Development of Antibodies for the Detection of the Toxin Anatoxin-by Immunoassay

Fresh water cyanobacteria harmful algal blooms (HAB), have potentially many adverse environmental impacts. For instance, bloom mats of filamentous algae are believed to reduce ambient light levels below those required for submerged aquatic vegetation to survive. Blue-green algae, form blooms that make a surface scum and have been associated with low levels of dissolved oxygen that can be lethal to fish and invertebrate species. In addition to the overt environmental impact, blue green algal blooms can also produce significant quantities of natural toxins. The toxins produced by these Cyanobacteria, are extremely toxic to many species. Cyanobacterial toxins (Anatoxin-a, Microcytins, Saxitonxin) have also been identified as potential biological weapons, these toxins can potentially be introduced into our water systems and since they are not removed efficiently by conventional water treatment systems, they could kill many people. Anatoxin-a is also know as ¿the very fast death factor¿ (LD50 for mice is 200 ug/Kg with 4-7 minute survival), wild and domestic animals poisoned through ingestion have been observed in the field to be staggering, gasping and suffering convulsions, following by death within minutes to hours. Effective environmental and health protection requires the sensitive and efficient detection of cyanobacterial toxins. Current analytical method for quantifying the concentration of cyanobacteria toxins in water and in biomass include the mouse bioassay, HPLC, and the phosphatase inhibition assay, some of these methods are not sensitive enough or require the use of many animals. While chromatographic methods are capable of detecting and identifying compounds, these methods are time consuming, labor intensive, and require the use of flammable and/or toxic solvent for sample extraction. Therefore, faster, sensitive and less expensive analytical methods such as WLISA for the detection of cyanobacterial toxins are ideal for the establishment of efficient and cost-effective screening programs that could be used on-site without the use of solvents in water samples, therefore allowing a more precise prophylactic and corrective treatments of water treatment facilities, ultimately benefiting the environment and public health. The proposed Phase I research, focus on creating polyclonal antibodies and hybridoma cell lines that produce monoclonal antibodies, which react with anatoxin-a with sufficient selectivity, affinity and avidity to be utilizes in a commercial immunoassay system. Once these antibodies are available, commercial immunoassays to detect this cyanobacterial toxin in water and other environmental matrices will be developed and validated (Phase II). This immunoassay will complement Abraxis other commercial cyanobacterial toxin ELIAS for Microcystims.