The use of serological assays in viral diagnostics is still the preferred method for robust detection assays. Nucleic acid-based detection is more sensitive and rapid, but the specificity of the reactions can limit detection, particularly within diverse virus species and those with high rates of recombination.
We are creating a system to generate recombinant single-chain variable fragment (scFv) antibodies, with properties that will allow their production and site-directed labeling. The specific chemistry used to label the scFvs should allow detection without alteration of the kinetics with which they bind to their antigen, an interaction that can be negatively affected by random crosslinking techniques that are currently employed in antibody-reporter linkage.
Two different vector systems, both based on the phage display library of pCANTAB-link (Sapats et al.2003) have been constructed. One has the labeling site, and an immobilized metal ion affinity chromatography (IMAC) purification tag, and the second has both of these and the addition of the sequence for the human kappa light chain region. Expression of scFvs with the human kappa light chain have been shown to form dimers which increase target specificity, and will also allow detection using anti-human labeled antibodies.
For a test system, the use of Potato Virus Y (PVY) was chosen. Purified PVY was injected into chickens, and the spleens harvested for RNA extraction. An initial scFv library was created in pCANTAB-Link, screened and two binders to the purified virus were isolated. The scFvs developed will be tested for utility in traditional ELISA and microsphere assays.
%8 09/02/2011