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Isolation of cell surface-specific human monoclonal antibodies using phage display and magnetically activated cell sorting: Applications in immunohematology
CURRENT LITERATURE In cases in which the antibody titer is high or rising and the father types Fy(a+b+), genotyping of amniocytes should be performed to help plan management of the pregnancy.
A New High-Molecular-Weight Glycophorin C Variant With a Duplication of Exon 2 in the Glycophorin C Gene. Uchikawa M, Tsuneyama H, Onodera T, et al. Transfus Med 7:305-309, 1997. Glycophorin C (GPC) and glycophorin D (GPD) are encoded by the same gene (GYPC) and carry the antigens of the Gerbich blood group system. The moleCular basis for the Gerbichnegative phenotypes has been determined. The Gerbich type ( G e : - 2 , - 3 , 4 ) is attributable to an absence of exon 3 as a consequence of an intragenic crossover. The reciprocal product of this molecular event is a duplication of exon 3 and is seen in the Ls(a+) phenotype. The Yus type (Ge:-2,3,4) is due to a deletion of exon 2 as a consequence of a different intragenic crossover. The reciprocal product had previously not been found but was predicted to contain two copies of exon 2 but not a novel antigen because the sequence of the junction of ex6n 2 to the duplicated exon 2 would be predicted to be the same as the sequence at the junction of exon 2 to exon 3. The paper describes serological, immunochemical, and molecular techniques used to study three unrelated donors with a high-molecular-weight variant of GPC. The red blood cells (RBCs) with the variant reacted more strongly with a monoclonal anti-GPC (CBC-96) when compared with wild-type controls. Immunoblotting' with anti-GPC showed a high-molecularweight band with an approximate M,. of 45,000. This compares with 46,000 for GPC.Ls a and 40,000 for GPC. The 510-bp products from GPC eDNA from the three donors were sequenced from nucleotides 18 to 447. A duplicated sequence from nucleotide 50 to 106 was found and shown to correspond to the entire sequence of exon 2 of GYPC. The variant glycophorins that are predicted to have a repeat of amino acids encoded by exon 2 have been named GPC.MAT and GPD.MAT. The monoclonal anti-GPC (CBC-96) was used in automated blood grouping machines to screen 91,000 samples from blood donors. Twenty-six samples were strongly reactive, were L s ( a - ) , and had a GPC variant band on immunoblotting of apparent Mr 45,000. Thus, the incidence of GRMAT in the Japanese population is approximately 0.02%.
B-Elimination of O-Glycans From Glycoproteins Transferred to Immobilon P Membranes: Method and Some Applications. Duk M, Ugorski M, Lisowska E, Anal Biochem 253:98-102, 1997 This paper describes a method for selective [3-elimination of O-glycans from glycoproteins on Immobilon P membranes. Experiments were performed with red blood cell membranes, in which glycophorins are the major poly-O-glycosylated components, and with lysates of human colon cancer cells CX-I.1. The effect o f O-glycan degradation was detected with lectins and monoclonal antibodies against peptidic, glycopetidic, and carbohydrate epitopes. With red blood cell membrane proteins, the O-glycans of glycophorins were undetectabte on blots after desialylation and heating in 0.055 mol/LNaOH for 16 hours at 40~ The N-glycans and peptidic epitopes were apparently
227 unchanged by this treatment. The method was used to show that most protein-linked sialyl-Le~ epitopes present on CX-I.1 cancer cells are located on O-glycosidic chains. It was also used to study the dependence of peptic epitopes on O-glycosylation. Most monoclonal antibodies specific for blood group antigen M or N required the presence of O-glycans. Monoclonal anti-M that cross-reacted with M g + red blood ceils reacted with glycophorin A carrying either M or N, as well as with glycophorin B on NaOH-treated blots. It is important to note that the conditions of treatment in alkali destroyed nitrocellulose paper but did not affect the stability of Immunobilon P membrane. The method is simple and inexpensive and provides a practical way to study the effect of O-glycosylation on proteins attached to Immunobilon.
