Patent Application
20090035786
The invention is concerned with affinity columns used for immunological screening for environmentally occurring toxins, for example, those found in food products, and is particularly directed to multi-analyte columns for detecting a plurality of toxins that may be present in a single sample.
Awareness of the incidence and effect of human and animal exposure to toxic substances by humans and other animals via food, water, and air is of critical importance to our survival. The detection of toxins such as aflatoxin, ochratoxin, zearalenone, deoxynivalenol, fumonisin and T-2 has become especially important. In particular, screening procedures for assessing the exposure of humans to such toxins may require the ability to quantify both the toxin and its metabolites.
Aflatoxins are a typical example of the compounds for which screening is desired. Aflatoxins are secondary fungal metabolites, mycotoxins, which are produced by Aspergillus flavus and Aspergillus parasiticus and are structurally a group of substituted coumarins containing a fused dihydrofurofuran moiety. Aflatoxins occur naturally in peanuts, peanut meal, cottonseed meal, corn, dried chili peppers, and the like. However, the growth of the mold itself does not predict the presence or levels of the toxin because the yield of aflatoxin depends on growth conditions as well as the genetic requirements of the species. A variety of aflatoxins, that is types B1, B2, G1, G2, M1 and M2, have been isolated and characterized. Aflatoxin B1 (“AFB1”) is the most biologically potent of these aflatoxins and has been shown to be toxic, mutagenic and carcinogenic in many animal species. This mycotoxin is a frequent contaminant of the human food supply in many areas of the world and is statistically associated with increased incidence of human liver cancer in Asia and Africa, in particular (Busby et al., in Food-Born Infections and Intoxications (Riemann and Bryan, Editors) Second Edition, Academic Press, Inc., 1979, pp. 519-610; Wogan, G. N. Methods Cancer Res. 7:309-344 (1973)).
AFB1 also forms covalently linked adducts with guanine in DNA after oxidative metabolism to a highly reactive 2,3-exo-epoxide, the major adduct product being 2,3-dihydro-2-(N7-guanyl)-3-hydroxy-aflatoxin B1 (“AFB1-N7-Gua”) (Lin et al., Cancer Res. 37:44304438 (1977); Essigman et al., Proc. Natl. Acad. Sci. USA 74:1870-1874 (1977); Martin et al., Nature (London) 267:863-865 (1977)). The AFB1-N-7-Gua adduct and its putative derivatives (2,3-dihydro-2-(N-5-formyl-2′,5′,6′-triamino-4′-oxo′N5-pyrimidyl)-3-hydroxy-aflatoxin B1) (“AF-N7-Gua”) have been identified in a wide variety of tissues and systems such as rat liver in vivo, cultured human bronchus and colon, and human lung cells in culture after acute or chronic administration (Haugen et al., Proc. Natl. Acad. Sci. USA 78:4124-4127 (1981)).
Some investigations regarding quantitation of aflatoxin B1 and its metabolites including its DNA adduct have been conducted using immunological techniques and monoclonal antibodies (Hertzog et al., Carcinogensis 3:825-828 (1982); Groopman et al., Cancer Res. 42:3120-3124 (1982); Haugen et al., Proc. Natl. Acad. Sci. USA 78: 412-44127 (1981)). Similar research has been conducted utilizing immunological techniques and reagents for other low molecular weight toxins found in our environment (Johnson et al., J. Analyt. Toxicol. 4:86-90 (1980); Sizaret et al., J.N.C.I. 69:1375-1381 (1982); Hu et al., J Food Prot. 47:126-127 (1984); and Chu, J. Food Prot. 47:562-569 (1984)).
U.S. Pat. No. 4,818,687 describes a general non-invasive screening procedure for assessing the exposure of humans and animals to environmentally occurring carcinogens. Therein, an affinity matrix and a method for the detection of low molecular weight compositions such as aflatoxins are provided utilizing specific monoclonal IgM antibody.
Affinity columns for detecting the presence of a single analyte, for example, one of aflatoxin, ochratoxin, zearalenone, deoxynivalenol or fumonisin, in a sample are well known. An affinity column for detecting both aflatoxin and ochratoxin in a single sample as well as an affinity column for detecting aflatoxin, ochratoxin and zearalenone have been commercially available. However, columns targeting higher numbers of chemical species necessarily must capture more diverse analytes. Aflatoxin is a large aromatic, multi-ring structure. Deoxynivalenol (DON) is a highly polar toxin that is smaller than a molecule of table sugar-sucrose. The lipid-like fumonisin shares structural characteristics with sphingolipids. Thus, the preparation of multi-analyte columns and their methods of use increase in complexity far out of proportion to the number of toxins being added for analysis. Column development must allow for treatment of all target analytes according to similar methods, in order that they all be analyzed with a single column.
