InVivoMAb anti-human Fy3

InVivoMAb anti-human Fy3 (CLONE: MIMA-29)

Khan R, Menard M, Jen CC, Chen X, Norris PAA, Lazarus AH (2020). "Inhibition of platelet phagocytosis as an in vitro predictor for therapeutic potential of RBC antibodies in murine ITP" Blood 135(26):2420-2424. PubMed

Polyclonal anti-D is a first-line therapy for immune thrombocytopenia (ITP). Monoclonal antibodies are desirable alternatives, but none have yet proven successful despite their ability to opsonize erythrocytes (or red blood cells, RBCs) and cause anemia. Here, we examined 12 murine erythrocyte-specific antibodies of different specificity and subtypes and found that 8 of these antibodies could induce anemia in antigen-positive mice. Of these 8 antibodies, only 5 ameliorated ITP. All antibodies were examined for their in vitro ability to support macrophage-mediated phagocytosis of erythrocytes. Antibodies which supported erythrocyte phagocytosis in vitro successfully ameliorated ITP in vivo. To examine the ability of each antibody to inhibit phagocytosis of platelets, the antibodies were used to sensitize erythrocytes in vitro and these were added to a platelet phagocytosis assay. Antibodies that inhibited platelet phagocytosis in vitro also all ameliorated ITP in vivo. We conclude that inducing anemia is not a sufficient condition for amelioration of ITP but that the antibody's ability to prevent platelet phagocytosis in vitro predicted its ability to ameliorate ITP. We suggest that inhibition of in vitro platelet phagocytosis may prove to be a valuable tool for determining which erythrocyte antibodies would likely be candidates for clinical use in ITP.

Gruber DR, Richards AL, Howie HL, Hay AM, Lebedev JN, Wang X, Zimring JC, Hudson KE (2020). "Passively transferred IgG enhances humoral immunity to a red blood cell alloantigen in mice" Blood Adv 4(7):1526-1537. PubMed

Antibodies are typically thought of as the endpoint of humoral immunity that occur as the result of an adaptive immune response. However, affinity-matured antibodies can be present at the initiation of a new immune response, most commonly because of passive administration as a medical therapy. The current paradigm is that immunoglobulin M (IgM), IgA, and IgE enhance subsequent humoral immunity. In contrast, IgG has a "dual effect" in which it enhances responses to soluble antigens but suppresses responses to antigens on red blood cells (RBCs) (eg, immunoprophylaxis with anti-RhD). Here, we report a system in which passive antibody to an RBC antigen promotes a robust cellular immune response leading to endogenous CD4+ T-cell activation, germinal center formation, antibody secretion, and immunological memory. The mechanism requires ligation of Fcγ receptors on a specific subset of dendritic cells that results in CD4+ T-cell activation and expansion. Moreover, antibodies cross-enhance responses to a third-party antigen, but only if it is expressed on the same RBC as the antigen recognized by the antibody. Importantly, these observations were IgG subtype specific. Thus, these findings demonstrate that antibodies to RBC alloantigens can enhance humoral immunity in an IgG subtype-specific fashion and provide mechanistic elucidation of the enhancing effects.

Hawksworth J, Satchwell TJ, Meinders M, Daniels DE, Regan F, Thornton NM, Wilson MC, Dobbe JG, Streekstra GJ, Trakarnsanga K, Heesom KJ, Anstee DJ, Frayne J, Toye AM (2018). "Enhancement of red blood cell transfusion compatibility using CRISPR-mediated erythroblast gene editing" EMBO Mol Med 10(6):e8454. PubMed

Regular blood transfusion is the cornerstone of care for patients with red blood cell (RBC) disorders such as thalassaemia or sickle-cell disease. With repeated transfusion, alloimmunisation often occurs due to incompatibility at the level of minor blood group antigens. We use CRISPR-mediated genome editing of an immortalised human erythroblast cell line (BEL-A) to generate multiple enucleation competent cell lines deficient in individual blood groups. Edits are combined to generate a single cell line deficient in multiple antigens responsible for the most common transfusion incompatibilities: ABO (Bombay phenotype), Rh (Rh_null), Kell (K₀), Duffy (Fy_null), GPB (S-s-U-). These cells can be differentiated to generate deformable reticulocytes, illustrating the capacity for coexistence of multiple rare blood group antigen null phenotypes. This study provides the first proof-of-principle demonstration of combinatorial CRISPR-mediated blood group gene editing to generate customisable or multi-compatible RBCs for diagnostic reagents or recipients with complicated matching requirements.

