Chemistry: molecular biology and microbiology – Measuring or testing process involving enzymes or... – Involving antigen-antibody binding – specific binding protein...
Reexamination Certificate
2000-01-24
2002-09-03
Nguyen, Bao-Thuy L. (Department: 1641)
Chemistry: molecular biology and microbiology
Measuring or testing process involving enzymes or...
Involving antigen-antibody binding, specific binding protein...
C435S007100, C436S547000, C436S548000, C530S388100
Reexamination Certificate
active
06444433
ABSTRACT:
This invention pertains to a method to detect 2-methylisoborneol using monoclonal antibodies and to a hybridoma cell line that produces such monoclonal antibodies.
Metabolites synthesized by some cyanobacteria, actinomycetes, and fungi are responsible for the earthy/musty or “off-flavor” taste in potable water and certain fish. See S. W. Krasner, “Analytical methods for the identification and quantification of earthy/musty flavors in drinking water: A review,” Water Quality Bulletin, vol. 13, pp. 78-83 (1988); and P. B. Johnsen et al., “Physiological approaches to the management of off-flavors in farm-raised channel catfish,
Ictalurus punctatus
,” in Recent Developments in Catfish Aquaculture, pp. 141-161 (1994). Two metabolites, which are the primary compounds responsible for this “off-flavor,” are geosmin (1&agr;, 10&bgr;-dimethyl-9&agr;-decalol) and 2-methylisoborneol (1-R-exo-1,2,7,7-tetramethyl-bicyclo-[2.2.1]-heptan-2-ol) (“MIB”). These two compounds cause problems for the potable water and aquaculture industries because of the low concentrations of the compounds required to produce the “off-flavor” taste and because of the ubiquitous nature of the organisms that produce these metabolites.
The problem with earthy/musty flavors constitutes a significant restriction to the growth of the catfish industry. C. S. Tucker et al., “Environment-related off-flavors in fish,” in Aquaculture and Water Quality, D. E. Brune and J. R. Tomasso, eds., pp. 133-179 (1991). Catfish cultured in ponds in the southern United States that are deemed unacceptable because of a “musty” flavor can be as high as 80% at any one time. J. F. Martin et al., “Pharmacokinetics and tissue disposition of the off-flavor compound 2-methylisoborneol in the channel catfish (
Ictalurus punctatus
), Can. J. Fish. Aquat. Sci., vol. 47, pp. 544-547 (1990); J. F. Martin et al., “Musty odor in chronically off-flavored channel catfish: Isolation of 2-methyleneboranane and 2-methyl-2-bornene,” J. Agric. Food Chem., vol. 36, pp. 1257-1260 (1988); and J. F. Martin et al., “2-methylisoborneol implicated as a cause of off-flavor in channel catfish,
Ictalurus punctatus
(Rafinesque), from commercial culture ponds in Mississippi,” Aquaculture and Fisheries Management, vol. 19, pp. 151-157 (1988). When “musty” fish cannot be harvested and brought to the market, the fish are held and fed until deemed “on-flavor” by an experienced human taster employed by the processing plant. However, during this time the fish grow beyond their optimal market size. Fish greater than about an 11 oz fillet are considered undesirable to both the producers and processors because they are more expensive to keep, the fat content of the fillet increases, automatic filleting is more difficult, and fish survival rates decrease as holding times increase.
Additionally, the earthy/musty flavors have been identified as a problem with trout and in potable water. See, for example, M. Yurkowski et al., “Identification, analysis, and removal of geosmin from muddy-flavored trout,” J. Fish. Res. Board Can., vol. 31, pp. 1851-1858 (1974); G. Izaquirre et al., “Geosmin and 2-methylisoborneol from cyanobacteria in three water supply systems,” Applied and Environmental Microbiology, vol. 43, pp. 708-714 (1981); and Aoyama, “Studies on the earthy-musty odors in natural water (IV). Mechanism of earthy-musty odor production of actinomycetes,” Journal of Applied Bacteriology, vol. 68, pp. 405-410 (1990).
Methylisoborneol (“MIB”) is a small terpenoid compound synthesized by cyanobacteria and actinomycetes and is illustrated below. It is fat-soluble and not easily volatilized.
