Patent Publication Number: US-2007117218-A1

Title: Afoz multi-analyte affinity column

Description:
CROSS-REFERENCES TO RELATED APPLICATIONS  
      The present application claims priority to U.S. Provisional Application 60/738,111, filed Nov. 17, 2005, and to European Patent Application 05028101.3, filed Dec. 21, 2005, the disclosures of each of which are incorporated herein by reference. 
    
    
     FIELD OF THE INVENTION  
      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.  
     BACKGROUND OF THE INVENTION  
      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 and fumonisin 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 B 1 , B 2 , G 1 , G 2 , M 1  and M 2 , have been isolated and characterized. Aflatoxin B 1  (“AFB 1 ”) 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)).  
      AFB 1 , 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-(N 7 -guanyl)-3-hydroxy-aflatoxin B 1 , (“AFB 1 -N7-Gua”) (Lin et al.,  Cancer Res.  37:4430-4438 (1977); Essigman et al.,  Proc. NatL. Acad. Sci. USA  74:1870-1874 (1977); Martin et al.,  Nature  (London) 267:863-865 (1977)). The AFB 1 -N7-Gua adduct and its putative derivatives (2,3-dihydro-2-(N5-formyl-2′,5′, 6′-triamino-4′-oxo′N5-pyrimidyl)-3-hydroxy-aflatoxin B 1 ) (“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 B 1  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: 4124-4127 (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 B 1 , B2, G 1 , G 2  and M 1  (Afla), deoxynivalenol (DON), fumonisin B 1 , B 2  and B 3 , 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.  
     SUMMARY OF THE INVENTION  
      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, fumonisin, ochratoxin and zearalenone. The muti-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 fumonisin, a third quantity of a third resin comprising an antibody having specificity for ochratoxin and a fifth quantity of a fifth 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 5min.  
      In one embodiment of the invention, a multi-analyte column capable of analyzing a single sample containing aflatoxin, fumonisin, ochratoxin 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, and about 2.8 to 3.2 units of resin containing antibody having specificity for fumonisin. As used herein, one unit of resin is defined as the quantity of resin containing antibody that will bind 50 ng of aflatoxin, 3300 ng of fumonisin, 50 ng of ochratoxin, 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 G 1 , G 2 , B 1 , B 2  and M 1 , fumonisins B 1 , B 2  and B 3 , ochratoxin A, 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, fumonisin, ochratoxin 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. 
    
