Fine Characterization of a Series of New Monoclonal Antibodies Directed against Glycophorin A

Rasamoelisolo, Michele; Czerwiński, Marcin; Bruneau, Vincent; Lisowska, Elwira; Blanchard, Dominique

doi:10.1046/j.1423-0410.1997.7230185.x

Cited by 20 publications

(5 citation statements)

References 34 publications

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“…11 We extended these studies using wellcharacterized anti-hGPA mAbs. [12][13][14][15]18,19 Hemagglutination with anti-M and anti-N mAbs (6A7 and N92, respectively) revealed that hGPA-Tg-mRBC expressed the M allele (Table 1). These results are particularly interesting because 6A7 recognizes a sialic aciddependent epitope, 15 and mouse and human RBC sialic acids differ.…”

Section: Resultsmentioning

confidence: 99%

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Mouse models of IgG- and IgM-mediated hemolysis

Schirmer

Song

Baliff

et al. 2006

Blood

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Well-characterized mouse models of alloimmune antibody-mediated hemolysis would provide a valuable approach for gaining greater insight into the pathophysiology of hemolytic transfusion reactions. To this end, mouse red blood cells (mRBCs) from human glycophorin A transgenic (hGPA-Tg) donor mice were transfused into non-Tg recipients that had been passively immunized with IgG or IgM hGPA-specific monoclonal antibodies (mAbs). In this novel murine "blood group system," mRBCs from hGPA-Tg IntroductionHemolytic transfusion reactions (HTRs) are dangerous complications of blood transfusions. IgM-mediated HTRs (IgM-HTRs) occur acutely, are usually due to ABO incompatibility, typically cause intravascular hemolysis, and can lead to shock, renal failure, coagulopathy, and death. 1 Although infrequent, IgM-HTRs have a high mortality rate. IgG-mediated HTRs (IgG-HTRs) are more common, but usually less severe, than IgM-HTRs; they can occur acutely or be delayed, typically cause extravascular hemolysis, and occasionally result in renal failure, coagulopathy, and death. 2 Symptomatic immune-mediated red blood cell (RBC) destruction also occurs in autoimmune hemolytic anemia (AIHA), 3 blood group incompatible transplantation, 4 and immune thrombocytopenic purpura (ITP) treated with Rh-immune globulin. 5 Although various different modalities are used to treat HTRs, there is no definitive evidence regarding efficacy. Given the sporadic nature of HTRs, designing human trials to evaluate treatment options is difficult. Therefore, to study the mechanisms underlying HTRs, to evaluate the efficacy of existing treatments, and to develop new treatments, a relevant, inexpensive, and tractable animal model is required. 6 A mouse model would be ideal for this purpose, 7 given the abundance of reagents and relevant knockout (KO) and transgenic (Tg) animals. Although mouse blood group polymorphisms exist, 8 and transfused incompatible mouse RBC (mRBCs) are rapidly cleared, 9,10 no one has exploited these findings to develop a model of HTRs. The availability of a Tg mouse expressing human glycophorin A (hGPA; gene symbol designation, GYPA) on mRBCs 11 offers an approach using a well-described glycoprotein target and well-characterized reagents.We describe initial studies validating mouse models of IgG-and IgM-mediated alloimmune hemolysis. Thus, mRBCs from hGPA-Tg donors were transfused into non-Tg recipients that were passively immunized with IgG or IgM hGPA-specific monoclonal antibodies (mAbs). The clearance of the transfused mRBCs was quantified and the roles of complement and Fc␥ receptors were evaluated. Materials and methods AntibodiesPurified mAbs NaM10-2H12 (IgG3), NaM26-3F4 (IgG1), and NaM10-6G4 (IgG2a), which recognize peptide epitopes on the M and N forms of hGPA, 12 were purchased from EFS (Nantes, France). Hybridomas producing mAb 6A7, an IgG1 specific for a sialic acid-dependent epitope on M-type hGPA, 13-15 10F7, an IgG1 recognizing a nonpolymorphic epitope on hGPA, 13,14 and J11d, a rat IgM recognizing CD24 on mRBCs, 16,17...

