Immunoassay Test Systems Research Articles

Results of studies on the development of an immuno-enzymed test system for diagnosing infectious enterotoxemia in animals

Research materials on the development of an enzyme-linked immunosorbent assay for the determination of specific antibodies to Cl. perfringens bacteria were presented. Strains of Cl. perfringens were used as production strains: No. 28 (type A), LD-1 (type B), No. 392 (type C), No. 213 (type D). To obtain antigen, a daily suspension of Cl perfringens bacteria, containing 20 billion microbial cells in 1 cm3 (5 billion microbial cells of each serotype), was sounded on an ultrasonic disintegrator at a frequency of 20 MHz for 15 minutes at 4 °C, then the endotoxin was precipitated with ammonium sulfate and purified by differential dissolution of the precipitate in 0.02 M phosphate buffer, followed by dialysis against tap water. Control positive serum with specific antibodies of at least 1:12800 to antigens of Cl. perfringens bacteria was obtained by hyperimmunization of cattle with corpuscular antigens and toxoids. The basic conditions of the ELISA reaction were standardized. Determination of the concentration of sorption of the antigen was carried out by parallel testing of various dilutions of the conjugate. The most acceptable titer of the blood serum of animals was found at a dilution of the conjugate of 1:2500 and a concentration of the sorbed antigen of 5-8 μg/cm3. The production test of the enzyme immunoassay test system was carried out in cattle farms in three regions of the Russian Federation, unfavorable for anaerobic enterotoxemia in calves.

TRIPTERIGIUM WILFORDII EXTRACT AS A REGULATOR OF SPONTANEOUS AND IN VITRO STIMULATED CYTOKINE PRODUCTION IN ATOPIC DERMATITIS

Background. The analysis of traditional, primarily Chinese, medicine is the subject of close attention of modern medicinal substances developers. One of the important, frequently used plant substrates is Tripterygium Wilfordii, which is used as an antiinflammatory agent, including for the treatment of skin diseases. However, the mechanisms of action of this agent, particularly in such a widespread disease as atopic dermatitis, still need to be investigated. Aim. To study of the effect of Tripterygium Wilfordii extract on in vitro cytokine production in normal and atopic dermatitis patients. Materials and Methods. In a culture of the effect of Tripterygium Wilfordii extract on spontaneous and phytohemagglutinininduced synthesis of IL-2, IL-4, IL-6, IL-10, IL-17, TNFα, IFNγ was evaluated in mononuclear cells culture obtained from healthy donors and patients with atopic dermatitis using enzyme immunoassay test systems Cloud-Clone Corp. (CCC, Wuhan) Results. It was found that the level of spontaneous cytokine synthesis in patients with atopic dermatitis was significantly increased, while the response to the mitogenic stimulus was sharply reduced, absent or inverted. Tripterygium Wilfordii extract in all samples, except for intact cells of healthy donors, showed a pronounced inhibitory effect. Conclusions. Under in vitro conditions, cultured mononuclear cells of patients with atopic dermatitis produce a greater amount of IL-2, IL-4, IL-6, IL-10, IL-17, TNFα, IFNγ, which indicates the activation of the cytokine function. Tripterygium Wilfordii extract has a pronounced inhibitory effect, especially on activated cells. The use of Tripterygium Wilfordii extracts seems reasonable for promising pharmaceutical substances.

