Modeling and characterization of single chain variable fragments (SCFV) specific for an isocyanate-based protein conjugate

Diisocyanates are highly reactive chemicals used widely in industry to make polyurethane foams, paints, coatings etc. Previous work described the isolation of ScFvs capable of catalytic hydrolysis of carbamate bonds within polyurethane made from diisocyanates. Characterization of the binding specificity of two of these catalytic antibodies, ScFvs #12 and #9, was one goal of the current work with the specific aim of improving catalysis. It is demonstrated that ScFv #12 recognizes and binds the tolyl group of toluene diisocyanate (TDI) but does not discriminate between a carbamate and a urea bond made by the reaction of an isocyanate with a protein. Binding specificity was tested using a set of TDI, p-tolylisocyanate (TMI) and butylisocyanate (BI)-human serum albumin (HSA) conjugates made while varying pH and molar ratio of diisocyanate and HSA. A characteristic biphasic recognition pattern was found in the binding of these varied conjugates with ScFv #12. The pattern showed maxima at pH 7.5 and above pH 8.0, the former ascribed to recognition of TDI polymers attached to HSA and the latter ascribed to high TDI modification through lysine modification on HSA. This same pattern was observed with sera from a small sampling of TDI-exposed and control humans. Molecular modeling and site-directed mutagenesis has been used to identify locations on ScFv #9 and #12 important for catalysis and binding respectively. Mutated positions on ScFv #12 important for binding have been tested for their effect on antigen recognition. Substitution of one of the selected positions, Y213, in the binding site with alanine shows lost of antigen recognition while replacement with cysteine recovers and improves the binding affinity of all mutants. Subsequent labeling of the cysteines with fluorescent dyes show no effect on target binding. Exploration of the use of these sites as possible attachment sites for environmentally sensitive dyes has provided inconclusive results thus far.