Tag Archives: specificity
Purification and physico-chemical properties of Bacillus sp. L9 protease with fibrin(ogen)olytic activity
O. V. Gudzenko1*, L. D. Varbanets1, V. O. Chernyshenko2, E. M. Stognii2
1D.K. Zabolotny Institute of Microbiology and Virology,
National Academy of Sciences of Ukraine, Kyiv;
2Palladin Institute of Biochemistry, National Academy of Sciences of Ukraine, Kyiv;
*e-mail: alena.gudzenko81@gmail.com
Received: 18 September 2025; Revised: 13 October 2025;
Accepted: 28 November 2025; Available on-line: 23 December 2025
Previously, we isolated a number of Bacillus sp. strains from the dry grass of the coastal zone of the Kinburn Spit, which may be promising for further research as producers of proteases with fibrinolytic and fibrinogenolytic activity. The aim of the work was to isolate, purify and study the properties of fibrin(ogen)ase from the Bacillus sp. L9 strain. The enzyme preparation was isolated from the supernatant of the Bacillus sp. L9 culture liquid. The yield of the purified enzyme was 1.8%, the specific fibrinogenolytic and fibrinolytic activities were 483 and 383 U/mg protein, respectively, the molecular weight of the enzyme was about 40 kDa, the optimum pH was 8.0, and the thermooptimum was 40°C. Bacillus sp. L9 fibrin(ogen)ase is a serine protease, in the active center of which is the carboxyl group of the C-terminal (aspartic or glutamic) amino acid. At some distance from the active site are localized sulfhydryl groups that do not participate in catalysis, but play an important role in maintaining the catalytically active conformation of the protein molecule. The enzyme from Bacillus sp. L9 hydrolyzed fibrin molecules much more slowly than fibrinogen, and showed the greatest specificity in the hydrolysis of bonds formed by the Aα-chain of fibrinogen. According to the specificity of action on fibrinogen, the enzyme was identified as α-fibrinogen(ogen)ase.
Development and characterization of highly informative ELISA for the detection of IgG and IgA antibodies to Сhlamydia trachomatis
O. Yu. Galkin1, Yu. V. Gorshunov1, O. B. Besarab1, O. M. Ivanova2
1National Technical University of Ukraine “Igor Sikorsky Kyiv Polytechnic Institute”;
e-mail: alexfbt@gmail.com;
2Xema Ltd., Kyiv
The goal of this work was developing of highly informative an enzyme-linked immunosorbent assay (ELISA) for the detection of IgG and IgA antibodies against to Chlamydia trachomatis, as well as comparative characterization of developed assay using standardized control materials. The study was conducted using: monoclonal antibodies (McAbs) to human IgA and IgG; recombinant Ch. trachomatis proteins – Pgp3, major outer membrane protein (MOMP); two panels of characterized sera and four reference ELISA kits. The study of immunochemical activity of peroxidase conjugates of McAbs was performed in comparison with conjugates of commercial analogues: anti-IgG McAb 2A11 and anti-IgA McAb AD3. About half of the conjugates from the received McAbs panel were more active compared to the reference antibody conjugates. It was quite justified to use the conjugates of antibodies that interact with different antigenic determinants. When IgG antibodies were detected to MOMP, it was justified 1.14-1.56 times more; when IgA antibodies were detected to MOMP, it was justified 1.16-1.37 times more. ELISA for detecting IgG/IgA antibodies to MOMP and Pgp3 of Ch. trachomatis were evaluated using appropriately described serum panels OCO-42-28-313-00 and OCO-42-28-314-00. Comparative studies of the developed ELISA for the detection of IgG and IgA antibodies to the MOMP and Pgp3 of Ch. trachomatis showed their prominent advantage over the commercial analogues, which more clearly demonstrates the difference in the ratio of average values of optical density of positive and negative samples of the described panel of sera: this indicator for commercial kits was 1.36-3.59 times less.
Characteristics of enzyme-linked immunosorbent assay for detection of IgG antibodies specific to Сhlamydia trachomatis heat shock protein (HSP-60)
O. Yu. Galkin, A. B. Besarab, T. N. Lutsenko
National Technical University of Ukraine “Igor Sikorsky Kyiv Polytechnic Institute”;
e-mail: alexbio@mail.ua
The goal of this work was to study sensitivity and specificity of the developed ELISA set for the identification of IgG antibodies against Chlamydia trachomatis HSP-60 (using biotinylated tyramine-based signal amplification system). The study was conducted using a panel of characterized sera, as well as two reference ELISA sets of similar purpose. According to the results of ELISA informative value parameters, the ELISA we have developed showed the highest specificity and sensitivity parameters (no false negative or false positive results were registered). In 4 out of 15 intralaboratory panel serum samples initially identified as negative, anti-HSP-60 IgG-antibodies test result in reference ELISA sets upon dilution changed from negative to positive. The nature of titration curves of false negative sera and commercial monoclonal antibodies А57-В9 against C. trachomatis HSP-60 after incubation for 24 h was indicative of the presence of anti-idiotypic antibodies in these samples. Upon sera dilution, idiotypic-anti-idiotypic complexes dissociated, which caused the change of test result. High informative value of the developed ELISA set for identification of IgG antibodies against C. trachomatis HSP-60 has been proven. Anti-idiotypic antibodies possessing C. trachomatis anti-HSP-60 activity and being one of the causes of false negative results of the relevant ELISA-based tests have been identified in blood sera of individuals infected with chlamydial genitourinary infection agents.
Biological and immunochemical properties of polyreactive immunoglobulins
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
A previously unknown phenomenon of acquired polyreactivity of serum immunoglobulins, which were subject to the effect of concentrated solutions of chaotropic ions, such as KSCN (3.0-5.0 M), low/high pH (pH 2.2-3.0), or heating to 58-60 °C, was originally described by the authors in 1990. Eleven years after that, similar data were published by J. P. Bouvet et al.(2001), which confirmed completely our results concerning the influence of either chaotropic ions or drastic shift of pH on polyreactive properties of immunoglobulins. Our further investigations (1993, 1995, 1998) of polyreactive serum immunoglobulins (PRIG) properties have revealed that the mechanism of nonspecific interaction between PRIG and antigens much differs from the mechanism of interaction between specific antibodies and corresponding antigens. Later we have shown that the increase in PRIG reactivity could be induced in vivo (1999) and PRIG are one of serum components of human or animal sera. Then, it could be suggested that PRIG may perform certain biological functions. Studying PRIG’s effect on the phagocytosis of microbes or on the tumor growth (S. A. Bobrovnik et al., 1995, 1998) have revealed that PRIG may play a certain role in protecting the body from infections and probably may influence the development of various pathological processes. Recently we also found (S. A. Bobrovnik et al., 2014) that IgG PRIG content significantly increases in aged people. These data demonstrate that further investigations of PRIG’s immunochemical properties and study of their biological role in organism protection from various diseases is very important.