Sensitive solid-phase immune electron microscopy double-antibody technique with gold-immunoglobulin G complexes for detecting rotavirus in cell culture and feces

Abstract

A new solid-phase immune electron microscopy double-antibody colloidal-gold technique (SPIEMDAGT) was developed and compared with direct electron microscopy, direct immune electron microscopy, and enzyme immunoassay for detecting rotavirus. Guinea pig and rabbit antirotavirus antisera were used as capture and detector antibodies, respectively, and goat anti-rabbit immunoglobulin G-gold complexes were employed as a label. Animal rotavirus in cell culture media and human virus in stool specimens were detected by this method. On average, SPIEMDAGT detected 800 times more virus particles than direct electron microscopy and 45 times more particles than direct immune electron microscopy and yielded 20% more positives than enzyme immunoassay. SPIEMDAGT could detect not only viral antigen associated with morphologically recognizable particles but also antigen present when whole virus particles were not visible.

Comparison of Polymerase Chain Reaction and Dot Hybridization with Enzyme‐Linked Immunoassay, Virological Examination and Polyacrylamide Gel Electrophoresis for the Detection of Porcine Rotavirus in Faecal Specimens

“…Rapid diagnosis of rotaviral infections is based on revealing the presence of rotaviral particles, their antigens or dsRNA in the clinical material samples at the early stage of infection. For its advantage, numerous methods were worked out, such as immunoenzymatic test using polyclonal and monoclonal antibodies (Dennehy et al, 1988;Pacini et al, 1988;Thomas et al, 1988;Brooks et al, 1989;Kok and Burrell 1989), agglutination using latex (Hughes et al, 1984), molecular hybridization (Arens and Swierkosz, 1989;Olive and Sethi, 1989), electrophoresis in polyacrylamide gel (Herring et al, 1982), the technique of direct electron microscopy as well as applying immunological aggregation of the virus (Benfield et al, 1984;Wu et al 1990) and recently RT-PCR technique (Gouvea et al, 1990;Wilde et al, 1990). Each of the above mentioned methods has its advantages and disadvantages.…”

Diversidade genética dos rotavírus humanos detectados em pacientes com diarréia aguda no Estado de São Paulo, no período de 1996 a 2006.

“…A MED foi a primeira técnica utilizada para a detecção de partículas de rotavírus pelo exame direto em amostras fecais DAVIS, 1973). A IME apresenta maior especificidade e sensibilidade comparada à MED, pois esta técnica utiliza anticorpos específicos para rotavírus (WU et al, 1990). A MED e IME são ferramentas importantes no diagnóstico dos rotavírus e outros vírus associados à doença diarréica, porém seu uso rotineiro se torna inviável devido à necessidade de técnicos altamente especializados e ao custo elevado do aparelho (DENNEHY et al, 1988 (YOLKEN et al, 1978;PEREIRA et al, 1985;DENNEHY et al, 1988).…”