title: Fast track antibody V-gene rescue, recombinant expression in plants and characterization of a PfMSP4-specific antibody.
abstract: BACKGROUND
  Monoclonal antibodies (mAbs) are essential tools in biological research, diagnosis and therapy, and are conventionally produced in murine hybridoma cell lines. Professional applications of mAbs depend on the steady supply of material. Because hybridoma cultures can stop producing the antibody or even die, preservation of the unique epitope specificity of mAbs by rescue of the sequences encoding the antibody variable domains (V regions) is important. The availability of these sequences enables not only the recombinant expression of the original antibody for further applications, but opens the road for antibody engineering towards innovative diagnostic or therapeutic applications. A time- and cost-efficient production system enabling the detailed analysis of the antibodies is an essential requirement in this context.
  
  
  METHODS
  Sequences were rescued from three hybridoma cell lines, subjected to sequence analysis, subcloned into binary expression vectors and recombinantly expressed as chimeric mAb (constant regions of human IgG1:k1) in Nicotiana benthamiana plants. The properties of the recombinant and the murine mAbs were compared using competition enzyme-linked immunosorbent assay (ELISA) and surface plasmon resonance (SPR) spectroscopy. The recognition of native PfMSP4 by the recombinant mAb was analysed by immunofluorescence staining of Pf 3D7A schizonts and by western blot analysis of merozoite extract.
  
  
  RESULTS
  The rescued sequences of all three hybridoma cell lines were identical. The recombinant mAb was successfully expressed as IgG in plants at moderate levels (45 mg/kg fresh leaf weight). Preservation of the original epitope was demonstrated in a competition ELISA, using recombinant mAb and the three murine mAbs. EGF_PfMSP4-specific affinities were determined by SPR spectroscopy to 8 nM and 10 nM for the murine or recombinant mAb, respectively. Binding to parasite PfMSP4 was confirmed in an immunofluorescence assay showing a characteristic staining pattern and by western blot analysis using merozoite extract.
  
  
  CONCLUSIONS
  As demonstrated by the example of an EGF_PfMSP4-specific antibody, the described combination of a simple and efficient hybridoma antibody cloning approach with the flexible, robust and cost-efficient transient expression system suitable to rapidly produce mg-amounts of functional recombinant antibodies provides an attractive method for the generation of mAbs and their derivatives as research tool, novel therapeutics or diagnostics.
authors: Fraunhofer Institute for Molecular Biology and Applied Ecology IME, Forckenbeckstraße 6, 52074, Aachen, Germany. Stephanie.Kapelski@ime.fraunhofer.de.|RWTH Aachen University, Institute for Molecular Biotechnology, Worringer Weg 1, 52074, Aachen, Germany. Stephanie.Kapelski@ime.fraunhofer.de.|Institute for Applied Medical Engineering at RWTH Aachen University and Hospital, Department of Experimental Medicine and Immunotherapy, Pauwelsstraße 20, 52074, Aachen, Germany. Stephanie.Kapelski@ime.fraunhofer.de.
authors: Stephanie
authors: S
authors: Kapelski
authors: Fraunhofer Institute for Molecular Biology and Applied Ecology IME, Forckenbeckstraße 6, 52074, Aachen, Germany. Alexander.Boes@ime.fraunhofer.de.
authors: Alexander
authors: A
authors: Boes
authors: Fraunhofer Institute for Molecular Biology and Applied Ecology IME, Forckenbeckstraße 6, 52074, Aachen, Germany. Holger.Spiegel@ime.fraunhofer.de.
authors: Holger
authors: H
authors: Spiegel
authors: Fraunhofer Institute for Molecular Biology and Applied Ecology IME, Forckenbeckstraße 6, 52074, Aachen, Germany. Melanie.Almeida@rwth-aachen.de.|RWTH Aachen University, Institute for Molecular Biotechnology, Worringer Weg 1, 52074, Aachen, Germany. Melanie.Almeida@rwth-aachen.de.
authors: Melanie
authors: M
authors: de Almeida
authors: Fraunhofer Institute for Molecular Biology and Applied Ecology IME, Forckenbeckstraße 6, 52074, Aachen, Germany. Torsten.Klockenbring@ime.fraunhofer.de.
authors: Torsten
authors: T
authors: Klockenbring
authors: Fraunhofer Institute for Molecular Biology and Applied Ecology IME, Forckenbeckstraße 6, 52074, Aachen, Germany. Andreas.Reimann@ime.fraunhofer.de.
authors: Andreas
authors: A
authors: Reimann
authors: Fraunhofer Institute for Molecular Biology and Applied Ecology IME, Forckenbeckstraße 6, 52074, Aachen, Germany. Rainer.Fischer@ime.fraunhofer.de.|RWTH Aachen University, Institute for Molecular Biotechnology, Worringer Weg 1, 52074, Aachen, Germany. Rainer.Fischer@ime.fraunhofer.de.
authors: Rainer
authors: R
authors: Fischer
authors: Fraunhofer Institute for Molecular Biology and Applied Ecology IME, Forckenbeckstraße 6, 52074, Aachen, Germany. Stefan.Barth@ime.fraunhofer.de.|Institute for Applied Medical Engineering at RWTH Aachen University and Hospital, Department of Experimental Medicine and Immunotherapy, Pauwelsstraße 20, 52074, Aachen, Germany. Stefan.Barth@ime.fraunhofer.de.
authors: Stefan
authors: S
authors: Barth
authors: Fraunhofer Institute for Molecular Biology and Applied Ecology IME, Forckenbeckstraße 6, 52074, Aachen, Germany. Rolf.Fendel@ime.fraunhofer.de.|RWTH Aachen University, Institute for Molecular Biotechnology, Worringer Weg 1, 52074, Aachen, Germany. Rolf.Fendel@ime.fraunhofer.de.|Institute for Applied Medical Engineering at RWTH Aachen University and Hospital, Department of Experimental Medicine and Immunotherapy, Pauwelsstraße 20, 52074, Aachen, Germany. Rolf.Fendel@ime.fraunhofer.de.
authors: Rolf
authors: R
authors: Fendel
mesh_terms: D000818:Animals; D000911:Antibodies, Monoclonal / Q000235:genetics / Q000276:immunology* / Q000302:isolation & purification; D000913:Antibodies, Protozoan / Q000235:genetics / Q000276:immunology* / Q000302:isolation & purification; D000953:Antigens, Protozoan / Q000276:immunology*; D015153:Blotting, Western; D004797:Enzyme-Linked Immunosorbent Assay; D015870:Gene Expression; D006801:Humans; D007135:Immunoglobulin Variable Region / Q000235:genetics / Q000276:immunology* / Q000302:isolation & purification; D008807:Mice, Inbred BALB C; D008856:Microscopy, Fluorescence; D030781:Organisms, Genetically Modified / Q000235:genetics / Q000378:metabolism; D015800:Protozoan Proteins / Q000276:immunology*; D011994:Recombinant Proteins / Q000235:genetics / Q000276:immunology / Q000302:isolation & purification; D020349:Surface Plasmon Resonance; D014026:Nicotiana / Q000235:genetics / Q000378:metabolism*
chemical_list: D000911:Antibodies, Monoclonal; D000913:Antibodies, Protozoan; D000953:Antigens, Protozoan; D007135:Immunoglobulin Variable Region; D015800:Protozoan Proteins; D011994:Recombinant Proteins; C118839:merozoite surface protein 4, Plasmodium
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