Biopharmaceutical properties of polyvalent bacteriophage capsules

Scientific relevance. Rational phage therapy is a highly effective way to combat bacterial infections, especially in conditions of steadily increasing antibiotic resistance. Most bacteriophage preparations are formulated as oral and topical solutions. However, oral administration of liquid phage preparations results in significant inactivation in the stomach. To shield active ingredients from the acidic environment, Sextaphag® Pyobacteriophage, polyvalent, has been formulated into capsules.Aim. This study evaluated the polyvalent bacteriophage preparation in capsules in terms of its potency and biopharmaceutical properties.Materials and methods. The study compared the polyvalent bacteriophage preparation formulated as capsules with the polyvalent bacteriophage preparation formulated as a solution. The potency was evaluated by the Appelmans method, and phage particles were quantified by the Gratia method. To evaluate the acid-neutralising capacity, the authors placed test samples of the bacteriophage preparation in 0.1 M hydrochloric acid and analysed their potency by the Appelmans method. Chinchilla rabbits were used to analyse anti-phage immune responses, and their antibody levels were measured using an enzyme immunoassay test system developed by the authors. The pharmacokinetic parameters were studied in outbred white mice after oral dosing.Results. The polyvalent bacteriophage preparation exhibited high lytic activity towards Escherichia coli, Klebsiella pneumoniae, Proteus mirabilis, Proteus vulgaris, Pseudomonas aegidinosa, Staphylococcus aureus, and Streptococcus pneumoniae, which accounted for a dilution factor of 10-6. Following oral administration of the polyvalent bacteriophage preparation in capsules to mice, the level of absorption was 3.1–3.7 times higher than that observed with the solution. Repeated oral administration of therapeutic doses did not induce anti-phage antibodies in rabbits. The stability study showed that the polyvalent bacteriophage preparation retained high lytic activity for 18 months.Conclusions. According to the study results, the polyvalent bacteriophage preparation in capsules exerts significant antibacterial activity against the studied microorganisms, has a high level of absorption, retains its lytic activity for a long time, and does not induce anti-phage antibodies after oral dosing, which confirms its safety and efficacy.

CHARACTERISTICS OF IMMUNOREACTIVITY IN PATIENTS WITH VARIOUS FORMS OF DIABETIC FOOT ULCER

The article presents a comparative assessment of the levels of cytokines that regulate the differentiation of various macrophage phenotypes, as well as immunological responses in patients with diabetes. We examined and treated 245 patients who were allocated to two groups. The first group comprised neuropathic patients (comparison subgroup 1A without DFU, n=30 and subgroup 1B with DFU, n=80). The second group included patients with neuroischemic type 2 diabetes (comparison subgroup 2A without DFU, n=35 and subgroup 2B with DFU, n=100). As an indicator of hyperglycemia, the glycated hemoglobin level (HbA1c) was used, which was determined on the DCA 2000 device by the method of latex immunoagglutination inhibition using the “Hemoglobin A1c Reagent kit”. The concentration of cytokines TNF-α, IFN-γ, IL-6, and OPG in blood serum was determined on the first day of hospitalization before surgery using enzyme immunoassay test systems (OOO “Cytokine”, St. Petersburg) in accordance with the manufacturer's instructions. An imbalance of cytokines was revealed, indicating a shift in immunomodulatory responses, aggravation of metabolic and vascular disorders in tissues, accompanied by an inadequate course of the inflammatory and reparative process and the formation of a diabetic foot ulcer.

Creation of Antibody Immunoassay Test Systems for Indication of Enteropathogenic Yersinia

3 immunoassay test systems based on antibodies obtained as a result of immunization of rabbits with one of the antigens: disintegrated membranes (DM) Y. enterocolitica, DM Y. pseudotuberculosis and lipopolysaccharide Y. pseudotuberculosis were created. The best diagnostic properties were possessed by a test system based on antibodies to Y. pseudotuberculosis DM. It showed high titers of antibodies to various serovars of Y. pseudotuberculosis (1:25600-1:51200), incl. to microbe strains isolated from farm animals, and low titers to Y. enterocolitica (1:200). In the accumulation media with pig excrements seeded with Yersinia, the experimental test system detected Y. pseudotuberculosis already on the 3rd day of “cold enrichment” provided that 50 or more Yersinia cells per ml were inoculated into the accumulation medium together with excrements.

Development of an immunoassay test system based on monoclonal antybodies and immunomagnetic particles for the detection of F. tularensis cells.