Arthur CM, Patel SR, Smith NH, Bennett A, Kamili NA, Mener A, Gerner-Smidt C, Sullivan HC, Hale JS, Wieland A, Youngblood B, Zimring JC, Hendrickson JE, Stowell SR (2017). "Antigen Density Dictates Immune Responsiveness following Red Blood Cell Transfusion" J Immunol 198(7):2671-2680. PubMed

Although RBC transfusion can result in the development of anti-RBC alloantibodies that increase the probability of life-threatening hemolytic transfusion reactions, not all patients generate anti-RBC alloantibodies. However, the factors that regulate immune responsiveness to RBC transfusion remain incompletely understood. One variable that may influence alloantibody formation is RBC alloantigen density. RBC alloantigens exist at different densities on the RBC surface and likewise exhibit distinct propensities to induce RBC alloantibody formation. However, although distinct alloantigens reside on the RBC surface at different levels, most alloantigens also represent completely different structures, making it difficult to separate the potential impact of differences in Ag density from other alloantigen features that may also influence RBC alloimmunization. To address this, we generated RBCs that stably express the same Ag at different levels. Although exposure to RBCs with higher Ag levels induces a robust Ab response, RBCs bearing low Ag levels fail to induce RBC alloantibodies. However, exposure to low Ag-density RBCs is not without consequence, because recipients subsequently develop Ag-specific tolerance. Low Ag-density RBC-induced tolerance protects higher Ag-density RBCs from immune-mediated clearance, is Ag specific, and occurs through the induction of B cell unresponsiveness. These results demonstrate that Ag density can potently impact immune outcomes following RBC transfusion and suggest that RBCs with altered Ag levels may provide a unique tool to induce Ag-specific tolerance.

Richards AL, Howie HL, Kapp LM, Hendrickson JE, Zimring JC, Hudson KE (2017). "Innate B-1 B Cells Are Not Enriched in Red Blood Cell Autoimmune Mice: Importance of B Cell Receptor Transgenic Selection" Front Immunol . PubMed

Autoimmune hemolytic anemia (AIHA) results from breakdown of humoral tolerance to RBC antigens. Past analyses of B-cell receptor transgenic (BCR-Tg) mice that recognize RBC autoantigens led to a paradigm in which autoreactive conventional B-2 B cells are deleted whereas extramedullary B-1 B cells escape deletion due to lack of exposure to RBCs. However, BCR-Tg mice utilized to shape the current paradigm were unable to undergo receptor editing or class-switching. Given the importance of receptor editing as mechanism to tolerize autoreactive B cells during central tolerance, we hypothesized that expansion of autoreactive B-1 B cells is a consequence of the inability of the autoreactive BCR to receptor edit. To test this hypothesis, we crossed two separate strains of BCR-Tg mice with transgenic mice expressing the BCR target on RBCs. Both BCR-Tg mice express the same immunoglobulin and, thus, secrete antibodies with identical specificity, but one strain (SwHEL) has normal receptor editing, whereas the other (IgHEL) does not. Similar to other AIHA models, the autoreactive IgHEL strain showed decreased B-2 B cells, an enrichment of B-1 B cells, and detectable anti-RBC autoantibodies and decreased RBC hematocrit and hemoglobin values. However, autoreactive SwHEL mice had induction of tolerance in both B-2 and B-1 B cells with anti-RBC autoantibody production without anemia. These data generate new understanding and challenge the existing paradigm of B cell tolerance to RBC autoantigens. Furthermore, these findings demonstrate that immune responses vary when BCR-Tg do not retain BCR editing and class-switching functions.