    
     DETAILED DESCRIPTION OF THE INVENTION  
     INCLUDING PREFERRED EMBODIMENTS  
      In accord with the present invention, a multi-analyte column capable of analyzing a single sample containing aflatoxin, fumonisin, ochratoxin 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. 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, 350 μl for fumonisin and 350 μl for zearalenone.  
      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, 3300 ng of fumonisin, 50 ng of ochratoxin, 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 and about 2.8 to 3.2 units of resin containing antibody having specificity for fumonisin.  
      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 are analyzed for the particular analytes using HPLC techniques equipped with in-line photochemical reactor, post column derivatizer, and fluorescent detectors.  
      Muti-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 and zearalenone, in combination with HPLC and/or mass spectrometry detection. The detection of the toxin can be illustrated, typically, by spiking a sample of a solid, extract, malt beverage or rice wine with toxins. If desired, the sample can be dried to eliminate the alcohol content. Then resuspend the dried sample in deoionized water or phosphate buffered saline (PBS) to a volume equal to the original sample, Dilute the resuspended sample 1:1 (v/v) in 1/10 diluted phosphate buffered saline 10× stock solution from VICAM (pH of sake and beer samples are roughly between 5.0 and 6.0). Load the sample onto the multi-analyte column at a speed of about 2 drops/second. Wash the column with deonized water or phosphate buffered saline). Elute the toxins from the column with methanol. Dry the aqueous methanolic eluate and reconstitute in methanol. Inject about a 30ul sample onto the HPLC. Preferably, the HPLC is equipped with in-line photochemical reactor (PHRED), post-column derivatizer, and fluorescence detectors. Aflatoxins are detected by fluoresence after post-column photochemical derivitization (post-column iodine may also be used). Fumonisin is derivatized with o-phthaldialdehyde and detected by fluoresence. Zearalenone is detected by fluoresence. Ochratoxin is detected by fluoresence. 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.  
      Alcoholic beverages can contain naturally occurring multiple mycotoxins. A single sample of an alcoholic beverage can be analyzed for aflatoxin, fumonisin, ochratoxin and zearalenone using the four analyte column of the present invention.  
      The following example illustrates detection of aflatoxins G 1 , G 2 , B 1 , B 2 , fumonisins B 1 , B 2 , B 3 , ochratoxin A, 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 and 100 μl of a fourth resin having an antibody specific for zearalenone, wherein each resin has approximately 5 mg/ml of antibody and toxin detection capability per unit described herein. Spiked samples were used to calculate recovery from the column.  
      Materials and Methods  
      Reqgents and Chemicals  
      The o-phthaldialdehyde (OPA) solid, OPA diluent (5.4% potassium borate in water), Thiofluor™ (N,N-Dimethyl-2-mercaptoethylamine hydrochloride) solid, and phosphoric buffer solution (P/N 1700-1108) can be obtained from Pickering Laboratories (Mountain View, Calif.). The 30% (w/v) Brij® 35 solution can be obtained from Sigma (St. Louis, Mo.). Acetonitrile and methanol (both Optima grade) can be obtained from Fisher Scientific (Pittsburgh, Pa.). Deionized water can be produced by a Millipore Milli-Q system (Bedford, Mass.). Amber glass ampules of aflatoxin B 1 , B 2 , G 1  &amp; G 2 , ochratoxin A, and zearalenone standards in appropriate organic solvents can be obtained from Supelco (Bellefonte, Pa.). Fumonisin B 1 , B 2  &amp; B 3  can be obtained from PROMEC of Medical Research Council (Tygersberg, South Africa). SurfaSil™ siliconizing fluid for surface treatment of in-house laboratory glassware can be obtained from Pierce Biotechnology (Rockford, Ill.).  
      Reagent Preparation  
      OPA reagent preparation: One 950-mL size bottle containing the OPA diluent is sparged with helium (inert gas) for 10 min. A 300-mg OPA portion is added into 50 mL beaker and dissolved in 10 mL methanol. A 2-g Thiofluor™ portion is then added into 50 mL beaker. The inert gas is turned off and the OPA solution and Thiofluor™ mixture are added to the sparged diluent. A 3-mL portion of 30% (w/v) Brij® 35 solution also is added and then mixed well with the rest. This specially prepared reagent is readily oxidized and should be kept under inert gas. Saran™ (polyvinylidene chloride) tubing for inert gas supply and reagent connections is used to minimize this problem.  
      Multi-toxin stock standard solution preparation: Accurately measured amounts of all four mycotoxin families are transferred into a silanized borosilicate glass volumetric flask. Accompanying organic solvents are dried and reconstituted with deionized water, and filled to the mark to prepare a known mixed-toxin stock standard solution to be used for multi-toxin standard calibration and sample spiking purposes.  
      Apparatus and Equipment  
      The complete system apparatus contained several instruments that are assembled in series (HPLC injector—analytical column—photochemical reactor—post-column derivatizer—fluorescence detector—waste). The HPLC set-up consists of Agilent 1100 Series quaternary pump and injection system, including a standard autosampler. The 1100 Series fluorescence and diode-array detector (DAD) from Agilent Technologies (Palo Alto, Calif.) are used.  
      Analytical Conditions  
      The MycoToX™, C 18  analytical column, 4.6×250 mm, 5 μm particle size, and a 5-μm guard column are from Pickering Laboratories (Mountain View, Calif.). Agilent&#39;s ChemStation software is used for data management. The mobile phase consists of combinations of three reagents. The HPLC gradient is as follows:  
               TABLE 1                          HPLC gradient                                         Phosphoric buffer                   Time   (P/N 1700-1108), %   Methanol, %   Acetonitrile, %                                                 0.0   85   0   15           5.0   85   0   15           5.1   57   28   15           20.0   57   28   15           23.0   40   60   0           40.0   40   60   0           50.0   0   100   0           60.0   0   100   0                      
 
      The flow rate is 1 mL/min with column temperature of 40° C. and injection volume of 30 μL. The equilibration time is 10 min.  
      Photochemical Reactor for Enhanced Detection (“PHRED”™)  
      The PHRED™ unit (Aura Industries, New York, N.Y.) is equipped with a 254 nm low pressure Hg lamp and the PTFE (poly-tetrafluoroethylene) knitted reactor coils. The 254-nm UV light is able to perform continuous photolytic derivatization to enhance the sensitivity and/or selectivity of fluorescence detection response. The photochemical reactor is placed between the HPLC analytical column and the detector.  
               TABLE 2                          Detection                             Analyte   Derivatization   Detection   Wavelength               Aflatoxins   Photolytic   Fluorescence   λ ex  = 365 nm           (PHRED ™)       λ em  = 455 nm       Fumonisins   Post-column   Fluorescence   λ ex  = 330 nm           (OPA)       λ em  = 465 nm       Ochratoxin A   None   Fluorescence   λ ex  = 335 nm                   λ em  = 455 nm       Zearalenone   None   Fluorescence   λ ex  = 275 nm                   λ em  = 455 nm                  
 
     
       
         
           
               
             
               
                 TABLE 3 
               
             
            
               
                   
               
               
                   
               
               
                 The wavelength settings on the fluorescence 
               
               
                 detector are as follows: 
               
            
           
           
               