There have been numerous reported incidences of naturally-occurring mycotoxins such as, aflatoxin B1, B2, G1, G2 and M1 (Afla), deoxynivalenol (DON), fumonisin B1, B2 and B3, ochratoxin A (OTA), and zearalenone (Zear) in various substrates. Malt beverages and wines can contain different multi-toxin combinations from fungi-infected grains and fruits used in the production. A desire still exists for competent multi-analyte columns for analyzing a plurality of toxins with a single column.
It is not possible to obtain satisfactory analytical results in a multi-analyte column by merely combining the quantities of resin used in a single analyte column to analyze each particular analyte. The invention is based, at least in part, on the discovery that satisfactory analytical results are possible by incorporating into the column antibodies that are specific for the analytes to be analyzed.
Thus the present invention provides a multi-analyte column capable of analyzing a single sample containing one or more of aflatoxin, deoxynivalenol (“DON”), fumonisin, ochratoxin, T-2 (and/or HT-2) and zearalenone. The multi-analyte columns in accord with the present invention comprise a first quantity of a first resin comprising an antibody having specificity for aflatoxin, a second quantity of a second resin comprising an antibody having specificity for deoxynivalenol, a third quantity of a third resin comprising an antibody having specificity for fumonisin, a fourth quality of a fourth resin comprising an antibody having specificity for ochratoxin, a fifth quantity of a fifth resin comprising an antibody having specificity for T-2 (and/or HT-2) and a sixth quantity of a sixth resin comprising an antibody having specificity for zearalenone.
It is desirable to obtain at least a 60%, preferably at least a 70% recovery from the column for each toxin in the sample. It also is desirable to have a column flow rate of at least 3 ml per minute, preferably so that a 10 ml sample will flow through the column in less than 5 min.
In one embodiment of the invention, a multi-analyte column capable of analyzing a single sample containing aflatoxin, deoxynivalenol, fumonisin, ochratoxin, T-2 (and/or HT-2) and zearalenone, comprises for each unit of resin containing antibody having specificity for ochratoxin, about 0.95 to 1.05 units of resin containing antibody having specificity for zearalenone, about 1.9 to 2.1 units of resin containing antibody having specificity for aflatoxin, about 2.35 to 2.65 units of resin containing antibody having specificity for fumonisin, about 2.8 to 3.2 units of resin containing antibody having specificity for T-2 (and/or HT-2) and about 4.7 to 5.3 units of resin containing antibody having specificity for deoxynivalenol. As used herein, one unit of resin is defined as the quantity of resin containing antibody that will bind 50 ng of aflatoxin, 500 ng of deoxynivalenol, 3300 ng of fumonisin, 50 ng of ochratoxin, 830 ng of T-2 (and/or HT-2) or 1140 ng of zearalenone, respectively. Such resin typically will contain about 5 mg antibody per ml of resin. However, any suitable loading of antibody on the resin can be used in accord with quantities and methods well known to those skilled in the art.
In a preferred embodiment, the multi-analyte column of the present invention is capable of analyzing a sample to detect aflatoxins G1, G2, B1, B2 and M1, DON, fumonisins B1, B2 and B3, ochratoxin A, T-2 and HT-2, and zearalenone in the analysis of a single sample applied to the column.
The invention also provides a method for analyzing a single sample for aflatoxin, deoxynivalenol, fumonisin, ochratoxin, T-2 (and/or HT-2) and zearalenone, the method comprising providing a multi-analyte column as described herein, applying liquid sample suspected of containing one or more of the specified toxins to bind any of the specified toxins to resins in the column, washing the column, eluting the resins and analyzing the eluant for the presence of each of the specified toxins. The liquid sample can be a liquid suspected of containing toxins or a liquid extract of a solid material suspected of containing toxins. Specific examples of sample materials that can be analyzed in accord with the columns of the present invention include fungi-infected grains and fruits, and alcoholic beverages such as, for example, malt beverages and wines.
