Characteristics of enzyme-linked immunosorbent assay for detection of IgG antibodies specific to Сhlamydia trachomatis heat shock protein (HSP-60)

Abstract

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

Modern Approaches to Quality Evaluation of Enzyme-Linked Immunoassay Diagnostic Kits

“…Monoclonal antibodies (McAbs) are used in ELISA kits in two forms: as a part of immunosorbent or in the form of conjugate with an enzyme (most frequently peroxidase). Modern diagnostic kits include McAbs-based conjugates, specific to various infectious and non-infectious antigens [5][6][7].…”

Effective Bioactivity Retention of Low-Concentration Antibodies on HFBI-Modified Fluorescence ICTS for Sensitive and Rapid Detection of PSA

“…The membrane-based immunochromatography test strip has been widely used for near-patient and point-of-care diagnostics due to its advantages of fast, convenient, low-cost, and single test. − However, the conventional gold nanoparticle (GNP)-based strip commonly just provided a qualitative (yes or no) or semiquantitative result with poorer detection sensitivity , and most fluorescence immunochromatography test strips (FICTS) hardly provide sensitive detection as low as the clinical laboratory medicine, such as enzyme-linked immunosorbent assay (ELISA) and electrochemiluminescence immunoassay (ECLIA) but the issues with ELISA and ECLIA mainly lie in the multistep processing of samples, long diagnostic times, and overall cost . Generally, FICTS mainly consists of the porous nitrocellulose membrane (PNM), Abs, and fluorescence-signal-generating reagents. , To improve the sensitivity, much effort is spent and cost incurred on the signal-generating reagents of FICTS, including the following: (1) utilization of new type of fluorescent probes, such as quantum dots (QDs) and upconverting nanoparticles, (2) packaging multiple fluorescent nanoparticles or dyes to form the high fluorescent nanoprobes, such as fluorescent or dye-doped nanospheres containing lots of fluorescers, , and (3) surface-enhanced Raman scattering. , Although the analytical sensitivity has been improved to some extent, it is still far below the detecting requirement for trace amounts of targets.…”

“…The membrane-based immunochromatography test strip has been widely used for near-patient and point-of-care diagnostics due to its advantages of fast, convenient, low-cost, and single test. 1−4 However, the conventional gold nanoparticle (GNP)based strip commonly just provided a qualitative (yes or no) or semiquantitative result with poorer detection sensitivity 5,6 and most fluorescence immunochromatography test strips (FICTS) hardly provide sensitive detection as low as the clinical laboratory medicine, such as enzyme-linked immunosorbent assay (ELISA) 7 and electrochemiluminescence immunoassay (ECLIA) 8 but the issues with ELISA and ECLIA mainly lie in the multistep processing of samples, long diagnostic times, and overall cost. 9 Generally, FICTS mainly consists of the porous nitrocellulose membrane (PNM), Abs, and fluorescence-signalgenerating reagents.…”

Biotechnology for obtaining hybrid positive control samples for immunoassay for detecting antibodies against Chlamydia trachomatis

“…Among the whole complex of methods of clinical laboratory diagnostics, there are serological methods, which are based on the identification of serological markers (antigens, allergens, antibodies) of infectious (viral, bacterial, fungal, and parasitic) and non-infectious (including autoimmune, allergic, endocrine and oncological) diseases. The most informative, universal and, as a consequence, widespread serology test is the enzyme-linked immunosorbent assay (ELISA) (Nikolaenko et al, 2005;Galkin et al, 2017). The possibility of detecting by means of ELISA specific antibodies of different classes can differentiate the primary infectious process and its remission, exacerbation or chronicization of the disease (that is, to conduct a differential diagnosis).…”