Abstract: 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.

Abstract: 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.

Abstract: 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 (VH) and the light (VL) chain antibody genes. Mab clone 1A4A1 in E. coli strain TG-1 was successfully cloned as ScFv. Results were reproduced in E. coli 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 VL 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 VL 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.

Abstract: 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.

Abstract: The invention discloses generation of a single chain variable fragment (ScFv) antibody from a well-characterized monoclonal antibody (Mab) against Venezuelan equine encephalitis (VEE) virus, by cloning variable regions of the heavy (VH) and the light (VL) chain antibody genes, connected by a DNA linker, in phagemid expression vector pCANTAB 5 E. Mab clone 1A4A1 was successfully cloned as ScFv in Escherichia coli strain TG-1 and expressed as a ˜30 KDa ScFv protein which was functional in recognizing VEE by ELISA. Results were reproduced in Escherichia coli strain HB2151, where the same clone, designated A116, was expressed mainly as soluble periplasmic protein. The 30 KDa A116 displayed weak binding specificity to VEE. Sequence analysis revealed a frame shift in the N-terminal region of the VL domain, upstream to the complementarity-determnining region 1, as the probable cause of reduced activity.