Production of Stable Human-Mouse Hybridomas Secreting Monoclonal Antibodies against Rh D and c Antigens

Rapaille, A.; François-Gérard, C.; Donnay, Danielle; Sondag-Thull, D

doi:10.1159/000462335

Cited by 7 publications

(8 citation statements)

References 24 publications

(48 reference statements)

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“…Various strategies have been used for the generation of human monoclonal anti‐Rh‐secreting cell lines, which include direct fusion of immune B cells to a myeloma of mouse or human origin, EBV transformation of the immune B cells and EBV transformation followed by back fusion to a myeloma fusion partner (McCann et al ., 1988; Hughes‐Jones & Parsons, 1992). Use of these approaches has enabled the production of a number of blood group‐specific human anti‐Rh MoAbs by investigators from different laboratories (Kumpel et al ., 1989; Thompson et al ., 1990; Rapaille et al ., 1993; Thompson et al ., 1994; Raache et al ., 1998). Successful establishment of cloned, specific Ab‐secreting cell lines has proved to be difficult using EBV transformation alone, as transformed cells are often unstable, have low cloning efficiency and cease to produce Ab because of overgrowth by nonproducing or unspecific cells or differentiate into nonproliferating plasma cell types (Thompson & Hughes‐Jones, 1990).…”

Section: Discussionmentioning

confidence: 99%

“…human anti-Rh MoAbs by investigators from different laboratories (Kumpel et al, 1989;Rapaille et al, 1993;Thompson et al, 1994;Raache et al, 1998). Successful establishment of cloned, specific Ab-secreting cell lines has proved to be difficult using EBV transformation alone, as transformed cells are often unstable, have low cloning efficiency and cease to produce Ab because of overgrowth by nonproducing or unspecific cells or differentiate into nonproliferating plasma cell types (Thompson & Hughes-Jones, 1990).…”

Section: Discussionmentioning

confidence: 99%

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Establishment of human heterohybridoma and lymphoblastoid cell lines specific for the Rh D and C antigens

Pasha

Roohi

Shokri

2003

Transfusion Medicine

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Human monoclonal antibodies specific for the D antigen of the Rh system are valuable tools for blood group typing and prevention of erythroblastosis. In this study, peripheral blood lymphocytes obtained from an Rh-negative woman immunized with Rh-positive fetuses were immortalized with Epstein-Barr virus (EBV), and transformed lymphoblastoid cell lines (LCLs) secreting antibodies to Rh antigens were generated. The presence of specific antibody was assessed by direct haemagglutination using Rh-positive, papain-treated red blood cells (RBCs), and the production of human antibody was assayed by enzyme-linked immunosorbent assay (ELISA). Specificities of the antibodies were determined by a panel of RBCs of known Rh phenotypes. Five LCLs produced antibody specific for the D antigen, and one LCL showed specificity towards the C antigen of the Rh blood group system. High-titre anti-Rh antibody-producing LCLs were subsequently selected and fused with a human x mouse heteromyeloma cell line. A hybridoma line producing human antibody of the immunoglobulin M (IgM) isotype, which strongly reacted with the D antigen, was established. The hybridoma was cloned, and the monoclone has been stable for growth and antibody production during 8 months of continuous culture, with a mean antibody concentration of 11.5 microg mL-1 and haemagglutination titre of 1/20 480. This antibody was not able to agglutinate a sample of native weak D RBCs (Du); however, agglutination was achieved with papain-treated Du RBCs. Immunoprecipitation of the D antigen by this antibody, followed by Western blot analysis, did not reveal any immobilized D-specific polypeptide. As this human antibody readily agglutinates D+ RBCs in saline, it has the potential to be used as an efficient reagent in routine blood group typing.

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Section: Discussionmentioning

confidence: 99%

Section: Discussionmentioning

confidence: 99%

Establishment of human heterohybridoma and lymphoblastoid cell lines specific for the Rh D and C antigens

Pasha

Roohi

Shokri

2003

Transfusion Medicine

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“…Cloning anti‐D alloantibodies from sensitized individuals was one of my first applications of phage display given the clinical significance of anti‐D and the technical difficulties inherent in use of conventional cellular approaches for human MoAb production 10 . At that time and despite heroic efforts, 11‐18 no more than approximately eight immunoglobulin G (IgG) anti‐D MoAbs had been produced from the immune repertoire of a single individual with tissue culture methods. Consequently, a comprehensive study of the genetic and serologic diversity in immune response to D within a given individual and between individuals could not have been performed.…”