de Wolski K, Fu X, Dumont LJ, Roback JD, Waterman H, Odem-Davis K, Howie HL, Zimring JC (2016). "Metabolic pathways that correlate with post-transfusion circulation of stored murine red blood cells" Haematologica 101(5):578-86. PubMed

Transfusion of red blood cells is a very common inpatient procedure, with more than 1 in 70 people in the USA receiving a red blood cell transfusion annually. However, stored red blood cells are a non-uniform product, based upon donor-to-donor variation in red blood cell storage biology. While thousands of biological parameters change in red blood cells over storage, it has remained unclear which changes correlate with function of the red blood cells, as opposed to being co-incidental changes. In the current report, a murine model of red blood cell storage/transfusion is applied across 13 genetically distinct mouse strains and combined with high resolution metabolomics to identify metabolic changes that correlated with red blood cell circulation post storage. Oxidation in general, and peroxidation of lipids in particular, emerged as changes that correlated with extreme statistical significance, including generation of dicarboxylic acids and monohydroxy fatty acids. In addition, differences in anti-oxidant pathways known to regulate oxidative stress on lipid membranes were identified. Finally, metabolites were identified that differed at the time the blood was harvested, and predict how the red blood cells perform after storage, allowing the potential to screen donors at time of collection. Together, these findings map out a new landscape in understanding metabolic changes during red blood cell storage as they relate to red blood cell circulation.

Richards AL, Kapp LM, Wang X, Howie HL, Hudson KE (2016). "Regulatory T Cells Are Dispensable for Tolerance to RBC Antigens" Front Immunol . PubMed

Autoimmune hemolytic anemia (AIHA) occurs when pathogenic autoantibodies against red blood cell (RBC) antigens are generated. While the basic disease pathology of AIHA is well studied, the underlying mechanism(s) behind the failure in tolerance to RBC autoantigens are poorly understood. Thus, to investigate the tolerance mechanisms required for the establishment and maintenance of tolerance to RBC antigens, we developed a novel murine model. With this model, we evaluated the role of regulatory T cells (Tregs) in tolerance to RBC-specific antigens. Herein, we show that neither sustained depletion of Tregs nor immunization with RBC-specific proteins in conjunction with Treg depletion led to RBC-specific autoantibody generation. Thus, these studies demonstrate that Tregs are not required to prevent autoantibodies to RBCs and suggest that other tolerance mechanisms are likely involved.

Tormey CA, Santhanakrishnan M, Smith NH, Liu J, Marschner S, Goodrich RP, Hendrickson JE (2016). "Riboflavin-ultraviolet light pathogen reduction treatment does not impact the immunogenicity of murine red blood cells" Transfusion 56(4):863-72. PubMed

Background: Ultraviolet (UV) illumination/pathogen reduction effectively inactivates white blood cells (WBCs) in whole blood. Given that cotransfused WBCs may impact recipient immune responses, we hypothesized that pathogen reduction of whole blood may alter responses to RBC antigens. Study design and methods: Transgenic mice expressing a model (HOD) antigen, authentic human (hGPA or KEL) antigens, or natural fluorescence (uGFP) on their RBCs were utilized as blood donors. Recipients were transfused with fresh whole blood to which riboflavin had been added or fresh whole blood treated by UV illumination/pathogen reduction treatment after the addition of riboflavin. Posttransfusion RBC recovery, survival, and alloimmunization were measured by flow cytometry. Results: UV illumination/pathogen reduction treatment did not alter RBC antigen expression, and recipients of treated syngeneic RBCs had persistently negative direct antiglobulin tests. Greater than 75% of treated and untreated syngeneic RBCs were recovered 24 hours posttransfusion in all experiments, although alterations in the long-term posttransfusion survival of treated RBCs were observed. Treated and untreated KEL RBCs induced similar recipient alloimmune responses, with all recipients making anti-KEL glycoprotein immunoglobulins (p > 0.05). Alloimmune responses to treated HOD or hGPA RBCs were no different from untreated RBCs (p > 0.05). Conclusion: Pathogen inactivation treatment of fresh whole murine blood with riboflavin and UV illumination does not impact the rate or magnitude of RBC alloimmunization to three distinct RBC antigens. Further, UV illumination/pathogen reduction appears safe from an immunohematologic standpoint, with no immunogenic neoantigens detected on treated murine RBCs. Future studies with fresh and stored human RBCs are warranted to confirm these findings.

