Quantitative Immunoblotting with Fusion Proteins

The development of vaccines against HIV-1 is currently hindered by incomplete understanding of correlates of protective immunity (1 –3 ). Experiments are necessary to measure immune responses in sufficiently fine detail that specific protective responses can be discerned from those that are irrelevant or harmful. When vaccines are tested as potential therapeutics, it is further necessary to differentiate induced responses from those associated with the infection itself. Measurement of humoral responses to well-defined antigens particularly lends itself to detailed mapping (4 ). Small synthetic antigens may be used in ELISA or BIAcore assays (5 –7 ). Larger antigens, such as fusion proteins, may require assays with more specificity, because of the possibility of immune-reactive contaminants. A particularly useful technique in this context is immunoblotting, because contaminating antigens are separated away during the electrophoresis step (8 ,9 ). In the authors’ laboratory, immunoblots employing fusion proteins of HIV-1 envelope sequences have been successfully used to quantitate new responses post immunization with a vaccine candidate in spite of a substantial baseline response to the whole antigen (10 ,11 ). The same technique was used to measure responses against vaccine candidates in small animal models (12 ,13 ).