Simplification and Standardization of Dot-ELISA for Human Schistosomiasis mansoni

Abstract

Dot-ELISA, a technique that shares the same principles as the enzyme immunoassay, is useful for detection of anti-Schistosoma mansoni antibodies in the sera of patients with Schistosoma mansoni infections. The antigens were fixed to the nitrocellulose strips, blocked with 1% bovine serum albumin in 0.05% Tween 20. Patient sera (40) and normal laboratory personnel sera (9) were applied to the sheet directly, without cutting the strips into small discs. The nitrocellulose sheets are kept in a humid chamber for 30 min and then washed. After incubation with peroxidase-conjugated goat anti-human antibody, washing, and addition of substrate, positive reactions appear as brown dots against the white background. The room temperature assay takes about 2 hr. The optimum antigen concentration is 20-80 ng per dot and the optimum serum dilution is 1:100-1:400. The sensitivity and specificity of the assay are 90-95% and 90%, respectively. The level of positivity of the dot-ELISA by an arbitrary scale compares with standard micro-ELISA. The single positive reaction in a normal serum sample in dot-ELISA is also positive in micro-ELISA. Cross-reactivity between the S. mansoni antigen and human fascioliasis sera was noticed in 2 out of 8 patient sera. Good correlation between the arbitrary level of dot-ELISA and the absorbance of standardized micro-ELISA shows that the dot-ELISA is useful both for laboratory and field studies.

Evaluation of the dot enzyme-linked immunosorbent assay in comparison with standard ELISA for the immunodiagnosis of human toxocariasis

“…We found that the best result to fix TES antigen to NC sheets was obtained with sodium bicarbonate buffer with 2% OVA which could be responsible for the need for lesser antigen concentration than previously reported (Camargo et al 1992). Simplification of the procedure can be achieved by the use of large NC strips (Boctor et al 1987), mass production of NC strips (Janitschke et al 1987), and the use of the commercially available Bio-dot apparatus (Chan & Ko 1988). Another advantage of this method is that it is simple, rapid (about 2 h), does not need expensive equipment, results can be read visually, a large number of samples can be assayed, and the test is performed at room temperature.…”

“…Dot-ELISA test, a modification of the standard ELISA test, offers a simple and less expensive tool for toxocariasis detection. The dot-ELISA has been successfully adapted for the detection of parasitic diseases in humans as leishmaniasis, schistosomiasis, toxoplasmosis, and hydatidosis (Pappas et al 1984, Boctor et al 1987, Rogan et al 1991, Elsaid et al 1995. A dot-ELISA for diagnosis of human toxocariasis was described (Camargo et al 1992).…”

“…Dot-ELISA procedure -The optimal conditions for dot-ELISA were standardized according to the procedures described by Boctor et al (1987) with some modifications. Strips of NC paper (Sigma), 1 cm wide and 6 cm long were placed on a microtiter 96-well plastic plate and after a slight pressure round areas were marked.…”

“…The cut-off point was set at the mean optical density (OD) of the negative reference serum, plus 3 standard deviation (SD). Serum samples with OD > 0.26 (cut-off) were considered as reactives.Dot-ELISA procedure -The optimal conditions for dot-ELISA were standardized according to the procedures described by Boctor et al (1987) with some modifications. Strips of NC paper (Sigma), 1 cm wide and 6 cm long were placed on a microtiter 96-well plastic plate and after a slight pressure round areas were marked.…”

Efficacy of Dot-ELISA using different antigens in detecting anti-schistosome antibodies among bovines in field conditions

“…Immunodiagnostic methods based on the detection of antibodies continued to be the most effective and practical method for diagnosis of imported schistosomosis (Tsang and Wilkins 1997). Serodiagnosis of S. haematobium (Khalil et al 1993), S. japonicum (Hwu et al 1978) and S.mansoni (Boctor et al 1987) have been attempted using various immunodiagnostic assays with antigens from adult worms and eggs. Enzyme linked immunosorbent assay (ELISA) is a simple, sensitive and rapid serodiagnostic technique which has been widely used for detection of the circulating antibodies and antigens related to many parasitic diseases (Voller et al 1976).…”