ABSTRACT
Murine monoclonal antibodies (MoAbs) directed against DAF (Decay Accelerating Factor, CD55 antigen) and MIRL (Membrane Inhibitor of Reactive Lysis, CD59 antigen) were used to identify the affected red cells (CD55-/CD59-) of PNH patients. MoAbs NaM16-4D3 (CD55, IgG2a) and NaM77-1E5 (CD59, IgG3) weakly agglutinate red cells and represent powerful tools to quantitate normal (PNHI) and abnormal (PNHII and PNHIII) cells from PNH patients by indirect flow cytometry. MoAbs NaM125-7H10 (CD55) and NaM123-6G12 (CD59), both IgM, were selected for their agglutinating properties and used for the separation of PNHI from PNHII and PNHIII red cells by the gel test technology. From analysis of artificial mixtures of DAF+ and DAF- cells, a direct relationship was established between fluorescent cells detected by flow cytometry, and erythrocytes agglutinated in microtyping cards. The method was further confirmed by analysis of ten blood samples from PHN patients and represent an alternative to classical hemolysis tests. On the basis of our experience we propose the following for the diagnosis of PNH: 1) agglutination test with NaCl microtyping cards using IgM CD55 and CD59; 2) flow cytometry analysis for accurate quantitation of CD55-/CD59- red cells.
Subject(s)
Hemoglobinuria, Paroxysmal/diagnosis , Agglutination Tests , Antibodies, Monoclonal , Antigens, CD/blood , CD55 Antigens , CD59 Antigens , Erythrocytes, Abnormal/immunology , Gels , Hemoglobinuria, Paroxysmal/immunology , Humans , Immunoglobulin M , Membrane Glycoproteins/blood , Time FactorsABSTRACT
Balb/c mice were immunized against papain-treated fetal erythrocytes and splenocytes were fused with Sp2/0-Ag-14 myeloma cells. Several hybrids secreting antibodies directed against antigenic determinants predominantly exposed on fetal and cord cells were selected and cloned twice. Antibodies NaM61-1A2 and NaM61-768 (IgM class) were shown to be specific for an endo-beta-galactosidase-sensitive oligosaccharide chain. The antigen, strongly expressed on fetal and cord cells, was identified as the i blood group antigen. The antibodies represent powerful blood group reagents to be use in conventional agglutination techniques as well as in the gel typing system and in indirect flow cytometry. The antibody NaM46-4A8 (IgG class) is specific for an antigenic structure expressed on fetal cells and accessible only after papain, ficin, bromelin and endo-beta galactosidase treatment. The antigen was not identified.
Subject(s)
Antibodies, Monoclonal/immunology , Erythrocyte Membrane/immunology , Fetal Blood/immunology , Glycoside Hydrolases , Hemagglutinins/immunology , Immunoglobulin G/immunology , Immunoglobulin M/immunology , Adult , Animals , Antibody Specificity , Bromelains/pharmacology , Carbohydrate Sequence , Erythrocyte Membrane/drug effects , Female , Ficain/pharmacology , Flow Cytometry , Hemagglutination Tests , Humans , Infant, Newborn/blood , Mice , Mice, Inbred BALB C/immunology , Molecular Sequence Data , Papain/pharmacology , Pregnancy , beta-Galactosidase/pharmacologyABSTRACT
A murine monoclonal antibody (NaM19-3C4, IgG1, Kappa) was produced from splenocytes of mice immunized with red blood cells. The antibody agglutinated untreated Ge:2,3,4 and Ge:-2,3,4 erythrocytes in indirect antiglobulin test but failed to agglutinate trypsin-treated cells. Gerbich-negative erythrocyte of the Leach- (Ge:-2,-3,-4) and of the Gerbich- (Ge:-2,-3,4) types were not recognized by the antibody. Immunoblotting experiments showed that the antibody bound to glycophorins C and D from control erythrocytes and to the abnormal glycophorin C identified in the Gerbich-negative cells of the Yussef type (Ge:-2,3,4). No binding to the altered glycophorin C from Ge:-2,-3,4 erythrocytes was observed, indicating that the antibody specifically recognized the Ge:3 epitope localized within residues 40-50 of glycophorin C.