Biochemical characterization of the neutrophil-specific antigen NB1

D. F. Stroncek, Keith M Skubitz, Jeffrey Mc Cullough

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96 Scopus citations

Abstract

Neutrophil-specific alloantibodies and the antigens they recognize are important in clinical medicine, but little is known about the structure of these antigens. Alloimmunization to the antigen NB1 is a clinically important cause of neonatal neutropenia and febrile transfusion reactions. To study the immunochemistry of the NB1 antigen, we prepared neutrophil plasma membranes and granules by nitrogen cavitation and differential centrifugation and then analyzed them by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and immunoblotting with alloantibodies to several neutrophil-specific antigens. Two different antisera to the neutrophil-specific antigen NB1 identified an approximately 55-Kd protien by immunoblotting on neutrophil membranes from four NB1-positive donors but not on neutrophil membranes from five NB1-negative donors. Four anti-NB1 antisera immonoprecipitated a 58- to 64-Kd protien from extracts of NB1-positive neutrophils surface-labeled with125l using lactoperoxidase, but not from similarly treated NB1-negative neutrophils. Normal human serum did not immunoprecipitate or immunoblot any protiens from these same neutrophil preparations. The NB1 antigen was detected by immunoblotting in secondary granules but was not found in primary granules. The electrophoretic mobility of the antigen was decreased slightly by reduction, suggesting that intrachain disulfide bonds were present. After reduction, the antigen could no longer be recognized by anti-NB1 antisera, but treatment of the antigen with periodate had no effect on the ability of anti-NB1 antisera to recognize the antigen, suggesting that it is not a carbohydrate. The data suggest that the neutrophil-specific antigen NB1 is present on a 58- to 64Kd surface glycoprotein that is also present in secondary granules, and that the NB1 epitope is not a carbohydrate but probably resides in the tertiary structure of the protien blckbone.

Original languageEnglish (US)
Pages (from-to)744-755
Number of pages12
JournalBlood
Volume75
Issue number3
StatePublished - Feb 1 1990

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