title: Recombinant single-chain Fv antibody fragment-alkaline phosphatase conjugate: a novel in vitro tool to estimate rabies viral glycoprotein antigen in vaccine manufacture.
abstract: The purpose of this study was to design a novel in vitro tool by using recombinant protein technology to qualify the whole reagent preparation procedure, to be used to quantify rabies viral antigen preparation in a simple and rapid format for potency control of rabies vaccines. 50AD1 is a neutralizing monoclonal antibody directed against the rabies virus glycoprotein that binds to native conformational antigenic site III. In the present study, the DNA fragments encoding the variable domains of 50AD1 were inserted into a prokaryotic expression vector so as to produce a single-chain Fv antibody fragment (scFv) genetically fused to the bacterial alkaline phosphatase (AP). The recombinant fusion protein preserved both the AP enzymatic activity and the antigen-binding activity against the rabies virus glycoprotein nearly identical to the parental antibody, and was used successfully in different assays including ELISA, dot-blot and cell culture tests. The present study shows that the genetic fusion protein provides a new tool for one-step rabies virus immunodetection, which can be produced in homogeneous bifunctional reagent, easily, quickly and reproducibly. In addition, this recombinant immunoconjugate is a promising alternative reagent for applications involving immunodetection, it presents a similar sensitivity and specificity to that obtained with classical reagents.
authors.affiliation: Laboratoire d'Immunopathologie, Vaccinologie et Génétique Moléculaire, Institut Pasteur de Tunis, 13 Place Pasteur BP74, 1002 Tunis-Belvédère, Tunisia. mohamed.mousli@pasteur.rns.tn
authors.forename: Mohamed
authors.initials: M
authors.lastname: Mousli
authors.forename: Imène
authors.initials: I
authors.lastname: Turki
authors.forename: Habib
authors.initials: H
authors.lastname: Kharmachi
authors.forename: Mohamed
authors.initials: M
authors.lastname: Saadi
authors.forename: Koussay
authors.initials: K
authors.lastname: Dellagi
mesh_terms: D000469:Alkaline Phosphatase / Q000235:genetics / Q000378:metabolism; D000595:Amino Acid Sequence; D000818:Animals; D000956:Antigens, Viral / Q000032:analysis*; D001483:Base Sequence; D002460:Cell Line; D007118:Immunoassay / Q000379:methods*; D007135:Immunoglobulin Variable Region / Q000235:genetics / Q000276:immunology; D008969:Molecular Sequence Data; D011818:Rabies / Q000821:virology; D011819:Rabies Vaccines; D011820:Rabies virus / Q000276:immunology* / Q000302:isolation & purification*; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000276:immunology; D014759:Viral Envelope Proteins / Q000032:analysis* / Q000276:immunology
chemical_list: D000956:Antigens, Viral; D007135:Immunoglobulin Variable Region; D011819:Rabies Vaccines; D011993:Recombinant Fusion Proteins; D014759:Viral Envelope Proteins; D000469:Alkaline Phosphatase