1Fedorova O., 1Zayarniuk N., 1Karpenko O., 2Stanishevskij Y.M., 2Gritskova I.A., 3Zagorij G., 4Ponomarenko V.M., 1Novikov V.P.
1 Lviv Polytechnic National University, 79013, Lviv, Bandery st., 12, phone/fax (032) 2582209, e-mail: firstname.lastname@example.org
2M.V.Lomonosov Moscow State Academy of Fine Chemical Technology
3 PJSC“Pharmaceutical company” Darnitsa” 02093, Ukraine, Kyiv, Borispilska Str., 13
4P.L. Shupic National Medical Academy of Post-Graduate Education, 04112, Ukraine, Kyiv, Dorogozhytska Str., 9
CREATION OF POLYMER TEST-systems for diagnostics of infectious, viral, autoimmunity diseases
In therapy of infectious, viral and autoimmune diseases a great role is given to diagnostics, as similar epizootological data, clinical symptoms and morphological changes are often observed in many diseases; there are cases of atypical and latent forms of the disease and the presence of mixed infections.
Medical practice has quite a wide range of diagnostic techniques, particularly immunodiagnostic ones including the use of indirect haemagglutination reaction (IHR), enzyme-linked immunosorbent assay (ELISA), radial immunodiffusion (RID), neutralization in cell culture. However, these methods have a number of disadvantages – lack of sensitivity and specificity, reproducibility from batch to batch, stability in the design of products, shelf life. Significant benefits arouse with the use of polymeric carriers – “polymer microspheres” (PM), which were used as bioligand carriers when creating test kits for the of latex agglutination reaction (LAR). Simplicity and the ability to test performance in virtually any environment without special equipment, high sensitivity, specificity, and reproducibility of the method RLA, the relative cheapness of analysis can improve quality diagnostics of diseases.
For the creation of highly sensitive diagnostic test systems we synthesized PM with the functional groups of a different nature on the surface (amino-, carboxy-, epoxy-, aldehyde, hydroxyl) that can interact with the functional groups of the assignable bioligand. The copolymer microspheres of styrene and sodium styrenesulfonate were obtained by the method of emulsifier-free polymerization; copolymer microspheres of styrene, styrenesulfonate and methacrylic acid, or glycidylmethacrylate, or acrolein were obtained by seed polymerization on bare polystyrene microspheres. In each specific method of PM production the conditions of synthesis were experimentally selected: the ratio of the monomer and aqueous phases, the amount of initiator, polymerization time, temperature regime. The theoretical analysis and experimental evaluation of physico-chemical characteristics of the polymer carriers, determining the necessary and optimal conditions of their use for diagnostic bioassay systems, was carried out. The PM diameter, size distribution and the magnitude of the surface charge was identified by the method of photon-correlation spectroscopy. It was found that an important factor of the suitability of the synthesized functional PM with a diameter of 0,85-1,55 µm and a narrow particle size distribution (coefficient of dispersion of 0,7-1 %), is their aggregate stability in the electrolyte solution, which is achieved through the introduction of hydrophilic surfactants (PVP, albumin) in the given concentrations (~ 0,1 mg/ml, based on the 0.1% polymer suspension solution) into interfacial adsorption layer.
When designing the bioassay systems for viral, infectious and autoimmune diseases specific immunoglobulins G (IgG), which were immobilized on the surface of the carrier via a spacer-protein A (strain Staphylococcus aureus) were used as bioligands. The selection of the spacer was stipulated by its ability to affinity binding with Fc-fragment of the molecule. In this case, the IgG Fab-fragment, carrying the active centers for the reaction with defined antigen, provides greater accessibility of binding to the antigenic determinants of potential pathogens.
The optimum concentration of protein A (0,05 mg / ml) on the PM surface and the minimum serum dilution at which the spontaneous aggregation of conjugate particles “PM-protein A” occurs, was determined. The stability of the conjugate “PM-protein A” allows its use for more than 6 months.
The following bioassay systems of varying specificity were obtained using high-titer sera and the universal conjugate “PM-protein A” received: yersiniosis system for Y. enterocolitica; salmonellosis system for S. pullorum; leptospirosis system for L. conicula; system for infectious bronchitis virus strain N120.
C-reactive protein (CRP) - the most famous reactant of the acute phase of inflammation in humans and animals, the level of which rapidly and significantly increases during the inflammations of various nature and localization of parasitic infections, traumas, tumors, accompanied by inflammation and necrosis of the tissues. Therefore the timely detection of CRP is one of the factors of the organism defense against infections. When creating the bioassay systems for this purpose the lipids of different nature were used as bioligands: egg phosphatidylcholine (EPC), hydrogenated egg phosphatidylcholine (EHPC) and cerebroside, which are able to interact with the CRP, as well as specific immunoglobulin G (IgG). The investigation of their impact and ways of immobilization on the PM surface on the RLA sensitivity was carried out.
The approbation of CRP detection with RLA method and with traditional radial immunodiffusion method (RID) in the sera of patients with coronary heart disease, rheumatologic diseases, and acute pancreatitis was carried out for the determination of the diagnostic significance of the developed bioassay system. The results of the CRP determination by the RLA method correlates well with the routine RID method, the correlation coefficient lies in the range from 0,71 to 1,0.
Autoimmune thyroid diseases, such as Hashimoto's thyroiditis, Graves' disease and others, are the problem of modern endocrinology. One of the major thyroid autoantigens is thyroglobulin (TG).
When designing a bioassay system for the detection of autoantibodies to TG the TG molecule immobilized on the surface of the functional PM (with a concentration of aldehyde groups 0,12 x10-3 mol/g) at different temperatures was used as the ligand. The analysis showed that the sensitivity of the developed RLA bioassay system is not inferior to IHR method used in clinical practice. Our system allows to define autoantibodies to TG in blood serum with low autoantibodies content – 10-80 IU/ml, moderate content – 81-640 IU/ml and high content – 641 IU/ml. The developed bioassay system will increase the exploitation time from 2-3 to 6 months, and also reduce the titration range of the analyzed serum by 80-90%, providing the rapidity of RLA method.