Tularemia is an especially dangerous infection caused by the gram-negative bacterium Francisella tularensis. It belongs to natural focal infections, and therefore is under continuous control by quarantine services. When carrying out their activities they use a whole range of diagnostic tools. The objective of this research is to develop an enzyme immunoassay based on highly specific monoclonal antibodies and immunomagnetic particles for monitoring the tularemia pathogen. To produce hybridomas mice were immunized with cells of the vaccine strain F. tularensis subsp. holarctica 15 NIIEG. After cell fusion hybridomas were selected by a solid-phase enzyme immunoassay (ELISA) using lipopolysaccharide (LPS) of the tularemia microbe. As a result, two hybridomas, 1C2 and 3F5, were produced. MABs of the hybridomas were obtained by using BALB / c mice. The MABs were purified by sepharose A affinity chromatography and used for conjugation with magnetic particles, and for biotinylation followed by matching a pair for ELISA. The pair of IMPs and MABs 3F5 as well as biotinylated FB11-x MABs was the best in detecting tularemia cells. The use of this MAB pair in ELISA allowed the identification of 105 microbial cells/ml in a 4 ml sample and 5×103 microbial cells/ml in a 45ml sample. Interaction with F. tularensis subsp. novicida Utah112 cells was absent.

Chromogranin A and chromogranin B in pancreatic neuroendocrine tumors

For the first time in Russia a comparative study of chromogranin A (CgA) and chromogranin B (CgB) in neuroendocrine tumors (NETs) of the pancreas was performed. We examined 50 primary patients with pancreatic NETs and 42 healthy people. The determination of CgA and CgB was performed in blood serum using standard enzyme-linked immunoassay test-systems (Chromogranin A NEOLISA, Eurodiagnostica; Human Chromogranin B, USCN). The levels of CgA and CgB in pancreatic NETs significantly differed from the control group. There was found the association between CgA levels and the dissemination of the process, while CgB demonstrated the properties of a marker independent from the tumor dissemination. The diagnostic sensitivity of CgA was 76 %, CgB – 68 %. Complex determination of CgA and CgB enhanced the diagnostic sensitivity to 84 %. Our data indicate the potential usefulness of complex CgA and CgB in the diagnosis of pancreatic NETs.

Иммунологические причины синдрома задержки развития плода у беременных с антифосфолипидным синдромом

Objective: to conduct a comparative analysis of the level of antibodies to phospholipids, the concentration of the pro-inflammatory cytokine TNF-α, as well as their correlation with the severity of fetal development retardation syndrome (FDS) and the outcome of pregnancy. Materials and methods. There was conducted a study of 285 pregnant women with FDS for the presence of antibodies to β2GPI as the most specific marker of the autoimmune process of APS in pregnancy 23–36 weeks. Group I included 103 (36.1%) women with a positive test for the presence of antibodies to β2GPI, group II (control) - 27 pregnant women of the same age and gestational age with physiological pregnancy. To study the immunological status, the titer of antibodies of the IgM and IgG class against cardiolipin was determined using enzyme immunoassay test systems produced by the «Genesis Diagnostics» company (Great Britain), and the titer of antibodies of the IgM class, IgG to β2GPI was tested using the «Biotech Inc» test systems (USA). Tests for the quantitative study of human TNF-α are based on the method of enzyme-linked immunosorbent assay using specific monoclonal antibodies TNF-α. Results. A statistically significant increase in all classes of antiphospholipid antibodies and TNF-α was revealed in pregnant women with FDS with an increase in gestational age compared with the control group. A significant increase in the levels of AKLA and antibodies to β2GPI IgG class in the II trimester (p <0.05) is also characteristic of pregnant women with FDS with a tendency to increase indicators in the III trimester. So, the average level of AKLA IgG in the II trimester was increased by 12.1 times; antibodies to β2GPI IgG - 7.4 times. In the III trimester, the average level of IgG AKLA was 11.7 times higher in comparison with the average level of similar indicators of the control group, and the average level of antibodies to β2GPI IgG class was 7.6 times higher than that in pregnant women with physiological pregnancy. A statistically significant direct correlation between the severity of FDS and the level of antibodies to β2GPI IgG (r = 0.61; p <0.01), SZRP and TNF-α (r = 0.41; p <0.01) was also revealed. Conclusion. The results of the study make it possible to predict the severity of FDS depending on the level of autoantibodies and indicators of the pro-inflammatory cytokine TNF-α, as well as determine the intensity and effectiveness of the treatment and preventive measures. Key words: pregnancy, antiphospholipid syndrome, fetal development retardation syndrome, tumor necrosis factor.

