The use of an excreted superoxide dismutase in an ELISA and Western blotting for the diagnosis of Leishmania (Leishmania) infantum naturally infected dogs

Abstract

An excreted iron superoxide dismutase of pI 3.75 and a molecular mass of approximately 25 kDa was partially purified by QAE Sephadex ion-exchange chromatography from the in vitro culture of Leishmania (Leishmania) infantum. This enzyme was detected by enzyme-linked immunosorbent assay and Western blot of anti-L. infantum antibodies in dog serum. For the determination of the sensitivity and specificity of this protein, the results using the complete-parasite antigen fraction were taken as references. For this, 39 sera were assayed in dogs from different Spanish provinces. By Western blot, at a dilution of 1:250, 82% of the sera were positive when superoxide dismutase excreted was used as the antigen, against 56.4% positivity when the complete parasite was used as the antigen. These findings support the results of a previous study, indicating that the superoxide dismutase excreted can be useful in diagnosing L. (L.) infantum.

Anticuerpos de Trypanosoma cruzi, Leishmania mexicana y Leishmania braziliensis en perros domiciliados de Tabasco, México

“…Previo al lavado, la membrana se incubó durante dos horas más a temperatura ambiente con el segundo anticuerpo, anti-perro de inmunoglobulina G conjugada con peroxidasa (Sigma Immunochemicals; dilución 1/1000). Esta última se lavó con sustrato Diaminobencidina (0.5 mg/ml en un tampón de Tris/HCl 0.1 M, pH 7.4 que contiene 1/5000 H 2 O 2 [10 v/v]) y se añadió a la reacción, que se detuvo con una serie de lavados con agua destilada de acuerdo a la técnica sugerida por Marín et al (13).…”

“…Before the washing, the membrane was incubated for two hours mass room temperature with the second antibody, anti-dog immunoglobulin G conjugated with peroxidase (Sigma Immunochemicals; dilution 1/1000. The latter was washed with diaminobenzidine substrate (0.5 mg/ml in Tris buffer/HCl 0.1M pH 7.4 containing H2O2 1/5000 [10 v/v]) and was added to the reaction, which was stopped with a number of washings with distilled water according to the technique suggested by Marin et al (13).…”

Trypanosoma cruzi: Seroprevalence Detection in Suburban Population of Santiago de Querétaro (Mexico)

“…Afterwards, the parasites were again grown in MTL medium without serum for 24 h; the supernatant was collected by centrifugation at 1500 rpm for 10 min and then passed through a filter of 0.45- μ m pore size, and solid ammonium sulphate added. The protein fraction, which precipitated at between 35 and 85% salt concentration, was centrifuged (9000 rpm for 20 min at 4°C), redissolved in 2.5 mL of 20 mM potassium phosphate buffer (pH 7.8) containing 1 mM EDTA (Buffer 2), and dialysed in a Sephadex G-25 column (Pharmacia, PD 10), previously balanced with Buffer 2, bringing it to a final volume of 2.5 mL (fraction SODeCRU) [16]. …”

An Iron-Superoxide Dismutase Antigen-Based Serological Screening of Dogs Indicates Their Potential Role in the Transmission of Cutaneous Leishmaniasis and Trypanosomiasis in Yucatan, Mexico

“…The protein content was determined using the Bio-Rad test, based on the Bradford method (Sigma Immunochemical), with bovine serum albumin as standard (Marín et al 2007). …”

“…One such candidate antigen is excreted iron-superoxide dismutase (Fe-SODe), which has been found to be highly immunogenic and specific, thus making it a useful molecular marker for diagnosing infection with these parasites. Indeed, this antigen has been shown to provide good results in the diagnosis of visceral canine leishmaniasis (Marín et al 2007) and both cutaneous and mucocutaneous leishmaniasis in human serum in Peru ).…”