In accord with the present invention, a multi-analyte column capable of analyzing a single sample containing aflatoxin, deoxynivalenol, fumonisin, ochratoxin, T-2 (and/or HT-2) and zearalenone can be prepared. Resins containing antibody having specificity for each of the toxins are included. Antibodies are raised by well known techniques and monoclonal antibodies are prepared having specificity for each toxin. Resins having each antibody bound thereto are prepared by techniques well known to those skilled in the art. Any resin material known by those skilled in the art to be useful for carrying attached antibodies can be used. A preferred resin material is Sepahrose® 4B available from Amersham Biosciences (Piscataway, N.J.). The antibodies are then attached to the resin using techniques well known to those skilled in the art. Preferably, about 5 mg of antibody is bound to one ml of resin. The resin preferably has a particle size range of about 45 to about 165 μm.
Columns are then prepared using appropriate quantities of each resin. For example, in one embodiment of the invention, in a 3 ml column having a diameter of 8.93 mm, a supporting porous disk, or the like, is positioned to support the resin bed while permitting flow out of the column. 200 μl of a first resin having an antibody specific for aflatoxin is layered on the disk. Then, 100 μl of a second resin having an antibody specific for ochratoxin is layered on the first resin. Then, 250 μl of a third resin having an antibody specific for fumonisin is layered on the second resin. Then, 100 μl of a fourth resin having an antibody specific for zearalenone is layered on the third resin. Then, 300 μl of a fifth resin having an antibody specific for T-2 (and/or HT-2) is layered on the fourth resin. 500 μl of a sixth resin having an antibody specific for DON is layered on the fifth resin. Finally, another porous disk, or the like, if desired, can be positioned to distribute the liquid sample across the column and/or hold the resin in place. Alternatively, the resins can be layered in any order or they can be mixed together and then loaded into the column as a mixture. Further, a suitable porous media such as, e.g., glass wool or the like, can be used in place of the porous disk.
For comparable size single analyte columns performing the same task, the same antibody/resins typically are loaded presently at 200-250 μl for aflatoxin, 200-250 μl for ochratoxin, 300 μl for T-2, 350 μl for fumonisin, 350 μl for zearalenone and 550 μl for DON.
In the above embodiment, 100 μl of resin is equal to one unit. Each unit of resin is capable of binding about 50 ng of aflatoxin, 500 ng of deoxynivalenol, 3300 ng of fumonisin, 50 ng of ochratoxin, 830 ng for T-2 (and/or HT-2) or 1140 ng of zearalenone, respectively. In accord with the invention, for each unit of resin having ochratoxin specific affinity, the column contains about 0.95 to 1.05 units of resin containing antibody having specificity for zearalenone, about 1.9 to 2.1 units of resin containing antibody having specificity for aflatoxin, about 2.35 to 2.65 units of resin containing antibody having specificity for fumonisin, about 2.8 to 3.2 units of resin containing antibody having specificity for T-2 (and/or HT-2) and about 4.7 to 5.3 units of resin containing antibody having specificity for deoxynivalenol.
The total amount of resin in the column should permit a sample fluid to flow through the column at a preferred rate of about 1-2 drops per sec.
For solid foods, preferably toxins are extracted from the food using a water-based or water compatible solvent such as, for example, water:methanol, water:acetonitrile, ethanol, water:ethanol, salt solutions, buffer solutions, and the like, etc. Such solvents are well known to those skilled in the art. Typically, in such solvents the organic component is greater. Extracts can be diluted with water prior to chromatography.
After loading the sample on the column, the column typically is washed to remove any extraneous materials that may be held up on the column so that only bound materials, i.e., the toxins, remain. The column generally can be washed with the water compatible solvent but typically having a greater water presence.
The column is eluted with solvents as is well known to those skilled in the art. The eluants preferably are analyzed for the particular analytes using HPLC/MS techniques.
Multi-analyte columns in accord with the present invention can be used as a clean-up step in analysis of extracts from solid materials or of liquid products such as alcoholic beverages for aflatoxins, fumonisins, ochratoxin A, deoxynivalenol, T-2 (and/or HT-2) and zearalenone, in combination with HPLC and/or mass spectrometry detection. LC/MS and LC/MS-MS methods for detection can also be used. Methods for detecting the toxins are well known to those skilled in the art.
