Photomodification of antibodies for radioimmunotherapy and western blot analysis.

摘要

Monoclonal antibodies are used as delivery vehicles for carrying radioactive compounds in vivo. Photolabeling of antibodies is a gentle way of modification of antibodies. Photomodified antibodies can be used for various purposes like radioimmunotherapy and western blot analysis useful in detection of proteins.; CC49 antibody was photolabeled under different conditions to obtain the optimum photolabeling conditions, and they were 20 mM, pH 8.0 Tris buffer with one minute photolysis in presence of 15μM [γ32P]2 N₃ATP. Also experiments were conducted to analyze the main hypothesis as to whether antigen recognition is affected by changing the ratio of photolabeling into the heavy chain versus light chain, and it was proved that photolabeling ratios into heavy chain versus light did not affect the antigen recognition.; An efficacious single antibody radioimmunoassay to detect glutamine synthetase in human lumbar cerebrospinal fluid samples was accomplished. Optimum conditions for western blot analysis of glutamine synthetase present in cerebrospinal fluid were developed which were found to be 12.5 μM [γ32p]2-N ₃ATP in presence of 20 mM phosphate buffer of pH 7.0. The blot was incubated with this photolabeled antibody in a 1:5000 dilution to get optimum results.; The importance of this technique lies in the fact that this methodology can be used for detection of various important antigens for diagnostic purposes in case of many neurological and other diseases.; Thermostable proteins are stabilized by various mechanisms of which oligomeric integrity is an important one. To understand the molecular mechanism of thermostability in glucose-6-phosphate dehydrogenase enzyme from the thermophile Aquifex aeolicus, computer modeling was performed to predict which of the cysteines present on the subunit interface have a possible role in disulfide bond formation. Site directed mutagenesis was performed on the enzyme to convert cysteines 184 and 352 to serines, and the resulting effect was dramatic with the half life of the enzyme decreasing from 24 hours to 3 hours at 90°C. This result proves that intersubunit interactions and oligomerization are very important for the thermostability of this protein.