Isolation of Cell Surface-Specific Human Monoclonal Antibodies Using Phage Display and Magnetically Activated Cell Sorting: Applications in Immunohematology. Siegel DL, Chang TY, Russell SL, et aL J Immunol Methods 206:73-85, 1997 This paper describes a method for the isolation of filamentous phage-displayed human monoclonal antibodies directed at cell surface molecules. To optimize capture of antigen-specific phage, a process using simultaneous positive and negative selection was used. Red blood cells bearing the antigen of interest are precoated with magnetic beads and diluted in an excess of unmodified antigen-negative red blood cells. After incubation, the antigen-positive cell population was retrieved using magnetically activated cell sorting. The antigen-specific Fab/phage were eluted and propagated in bacterial culture. Using this approach, 50 unique, clinically useful IgG1K and IgGlk anti-D were isolated from a single alloimmunized donor. The anti-D were tested against red blood cells with partial D antigens (D IIIa, D IVy,D Va, D VI, D vIII) and shown to be specific for one of five distinct epitopes (D1, D2, D3, D5, and D6/7). The Fab/phage antibodies can be used in microplates or by column technology using anti-M13 in place of antiglobulin serum. The potential Of this approach for making monoclonal antibodies that have application in immnnohematology is enormous.
Intrauterine Transfusions Affect Fetal T-cell Immunity. ViCtor HE, Hawes GE, van den Oever C, et al. Blood 90:2492-2501, 1997. Pregnancy complicated by severe hemolytic disease can be successfully treated with intrauterine transfusion, and such transfusions have become an established procedure yielding excellent survival rates for affected individuals. Normally, the fetal immune system develops in a sterile environment, and the fetus is only exposed to minimal levels of maternal transplantation antigens present on transplacental lymphocytes. Somehow, interactions between the fetal and maternal immune systems do not induce adverse immune stimulation. Because blood transfusion in adults affects the immune system by inducing immune stimulation or immune suppression, it was hypothesized that intrauterine transfusion may adversely affect the unprimed fetal immune system. To evaluate semiquantitatively the effects of intrauterine transfusion on the fetal immune system, these authors have examined the T-cell receptor variable chain usage in five transfused subjects versus five controls; the control subjects underwent fetal blood sampling but did not receive
A New High-Molecular-Weight Glycophorin C Variant With a Duplication of Exon 2 in the Glycophorin C Gene. Uchikawa M, Tsuneyama H, Onodera T, et al. Transfus Med 7:305-309, 1997. Glycophorin C (GPC) and glycophorin D (GPD) are encoded by the same gene (GYPC) and carry the antigens of the Gerbich blood group system. The moleCular basis for the Gerbichnegative phenotypes has been determined. The Gerbich type ( G e : - 2 , - 3 , 4 ) is attributable to an absence of exon 3 as a consequence of an intragenic crossover. The reciprocal product of this molecular event is a duplication of exon 3 and is seen in the Ls(a+) phenotype. The Yus type (Ge:-2,3,4) is due to a deletion of exon 2 as a consequence of a different intragenic crossover. The reciprocal product had previously not been found but was predicted to contain two copies of exon 2 but not a novel antigen because the sequence of the junction of ex6n 2 to the duplicated exon 2 would be predicted to be the same as the sequence at the junction of exon 2 to exon 3. The paper describes serological, immunochemical, and molecular techniques used to study three unrelated donors with a high-molecular-weight variant of GPC. The red blood cells (RBCs) with the variant reacted more strongly with a monoclonal anti-GPC (CBC-96) when compared with wild-type controls. Immunoblotting' with anti-GPC showed a high-molecularweight band with an approximate M,. of 45,000. This compares with 46,000 for GPC.Ls a and 40,000 for GPC. The 510-bp products from GPC eDNA from the three donors were sequenced from nucleotides 18 to 447. A duplicated sequence from nucleotide 50 to 106 was found and shown to correspond to the entire sequence of exon 2 of GYPC. The variant glycophorins that are predicted to have a repeat of amino acids encoded by exon 2 have been named GPC.MAT and GPD.MAT. The monoclonal anti-GPC (CBC-96) was used in automated blood grouping machines to screen 91,000 samples from blood donors. Twenty-six samples were strongly reactive, were L s ( a - ) , and had a GPC variant band on immunoblotting of apparent Mr 45,000. Thus, the incidence of GRMAT in the Japanese population is approximately 0.02%.
