In Vivo and in Vitro Stimulation of Murine Splenocytes With Brevetoxin-2 Immunogenic Conjugates for the Preparation of Monoclonal Antibodies Against Ciguatoxin-Related Compounds.
Date of Award
Doctor of Philosophy (PhD)
School of Nutrition and Food Sciences
Douglas L. Park
The proliferation of toxic algae is a natural phenomenon known as a harmful algal bloom. Ciguatera fish poisoning and neurotoxic fish poisoning are syndromes associated with this phenomenon. Current methods for the detection of their toxins (ciguatoxins and brevetoxins) are expensive, time-consuming, require trained personnel and special laboratory equipment. In the case of ciguatoxins, the lack of standard material worsens the problem. Specific objectives of this study included the use of brevetoxin-2 (PbTx-2) for the preparation of antibodies which could cross-react with ciguatoxins, the use of different PbTx-2 immunogenic conjugates, the use of in vivo versus in vitro stimulation of murine splenocytes, and the use of alternative screening assays to detect anti-PbTx-2 antibodies. Three different immunogenic conjugates were prepared for immunization (BSA-PbTx-2, KLH-PbTx-2, and LPH-PbTx-2) and given to three groups of mice. After several injections, murine anti-PbTx-2 IgG antibodies were detected in the serum of the mice using experiments such as measurement of titer, competition assays, and isotyping experiments. However, after fusion of the spleens of 12 mice, no monoclonal antibodies were obtained which could be used for detection of ciguatoxin-related compounds. Difficulties associated in producing antibodies against brevetoxin-2 are its size and hydrophobicity. Moreover, intrinsic problems in the production of monoclonal antibodies include the overgrowth of antibody-producing hybridomas by non-producer cells or fibroblasts in the same well and loss of chromosomes involved in the synthesis of heavy and/or light chains. The use of in vitro stimulation of murine splenocytes with the BSA-PbTx-2 conjugate failed to yield monoclonal anti-PbTx-2 antibodies. However, this method should be further explored using different doses of stimulation and carrier-specific T cell lines to increase the probability of obtaining antibodies of the desired specificity. To assure that anti-PbTx-2 antibodies had not been missed in the tissue culture supernatants, a highly sensitive modified cytotoxicity assay was used. However, this assay failed to detect antibodies capable of neutralizing the PbTx-2 activity on Neuro-2a cells. This may have been due to the lack of anti-PbTx-2 antibodies in the supernatants or to their low affinity which was insufficient to compete with the sodium channels on Neuro-2a cells for free PbTx-2.
Guzman perez, Sonia Edith, "In Vivo and in Vitro Stimulation of Murine Splenocytes With Brevetoxin-2 Immunogenic Conjugates for the Preparation of Monoclonal Antibodies Against Ciguatoxin-Related Compounds." (1999). LSU Historical Dissertations and Theses. 6891.