Structure of MIB
MIB is believed to be taken up by fish through their gills and stored in fatty tissues. Fish with higher fat reserves are more prone to a “musty” flavor. Research related to quality control of catfish flavor has focused on the pond management by studying factors that influence the growth of algae or fungi populations, such as water quality, pH, oxygen levels, soil quality, fish density, feed quality and application rates, and water temperature. See, e.g., H. G. Peterson et al, Physiological toxicity, cell membrane damage, and the release of dissolved organic carbon and geosmin by
Aphanizomenon flos
-
aquae
after exposure to water treatment chemicals,” Wat. Res. Vol. 29, pp. 1515-1523 (1995); C. P. Dionigi et al., “Copper-containing aquatic herbicides increase geosmin biosynthesis by
Streptomyces tendae
and
Penicillium expansum
,” Weed Science, vol. 43, pp. 196-200 (1995); and R. Velzeboer et al., “Release of geosmin by
Anabaena circinalis
following treatment with aluminum sulphate,” Wat. Sci. Tech., vol. 31, pp. 187-194 (1995). Although many approaches to eliminate algal growth have been tried, some algal growth is beneficial to maintain an adequate oxygen content in the water during daylight hours, especially when the temperature of the water is high. Because of this interaction between algae and oxygen and because of the large numbers of factors that affect both algal and fungal growth, controlling the “off-flavor” by pond management to control the algal density has proven difficult.
Another problem compounding the problem of “musty flavor” in aquaculture products is the difficulty in easily measuring the metabolites responsible. The “musty flavor” is caused by metabolites that are present at extremely low concentrations. The two methods used currently to detect both geosmin and MIB are human tasters and gas chromatography (“GC”). The human gustatory threshold of geosmin or MIB is approximately 10 to 30 ng/L in pure water. See, e.g., J. A. Maga, “Musty/earthy aromas,” Food Reviews International, vol. 3, pp. 269-284 (1987); P. -E. Persson, “Sensory properties and analysis of two muddy odor compounds, geosmin and 2-methylisoborneol, in water and fish,” Water Research, vol. 14, pp. 1113-1118 (1980); and S. F. Wood et al., “2-Methylisoborneol, improved synthesis and a quantitative gas chromatographic method for trace concentrations producing odor in water,” J. Chromatography, vol. 132, pp. 405-420 (1977). Human sensory evaluation is the most sensitive method of detection currently available, but this method is subjective with a large degree of variation between tasters and even between tests by the same tester. Despite this problem, necessity has dictated the use of human flavor-tasters at catfish processing plants who have the authority to accept or reject catfish prior to harvest or delivery.
Gas chromatography (“GC”) has been used to measure geosmin and MIB in analytical laboratories, but the use of this method by industry is limited. See, e.g., H. P. Dupuy et al., “Analysis for trace amounts of geosmin in water and fish,” JAOCS, vol. 63, pp. 905-908 (1986). Analysis by GC requires that all hydrophilic species be completely removed from the sample. Thus, fish samples must be extracted and prepared using multi-step procedures that give low recovery rates and poor reproducibility. Recovery rates for known controls can be as low as 40 to 50%. More importantly, GC analysis requires skill and training, is costly, and is not adaptable to mass screening.
There is an unfilled need for a method to measure MIB that is more reliable and facile than either the sensory or gas chromatographic analyses currently available.
An immunoassay method to measure MIB has been tried. Polyclonal antibodies (PAb) were produced by binding compounds similar in structure to geosmin and MIB (argosmin and camphor, respectively) to a carrier protein to create a conjugate. The conjugate was then used to elicit an immune response and subsequent production of polyclonal antibodies. See S. -Y. Chung et al., “Development of an ELISA using polyclonal antibodies specific for 2-methylisoborneol,” J. Agric. Food Chem., vol. 38, pp. 410-415 (1990); Chung et al., “Development of an enzyme-linked immunosorbent assay for geosmin,” J. Agric. Food Chem., vol. 39, pp. 764-769 (1991); and Chung et al., “Attempts to improve the sensitivity of an enzyme-linked immunosorbent assay for 2-methylisoborneol,” Water Sc
Park Eun-Sung
Plhak Leslie C.
Board of Supervisors of Louisiana State University and Agricultu
Davis Bonnie J.
Nguyen Bao-Thuy L.
Runnels John H.
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