Grantee Research Project Results
Final Report: Development of Antibodies for the Detection of the Toxin Anatoxin by Immunoassay
EPA Contract Number: EPD06014Title: Development of Antibodies for the Detection of the Toxin Anatoxin by Immunoassay
Investigators: Rubio, Fernando M.
Small Business: Abraxis, LLC
EPA Contact: Richards, April
Phase: I
Project Period: March 1, 2006 through August 31, 2006
Project Amount: $70,000
RFA: Small Business Innovation Research (SBIR) - Phase I (2006) RFA Text | Recipients Lists
Research Category: Watersheds , SBIR - Water and Wastewater , Small Business Innovation Research (SBIR)
Description:
The purpose of this Phase I research was to show the feasibility on the development of polyclonal antibodies and hybridoma cell lines to be used for the detection of the toxin anatoxin-a by immunoassay (ELISA). The antibodies that were to be generated needed to have sufficient selectivity, affinity, and avidity to be usable in a commercial ELISA system to be developed and validated during Phase II.
Summary/Accomplishments (Outputs/Outcomes):
Synthetic anatoxin-a was purchased from TECRA. It was conjugated chemically to ovalbumin, bovine serum albumin, and thyroid binding globulin to produce immunogens and coating conjugates. In addition a KLH-anatoxin-a coating conjugate and a sample of anatoxin-a produced naturally by algae was obtained for testing from Dr. Geoffrey Codd at the University of Dundee, Scotland.
Rabbits and mice were immunized with the synthetized immunogens, and bleeds were collected monthly during the project. Antibody screening assays and quantitative assays were developed. All immunized rabbits (6) produced antibody titers, although the displacement (standard curves) using both synthetic and natural anatoxin-a obtained exhibited poor displacement (low sensitivity). One of the mice (ABX M-9) exhibiting the highest antibody titer was chosen for fusion with myeloma cells to produce the hybridoma cell lines. Approximately 100 wells were found to contain hybridoma cell lines producing some type of antibody (IgG); the positive wells were further screened for the target antibodies using antigen (anatoxin-a) conjugates. Unfortunately, we could not obtain any specific monoclonal antibodies against anatoxin-a.
Various telephone conferences calls were conducted with Foresight Technologies to helps us obtain information for a technology niche analysis and commercialization plan for this technology in case a decision was made to submit a proposal for Phase II funding. A technology niche analysis report was produced by Foresight Technology, indicating the true need on the marketplace for the proposed assay.
Conclusions:
We have demonstrated that polyclonal antibodies against anatoxin-a could be produced; however, the antibodies produced did not have sufficient avidity or affinity to produce a viable commercial ELISA kit product. We could not obtain a hybridoma cell line against anatoxin-a. Our research efforts will continue for a period of time because of the scientific and commercial importance to have a rapid analytical method for anatoxin-a. Future research will be funded by Abraxis.
Supplemental Keywords:
small business, SBIR, harmful algal blooms, HAB, cyanobacterial toxins, biological weapons, drinking water, water treatment, biohazard, blue-green algae, anatoxin-a, microcytins, saxitoxin, water quality, cyanobacterial toxin detection, public health, environmental toxins, monoclonal antibodies, EPA, ecosystem protection/environmental exposure & risk, scientific discipline, water, ecological risk assessment, ecology and ecosystems, environmental monitoring, algal blooms, ecology, algal toxins, biotoxin risk, cyanobacteria, mouse bioassay, polyclonal antibodies,, RFA, Scientific Discipline, Water, Ecosystem Protection/Environmental Exposure & Risk, algal blooms, Environmental Monitoring, Ecological Risk Assessment, Ecology and Ecosystems, toxic cyanobacteria, HAB ecology, mouse bioassay, biotoxin risk, polyclonal antibodies, algal toxinsThe perspectives, information and conclusions conveyed in research project abstracts, progress reports, final reports, journal abstracts and journal publications convey the viewpoints of the principal investigator and may not represent the views and policies of ORD and EPA. Conclusions drawn by the principal investigators have not been reviewed by the Agency.