Immunoassays for 2-methylisoborneol (MIB) and monensin in water were developed, devised and tested to see if the sensitivity could be established and improved. MIB and monensin are hydrophobic haptens with molecular weights of 168 and 671 Da, respectively. Rabbits were immunized with (-) camphor-BSA and (-) borneol-BSA for the production of polyclonal antibodies (pAbs) to MIB. Monoclonal antibodies (mAbs) were produced in Mus musculus using (-) camphor-BSA as immunogen. (+) Bornylamine-thyroglobulin (TG) and MIB-TG were synthesized and used as plate coatings. For the monensin immunoassay, monensin was conjugated to BSA and OVA for immunogen and plate coating, respectively. Several physical parameters that affect the sensitivity of immunoassays including pre-incubation of antibody and antigen, incubation time and temperature, detergent, organic solvents, and ionic strength were evaluated. Improvement of immunoassay sensitivity was also performed by reducing the concentrations of coating antigen and antibodies and using alternative reporter systems such as chemiluminescence (CICL-ELISA), tyramide signal amplification (TSA) and biotin-streptavidin. Different assay formats, i.e., competitive indirect and competitive direct were also compared. Usability of both pAb-based immunoassays for MIB and monensin was evaluated in fortified water samples.
A polyclonal-based (pAb) ELISA for MIB had a detection limit of 4.8 ng mL-1 and an IC50 of 105 ng mL-1. Rabbits immunized with (-) camphor-BSA showed a higher immune response than rabbits immunized with (-) borneol-BSA. One clone (i.e., 4F11) of fourteen characterized clones was used to create the monoclonal antibody (mAb)-based ELISA, which had an IC50 of 100.2 ng mL-1 and an LOD of 1.9 ng mL-1. The pAb- and mAb-based CI-ELISA were not specific to MIB alone and cross reacted with camphor and camphor-like compounds. Meanwhile, a pAb-based ELISA for monensin produced a detection limit of 0.1 ng mL-1 and had an IC50 of 1.056-1.090 ng mL-1 with high specificity to monensin. Other reporter systems did not improve the sensitivity of the immunoassays significantly. MIB and monensin polyclonal-based assays showed good correlation to analytical instrumental methods (i.e., GC-MS and LC-MS) in fortified water samples.
With a detection limit of ca. 5 ng mL-1 and 0.1 ng mL-1 for MIB and monensin, respectively, both polyclonal-based assays can be used for detection of these analytes in water from different sources and employed as screening tools to complement GC/HPLC-MS instrument methods.
| Identifer | oai:union.ndltd.org:LACETR/oai:collectionscanada.gc.ca:OGU.10214/7447 |
| Date | 03 September 2013 |
| Creators | Sukor, Rashidah |
| Contributors | Hall, J. Christopher |
| Source Sets | Library and Archives Canada ETDs Repository / Centre d'archives des thèses électroniques de Bibliothèque et Archives Canada |
| Language | English |
| Detected Language | English |
| Type | Thesis |
| Rights | Attribution 2.5 Canada, http://creativecommons.org/licenses/by/2.5/ca/ |
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