title: Pre-screening for antigen detectability in cells: a TEM-based solid phase digital immunogold detection method utilizing ultra low volumes of reagents.
abstract: Rapid and sensitive pre-screening for the presence of antigens in cell samples and confirmation of reactivity of antibodies, before proceeding with electron microscopy, is highly desirable. Most of the methods developed for this purpose are generally not very efficient and suitable for dealing with very small volumes of sample and reagents. In this work we present a simple, sensitive and rapid solid phase transmission electron microscope (TEM) based method for the detection of picogram (pg) levels of soluble antigens using as little as 10 micro L of reagents. Protein was adsorbed onto grids coated with polystyrene films to form the solid phase. The presence of antigen was detected using immunogold labelling. Gold particles adhering to the film were visualized and counted in a TEM providing a digital signal. This method was 100-fold more sensitive than dot blot in detection of rabbit IgG. We have demonstrated the utility of this technique by screening for Vitreoscilla haemoglobin (VHb) antigen in cell lysates and confirming the results directly with immunogold labelling transmission electron microscopy of cell sections.
authors.affiliation: Institute of Microbial Technology, Sector 39A, Chandigarh 160036, India.
authors.forename: R
authors.initials: R
authors.lastname: Kaur
authors.forename: K L
authors.initials: KL
authors.lastname: Dikshit
authors.forename: M
authors.initials: M
authors.lastname: Raje
mesh_terms: D000818:Animals; D000906:Antibodies; D000954:Antigens, Surface / Q000032:analysis*; D004797:Enzyme-Linked Immunosorbent Assay; D006046:Gold / Q000737:chemistry* / Q000276:immunology; D015151:Immunoblotting; D007074:Immunoglobulin G; D007158:Immunologic Techniques; D008854:Microscopy, Electron / Q000379:methods*; D016253:Microscopy, Immunoelectron / Q000379:methods; D011817:Rabbits
chemical_list: D000906:Antibodies; D000954:Antigens, Surface; D007074:Immunoglobulin G; D006046:Gold