Specific antibodies against binase: Preparation and application

Abstract

Secreted ribonuclease of Bacillus pumilus (binase) is a promising antitumor agent with established selective cytotoxicity against various types of cancer cells expressing specific oncogenes. However, molecular mechanisms behind the apoptosis-inducing action of binase remain unclear. It was shown that catalytic activity, protein charge, oligomerization, and interaction with RAS oncogene contribute to binase cytotoxicity. To identify the intracellular targets of binase antitumor activity, binase-specific molecular tools could be of great assistance. In this article, a specific polyclonal rabbit serum was obtained by rabbit immunization with the industrial binase preparation using four subcutaneous protein injections with Freund adjuvant and revaccination. Isolation and purification of antibodies against ribonuclease from the serum were performed using affinity chromatography. Affinity resin was prepared by the immobilization of binase in CNBr-activated Sepharose 4. The specificity of the serum and antibodies was confirmed by agar gel immunodiffusion assay and western blotting. It was demonstrated that the antibodies can be successfully applied for testing native and inactivated forms of binase, as well as binase within cell lysates and protein complexes. The obtained specific antibodies will help to reveal the localization of binase in eukaryotic cells. They will also be useful for determining binase interaction with surface and intracellular targets, including viral components.

Keywords

• ribonuclease
• binase
• antitumor activity
• obtaining of specific antibodies