Assessment of progression of atrial fibrillation in middle-aged individuals with hypertension in combination with comorbid extracardial diseases

Purpose. To assess the progression of atrial fibrillation in middle-aged people with hypertension in combination with comorbid extracardiac diseases.Materials and methods. In an observational cohort study, 308 patients aged 45–65 years with atrial fibrillation (paroxysmal and persistent forms) with hypertension in combination with extracardiac pathology were observed: diabetes mellitus (n = 40), thyrotoxicosis (n = 42), hypothyroidism (n = 59), abdominal obesity (n = 64) and chronic obstructive pulmonary disease (n = 47). The control group consisted of 56 patients with hypertension + AF without concomitant extracardiac disease. The level of MMP-9 was determined using the Human MMP-9 (total) Immunoassay test-system (USA); NT-proBNP — using the NTproBNP-IFA-Best reagent kit; galectin-3 — with ELISA — Bender MedSystems (Austria). Echocardiography was performed using an Acuson Aspen apparatus (USA), 24-hour ECG monitoring with Schiller Medilog Holter system. All statistical calculations were carried out in the program Rstudio 0.99.879 (RStudio, USA).Results. In patients with hypertension and atrial fibrillation, the combination of diabetes mellitus (p = 0.041) and abdominal obesity (p = 0.004) is the most prognostic factor for AF progression. In groups of patients with diabetes mellitus, hypothyroidism and abdominal obesity, the most pronounced indicators of diastolic dysfunction of the left ventricle were: E/A, LVMI (men); the size of the left atrium and the end-diastolic size of the left ventricle are increased in all clinical groups. The prognostic value of biomarkers of fibrosis and remodeling of galectin-3 and MMP-9, and NT-proBNP in the progression of atrial fibrillation in patients with hypertension in combination with extracardial diseases is shown.Conclusion. There is no doubt that in case of hypertension, the decompensation of long-term hypertrophic myocardium is based on a violation of the balanced growth of its various structures and the formation of fibrosis and myocardial dystrophy, which was confirmed in the present study. The detected elevated levels of MMP-9, galectin-3 and NT-proBNP, as well as ultrasonic signs of myocardial remodeling, confirmed that they statistically significantly affect the progression of atrial fibrillation. In this regard, a personalized approach to a patient with atrial fibrillation in combination with comorbid pathology is required, especially in middle-aged people at the stage of comorbidity formation, under the conditions of received diagnostic information on the electrical function of the heart and assessment of the functional capabilities of CVS during its transition to various levels of functioning.

Antiviral effect of «Kagocel» substance in vitro on influenza viruses H1N1, H1N1pdm09 and H3N2.

Active circulation of pandemic influenza and new variants of influenza H3N2 strains requires monitoring of antiviral efficacy of drugs permitted for influenza therapy in the Russian Federation. Assessment of antiviral efficacy of «Kagocel» substance against influenza viruses H1N1, H1N1pdm09 and H3N2 in vitro. Cytotoxic effect of «Kagocel» substance on MDCK cells had been determined by stained with MTS. Antiviral efficacy of «Kagocel» substance against influenza infection has been studied in vitro in the culture of MDCK cells infected with influenza virus strains: A/Puerto Rico/8/34 (H1N1), А/California/7/2009 (H1N1)pdm09, А/Hong Kong/1/68 (H3N2) and А/ Hong Kong/4801/2014 (H3N2). The antiviral activity of «Kagocel» substance was tested by its effect on the infectious titer of the influenza viruses and on its impact on the expression level of viral antigens in the enzyme immunoassay test system. «Kagocel» substance had low toxicity for MDCK cells. «Kagocel» inhibited the infection titer of influenza virus strains A/Puerto Rico/8/34 (H1N1), А/California/7/2009 (H1N1)pdm09, А/Hong Kong/1/68 (H3N2) and А/ Hong Kong /4801/2014 (H3N2) in the MDCK cell culture with equal efficacy. Study of the impact of «Kagocel» substance on the expression level of viral antigens by ELISA also revealed its antiviral efficacy for all tested strains. Dose dependence was observed from concentration of substance and from infective dose of virus. Effective suppression of the reproduction of influenza virus strains A(H1N1), A(Н1N1)pdm09 and A(H3N2) in the different sublines of MDCK cells with «Kagocel» was shown by the different methods. These results give the possibility to suggest that along with the ability to induce interferons, «Kagocel» can impact on the reproduction of influenza virus, but the further research is needed. «Kagocel» substance effectively inhibits the reproduction of influenza virus strains A(H1N1), A(Н1N1)pdm09 and A(H3N2) in vitro. At the same time, the selectivity index is quite high.

