Rat X Mouse Hybridoma Secreting Monoclonal Antibody to Mouse Alpha-1-Protease Inhibitor: Development and Characterization

Abstract

<p>In our investigations concerning the control of synthesis of mouse alpha-1-protease inhibitor (α1Pi), also called alpha-1-antitrypsin (α1AT), a major inhibitor of serum proteases, we had reason to attempt to develop a monoclonal antibody to this molecule to clarify some apparent molecular heterogeneity, as conventional approaches to separate the isoproteins had met with limited success. We immunized Lewi rats with purified mouse α1Pi and fused the immune spleen cells with mouse SP2 plasmacytoma cells after optimizing some of the fusion parameters such as the source of polyethylene glycol (PEG), its concentration and exposure time to cells. In addition, we examined two different fusion protocols for their ability to produce xenogenic rat X mouse hybrid cells. Several hybridomas were produced, one in particular secreting rat IgM specific for mouse α1Pi. Screening of the positive clones was carried out by modified ELISA and radioimmunoassays. Characterization of the cell product was completed by SDS-PAGE and radioimmunoelectrophoresis. Specificity of the secreted immunoglobulin (D7-IgM) was determined using crossed radioimmunoelectrophoresis with ³⁵S-methionine labelled monoclonal antibody. The establishment of a mouse ascitic form of the hybridoma was not successful even after prolonged subcutaneous adaptation of the hybridoma line in ALS treated, bone marrow reconstituted, X-irradiated Balb/c recipients. Mice bearing the solid tumor exhibited pathology in the liver in the form of discrete necrotic lesions suggesting metastatic localization of the hybridoma in the liver and local production and interaction of the monoclonal antibody with the hepatocyte via one of its secreted molecules, alpha-1-protease inhibitor.</p>