Art ides Anal. Chem. 1994,66, 9-15
Affinity Probe Capillary Electrophoresis: Analysis of Recombinant Human Growth Hormone with a Fluorescent Labeled Antibody Fragment Klyohlto Shlmurat and Barry L. Karger' Barnett Institute, Northeastern University, Boston, Massachusetts 02 1 15 A new highly sensitive microscale analytical procedure called affinity probe capillary electrophoresis (APCE) is presented. One of the two species, which can form a biospecific complex, is labeled with a fluorescent dye. The affinity probe is used to detect the analyte as a complex after the separation of the excess free probe by capillary electrophoresis. As an example of this approach, an FAB' fragment of a mouse monoclonal antibody (anti-human growth hormone) was labeled with tetramethylrhodamine-iodoacetamideat a hinge region thiol group. Samples were mixed with the purified labeled antibody fragment, and the associated complex was separated by capillary isoelectric focusing with detection by laser-induced fluorescence. In these preliminary results, methionyl recombinant human growth hormone (met-rhGH) could be successfully determined down to detection levels of -5 X 10-l2M. Mono- and dideamidated variants of met-rhGH were detected simultaneously with the nondeamidated form of the antigen as separate peaks.
Capillary electrophoresis (CE) is rapidly becoming an important procedure for the analysis of biological molecules.'92 In conjunction with laser-induced fluorescence detection, detection limits of 10-19-10-20mol (lO-lo-lO-ll M) or lower of a fluorescent labeled analyte are possiblea3 However, at such levels, the analysis of real samples, which often requires covalent labeling of analyte with fluorescent dyes, can be problematic due to nonquantitative derivatization in complex sample matrices, as well as detector interferences resulting from an excess of the labeling reagent. In contrast to covalent derivatization, complex formation based on biospecific recognition has traditionally been used in complex systems, as witnessed by the importance of immunoassay methods. Changes in the electrophoretic pattern as a result of biospecificcomplexationhave attracted a great deal of interest * Author to whom correspondence should be addressed. t On leave from Faculty of Pharmaceutical Sciences, Teikyo University,
Sagamiko, Kanagawa 199-01, Japan. (1) Karger, B. L.; Foret, F. In Capillary Electrophoresis Technology; Guzman, N. A., Ed.; Marcel Dekker: New York, 1993; pp 3-64. (2) Landers, J. P.;M a , R. P.; Spelsberg, T. C.; Nolan, J. A,; Ulfelder, K. J. BioTechniqves 1993, 14, 98-1 11. (3) Cheng, Y.-F.; Dovichi, N. J. Science 1988,242, 562-564. 0003-2700/94/0366-0009$04.50/0 0 1993 American Chemical Society
as forms of affinity electrophoresisk7or, more recently, affinity capillary electrophoresis (ACE) .8-12 Mobility shift assays are also based on this principle.13 CE can detect small changes in electrophoretic behavior upon complexation which might otherwise be difficult to observe by conventional slab gel electrophoresis. Furthermore, free-solution CE can in principle utilize biospecific binding directly, without the disadvantages of nonspecific interactions with insoluble matrices. On-line quantitation, microscale analysis, and automatic instrumentation are also important features of ACE. Fluorescent labeling of one of the interacting molecules in a biospecific complex can provide a sensitive probe for trace determination of an analyte. Any CE mode of separation can be used; however, with respect to low detection limits, isoelectric focusing (IEF)14 and isotachophoresis (ITP) followed by CZE15J6 are especially attractive because of simultaneous concentration and separation during analysis. The use of labeled reagents should be applicable to any type of biospecific interaction, including enzyme-inhibitor, enzyme-regulator protein, lectin
