Gel fiberglass as a new matrix of affinity chromatography columns for isolation of colon cancer-associated antigens

Gel fiberglass (GFG), a new affinity biosensor, was used to isolate human p53 antigen with rabbit anti-rat p53 IgG. The biosensor was prepared as a membrane from glass fibers covered with oxysilanes. A thin layer of protein, trapped in gel glass during its preparation, is deposited on the surface of a lattice of glass fibers. In such conditions, a maximum number of protein molecules may contact external agents percolated through a membrane. The membranes demonstrate high stability and can be stored in dry conditions or several months at room temperature. Columns for affinity chromatography were prepared from the GFG membranes and were used to isolate various proteins, including the tumor-associated antigens (TAA). The capacity of such columns was calculated as the amount mg of protein isolated from 1 ml of TAA-containing serum. In colon cancer patients, up to 5-6 mg TAA were extracted from 1 mi of sera. Two main components of cytoplasmic TAA isolated in our experiments were p64 and p53 proteins. Their concentration was determined by HPLC. The p53 protein has been isolated from the serum of cancer patients in the highest concentration yet reported, up to 3-4 mg/ml. In our previous studies, isolation of p53 protein was based on its affinity reaction with anti-p53 IgG generated against antigens of the same species. Herein, we report for the first time the capability to isolate human p53 antigen using GFG columns with entrapped anti-rat p53 IgG. Blood levels of p53 antigen isolated were very similar in both experiments. This has both theoretical and practical significance, demonstrating that the GFG membranes have great potential for isolating macromolecules utilizing various ligands. The finding facilitates an easy and highly effective method to isolate antigens from different organs, both animal and human, which can be used for important goals including diagnosis, therapy and generation of specific antibodies.