Immunolabeling of CD3-Positive Lymphocytes with a Recombinant Single-Chain Antibody/Alkaline Phosphatase Conjugate
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Philippe Bourin
Abstract
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 antigenbinding specificity of antibody G3(3). A gene encoding a singlechain 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.
Copyright © 2000 by Walter de Gruyter GmbH & Co. KG
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Articles in the same Issue
- The Twin-Arginine Translocation System: A Novel Means of Transporting Folded Proteins in Chloroplasts and Bacteria
- De Novo DNA Methylation at Nonrandom Founder Sites 5' from an Unmethylated Minimal Origin of DNA Replication in Latent Epstein-Barr Virus Genomes
- Trichostatin A Modulates Expression of p21waf1/cip1, Bcl-xL, ID1, ID2, ID3, CRAB2, GATA-2, hsp86 and TFIID/TAFII31 mRNA in Human Lung Adenocarcinoma Cells
- Effects of the Active Aldehyde Group Generated by RNA N-Glycosidase in the Sarcin/Ricin Domain of Rat 28S Ribosomal RNA on Peptide Elongation
- Responses to Peroxynitrite in Yeast: Glyceraldehyde-3-Phosphate Dehydrogenase (GAPDH) as a Sensitive Intracellular Target for Nitration and Enhancement of Chaperone Expression and Ubiquitination
- Enhancing the T R Transition of Insulin by Helix-Promoting Sequence Modifications at the N-Terminal B-Chain
- Structure-Dependent Effects of Glucose-Containing Analogs of Platelet Activating Factor (PAF) on Membrane Integrity
- Two Different Mechanisms for Activation of Cyclic PIP Synthase: by a G Protein or by Protein Tyrosine Phosphorylation
- 3-Hydroxybenzoate:Coenzyme A Ligase from Cell Cultures of Centaurium erythraea: Isolation and Characterization
- Gel Chromatographic Characterization of the Hydrophobic Interaction of Glycosylphosphatidylinositol-Alkaline Phosphatase with Detergents
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