Epitope specificity of two anti-morphine monoclonal antibodies: In vitro and in silico studies.

Monoclonal antibodies (mAbs) against morphine are important in the development of immunotherapeutic and diagnostic methods for the treatment and prevention of drug addiction. By the surface plasmon resonance (SPR) and enzyme immunoassay techniques, we characterized two previously obtained mAbs 3K11 and 6G1 and showed their ability to recognize free morphine and morphine-containing antigens in different ways because of the epitope specificity thereof. Using the defined amino acid sequences, we obtained three-dimensional models of the variable regions of Fab fragments of these antibodies and compared them with the known sequence and spatial structure of the anti-morphine antibody 9B1. Docking simulations are performed to obtain models of the antibodies complexes with morphine. Differences in the models of 3K11 and 6G1 complexes with morphine correlate with their experimentally detected epitope specificity. The results, in particular, can be used for the structure-based design of the corresponding humanized antibodies. According to our modeling and docking results, the very different modes of morphine binding to mAbs 3K11 and 6G1 are qualitatively similar to those previously reported for cocaine and two anti-cocaine antibodies. Thus, the obtained structural information brings more insight into the hapten recognition diversity.

Chromatography under critical conditions: An analogy between functionalized and partially cyclic polymers.

An analogy is established between functionalized polymers and partially cyclic macromolecules (PCMs) in the liquid chromatography at critical conditions (LCCC). Application of the functionalized chain analogy (FCA) for prediction of the behavior of complex multi-cyclic PCMs in the LCCC mode is demonstrated. By using FCA, we discuss possibilities of LCCC to separate multi-cyclic PCMs by the number of cycles, and with respect to molecular topology. FCA is also extended to describe PCMs with specifically adsorbing groups; this results in a simplified theory of LCCC of functionalized PCMs. By simulating chromatograms of heterogeneous functionalized PCMs at the conditions of LCCC, we show possible dramatic effects of functional groups on the topological separation of PCMs: even the retention order of components may change to opposite.

Theory of chromatography of partially cyclic polymers: Tadpole-type and manacle-type macromolecules.

A theory of chromatography is developed for partially cyclic polymers of tadpole- and manacle-shaped topological structures. We present exact equations for the distribution coefficient K at different adsorption interactions; simpler approximate formulae are also derived, relevant to the conditions of size-exclusion, adsorption, and critical chromatography. Theoretical chromatograms of heterogeneous partially cyclic polymers are simulated, and conditions for good separation by topology are predicted. According to the theory, an effective SEC-radius of tadpoles and manacles is mostly determined by the molar mass M, and by the linear-cyclic composition. In the interactive chromatography, the effect of molecular topology on the retention becomes significant. At the critical interaction point, partial dependences K(Mlin) and K(Mring) are qualitatively different: while being almost independent of Mlin, K increases with Mring. This behavior could be realized in critical chromatography-for separation of partially cyclic polymers by the number and molar mass of cyclic elements.

Two-dimensional liquid chromatography of diblock copolymers: simulation at various adsorption interaction conditions.

Diblock copolymers, which are heterogeneous in both molar mass and composition, can be fully characterized by using two-dimensional chromatography. Since the size-exclusion, the adsorption, and the critical interaction based modes of chromatography are possible for each of the polymers A and B, this leads to a variety of options for 2D-chromatography of copolymers AB. Using the theory of chromatography of block copolymers, 2D-chromatograms are simulated that correspond to the most interesting of these options. Orthogonal 2D-chromatograms are expected, if in the 1st dimension the critical condition is created for A, while in the 2nd dimension--for B. The situations, where A and B are both adsorbable, as well as those where the conditions of adsorption for A and SEC for B are created, are also considered. In particular, it is shown that the 2nd dimension combination of the critical condition for A and SEC--for B is preferable than that with SEC condition for both A and B. The simulated 2D-chromatograms of low- and high molar mass diblock copolymers, as well as of copolymers with one short block are compared with the reported real ones; it is concluded that the corresponding virtual and real 2D-chromatograms are qualitatively very similar.

Calibration of chromatographic systems for quantitative prediction of chromatography of homopolymers.

A method is proposed for obtaining from the experimental data the main parameters, which determine the chromatographic behavior of homopolymers: a dependence of the adsorption interaction parameter on the mobile phase composition, and the pore volume and pore size values. The interaction parameter can be determined by this method in both adsorption, critical, and SEC-type regimes of chromatography. The method is tested in 'mathematical experiments' with theoretical data that mimic experiments, and is applied to calibrate real chromatographic systems. With the obtained calibrations quantitative modeling of chromatograms of PEG samples on two typical ODS columns with different pore size in methanol/water and acetone/water mixed solvents is performed; the simulation results are compared with real chromatograms.

Liquid chromatography of theta-shaped and three-armed star poly(tetrahydrofuran)s: theory and experimental evidence of topological separation.

A theoretical background is provided for the methods of the analysis and topological separation of complex macrocyclic polymers by means of size exclusion chromatography (SEC), liquid adsorption chromatography, and liquid chromatography at the critical condition. We focus on a particular problem of the separation of theta-shaped polymers from its three-armed star analogues. Based on the theory, we simulate chromatograms for model mixtures of polydisperse theta- and star-polymers and analyze conditions for good separation of these polymers by topology. A theory is compared with the behavior of star- and theta-shaped poly(THF)s in size exclusion and interactive chromatography, and a good qualitative agreement between theory and experiment is observed. In particular, it is shown that in both SEC and interactive chromatography theta-polymers elute after the corresponding stars of the same molar mass. According to theory and experiment, chromatography under the critical and near-critical interaction conditions is especially promising for the separation of polydisperse macrocyclic polymers from their linear or branched analogues.

Theory of chromatographic separation of linear and star-shaped binary block-copolymers.

Equations for the distribution coefficient of heteroarm stars are derived by using a model of an ideal chain in a slit-like pore; these equations together with those previously reported for linear block-copolymers are applied to describe chromatography of such copolymers. According to the theory, the retention generally depends on molar mass, composition, and architecture (microstructure and topology) of copolymers, on pore size and on adsorption interaction of chain units A and B. Three special modes of chromatography are studied in detail. (i) If interactions for A and B are close to the critical point of adsorption (CPA), the retention practically does not depend on architecture, and high molar mass copolymers can be separated by composition. (ii) At SEC condition for B and strong adsorption for A, copolymers in principle can be separated by architecture; better separation is expected in wide pores. Retention of linear block-copolymers decreases with increasing of the number of blocks; for heteroarm stars the theory predicts retention decreasing as: AB > StarAAB > StarABB; StarAAAB > StarABBB > StarAABB; StarAAAAB > StarABBBB > StarAAABB > StarAABBB. (iii) At the CPA for B copolymers AB, BAB and heteroarm stars regardless molar mass of B, M(B), can be separated by M(A). The same is true for ABA and ABAB...A in narrow pores. While the retention of AB, BAB, Star AB...B and StarAAB...B is the same, copolymers AB, ABA and linear multiblock-copolymers can be separated, as well as symmetric and very asymmetric triblock-copolymers ABA.