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the formation and presence of several complexes. Multiple complexes reduce sensitivity and specificity of the final assay, and only complicate the identification of a single, individual Ag, possibly present together with cross-reacting analogs that might also form Ag–Ab complexes with the same FL-tagged Ab (Fab′) species. Thus, though use of Fab′ species for the immunorecognition step does reduce microheterogeneity problems by removing the carbohydrate region of the Ab, solution tagging with common FL reagents, such as tetramethylrhodamine (TR), often leads to multiple products, which again complicate the final CE patterns (62,63).

There are several reports in the literature that have utilized CE-based techniques for recognition of a specific antigen and then performed an immunoassay on the now-isolated species. These have all involved a form of ACE, usually immuno-ACE, and thus could be applicable for combinatorial library searching in the future.

Immunoassays are based on the bioaffinity between an Ag and its corresponding Ab. Quantitation of Ag requires the ability to detect and discriminate between the Ag–Ab complex and either the free Ab or Ag. The high separating power of CE makes this a logical candidate for the development of immunoassays. In comparison with conventional immunoassays, a CE-based immunoassay has the following advantages: (a) It is fast and easy to automate. Fast complexation rates for a homogeneous solution system (preinjection) require less incubation time than for a heterogeneous, solution-solid phase system (conventional immunoassays on a plate/tube). (b) Multiple washing steps are eliminated. (c) Less sample is needed. (d) The high separation power of CE can discriminate between specific and nonspecific binding, protein variants, metabolites, and so forth. This last advantage is potentially significant when compared with current methods for performing immunoassays on a plate or in a tube or even by immunodetection in a flow injection mode (64). That is, in CE, there is the potential for baseline resolution and thus identification among the various possible antigens that could recognize a given Ab, such as protein variants, metabolites, decomposition products, and deamidation products. This is not possible in any of the current batch-type immunoassays.

For CE-based immunoassays, Fab fragments are usually preferred to the intact Abs, due to the multiple Ag binding sites on Abs and microheterogeneity of the Fc (crystalline or carbohydrate) portion of the Ab caused by the variations in glycosylation. That is why in almost all of the reported CZE electropherograms for intact Abs, such peaks are usually quite broad, no matter the specific separation conditions. Fab or Fab′ fragments are often, under very

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similar HPCE conditions, much sharper and better resolved, especially in CZE and CIEF. Papain and pepsin are the most commonly used reagents for Ab digestion. Even after digestion, it is possible that more than a single Fab or Fab′ species will be obtained. In the work of Schmalzing et al., three Fab fragments were observed, due to the fact that papain cleavage is not very specific, or derived from the microheterogeneity in the Fab fragments (65). Cation exchange separation was therefore performed to achieve a single Fab species.

Since Ag–Ab complex formation is a reversible reaction, a certain degree of complex dissociation will take place inside of the capillary. Once the complex dissociates, the Ag and Ab will rapidly move apart due to the differences in their electrophoretic mobilities. The level of the complex detected may actually be lower than that formed in the original mixture. The amount of decrease depends on the dissociation kinetics and the time required for separation. To obtain maximal complex signal, a short separation time is normally recommended (61,66).

At present, most CE-based immunoassays can be divided into two categories: competitive and direct immunoassays. For competitive immunoassays, the Ag molecules are first labeled with FL tags. Labeled Ag and a limited amount of Ab are then added to the sample to be analyzed. The labeled Ag will compete with the original/sample Ag in the sample for the limited binding sites on the Ab. After incubation, a small amount of the Ab-Ag mixture is injected into the capillary. Upon separation, using fluorescence detection, two peaks will appear in the electropherogram, one coming from the labeled Ag and the other from the Ab–Ag complex. The free Ag may elute together with the labeled Ag if the FL tag is a small molecule, or it may be separated from the labeled Ag, but under both conditions, no free Ag can be observed by FL detection. The amount of Ag in the sample is directly proportional to the free, FL-labeled Ag signal, and inversely proportional to the complex Ab-Ag (FL) signal. In principle, both signals can be used for quantitation. Most of the competitive immunoassays described are carried out on small molecules with an FL tag.

