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.

The goal of the work was justification of standardization parameters of the product based on recombinant human

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.

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 (differential diagnosis). This approach is implemented in the methodology for evaluation of patients for the presence of humoral immune response to TORCH-infections pathogens (toxoplasmosis, rubella, cytomegalovirus, herpes simplex viruses’ infections, and some others). Therefore, testing for presence of specific IgG and IgM antibodies against TORCH-infections pathogens in blood serum is an important element of motherhood and childhood protection. The essential problem in the production of IgM-capture ELISA diagnostic kits is obtaining of positive control. The classic version of positive control is human blood serum (plasma) containing specific antibodies. But specific IgM-positive sera are insignificant raw materials. This fact can seriously restrict the production of diagnostic kits, especially in the event of large-scale production. We have suggested the methodological approach to using of synthetic positive controls in IgM-capture ELISA kits based on conjugate of normal human IgM and monoclonal antibodies against horseradish peroxidase. It is found that this task can be fulfilled by means of NHS ester-maleimide-mediated conjugation (by sulfosuccinimidyl-4-(N-maleimidomethyl)cyclohexane-1-carboxylate), reductive amination-mediated conjugation (by sodium periodate) and glutaraldehyde-mediated conjugation. It was found that conjugates of normal human IgM and monoclonal antibodies against horseradish peroxidase obtained using NHS ester-mediated maleimide conjugation and periodate method were similar by molecular weight, whereas conjugate synthesized by glutaraldehyde method comprised at least three types of biopolymers with close molecular weight. It was found that synthetic positive control obtained by different methods was characterized by higher titer compared to IgM-positive high-titer serum. However, positive control obtained by NHS ester-mediated maleimide conjugation had the best titration profile characteristics.

The development of the English language in the XX-XXI centuries made linguists correct and modify their ideas as to productivity of word-formation means and derivational patterns. The authors of the article claim that there are solid grounds and strong reasons to refer abbreviation to the category of major word-building means. Changes in the vocabulary provoked by the emergence and formation of new

To address a number of fundamental and applied problems in immunology, molecular and cellular biology and biotechnology it is necessary to obtain Fc-fragments of immunoglobulins. Fc-fragments may be used for studying of the effector functions of antibodies which are mediated by these areas. They are often used as an immunogen to produce anti-specie (based on so-called secondary antibody) conjugate in the development of serological tests for diagnostics (predominantly such conjugate based on monoclonal antibodies). The work is aimed to develop improved methods of obtaining and allocation of Fc-fragments of human IgA. To achieve this objective, optimization of hydrolysis of IgA with subsequent purification of Fс-fragments have been carried out. Improved method of obtaining Fc-fragments of IgA provides: papain hydrolysis of immunoglobulin in the environment of nitrogen for 4 h, allowing to achieve maximum output of Fc-fragments without their further degradation: isolation and purification of Fc-fragments of human IgA by one-stage gel filtration on sephadex G-100; control of purity of the target product by electrophoresis in polyacrylamide gel with sodium dodecyl sulfate and Ouchterlony immunodiffusion. Enzymatic hydrolysis was carried out at the optimal temperature of papain (37 °C). As the oxygen in the air may have inhibitory effect on enzymatic hydrolysis reaction, the reaction mixture was incubated in the nitrogen atmosphere to prevent inactivation of papain. To reduce the incident degradation of immunoglobulin molecules, papain hydrolysis was carried out without using an enzyme activator (cysteine). Usage of the proposed scheme allows obtaining Fc-fragments of human IgA of high purity. Outcome of Fc-fragments after all stages of purification was about 18% of the initial amount of IgA in the preparation. Molecular weight from Fc-fragments of human IgA was equal to approximately 70 kDa.

No abstract