodies. The valves also block pas- tification. Relatively large amounts of protein were required, and sage of the sample to the fused-silica capillaries, thereby avoiding only a limited number of samples could be processed because of contamination of the internal wall of the capillaries and extend- cumbersome sequencing procedures. Although 2-D polyacryl66 A
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amide gel electrophoresis (2D-PAGE) followed by MS permits characterization of thousands of new proteins and peptides, these techniques do not provide sufficiently high throughput to characterize the hundreds of thousands of proteins, or more, that are believed to make up the human proteome. The challenge in proteomics is to combine the correct techniques with the proper samples and analytes. 2D-PAGE has proved to be of great use in protein characterization and is still in use in many laboratories. Alternative approaches are becoming popular, such as those based on liquid separation in one or more dimensions followed by MS or MS/MS; these methods are generally much faster and easier to automate than 2D-PAGE. Furthermore, an urgent need exists for better tools for qualitative and quantitative, highthroughput proteomics studies. Affinity techniques coupled to CE may considerably reduce the time, cost, and quantity of required material and may significantly increase the rate at which proteins and peptides can be analyzed. Once this dynamic process is completely automated, the key issues in high-throughput procedures can be addressed. Moreover, the availability of specific antibodies targeted to peptides containing co- and posttranslational modifications will facilitate the identification and characterization of covalent modifications crucial to protein function and activity. IACE is growing rapidly to become a powerful tool in the identification of the large number of unknown human proteins—as many as 50% of the total—that still have unknown functions. The human proteome holds the promise of a revolution in disease diagnosis, prognosis, and treatment. IACE may play a major role in the advancement of proteomics research and may contribute to improved efficiency and effectiveness in the clinical laboratory. However, as for most other techniques for protein characterization, a few drawbacks need to be overcome to make IACE a successful, routine technique in proteomics research. Norberto A. Guzman is a senior research fellow at Johnson & Johnson Pharmaceutical Research & Development. Terry M. Phillips is the chief of ultramicro-analytical immunochemistry in the division of bioengineering and physical sciences at the National Institutes of Health. Their research interests include development of miniaturized platforms for studying immunoaffinity interactions and molecular recognition reactions between proteins and other molecules. Address correspondence about this article to Guzman at Bioanalytical Drug Metabolism, JJPRDUS-B356, 1000 Route 202, Box 300, Raritan, NJ 08869 (nguzman@ prdus.jnj.com) or to Phillips at UAIR, ORS, OD, Bldg. 13, 3N17, National Institutes of Health, Bethesda, MD 20892 (
[email protected]).
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