ANALYSIS OF THE EXPRESSION OF NB1 ANTIGEN USING 2 MONOCLONAL-ANTIBODIES

Background: Neutrophil-specific antigen NB1 is expressed on neutrophil subpopulations in 97 percent of healthy individuals and is located on 56- to 64-kDa glycoprotein. While the molecule carrying NB1 has been identified, the nature of the NB1 epitope has not been well characteri zed. Study Design and Methods: Two monoclonal antibodies (MoAbs), 1B5 and the recently produced 7D8, and four alloantibodies, all specific f ar NB1, were used to investigate the expression of NB1 on neutrophils from several donors. Results: MoAb 708 was shown to be specific for NB I, It reacted with NB1-positive neutrophils from 52 donors in the gran ulocyte immunofluorescence assay and did not react with NB1-negative n eutrophils from 8 donors. MoAb 708 immunoblotted a 56- to 64-kDa molec ule on neutrophils from eight NB1-positive donors and did not react wi th this molecule on NB1-negative neutrophils from two donors. When 708 was tested in the monoclonal antibody immobilization of granulocyte a ntigens assay, it reacted with two NB1 alloantibodies, but not with NA 1 or NA2 alloantibodies. To determine if MoAbs 708 and 1 B5 recognized the same epitope, both were tested against the same NB1-positive neut rophils and the cells were analyzed by two-color flow cytometry. Both antibodies bound independently to neutrophils, which indicated that th e antibodies recognized different epitopes. When similar studies were performed with MoAb 708 and three NB1 alloantibodies, 708 partially in hibited the binding of two of the alloantibodies. The size of the NB1- positive subpopulation was analyzed in 25 people using flow cytometry with both MoAbs and three alloantibodies, The subpopulation of antigen -positive cells was similar in all donors when 708 and the three NBI a lloantibodies were tested; however, the subpopulation recognized by Mo Ab 1 B5 was smaller iri two of the donors. Neutrophils from one of the se people were analyzed by immunoblotting, and no differences were det ected in the molecule carrying NB1 in those neutrophils and that molec ule in control neutrophils. Conclusion: NB1 specificity is made up of at least two separate epitopes. The expression of NBI varied among ant igen-positive individuals. While NB1 is expressed by a 56- to 64-kDa g lycoprotein, the structure of this protein on antigen-negative cells h as not been determined.