The object of this proposal is to develop a new analytical method (lateral flow immunochromatographic device) for the rapid and on-site detection of pyridine carboxylic acid herbicides (aminopyralid, clopyralid, picloram and triclopyr) in compost and animal waste. The availability of a screening method to determine if compost is safe to be used in crops and ornamental flowers will greatly help protect the composting industry from law suits and growers from damaged crops. This project will also help reduce the adverse impact of animal manure on the environment, assure that crops labeled as organic are free of pyridine carboxylic acid herbicide chemical inputs, and to help optimize manure management. Current analytical methods for quantifying the concentration of pyrimidine carboxylic acid herbicides in compost and waste products, include solvent extraction, followed by instrumental analysis. These methods are time consuming, labor intensive, and require in some cases the use of flammable and/or toxic solvents. A faster, customer/environmentally friendly and less expensive method such as a lateral flow immunochromatographic device (dipsticks) for the detection of pyrimidine carboxylic acid herbicides could find a broad application in the composting, agricultural (crops) and environmental monitoring (water) markets. The proposed analytical test will allow establishment of efficient and cost-effective screening programs that could be used quickly on-site for the analysis of compost ad other environmental matrices, therefore protecting compost producers, users, and the environment. The strategy in this proposal will be performed in 2 phases. Phase I, focuses on creating polyclonal antibodies and several hybridoma cell lines that produce monoclonal antibodies, which react with the pyridine carboxylic acid herbicides of interest with sufficient selectivity, affinity and avidity to be utilized in a commercial dipstick type immunoassay system. Once these antibodies are developed, phase II will be started, this phase will involve the development and validation of a commercial dipstick immunoassay format for on-site testing, to detect the target herbicides in animal waste such as compost, solid manure, farmyard waste and manure slurry, as well the required extraction procedures to be able to quickly and easily analyze the extracts from those matrices. One of our approaches to obtain broad selectivity of the immunoreagents towards the 4 target analytes will be to prepare antibody cocktails. The proposed research is in line with several of USDA's strategic goals: 1) will expand domestic marketing opportunities for compost and manure byproducts and strengthen risk management by allowing to monitor those product for contamination with pyridine carboxylic acid herbicides that have potential to destroy crops, therefore, helping farmers, researchers and compost facilities in their decision-making process; 2) develop alternative markets for agricultural byproducts; 3) safeguard agriculture from herbicide threats i. e. if contaminated manure were to be used as fertilizer. OBJECTIVES: The object of this project is to develop a new analytical method (immuno-chromatographic devices or dipsticks) for the rapid and on-site detection of pyridine herbicides in compost and animal waste. The proposed analytical tests are will allow the establishment of efficient and cost-effective screening programs that could be used quickly on-site for the analysis of compost and other environmental matrices, therefore protecting compost producers and users. The proposed strategy in this proposal will be performed in 2 phases. Phase I, focuses on creating polyclonal antibodies and several hybridoma cell lines that produce monoclonal antibodies, which react with the pyridine carboxylic acid herbicides of interest (aminopyralid, clopyralid, picloram, triclopyr) with sufficient selectivity, affinity and avidity to be utilized in a commercial dipstick type immunoassay system. Once these antibodies are developed, phase II will be started, this phase will involve the development and validation of a commercial dipstick immunoassay format for on-site testing, to detect the target herbicides in animal waste such as compost, solid manure, farmyard waste and manure slurry, as well the required extraction procedures to be able to quickly and easily analyze the extracts from those matrices. One of our approaches to obtain broad selectivity of the immunoreagents towards the 4 target analytes will be to prepare antibody "cocktails". The primary objective for Phase I, is to produce polyclonal antibodies, and hydridoma cell lines that provide monoclonal antibodies suitable for a commercial dipstick immunoassay for the detection of aminopyralid, clopyralid, picloram and triclopyr at a sensitivity of < 2 ppb with little or no cross-reactivity with other acid herbicides (fluroxypyr, imazapyr), and to document the production method. To accomplish this primary objective, the following goals will be achieved: ? Synthesize/procure target pyridine carboxylic acid ligands (weeks 1-2) ? Synthesize ligand-protein conjugates to be used as immunogens and as screening conjugates (weeks 1-2) ? Immunize mice (weeks 2-14) ? Immunize rabbits (weeks 2-14) ? Perform cell fusions and produce hybridoma cell lines derived from immunized mice (weeks 15-23) ? Produce, screen and evaluate polyclonal antibodies derived from rabbits (weeks 15-26) ? Screen evaluate and select monoclonal antibodies for use in an immunoassay (weeks 23-26) ? Screen evaluate and select polyclonal antibodies for use in an immunoassay (weeks 23-26) After we elicit antibody formation, screening will be performed to select the antibodies exhibiting 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 profile (aminopyralid, clopyralid, picloram and triclopyr, as well as other acid herbicides and other pyridine carboxylic acid herbicides) and for potential product development. APPROACH: The Phase I work plan consists of 3 stages: 1) synthesis of pyridine carboxylic acid ligands and preparation of immunogens by coupling ligands to protein carriers: BSA, OVA, KLH; 2) inoculation of rabbits/mice with immunogens, followed by a screen to monitor the development of antibodies; 3) production of hybridomas and polyclonals antibodies. The steps are sequential and are anticipated to take 7 months to complete. The schedule listed is approximate due to the nature of the immunological response of animals. Pyridine carboxylic acid ligands will be synthesized or obtain commercially and reacted with proteins through their COOH or amine reactive group(s) using techniques commonly used at Abraxis. The amount of substitution of the ligands on carrier proteins will be determined by colorimetric techniques. Procedures for raising monoclonal antibodies are well established, standard methodologies will be used. Immunogens will be injected into several sets of mice. 7-10 days following each round of injections, serum will be drawn and tested for antibody production and titer. The testing will be accomplished by a competitive indirect ELISA which will be developed utilizing a ligand-conjugate linked to OVA. The presence of specific antibody will be detected with HRP-labeled anti-mouse, and further characterized by demonstrating inhibition with the herbicides of interest. After positive titers have been established (approx. 3 months from first injection) the third stage of Phase I, will start. Nine rabbits will be injected with the various immunogens. Approx. 14 days following each round of injections, serum will be drawn and tested by a competitive indirect ELISA for antibody production and titer. Starting with week 14, and every 4 weeks thereafter, production bleeds will be collected, screened against immunogens, and competition experiments against target and non-target compounds. Standard curves will be characterized for LOD and EC50s. Patterns of reactivity for each bleed against target and non-target compound and standard curve sensitivity will be obtained to identify and assess those production bleeds containing antibodies with the potential for development of a commercial pyridine carboxylic acid herbicide dipstick immunoassay which will be undertaken in Phase II. To develop hybridomas for monoclonal antibody development, splenocytes from the positive-titer mice will be fused with a myeloma cell line. Approx. 7-14 days after fusion, supernatants from colonies will be screened for IgG production. Colonies that are IgG-positive will be expanded and screened for positive titers. Hybridoma cells showing positive titers will be cloned by the limiting dilution technique. Supernatant from a monoclonal will be collected for screening against pyridine carboxylic acid immunogens and against both target and non-target compounds as decribed above (rabbit section) to identify and assess those clones producing antibodies with the potential for development of a commercial pyridine carboxylic acid herbicides dipstick immunoassay (Phase II). Subsequent antibody scale-up would be through cell culture or ascites production
