| Since the Industrial Revolution many different organic contaminants (e.g., drugs and pesticides) with molecular weights less than 1000 Da (i.e., haptens) have been deposited into the environment, thereby affecting non-target organisms. Consequently, there is a need to develop sensitive assays for the detection of these contaminants to determine their fate, persistence and impact in the environment. Immunoassays have been shown to be one of the most sensitive and reliable detection methods. This thesis addresses some of the challenges associated with isolating antibody fragments and developing immunoassays against two hydrophobic haptens, which are water contaminants, i.e. the agricultural fungicide azoxystrobin (MW 403 Da; H2O solubility 6 mg L -1) and the veterinary antibiotic monensin (MW 671 Da; H2O solubility 50 mg L-1). To isolate anti-azoxystrobin V HHs, a hyper-immunized library was constructed by isolating the V HH-coding genes from lymphocytes collected from a llama immunized with azoxystrobin conjugated to bovine serum albumin (BSA). Six rounds of panning were performed against azoxystrobin conjugated to either ovalbumin (OVA) or rabbit serum albumin (RSA) to enrich clones expressing VHH specific to the hapten. After screening 100 clones, four VHH-antibodies with different amino acid sequences were identified and expressed in soluble format. Indirect competitive enzyme-linked immunosorbent assay (CI-ELISA) showed three of the four VHHs were specific for azoxystrobin. The IC50 values of these VHHs were between 2 and 11.3 muM.;Anti-monensin scFvs were isolated from another hyperimmunized-phage-displayed library originating from splenocytes of a mouse immunized with monensin conjugated to BSA. Three rounds of selection were performed against monensin conjugated to OVA and keyhole limpet hemocyanin (KLH), alternatively. A total of 376 clones from rounds two and three were screened for their specific binding to monensin conjugates and the positive clones were sequenced. After soluble expression of positive scFvs, an indirect competitive fluorescence polarization (CI-FP) assay showed that four of these scFvs bound to free monensin. The IC50 values ranged from 0.031 to 231 muM. Antibody cross reactivity studies using salinomycin, lasalocid A, kanamycin and ampicillin revealed that the two best scFvs were highly specific for monensin. |