Validation of Enzyme Immunoassay for Preclinical Pharmacokinetic Trials of Rituximab

An integral part of preclinical pharmacokinetic studies is the development of a bioanalytical method for determination of the drug in a biological fluid.The aim of the research was to assess the suitability of the test system based on enzyme-linked immunosorbent assay (ELISA) for quantitative determination of rituximab in the blood serum of laboratory animals after intravenous administration of rituximab at a dose corresponding to the therapeutic dose in humans. Th test system was developed by the Scientific and Production Center Probiotech.Materials and methods: the determination of rituximab in biological samples was carried out using a two-stage sandwich-type ELISA, followed by detection based on horseradish peroxidase. The ELISA results were recorded using a microplate photometer at a wavelength of 450 nm.Results: the experiments helped to establish the detection limit (0.24 ng/mL) and the lower limit of quantitation (1.00 ng/mL) of rituximab in rabbit blood serum, they also demonstrated high selectivity of analyte determination in a multicomponent biological matrix. The mean rituximab concentration was within 14 % of the nominal value in the entire working range of the method. The within-run and between-run precision of the assay did not exceed 7.4 %, the total error of the method did not exceed 20.1 %. The linearity of dilution makes it possible to use the assay for the analysis of biological samples with a wide range of rituximab concentrations. The stability of the analyte in the rabbit blood serum was confirmed by storing samples for 6 hours at room temperature, for 50 days at —35 °C, and after 3 freeze-thaw cycles. The validated immunoassay was successfully used to determine the rituximab concentration in biological samples obtained in the rituximab pharmacokinetic trial in rabbits. The accuracy of the results was confirmed for the entire range of the determined concentrations; parallelism was demonstrated between the calibration curve and the results of analysis of serially diluted rabbit serum samples with the maximum concentration of rituximab.Conclusions: the proposed enzyme immunoassay test system can be used for quantitative determination of rituximab in the blood serum of laboratory animals, as it meets acceptance criteria for all validation parameters described in the international guidelines on validation of bioanalytical methods.

Вибір хірургічної тактики у постраждалих із скелетно-абдомінальною травмою з врахуванням імунологічного статусу

Мета роботи: зниження летальності та хірургічних ускладнень постраждалих зі скелетно-абдомінальною травмою шляхом оптимізації лікувально-діагностичної тактики з врахуванням даних імунологічного статусу.
 Матеріали і методи. Концентрацію цитокінів, а саме інтерлейкіну 2 (ІЛ-2), інтерлейкіну 10 (ІЛ-10), фактора некрозу пухлин альфа (ФНП-α) та інтерферону гама (ІФН-γ) у сироватці крові визначали за допомогою імуноферментних тест-систем. Дослідження імунологічного статусу проводили до постраждалих при госпіталізації в стаціонар та на 7 і 14 доби.
 Результати досліджень та їх обговорення. При госпіталізації постраждалих зі скелетно-абдомінальною травмою концентрація прозапальних цитокінів ФНП-α та ІФН-γ зростали, відповідно, у 4,5 та у 2,2 раза. Одночасно ми спостерігаємо падіння протизапальних внутрішньоклітинних месенджерів ІЛ-2 у 4,1, а ІЛ-10 – у 9,5 разів. На 7-му добу все ще спостерігається зростання вмісту ФНП-α на 16,7 %. Вміст протизапальних захисних цитокінів ІЛ-2 та ІЛ-10 на 7 добу підвищувався на 79,8 % та 147,6 %. На 14-ту добу концентрація ФНП-α залишалася на 96,3 % вище порівняно з показниками на день госпіталізації. Вміст ІФН-γ на 51,9 % відрізнявся від даних у день госпіталізації. Концентрація ІЛ-2 на 14 добу була вище на 163,9 % порівняно з первинними даними та на 46,8 % порівняно з показниками пацієнтів на 7 добу. Вміст ІЛ-10 зростав на 159,0 % порівняно із 7-ю добою та був на 541,3 % (у 5,4 раза) вище від показників на час госпіталізації аналіз імунологічного статусу підтвердив високу ймовірність смерті хворого скелетно-абдомінальною травмою в результаті інтраопераційних порушень метаболізму при тривалих оперативних втручаннях, ніж в результаті цілеспрямованого скорочення операції.

