Her Majesty the Queen in Right of Canada, As Represented by the Minister of National Defence

Nexus Dx, Inc.

Azhar Alvi

Leslie P Nagata

R Elaine Fulton

Weigang Hu

George Jackowski

William Yajima

Roshana Sikora

Mee-Ra Hong

The invention provides a method to efficiently express high levels of a recombinant untagged NT-proBNP for use as a calibrator in NT-proBNP immunoassays.

Calibrator for NT-proBNP immunoassay

Construction of a recombinant gene fusion encoding a human IgG1 heavy chain constant region and a single-chain variable fragment antibody of 1A4A1 monoclonal antibody is disclosed. The recombinant antibody of the present invention confers human immune effector functions on murine antibodies. After expression in bacteria as inclusion bodies, the recombinant antibody was purified and refolded in vitro. The recombinant soluble antibody retains high antigen-binding affinity to VEE and possesses some human IgG crystallizable fragment domain functions. On non-reducing gel electrophoresis analysis, disulfide bond formation was found in the hinge region of the recombinant antibody. The present invention shows that the recombinant antibody is in a native, functionally active form and it provides the basis to characterize the recombinant antibody for efficacy in vivo.

Fusion protein of human IgG1 heavy chain constant region and scFv antibody against equine encephalitis virus

A recombinant gene encoding a single-chain variable fragment (scFv) antibody against Venezuelan equine encephalitis virus (VEE) being cloned into a prokaryotic T7 RNA polymerase-regulated expression vector was disclosed. A streptavidin-binding peptide (SBP) sequence fused to a 6His tag is then attached downstream to the scFv gene. The recombinant fusion protein is expressed in bacteria and then purified by immobilized metal affinity chromatography. 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, but also possessed streptavidin-binding activity. This discovery obviates the need for chemical biotinylation of antibodies and the risk associated with antibody denaturation and provides a stable and reproducible reagent for rapid and efficient immunoassay of VEE.

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

A genetically biotinylated single chain fragment variable (scFv) antibody against Venezuelan equine encephalitis virus (VEE) being applied in a system consisting of an immunofiltration-enzyme assay (IFA) with a light addressable potentiometric sensor (LAPS) for the rapid identification of VEE is disclosed. The IFA entails formation of an immunocomplex sandwich consisting of VEE, biotinylated antibody, fluoresceinated antibody and streptavidin, capturing the sandwich by filtration on biotinylated membrane, and detecting the sandwich by anti-fluorescein urease conjugate. The concentration ratio of biotinylated to fluoresceinated antibodies is investigated and optimized. The IFA/LAPS assay sensitivity was approximately equal to that of a conventional enzyme-linked immunosorbant assay utilizing polystyrene plates and a chromogenic substrate, however, less time and effort were required for performance of the IFA/LAPS assay.

Genetically biotinylated recombinant antibody in immunofiltration assay by light addressable potentiometric sensor for identification of Venezuelan equine encephalitis virus

A single chain variable fragment (ScFv) antibody from a monoclonal antibody (Mab) against Venezuelan equine encephalitis (VEE) virus, is generated by cloning linked variable regions of the heavy (V ) and the light (V ) chain antibody genes. Mab clone 1A A in strain TG-1 was successfully cloned as ScFv. Results were reproduced in strain HB2151, expressing the same clone, A116, though displaying weak binding specificity to VEE due to a frame shift in the N-terminal region of the V domain, upstream to the complementarity-determining region 1. A PCR-based site-directed mutagenesis approach was adopted to re-introduce the three single-base deletions in the 5 prime region of the V gene of A116, corresponding to framework-1 region, producing mutant MA116, correcting a localized frame-shift in framework-1 region to consensus framework-1 amino acid sequence. A MA116 clone, MA116-15, shows comparable reactivity to the parental Mab in recognizing VEE antigen.

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Mississauga, Canada

Azhar Alvi