A solid-phase method for evaluation of gold conjugate used in quantitative detection of antigen by immunogold-labeling electron microscopy Ramandeep Kaur 1 , Manoj Raje * Institute of Microbial Technology, Sector 39 A, Chandigarh 160036, India Received 14 March 2003; received in revised form 15 May 2003; accepted 10 June 2003 Abstract 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. D 2003 Elsevier B.V. All rights reserved. Keywords: Gold conjugate; Immunogold labeling; Transmission electron microscopy; Optimization 1. Introduction Immunogold-labeling electron microscopy is a powerful technique for the localization and quantifi- cation of antigens in cells and tissues. A crucial factor for the successful application of this method is in the ability to decide upon the use of active reagents at suitable concentrations yielding minimum nonspecific binding (NSB) while retaining a high level of signal. As immuno-electron microscopy experiments are tedious, it is usually a common practice in most laboratories to carry out some level 0022-1759/$ - see front matter D 2003 Elsevier B.V. All rights reserved. doi:10.1016/S0022-1759(03)00254-0 Abbreviations: TEM, transmission electron microscope; NSB, nonspecific binding; S/N ratio, signal-to-noise ratio; VHb, Vitre- oscilla Hemoglobin protein; anti-VHb, polyclonal antiserum against VHb protein; PBS, phosphate-buffered saline; PBST, PBS contain- ing 0.01% Tween-20; GP/l 2 , gold particles/l 2 . * Corresponding author. Tel.: +91-172-695215; fax: +91-172- 690632/690585. E-mail address: manoj@imtech.res.in (M. Raje). 1 Current address: Department of Biotechnology, Guru Nanak Dev University, Amritsar-143005, India. www.elsevier.com/locate/jim Journal of Immunological Methods 279 (2003) 33 – 40