Analysis of Proteins by Immunoblotting

In immunoblotting (western blotting), proteins are first separated by SDS-PAGE and then transferred electrophoretically from the gel onto a support membrane that binds proteins tightly. After the unreacted binding sites of the membrane are blocked to suppress nonspecific adsorption of antibodies, the immobilized proteins are reacted with a specific polyclonal or monoclonal antibody. Antigen–antibody complexes are visualized using chromogenic, fluorescent, or chemiluminescent reactions. Immunoblotting protocols are reagent specific and, owing to the wide assortment of equipment, reagents, and antibodies available, highly diverse. Presented here is an example of a workable protocol for developing a blot using horseradish peroxidase (HRP)–conjugated secondary antibody and enhanced chemiluminescence (ECL). ECL is based on the emission of light during the HRP-catalyzed oxidation of luminal or other substrates. Emitted light is captured on film or by a CCD camera, for qualitative or semiquantitative analysis. Because ECL is so sensitive, it has become a popular detection method. This protocol can be modified for different membranes, antibodies, and detection systems. Optimal dilutions of the primary and secondary antibodies need to be determined empirically, but recommendations provided by the manufacturer are usually a good starting point.

Study of the Level and Structure of Population Immunity to SARS-CoV2 in the Population of the Republic of Tatarstan during the Second Peak of the Spread of COVID-19

Background. The COVID-19 pandemic has become a serious challenge for all of humanity due to the rapid global spread, high frequency of severe forms, increased mortality and required the development of new approaches to managing epidemiological processes. Serological studies are the most important tool for monitoring the infectious process, identifying risk groups, assessing the effect of vaccines used and epidemiological projections.Purpose. Conducting serological monitoring in relation to the modern transferred new coronavirus infection determining the level and structure of population immunity to SARS-CoV-2 in the population of the Republic of Tatarstan; maintaining the period of spread of COVID-19 from August to December 2020.Materials and methods. The study involved 41 444 residents of the Republic of Tatarstan, who filled out questionnaires that included clinical, anamnestic data and an epidemiological history in relation to COVID-19, who were tested for the presence of common antibodies (IgG, IgA and IgM) to the SARS coronavirus. CoV-2 by the method of enhanced chemiluminescence on the VITROS 3600 analyzer using the VITROS Anti-SARS-CoV-2 Total Reagent Pack test systems.Statistical processing was carried out by methods of variation statistics and correlation analysis according to the Pearson method using MS Excel and WinPepi.Results. Seropositivity to SARSCoV-2 in the population of the Republic of Tatarstan averaged 35.8 ± 0.235%. An increase in the level of seropositive persons was noted from 29.95 ± 0.674% in August to 68 ± 9.33% in December. The highest proportion of seropositive individuals was found in was found in the group of the able-bodied population aged 18–59 years. The average geometric titer of antibodies was 4.2 (4.09–4.31), among seropositive – 89.29 (88.13–90.46). In the social and professional structure of the population, the largest proportion of seropositive individuals was found in was found among production workers 40.35 ± 2.177, creative professions – 40 ± 9.798; health care 35.24 ± 0.389; 34.26 ± 1.218 unemployed and 33.06 ± 2.479 civil servants. Among the residents of the Republic of Tatarstan, seropositive to the SARS-CoV-2 virus, the proportion of asymptomatic forms of infection was 82.59 ± 0.446%.Сonclusions. There is a positive dynamics of seropositivity among the population of the Republic of Tatarstan. The results of sero-epidemiological monitoring can be used to predict the epidemiological situation, plan measures for specific and non-specific prophylaxis of COVID19.