B-Elimination of O-Glycans From Glycoproteins Transferred to Immobilon P Membranes: Method and Some Applications. Duk M, Ugorski M, Lisowska E, Anal Biochem 253:98-102, 1997 This paper describes a method for selective [3-elimination of O-glycans from glycoproteins on Immobilon P membranes. Experiments were performed with red blood cell membranes, in which glycophorins are the major poly-O-glycosylated components, and with lysates of human colon cancer cells CX-I.1. The effect o f O-glycan degradation was detected with lectins and monoclonal antibodies against peptidic, glycopetidic, and carbohydrate epitopes. With red blood cell membrane proteins, the O-glycans of glycophorins were undetectabte on blots after desialylation and heating in 0.055 mol/LNaOH for 16 hours at 40~ The N-glycans and peptidic epitopes were apparently
227 unchanged by this treatment. The method was used to show that most protein-linked sialyl-Le~ epitopes present on CX-I.1 cancer cells are located on O-glycosidic chains. It was also used to study the dependence of peptic epitopes on O-glycosylation. Most monoclonal antibodies specific for blood group antigen M or N required the presence of O-glycans. Monoclonal anti-M that cross-reacted with M g + red blood ceils reacted with glycophorin A carrying either M or N, as well as with glycophorin B on NaOH-treated blots. It is important to note that the conditions of treatment in alkali destroyed nitrocellulose paper but did not affect the stability of Immunobilon P membrane. The method is simple and inexpensive and provides a practical way to study the effect of O-glycosylation on proteins attached to Immunobilon.
Isolation of Cell Surface-Specific Human Monoclonal Antibodies Using Phage Display and Magnetically Activated Cell Sorting: Applications in Immunohematology. Siegel DL, Chang TY, Russell SL, et aL J Immunol Methods 206:73-85, 1997 This paper describes a method for the isolation of filamentous phage-displayed human monoclonal antibodies directed at cell surface molecules. To optimize capture of antigen-specific phage, a process using simultaneous positive and negative selection was used. Red blood cells bearing the antigen of interest are precoated with magnetic beads and diluted in an excess of unmodified antigen-negative red blood cells. After incubation, the antigen-positive cell population was retrieved using magnetically activated cell sorting. The antigen-specific Fab/phage were eluted and propagated in bacterial culture. Using this approach, 50 unique, clinically useful IgG1K and IgGlk anti-D were isolated from a single alloimmunized donor. The anti-D were tested against red blood cells with partial D antigens (D IIIa, D IVy,D Va, D VI, D vIII) and shown to be specific for one of five distinct epitopes (D1, D2, D3, D5, and D6/7). The Fab/phage antibodies can be used in microplates or by column technology using anti-M13 in place of antiglobulin serum. The potential Of this approach for making monoclonal antibodies that have application in immnnohematology is enormous.
Intrauterine Transfusions Affect Fetal T-cell Immunity. ViCtor HE, Hawes GE, van den Oever C, et al. Blood 90:2492-2501, 1997. Pregnancy complicated by severe hemolytic disease can be successfully treated with intrauterine transfusion, and such transfusions have become an established procedure yielding excellent survival rates for affected individuals. Normally, the fetal immune system develops in a sterile environment, and the fetus is only exposed to minimal levels of maternal transplantation antigens present on transplacental lymphocytes. Somehow, interactions between the fetal and maternal immune systems do not induce adverse immune stimulation. Because blood transfusion in adults affects the immune system by inducing immune stimulation or immune suppression, it was hypothesized that intrauterine transfusion may adversely affect the unprimed fetal immune system. To evaluate semiquantitatively the effects of intrauterine transfusion on the fetal immune system, these authors have examined the T-cell receptor variable chain usage in five transfused subjects versus five controls; the control subjects underwent fetal blood sampling but did not receive