Analysis of the expression of NB1 antigen using two monoclonal antibodies

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Abstract
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 characterized. Two monoclonal antibodies (MoAbs), 1B5 and the recently produced 7D8, and four alloantibodies, all specific for NB1, were used to investigate the expression of NB1 on neutrophils from several donors. MoAb 7D8 was shown to be specific for NB1. It reacted with NB1-positive neutrophils from 52 donors in the granulocyte immunofluorescence assay and did not react with NB1-negative neutrophils from 8 donors. MoAb 7D8 immunoblotted a 56- to 64-kDa molecule on neutrophils from eight NB1-positive donors and did not react with this molecule on NB1-negative neutrophils from two donors. When 7D8 was tested in the monoclonal antibody immobilization of granulocyte antigens assay, it reacted with two NB1 alloantibodies, but not with NA1 or NA2 alloantibodies. To determine if MoAbs 7D8 and 1B5 recognized the same epitope, both were tested against the same NB1-positive neutrophils and the cells were analyzed by two-color flow cytometry. Both antibodies bound independently to neutrophils, which indicated that the antibodies recognized different epitopes. When similar studies were performed with MoAb 7D8 and three NB1 alloantibodies, 7D8 partially inhibited 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 7D8 and the three NB1 alloantibodies were tested; however, the subpopulation recognized by MoAb 1B5 was smaller in two of the donors. Neutrophils from one of these people were analyzed by immunoblotting, and no differences were detected in the molecule carrying NB1 in those neutrophils and that molecule in control neutrophils. NB1 specificity is made up of at least two separate epitopes. The expression of NB1 varied among antigen-positive individuals. While NB1 is expressed by a 56- to 64-kDa glycoprotein, the structure of this protein on antigen-negative cells has not been determined.