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

Fresh water cyanobacteria harmful algal blooms potentially have 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 algae blooms also can produce significant quantities of natural toxins. The toxins produced by these cyanobacteria are extremely toxic to many species. Cyanobacterial toxins (anatoxin-a, microcytins, saxitoxin) also have been identified as potential biological weapons. If these toxins were to be introduced into our water systems, they could not be removed efficiently by conventional water treatment systems and potentially could kill many people. Anatoxin-a also is known as “the very fast death factor” (LD50 for mice is 200 mg/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, followed by death within minutes to hours. Effective environmental and health protection requires the sensitive and efficient detection of cyanobacterial toxins. Current analytical methods for quantifying the concentration of cyanobacterial toxins in water and in biomass include the mouse bioassay, high-performance liquid chromatography, and the phosphatase inhibition assay. Some of these methods are not sensitive enough or require the use of many animals. Although 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, more sensitive, and less expensive analytical methods such as enzyme-linked immunosorbent assay (ELISA) for the detection of cyanobacterial toxins are ideal for the establishment of efficient and cost-effective screening programs that could be used onsite without the use of solvents in water samples. ELISA allows more precise prophylactic and corrective treatment of water at treatment facilities, ultimately benefiting the environment and public health. This Phase I research focuses on creating polyclonal antibodies and hybridoma cell lines that produce monoclonal antibodies. These antibodies react with anatoxin-a with sufficient selectivity, affinity, and avidity to be utilized 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 ELISA for microsystems.