R. Elaine Fulton

Bispecific and bifunctional single chain recombinant antibodies

Bispecific and bifunctional monoclonal antibodies as second generation monoclonals, produced by conventional chemical or somatic methods, have proved useful in the immunodiagnosis and immunotherapy of cancer and other diseases. Recombinant antibodies produced by genetic engineering techniques have also become available for use in preclinical and clinical studies. Furthermore, through genetic engineering, it is possible to remove or add on key protein domains in order to create designer antibody molecules with two or more desired functions. This review summarizes the strategies for development of single chain variable fragment (scFv) bifunctional and bispecific antibodies. The advantages and disadvantages as well as the problems of generating the various bispecific and bifunctional antibody constructs are reported and discussed. Since conventionally prepared bispecific and bifunctional monoclonal antibodies have already shown promise in clinical trials and results from preclinical studies of recombinant bispecific antibodies are encouraging, clinical trials in humans of recombinant bispecific and bifunctional antibodies, as a new generation of biologicals, are likely to be the thrust in the next decade and beyond.

Recombinant anti-botulinum neurotoxin A single-chain variable fragment antibody generated using a phage display system.

A recombinant single-chain fragment variable antibody (scFv) to botulinum A neurotoxin (BoNT/A) was developed. BALB/C mice were immunized with BoNT/A. Splenomic RNA was isolated from the hyperimmune mice and used to prepare a cDNA library, from which the variable regions of the heavy and light chain antibody genes were generated and connected by a DNA linker. The resulting scFv genes were cloned into the phagemid vector pCANTAB5 in order to construct phage display scFv libraries. Individual anti-BoNT/A phage clones were isolated from the phage display libraries by immunoaffinity selection using immobilized BoNT/A and further evaluated by enzyme-linked immunosorbant assay, immunoprecipitation and Western blotting. Forty-eight clones were found to be BoNT/A-reactive. The most reactive clone, designated D12, was selected for further study. The scFv gene of D12 was subcloned into a Pichia pastoris vector, and expression in yeast was evaluated.

Genetic engineering of streptavidin-binding peptide tagged single-chain variable fragment antibody to venezuelan equine encephalitis virus

A recombinant gene encoding a single-chain variable fragment (scFv) antibody against Venezuelan equine encephalitis virus (VEE) was cloned into a prokaryotic T7 RNA polymerase-regulated expression vector. A streptavidin-binding peptide gene fused to a 6His tag was attached downstream to the scFv gene. The recombinant fusion protein was expressed in bacteria as inclusion bodies that were subsequently solubilized with 8 M urea and renatured by an arginine system. Purification of the fusion protein was achieved by immobilized metal affinity chromatography. Enzyme-linked immunosorbent assay (ELISA) and Western blotting results revealed that the fusion protein not only retained VEE antigen binding and specificity properties similar to those of its parent native monoclonal antibody (MAb), but also possessed streptavidin-binding activity. This experimental approach can eliminate the need for chemical biotinylation of antibodies and the risk associated of antibody denaturation and can provide a stable and reproducible reagent for rapid and efficient immunoassay of VEE when detected by horseradish peroxidase (HRP)-conjugated streptavidin.

Microbead electrochemiluminescence immunoassay for detection and identification of Venezuelan equine encephalitis virus.

An electrochemiluminescence (ECL) immunoassay, incorporating chemically biotinylated and ruthenylated antibodies down-selected from a panel of monoclonal and polyclonal reagents, was developed to detect and identify Venezuelan equine encephalitis virus (VEEV). The limit of detection (LOD) of the optimized ECL assay was 10(3)pfu/ml VEEV TC-83 virus and 1 ng/ml recombinant (r) VEEV E2 protein. The LOD of the ECL assay was approximately one log unit lower than that of a sandwich enzyme-linked immunosorbent assay (ELISA) incorporating the same immunoreagents. Repetition of ECL assays over time and by different operators demonstrated that the assay was reproducible (coefficient of variation 4.7-18.5% month-to-month; 3.3-8.8% person-to-person). The VEEV ECL assay exhibited no cross-reactivity with two closely related alphaviruses or with 21 heterologous biological agents. A genetically biotinylated recombinant VEEV antibody, MA116SBP, was evaluated for utility for detection of rE2; although functional in the ECL assay, the LOD was two log units higher (100 ng/ml vs 1 ng/ml) using MA116SBP than when chemically biotinylated antibody was used. The ECL assay detected VEEV at the lowest LOD (highest sensitivity) hitherto reported in the published literature and ECL assay results were generated in ∼60 min compared to a 6-8h period required for ELISA. Results have demonstrated a sensitive, rapid, and fully automated ECL immunoassay for detection and identification of VEEV.

A Suspension Array Immunoassay for the Toxin Simulant Ovalbumin

Abstract A microsphere-based suspension array (SA) system was used for the development and characterization of an immunoassay for the toxin simulant ovalbumin. Results obtained by SA immunoassay were compared with those obtained by enzyme-linked immunosorbent assay (ELISA) using the same immunoreagents. The limit of detection (LOD) for the SA ovalbumin assay was 4.9 ng/mL, compared to a LOD of 0.01 ng/mL for the ovalbumin ELISA. Although the ELISA LOD exceeded that of the SA assay, the SA assay was simple and rapid to perform, with assays being completed in half the time of the traditional ELISA. The well-to-well reproducibility (coefficient of variation (CV)) of the ELISA and the SA assay was 4.9% and 5.1%, respectively. The ELISA and SA assay plate-to-plate reproducibility was 14.8% and 6.1%, respectively. The protocols used to develop the SA assay for ovalbumin may be used as a template for development of other SA assays for toxins, bacteria, and viruses.