Yu H, Stowell SR, Bernardo L, Hendrickson JE, Zimring JC, Amash A, Uchikawa M, Lazarus AH (2014). "Antibody-mediated immune suppression of erythrocyte alloimmunization can occur independently from red cell clearance or epitope masking in a murine model" J Immunol 193(6):2902-10. PubMed

Anti-D can prevent immunization to the RhD Ag on RBCs, a phenomenon commonly termed Ab-mediated immune suppression (AMIS). The most accepted theory to explain this effect has been the rapid clearance of RBCs. In mouse models using SRBC, these xenogeneic cells are always rapidly cleared even without Ab, and involvement of epitope masking of the SRBC Ags by the AMIS-inducing Ab (anti-SRBC) has been suggested. To address these hypotheses, we immunized mice with murine transgenic RBCs expressing the HOD Ag (hen egg lysozyme [HEL], in sequence with ovalbumin, and the human Duffy transmembrane protein) in the presence of polyclonal Abs or mAbs to the HOD molecule. The isotype, specificity, and ability to induce AMIS of these Abs were compared with accelerated clearance as well as steric hindrance of the HOD Ag. Mice made IgM and IgG reactive with the HEL portion of the molecule only. All six of the mAbs could inhibit the response. The HEL-specific Abs (4B7, IgG1; GD7, IgG2b; 2F4, IgG1) did not accelerate clearance of the HOD-RBCs and displayed partial epitope masking. The Duffy-specific Abs (MIMA 29, IgG2a; CBC-512, IgG1; K6, IgG1) all caused rapid clearance of HOD RBCs without steric hindrance. To our knowledge, this is the first demonstration of AMIS to erythrocytes in an all-murine model and shows that AMIS can occur in the absence of RBC clearance or epitope masking. The AMIS effect was also independent of IgG isotype and epitope specificity of the AMIS-inducing Ab.

Stowell SR, Liepkalns JS, Hendrickson JE, Girard-Pierce KR, Smith NH, Arthur CM, Zimring JC (2013). "Antigen modulation confers protection to red blood cells from antibody through Fcγ receptor ligation" J Immunol 191(10):5013-25. PubMed

Autoantibodies and alloantibodies can damage self-tissue or transplanted tissues through either fixation of complement or ligation of FcγRs. Several pathways have been described that imbue self-tissues with resistance to damage from complement fixation, as a protective measure against damage from these Abs. However, it has been unclear whether parallel pathways exist to provide protection from FcγR ligation by bound Abs. In this article, we describe a novel pathway by which cell surface Ag is specifically decreased as a result of Ab binding (Ag modulation) to the extent of conferring protection to recognized cells from Fcγ-dependent clearance. Moreover, the Ag modulation in this system requires FcγR ligation. Together, these findings provide unique evidence of self-protective pathways for FcγR-mediated Ab damage.