Dried distiller's grains can contain naturally occurring multiple mycotoxins. A single sample of an alcoholic beverage can be analyzed for aflatoxin, deoxynivalenol, fumonisin, ochratoxin, T-2 (and/or HT-2) and zearalenone using the six analyte column of the present invention.
The following example illustrates detection of aflatoxins G1, G2, B1, B2, DON, fumonisins B1, B2, B3, ochratoxin A, T-2 and zearalenone using a column containing 200 μl of a first resin having an antibody specific for aflatoxin, 100 μl of a second resin having an antibody specific for ochratoxin, 250 μl of a third resin having an antibody specific for fumonisin, 100 μl of a fourth resin having an antibody specific for zearalenone, 300 μl of a fifth resin having an antibody specific for T-2 and/or HT-2, and 500 μl of a sixth resin having an antibody specific for DON, wherein each resin has approximately 5 mg/ml of antibody and toxin detection capability per unit described herein. Spiked samples are used to calculate recovery from the column.
A Shimadzu 2010 LC is fitted with an autosampler and a dual Mass Selective and UV Detector. The Shimadzu 2010 is used in two operating modes, positive for Aflatoxin (B1, B2, G1, G2), Fumonisin (B1, B2, B3), Zearalenone, DON (Vomitoxin), and T2 and then switched to the negative mode for Ochratoxin. (In the positive mode, the ions are positively charged; in the negative mode, the ions are negatively charged. Ochratoxin is more readily detected in the negative mode.) The instrument is capable of operating in APCI (Atmospheric Pressure Chemical Ionization) or ESI (Electro Spray Ionization). Only the APCI was used for mycotoxins. The mobile phase differs between the two modes, with ammonium acetate/triethylamine in the negative mode and ammonium acetate/formic acid in the positive mode. The LC column is a C18 5μ pore that is 5 cm in length and 2.1 mm in diameter.
The individual analytes are scanned to obtain major ions, and then an ion is selected that is free from interference and shows a linear response to increasing concentrations. Generally, a five (5) point curve is set up. The standards are stored in two sets. One set is just Ochratoxin, while the other is the combination of the other five standards. The run time is; about ten (10) minutes for the positive mode mycotoxins and about five (5) minutes for Ochratoxin. The instrument is capable of achieving<1 ppb levels for the four aflatoxins and 100 ppb for the fumonisins.
The samples are extracted using a methanol-water extraction solution and the extract is cleaned up and then put through Vicam affinity chromatography columns. The columns are eluted and the eluent analyzed by liquid chromatography/mass spectrometry (LC-MS).
As used in the specification and claims, the singular forms “a”, “an” and “the” include plural references unless the context clearly dictates otherwise. For example, the term “a molecule” also includes a plurality of molecules.
As used herein, an “analyte” is the element of the sample to be detected or isolated. An analyte includes, but is not limited to, a toxin, a toxoid, a toxic substance, a poisonous substance, a poison, or a specific impurity of interest. An analyte may be of biological or non-biological origin. In some embodiments, the analyte specifically binds a binding reagent. In some embodiments, the presence or absence of the analyte may be used to detect, for example, contamination of a sample. Alternatively, the presence or absence of the analyte may be used to determine the physiological condition of an organism from which the sample was obtained. A wide range of other uses will occur to one of skill in the art.
As used herein, a “toxin” includes a toxoid, a toxic substance, a poisonous substance, or a poison of biological or non-biological origin. In some embodiments, a toxin causes damage or disease to a cell or an organism.
As used herein, “specificity” refers to the ability of an antibody to discriminate between antigenic determinants. It also refers to the precise determinants recognized by a particular receptor or antibody. It also refers to the ability of a receptor to discriminate between substrates, such as drugs. With respect to nucleic acids, it refers to identity or complementarity as a function of competition or recognition/binding, respectively. “Specificity” of recognition or binding may be affected by the conditions under which the recognition or binding takes place (e.g., pH, temperature, salt concentration, and other factors known in the art).
As used herein, a “ligand” is a molecule or molecular complex that can be bound by another molecule or molecular complex. The ligand may be, but is not limited to, a molecule or molecular complex bound by a receptor, or it may be a complementary fragment of nucleic acid.
