A solid-phase method for evaluation of gold conjugate used in quantitative detection of antigen by immunogold-labeling electron microscopy.
Rapid and sensitive screening for confirming the reactivity of reagents, before proceeding for electron microscopy, is highly desirable. ELISA-based methods have been shown to be highly efficient and successful for rapid prescreening and optimization of immunological as well as sample-processing reagents for the sensitive detection and quantitation of antigen by electron microscopy. The drawback of these methods lies in their inability to provide any information regarding the gold conjugate used for the final observed and measured signal. In this work, we demonstrate a simple and rapid, solid-phase method in ELISA format that is also suitable for evaluation and optimization of the gold conjugate. We have demonstrated the utility of this technique by screening for Vitreoscilla hemoglobin (VHb) antigen in cell lysates and confirming the results directly with immunogold-labeling transmission electron microscopy (TEM) of cell sections. The sensitivity of detection and quantitation of antigens by immuno-electron microscopy depends upon the assay procedure being optimized to obtain the best possible signal. Our study indicates that evaluation of gold conjugate by the solid-phase assay could help in the rapid optimization of this reagent for immunogold localization and quantification of antigens by TEM.
Kaur
Ramandeep
R
Institute of Microbial Technology, Sector 39 A, 160036 Chandigarh, India.
Raje
Manoj
M
D000941:Antigens / Q000032:analysis*; D004926:Escherichia coli / Q000648:ultrastructure; D006046:Gold; D007150:Immunohistochemistry / Q000379:methods*; D008854:Microscopy, Electron / Q000379:methods*
D000941:Antigens; D006046:Gold