Immunolabeling of CD3-positive lymphocytes with a recombinant single-chain antibody/alkaline phosphatase conjugate.
G3(3) is a novel murine monoclonal antibody directed against the CD3 antigen of human T lymphocytes which could be used to analyze lymphoid malignancies. We have produced and characterized a recombinant colorimetric immunoconjugate with the antigen-binding specificity of antibody G3(3). A gene encoding a single-chain antibody variable fragment (scFv) was assembled using the original hybridoma cells as a source of antibody variable heavy (VH) and variable light (VL) chain genes. The chimeric gene was introduced into a prokaryotic expression vector in order to produce a soluble scFv fused to bacterial alkaline phosphatase. DNA sequencing and Western blotting analyses demonstrated the integrity of the soluble immunoconjugate recovered from induced recombinant bacteria. The scFv/AP protein was bifunctional and similar in immunoreactivity to the parent G3(3) antibody. Flow cytometry and immunostaining experiments confirmed that the activity of the scFv/AP protein compares favourably with that of the parent antibody. The scFv/AP conjugate was bound to CD3 antigen at the surface of T cells and was directly detected by its enzymatic activity. Thus this novel fusion protein has potential applications as an immunodiagnostic reagent.
Bourin
P
P
Centre de Transfusion Sanguine des Armées, Clamart, France.
Servat
A
A
Lataillade
J J
JJ
Goyffon
M
M
Vaux
D
D
Billiald
P
P
D000469:Alkaline Phosphatase / Q000737:chemistry* / Q000276:immunology; D000595:Amino Acid Sequence; D000911:Antibodies, Monoclonal; D000915:Antibody Affinity; D001483:Base Sequence; D015153:Blotting, Western; D017252:CD3 Complex / Q000032:analysis* / Q000276:immunology; D002460:Cell Line; D002846:Chromatography, Affinity; D005434:Flow Cytometry; D005822:Genetic Vectors / Q000138:chemical synthesis; D006801:Humans; D006825:Hybridomas; D007135:Immunoglobulin Variable Region / Q000737:chemistry* / Q000276:immunology; D007150:Immunohistochemistry; D008969:Molecular Sequence Data; D009000:Monocytes / Q000276:immunology; D014066:Palatine Tonsil / Q000166:cytology / Q000276:immunology; D015202:Protein Engineering; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000276:immunology; D013601:T-Lymphocytes / Q000737:chemistry / Q000276:immunology*
D000911:Antibodies, Monoclonal; D017252:CD3 Complex; D007135:Immunoglobulin Variable Region; D011993:Recombinant Fusion Proteins; D000469:Alkaline Phosphatase