This project addresses the commercial potential of performing cell surface marker analysis using monoclonal antibodies tagged with a variety of heavy metal colloids of varying size and color. Currently, cell marker analysis, proven to be clinically important in the detection, diagnosis, and monitoring of human diseases, are performed either manually via fluorescent/light microscopy with its imprecise identification and statistical counting errors or by flow cytometric analysis with a costly capital equipment outlay. The commercial availability of easily visualized, nonfluorescent monoclonal reagents will permit clinical hospital laboratories to perform cell marker studies on existing capital equipment and image analysis systems, which perform routine blood cell differentials in many labs. These image analysis systems are not capable of fluorescence detection which negates their use with existing fluorescent monoclonals.One aspect of this project will involve the development of such nonfluorescent heavy metal colloid tags that, because of their variable size and color differences, may be used in combination, resulting in savings to the customer. These will be coupled to a variety of commonly used and specialized monoclonal reagents against human leukocytes. This technique will permit direct visualization of the cellular elements spread in a monolayer or thin section manner on standard microscopic slides, and will permit instant cytomorphologic correlation with the cell in question. All new reagents developed will be evaluated by manual and flow cytornetric methodologies in a double bind test. Once developed the reagents will be marketed for cell marker analysis using direct visual assessment (manual and/or image analysis systems) as reagents or in kit form for use in the routine clinical laboratory.A second aspect of this project will attempt to develop and characterize selected murine monoclonal antibodies to human tumor-specific antigens. All resultant monoclonals will be carefully screened and correlated with histopathologic interpretations. The resultant monocionals will be tagged with the new colloids and with traditional fluorescent dyes for use in manual, flow, and image cytornetric systems.These studies have the potential to improve the level of testing sensitivity for cell marker analysis by manual methodologies through the elimination of problems related to human (eye) detection of fluorescence intensity, as the colloids are assessed by light techniques, and will use existing clinical lab equipment.National Institute Of General Medical Sciences
