Nenad M. Grubor 1 Daniel W. Armstrong 1, 2 Ryszard Jankowiak 1, 3 1 Ames Laboratory – USDOE, Department of Chemistry, Iowa State University, Ames, Iowa, IA, USA 2 Department of Chemistry and Biochemistry, University of Texas at Arlington, Arlington, TX, USA, 3 Department of Chemistry, Kansas State University, Manhattan, KS, USA Received September 6, 2005 Revised November 21, 2005 Accepted November 22, 2005 Research Article Flow-through partial-filling affinity capillary electrophoresis using a crossreactive antibody for enantiomeric separations It is demonstrated that the separation of diastereoisomers and enantiomers can be accomplished by the flow-through partial-filling affinity CE using a crossreactive mAb. This approach revealed differences in the binding strength of the (6)-cis- and (6)-trans-benzo[a]pyrene tetrols with the anti-polycyclic aromatic hydrocarbon mAb and demonstrated that (1)-enantiomers are more strongly immunocomplexed than their (2)-counterparts. It is proposed that crossreactive monoclonal antibodies (i.e. mAb raised against achiral molecule and possessing limited selectivity) could be effectively utilized for specific stereoisomeric differentiation and chiral separations. Keywords: Affinity capillary electrophoresis / Benzo[a]pyrene tetrols / Chiral separa- tions / Crossreactive antibodies / Enantiomers DOI 10.1002/elps.200500660 1 Introduction Antibody–antigen interactions are among the most spe- cific molecular recognition phenomena in nature [1, 2]. Interactions responsible for this selective binding are hydrogen bonds and electrostatic and hydrophobic inter- actions. Because of the chiral nature of proteins, it is possible for enantiomers to interact with an antibody in a qualitatively (i.e. different complex geometry) and/or quantitatively (i.e. with different binding strength) different fashion [3]. However, examples of nonspecific (crossreactive) anti- bodies are numerous. Structural and mechanistic studies have revealed two distinct mechanisms that are respon- sible for the crossreactive interaction of antibodies: (1) antibody conformational diversity, i.e. the pre-existing equilibrium of antibody conformers, each capable of binding structurally different antigens [4, 5] and (2) induced fit mechanism, i.e. the flexibility of an antibody binding pocket, usually operable for crossreactivity in the case of shared ligand chemistry (molecular mimicry) [6, 7]. However, promiscuous antibodies can exhibit different degrees of heterospecificity [8], that is, broad specificity within a family of similar molecules, but significant inter- action with unrelated antigen(s) [9]. Our recent work has been focused on developing appli- cations of crossreactive antibodies for highly specific analyses using multidimensional spectroscopic detec- tion, primarily high-resolution, fluorescence line-narrow- ing spectroscopy (FLNS) [10, 11]. We have demonstrated that the highly promiscuous anti-PAH mAb can be used for capturing structurally related molecules, such as fluoranthene, pyrene, benzo[a]pyrene, as well as diaster- eomeric benzo[a]pyrene tetrols (BPTs) [12]. FLNS detec- tion is used to directly probe the molecules immunocom- plexed with the antibody, enabling their unambiguous positive identification. Moreover, we have shown that geometrically different interactions of stereoisomeric cis- BPT and trans-BPT with anti-PAH mAb can be spectro- scopically distinguished allowing for chiral differentiation (in preparation). Protein–ligand, as well as protein–protein interactions are studied by various chromatographic and electrophoretic methods, predominantly HPLC and CE using various operational modes: zonal elution, frontal analysis, vacancy methods, etc. [13, 14]. These methodologies are capable of revealing the information on the binding con- stants and number of binding sites for a solute–protein system, as well as the thermodynamic parameters, rate Correspondence: Professor Ryszard Jankowiak, Department of Chemistry and Terry C. Johnson Center for Basic Cancer Research Kansas State University, Manhattan, KS 66502, USA E-mail: ryszard@ksu.edu Fax: 11-785-532-6666 Abbreviations: BPT , benzo[a]pyrene tetrol; cis-BPT , r -7,t-8,t-9,t-10- tetrahydroxy-7,8,9,10-tetrahydrobenzo[a]pyrene; FTPFACE, flow- through partial-filling affinity CE; trans-BPT , r -7,t-8,9,c-10-tetrahy- droxy-7,8,9,10-tetrahydrobenzo[a]pyrene 1078 Electrophoresis 2006, 27, 1078–1083  2006 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim www.electrophoresis-journal.com