Anti-metatype antibodies in immunoassays

In this review anti-metatype antibodies are described invoking new principles in immunoassay development. Anti-metatype antibodies are immunological reagents specific for the conformation of the liganded antibody active site which do not interact with bound ligand or unliganded antibody. Relationships between anti-metatype antibody reactivity and the ligand-induced conformational state of monoclonal antibodies are reviewed with emphasis on the fluorescein hapten as a small molecule model system. One characteristic result of the interaction of anti-metatype antibodies with liganded antibodies is a significant delay in the dissociation rate (k₂) of the ligand bound within the primary immune complex. The latter is an important consideration for assay development. Polyclonal and monoclonal anti-metatype antibody reagents are characterized in terms of their differential effects on the ligand dissociation rate. Anti-metatype antibody reactivity is further discussed in terms of protein-protein specificity patterns and relative interactions with idiotype-family members, structural derivatives, and site-specific mutants. Incorporation of principles inherent in the anti-metatype concept and their application to assay development are summarized.Abbreviations D₂O deuterium oxide - Fab 50 kd antibody fragment containing V_HC_H1 + V_LC_L domains - FITC(I) fluorescein isothiocyanate (isomer I) - Fv 26 kd fragment of the antibody molecule containing the variable domains of the H and L chains - Ig immunoglobulin - IgG immunoglobulin G with a mol. wt. of 150 kd. - IgM immunoglobulin M with a mol. wt. of 10⁶d - Id idiotype - Ka antibody affinity (k₁/k₂) in M^–1 - k₁ second order rate of ligand association in M^–1s^–1 - k₂ first order rate of ligand dissociation in s^–1 - K_D dissociation constant or the reciprocal of the affinity constant (1/Ka) - Mab monoclonal antibody - Met metatype - NMR nuclear magnetic resonance - SCA single chain Fv derivative containing a synthetic linker between the two variable domains - V_H variable domain of the antibody H chain - V_L variable domain of the antibody L chain     

Development of a capillary electrophoretic method for the separation of diastereoisomers of a new human immunodeficiency virus protease inhibitor

A capillary electrophoretic (CE) method was developed for the separation of diastereoisomers of a new human immunodeficiency virus (HIV) protease inhibitor TMC114. In total 16 isomers of this drug have been synthesized (eight pairs of enantiomers). We succeeded in the separation of the eight diastereoisomers, but no enantiomers could be separated. Because of the high similarity and water-insolubility of these isomers, the separation is a real challenge. Different CE modes were tried out: capillary zone electrophoresis (CZE), nonaqueous capillary electrophoresis (NACE), micellar electrokinetic capillary chromatography (MEKC), and microemulsion electrokinetic capillary chromatography (MEEKC). Only MEEKC offered resolution of these compounds.     

A 13-membered crown ether with an azoxy subunit in the macrocycle: Synthesis and X-ray structure

The presented azoxy compound is an example of a new crown ether analogue. It has been synthesized by the reduction of an open chain dinitro compound with stannite under strongly alkaline conditions. A method for the separation of the azo and azoxy compounds formed simultaneously has been proposed. The structures of two crystallographically independent molecules of compound2 have been determined. In spite of the small size of the macroring in compound2, the phenyl residues around the azoxy group have atrans orientation. Supplementary Data relating to this article have been deposited with the British Library as Supplementary Publication No. 82157 (11 pages).     

Aggregation behavior of p-n-alkylbenzamidinium chloride surfactants

The aggregation behavior of a novel class of surfactants, p-n-alkylbenzamidinium chlorides, has been investigated. The thermodynamics of aggregation of p-n-decylbenzamidinium chloride mixed with cationic and anionic cosurfactants has been studied using isothermal titration calorimetry. For mixtures of p-n-decylbenzamidinium chloride with n-alkyltrimethylammonium chlorides, the aggregation process is enthalpically more favorable than for the pure n-alkyltrimethylammonium chlorides, probably caused by diminished headgroup repulsion due to charge delocalization in the amidinium headgroup. A critical aggregation concentration between 3 and 4 mM has been extrapolated for p-n-decylbenzamidinium chloride at 40 degrees C, around two times lower than that of similar surfactants without charge delocalization in the headgroup and well comparable to that of similar surfactants with charge delocalization in the headgroup. In mixtures of p-n-decylbenzamidinium chloride with either sodium n-alkylsulfates or sodium dodecylbenzenesulfonate, evidence is found for the formation of bilayer aggregates by the pseudo-double-tailed catanionic surfactants composed of p-n-decylbenzamidinium and the anionic surfactant. These aggregates are solubilized to mixed micelles by excess free anionic surfactant at the measured critical aggregation concentration.     

Studies on the Distribution of Phenoxyacetic Acid in Two-Phase Systems: <Emphasis Type="Italic">n</Emphasis>-Aliphatic Hydrocarbon – Water

Summary. Phenoxyacetic acid distribution in two-phase systems n-aliphatic hydrocarbon (C₅–C₈) – water and its dimerization in organic phase were investigated. The values of distribution coefficient (D _HR), distribution constant (K _D), and dimerization constant (K _dim) of acid were obtained. The empirical correlations of these quantities with Hildebrand solubility parameter of organic solvents were established. The influence of pH of the aqueous phase as well as the polarity of the applied organic solvents on phenoxyacetic acid physical chemistry in the two-phase systems was described.     

Mapping of possible prion protein self-interaction domains using peptide arrays

Background  

The common event in transmissible spongiform encephalopathies (TSEs) or prion diseases is the conversion of host-encoded protease sensitive cellular prion protein (PrP^C) into strain dependent isoforms of scrapie associated protease resistant isoform (PrP^Sc) of prion protein (PrP). These processes are determined by similarities as well as strain dependent variations in the PrP structure. Selective self-interaction between PrP molecules is the most probable basis for initiation of these processes, potentially influenced by chaperone molecules, however the mechanisms behind these processes are far from understood. We previously determined that polymorphisms do not affect initial PrP^C to PrP^Sc binding but rather modulate a subsequent step in the conversion process. Determining possible sites of self-interaction could elucidate which amino acid(s) or amino acid sequences contribute to binding and further conversion into other isoforms. To this end, ovine – and bovine PrP peptide-arrays consisting of 15-mer overlapping peptides were probed with recombinant sheep PrP^C fused to maltose binding protein (MBP-PrP).     

Simultaneous propane dehydrogenation andco2hydrogenation overCrOx/SiO2catalyst

Summary Single reverse water-gas shift (RWGS) and dehydrogenation of propane with CO₂(DH-CO₂) reactions in the presence and absence of the CrO_x/SiO₂ catalyst have been studied between 673 and 873 K. It was found that the CrO_x/SiO₂ catalyst is active both in the dehydrogenation of propane and in the RWGS reactions. The obtained results suggest that the dehydrogenation of propane to propene in the presence of CO₂on CrO_x/SiO₂can be facilitated by the RWGS reaction.</o:p>