               
               
            
               
                 Time 
                 λ ex   
                 λ em   
               
               
                   
               
            
           
           
               
               
               
            
               
                 0.0 
                 365 
                 455 
               
               
                 26.0 
                 365 
                 455 
               
               
                 26.1 
                 330 
                 465 
               
               
                 36.0 
                 330 
                 465 
               
               
                 36.1 
                 335 
                 455 
               
               
                 41.0 
                 335 
                 455 
               
               
                 41.1 
                 275 
                 455 
               
               
                 44.0 
                 275 
                 455 
               
               
                 44.1 
                 330 
                 465 
               
               
                 60.0 
                 330 
                 465 
               
               
                   
               
            
           
         
       
     
      The highest detector sensitivity level at PMT gain 16 is selected. All gradient and wavelength changes are programmed through the ChemStation software.  
      Post-Column Conditions  
      The PCX5200 post-column derivatization system is equipped with Control Software from Pickering Laboratories (Mountain View, Calif.). The reactor volume and temperature are set at 1.4 ml and 65° C., respectively. The derivatizing reagent is a specially prepared OPA reagent. The flow rate is set at 0.3 ml/min. The post-column pump program was activated using the PCX5200 Control Software, which turns the pump on at 23.0 min, then off at 34.5 min and on again at 43.5 min and finally off at 60.0 min.  
      Sample Preparation and SPE Column Clean-Up Protocols  
      The Visiprep® 24-port SPE vacuum manifold and Visidry® drying attachment from Supelco (Bellefonte, Pa.) or the RapidTrace® automated SPE workstation from Zymark/Caliper LifeSciences (Hopkinton, Mass.) are used for sample preparations. The alcoholic beverage sample is dried to remove alcohol and other volatile organic constituents, and then reconstituted to its original volume in one-tenth diluted PBS concentrate solution. Either a 5-ml aliquot of alcoholic beverage in one-tenth diluted PBS concentrate solution spiked with multi-toxin standards (sample) or a one-tenth diluted PBS solution spiked with multi-toxin standards (control) is passed through the multi-toxin antibody-based SPE column. Larger sample volumes can be added if a mycotoxin pre-concentration step is desired. The IA column is washed with 4 ml deionized water. Target mycotoxins are eluted with 3 ml methanol. The water-washing step is done at a flow rate of about 2 drops/sec, but the sample loading and methanol-elution steps are performed at a slower rate (≦1 drop/sec). Then, the methanol eluate collected in a silanized borosilicate culture tube is dried and reconstituted in 3 ml methanol. The methanol eluate is either dried down at ambient temperature using air or at 40° C. under nitrogen. During the enrichment step the dried mycotoxins are then reconstituted with a smaller amount of methanol compared to the original sample volume loaded on to the column. The process is done in a silanized borosilicate tube tightly covered by a piece of Parafilm® film or a plastic cap, and mixed well using the Vortex-Genie™ vortexer from Scientific Industries (Bohemia, N.Y.). Thirty microliters of the prepared sample solution is injected into the HPLC.  
      This HPLC method simultaneously analyzes aflatoxins, fumonisins, ochratoxin A and zearalenone with post-column photochemical and o-phthaldialdehyde (OPA) derivatizations. The ruggedness of separation and detection is established on a representative multi-toxin mid-level calibration standard chromatogram. Generated 5-point multi-toxin standard calibration curves preferably show linear regression correlation coefficients ≧0.999.  
      The photochemical reactor is strategically placed in series before the post-column derivatizer hardware for the photolytic derivatization of aflatoxins. The method allows for fluorescent detection of the fumonisins using the prepared OPA reagent, aflatoxins via photolysis and the natural-fluorescence of zearalenone and ochratoxin A. Fumonisins have a primary amine group that is derivatized post-column with OPA and a mercaptan to form a highly-fluorescent adduct, 1-alkyl-2-thioalkyl-subsitituted isoindole exhibiting optimal excitation at 330 nm and maximal emission at 465 nm. The OPA reagent flow is started after the aflatoxins elution and stopped after the fumonisin B 1 , peak elution. Sufficient delay time is allocated to flush the OPA from the tubing prior to the ochratoxin A and zearalenone elution. Then, the OPA reagent flow is turned back on during the fumonisin B 3  and B 2  elution. The fluorescence detector is time-programmed to change excitation and emission wavelengths for multi-toxin response optimization.  
      Alternatively, all of the toxins can be analyzed using LC-MS in accord with procedures well known to those skilled in the art.  
      The multi-toxin recoveries in spiked PBS control and alcoholic beverage samples with the enrichment step in the silanized borosilicate tube preferably exceed 70% with RSD≦10%. Acceptable multi-toxin spike recovery ranges demonstrate the 4 analyte IA column&#39;s ability to effectively and selectively bind with the targeted mycotoxins.  
      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.  
      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.