Tag Archives: phage display

Comparative characteristic of the methods of protein antigens epitope mapping

O. Yu. Galkin

National Technical University of Ukraine “Kyiv Polytechnic Institute”;
e-mail: alexfbt@mail.ru

Comparative analysis of experimental methods of epitope mapping of protein antigens has been carried out. The vast majority of known techniques are involved in immunochemical study of the interaction of protein molecules or peptides with antibodies of corresponding specifici­ty. The most effective and widely applicable metho­dological techniques are those that use synthetic and genetically engineered peptides. Over the past 30 years, these groups of methods have travelled a notable evolutionary path up to the maximum automation and  the detection of antigenic determinants of various types (linear and conformational epitopes, and mimotopes). Most of epitope searching algorithms were integrated into a computer program, which greatly facilitates the analysis of experimental data and makes it possible to create spatial models. It is possible to use comparative epitope mapping for solving the applied problems; this less time-consuming method is based on the analysis of competition between different antibodies interactions with the same antigen. The physical method of antigenic structure study is X-ray analysis of antigen-antibody complexes, which may be applied only to crystallizing­ proteins, and nuclear magnetic resonance.

Recombinant single chain variable fragment antibodies (scFv) against Pro(144)-Leu(155) fragment of human protein C

O. S. Oliinyk, K. O. Palyvoda, N. E. Lugovskaya,
D. V. Kolibo, E. V. Lugovskoy, S. V. Komisarenko

Palladin Institute of Biochemistry, National Academy of Sciences of Ukraine, Kyiv;
e-mail: lenaoliinyk@mail.ru

The aim of this work was to obtain the recombinant single chain variable fragments of antibodies (scFv) against human protein C, the key component of blood anticoagulation system. For this purpose a peptide that mimics a Pro144-Leu155 sequence of protein C was synthesized and the murine immune scFv library against this peptide was constructed. The protein C specific scFv 9E were selected from the constructed library by the phage-display method. The scFv 9E dissociation constant was found to be 2∙10-9 М. It was shown that scFv 9E were suitable for protein C detection by ELISA and Western blotting. Selected scFv could be further used for protein C investigation and for the development of quantitative methods for protein C detection in human blood.