Highly specific and sensitive anti-Strongyloides venezuelensis IgY antibodies applied to the human strongyloidiasis immunodiagnosis.

Due to the epidemiological problem of the neglected condition of human strongyloidiasis, rapid and effective diagnosis is extremely important, with the development of new diagnostic tools being essential to reduce infections and chronic cases. Avian immunoglobulin Y (IgY) technology is an alternative for antibody production that has high specificity and profitability. This study aimed to produce and fractionate IgY antibodies from the egg yolks of hens that were immunized with the total antigenic extracts of Strongyloides venezuelensis infectious filariform larvae (iL3) and parthenogenetic females (pF). IgY antibodies were then evaluated by their recognition of antigenic proteins, evolutive helminth forms, and serological diagnosis of human strongyloidiasis by the detection of immune complexes in serum samples. Egg yolks were fractionated to obtain IgY antibodies by thiophilic interaction chromatography. Immune complex detection in serum samples showed diagnostic values for anti-iL3 IgY and anti-pF IgY antibodies at 95.56% and 88.89% sensitivity and 95.56% and 91.11% specificity, respectively. Therefore, IgY technology is a promising tool for the detection of blood circulating Strongyloides antigens, with possible application as a serological diagnostic method.

Structure-Based Discovery of Thiosemicarbazone Metalloproteinase Inhibitors for Hemorrhage Treatment in Snakebites.

The venoms of snakes are composed by many toxins, which are responsible for various toxic effects including intense pain, bleeding disorders, and local tissue damage caused by hemorrhage and necrosis. The snake venom metalloproteinases (SVMPs) are proteolytic zinc-dependent enzymes acting in different hemostatic mechanisms. In this work, a structure-based molecular modeling strategy was used for the rational design, by means of a homology 3D model of an SVMP isolated from Bothrops pauloensis venom (BpMP-I), followed by synthesis and in vitro evaluation of new thiosemicarbazones as the first inhibitors of the B. pauloensis SVMP. Besides being effective for the SVMP inhibition, two molecules were shown to be effective also in vivo, inhibiting hemorrhage caused by the B. pauloensis whole venom. Docking studies on metalloproteinases from other snake species suggest that the thiosemicarbazones activity is not confined to BpMP-I, but seems to be a common feature of metzincins.

A New Phospholipase A2 from Lachesis muta rhombeata: Purification, Biochemical and Comparative Characterization with Crotoxin B.

Phospholipases A2 (PLA2s) are enzymes responsible for inflammatory effects, edema formation, myotoxicity, neurotoxicity and other manifestations from envenoming. In this paper we report the isolation and biochemical characterization of Lmr-PLA2, the first acidic PLA2 found in Lachesis muta rhombeata venom. Furthermore, this study compared biological effects of Lmr-PLA2 and crotoxin B (CB), a PLA2 from Crotalus durissus terrificus venom. Lmr-PLA2 was isolated by molecular exclusion and reversed phase chromatography. The purified enzyme showed a molecular mass of 13,975 Da, pI of 5.46 and its partial amino acid sequence showed a high identity with PLA2s already described in the literature. In addition, this enzyme possesses the residue D49 in its amino acid sequence, indicating that it is a catalytically active PLA2. Lmr-PLA2 presented high phospholipase activity and was able to inhibit platelet aggregation. Studies of biochemical characterization of new PLA2s, as Lmr-PLA2, are relevant since they help to clarify the structure-function relationship of this important class of toxins.

Produção e purificação de imunoglobulinas Y policlonais anti-Leptospira spp / Production and purification of polyclonal anti-Leptospira immunoglobulin Y

Objetivou-se verificar se galinhas imunizadas com uma solução de Leptospira interrogans inativadas e proteínas de membrana externa do sorovar Hardjo, poderiam produzir anticorpos policlonais específicos anti-leptospiras, detectáveis em testes ELISA. Foram imunizados oito galinhas com 25 semanas de idade, da raça White Leghorn, sendo três imunizadas com uma suspensão de leptospiras inativadas, três com uma solução de proteínas de membrana externa extraída do sorovar Hardjo e duas controle. Coletas de sangue foram realizadas quinzenalmente e de ovos diariamente. A IgY foi purificada a partir da gema dos ovos utilizando para a delipidação o método de diluição em água ácida e a precipitação com sulfato de amônio. Nos testes ELISA realizados para verificar a especificidade da IgY, foi demonstrada a produção de anticorpos anti-Leptospira, tanto no soro quanto nas gemas purificadas. O pico de produção de anticorpos específicos ocorreu na 5º semana após a primeira imunização. Ficou demonstrada a possibilidade da indução da produção de anticorpos específicos em galinhas imunizadas com leptospiras do sorovar Hardjo inativadas, bem como, com proteínas de membrana externa (PME) extraidas desse sorovar. As galinhas imunizadas com uma suspensão de leptospiras inativadas ou com PME de Leptospira interrogans do sorovar Hardjo produziram anticorpos reativos a PME Hardjo detectáves por teste ELISA.

The aim was to determine whether hens immunized with an inactivated suspension of Leptospira and a solution of outer membrane proteins extracted from the serovar Hardjo, could produce specific polyclonal antibodies to Leptospira, detected in ELISA assay. Eight hens White Leghorn race with 25-weeks-old were immunized, three with an inactivated suspension of Leptospira, three with a solution of outer membrane proteins (OMP) extracted from the serovar Hardjo and two controls immunized with saline. Blood samples were collected fortnightly and eggs daily. The IgY was purified from the egg yolk using the method for the delipidation of dilution with water acidic and ammonium sulfate precipitation. The ELISA assay was performed to verify the specificity of the IgY, these was possible to observe the production of specific antibody to Leptospira both in serum and purified egg yolk. The specific antibody titers peaked in the fifth week post immunization. The production of polyclonal IgY was effective for producing high titers of specific antibodies.