Linker modification introduces useful molecular instability in a single chain antibody.

SCA 4-4-20/212, is a recombinant single chain antibody directed against fluorescein (Fl) composed of the variable light (VL) and variable heavy (VH) domains of the monoclonal antibody 4-4-20, tethered by a 14 amino acid linker. Binding of SCA 4-4-20/212 to Fl quenches its fluorescence, thus enabling the distinction between bound and free Fl. This was used to follow antibody denaturation which followed a two-step process: rapid selected and restricted denaturation followed by slow and progressive denaturation. This two-phase phenomenon might reflect selective susceptibility of the CDR loops to denaturation. Furthermore, a new SCA, SCA 4-4-20/9, was constructed by site-directed mutagenesis of SCA 4-4-20/212 using PCR methodology. SCA 4-4-20/9 was similar to SCA 4-4-20/212, but for a nine residue linker. The two SCAs were compared for Fl binding, heat stability, the effect of denaturing agents and susceptibility to proteolysis. The modification of the linker caused a general conformational rearrangement in the SCA molecule, rendering it more sensitive to denaturation and proteolysis. This molecular instability may find utility in the application of SCAs in analytical systems or as the recognition component in biosensors.