Evaluation of immunoadsorbent electron microscopic techniques for detection of Sindbis virus.
Two immunosorbent electron microscopic techniques (ISEM), the protein A coated grid technique (PA-CGT) and the antibody coated grid technique (AB-CGT) were applied and evaluated for the detection of Sindbis virus from infected tissue culture fluids. At optimal conditions, the efficiency of trapping the virions was only about 1.5 higher with the PA-CGT as compared to the AB-CGT, but the PA-CGT was less dependent on the antiserum dilution used in the test. Both methods were suitable for quantitation experiments, since the number of virions trapped was proportional to the virus concentration. The influence of virus incubation time and temperatures, staining solutions, buffers and washing procedures on the trapping efficiency and specificity was further studied with the PA-CGT. Maximal trapping on coated grids was obtained after 3 h incubation of the virus. At room temperature, less debris was found on the grids, as compared to 37 degrees C, and the numbers of virions counted were only slightly lower. The optimal staining solution was alcohol uranyl acetate. The specificity of the PA-CGT was dependent on washing steps with phosphate buffered saline containing bovine serum albumin. With the standard procedure, at room temperature around 3 X 10(7) virions/ml (1 X 10(6) PFU/ml) were specifically detected in about 1.5 h.
D000818:Animals; D007163:Immunosorbent Techniques; D008854:Microscopy, Electron; D011817:Rabbits; D012845:Sindbis Virus / Q000302:isolation & purification*; D013194:Staining and Labeling; D013205:Staphylococcal Protein A; D013696:Temperature; D013997:Time Factors
D013205:Staphylococcal Protein A