Perfluorooctanoic acid (PFOA) is a synthetic chemical which does not occur naturally in the environment,it has been used in many commercial applications. A number of scientific findings since the late 1990's have elevated PFOAs to the category of great environmental concern beacuse of its persistence, and the potential for bioaccumulation and biomagnification. Effective environmental and health protection requires the sensitive and efficient detection of PFOA in various sample matrices such as foods, water, soil, sediment, blood, etc. Therefore, faster and less expensive analytical methods such as ELISA for the detection of PFOA are ideal for the establishment of efficient and cost-effective screening programs for these compounds. The purpose of this project is to develop the immunoreagents (polyclonal and monoclonal antibodies) which react with PFOAs with sufficient selectivity, affinity and avidity to be utilized in the development of a commercial immunoassay system. OBJECTIVES: The primary objective for Phase I, is to produce polyclonal antibodies, and hydridoma cell lines that provide monoclonal antibodies suitable for a commercial perfluorooctanoic acid (PFOA) immunoassay, and to document the production method. To accomplish this primary objective, the following goals need to be achieved: 1)Synthesize PFOA ligands, 2)Synthesize ligand-protein conjugates to be used as immunogens, 3)Produce hybridoma cell lines derived from immunized mice, 4)Screen evaluate and select monoclonal antibodies for use in an immunoassay, 5)Produce, screen and evaluate polyclonal antibodies derived from rabbits. Feasibility depends on choosing the appropriate immunogens to elicit antibody formation and a screening strategy to select the desired selectivity, affinity, and avidity profile. Once candidate antibodies have been selected from viable hybridoma cells, clonal cells will be isolated and expanded to harvest antibodies for further characterization. Polyclonal and monoclonal antibodies will be screened for cross-reactivity (pesticides, industrial chemicals, and other compounds generally found in water) and for potential product development. Furthermore, antibodies produced in the course of this work could be used in affinity columns for sample concentration prior to analysis with immunoassay or traditional methods. The work plan for Phase I consists primarily of three stages. The first stage is the synthesis of PFOA ligands and the preparation of immunogens by covalently conjugating the PFOA ligands to protein carriers such as bovine serum albumin, ovalbumin, Keyhole Limpet Hemocyanin (KLH). The second stage is the inoculation of rabbits and BALB/c mice with the immunogens followed by a screen to monitor the development of polyclonal anti-PFOA antibodies. The screening for antibodies against PFOA will be accomplished by competitive indirect enzyme-linked immunosorbent assay (CI-ELISA) utilizing a second PFOA conjugate linked to ovalbumin or other protein unrelated to the immunogen. The third stage is the production of hybridomas. Once measurable anti-PFOA antibody is detected in the mice, one individual will be selected for fusion with a myeloma cell line to produce hybridomas and subsequent screening of the cells for anti-PFOA antibody production. Repeated screening and re-cloning will insure the yield of clones that secrete antibodies with the desired reactivity, affinity and avidity, suitable for the development of a commercial immunoassay. Ligand synthesis, immunogen conjugation, and assay development will be performed by Abraxis at its facility. Immunization and fusions are to be performed by Dr. Weilin Shelver, USDA-ARS-Fargo. All materials and processes are to be developed under joint decision of Abraxis and USDA-ARS, and under the direction of Abraxis LLC. APPROACH: PFOA ligands are to be prepared for use in the conjugation to proteins. In this work, PFOA and structurally similar compound to PFOA will be reacted with succinic anhydride or other suitable compound to produce a derivative of PFOA. The resultant carboxylic acid derivative of PFOA or PFOA alone is then activated using EDC/NHS for conjugation to the carrier proteins, BSA, KLH, ovalbumin. Other derivatives with varying chain length will also be synthesized. Immunogens synthesized from derivatized PFOA haptens are to be injected into mice (BALB/c) and rabbits on a schedule designed to elicit and maintain antibody production by the mouse and rabbit immune system. Approximately 7 to 10 days following each subsequent round of injections, serum will be drawn from the mice and rabbits, and tested for antibody production and titer. The testing (screen) will be accomplished by a competitive indirect ELISA which is to be developed utilizing a PFOA-conjugate linked to ovalbumin. The presence of specific antibody can then be detected with an enzyme-labeled anti-mouse antibody or by the use of goat anti-rabbit labeled antibodies in the case of rabbits, and further characterized by demonstrating displacement with PFOA. After positive titers have been established, the third stage of Phase I can be started. Starting with week 14 and every 4 weeks thereafter production bleeds are collected from the immunized rabbits, screened against immunogens, and competition experiments conducted against target compound (PFOA) and other PFCs. Standard curves will be characterized for the least detectable dose and EC50s. Patterns of reactivity for each production bleed against the target and non-target compound as well as standard curve sensitivity will be mapped to identify and assess those production bleeds containing antibodies with the potential for development of a commercial PFOA immunoassay which will be undertaken in Phase II. To develop hybridomas for monoclonal antibody development, splenocytes from the positive-titer mice are fused with a myeloma cell line (Sp2/0.Ag14), and then incubated in mammalian cell culture conditions. Approximately 7-14 days after fusion, supernatants from colonies will be screened for IgG production. Cell colonies that exhibit IgG-positive responses are expanded. Hybridoma cells showing positive titers to immunogens will be cloned. Supernatant from a hybridoma will be collected for screening against the PFOA immunogens and against both the target compound (PFOA) and other PFCs. Competition experiments will be conducted with PFOA and other PFC compounds and the standard curves will be characterized for the least detectable doses and EC50s. Patterns of reactivity for each monoclonal against the target, and non-target compounds as wells as the standard curve sensitivity will be mapped to identify and assess those clones producing antibodies with the potential for development of a commercial PFOA immunoassay which will be undertaken in Phase II. Subsequent antibody scale-up would be through ascites production
