Chemistry: molecular biology and microbiology – Measuring or testing process involving enzymes or... – Involving antigen-antibody binding – specific binding protein...
Reexamination Certificate
1999-09-02
2002-12-03
Le, Long V. (Department: 1641)
Chemistry: molecular biology and microbiology
Measuring or testing process involving enzymes or...
Involving antigen-antibody binding, specific binding protein...
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Reexamination Certificate
active
06489132
ABSTRACT:
FIELD OF THE INVENTION
The present invention pertains generally to methods and apparatus for analytical chemistry, and more particularly to test kits and methods for qualitatively or quantitatively determining one or more analytes present within a matrix such as a food substance or biological fluid.
BACKGROUND OF THE INVENTION
It is routinely desirable to test for the presence of specific analytes in substances which are intended for human consumption or application to the human body (e.g., foods, beverages, cosmetics, toiletries, topical solutions, contact lens solutions, pharmaceutical preparations, etc.) to confirm that such substances are fresh (i.e., not degraded), pure and free of contamination. Additionally, it is often desirable to test for the presence of specific analytes in samples of biological fluids (e.g., blood, plasma, serum, urine, saliva, bile, lymph, etc.) which have been extracted from the human body.
However, the analytical techniques which have heretofore been utilized to quantitatively or qualitatively test for specific analytes in complex matrices are often problematic, due to the fact that such substances may contain many diverse physical and/or chemical species, some or all of which may interfere with the intended analysis. Thus, it is frequently necessary for the test substance to be subjected to extensive sample preparation steps, in order to isolate and/or concentrate the particular analyte(s) of interest, prior to actually proceeding with analytical determination of the desired analyte(s). Moreover, in instances where the test substance is a solid material (e.g., food) it is often necessary to chop or grind the solid material into particles, and to extract the desired analyte(s) from such particles by adding one or more liquid digestants, solvents or other fluids to form a slurry or suspension, and thereafter performing a “clean up” of the slurry or suspension by filtration or centrifugation to separate the analyte containing liquid from the extraneous solid matter.
In instances where multiple analytes are to be determined, it is often necessary to perform several separate, time consuming, analytical procedures (e.g., gas chromatography (GC), high performance liquid chromatography (HPLC) or other analytical chemistry procedures) on aliquots or extracts of the test substance, in order to generate the desired multiple analyte data.
Thus, the traditional methods for determining the presence of, or detecting specific analyte(s) in complex matrices (e.g., substances which contain matter other than the desired analyze(s)) can be quite time consuming, skill intensive and expensive.
A. Testing of Foods to Ensure Purity and Wholesomeness
It is frequently desirable to detect or quantify, in foods, one or more particular analyte(s) which are indicative of the freshness or quality of the food. In routine quality control testing of foods ,it is common practice to test for the presence of various contaminates, additives, degradation products, and/or chemical markers of microbial infestation (e.g., bacterial endotoxins, mycotoxins, etc . . . ). However, the current methods by which such quality control testing of food is accomplished are typically either: a) complex and skill-intensive analytical chemistry procedures or b) highly subjective and qualitative sensory evaluations (e.g., smell test, taste test, appearance, etc.).
B. Oxidative Degradation of Fatty Foods
As fatty acids within foods oxidize, relatively unstable lipid hydroperoxides are formed. The presence of these lipid hydroperoxides typically do not affect the smell or flavor of the food in any discernible way. These lipid hydroperoxides then further decompose to form relatively stable lipid aldehydes (e.g., malonaldehyde). The accumulation of lipid aldehydes within the food can give rise to off-odors and off-flavor of the food. Thus, it is difficult or impossible to detect the presence of abnormally high lipid hydroperoxide levels in foods by smell or taste testing, despite the fact that such elevated lipid hydroperoxide levels may indicate that the fats of the food have begun to undergo oxidative degradation and are becoming rancid. Moreover, inadvertent consumption of. these undetected lipid hydroperoxides may adversely affect the health of human beings due to the fact that such hydroperoxides are believed to play a significant role in the pathogenesis of atheroschlortic vascular disease and/or other health problems.
