Quantitation of immunogold with an interactive image analysis system. A new, practical approach to antibody-induced modulation, internalization and intracellular transport of surface antigens in viable hematopoietic cells.
This work aimed to evaluate the value of computerized quantitative analysis in ultrastructural studies on internalization and intracellular transport of leukocyte antigens traced with immunogold in viable lymphoid cells. The time of analysis and the results were compared with those obtained with a standard method based on counting gold particles on electron micrographs. A commercially available, personal computer-based, single-screen system was used for capturing and processing the ultrastructural images, counting gold particles and presenting data. The viable lymphoblasts of RAJI and NALM-6 cell lines were labeled with B4 (anti-CD19) murine monoclonal antibody followed by rabbit antimouse immunoglobulins coupled to 12.8-nm colloidal gold, cultured for six hours to allow internalization of CD19 antigen, collected after various periods of time and processed for electron microscopy. Each sample was analyzed by counting the gold particles on 30 randomly chosen, equatorial cell sections. Surface-bound particles and those located within certain intracellular compartments (that is, cytoplasmic vesicles, multivesicular bodies and lysosomes) were counted separately. No significant differences were found between the results obtained using electron micrographs and those yielded by on-screen analysis of digitized images. However, the time of analysis varied considerably--approximately eight and two hours, respectively. Also, using computerized analysis saved the cost of photographic processing. In conclusion, the system provided a reliable, rapid and inexpensive alternative for quantitation of immunogold-labeled leukocyte antigens at the ultrastructural level.
Pulczynski
S
S
University Department of Pathology, Aarhus Amtssygehus, Denmark.
Jensen
O M
OM
D000906:Antibodies / Q000276:immunology; D015703:Antigens, CD / Q000032:analysis* / Q000276:immunology; D018941:Antigens, CD19; D000944:Antigens, Differentiation, B-Lymphocyte / Q000032:analysis* / Q000276:immunology; D001402:B-Lymphocytes; D002460:Cell Line; D006410:Hematopoiesis; D006801:Humans; D007091:Image Processing, Computer-Assisted; D007150:Immunohistochemistry
D000906:Antibodies; D015703:Antigens, CD; D018941:Antigens, CD19; D000944:Antigens, Differentiation, B-Lymphocyte