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.
The neutrophil-specific NB antigen system has been serologically characterized with human alloantisera. Two alleles, NB1 and NB2, have been described; however, there may be important quantitative or qualitative variation in the expression of NB1 and NB2. Human alloantibodies have been used to identify the 58- to 64-kDa glycoprotein (GP) on which NB1 antigen is located, but an NB2 antigen-bearing molecule has not yet been identified. To identify the NB2 molecule, human alloantibody to NB1 was used to isolate the 58- to 64-kDa NB1 GP, and rabbits were immunized with this GP. Two rabbit antisera were produced. Both antisera immunoblotted and immunoprecipitated the 58- to 64-kDa GP on which NB1 is located, but neither identified the molecule on which NB2 is located. The inability of two rabbit polyclonal antibodies specific for the NB1 molecule to react with the NB2-bearing molecule suggests that considerable differences may exist between these two molecules or that NB2 as currently defined is not related to NB1.
We have previously described a 24-year-old woman with quinine-dependent antibodies that reacted with neutrophils, red blood cells (RBCs), platelets, and T lymphocytes. The drug-dependent neutrophil antibody was found to react with 85- and 60-Kd neutrophil membrane molecules. In these studies, we further characterized these molecules and found that both were glycosyl-phosphatidylinositol (GPI)-linked and contained sialic acid residues and N-linked carbohydrate side chains, but neither contained O-linked carbohydrates. The protein backbone of the 60-Kd molecule was 45 Kd, and the 85 Kd glycoprotein (GP) was made up of 33- and 31-Kd proteins. While some GPI-anchored neutrophil GPs are released by stimulated neutrophils, neither the 85- nor the 60-Kd GP was released by neutrophil stimulated with C5a, f-met-leu-phe (FMLP), or phorbol myristate acetate (PMA). Neutrophil-specific antigen NB1 is located on a 58- to 64-Kd GP. To determine if the quinine-dependent antibody and anti-NB1 recognize the same GP, immunoprecipitation studies were performed with the quinine-dependent antibody using neutrophils with varying NB1 phenotypes. The 60-Kd GP was detected on NB1-positive neutrophils from 11 of 12 donors tested, but not on NB1- negative neutrophils from two donors tested. After solubilized 125I- labeled neutrophils were absorbed with anti-NB1, the quinine-dependent antibody immunoprecipitated the 85-Kd GP, but not the 60-Kd GP. These results indicate that anti-NB1 and the quinine-dependent antibody identified the same GP. The 85-Kd GP was detected on neutrophils from all 14 donors tested. The electrophoretic mobility of the 85-Kd GP was similar to the electrophoretic mobility of the major 125I-labeled neutrophil protein.
We have previously described a 24-year-old woman with quinine-dependent antibodies that reacted with neutrophils, red blood cells (RBCs), platelets, and T lymphocytes. The drug-dependent neutrophil antibody was found to react with 85- and 60-Kd neutrophil membrane molecules. In these studies, we further characterized these molecules and found that both were glycosyl-phosphatidylinositol (GPI)-linked and contained sialic acid residues and N-linked carbohydrate side chains, but neither contained O-linked carbohydrates. The protein backbone of the 60-Kd molecule was 45 Kd, and the 85 Kd glycoprotein (GP) was made up of 33- and 31-Kd proteins. While some GPI-anchored neutrophil GPs are released by stimulated neutrophils, neither the 85- nor the 60-Kd GP was released by neutrophil stimulated with C5a, f-met-leu-phe (FMLP), or phorbol myristate acetate (PMA). Neutrophil-specific antigen NB1 is located on a 58- to 64-Kd GP. To determine if the quinine-dependent antibody and anti-NB1 recognize the same GP, immunoprecipitation studies were performed with the quinine-dependent antibody using neutrophils with varying NB1 phenotypes. The 60-Kd GP was detected on NB1-positive neutrophils from 11 of 12 donors tested, but not on NB1- negative neutrophils from two donors tested. After solubilized 125I- labeled neutrophils were absorbed with anti-NB1, the quinine-dependent antibody immunoprecipitated the 85-Kd GP, but not the 60-Kd GP. These results indicate that anti-NB1 and the quinine-dependent antibody identified the same GP. The 85-Kd GP was detected on neutrophils from all 14 donors tested. The electrophoretic mobility of the 85-Kd GP was similar to the electrophoretic mobility of the major 125I-labeled neutrophil protein.
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