Liepkalns JS, Hod EA, Stowell SR, Cadwell CM, Spitalnik SL, Zimring JC (2012). "Biphasic clearance of incompatible red blood cells through a novel mechanism requiring neither complement nor Fcγ receptors in a murine model" Transfusion 52(12):2631-45. PubMed

Background: Antibody binding to red blood cells (RBCs) can induce potentially fatal outcomes, including hemolytic transfusion reactions (HTRs), hemolytic disease of the fetus and newborn, and autoimmune hemolytic anemia. The mechanism(s) of RBC destruction following antibody binding is typically thought to require complement activation and/or the involvement of Fcγ receptors (FcγRs). In the current report, we analyzed mechanisms of HTRs during incompatible transfusions of murine RBCs expressing human glycophorin A (hGPA) into mice with anti-hGPA. Study design and methods: C3 and Fcγ receptor knockout, splenectomized, Fcγ receptor blocking antibody-treated, and clodronate-treated mice were passively immunized with anti-hGPA (10F7 or 6A7) and transfused with RBCs expressing the hGPA antigen. Posttransfusion blood and serum were collected and analyzed via flow cytometry and confocal microscopy. Results: This HTR model results in both rapid clearance and cytokine storm. Neither complement nor FcγRs were required for RBC clearance; in contrast, FcγRs were required for cytokine storm. Circulating aggregates of hGPA RBCs were visible during the HTR. Splenectomy and phagocyte depletion by clodronate had no effect on acute RBC clearance; however, incompatible RBCs reentered over 24 hours in clodronate-treated mice. Conclusion: These data demonstrate a biphasic HTR, the first phase involving sequestration of incompatible hGPA RBCs and the second phase involving phagocytosis of sequestered RBCs. However, the mechanism(s) of phagocytosis in the second phase required neither C3 nor FcγRs. These findings demonstrate novel mechanistic biology of HTRs.

Grodecka M, Bertrand O, Karolak E, Lisowski M, Waśniowska K (2012). "One-step immunopurification and lectinochemical characterization of the Duffy atypical chemokine receptor from human erythrocytes" Glycoconj J 29(2-3):93-105. PubMed

Duffy antigen/receptor for chemokines (DARC) is a glycosylated seven-transmembrane protein acting as a blood group antigen, a chemokine binding protein and a receptor for Plasmodium vivax malaria parasite. It is present on erythrocytes and endothelial cells of postcapillary venules. The N-terminal extracellular domain of the Duffy glycoprotein carries Fy(a)/Fy(b) blood group antigens and Fy6 linear epitope recognized by monoclonal antibodies. Previously, we have shown that recombinant Duffy protein expressed in K562 cells has three N-linked oligosaccharide chains, which are mainly of complex-type. Here we report a one-step purification method of Duffy protein from human erythrocytes. DARC was extracted from erythrocyte membranes in the presence of 1% n-dodecyl-β-D-maltoside (DDM) and 0.05% cholesteryl hemisuccinate (CHS) and purified by affinity chromatography using immobilized anti-Fy6 2C3 mouse monoclonal antibody. Duffy glycoprotein was eluted from the column with synthetic DFEDVWN peptide containing epitope for 2C3 monoclonal antibody. In this single-step immunoaffinity purification method we obtained highly purified DARC, which migrates in SDS-polyacrylamide gel as a major diffuse band corresponding to a molecular mass of 40-47 kDa. In ELISA purified Duffy glycoprotein binds anti-Duffy antibodies recognizing epitopes located on distinct regions of the molecule. Results of circular dichroism measurement indicate that purified DARC has a high content of α-helical secondary structure typical for chemokine receptors. Analysis of DARC glycans performed by means of lectin blotting and glycosidase digestion suggests that native Duffy N-glycans are mostly triantennary complex-type, terminated with α2-3- and α2-6-linked sialic acid residues with bisecting GlcNAc and α1-6-linked fucose at the core.