For example, Schultz et al. demonstrated a competitive immunoassay of insulin with a sensitivity around 10 9 M (66,67). Using this assay, the insulin content of single islets of Langerhans was determined. Fluorescein Isothiocyanate (FITC) tagging of insulin yielded at least three distinct products, all of which were separated by CZE, as illustrated in Fig. 5 (66). When these FL-tagged insulin species were then complexed with their Fab, at least two complexes were formed, appearing together with uncomplexed, FL-tagged in-

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Figure 5 (Top) Electropherogram of 100 nM FITC-insulin under HPCE conditions that employed: uncoated capillaries, 25 m i.d. and 150 m o.d., total lengths of 25– 30 cm, length to detector 12–15 cm, buffer of 0.05 M sodium phosphate with 0.025 M K2SO4 at pH 7.5, applied voltage 1000 V/cm, hydrostatic injection. (Bottom) Electropherogram of 100 nM FITC-insulin and 50 nM Fab. Peaks 2, 3, and 5 are FITCinsulin, while peaks 1 and 4 are due to the formation of the complex of Fab with FITCinsulin in peaks 2 and 5, respectively. An He-Cd laser was used as the excitation source (66). (Reproduced with permission from the copyright holder, American Chemical Society and Analytical Chemistry.)

sulin (FL detection) (Fig. 5). Perhaps these results highlight some of the lingering problems in using immunoassays and/or immunorecognition methods in HPCE, whereby contrary to batch-type immunoassays, the presence of several FLor otherwise tagged Ag or Ab species in CE can lead to multiple, complex peaks, complicating the final results. These limitations, especially with larger Ags, also with tagged Abs, have not been resolved (62,63).

Schmalzing et al. developed a similar method to that above, but now for cortisol in serum (57). In this case, it was fairly easy to FL-tag the small cortisol molecule for this FL based CE-immunoassay, whereby a single final tagged antigen formed. This is an excellent example of how the combination

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of immunorecognition and immunoassay-based methods can be routinely applied with CZE separations for improved, perhaps absolute, analyte identification and full quantitation, using excellent calibration plots and validated methods.

Chen and Pentoney have reported a competitive immunoassay method for digoxin in serum with sensitivity in the low 10 11 M range (68). The high-resolution power of CE makes the simultaneous analysis of two, or more than two, species feasible, whereas this may not be the case in most other separation modes (e.g., HPLC). Chen and Evangelista also reported a simultaneous competitive immunoassay of morphine and phencyclidine in urine (69). The immunoassay could be performed routinely and reproducibly in less than 5 min with detection limits of 4 nM for phencyclidine and 40 nM for morphine.

The problem with labeling big molecules, such as proteins and antibodies, is that multiple products are typically obtained (as above) because most of the labeling reactions utilize the primary amino groups on the proteins and antibodies. The tags can bind in different amounts and at different locations to the proteins (62,70–73). In one illustrative example, this problem was avoided by using thiol groups at the hinge region of the Fab’ fragment to react with the label (61), although several chemical reactions were involved to achieve the monothiol group on Fab’. Even here, two separate, tagged Fab’ species were produced (Fig. 4), still complicating the overall CE assay and specificity.

Multiple labeled Ags (or Abs) lead to multiple peaks or, at times, a broad peak in the CE electropherograms. These species can then form more than one Ab–Ag complex, which complicates the analysis, due to perhaps insufficient resolution of the labeled Ags and the Ab–Ag complexes. Sometimes chemical modifications to the Ag (or Ab) are necessary to facilitate the separation by changing the final mass/charge ratio (74–76).

The principle of direct immunoassays is that Abs are first tagged with FL labels and then mixed with the Ag sample of interest. The Ab should be in excess. After incubation, the mixture is injected into the capillary, and the amount of Ag can be determined by the Ab–Ag complex signal. Competitive immunoassays are usually preferred for small Ags because the separation of the Ab–Ag complex from the Ab can be very difficult for small Ags. As above, Shimura and Karger reported a direct immunoassay of hGH (61). Due to the focusing effect of CIEF (Fig. 4) and the high sensitivity of CE–laser- induced fluorescence (LIF), a detection limit of 0.1 ng/ml was achieved. Similarly, Chen demonstrated the direct immunoassay of IgG with a detection limit of 6 10 10 (74).

