PURIFICAÇÃO E CARACTERIZAÇÃO DE SUBTIPOS SLOW-MOVING E FAST-MOVING DE IMUNOGLOBULINA G A PARTIR DE SORO DE COELHO

A imunoglobulina G é a principal classe de anticorpos constituintes dos fluidos sanguíneos animais, tem massa de 150kDa e representa uma das estruturas mais importantes no sistema de defesa. Estas moléculas são largamente utilizadas em aplicações terapêuticas, imunodiagnósticos e purificação de outros anticorpos e antígenos em processos biomédicos, já que a purificação de proteínas limita os riscos de efeitos colaterais nos seres inoculados. Neste trabalho, imunoglobulinas G provenientes de soro de coelho foram obtidas por precipitação com sulfato de amônio saturado. A massa de IgG obtida correspondeu à cerca de 72,5% de todas as classes de imunoglobulinas presentes no soro. Em seguida, a IgG pura foi submetida à cromatografia de troca iônica em coluna de DEAE-Sepharose, processo que segregou os subtipos slow-moving (durante a eluição com tampão fosfato pH6,3) e fast-moving (fração eluída com tampão fosfato pH8,0). A caracterização da imunoglobulina G pura foi dada por eletroforese em gel SDS-PAGE 10%, onde a amostra não reduzida revelou banda única de peso molecular igual a 150kDa e a amostra reduzida com 2-mercapto-etanol revelou banda de 50kDa referente à cadeia pesada e 25kDa das cadeias leves.AbstractImmunoglobulin G is the main class of antibodies of the animal blood components, its mass is equal to 150kDa and it represents one of the most important structures of defense system. These molecules are widely applied in therapeutic uses, immunodiagnostics and in other antibodies and antigens purification, since proteins purification limits risks of collateral effects in inoculated bodies. In this work, immunoglobulins G from rabbit serum were obtained by precipitation with saturated ammonium sulfate. The IgG's mass obtained corresponded to about 72,5% of all the immunoglobulins' classes of the serum. After, the pure IgG was submitted to a ion-exchange chromatography in a DEAE-Sepharose column, which segregates the slow-moving piece (during the phosphate buffer pH6,3 elution) and fast-moving piece (fraction eluted with phosphate buffer pH8,0). The characterization of the pure immunoglobulin G was carried out by a 10% SDS-PAGE electrophoresis, where the non-reduced sample showed a single band with molecular weight of 150kDa and the sample that was reduced with 2-mercaptoethanol revealed bands with 50kDa related to the heavy chains and 25kDa of the light chains

Padronização de uma técnica de congelamento de sêmen em cães

Várias pesquisas têm sido desenvolvidas com o intuito de melhorar a técnica da criopreservação de sêmen em cães. O objetivo deste trabalho foi estabelecer o meio diluidor de congelamento do sêmen canino mais adequado para um protocolo de congelamento realizado em máquina computadorizada, além de otimizar a temperatura de descongelamento. Foram usados cinco cães e coletados três ejaculados/animal. Foram testados os meios Tris-cítrico (G1), Lactose-gema (G2) e INRA 82 (G3) com 5% de glicerol. Alíquotas de sêmen foram diluídas em cada meio e foram congeladas em máquina computadorizada programada para realizar uma curva de resfriamento de -0,5ºC/min, de 25-29ºC até 5ºC, período de equilíbrio de 1h a 5ºC, seguida da curva de congelamento de -20ºC/min, de 5ºC até -120ºC. Foram testadas duas temperaturas de descongelamento: A) 37ºC/30s e B) 52ºC/10s, seguida de 37ºC/30s. Os espermatozóides foram avaliados quanto à motilidade progressiva, morfologia, reatividade ao teste hiposmótico e integridade de membranas avaliadas pelo uso de fluorescência (CFDA/IP). Os meios de congelamento Tris-cítrico e Lactose-gema foram superiores ao meio INRA quanto aos parâmetros de motilidade e morfologia espermática (p0,05). O descongelamento a 52ºC resultou no aparecimento de maior porcentagem de anormalidades acrossomais que o descongelamento a 37ºC (

Non-Enzymatic Impedimetric Sensor Based on 3-Aminophenylboronic Acid Functionalized Screen-Printed Carbon Electrode for Highly Sensitive Glucose Detection

