Cookies on this website

We use cookies to ensure that we give you the best experience on our website. If you click 'Accept all cookies' we'll assume that you are happy to receive all cookies and you won't see this message again. If you click 'Reject all non-essential cookies' only necessary cookies providing core functionality such as security, network management, and accessibility will be enabled. Click 'Find out more' for information on how to change your cookie settings.

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.

Original publication

DOI

10.1515/BC.2000.023

Type

Journal article

Journal

Biol Chem

Publication Date

02/2000

Volume

381

Pages

173 - 178

Keywords

Alkaline Phosphatase, Amino Acid Sequence, Antibodies, Monoclonal, Antibody Affinity, Base Sequence, Blotting, Western, CD3 Complex, Cell Line, Chromatography, Affinity, Flow Cytometry, Genetic Vectors, Humans, Hybridomas, Immunoglobulin Variable Region, Immunohistochemistry, Molecular Sequence Data, Monocytes, Palatine Tonsil, Protein Engineering, Recombinant Fusion Proteins, T-Lymphocytes