Effects of Short-term and Long-termUse of Lithium on Thyroid Hormones in Nepalese Patients with Bipolar Disorder

Background: Lithium has been used for decades as mood-stabilizing agents in the management of bipolar disorder and other condition with a manic component. However, some studies have also reported varying degrees of thyroid abnormalities associated with lithium therapy and effect of such therapy on thyroid function is unclear in this part of world. Therefore, we aimed to determine the effect of long term use of lithium on thyroidfunctionin the individual with bipolar disorder receiving lithium therapy. Methods: A total of 75 bipolar disorder patients (24 males, 51 females) who are under lithium therapy and equal number of control were recruited for this study. Diagnosis of bipolar disorder was made by psychiatrist according to ICD-10-DCR guidelines and DSM-IV criteria. Serum fT3, fT4 and TSH were measured by enhanced chemiluminescence immunoassay. Statistical analysis was performed using SPSS 20.0 version. Results: The prevalence of primary hypothyroidism and subclinical hypothyroidism were found significantly increased in lithium treated group (12% and 17%, respectively) which was further increased with duration of treatment. The mean fT3 and fT4 concentration is low in lithium treated group compared to control group.Butmean TSH level was found significantlyhigher in lithium treated group compared to control (9.67±12.47 vs. 3.41±3.69, p<0.005). Conclusion: Our findings indicate that use of lithium therapy is associated with higher degree of primary hypothyroidism and subclinical hypothyroidism and female are more susceptible for the thyroid dysfunction associated with lithium therapy.

Turn on chemiluminescence-based probes for monitoring tyrosinase activity in conjunction with biological thiols

We report a chemiluminescent probe that permits the paired detection of tyrosinase (Tyr) and biological thiols. The Tyr-formed benzoquinone intermediate reacts with GSH and produces an enhanced chemiluminescence response.

Enhanced chemiluminescence determination of paracetamol

Due to the severe consequences of potential overdoses of paracetamol (PCM) on the human body, the measurement of PCM in pharmaceutical and biological samples is essential.

ROS Inhibitory Activity and Cytotoxicity Evaluation of Benzoyl, Acetyl, Alkyl Ester, and Sulfonate Ester Substituted Coumarin Derivatives

Background: A number of non-steroidal anti-inflammatory drugs (NSAIDs) including aspirin, indomethacin, ibuprofen, flufenamic acid, and phenylbutazone are being clinically used to treat inflammatory disorders. These NSAIDs are associated with serious side effects such as gastric ulceration, nephrotoxicity, and bleeding. Therefore, the identification of potent and safe therapy for inflammatory disorders is still of great interest to the medicinal chemist. Methods: A series of varyingly substituted benzoyl, acetyl, alkyl ester, and sulfonate ester substituted coumarins 1-64 were screened for the inhibition of ROS, generated from zymosan activated whole blood phagocytes, using luminol-enhanced chemiluminescence technique. Results: Among all tested compounds, 8 (IC50 = 65.0 ± 3.1 μM), 24 (IC50 = 41.8 ± 1.5 μM), 26 (IC50 = 10.6 ± 2.8 μM), 28 (IC50 = 20.9 ± 1.5 μM), and 41 (IC50 = 4.6 ± 0.3 μM) showed good anti- inflammatory potential as compared to standard antiinflammatory drug ibuprofen (IC50 = 54.3 ± 1.9 μM). Specifically, compounds 24, 26, 28, and 41 showed superior activity than standard antiinflammatory drug. Furthermore, compounds 12 (IC50 = 219.0 ± 1.4 μM), 14 (IC50 = 216.5 ± 6.2 μM), 16 (IC50 = 187.4 ± 2.2 μM), and 20 (IC50 = 196.2 ± 2.0 μM) showed moderate ROS inhibitory activity. Limited SAR study revealed that the hydroxy-substituted compound showed better ROS inhibition potential in case of 3-benzoyl and 3-ethylester coumarin derivatives. Whereas, chloro substitution was found to be important in case of 3-acetyl coumarin derivatives. Similarly, in case of sulfonate ester, chloro, and nitro groups especially at positions -4 and -3 of ring “R” played vital role in ROS inhibition. Furthermore, cytotoxicity of all active compounds was also checked on NIH-3T3 cell line. Compounds 12, 14, and 20 were found to be non-cytotoxic. Whereas, 8, 16, 24, 26, 28, and 41 were found to be very weak cytotoxic as compared to standard cycloheximide (IC50 = 0.13 ± 0.02 μM). Conclusion: Identified ROS inhibitors offer the possibility of additional modifications that could give rise to lead structures for further research in order to obtain more potent, and safer antiinflammatory agent.