Section: Use Of Phage Display To Study the Human Anti‐d Immune Responsementioning

confidence: 99%

Developing phage display tools for use in transfusion medicine

Siegel

2005

Transfusion

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GETTING A STARTn surveying the list of the more than 100 past and present NBF awards, what is as striking as the diversity in transfusion medicine topics is the diversity of recipients in terms of both their medical or scientific background and their stage of career ranging from trainee to senior level academician. For each of them, it would appear that funding through the NBF played a particularly personal and unique role in his or her professional development. My case is no exception. Although I had gained much experience in basic science research as a graduate student in biophysics in the late 1970s and early 1980s, my subsequent 7 years of clinical training comprising medical school, a clinical pathology residency, and a blood banking-transfusion medicine fellowship had placed me, in 1991, somewhat out of touch with the world of bench research.During my time away from the lab, everything suddenly became "molecular" and available as a kit. Techniques like polymerase chain reaction (PCR) and DNA sequencing had been invented and became routine laboratory tools, and researchers purchased buffer concentrates instead of making reagents themselves with a balance and pH meter. I was now about to start my academic career and before even considering the notion of applying for federal funding, I was in need of transitional support to afford me the opportunity to become "retooled" as well as to help me define my new area of research investigation.Having devoted my graduate work to the study of the human red cell (RBC) membrane cytoskeleton, 1-3 I decided to stick with the RBC but let my newly kindled I interest in immunohematology lead me to travel approximately 100 Å away from the membrane cytoskeleton through the phospholipid bilayer to the extracellular surface of the RBC where the antigens reside. In particular, I was interested in studying the immune response to RBC antigens as a way of gaining a better understanding of human humoral immune responses, notably those that can distinguish between closely related polymorphic structures. At this point in time, much had been known about the murine immune system because of the ease with which laboratory animal experimentation could be conducted, but ethical issues and other constraints put a damper on the at-will intravenous administration of foreign substances into humans to study the development of humoral immunity. It was apparent though, that the transfusion of allogeneic blood for therapeutic purposes and the undesirable sensitization that can often occur could provide a unique opportunity to ask basic questions about human antibody formation such as clonal relatedness, somatic mutation, affinity maturation, and others.To study human RBC antibodies on a molecular genetic level, I needed to be able to isolate and produce human monoclonal antibodies (MoAbs) from the peripheral blood B cells of sensitized individuals. Technical difficulties inherent in the immortalization of human B cells led me to the use of molecular approaches that obviated the need for cellular trans...

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“…Currently, human anti-D is obtained either from women who have been inadvertently immunized through transfusion, or from immunized volunteers. In an attempt to ensure reliable and reproducible supply of this important therapeutic and diagnostic reagent, cell lines secreting human anti-D monoclonal antibodies have been developed using strategies such as EBV transformation of immune B cells followed by fusion to a B cell line (Bron et al, 1984;Blancher et al, 1994), direct fusion of immune B cells to B cell lines of mouse or human origin (Rapaille et al, 1993) and EBV transformation of immune B cells followed by cloning or antigen enrichment (Kumpel et al, 1989;del Aguila et al, 1994). The production of human antibodies and antibody fragments by recombinant DNA techniques offers an alternative to this approach.…”

Section: Introductionmentioning

confidence: 99%

Expression and functional analysis of recombinant scFv and diabody fragments with specificity for human RhD

Asvadi

Fletcher

Raison

2002

J of Molecular Recognition

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In an attempt to generate recombinant anti-D reagents for possible diagnostic and therapeutic use we cloned the genes encoding the variable (V) domains of a human anti-D antibody secreted by the lymphoblastoid cell line BTSN4. A single-chain Fv (scFv) fragment was constructed using a 21 amino acid linker to join the genes encoding the variable domains of the BTSN4 heavy (VH) and light chains (VL). A diabody construct was also generated by reducing the length of the scFv linker from 21 to 10 residues. The scFv and diabody constructs were cloned into the pFLAG-CTS vector, expressed in E. coli host cells and the recombinant proteins were affinity-isolated from bacterial culture medium. Analysis of the recombinant proteins indicated that they retained the D antigen binding specificity of the parental BTSN4 IgG. Furthermore, both fragments mediated agglutination of papain-treated D positive erythrocytes in the absence of a cross-linking second antibody. While the agglutinating property of BTSN4 diabody was readily explained by the non-covalent association of this protein as a bivalent dimer, oligomeric forms of BTSN4 scFv were not detected when the protein was analysed by size exclusion chromatography. Thus, the agglutinating property of the scFv is not the result of the formation of non-covalently associated multimeric forms of the antibody fragment.

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Production of Stable Human-Mouse Hybridomas Secreting Monoclonal Antibodies against Rh D and c Antigens

Cited by 7 publications

References 24 publications

Establishment of human heterohybridoma and lymphoblastoid cell lines specific for the Rh D and C antigens

Establishment of human heterohybridoma and lymphoblastoid cell lines specific for the Rh D and C antigens

Developing phage display tools for use in transfusion medicine

Expression and functional analysis of recombinant scFv and diabody fragments with specificity for human RhD

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