As used herein, an “antibody” (Ab) is protein that binds specifically to a particular substance, known as an “antigen” (Ag) (described infra). An “antibody” is any immunoglobulin, including antibodies and fragments thereof, that binds a specific epitope. The term encompasses polyclonal, monoclonal, and chimeric antibodies (e.g., multispecific antibodies).
As used herein, an “antigen” (Ag) is any substance that reacts specifically with antibodies or T lymphocytes (T cells). An “antigen-binding site” is the part of an immunoglobulin molecule that specifically binds an antigen.
As used herein, “biological sample” includes samples of tissues, cells, blood, fluid, milk, or other materials obtained from a biological organism. It also includes a biological organism, cell, virus, or other replicative entity.
As used herein, “food” includes any substance or product intended for human or animal consumption, regardless of nutritional value. “Food” includes beverages, including milk and water. It also includes ingredients or substances used in the preparation of food (e.g., oil, fat, spices, flavorings, etc.). “Food” may be processed, partially processed, or unprocessed. It can include artificial and/or natural ingredients.
As used herein, “beverage” or “drink” includes any liquid or colloidal substance intended for human or animal consumption, regardless of nutritional value. “Beverages” include milk and other dairy drinks (e.g., flavored milk, cream, eggnog, milkshakes, frappes), water, coffee and coffee-based beverages, tea and tea-based beverages, other infusions (e.g., herbal tea/tisane, roasted grain or nut beverages), beer, wine, other alcoholic substances (e.g., whisky, vodka, gin, brandy, rum, schnapps, liqueurs), fruit and vegetable juices and ciders, sap, soft drinks regardless of degree of carbonation (e.g., mixed juice beverages, syrup and powdered drinks, sodas and colas, sports and energy drinks), non-dairy milks (e.g., soy milk, rice milk, coconut or almond milk), hot chocolate, and hot cocoa. “Beverages” may be processed, partially processed, or unprocessed. Wine and beer may be alcoholic beverages, may have reduced alcohol or may be non-alcoholic. Coffee and tea may be undecaffeinated (i.e., “regular”) or partially or wholly decaffeinated. Tea may be non-oxidized, partially oxidized, or fully oxidized. “Beverages” can include artificial and/or natural ingredients. “Beverages” may be enriched with vitamins and minerals or may be flavored with spices, herbs, seasonings, juices, flowers, berries, or other flavorings or substances (e.g., toasted rice).
When not otherwise stated, “substantially” means “being largely, but not wholly, that which is specified.”
1.0 Liquid Chromatography/Mass Spectrometry (LC-MS):
2.0 Sample Extraction:
3.0 Sample Clean-up
4.0 Column Chromatography
The procedure described above was performed on ten (10) replicates of a sample of dried distiller's grains (DDG) with solubles spiked at the indicated levels. Ochratoxin was analyzed in the negative mode. All other toxins were analyzed in the positive mode.
Precisions and recoveries showed a good correlation with theoretical levels (parts per billion [ppb] or parts per million [ppm]) based on spiking. All recovery values were above 70%, with most above 90%. The average values and percentages of residual standard deviation (% RSD) are provided.
Throughout this application, various publications including United States patents, are referenced by author and year and patents by number. The disclosures of these publications and patents in their entireties are hereby incorporated by reference into this application in order to describe more fully the state of the art to which this invention pertains.
The invention has been described in an illustrative manner, and it is to be understood that the terminology which has been used is intended to be in the nature of words or description, rather than of limitation.
Modifications and variations of the present invention are possible in light of the above teachings. It is, therefore, to be understood that within the scope of the described invention, the invention may be practiced otherwise than as specifically described.
The present inventions have been described in detail including preferred embodiments thereof. However, it should be appreciated that those skilled in the art, upon consideration of the present disclosure, may make modifications and improvements within the spirit and scope of the present inventions.
| Number | Date | Country | Kind |
|---|---|---|---|
| 50281021.1 | Dec 2005 | EP | regional |
The present application claims priority to U.S. Provisional Application 60/738,330, filed Nov. 17, 2005, and to European Patent Application 05028102.1, filed Dec. 21, 2005, the disclosures of each of which are incorporated herein by reference.
| Filing Document | Filing Date | Country | Kind | 371c Date |
|---|---|---|---|---|
| PCT/US06/44739 | 11/17/2006 | WO | 00 | 9/12/2008 |
| Number | Date | Country | |
|---|---|---|---|
| 60738330 | Nov 2005 | US |
