Immunoaffinity capillary electrophoresis

Guzman, N. A. (2004). Immunoaffinity capillary electrophoresis applications of clinical and pharmaceutical relevance. Anal. Bioanal. Chem. 378(1), 37-39. 

NA Guzman. Determination of immunoreactive gonadotropin-releasing hormone in serum and urine by on-line immunoaffinity capillary electrophoresis coupled to mass spectrometry. J Chromatogr B 749 197—213, 2000. 

Guzman, N. A., Improved solid-phase microextraction device for use in on-line immunoaffinity capillary electrophoresis. Electrophoresis 24(21), 3718-3727, 2003. 

Immunoaffinity capillary electrophoresis is rapidly growing in the field of pharmaceutical and diagnostic applications. Several applications have been developed to affinity concentrate proteins and peptides found at low concentrations in simple and complex matrices in order to enhance analyte detectability when separated by CE (68,120,407,435). Figure 12 shows a microphotograph... 

TM Phillips, BF Dickens. Analysis of recombinant cytokines in human body fluids by immunoaffinity capillary electrophoresis. Electrophoresis 19 2991-2996, 1998. 

TM Phillips, JJ Chmielinska. Immunoaffinity capillary electrophoresis analysis of cyclosporin in tears. Biomed Chromatogr 8 242-246, 1994. 

Phillips, T.M., Kennedy, L.M., and DeFabo,E.C. (1997) Microdialysis immunoaffinity capillary electrophoresis studies on neuropeptide induced lymphocyte secretion. Journal of Chromatography B, 697, 101 109. 

Bomemann, C. et al., Fluorescence-labelled antigen-binding fragments (Fab) from monoclonal antibody 5F12 detect human erythropoietin in immunoaffinity capillary electrophoresis, Anal. Bioanal. Chem., 376,1074, 2003. 

Phillips, T.M. Smith, P. Analysis of intracellular regulatory proteins by immunoaffinity capillary electrophoresis coupled with laser-induced fluorescence detection. Biomed. Chromatogr. 2003,17, 182. 

Capillary zone electrophoresis (CZE), the most widely used CE mode, is also the mode most frequently used for performing CE immunodetection. The first assay of immunoaffinity capillary electrophoresis described was carried out with CZE in noncompetitive format (Fig. 8a) [79,80]. This format has been used to determine a protein in a matrix as complex as human serum by incubating the sample with specific antibody for 1 hr before the introduction onto the CE column [81]. The size of the immunocomplex peak was seen to increase with incubation time. One drawback of this method is the long incubation time needed because of the slow reaction kinetics of formation of Ab-Ag complex. To prevent noticeable complex dissociation during the analysis, conditions to achieve a separation time shorter than 3 min were chosen. 

Phillips, T.M., Wellner, E.F. Measurement of naproxen in human plasma by chip-based immunoaffinity capillary electrophoresis. Biomed. Chromatogr. 2006 20 662-667. 

E. J. Cole and R. T. Kennedy, Seleaive preconcenti ation for capillary zone electrophoresis using protein G immunoaffinity capillary chi omatography . Electrophoresis 16 549-556(1995). 

Immunoaffinity procedures have also been developed to selectively extract corticosteroids from different sample matrices. Thus, Seymour et al. demonstrated the higher efficiency of the immunoaffinity methods compared with the conventional extraction procedures using organic solvents [177]. Immunosorbents have also been used for online procedures followed by HLPC-UV [178, 179], HPLC-APCI-MS [179,180], GC-MS [176,181], or capillary electrophoresis [182]. Poly(hydroxyethyl methacrylate) (HEMA) was evaluated as a support material for the anti-dexamethasone antibodies used in IAC. The online IAC-HPLC-MS allowed determination of dexamethasone and flumethasone in equine urine with LODs in the range 3-4 ng mL-1 [180]. The cross-reactivity values obtained in the ELISA and the recoveries of an IAC-HPLC procedure are presented in Table 7. Bagnati et al. developed an immunoaffinity extraction... 

An immunoassay using immobilized antibodies was described by Phillips and Chmielinska (14). By means of this approach the determination of the immunosuppressant cyclosporin A (CsA) in tear fluid was obtained using UV detection. Although the detection limit is sufficient using only immu-noaffinity capillary electrophoresis, overlapping signals can be avoided. A set of tear samples was quantified. Immunoaffinity CE was able to detect... 

The technique has been named immunoaffinity electrochromatography (1ACEC) [180]. In principle, the solute diluted in a large volume is applied to a capillary column packed with a support containing anti-solute IgG. The solute is selectively extracted by the immunoaffinity column, and the bound analyte is eluted and separated further (if desired) by capillary electrophoresis. 

The majority of reports have used electrospray ionization mass spectroscopy (ESI-MS) as an analytical detection method because of its sensitivity and the soft namre of its ionization procedure, which generally only leads to the detection of the molecular ions of the positive library members. Many separation techniques have been coupled to ESI-MS, including affinity chromatography (49), size exclusion chromatography (50, 51), gel filtration (52), affinity capillary electrophoresis (53-58), capillary isoelectric focusing (59), immunoaffinity ultrafiltration (60), and immunoaffinity extraction (61). ESI-MS has also been used alone (62) to screen a small carbohydrate library. Other examples reported alternative analytical techniques such as MALDI MS, either alone (63, 64) or in conjunction with size exclusion methods (65), or HPLC coupled with immunoaffinity deletion (66). 

The specificity of IA and antibody immobilization on solid-phase support techniques can be utilized for sample clean-up and concentration before capillary electrophoresis and mass spectrophotometry. For example, analysis of prostacyclins requires sensitivity in the pg/mL range and selectivity from multiple endogenous prostaglandin compounds. Immunoaffinity sample clean-up before GCMS has been used for thromboxane B2 analogs (244-246), iloprost (247), and nocloprost (248). Immunoaffinity sample clean-up before HPLC determinations was reported for clenbuterol (249) and pravastatin (250). 

Just as in the computer and Internet revolution, other successful technologies, such as capillary electrophoresis, offer proven and open solutions, deliver robust results, are cost effective and easy to implement, and yield high value. Currently, capillary electrophoresis can quantitate concentration detection limits in the range of 1 ng/mL to 1 pg/mL using on-line immunoaffinity preconcentration with UV or conventional laser-induced fluorescence detection systems (58,... 

DH Thomas, DJ Rakestraw, JS Schoeniger, V Lopez-Avila, J Van Emon. Selective trace enrichment by immunoaffinity capillary electrochromatography on-line with capillary zone electrophoresis-laser induced fluorescence. Electrophoresis 20 57-96, 1999. 

Thomas, D. H., Rakestraw, D. J., Schoeniger, J. S., Lopez-Avila, V., and Van Emon, J., Selec-hve trace enrichment by immunoaffinity capillary electrochromatography on-hne with capillary zone electrophoresis—Laser-induced fluorescence. Electrophoresis, 20,57-66, 1999. 

An elegant study was presented by Thomas et al. who employed selective solid-phase extraction by immunoaffinity CEC (IACEC) to enhance detection limits. A model compound, fluorescein isothiocyanate biotin, was electrokinetically applied to a capillary column packed with an immunoaffinity stationary phase. The analyte was first selectively bound to the stationary phase, then eluted, migrated by zone electrophoresis, and detected by LIF [79],... 

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