Date of Award

1999

Document Type

Dissertation

Degree Name

Doctor of Philosophy (PhD)

Department

Environmental Sciences

First Advisor

Leslie C. Plhak

Abstract

2-Methylisoborneol (MIB), a secondary metabolite of cyanobacteria and fungi, causes earthy-musty taste and odor. This is a significant problem in the aquaculture industry and large scale water supplies. Especially it is a major problem for the channel catfish industry. To monitor the levels of this compound for quality control and abatement, rapid, sensitive and inexpensive methods are needed. This research reports the development of an indirect enzyme linked immunosorbent assay (ELISA) for MIB using monoclonal antibodies. For the preparation of monoclonal antibodies against MIB, MIB-Limulus polyphemus hemocyanin (LPH) and borneol-LPH were synthesized as an immunogen. Borneol-bovine serum albumin (BSA), isoborneol-BSA, and MIB-BSA were synthesized as a solid phase protein conjugate to compare and optimize the effect of solid phase conjugate structure. To produce monoclonal antibodies, two mice were immunized with borneol-LPH protein conjugate and another two mice were immunized with MIB-LPH. Hybridoma cells were made by the fusion of myeloma cells and spleen cells of the mice that showed high antibody titer and specificity. Hybridoma cells secreting high affinity monoclonal antibodies were cloned by the limiting dilution method three times to ensure clonality. For the optimization of ELISA, different Ab concentrations and different solid phase conjugate concentrations were tested. At lower concentrations of antibody and solid phase conjugate, the sensitivity was greatest, however the signal also began to decrease. The effect of solid phase conjugate structure was studied by comparing the sensitivity of ELISA using borneol-BSA, isoborneol-BSA, and MIB-BSA as a solid phase conjugate. When MIB-BSA was used as a solid phase conjugate, the affinity for the free MIB was improved against the affinity to MIB-BSA and the sensitivity of ELISA was greatest. By using anti-borneol monoclonal antibody (1/250) and MIB-BSA (0.5mug/ml) as a solid phase conjugate, standard curve was constructed from 100mg/L to 0.1ng/L range. The linear range of this standard curve was found to occur between 1 ng/L and 1 mg/L MIB. The detection limit, defined as the MIB concentration giving an A/Ao value of 0.8, was found to be approximately 1ng/L.

ISBN

9780599548756

Pages

139

DOI

10.31390/gradschool_disstheses.7054

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