The present work describes the isolation and purification of two Leishmania chagasi (= syn. Leishmania infantum) recombinant proteins, rLci2B and rLci1A, and their use in the development of an immunoassay for the diagnostic of canine leishmaniasis. After protein expression and cell disruption, rLci2B was purified by immobilized metal affinity chromatography followed by size exclusion chromatography, whereas rLci1A, expressed as an inclusion body, was treated with urea and purified by anion-exchange chromatography. Homogeneities were ascertained by denaturing gel electrophoresis (MW (rLci2B) = 46,370; MW(rLci1A) = 88,400), isoelectric focusing (pI (rLci2B) = 5·91; pI (rLci1A) = 6·01) and Western blot. An indirect ELISA was developed using the purified antigens rLci2B and rLci1A and a leishmaniasis canine serum panel (n = 256). The ELISA showed 100% sensitivity and 95% specificity for rLci2B and 96% sensitivity and 92% specificity for rLci1A. The purified proteins did not present cross-reactivity with sera from dogs infected with Trypanosoma caninum, Babesia canis and Ehrlichia canis. Cross-reaction was verified with sera from dogs infected with Leishmania brasiliensis (11·7% for rLci2B and 2·9% for rLci1A). Based on ELISA results, it is suggested the use of rLci2B and rLci1A as antigens in an alternative serological assay for diagnostic of canine leishmania.
In Brazil, polysaccharide-protein conjugate vaccine against Neisseria meningitidis group C (MenCPS-TT) using hydrazine-activated-tetanus toxoid (TT) as a carrier protein has been developed. Because of the toxicity of hydrazine in humans, it is necessary to monitor this substance's process control step during the vaccine production. The electroanalytical methodology was developed and validated for the determination of hydrazine during the process control of MenCPS-TT vaccine production by differential pulse polarography. The reduction potential was -0.95 V in acetone and sulphuric acid solution. The method presented linear range between 30 and 150 microgL(-1)and recovery of 93.5+/-0.8%.
Introduction: Coronavirus 2019 disease , caused by the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is considered the worst pandemic disease of the current millennium. In some cases, it causes severe neurological complications, such as encephalitis and Guilain-Barré syndrome. Therapeutic strategies that clearly inhibit the effects of this virus in the brain still need to be achieved. Therefore, polymeric nanoparticles (PNPs) have been shown to be a promising material in the biomedical area due to the targeted administration of therapeutics (e.g. antivirals) for specific areas of the body such as the brain. So, this work describes development of encapsulated polycaprolactone (PCL) nanoparticles against SARS-CoV-2 on infected brain cells.Objective: Synthesize PCL-carrier nanoparticles against the SARS-CoV-2 virus and evaluate their biological activities.Methodology: 1) Synthesis: The PNPs suspensions were obtained by the unique method of emulsion and solvent evaporation, using a 4:1 ratio of polymer and drug, which was selected in previous studies. The organic solvent was then removed by vacuum evaporation and the PNPs were washed by the ultrafiltration method. 2) Characterization: The mean diameter and zeta potential of the nanoparticles were determined by Dynamic Light Scattering method using Zetasizer TM Nano ZS 90 equipment. The amount of free antiviral was estimated by UV-visible spectroscopy and the encapsulation efficiency (EE%) was calculated by subtraction the amount of free drug released from the total of the inserted drug. Biologic function was evaluated in vitro by using Vero E6 cells. Results:The average size of PNPs was estimated as 173.3 ± 0.08 nm with a polydispersivity index (PDI) of 0.07 suggesting a narrow size distribution and high homogeneity. In addition, zeta potential was slightly negative due to dissociation of the PCL functional groups on the particle surface. The concentration of the free drug releasing, calculated as encapsulation efficiency was estimated as 69.0%. Also, in vitro assay showed to be non toxic and able to inhibit viral replication by 40%. Conclusion:The production of PNPs by the single emulsion and solvent evaporation method was efficient for the production of carrier particles with nanometric scale. The sample showed size within desired range which would allow targeting to the brain. In addition, the encapsulation efficiency showed that high level of the drug remains encapsulated. Therefore, we were able to obtain compatible nanoparticles for use in the brain in which preliminary in vitro tests proved to be non-toxic and able to inhibit viral replication even at low doses of antiviral.
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