A highly sensitive glucose sensor was prepared by a one-step method using 3-aminophenyl boronic acid as a unit of recognition and a screen-printed carbon electrode (SPCE) as an electrochemical transducer. Scanning Electron Microscopy confirmed the success of the functionalization of the SPCE due to the presence of clusters of boronic acid distributed on the carbon surface. In agreement with the Electrochemical Impedance Spectroscopy (EIS) tests performed before and after the functionalization, Cyclic Voltammetry results indicated that the electroactivity of the electrode decreased 37.9% owing to the presence of the poly phenylboronic acid on the electrode surface. EIS revealed that the sensor was capable to selectively detect glucose at a broad range of concentrations (limit of detection of 8.53 × 10−9 M), not recognizing fructose and sucrose. The device presented a stable impedimetric response when immediately prepared but suffered the influence of the storage time and some interfering species (dopamine, NaCl and animal serum). The response time at optimized conditions was estimated to be equal to 4.0 ± 0.6 s

IMMUNOSENSOR BASED ON INK PRINTED ELECTRODE FOR STAPHYLOCOCCAL ENTEROTOXIN DETECTIONdoi: http://dx.doi.org/10.5892/ruvrd.v12i1.1400

Staphylococcal enterotoxin is one of the more aggressive enterotoxins produced by Staphylococcus aureus strains and it is a common cause of food poisoning. Analytical methods that are sensitive, low cost and easy to use are needed to evaluate the food quality. This work describes the development of a label free immunosensor based on screen-printed AuNPs/carbon and the characterization of its analytical response for staphylococcal enterotoxin B (SEB) detection. The biosensor was constructed from self-assembled monolayer of thiols and protein A for the oriented immobilization of the polyclonal antibodies against SEB. As electrons mediator, potassium ferrocyanide was used. The electrochemical detection was direct with the parameters following: -0.2 to 0.6 V with the pulse amplitude of 0.075 V and the pulse width of 75 ms. The immunosensor showed detection and quantification limits of 0.4 µg mL-1 and 1.6 µg mL-1,respectively. The immunosensor showed quite satisfactory performance in contaminated and non-contaminated cheese samples

Fractionation of Apis mellifera venom by means of ultrafiltration: removal of phospholipase A 2

<div><p>Abstract The fractionation of apitoxin (bee venom) by means of a commercial 10 kDa ultrafiltration membrane was investigated aiming at the removal of phospholipase A2, the main allergenic substance. The feed content was varied from 1 to 50 g apitoxin/L, in deionized water, and caused changes in membrane flux and rejection, due to concentration polarization. The increase in pressure difference and stirring rate improved the flux through the membrane. The best result was achieved for 1 g apitoxin/L in feed stream, with a pressure difference of 220 kPa, and 750 rpm, with a permeate flux of 103 kg/m2h. The use of ultrafiltration was efficient to improve the permeate safety since biological tests revealed that the remaining enzyme lost its ability to catalyze the hydrolysis of phospholipids.</p></div

<b>Antibacterial activity of different types of snake venom from the Viperidae family against <i>Staphylococcus aureus

Toxins and venoms produced by living organisms have exhibited a variety of biological activities against microorganisms. In this study, we tested seven snake venoms from the family Viperidae for antibacterial activity and the activities of reversal of antibiotic resistance and inhibition of biofilm formation against 22 clinical isolates of Staphylococcus aureus. Bothrops moojeni venom exhibited anti staphylococcal activity with the lowest mean value of minimum inhibitory concentration (MIC). Moreover, reversal of antibiotic resistance was observed for combinations of B. moojeni venom (½ x MIC) and norfloxacin or ampicillin (both ½ x MIC) for 86.4% and 50% of the isolates, respectively. B. moojeni venom alone at ½ MIC inhibited 90% of biofilm formation, whereas in combination with ciprofloxacin, both at ½ MIC, a reduction on the NorA efflux pump activity was observed. The detection of in vitro mutants colonies of S. aureus resistant to B. moojeni venom was low and they did not survive. A phospholipase A2 was purified from the venom of B. moojeni and displayed anti-staphylococcal activity when tested alone or in combination with ciprofloxacin. The results presented here will contribute to the search for new antimicrobial agents against resistant S. aureus.