Single-chain variable antibody fragments (scFvs) are molecules with immense therapeutic and diagnostic potential. Knowledge of their three-dimensional structure is important for understanding their antigen-binding mode as well as for protein-engineering approaches such as antibody humanization. A major obstacle to the crystallization of single-chain variable antibody fragments is their relatively poor homogeneity caused by spontaneous oligomerization. A new approach to optimization of the crystallizability of single-chain variable antibody fragments is demonstrated using a representative single-chain variable fragment derived from the anti-CD3 antibody MEM-57. A Thermofluor-based assay was utilized to screen for optimal conditions for antibody-fragment stability and homogeneity. Such an optimization of the protein storage buffer led to a significantly improved ability of the scFv MEM-57 to yield crystals.

• Klíčová slova
• Thermofluor assay, crystallizability optimization, crystallization, differential scanning fluorimetry, oligomerization, single-chain antibody fragment,
• MeSH
• gelová chromatografie MeSH
• jednořetězcové protilátky chemie   MeSH
• krystalizace * MeSH
• lidé MeSH
• molekulární sekvence - údaje MeSH
• sekvence aminokyselin MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• jednořetězcové protilátky MeSH

This study compares the toxic effects of native cyclosporia A (CyA) with those of targeted CyA that is conjugated with the anti-rat-thymocyte antibody of rabbit origin via the N-(2-hydroxypropyl)methacrylamide (HPMA) carrier bearing digestible, reactive oligopeptide side chains. Ten toxic doses of native CyA (50 mg/kg i.p.) given to young adult rats in the course of 14 d produced a severe renal lesion-diffuse microvacuolization of the proximal tubules in the deep cortex, and hypergranulation of juxtaglomerular regions. Severe atrophy of the thymic medulla was documented by morphometry. In the cortex the epithelial reticular (but not deep interdigitating) cells showed ultrastructural signs of severe degeneration and lysis. The immature CD4+8+ double-positive cortical lymphocytes were preserved whereas the single-positive medullary thymocytes were greatly depleted; there was also a restriction of MHC class II antigen expression in the medulla. The number of medullary B cells was increased. The cytokeratin net was focally shrunken in the cortex and almost negative in the medulla, with loss of Hassall's corpuscles. After ten corresponding doses of antibody-targeted conjugated CyA no damage to the renal tubules and arterioles appeared and the antiGBM or immune-complex deposition was absent. The thymus had a normal medulla with numerous mature thymocytes and the cortical epithelial reticulum remained well preserved. Thus, the main toxic effects of CyA could be eliminated by targeting. The T-cell-targeted drug was tested for preserved immunosuppressive properties and non-toxic character of HPMA copolymer carrier.

• MeSH
• cyklosporin toxicita   MeSH
• cyklosporiny toxicita   MeSH
• imunokonjugáty toxicita   MeSH
• imunosupresiva toxicita   MeSH
• krysa rodu Rattus MeSH
• ledviny účinky léků   MeSH
• nosiče léků toxicita   MeSH
• potkani Wistar MeSH
• T-lymfocyty účinky léků   imunologie   MeSH
• thymus účinky léků   MeSH
• zvířata MeSH
• Check Tag
• krysa rodu Rattus MeSH
• mužské pohlaví MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• srovnávací studie MeSH
• Názvy látek
• cyklosporin MeSH
• cyklosporiny MeSH
• imunokonjugáty MeSH
• imunosupresiva MeSH
• nosiče léků MeSH