Various analytical techniques have previously been available to detect the presence of the lipid hydroperoxides and/or lipid aldehydes in foods, many of which involve the separate steps of a) extraction, b) clean-up, c) derivitization, d) analysis and e) detection. These previously utilized analytical techniques for detecting lipid hydroperoxides and lipid aldehydes in foods are typically expensive, time consuming, and require considerable expertise and training.
In particular, one frequently used analytical procedure for lipid aldehydes, known as the thiobarbituric acid (TBA) assay, requires that the lipid aldehydes be extracted and isolated in an analytical solution and subsequently reacted with thiobarbituric acid to give a red fluorescent adduct, which exhibits maximum UV absorbance at 532 nm. The initial extraction and isolation of the lipid aldehydes frequently requires laborious sample preparation steps. Moreover, the TBA assay is not specific for malonaldehyde (the primary lipid aldehyde in rancid fats), but rather may react with other aldehydes or other chemical species which are not indicative of rancidity. Thus, the reliability and meaningfullness of the TBA assay for assessing rancidity in foods is controversial.
Other, more complicated analytical methods have been utilized to detect lipid hydroperoxides and/or rancidity-indicating aldehydes in foods, including procedures based on electron spin resonance, high-performance liquid chromatography, and liquid chromatography-chemiluminescence techniques. However, these other analytical methodologies for assessing rancidity of fats can be extremely expensive, time consuming, and labor-intensive.
Examples of previously-known analytical techniques or other evaluations for determining lipid aldehy des in foods or other complex matrices include those described in the following publications: Nollet, L.ML.(ed.),
Handbook of Food Analysis
, Marcel Decker, Inc. (1996); Warner, K.,
Sensory Evaluations Based on Odor and Flavor: Methods to Assess Quality and Stability of Oils and Fat Containing Foods
, Pgs. 49-75, AOCS Champaign Il. (1995); Evans, C. D.,
Analysis of Headspace Volatiles by Gas Chromatography
, Proceedings of AOCS October Meeting (Pg. 15-18) (1967); Dugan, L.,
Kreis Test for C═O Groups With Phloroglucinol
, Journal of the American Oil Chemists Society 32, Pg. 605 (1955).
Examples of previously-known methods for determining lipid peroxides in foods or other complex matrices include those described in the following publications: Nollet, L. M. L. (ed),
Handbook of Food Analysis
, Marcel Decker, Inc. (1996);
Methods to Determine Lipid Peroxides by Titration Method
, Journal of the American Oil Chemists Society, Vol. 26, Pg., 345 (1949); Gray, J. I.,
Conjugated Diene Measurements at
230-375 nm, Journal of the American Oil Chemists Society, Vol. 45, Pg. 632 (1978), Halliwell B, Gutteridge J M C.
Free radicals in biology and medicine
, 2nd ed. Oxford,d UK: University Press, 1989:543pp; Gutteridge J M C, Halliwell B.
The measurement and mechanisms of lipid peroxidation in biological systems
, Trends Biochem Sci 1990;15:129-35; Gutteridge J M C.
Lipid peroxidation: some problems and concepts
, in ed. (
Oxygen radicals and tissue injury
). Halliwell B., Bethesda, M D:FASEB, 1977:9-19; Gutteridge J M C, Kerry P J.
Detection by fluorescence of peroxides and carbonyls in samples of aracyidonic acid
. Br J Pharmacol 1982;76:459-61; Gutteridge J M C.
Iron promoters of the Fenton reaction and lipid peroxidation can be released from haemoglobin by Peroxides
. FEBS Lett 1986;20:291-5. ; Gutteridge J M C, Beard A P C, Quinla
Elias John F.
Gordon Virginia C.
Mirhashemi Soheila
Mittelstein Michael
Sorensen John T.
Buyan Robert D.
Le Long V.
Padmanabhan Kartic
Safety Associates, Inc.
Stout, Uxa Buyan & Mullins, LLP
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