Generation of high-affinity rabbit polyclonal antibodies to the murine urokinase receptor using DNA immunization.

ABSTRACT

The urokinase receptor (uPAR) is a glycolipid anchored cell surface glycoprotein that plays a central role in extracellular proteolysis during tissue remodeling processes and cancer invasion. By intramuscular (i.m.) injection of rabbits with plasmid DNA coding for a carboxy-terminally truncated secreted form of the murine uPAR (muPAR), specific anti-sera with a titer of 64,000, as measured by ELISA, have been obtained. Rabbits received a total of 10 monthly injections of 1 mg DNA in phosphate-buffered saline. The antibody titer peaked between the 5th and 7th injection and slowly declined after the 8th injection. After the final immunization the immune response persisted for at least 6 months without further injections. The antibodies generated by DNA immunization were useful for immunohistochemistry and immunoblotting, recognizing the antigen both in its native and in its reduced and alkylated form. Using the antibodies in immunoblotting muPAR was identified in lysates of peritoneal macrophages, spleen and lung tissue. Both the intact and cleaved form of muPAR were identified in lysates of a murine monocyte cell line P388D.1. No cross-reaction with human uPAR was observed. In immunohistochemical analysis of normal mouse lung tissue uPAR immunoreactivity was located in the alveoli and pulmonary vessels, whereas the bronchial epithelium was negative. These results demonstrate that DNA immunization of rabbits using i.m. injection is a very effective and easy method to raise polyclonal antibodies which can be used for characterization and localization of muPAR in mouse tissue.