Enhancement of Labeled Alpha-fetoprotein Antibodies and Antigen-antibody Complexes Fluorescence with Silver Nanocolloids

A model immunoassay test system is proposed based on metal enhanced photoluminescence to analyze low concentrations of alpha-fetoprotein, a tumor marker. Antigen-antibody reaction was performed on polystyrene plates coated with silver nanoparticles to increase sensitivity of fluorescent immunoassay and signal-to-noise ratio as compared to silver-free system. The proposed test system model uses layer-by-layer assembly approach, LED excitation and nanowatt photodetection set-up, so it is characterized by smaller probe volume, fast analysis and simplicity. The proposed model system offers alpha-fetoprotein detection at concentrations used in clinical practice. Photostability and photoluminescence enhancement for labeled alpha-fetoprotein antibodies on a silver substrate were found to depend on antibodies concentration. Labeled antibodies photostability on substrates was found to deviate within 20% as compared to silver-free samples whereas photoluminescence intensity enhancement is typically in the range of 6-8 times.

Recovery and Stability of Δ9-Tetrahydrocannabinol Using the Oral-Eze® Oral Fluid Collection System and Intercept® Oral Specimen Collection Device.

Oral fluid (OF) is increasingly used for clinical, forensic and workplace drug testing as an alternative to urine. Uncertainties surrounding OF collection device performance, drug stability and testing reproducibility may be partially responsible for delays in the implementation of OF testing in regulated drug testing programs. Stability of Δ(9)-tetrahydrocannabinol (THC) fortified and authentic specimens was examined after routine collection, transport and laboratory testing. Acceptable recovery and stability were observed when THC-fortified OF (1.5 and 4.5 ng/mL) was applied to Oral-Eze devices. Neat OF samples collected with Oral-Eze, processed per the package insert, and fortified with THC (3 and 6 ng/mL) were stable (±20%) at room temperature (21-25°C), refrigerated (2-8°C) and frozen (-25 to -15°C) conditions up to 1 month, while samples collected with Intercept devices showed decreases at refrigerated and room temperatures. After long-term refrigerated or frozen storage, maximum reductions in THC concentrations were 42% for Oral-Eze and 69% for Intercept. After ≥1 year frozen storage, 80.7% of laboratory specimens positive for THC (3 ng/mL cut-off) by GC-MS were reconfirmed positive (within 25%), with an average THC decrease of 4.2%. Specimens (n = 47) processed with Oral-Eze (diluted) and tested via enzyme immunoassay were concordant with LC-MS-MS results and showed 100% sensitivity and 95% specificity. Paired specimens collected with Oral-Eze and Intercept exhibited 98% overall agreement between the immunoassay test systems. Collectively, these data demonstrate consistent and reproducible recovery and stability of THC in OF after collection, transport and laboratory testing using the Oral-Eze OF Collection System.

The hustle and bustle of city life: monitoring the effects of urbanisation in the African lesser bushbaby.

