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Fig. 1. CE-LIF competitive immunoassay of four drugs in urine. This figure refers to the separation of four drugs in urine. A fused silica capillary 27 cm x 20 was used for separation. Buffer was 200 mM borate, pH 10.2, with LIF detection (633 mn excitation an 665 nm detection), voltage was 15 kV. The top figure is blank urine and the bottom figure is a urine spiked with the four drugs of interest. Cy5 is a negatively charged fluorescent cyanine dye. The drug concentrations were 73 nM (25 ng/mL) for THC-COOH, 100 nM (243 ng/mL) for PCP, 1 mM (285 ng/mL) for morphine, and 860 nM (248 ng/mL) for benzoylecgonine. Reproduced with permission from A. Chen, Beckman Coulter, Inc.

Although both of these methods will probably be developed, the underlying principle will be the same. Thus this chapter will focus only on the principles behind the technique along with examples of how it has been used.

2.1. Competitive Binding Immunoassay

The competitive binding assay is the most widely used immunoassay format used with CE. In this method, the sample containing the analyte, antibody or antibody fragment, and fluorescent-labeled tracer are mixed and incubated. The tracer competes with the analyte in the sample for a limited

Thc Capillary Electrophoresis

Fig. 1. CE-LIF competitive immunoassay of four drugs in urine. This figure refers to the separation of four drugs in urine. A fused silica capillary 27 cm x 20 was used for separation. Buffer was 200 mM borate, pH 10.2, with LIF detection (633 mn excitation an 665 nm detection), voltage was 15 kV. The top figure is blank urine and the bottom figure is a urine spiked with the four drugs of interest. Cy5 is a negatively charged fluorescent cyanine dye. The drug concentrations were 73 nM (25 ng/mL) for THC-COOH, 100 nM (243 ng/mL) for PCP, 1 mM (285 ng/mL) for morphine, and 860 nM (248 ng/mL) for benzoylecgonine. Reproduced with permission from A. Chen, Beckman Coulter, Inc.

number of antibody binding sites. After equilibrium is reached, the mixture is injected onto a column, where the free tracer and the Ab-Ag complexes (including fluorescent Ab-Ag complexes) are electrophoretically separated and detected by on-column LIF. For this type of immunoassay, the concentrations of tracer and antibody injected onto the CE column are essential for valid quantitation of the analyte (15).

The immunoassay of insulin was the first reported competitive CE-based immunoassay using an antibody fragment (fab) (16,17). However, the microheterogeity of the fab fragments produced two peaks during separation, making it difficult to interpret the assay results. In addition, the detection limit (3 nmol) could not be improved because the covalent attachment of the label to the amino acid on insulin reduced the affinity of the antibody for the tracer. Optimization of the assay subsequently led to the development of an on-line CE immunoassay device. This allowed for analysis of the insulin content of single islets of Langerhans in addition to the quantitation of glucose-stimulated insulin release (18). For this assay, three independent channels were used, one each for the labeled insulin, intact monoclonal antibody (MAb), and sample containing unknown levels of insulin. The contents of the channels were mixed where the channels intersect. The mixture was then directed to the reaction capillary through a stopper to a flow-gated interface. Incubation took place at the reaction capillary and the samples injected onto the column. The electropherogram typical of competitive immunoassays was obtained for insulin with a clear separation of the Ab-Ag peak and the fluorescent-labeled insulin peak. Because of the competition between the insulin in the sample and the labeled insulin for a limited number of antibody binding sites, quantitation of the insulin was possible. This assay generated a standard curve typical of a competitive binding immunoassay where high insulin levels produce a small Ab-Ag peak, whereas low insulin levels generate a large Ab-Ag peak.

In a manner similar to insulin, multianalyte drug assays have been developed using the competitive- based CE immunoassay format. In this type of assay, clear separation of free and bound tracer was observed (14) (see Fig. 1). Similar to the insulin immunoassay, the peak heights of the free tracers were also found to be concentration-dependent. Most of these assays have used the fluorescein-labeled tracer from fluorescence-polarization assay kits. Employing equal volumes of urine, tracer, and antibody CE-based immu-noassays for methadone (19), benzoylecgonine, and amphetamine/metham-phetamine (20) have been developed. An immunoassay that can separate the four analytes (methadone, morphine, benzoylecgonine and D-amphetamine) with sensitivity comparable to Abbott's TDx FLx FPIA assays has also been developed (21).

Benzoylecgonine

TIME (min)

Fig. 2. MOCA multianalyte immunoassay. This figure refers to the separation of methadone (M), morphine (O), benzoylecgonine (C), and D-amphetamine (A). The L and M refer to low- and medium-level multiconstituent controls, respectively. A fused silica capillary, 47 cm (40 cm to detector) x 75 was used for the separation. Buffer was 50 mM borate, pH 9.3, with LIF detection (488 nm excitation and 520 nm emission), voltage was 13 kV, and the capillary was kept at 20°C. Reproduced with permission from ref. (22).

