Progress 07/01/97 to 06/30/04
Outputs
Beta-cyclocitral-PPD conjugate was prepared and used to immunize two chickens and two mice for the production of polyclonal and monoclonal antibodies against beta-cyclocitral, respectively. Monospecific polyclonal antibodies were purified from the eggs laid by the immunized chickens, using affinity chromatography. For the production of monoclonal antibodies, hybridoma cells were made by fusion of myeloma cells and spleen cells of the mice that showed high antibody titer and specificity. Hybridoma cells that secreted high affinity monoclonal antibodies were cloned by the limiting dilution method and at least 10 hybridoma cell lines positive for anti-beta cyclocitral antibodies were established. ELISA based on chicken IgY and mouse IgG were developed and had limit of detection of 1.0 ng/ml and respective I50 of 3.93 and 7.98 ng/ml. Two enzyme-linked-immunoflow (ELIFA) assays were developed based on the ELISAs. The ELIFA protocols were easy to perform but were less
sensitive than the ELISAs as shown by their I50 of 46 micrograms/ml for IgY-based ELIFA and 93 micrograms/ml for IgG-based ELIFA.
Impacts
A rapid method that allows rapid determination of beta-cyclocitral in catfish pond will allow catfish farmers to better manager pond water. As a result, more catfish will be available for consumption and catfish farmers will generate additional income.
Publications
- Munene, Cate. Development of chicken polyclonal and mouse monoclonal-based enzyme immunoassays for the detection of beta-cyclocitral in catfish pond water. Ph.D. Thesis. Louisiana State University, 2004.
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Progress 01/01/03 to 12/31/03
Outputs
The catfish industry supplies yearly about 600 million dollars of edible products of which 300 million dollars are considered unmarketable because of the off-flavor defect which imparts a muddy and/or musty, or woody odor to pond-raised catfish. Off-flavor generating compounds -geosmin, 2-methylisoborneol, and beta-cyclocitral- are known to be rapidly embedded in the fatty tissues of the fish. Geosmin, 2-methyl-isoborneol, and beta-cyclocitral are semi volatile and fat soluble. Even at concentrations as low as 0.7 microgram of off-flavor compound per kg of fish, the fish is perceived as off-flavor and is rejected by processors. It has been reported that there are almost no economical means to remove off-flavor compounds from catfish ponds. An inexpensive and rapid method that can qualitatively and quantitatively detect geosmin or beta-cyclocitral will improve economic yield for catfish farmers and economic opportunities for catfish farmers and processors, reduce the
cost of catfish on the market and enhance the competitiveness of US catfish around the world. Immunochemical methods have the advantage of being sensitive, simple, inexpensive, and relatively fast. An attempt to prepare a geosmin analog for injection into chicken or mice was not successful. Two immunogens (beta-cyclocitral-KLH and beta-cyclocitral-PPD conjugates) and one solid phase conjugate (beta-cyclocitral-BSA), were prepared. A hydroxyl- and sulfhydryl-reactive heterobifunctional cross-linker, p-Maleimidophenyl Isocyanate (PMPI), was used to couple the hydroxyl containing hydroxycyclocitral to thiolated KLH and to thiolated PPD. KLH and PPD were thiolated using N-Succinimidyl S-Acetylthioacetate (SATA, Pierce), an amine-reactive heterobifunctional cross-linker, and then reacted with the thiol-reactive group (maleimide) on the PMPI-activated hydroxycyclocitral, to give hydroxycyclocitral-KLH/PPD conjugate containing an intact beta-cyclocitral structure as the hapten. Since the
conjugation protocol preserved the integrity of the antigen structure, the prepared immunogens will ensure production of beta-cyclocitral-specific antibodies that will result in sensitive, specific, and accurate immunochemical assays. In order to select only those antibodies that are formed against the hapten (beta-cyclocitral) and not the carrier proteins (KLH or PPD) or the linker (PMPI), BSA was coupled to hydroxycyclocitral by succinylation to give the solid phase conjugate, beta-cyclocitral-BSA. Hens have been immunized and eggs are being collected for polyclonal antibodies isolation. Mice have been immunized for monoclonal antibodies production. The project will be ended in December 2004.
Impacts
A rapid method that allows rapid determination of beta-cyclocitral in catfish pond will allow catfish farmers to better manager pond water. As a result,more tasty catfish will be available for consumption.