Hendrickson JE, Hod EA, Cadwell CM, Eisenbarth SC, Spiegel DA, Tormey CA, Spitalnik SL, Zimring JC (2011). "Rapid clearance of transfused murine red blood cells is associated with recipient cytokine storm and enhanced alloimmunogenicity" Transfusion 51(11):2445-54. PubMed

Background: Fourteen-day stored red blood cells (RBCs) containing an RBC-specific transgenic antigen (HOD) induce a recipient proinflammatory cytokine storm and are significantly more immunogenic compared to fresh RBCs. Given that recipient mice clear transfused stored RBCs more rapidly than fresh RBCs, we hypothesized that rapid RBC clearance was associated with adverse transfusion outcomes. Study design and methods: HOD RBCs were treated by two distinct methods known to lead to rapid posttransfusion RBC clearance: phenylhydrazine or heat. HOD antigen expression was analyzed on the treated cells before transfusion, and RBC recovery, recipient cytokine response, and recipient anti-HOD alloimmunization response were measured after transfusion. Results: Phenylhydrazine and heat treatment each led to near complete RBC clearance in recipients by 24 hours posttransfusion, without significantly altering HOD antigen expression on the transfused RBCs. Recipients of phenylhydrazine- or heat-treated RBCs had elevated circulating levels of keratinocyte-derived chemokine/CXCL-1, monocyte chemoattractant protein-1, and interleukin-6 after transfusion. Furthermore, phenylhydrazine- or heat-treated RBCs were significantly more immunogenic than control RBCs, with a mean 25.1- and 10.3-fold enhancement, respectively, of anti-HOD alloimmunization magnitude by flow cytometric crossmatch. Conclusions: Three separate insults to RBCs (storage, phenylhydrazine, or heat treatment) result in rapid posttransfusion clearance, with a recipient proinflammatory cytokine storm and enhanced alloimmunogenicity. These data are consistent with the hypothesis that rapid clearance of RBCs is causally involved in these outcomes and suggest that human donor RBCs with favorable posttransfusion clearance profiles may be less immunogenic.

Hendrickson JE, Hod EA, Hudson KE, Spitalnik SL, Zimring JC (2011). "Transfusion of fresh murine red blood cells reverses adverse effects of older stored red blood cells" Transfusion 51(12):2695-702. PubMed

Background: Although a subset of recent studies have suggested that red blood cell (RBC) storage length is associated with adverse patient outcomes, others have shown no such relationship. Adults may be transfused with RBC units of different storage lengths, and existing studies do not take into consideration that fresh RBCs may alter responses to concurrently transfused stored RBCs. To test this possibility, we utilized a murine model and investigated transfusion outcomes of fresh, stored, or fresh-plus-stored RBCs. Study design and methods: Fresh, 14-day-stored or fresh plus 14-day-stored leukoreduced RBCs from HOD-transgenic donors (with RBC-specific expression of hen egg lysozyme, ovalbumin, and human Duffy(b)) were transfused into naïve C57BL/6 recipients. Serum cytokines and anti-HOD alloimmunization were evaluated after transfusion. Results: In six of six experiments (n = 90 mice total), a proinflammatory serum cytokine storm of interleukin-6, keratinocyte-derived chemokine/CXCL1, and monocyte chemoattractant protein-1 was observed in transfusion recipients of stored but not fresh RBCs, along with high degrees of anti-HOD alloimmunization. However, concurrent transfusion of fresh HOD RBCs along with stored HOD RBCs significantly decreased these adverse outcomes (p < 0.05). Conclusions: These results are consistent with fresh murine HOD RBCs losing protective properties during storage, and introduce a previously unrecognized variable in RBC storage studies. If translatable to humans, uniform "old blood" groups may be needed in future clinical studies to more accurately investigate the biologic effects of older RBC units.

Desmarets M, Cadwell CM, Peterson KR, Neades R, Zimring JC (2009). "Minor histocompatibility antigens on transfused leukoreduced units of red blood cells induce bone marrow transplant rejection in a mouse model" Blood 114(11):2315-22. PubMed

