Generation of Monoclonal Antibodies to Conus Venom Peptides c1e and c4a

Abstract

Peptides found within cone snail venom (i.e. conotoxins) have neuroexcititory or neuroinhibitory activity, with individual conotoxins targeting a particular isoform of a receptor or ion channel. Because of their specificity and potentially lethal neurotoxicity, it is important to better understand the complex nature of these peptides that make up cone snail venom. Monoclonal anti-venom peptide antibodies enable us not only to conduct localization studies on the snail and prey, but also to assess the possibility of anti-venoms as treatment for exposure to the venom. The two conotoxins under investigation are: alpha-conotoxin c1e, a putative ionotropic acetylcholine receptor blocker, and c4a, a neuroexcitatory peptide whose molecular target remains unknown. Various techniques were learned in preparation for generating monoclonal antibodies against c1e and c4a, such as the Zebrafish Spinal Motility Assay, a test for peptide neurotoxicity prior to immunization of mice. An appropriate conjugation protocol was also determined for peptide conjugation to KLH and BSA, which will assist with antibody titer and ELISA testing following immunization. Other techniques include the Enzyme-linked Immunosorbent Assay (ELISA), an indicator of the presence and concentration of a specific antibody, as well as hybridoma cell culture and subcloning. Hybridoma cell culture involves the growing and fusion of antibody-producing cells to myeloma cells, ensuring that the resulting cell line remains immortal. After the hybridomas reach a healthy state of growth, the ELISA is employed to assess whether the appropriate antibodies are being produced. If so, the line is subcloned to produce monoclonal antibodies.