While the molecular basis of granulocyte function is largely unknown, glycoproteins of the plasma membrane are believed to play a prominent role in many of the cell's functions. The study of granulocyte membrane structure and function has been hindered by difficulty in obtaining membrane components of high purity. Our long-term goal is to identify, purify, and characterize a variety of granulocyte membrane proteins and to examine their roles in granulocyte structure and function. This goal will be approached by developing a variety of monoclonal antibodies directed against specific granulocyte membrane antigens. We have already developed several such antibodies and begun characterizing their respective antigens. One of these antibodies, AHN-1, selectively inhibits phagocytosis by neutrophils and reacts with two distinct membrane proteins. We plan to purify these proteins, produce new monoclonal antibodies which react with unique epitopes on these proteins, and use these antibodies to further characterize their role in cell function. We have also found that the vast majority of monoclonal antibodies produced from mice immunized with human granulocytes are directed against the same highly antigenic carbohydrate recognized by AHN-1. Therefore granulocyte membranes will be solubilized and passed over an affinity column containing immobilized AHN-1; the column effluent containing other antigens of interest will be used as the immunogen for the production of more monoclonal antibodies. We will characterize these antibodies as we have AHN-1. Proteins bearing antigens recognized by these antibodies will be identified by polyacrylamide gel electrophoresis of proteins immunoprecipitated from detergent extracts of radiolabeled cells. To define the role of these antigens in granulocyte development and function, we will study antigen expression on hemopoietic progenitor cells and test the effects of the antibodies on measurable neutrophil functions. Proteins of interest will be purified by monoclonal antibody affinity chromatography, and, if necessary, further chromatographic techniques. These purified proteins will be used for further biochemical characterization as well as immunogens for the production of polyclonal antisera, which will recognize a variety of different antigens present on the molecule. Antibodies produced in this project should provide useful tools to study the molecular nature of normal and abnormal granulocyte surfaces and to probe the role of these antigens in cell development and function. (A)
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