TIME (min)

Fig. 2. MOCA multianalyte immunoassay. This figure refers to the separation of methadone (M), morphine (O), benzoylecgonine (C), and D-amphetamine (A). The L and M refer to low- and medium-level multiconstituent controls, respectively. A fused silica capillary, 47 cm (40 cm to detector) x 75 was used for the separation. Buffer was 50 mM borate, pH 9.3, with LIF detection (488 nm excitation and 520 nm emission), voltage was 13 kV, and the capillary was kept at 20°C. Reproduced with permission from ref. (22).

Caslavka et al. (22) developed a CE-based immunoassay using 50 mM borate buffer, pH 9.3, for the simultaneous detection and quantitation of methadone (M), morphine (O), benzoylecgonine (C), and D-amphetamine (A). Using the fluorescein tracer from a tricyclic antidepressant FPIA assay kit (Abbott Diagnostics) as the internal standard (I.S.), the electrophero-grams in Fig. 2 were obtained for a blank urine, low-level control urine (19fold dilution), low-level control urine (10-fold dilution), low-level control urine, and medium-level control urine (from bottom to top). These data reveal that peak heights for all free tracers increase with increasing drug concentration. The ratios of the tracer peak heights with peak height of the I.S were used for multilevel internal calibration. The typical calibration

Concentration Graph Drug
Fig. 3. Calibration graph of the MOCA multianalyte immunoassay. Data are based on peak height ratios divided by the peak height of the internal standard. Reproduced with permission from ref. (22).

curves are shown in Fig. 3. This assay was found to be more sensitive than the enzyme multiplied immunoassay technique (EMIT) d.a.u. assay used in the authors' routine drug-assay laboratory.

Other examples of CE-based multianalyte immunoassay include analysis of urinary morphine, PCP, THC, benzoylecgonine (23), salicylate and paracetamol (24), theophylline and quinidine in serum (24), morphine and PCP in urine. The main limitation in multianalyte assays is the ability of CE to separate labeled antigen from each other and from the antibody-antigen complex. Using Abbott's TDxFLx FPIA reagents, as in the MOCA assay, a significantly increased response for free tracer was obtained. Ninefold dilution of all mixed fluids showed decreased responses when compared to threefold dilution.

2.2. Direct CE

For direct CE-based immunoassay, fluorescent- or enzyme-labeled antibody or antibody fragments are added in excess to a sample. After incubation, a small aliquot of mixture is injected onto the CE column and the fluorescent complex (antigen-antibody) is separated from excess-labeled antibody with detection by LIF. Only the signal for the complex (Ab-Ag) is used for quantitation and total amount of antigen present is determined by comparing the peak height and or peak area of complex to the linear range of the calibration curve. Interferences have been found to be minimal since CE separates the potential interfering substances from the Ab-Ag complex. In addition, the different spectroscopic properties of the components relative to the Ab-Ag complex allow interfering substances to be separated and identified (25). A variant of the sandwich format was also introduced to improve the separation. This involves using a matched pair of antibodies to perform independent binding to the analyte and subsequent mobility tailoring (26). The first antibody is the tracer (fluorescent- or enzyme-labeled) is for detection whereas the second is to modify mobility (highly charge modified). After equilibrium is reached the complex (Ab-Ag-Ab) is quantitated. Another version of the direct assay uses fluorescein-labeled protein G to quantitate IgG in human serum (27).

2.3. Microchip-Based CE

Microchip-based CE was developed by Manz and Harrison in 1992 (28,29). The procedure is based on micro-machining technology and involves chemical etching, photolithography, and water binding to build channels in glass or fused silica substrates. The chip can then be used to electrophoretically separate analytes. This is faster, more flexible, and can have an enormous sample throughput relative to conventional CE. The first adaptation of immunoassay on the microchip was by Koutny et al. (30). Using microchips, the authors developed an assay that could quantitate the levels of serum cortisol. Microchips provide have many performance-enhancing features (such as lower reagent consumption), faster separations, integrated sample handling steps (such as on-chip mixing, dilution, and labeling), as well as parallel multianalyte analysis by microfabrication of a multichannel device (31). High performance on-chip separations by CE have also been readily achieved using glass substrates (32,33). With the microchip, it is possible to control the flow of fluid at channel intersections using the electrokinetic and electroosmotic flow (EOF). Electrical and reagent leakages at intersections were also eliminated by application of bias voltages (34,35). Complex biochemical quantitations and reactions can be affected with microchips. Various modifications have been made in an attempt to adapt the microchip CE technology to the clinical laboratory including interfacing multiple-channel glass chips to an electrospray ioniza-tion mass spectrometer (36).

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