Urbanisation has become a severe threat to pristine natural areas, causing habitat loss and affecting indigenous animals. Species occurring within an urban fragmented landscape must cope with changes in vegetation type as well as high degrees of anthropogenic disturbance, both of which are possible key mechanisms contributing to behavioural changes and perceived stressors. We attempted to elucidate the effects of urbanisation on the African lesser bushbaby, Galago moholi, by (1) recording activity budgets and body condition (body mass index, BMI) of individuals of urban and rural populations and (2) further determining adrenocortical activity in both populations as a measure of stress via faecal glucocorticoid metabolite (fGCM) levels, following successful validation of an appropriate enzyme immunoassay test system (adrenocorticotropic hormone (ACTH) challenge test). We found that both sexes of the urban population had significantly higher BMIs than their rural counterparts, while urban females had significantly higher fGCM concentrations than rural females. While individuals in the urban population fed mainly on provisioned anthropogenic food sources and spent comparatively more time resting and engaging in aggressive interactions, rural individuals fed almost exclusively on tree exudates and spent more time moving between food sources. Although interactions with humans are likely to be lower in nocturnal than in diurnal species, our findings show that the impact of urbanisation on nocturnal species is still considerable, affecting a range of ecological and physiological aspects.

Markers of intravascular microinflammation and intima-media complex thickness of carotid arteries in individuals with psychoemotional disorders

Purpose: To examine the markers of intravascular microinflammation and thickness of the intimae-media complex (IMT) of the carotid arteries in healthy individuals with reference to psychoemotional disorders. Methods: A cohort of 173 healthy individuals, age range from 30 to 60, were examined for a psychoemotional state on a hospital anxiety and depression scale. The symptoms of depression, anxiety and asthenia were accumulated to make up an index of psychoemotional disorders (PED). The level of C-reactive protein (CRP) and cytokines: tumor necrosis factor alpha (TNF-α), interleukins (IL) -1, -4, -8, have been studied by enzyme immunoassay test systems (SeroELISA, DSL, USA). The IMT of the carotid arteries was determined with the help of apparatus Aloka 5000 with 07-10 MHz linear transducer in B-mode. Results: All subjects were split into 2 groups with and without psychoemotional disorders. Group 1 consisted of 98 people (45 men and 53 women, average age 43,9±0,9). The anxiety symptoms were found in 59 subjects (60,2%), depression - in 68 (69,4%), asthenia - in 74 (75,5%) of the group. Average index of PED in this group was 3,8±0,1 points. Group 2 included 75 individuals (32 men and 43 women, average age 42,1±0,8, p1-2=0,32) with no psychoemotional disorders. IMT in group 1 was 0,81±0,02 mm, in group 2 - 0,62±0,01 mm (p=0,001). Besides, group 1 was characterized by higher levels of CRP (3,70±0,01 mcg/ml) versus group 2 (1,01±0,04 mcg/ml, p=0,01). Group 1 has also demonstrated an increase of proinflammatory cytokines: IL-1 - 28,0±3,1 pg/ml, IL-8 - 271,8±57,3 pg/ml, TNF-α - 74,7±8,9 pg/ml compared to group 2 (p=0,03-0,001). Correlation analysis of the data indicated a close link between psychoemotional factors and inflammatory markers in group 1. Thus the amount of CRP is directly correlated to the level of anxiety (r=0,51, p<0,05) and asthenia (r=0,46, p<0,05). TNF-α is correlated to severity of depression (r=0,56, p<0,05) and anxiety (r=0,39, p<0,05) level. The research revealed direct correlation between IMT and depressive symptoms (r=0,65, p<0,05) and the degree of anxiety (r=0,74, p<0,05). However, these significant correlations were not found in group 2. Conclusions: The individuals with psychoemotional disorders have shown an increase in blood concentrations of markers of intravascular microinflammation and increased carotid IMT. The symptoms of depression and anxiety identified in these individuals are directly correlated to inflammatory markers and IMT.

Metrological complex for existing and developing immunoassay test systems: The method of calibration curves for immunoassay test systems

In this paper the metrological complex developed for the certification of immunoassay test systems is described. This complex allows measuring the calibration curves, the probabilities of false results, and the parameters of immunoassay test-system components. The use of this complex offers metrological support for the results of immunoassay analysis. Also in this paper the algorithm of plotting and measuring the calibration curves for immunoassay test systems is described in detail. This algorithm is described in the measurement guideline included in the metrological complex. The calibration curves for benzodiazepines and tuberculosis markers, measured by the developed algorithm, are used as an experimental base for the described method.

