Immunolabeling of CD3-positive lymphocytes with a recombinant single-chainantibody/alkaline phosphatase conjugate

G3(3) is a novel murine monoclonal antibody directed against the CD3 antige n of human T lymphocytes which could be used to analyze lymphoid malignanci es. We have produced and characterized a recombinant colorimetric immunocon jugate with the antigen-binding specificity of antibody G3(3). A gene encod ing a single-chain antibody variable fragment (scFv) was assembled using th e original hybridoma cells as a source of antibody variable heavy OIH) and variable light (VL) chain genes. The chimeric gene was introduced into a pr okaryotic expression vector in order to produce a soluble scFv fused to bac terial alkaline phosphatase, DNA sequencing and Western blotting analyses d emonstrated the integrity of the soluble immunoconjugate recovered from ind uced recombinant bacteria. The scFv/AP protein was bifunctional and similar in immunoreactivity to the parent G3(3) antibody. Flow cytometry and immun ostaining experiments confirmed that the activity of the scFv/AP protein co mpares favourably with that of the parent antibody. The scFv/AP conjugate w as 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 ap plications as an immunodiagnostic reagent.