Publications
- No publications reported this period
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Progress 01/01/02 to 12/31/02
Outputs
1. Preparation of geosmin-protein conjugate: Cybullol was identified as an analog of geosmin that can be used for conjugation to a protein carrier. However, cybullol is not commercially available. Cybullol will be isolated from the growth culture of Cyathus bulleri. Three strains (38347, 38351, 38355) of C. bulleri were obtained from ATCC and were grown on liquid medium at 25 C without agitation. The still cultures were allowed to mature for 60 days after which the mycelium was removed by filtration through cheesecloth. The clear yellow broth was extracted with ethyl acetate and the extract concentrated in vacuo to give the crude metabolites as a yellowish-brown semi-solid (50mg/l of culture broth). Thin layer chromatography of the crude metabolites revealed numerous spots which will be identified by GC/MS after purification with preparative TLC. The crude metabolites are stored at -20 C until use. 2. Preparation of beta-cyclocitral-PPD conjugate or beta-cyclocitral
KLH conjugate: A direct conjugation of beta-cyclocitral ( 2,66-trimethyl-1-cyclohexene-1-carboxyaldehyde) to a carrier protein such as PPD or KLH is not possible. Therefore, an analog to beta-cyclocitral was needed in order to provide a functional group for conjugation to a protein carrier. An analog of beta-cyclocitral with available OH group was synthesized in order to incorporate an extra functional group through which the coupling could be done and therefore preserve the integrity of the hapten. Spectroscopic properties of the conjugate including NMR, IR, MALDI, MP have confirmed that conjugation of beta-cyclocitral analog to a carrier protein (PPD or KLH) was successful. A solid phase immunogen involving the conjugation of hydroxycyclocitral to bovine serum albumin has also been prepared that will assist in purifying monospecific antibodies to beta-cyclocitral. Current research activity involves the preparation for chicken and mice immunization with the beta-cyclocitral- KLH or
beta cyclocitral-PPD conjugate.
Impacts
A rapid method that allows rapid determination of geosmin and/or beta-cyclocitral in catfish pond water will allow catfish farmers to a better manage their ponds. As a result, more palatable catfish at better price will be available for consumption.
Publications
- No publications reported this period
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Progress 01/01/01 to 12/31/01
Outputs
Geosmin is a tertiary alcohol and does not easily undergo oxidation to allow its facile conjugation with a protein carrier. Previous approaches used to conjugate geosmin to PPD were not straight forward as geosmin could not bind covalently to the protein. An analog of geosmin was sought to perform the conjugation. The analog is not commercially available. Cultures were obtained from ATCC to produce the analog, identify it, purify it, and conjugate it to a protein carrier before raising antibodies. An initial mass spectrometry analysis of the analog has given a peak at the m/z region of interest. More work needs to be carried out to identify the purified analog before coupling it to a protein. Beta-cyclocitral is another off-flavor generating compound which is under investigation. Beta-cyclocitral is an aldehyde. The chemistry of beta-cyclocitral conjugation with a protein is being developed to avoid the loss of antibody recognition of the off-flavor generating site on
the molecule. A direct conjugation of beta-cyclocitral with a protein, by reductive amination, gave a conjugate that could not elicit an immune response that recognizes the original beta-cyclocitral molecule. Beta-cyclocitral is being modified to provide a reaction group for coupling to a protein.
Impacts
The development of a quick test that allows catfish farmers and processors to check for the presence of off-flavor geosmin or beta-cyclocitral carries significant economic and social benefits to the people of Louisiana. Catfish farmers will be able to manage the quality of pond waters and fish and avoid losses associated with off-flavor fish that become unmarketable or have to be sold after extensive cleaning. Processors will buy on-flavor fish. A low cost production will trickle down to consumer who will not have to pay a high price for catfish. More on-flavor catfish will translate into more money in the pocket of farmers, processors, and consumers.
Publications
- No publications reported this period
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Progress 01/01/00 to 12/31/00
Outputs
Geosmin (1,10-trans-dimethyl-trans-9 decalol) is a musty earthy off-flavor compound generated by blue green algae and microorganisms in catfish ponds.Polyclonal and monoclonal antibodies are being developed against geosmin to detect ppt concentrations of geosmin in catfish filets. Polyclonal and monoclonal antibodies are also being developed against beta-cyclocitral (2,6,6-trimethyl-1-cyclohexene-1-carboxaldehyde), a compound responsible for woody flavor in pond-raised catfish. ELISA methods for rapid detection of geosmin or beta-cyclocitral are being developed.
Impacts
The development of rapid and inexpensive analyical methods for geosmin or beta-cyclocitral detection will assist farmers and producers in managing catfish ponds for water quality and fish quality.
