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ELISA (Enzyme-linked ImmunoSorbent Assay), as one possible application of antibody-based methods, has proved to be a good candidate for the determination of proteins in works of art due to the low limit of detection, high sensitivity (in the nanogram range), specificity and micro invasiveness of required sampling. Despite the advantages of this method, interference by metal ions present in pigments, lipids, saccharides and in other components of paints, grounds, and varnishes may cause several problems for incontestable identification of proteins. To overcome the drawbacks of ELISA, a novel way to purify the extracted mixtures to predominantly protein fractions, by the DEAE-modified (weak anion exchange) CIM® (Convective Interaction Media) chromatographic monoliths was introduced. Utilisation of monolithic chromatographic supports greatly enhanced the ELISA detection of ovalbumin in the model paint samples (aged and non-aged), for at least one tenfold dilution of a sample. In several cases the purification enabled detection of the target protein, otherwise not possible with non-purified samples. For the first time in the field of Cultural Heritage, the combined approach of ELISA and CIM® monolith-supported extraction was successfully tested also for the detection of protein in real-case paint layers. [ABSTRACT FROM AUTHOR]

Three corrosion inhibitors for copper-3-amino-1,2,4-triazole (ATA), benzotriazole (BTAH), and 1 -hydroxybenzotriazole (BTAOH)-were investigated by corrosion experiments and atomistic computer simulations. The trend of corrosion inhibition effectiveness of the three inhibitors on copper in near-neutral chloride solution is determined experimentally as BTAH ≳ ATA ⪢ BTAOH. A careful analysis of the results of computer simulations based on density functional theory allowed to pinpoint the superior inhibiting action of BTAH and ATA as a result of their ability to form strong N-Cu chemical bonds in deprotonated form. While these bonds are not as strong as the Cl-Cu bonds, the presence of solvent favors the adsorption of inhibitor molecules onto the surface due to stronger solvation of the Cl anions. Moreover, benzotriazole displays the largest affinity among the three inhibitors to form intermolecular aggregates, such as [BTA-Cu]n polymeric complex. This is another factor contributing to the stability of the protective inhibitor film on the surface, thus making benzotriazole an outstanding corrosion inhibitor for copper. These findings cannot be anticipated on the basis of inhibitors' molecular electronic properties alone, thus emphasizing the importance of a rigorous modeling of the interactions between the components of the corrosion system in corrosion inhibition studies. [ABSTRACT FROM AUTHOR]

• Multimodal metal affinity chromatography separated empty and full AAV capsids. • The method employed a positively charged metal affinity ligand. • Separation was observed with many immobilized metal ion species. • Retention of empty capsids was affected more than full capsids. • Different metal ion species bound different contaminant subsets. Separation of empty and full adeno-associated virus capsids by multimodal metal affinity chromatography was investigated using a positively charged metal affinity ligand. A subpopulation of empty capsids eluted first, followed by full capsids, and later by more empty capsids and debris. Empty and full capsid composition of chromatography fractions was evaluated by cesium chloride density gradient centrifugation followed by stratigraphic flow analysis of the centrifuge tube contents, monitored by intrinsic fluorescence. Columns charged with barium, calcium, magnesium, zinc, manganese, and ferric ions gave similar results with respect to capsid separation. Charging with cupric ions maintained resolution between early-eluting empty capsids and full capsids but caused them to elute at lower conductivity. Empty and full capsids were fractionated with Tris-borate gradients, sodium chloride gradients, and magnesium chloride gradients. Recovery of full serotype 9 capsids was 100% with complete elimination of empty capsids. All metal ions bound contaminant subsets that required sodium hydroxide for removal. Columns charged with ferric iron and manganese bound more contaminants than all other metals. Columns charged with calcium, magnesium, barium, and copper bound the least. Contaminant binding on zinc-charged columns was intermediate between the two groups. [ABSTRACT FROM AUTHOR]

HPLC is established as a fast convenient analytical technology for characterizing the content of empty and full capsids in purified samples containing adeno-associated virus (AAV). UV-based monitoring unfortunately over-estimates the proportion of full capsids and offers little value for characterizing unpurified samples. The present study combines dual-wavelength UV monitoring with intrinsic fluorescence, extrinsic fluorescence, and light-scattering to extend the utility of HPLC for supporting development of therapeutic AAV-based drugs. Applications with anion exchange (AEC), cation exchange (CEC), and size exclusion chromatography (SEC) are presented. Intrinsic fluorescence increases sensitivity of AAV detection over UV and enables more objective estimation of empty and full capsid ratios by comparison of their respective peak areas. Light scattering enables identification of AAV capsids in complex samples, plus semiquantitative estimation of empty and full capsid ratios from relative peak areas of empty and full capsids. Extrinsic Picogreen fluorescence enables semiquantitative tracking of DNA with all HPLC methods at all stages of purification. It does not detect encapsidated DNA but reveals DNA associated principally with the exteriors of empty capsids. It also enables monitoring of host DNA contamination across chromatograms. These enhancements support many opportunities to improve characterization of raw materials and process intermediates, to accelerate process development, provide rapid in-process monitoring, and support process validation. [ABSTRACT FROM AUTHOR]