Tag Archives: epitope mapping

New monoclonal antibodies to the Chlamydia trachomatis main outer membrane protein and their immunobiological properties

O. Yu. Galkin1,2, O. B. Besarab1, Yu. V. Gorshunov1, O. M. Ivanova3

1National Technical University of Ukraine “Igor Sikorsky Kyiv Polytechnic Institute”;
e-mail: alexfbt@gmail.com;
2Propharma Plant Ltd., Kyiv;
3Xema Ltd., Kyiv

Received: 18 July 2018; Accepted: 14 March 2019

One of the methods that have been widely used in the diagnosis of urogenital chlamydia is an enzyme-linked immunosorbent assay (ELISA), the use of which allows for differential diagnosis. In order to increase the efficiency of ELISA test kits production, for the kits for the diagnosis of urogenital chlamydia, based on the principle of indirect modification, following synthetic positive controls (PCs) can be used: a conjugate of IgM (IgA) normal immunoglobulins and monoclonal antibodies (McAbs) to C. trachomatis major outer membrane protein (MOMP). The goal of this work was to obtain high active and affinity McAbs to the C. trachomatis MOMP as well as the study of its immunobiological properties which are important for future biochemical approaches. The study was conducted using: polyclonal antibodies (PcAbs) to C. trachomatis; recombinant major outer membrane protein (MOMP) (191-354 a.r.; W4-W5); epitope mapping based on phage display technology. The original set from 16 clones of hybridomas, producers of McAbs to the C. trachomatis MOMP has been obtained. More than half of the tested McAbs (8 out of 14) were characterized by a rather high titer (≥1:800), and three of them had a titer of ≥1:1600. In general, the McAbs titer was correlated with the value of the affinity constant: McAbs with higher titles were characterized by a high value of the affinity constant. For McAbs with a titer of <1:800, the average Ka is 5.2×109 M-1, while for McAbs with a titer ≥1:800 – Ka = 10.7×109 M-1. Antigenic determinants of two McAbs 293F4 and 291F8 that actively competed with PcAbs are represented by two linear sequences of 320-325 a.r. and 326-330 a.r., respectively. The epitope, which interacts with McAb 296G2, is represented by a linear sequence of 347-352 a.r. McAb 296G2 did not show active competition with serum PcAbs. The resulting set of data allows selecting McAbs for use in PCs of the ELISA kit for the detection of IgA or IgM antibodies to C. trachomatis.

Obtaining and study of properties of new monoclonal antibodies against human IgE

O. Yu. Galkin1,2, A. A. Savchenko1, K. I. Nikitina1, O. M. Dugan1

1National Technical University of Ukraine “Kyiv Polytechnic Institute”;
2Hema Ltd., Kyiv, Ukraine;
e-mail: alexfbt@mail.ru

The original set of 12 clones of hybridomas, producers of monoclonal antibodies (mAbs) against human IgЕ, has been obtained. The study of biological properties of antibodies has been conducted: their specificity, affinity constant and titer in a cultural medium have been established. The obtained mAbs are directed to the two epitope regions on IgE molecule. The first group of mAbs relates to epitope region, represented by three epitopes (two of them overlap and one that does not overlap with others). The second epitope region is represented by only one epitope.

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.