Publications
- No publications reported this period
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Progress 01/01/99 to 12/31/99
Outputs
Monoclonal antibodies were developed against 2-methylisoborneol, an off-flavor generating compound that affects the sensory quality of catfish. MIB-LPH and borneol-LPH conjugates were synthesized as immunogens. Borneol-BSA, isoborneol-BSA,and MIB-BSA were synthesized as solid phase protein conjugates to study the effect of solid phase conjugate structure on the sensitivity of ELISA and the affinity for free MIB. By using anti-borneol monoclonal antibody (1/250) and MIB-BSA (0.5 microgram/mL) as solid phase conjugate a detection limit of MIB was found to be about 1 ng/mL. The ELISA protocol developed was optimized and applied to water samples. The lowest detection limit obtained was 0.1 ng/mL. In a related study, off-flavored fillets from geosmin and MIB contaminated catfish were cut into small pieces and vacuum tumbled for 10 min with 0.5-2% citric acid solution. Attributes such as mustiness/earthiness, sourness, tenderness, and overall acceptability were evaluated by
consumer sensory evaluation. MIB concentrations decreased in acid treated samples. Color of treated samples was lighter than the control as reflected by higher L* values. Sourness was more pronounced in acid treated samples. An aspect of toxicology was assessed by studying the impact of pure azadirachtin (AZA) and neem-based insecticides (Neemix and Bioneem) on eight aquatic animals (crayfish, white shrimp, grass shrimp, blue crab megalopes, water fleas, oyster eggs, freshwater snails, and mosquito larvae) and two cell (hybridoma and oyster) lines. Toxicities were assessed by short term tests for 48 and 96 h. The LC50 (in vivo bioassay) and IC50 (in vitro bioassays) were determined for each species. AZA showed less toxicity than Neemix and Bioneem on all species tested. Among the test animals, water fleas were the most sensitive to Neemix, Bioneem, and pure AZA with LC50's of 0.071, 0.034, and 0.382 microgram AZA/mL, respectively. Next to water fleas, mosquito larvae, oyster eggs and
blue crab megalopes were found to be very sensitive to Neemix and Bioneem. Crayfish and freswater snails showed the least sensitivity to Neemix, Bioneem, and pure AZA. The toxicity of both insecticides decreased with higher temperature, light, and time but Bioneem remained more toxic at 37 C than Neemix and appeared to be less sensitive to environmental factors. The results suggest that increased use of neem-based insecticides may have direct adverse effects on aquatic organisms, contrary to the common belief that plant-derived insecticides (e.g. Neemix and Bioneem) pose no risk to the ecosystem.
Impacts
Rapid methods such as ELISA will help catfish farmers control the level of off-flavor compounds in ponds and improve the sensory quality and marketability of catfish.
Publications
- Goktepe, I. 1999. Toxicity of neem-based insecticides on aquatic animals and cell lines. Ph.D. Dissertation, Louisiana State University.
- Park, E.S. 1999. Development of monoclonal antibody and enzyme linked immunosorbent assay for detection of off-flavor compound 2-methylisoborneol. Ph.D. Dissertation, Louisiana State University.
- Forrester, P.G.N. 1999. Reduction of off-flavor compounds in catfish fillets by acid treatment. M.Sc. Thesis, Louisiana State University.
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Progress 01/01/98 to 12/31/98
Outputs
An enzyme immunoassay for 2-methylisoborneol (MIB), a musty-flavored compound produced by some types of aquatic microrganisms, was optimized to give a detection limit of 0.05 ppb MIB. Direct and indirect immunoassay formats are currently being compared for this analysis. Polyclonal antibodies (PAb) and MAb have been produced to bind gossypol, a toxic compound from cottonseed. Antibodies were found to measure gossypol only when bound to a carrier protein or derivatized using amino-containing compounds. Cottonseed samples are currently being tested to correlate results to chemical, chromatographic and bioavailability assays. Azadirachtin (AZA) and neem-derived pesticides have been studied using various aquatic animal systems as well as cell culture. Toxicities using pure AZA, compared to pesticides at similar AZA concentrations, was found to be much lower in all studies conducted, indicating that AZA is not the only factor responsible for toxicity in neem pesticides.
Impacts
(N/A)
Publications
- Goktepe, I. and Plhak, L. 1998. Toxicity of azadirachtin pesticides on aquatic species. In: Water Resources Engineering 98. Proceedings of the International Water Resources Engineering Conference. (S.R. Abt, J. Young-Pezeshk and C.C. Watson, Eds.) American Society of Civil Engineers. pp. 1248-1253.
- Wang, X. and Plhak, L.C. 1998. Development of Immunochemical Methods for the Assessment of Gossypol. In: Proceedings of the 47th Annual Oilseed Conference, New Orleans, LA., Feb 1998.
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Progress 01/01/97 to 12/31/97
Outputs
Monoclonal antibodies (MAb) have been produced for 2-methylisoborneol (MIB), a musty-flavored compound produced by some types of algae and fungi. In competitive indirect ELISA the detection limit for anti-borneol MAb was found to be approximately 10 ppb MIB. More hybridoma are being produced with sensitivities to MIB and MIB-like compounds as well as geosmin. Polyclonal antibodies (PAb) have been produced in rabbits that recognize gossypol, a toxic pigment from cottonseed, when bound to protein via Schiff base intermediates. These PAb, as well as MAb that are currently being developed for bound gossypol, will be used to study the relationship between bound gossypol fractions and bioavailability. Hybridoma are also being produced for brevetoxin, okadaic acid, and ciguatoxin-like compounds. Cell culture assays are being developed for measuring the cytotoxicity of gossypol (using various cell types), azadirachtin-related compounds (using primary oyster cells and insect
cell lines), aflatoxin and fumonisin (using clone 9 rat liver cells). The effects of aflatoxin and fumonisin, either alone or in combination, on gap junctional cell-cell communication are being investigated using clone 9 rat liver cells.
Impacts
(N/A)
Publications
- Plhak, L.C. and Sporns, P. 1997. Biological activities of potato glycoalkaloids. In: Antinutrients and Phytochemicals in Food. (F. Shahidi, Ed.) American Chemical Society, Washington, DC. pp.115-126
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