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e x p e r i m e n t a l w o r k s e x p e r i m e n t a l w o r k s UDC 573.6.083.3+573.6.086.8 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 registered). In 4 out of 15 intralaboratory panel serum samples initially identified as nega tive, 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. K e y w o r d s: enzyme-linked immunosorbent assay, Chlamydia trachomatis, heat shock protein, anti-idiotypic antibodies, sensitivity, specificity.C hlamydial genitourinary infection (GUI) is one of the most common sexually transmitted infections. According to World Health Organization, Chlamydia trachomatis infects about 90 million people each year by sexual transmission. In Ukraine, the rate of chlamydial GUI is 80 cases per 100,000 population. Nearly 16% of pregnant women are infected with C. trachomatis. About 50-60% of tubular infertility cases are caused by chlamydial infection. A quarter of all ophthalmic and respiratory diseases in newborn and younger children are associated with chlamydial infection [1,2].Effective diagnosis is one of important components of chlamydial GUI control, which can be realized through both direct (detection of antigens, nucleic acids, pathogenic agent microscopic examination and cultivation) and indirect (specific antibo dies detection) techniques. One of the commonly used methods in diagnostics is enzyme-linked immunosorbent assay (ELISA) that allows differential diagnosis -determination of disease stage and course, which is especially important in chronic conditions. For that purpose blood serum (plasma) and human biological fluids are tested for the presence of IgM, IgA and IgG classes of specific antibodies to pathogen's antigens [2,3].Prolonged persistency of C. trachomatis increases the expression of heat shock protein with molecular mass 60 kDa (HSP-60), which is highly

The enzyme-linked immunosorbent assay (ELISA) is the most informative and versatile method of serological diagnostics. The possibility of detecting by ELISA specific antibodies of different classes allow to differentiate primary infectious process and its remission, exacerbation and chronic disease (holding of differential diagnosis). This approach is implemented in the methodology for evaluation of patients for presence of humoral immune response against the causative agent of urogenital chlamydiosis. As with other infections immediately after Chlamydia trachomatis infection the specific IgM antibodies are formed, and subsequently basic projective antibodies of IgG class are synthesized. However, at exacerbation of chronic urogenital chlamydiosis specific IgA antibodies can be synthesized. That is why comprehensive evaluation of patients for presence of humoral immune response to Ch. trachomatis involves plasma testing of specific antibodies of all three classes. The essential problem in the production of ELISA diagnostic kits is obtaining of positive control. The classic version of positive control is human blood plasma containing specific antibodies. But specific IgM- and IgA-positive sera are deficit raw materials. This fact can significantly limit the production of diagnostic kits, especially in case of large-scale manufacture. We have suggested methodological approach to use of synthetic positive controls in indirect ELISA kits based on conjugate of normal human IgM (IgA) and monoclonal antibodies against major outer membrane protein of Ch. trachomatis. It was found that it’s possible to realize such task by means of NHS ester-maleimide-mediated conjugation (by sulfosuccinimidyl-4-(N-maleimidomethyl)cyclohexane-1-carboxylate) and reductive amination-mediated conjugation (by sodium periodate). It was found that synthetic positive controls obtained by different methods are characterized by higher titer compared to IgM- and IgA-positive high-titer serum. However, positive control obtained by NHS ester-mediated maleimide conjugation has the best titration profile characteristics, at the release time and after one-week storage at37 °C.

In this review article an analysis of the biochemical and biophysical aspects of modern magnetic immunoassay (MIA) is conducted and additionally the problems and perspectives of its application in biology, biotechnology and medicine are defined. Magnetic immunoassay should be considered as an evolutionary extension of the classical immunoassay. MIA can have many variants of modifications, similar to the classic immunoenzymatic assay. The key distinctive element of the MIA is the use of magnetic particles (MPs), which are usually nanoparticles. MPs in the MIA can act as a marker for detection, or the solid phase at which the immunochemical reaction takes place. MIA possesses basic advantages over classical immunoassay methods: thanks to the unique magnetic properties of the MPs and the ability to manipulate it in the external magnetic field, it is possible to increase the informative value of the analysis (first of all, sensitivity and specificity), as well as the rigid requirements for “purity” of tested samples. For the purposes of immunoassay, magnetic particles of size from 10 to 200 nm are important, since such particles are in a superparamagnetic state, in the absence of strong magnetic fields; they are not agglomerated in a liquid medium. The size of the spherical particle determines the rate of sedimentation and mobility in the solution. The outer polymeric membrane serves as a matrix in which the surface functional groups are added, and also protects the core of the metal from the external environment. The outer shell may also consist of agarose, cellulose, porous glass, silicon dioxide etc. There are several strategies for the synthesis of nanoparticles: mechanical (dispersion), physical (gas phase deposition), wet chemical methods (chemical comprecipitation, thermal decomposition, methods of micro emulsion, hydrothermal reactions) and physico-chemical methods. Also used are magnesite nanoparticles of biogenic origin. Magnetic particles may function, and this is important for immunoassay. Surface functional groups include carboxylic, amino, epoxy, hydroxyl, tosyl, and N-hydroxysuccinate-activated groups. Magnetic spherical particles usually interact with surface molecules such as streptovidine, biotin, protein A, protein G, and immunoglobulin etc. Directions and prospects of the development of methods of magnetic immunoassay are determined, mainly, by the development of methods for detecting or influencing magnetic particles. In this case, the classical methods of detection are electrochemical methods, electrochemiluminescence, fluorescence. More modern ones include giant magnetoresistance, superconducting quantum interference devices, surface-enhanced Raman spectroscopy, biosensors based on nonlinear magnetization, magneto-PCR immunoassay. The current trend is to combine or integrate the application of various biochemical, physical, molecular and genetic, physico-chemical detection methods. In fact, all of these benefits undoubtedly open up broad prospects for the practical application of MIA in biology, biotechnology and medicine.

Original set of 15 clones of hybridomas producers of monoclonal antibodies (mAbs) to prostate-specific antigen (PSA) (using as a source of lymphocytes of mice of inbred strains NZB and Balb/c) was obtained. The activity in ELISA, affinity constant and titer in the culture fluid of the mAbs obtained from NZB mice are higher compared to the mAbs obtained from the Balb/c mice splenocytes. Conjugates of obtained mAbs with horseradish peroxidase were synthesized, and it allowed performing comparative epitope characterization of the resulting set of mAbs. Studied mAbs are directed to 4 epitopes of PSA molecule: 2 mAbs interacting with one of the epitopes showed cross-activity with PSA-related protein – human kallikrein 2; 2 mAbs of the same epitope specificity blocked the enzymatic activity of PSA; mAbs of the remaining two epitopes either did not affect PSA chymotrypsin activity at all, or this inhibitory effect was not significant. The obtained mAbs recognize antigenic determinants that are not screened when interacting with α1-antichymotrypsin, as well as those that are localized in the places where α1-antichymotrypsin interacts with PSA. Within each of the given two mAbs groups, there are antibodies belonging to different epitopes, which give prospects for the further use of the obtained mAbs in immunoassay and immunobiotechnology. Two alternative methods for determining of the mAbs constants of affinity (by Friguet and Scatchard) are comparable (for determination of the affinity constants of anti-PSA antibodies): coefficient of linear correlation between the affinity constants defined by different methods was 0.90.