Mouse IgG1 Lambda Isotype Control Antibody (Low Endotoxin, Azide-Free) Functional Grade

Purpose To evaluate the stability of a model Mab1 and Fab1 under in vitro vitreal conditions in the presence of various metabolites and in the presence of light at lambda > 400 nM. Methods A full length IgG1 monoclonal antibody (Mab1) and a fab fragment (Fab1) were formulated with various metabolites typically found in the vitreous humor and subjected to visible light treatment. Both proteins were analyzed using a variety of biochemical techniques. Furthermore, Fab1 was also tested for antigen binding ability using surface plasmon resonance. Results Our data shows that some vitreal metabolites such as riboflavin and ascorbic acid affect protein stability, via formation of reactive oxygen species (ROS) and advanced glycation end products (AGE) s respectively. In contrast, metabolites such as glutathione may protect these proteins from light-induced degradation to some extent. Conclusions Ascorbic acid and riboflavin were found to photosensitize therapeutic proteins especially when exposed to light. Ascorbic acid reacted with proteins even in the absence of light. Antioxidants such as glutathione helped limit photooxidation under ambient or blue light exposure. Since antioxidant capacity in the eye decreases with age we recommend understanding long term stability, including photooxidation and photosensitization, of new candidate proteins in the context of controlled or sustained release technologies for ocular diseases.

A group of six mouse monoclonal antibodies (MoAbs) with the unusual lambda-type light chain were generated by fusion of NS-1 cells with splenic cells derived from BALB/c mice immunized with crude extracts of Bermuda grass pollen (BGP). Four of them were IgG1, one was IgG2b, and one was IgG3. Binding inhibition assay showed that they recognized the same (or very similar) epitope. Using sera from BGP-allergic patients, it was found that the specific binding between the IgE antibodies and the MoAb 26-11-fixed antigen could be blocked by MoAb 26-11 itself and another MoAb 9-13 in a dose-dependent manner. It appears that the epitope recognized by the lambda-type MoAbs is a human IgE-binding antigenic determinant. Further physico-chemical analyses showed that this epitope was stable under heat but sensitive to treatments of sodium periodate and proteinase K. Results from these studies indicate that this unique epitope which leads to the generation of lambda-type MoAbs is part of a glycoprotein.