Tag Archives: ELISA

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.

Specificity and sensitivity of the new test for serological evaluation of tuberculosis using MPT83-MPT63 fusion antigen

A. A. Siromolot1,2, T. O. Chudina2, I. S. Danilova3,
O. M. Rekalova4, D. V. Kolybo1,2, S. V. Komisarenko2

1ESC Institute of Biology and Medicine, Taras Shevchenko National University of Kyiv, Ukraine;
2Palladin Institute of Biochemistry, National Academy of Sciences of Ukraine, Kyiv;
3NSC Institute of Experimental and Clinical Veterinary Medicine, Kharkiv, Ukraine;
4SI National Institute of Phthisiology and Pulmonology named after F. G. Yanovsky National Academy of Medical Sciences of Ukraine, Kyiv;
e-mail: saa0205@ukr.net

The aim of this work was to characterize the experimental test-system based on MPT83-MPT63 fusion antigen with reference commercial diagnostics and investigate the basic characteristics of enzyme-linked immunosorbent assay (ELISA) tests such as specificity and sensitivity. In addition, we investigated the correlation of biochemical and immunological parameters of blood samples of patients with tuberculosis, which was correctly diagnosed by those test-systems. It was shown that the developed test-system match the existing ones regarding the criteria of reliability and the basic requirements of ELISA kits. Recommendations for the testing of serum samples and indications for the use of the proposed serological diagnostic methods has been suggested.

Avidity of bivalent antibodies. Problems of its experimental determination and theoretical evaluation

S. A. Bobrovnik, M. A.Demchenko, S. V. Komisarenko

Palladin Institute of Biochemistry, National Academy of Sciences of Ukraine, Kiyv;
e-mail: s-bobrov@bk.ru

Some problems of experimental determination or theoretical evaluation of antibody avidity are considered. It was shown that in order to determine the fraction of nonoccupied antibodies in their mixture with the excess of the corresponding antigen which is required to estimate avidity the methods should be used which are more sensitive than ELISA. The available methods did not allow determining the avidity of bivalent antibodies because of many reasons. However, in the recent years new methods were suggested that make it possible to evaluate the avidity of bivalent antibodies and that of the receptors which consist of two binding sites connected by a flexible linker of the known length. Thus, there are all possibilities now for determining the avidity of bivalent antibodies in experiments or by theoretical methods.

Analysis of titration curves of serum antibodies obtained by ELISA

S. A. Bobrovnik

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

Different forms of titration curves obtained by ELISA for serum antibodies, monoclonal antibodies­ and polyreactive immunoglobulins were considered. A new interpretation of dose-dependent­ titration curves was suggested. It was shown that our interpretation of dose-dependent titration curves for antibodies which are presented in some biological liquids allow obtaining additional information about properties of the samples. This information was not obtained earlier because of the wrong understanding of the considered problem.

Age changes of human serum polyreactive immunoglobulins (PRIG) activity

S. A. Bobrovnik, M. A. Demchenko, S. V. Komisarenko

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

It has been determined that activity of serum polyreactive immunoglobulins (PRIG) changes with age in practically healthy people at the age from twenty five to seventy. Therewith, the activity of serum IgG PRIG increases most of all (about 3-4 times), while IgM PRIG activity, on the contrary, does not increase, but sometimes even decreases with age. IgA PRIG activity varies significantly more than IgG PRIG activity and, besides, IgА PRIG significantly less depends on age than IgG PRIG. The age changes in the activity of human serum PRIG, belonging to different types of immunoglobulins, may evidence for the important functional role of these immunoglobulins that has to be clarified.

Interaction peculiarities of polyreactive immunoglobulins and various antigens

S. A. Bobrovnik, M. O. Demchenko, S. V. Komisarenko

Palladin Institute of Biochemistry, National Academy of Sciences of Ukarine, Kyiv;
е-mail: s-bobrov@bk.ru

The influence of twin 20, lysozyme and protamine on the capability of polyreactive immunoglobu­lins (PRIG) to attach to various antigens was investigated. Twin 20 can inhibit the binding of PRIG to antigens on immunological plates but lysozyme and protamine can enhance it. As far as the mixture of the optimal concentrations of lysozyme and protamine cannot increase PRIG-antigen interaction in comparison to the optimal dose of protamine, we have concluded that the mechanism of their effect on PRIG binding is similar. Of special interest­ is the fact that twin 20 at optimal concentration of lysozyme or protamine does not decrease PRIG binding to various antigens but, on the contrary, increases PRIG-antigen interaction.