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A more universal immunoassay method in CE was developed by Reif et al. (76). In this approach, generic protein G was first tagged at several locations with solution FITC reagent. The heavily FITC-tagged protein G was then combined with the Ab, due to the affinity between protein G and the Fc portion of the antibody. This FITC, dual-protein reagent was then complexed with the Ag (analyte) in a CE-based assay. In this fashion, FITC-tagged protein G behaved as an FL label. The Ab did not have to be labeled for detection. This may have been the only sandwich immunoassay reported in CE. The term sandwich here refers to an approach that utilizes two antibodies surrounding the antigen, for improved specificity and identification of the correct analyte/antigen.

All of the work described above used an Ab-Ag reaction in solution. The same reaction can occur when Ab is immobilized onto the inner walls of the capillary. For example, Phillips and Chmielinska reported the analysis of cyclosporin in tears using this CE-based method (77). One third of the capillary was immobilized with the purified Ab. The sample to be analyzed was introduced into the capillary and incubated for 10 min. After rinsing the capillary with the buffer to remove the unbound materials, the buffer in the reservoirs was changed to an acidic solution. Under these conditions, the Ab–Ag immobilized complexes were broken. The active Ab remained immobilized on the CE capillary column, whereas the Ag was brought to the detector by EOF. Different from conventional, batch-type immunoassays, cyclosporin A (Cys A) and its metabolites could be differentiated due to the differences in their relative electrophoretic mobilities. As shown in Fig. 6, this is perhaps an ideal illustration of the power of combining Ab-based immunoassays with CE separations, and the inherent ability to resolve very similar species after a first immunorecognition step. It is apparent that this entire approach could be readily utilized for combinatorial library searching. In this particular instance, the assay did not use En or FL enhancement for detection, but rather represents a form of immunoaffinity extraction and preconcentration. This was then followed by the high resolving power of CE for whatever species (untagged) were first recognized by the immobilized Ab. It would, of course, be possible to first tag the Ag analytes in a sample and then introduce these into the immobilized Ab-CE system for improved isolation, separation, and detection. This approach, in general, is quite similar to the combination of immunoaffinity sample preparation followed by HPLC separation-detection methods (20,78–84).

Like conventional immunoassays, immunoassays in CE can be performed in different modes, such as sandwich, double sandwich, competitive, or direct on/off. However, the more species (analytes) that are possibly pres-

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Figure 6 Immunoaffinity or immunoassay CE profiles of tear fluid obtained from

(a) a patient with no clinical signs of CyA toxicity and (b) a patient during an episode of systemic toxicity. Relevant peaks: CyA, cyclosporin A; peaks 1–4 represent cyclosporin metabolites: 1, AM1, 2, AM9, 3, AM1c, 4, AM4N (77). (Reproduced with permission from the copyright holder, John Wiley & Sons, Ltd. and Biomedical Chromatography.)

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ent, the more challenging and difficult the needed separation may become. Other than FL tags, CL, EC, and enzyme labels can also be employed. Much less has been described using such enhanced (signal-amplified) approaches in immunorecognition or immunoassays combined with CZE. Because of the amplification effect of enzyme–substrate reactions, enzyme labels should provide lower (better) detection limits (85).

IV. SPECIFIC APPLICATIONS OF HPCE TO COMBINATORIAL CHEMISTRY ANALYSIS

A search of the recent literature on the use of HPCE for the analysis of combinatorial libraries shows that as of today not a great deal of work has been done in this area. In fact, only seven published papers could be found, four of which are from the same group (86–88). However, as three of the seven were published in 1996 (88–90), it can be suggested that perhaps this research area is now picking up and will show more results in the near future.