When successful, human leukocyte antigen (HLA)-matched bone marrow transplantation with reduced-intensity conditioning is a cure for several nonmalignant hematologic disorders that require chronic transfusion, such as sickle cell disease and aplastic anemia. However, there are unusually high bone marrow transplant (BMT) rejection rates in these patients. Rejection correlates with the number of transfusions before bone marrow transplantation, and it has been hypothesized that preimmunization to antigens on transfused blood may prime BMT rejection. Using a novel mouse model of red blood cell (RBC) transfusion and major histocompatibility complex-matched bone marrow transplantation, we report that transfusion of RBC products induced BMT rejection across minor histocompatibility antigen (mHA) barriers. It has been proposed that contaminating leukocytes are responsible for transfusion-induced BMT rejection; however, filter leukoreduction did not prevent rejection in the current studies. Moreover, we generated a novel transgenic mouse with RBC-specific expression of a model mHA and demonstrated that transfusion of RBCs induced a CD8(+) T-cell response. Together, these data suggest that mHAs on RBCs themselves are capable of inducing BMT rejection. Cellular immunization to mHAs is neither monitored nor managed by current transfusion medicine practice; however, the current data suggest that mHAs on RBCs may represent an unappreciated and significant consequence of RBC transfusion.

Wasniowska K, Lisowska E, Halverson GR, Chaudhuri A, Reid ME (2004). "The Fya, Fy6 and Fy3 epitopes of the Duffy blood group system recognized by new monoclonal antibodies: identification of a linear Fy3 epitope" Br J Haematol 124(1):118-22. PubMed

Four new anti-Duffy murine monoclonal antibodies (MAbs): two anti-Fy6 (MIMA-107 and MIMA-108), one anti-Fya (MIMA-19) and one anti-Fy3 (MIMA-29) were characterized. Identification of epitopes by means of synthetic peptides (Pepscan) showed that the anti-Fy6 reacted most strongly with peptides containing the sequence 19QLDFEDV25 of the Duffy glycoprotein, and less strongly with peptides containing LDFEDV (MIMA-107) or LDF only (MIMA-108). The anti-Fya recognized epitope 38DGDYGA43 containing the Gly42 residue, which defines the Fya blood group antigen. MIMA-29 is the first anti-Fy3 reactive with a linear epitope 281ALDLL285 located in the fourth extracellular domain (ECD4, loop 3) of the Duffy glycoprotein. The four new antibodies extend the list of six anti-Fy MAbs formerly characterized by Pepscan analysis that allow some general conclusions. Fine specificities of various anti-Fya, or anti-Fy6 are not identical, but all of them recognize linear epitopes located around, respectively, Gly42 or between two potential N-glycosylation sites at Asn16 and Asn27. Anti-Fy3 recognize either a linear epitope located in ECD4, or a conformational epitope that includes amino acid residues of ECD4 and of other ECDs.

Halverson GR, Chaudhuri A, Huang T, Yazdanbakhsh K, Reid ME (2001). "Immunization of transgenic mice for production of MoAbs directed at polymorphic blood group antigens" Transfusion 41(11):1393-6. PubMed

Background: Antibodies of human origin for blood typing are increasingly difficult to obtain, and, despite aggressive efforts, MoAbs with specificities to several blood group polymorphisms have eluded production. As an approach for the generation of MoAbs with defined specificities, the feasibility of immunizing mice that are transgenic for the target polymorphism, Fy(a)/Fy(b) of the Duffy blood group system, was tested with a source of the antithetical antigen. Study design and methods: Nontransgenic mice were immunized with recombinant Fy(b), and transgenic mice expressing human Fy(b) were immunized with recombinant Fy(a). Results: Immunization of the nontransgenic mice resulted in the production of MoAbs to the Duffy protein, but not to the Fy(a)/Fy(b) blood group polymorphism. However, immunization of the transgenic mice resulted in production of the first example of murine Fy(a) MoAb (MIMA-19). This antibody is being used to screen for Fy(a-) blood donors and has been evaluated by many laboratories in an international workshop. Conclusion: This approach provides an effective method for producing MoAbs with specificities to polymorphic epitopes. These MoAbs are needed in transfusion medicine to identify antigen-negative donors and to alleviate the critical shortage of blood bank typing reagents, which currently are available only from human-derived sources.