Development of single-chain variable fragment (scFv) antibodies against hapten benzo[a]pyrene: A binding study

Due to its highly carcinogenic and mutagenic effect on humans, a maximum tolerable limit of 10 ng/L of benzo[a]pyrene (B[a]P) in drinking water was set by the European Commission (Council Directive 98/83/EC). Although several polyclonal and monoclonal antibodies (mAb) for the detection of B[a]P and other polycyclic aromatic hydrocarbons (PAH) have been developed by others, a traditional enzyme-linked immunosorbent assay (ELISA) with a limit of quantification of 10 ng/L for monitoring B[a]P has not been developed. With this in mind, several single-chain variable fragment (scFv) antibodies were created using existing mAbs against the extremely hydrophobic hapten B[a]P, and their heavy and light chains recombined to make unique variable light (V _L) and heavy (V _H) chain combinations. Their binding behaviour was investigated using microtiter plate ELISA and surface plasmon resonance techniques. Specifically, the coding sequences for V _L and V _H chains of 10 murine anti-B[a]P antibody producing hybridoma cell lines were isolated by degenerate oligonucleotide primer sets, cloned in phagemid pIT2 and transferred into Escherichia coli HB2151. To systematically investigate the interaction of the V _L and V _H domains, three high-affinity B[a]P-specific and one nonspecific clone were selected and recombined to build a set of 16 different V _L and V _H combinations. On the basis of our data, it was shown that the V _H plays the major role for specific binding of B[a]P, whilst the V _L can, in some cases, increase the final sensitivity of the assay by one order of magnitude. Furthermore, the sequence analysis of scFvs indicates that the complementarity determining region H3 plays a major role in affinity, whilst cross-reactivity to seven other PAHs demonstrates the importance of the V _L in providing cross-reactivity. [Figure not available: see fulltext.]