Antibodies, proteolytic activity

The classic pathway is triggered by the formation of factor Cl at IgG or IgM on the surface of microorganisms (left). Cl is an 18-part molecular complex with three different components (Clq, Clr, and Cls). Clq is shaped like a bunch of tulips, the flowers of which bind to the Fc region of antibodies (left). This activates Clr, a serine proteinase that initiates the cascade of the classic pathway. First, C4 is proteolytically activated into C4b, which in turn cleaves C2 into C2a and C2b. C4B and C2a together form C3 convertase [1], which finally catalyzes the cleavage of C3 into C3a and C3b. Small amounts of C3b also arise from non-enzymatic hydrolysis of C3. 

Pores formed by PFTs are usually fairly well defined in size, so that judicious choice of toxin and of experimental conditions will generally enable one to produce stable membrane lesions ranging from approximately 8 A to 300 A in functional diameter. It becomes possible to manipulate the intracellular ionic milieu, to introduce small molecules such as nucleotides into the cells, or to apply large molecules such as antibodies to the cytoplasm. Since pure preparations of PFT are devoid of enzymatic (proteolytic) activity, they cause no direct alterations of cell constituents. In the following, simple strategies for utilization of PFTs will be outlined. 

Scheohter, I., Schechter, B., and Sela, M., Combining sites of antibodies with L-alanine and n-alanine peptide specificity and the effect of serum proteolytic activity on their estimation. Biochim. Biophys. Ada 127, 438-456 (1966). 

In the ellipsometer, plasma even when diluted about 150-fold, deposits immunologically identifiable fibrinogen but the film loses its ability to adsorb antibody to fibrinogen within minutes under these conditions. The loss is probably not caused by the plasma s proteolytic activity (18) and occurs more slowly at IOC than at 37C (19). At room temperature, undiluted plasma deposits fibrinogen within about 2 sec and the loss occurs within 30 sec. 

Cell culture products, for example, monoclonal antibodies, are mostly secreted into the medium. Therefore, separation of cells as solid particles from the medium containing the desired product is comparatively simple, as it does not require any cell disruption. The product itself often has a complex structure, especially in case of glycosylated proteins, and high sensitivity against temperature, shear forces, extreme pH, or any proteolytic activity. Product concentrations in cell culture processes were regarded as low ( lgl ) for a long time due to low cell... 

Purification entails use of an immunoaffinity column containing immobilized murine antifactor VII antibody. It is initially produced as an unactivated, single-chain 406 amino acid polypeptide, which is subsequently proteolytically converted into the two-chain active factor Vila complex. After sterilization by filtration, the final product is aseptically filled into its final product containers, and freeze-dried. 

Purification entails use of an immunoaffinity column containing immobilized murine antifactor VII antibody. It is initially produced as an unactivated, single chain 406 amino acid polypeptide, which is subsequently proteolytically converted into the two-chain active factor Vila complex. After sterilization by filtration, the final product is aseptically filled into its final product containers and freeze-dried. The excipients present in the product include sodium chloride, calcium chloride, polysorbate 80, mannitol and glycylglycine. When freeze-dried in the presence of these stabilizing substances and stored under refrigerated conditions, the product displays a shelf-life of at least 2 years. It has proved effective in the treatment of serious bleeding events in patients displaying anti-factor VIII or IX antibodies. 

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