The first published report on the use of HPCE to analyze a combinatorial library came in 1993 by Chu et al. (86). They evaluated using affinity capillary electrophoresis (ACE) for the identification, based on competitive binding, of tight-binding ligand(s) for a receptor in mixtures of equimolar ligand libraries. This has become the general approach in HPCE for determining binding constants, biologically recognized and presumably active, interacting partners, and as a general, potentially automatable, on-line screen of combinatorial libraries for active lead/target compounds. In this first example of using ACE to find active target compounds, vancomycin was used as the receptor and a small library was created consisting of 32 peptides to examine feasibility. This approach of library searching has now become the basis of a general method to perform drug screening and has since been commercialized (91). Of course, since this generalized approach appears to work for potential, lead drug candidates, it can be readily extended in the search for active agricultural chemicals, fragrances, perfumes, insect pheromones, affinity ligands, and so forth. The method is not limited to identifying only drug candidates. That is, it could just as readily be utilized for seeking agricultural compounds, perfumes, veterinary products, or any bioactive chemical compound.

Using this approach, vancomycin was first run in the electrophoresis buffer with a probe ligand, Fmoc-Gly-D-Ala-D-Ala (L), that showed high binding affinity for the target substrate, vancomycin. It was shown that the concentration of vancomycin affected the electrophoretic mobility of L (Fig.

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Figure 7 Illustration of CE-UV for identification of peptides binding to vancomycin by using affinity recognition in CE. How the concentration of vancomycin in the electrophoresis buffer (20 mM phosphate, pH 7.4) affects the electrophoretic mobility of Fmoc-Gly-D-Ala-D-Ala (L, black circles) but not Fmoc-Gly-L-Ala-L-Ala (open circles). Specific conditions indicated elsewhere (86a). (Reproduced with permission of the copyright holder, publisher and Journal of Organic Chemistry.)

7a, b), as can be seen in the shift of the L peak. When the peptide library was added, the electrophoretic mobility of L again was changed due to one or more of the peptides (L′) competing with L for vancomycin. Through the use of subsets of the library, it was possible to then specify (narrow possibilities) which of the peptide(s) was L′ (Fig. 8). Figure 9 shows how the mixture of 32 peptides was split into 2 groups of 16 peptides each and analyzed by CE. One set of 16 could then be eliminated as no shift was seen. The remaining 16 were split into 2 groups of 8 peptides each and the above was repeated. Eleven experiments were necessary to determine the one peptide that was a

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Figure 8 Stepwise elimination of noninteracting peptides from a mixture of 32 peptides and identification of one tight-binding ligand for vancomycin. Interpretation of each electropherogram is described in the text. Specific experimental conditions are described elsewhere (86a). (Reproduced with permission of the copyright holder, publisher and Journal of Organic Chemistry.)

tight binding receptor. In this way it was determined that L′ was a,e-Ac2-L- Lys-D-Ala-D-Ala (Fig. 7c, d). Thus, it was shown that HPCE could be used to identify ligands from small libraries that bind most tightly to a receptor, such as vancomycin (86).

In 1995, a report using ACE-MS was published (87). Chu et al. reported on the development of a simple, one-step procedure for the on-line separation and identification of ligands that again bind most tightly to a receptor. Vancomycin was again chosen as the receptor, and it was used in the electrophoretic buffer to completely fill the capillary. In order to prevent vancomycin from flowing into the MS, the electrophoretic buffer pH was chosen to prevent the vancomycin from migrating. A neutral, hydrophilic, polymer-coated capillary was used to minimize EOF in the capillary and to therefore reduce the vanco-

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Figure 9 Affinity CE-MS (ACE) of a synthetic, all-D, Fmoc-DDXX library of 100 tetrapeptides using vancomycin as the receptor (A–D). Selected ion electropherograms for the masses are indicated; (E) reconstructed ion electropherogram for runs without (left) and with (right) vancomycin in the electrophoresis buffer. Specific ACE and MS conditions are indicated elsewhere (87). (Reproduced with permission of the copyright holder, the publisher and the Journal of the American Chemical Society.)

mycin flow to the MS. The idea of the research was that ligands that bound tightly to the receptor would be retained in the capillary for a longer period. The later eluting peptides could then be identified by MS. From a known 100tetrapeptide all-D Fmoc-DDXX library, three peptides (Fmoc-DDYA, FmocDDFA, and Fmoc-DDHA) were identified that bound tightly to the vancomy-