Solution of the problem of nonspecific binding in solid-phase noninstrumental dot immunoassay

In this work, we evaluated blocking properties of various reagents used in dot immunoassay test systems on nitrocellulose membranes employing carbon-based diagnosticum to determine antibodies to tetanus anatoxin, thermostable toxin of Yersinia pseudotuberculosis, and HIV-I. The results support the usage of a nonionic detergent Tween 20 which efficiently inhibited nonspecific protein binding to the solid-phase reagent surface and did not decrease sensitivity of the analysis.

Determination of Pharmaceuticals Based on Indole Alkaloids with Amperometric DNA-Sensors and Enzyme Immunoassay Test-System

Bioaffine methods were developed for determining indole-containing alkaloids ajmaline and vincristine. These methods are based on the proposed amperometric DNA-sensors and on immunoenzyme test-system with a spectrophotometric indication of the analytical signal. The complexing between ajmaline and immobilized native DNA (n-IDNA) allows effective preliminary concentration from test solutions on the biosensor. The time of analysis is 25–30 min, limit of detection (LOD) for ajmaline is 1.0 × 10−10 M. The test-system utilized the immunological reaction of ajmaline with its antibodies and the enzyme marker, horseradish peroxidase, LOD is 1.5 × 10−9 M. Anti-cancer vincristine interaction with immobilized renatured DNA (r-IDNA) was studied and determination was carried out using an amperometric DNA-sensor. Bioaffine membrane drug concentration and reactivation of the sensor was performed. The affinity binding constant Kbind for vincristine-r-IDNA complex calculated by Scatchard's method was found to be high enough [(5.0 ± 0.4) × 105 l/mol] confirming high specificity of the complexing with r-IDNA. The duration of the assay is 40 min. The developed method is characterized with an LOD of 1.1 × 10−9 M, with the lack of the need for long sample processing. The pharmaceuticals were determined by those methods in model solutions of blood serum and in tablets and solutions for injections.

Interaction between heparin and cardiac troponin T and troponin I from patients after coronary bypass surgery

Objectives: Heparin is thought to play a crucial role in the clinical monitoring of patients with acute coronary syndrome as well as after coronary bypass surgery in that it interferes with different commercial immunoassay test systems for cardiac troponin T (cTnT) and troponin I (cTnI). The mechanism, however, by which heparin apparently affects the cTnT and cTnI levels in plasma is not yet resolved. Design and methods: We analyzed the effect of heparin by simultaneously collecting serum and heparin plasma samples from 32 patients after coronary bypass surgery. The cTnT and cTnI levels were determined using the Roche/Elecsys, the Dade-Behring/Opus and the Bayer/ACS:Centaur immunoassay systems in the absence or in the presence of either heparinase or protamine. Association between the cardiac troponins and the anticoagulant has been demonstrated by affinity chromatography using heparin as the ligand. Results: The data obtained indicate that heparin produces an apparent decrease in cTnT as well as of cTnI levels, analyzed either by the Elecsys, the Opus or the ACS:Centaur immunoassay systems. Individual patients show a wide variation in the discrepancies between serum and heparin plasma troponin especially in the cTnT immunoassay. Pretreatment of the heparin plasma samples either with heparinase or protamine cannot completely reverse the heparin-induced decrease in cTnT and cTnI levels and therefore addition of these reagents to the commercial test systems could not significantly improve the performance of the assay. When serum is supplemented with increasing concentrations of heparin, and cardiac troponin levels were reanalysed, significantly lower recoveries for the cTnT than for the cTnI immunoassays were detectable. Affinity chromatography with heparin Sepharose demonstrates that cTnT and cTnI interact differentially with the negatively charged ligand. Whereas cTnI shows minor affinity to the immobilized heparin and is eluted at near physiological conditions, cTnT is bound and can be quantitatively recovered only by solutions of high ionic strength. Conclusions: We conclude, therefore, that the apparent decrease in cTnT values by addition of heparin is a result of direct molecular interaction between the negatively charged glycosaminoglycan and clusters of basic residues within the sequence of the cardiac protein. In contrast, the effect of heparin on the cTnI immunoassay systems, is primarily indirect, most possibly induced by changes within the sample matrix itself.