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Section: Resultsmentioning

confidence: 99%

“…Wild-type mice can be actively immunized with hGPA 53 and many mouse mAbs are available. [12][13][14][15]18,19,53,[60][61][62][63] This type of blood group polymorphism (ie, the presence or absence of an entire protein) also occurs in humans: En(a Ϫ ) RBCs lack hGPA, S Ϫ s Ϫ U Ϫ RBCs lack human glycophorin B, and "Rh-negative" RBCs lack the Rh(D) protein. …”

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confidence: 99%

Mouse models of IgG- and IgM-mediated hemolysis

Schirmer

Song

Baliff

et al. 2006

Blood

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“…We were able to find only three reports of such antibodies. One of these, 2H12, recognizes glycophorin A 44 . In this case, the ability of IgG3 to agglutinate did not cause much surprise because glycophorin A protrudes significantly from the erythrocyte glycocalyx; thus, the distance between two epitopes on two cells is much smaller than the distance between cell surfaces.…”

Section: Discussionmentioning

confidence: 99%

Agglutinating mouse IgG3 compares favourably with IgMs in typing of the blood group B antigen: Functionality and stability studies

Klaus

Bzowska

Kulesza

et al. 2016

Sci Rep

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Mouse immunoglobulins M (IgMs) that recognize human blood group antigens induce haemagglutination and are used worldwide for diagnostic blood typing. Contrary to the current belief that IgGs are too small to simultaneously bind antigens on two different erythrocytes, we obtained agglutinating mouse IgG3 that recognized antigen B of the human ABO blood group system. Mouse IgG3 is an intriguing isotype that has the ability to form Fc-dependent oligomers. However, F(ab′)2 fragments of the IgG3 were sufficient to agglutinate type B red blood cells; therefore, IgG3-triggered agglutination did not require oligomerization. Molecular modelling indicated that mouse IgG3 has a larger range of Fab arms than other mouse IgG subclasses and that the unique properties of mouse IgG3 are likely due to the structure of its hinge region. With a focus on applications in diagnostics, we compared the stability of IgG3 and two IgMs in formulated blood typing reagents using an accelerated storage approach and differential scanning calorimetry. IgG3 was much more stable than IgMs. Interestingly, the rapid decrease in IgM activity was caused by aggregation of the molecules and a previously unknown posttranslational proteolytic processing of the μ heavy chain. Our data point to mouse IgG3 as a potent diagnostic tool.

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“…As the role of monoclonal antibodies in biology and medicine becomes increasingly important, so does the precise evaluation of the epitopes being recognized by the antibodies. There are many methods allowing to precisely define amino acid residues that are involved in antibody binding, such as evaluation of binding of the antibody to synthetic peptides (Pepscan analysis) or to recombinant peptides expressed either in bacteria on in eukaryotic cells [ 26 , 38 – 43 ]. Recently, saturation transfer difference spectroscopy NMR (STD-NMR) has been increasingly used in epitope-antibody studies, although majority of antibodies evaluated by this method bind carbohydrate antigens [ 44 – 46 ].…”

Section: Resultsmentioning

confidence: 99%

Studies of a Murine Monoclonal Antibody Directed against DARC: Reappraisal of Its Specificity

et al. 2015

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Duffy Antigen Receptor for Chemokines (DARC) plays multiple roles in human health as a blood group antigen, a receptor for chemokines and the only known receptor for Plasmodium vivax merozoites. It is the target of the murine anti-Fy6 monoclonal antibody 2C3 which binds to the first extracellular domain (ECD1), but exact nature of the recognized epitope was a subject of contradictory reports. Here, using a set of complex experiments which include expression of DARC with amino acid substitutions within the Fy6 epitope in E. coli and K562 cells, ELISA, surface plasmon resonance (SPR) and flow cytometry, we have resolved discrepancies between previously published reports and show that the basic epitope recognized by 2C3 antibody is 22FEDVW26, with 22F and 26W being the most important residues. In addition, we demonstrated that 30Y plays an auxiliary role in binding, particularly when the residue is sulfated. The STD-NMR studies performed using 2C3-derived Fab and synthetic peptide corroborated most of these results, and together with the molecular modelling suggested that 25V is not involved in direct interactions with the antibody, but determines folding of the epitope backbone.

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Fine Characterization of a Series of New Monoclonal Antibodies Directed against Glycophorin A

Abstract: These antibodies represent precise tools to investigate GPA and related molecules in different cells and tissues.

Cited by 20 publications

References 34 publications

Mouse models of IgG- and IgM-mediated hemolysis

Mouse models of IgG- and IgM-mediated hemolysis

Agglutinating mouse IgG3 compares favourably with IgMs in typing of the blood group B antigen: Functionality and stability studies

Studies of a Murine Monoclonal Antibody Directed against DARC: Reappraisal of Its Specificity

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