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Figure 5 (Top) Electropherogram of 100 nM FITC-insulin under HPCE conditions that employed: uncoated capillaries, 25 |J,m i.d. and 150 |lm o.d., total lengths of 2530 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 FITC-insulin, while peaks 1 and 4 are due to the formation of the complex of Fab with FITC-insulin 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 FL- or 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
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 X10-10 (74).
Krull et al.
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.