Title page for ETD etd-11102004-101210


Type of Document Dissertation
Author Munene, Cate Nakaweesa
Author's Email Address cmunene@lsu.edu
URN etd-11102004-101210
Title Development of Chicken Polyclonal and Mouse Monoclonal-Based Enyzyme Immunoassays for the Detection of Beta-Cyclocitral in Catfish Pond Water
Degree Doctor of Philosophy (Ph.D.)
Department Food Science
Advisory Committee
Advisor Name Title
Jack N. Losso Committee Chair
Casey C. Grimm Committee Member
J. Samuel Godber Committee Member
Ralph J. Portier Committee Member
Robert E. Truax Committee Member
Robert P. Hammer Committee Member
Huangen Ding Dean's Representative
Keywords
  • catfish
  • beta-cyclocitral
  • immunoassays
  • ELIFA
  • ELISA
  • antibodies
  • off-flavor
Date of Defense 2004-10-25
Availability unrestricted
Abstract
The Catfish industry faces a problem of off-flavors due to odorous compounds produced by cyanobacteria and blue-green algae. Beta-Cyclocitral imparts a hay-woody odor to pond water and fish tissues. At present there are no reliable pond treatment methods available to control these off-flavors. To monitor the levels of this compound for quality control, rapid, sensitive and inexpensive methods are needed. The major goal of this study was to develop enzyme-linked immunoflow assays based on monoclonal and polyclonal antibodies that are specific and sensitive enough to detect beta-cyclocitral in catfish pond water.

Beta-Cyclocitral-PPD conjugate was prepared and used to immunize two chickens and two mice for the production of polyclonal and monoclonal antibodies against beta-cyclocitral respectively. Monospecific polyclonal antibodies were purified from the eggs laid by the immunized chickens, using affinity chromatography. For the production of the monoclonal antibodies, hybridoma cells were made by fusion of myeloma cells and spleen cells of the mice that showed high antibody titer and specificity. Hybridoma cells that secreted high affinity monoclonal antibodies were cloned by the limiting dilution method and at least 10 hybridoma cell lines positive for anti-beta-cyclocitral antibodies were established. Immunochemical methods based on anti-beta-cyclocitral IgY and IgG were developed. The two ELISAs based on IgY and IgG had a limit of detection of 1.0 ng/mL and respective I50 values of 3.93 and 7.98 ng/mL. Two enzyme-linked-immunoflow (ELIFA) assays were developed based on the ELISAs. The ELIFAs were very easy to perform but were less sensitive than the ELISAs as shown by their I50 of 46 ug/mL for the IgY-based ELIFA and 93 ug/mL for the IgG-based ELIFA. Further investigations are required for the more efficient recovery of the monoclonal antibodies, to validate the ELISAs, to improve the sensitivity of the ELIFAs and to determine the potential to adapt the developed assays to a kit format for use by the catfish industry and water treatment industries as well as other aquaculture industries.

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