Patent Publication Number: US-2010112604-A1

Title: Home food test kit and method of use

Description:
TECHNICAL FIELD 
     This invention relates to a test kit for detecting toxins formed by the use of melamine ingredients in food products for human or pet consumption, more particularly a complete home test kit for use by consumers in the absence of sophisticated equipment or laboratory analysis and that is designed for economical manufacturing to ensure the end product is affordable to a wide audience of consumers. 
     BACKGROUND OF THE INVENTION 
     Control of ingredients used in the manufacture of food products for human or pet consumption has relied on industry compliance and government regulatory agencies. 
     Within particular regions these standards are rigorously maintained, while in other locations the controls are lacking and often ignored. This creates a risk to people and pets not only within the region producing the food products, but also to any location that imports these food products for consumption. 
     Emerging food exporting markets such as China and India among others value low cost manufacturing and have been found to use substitute low cost filler ingredients to further reduce cost and increase profits. In processed foods the ability to detect this practice is often costly and, in some parts of the world, is currently unattainable. 
     In dairy products and other food products a composition of melamine is added to the food product. Melamine enables the food product to mimic high protein pure dairy products, but in fact has no nutritional value. This practice was discovered in baby formula, white chocolates and dairy creamers all originally produced in China. Naturally diluting infant formula in and of itself is simply wrong in that infants are most in need of proper nutrition and the harm caused is irreparable. The problem is more severe in that the filler product, melamine, combines easily and quickly with acids in particular cyanuric acid (2,4,6 trihydroxy-1,3,5 triazine) to create a lethal toxin unfit for consumption. This by-product forms in aged or less refined melamine and when consumed the kidney rapidly produces kidney stones and can lead to kidney failure. In China thousands of infants were exposed to this toxin, several children died in a rather short time, and others have degraded liver and kidney functions that will never recover back to normal. 
     In the United States, the problem first exposed itself in pet food. When across the pet food industry millions of dogs and cats were exposed to harmful levels of melamine in pet food. Thousands of animals died and many more were so damaged they had to be euthanized. The problem has gotten so bad the Chinese government has asked for new rapid testing procedures and interest in Melamine detection methods has sky rocketed. 
     The current state of food testing relies on laboratories taking samples and conducting tests using costly and complex equipment. The test equipment employed often includes gas chromatography, mass spectrometers or spectrophotometer analyzers each costing up to several hundreds of thousands of dollars. Although many laboratories offer these testing services to the public, a single sample tested for the presence of melamine is time consuming and often cost prohibitive for the average consumer. More importantly by the time a food product recall can be announced large amounts of the toxic products have been consumed and irreparable damage has already occurred. The problem is this use of laboratories has been ineffective in stopping the trafficking of such dangerous food products. 
     It is an objective of the present invention to provide a home test kit for the detection of melamine in consumable food and beverage products. This home test kit can be conducted by consumers without requiring any expensive equipment and/or special laboratory training. 
     SUMMARY OF THE INVENTION 
     This invention is a system, protocol, method and apparatus to enable a person unskilled in chemical analysis and without the use of laboratory equipment, to test food and beverage products for the presence or absence of the chemical Melamine. 
     The invention includes a pre-assembled comprehensive home test kit to be used for testing raw or finished food or beverage products that may be contaminated with Melamine. The home test kit, including simple, safe and disposable materials, is designed to be affordable, non-hazardous, straightforward and easy to use. 
     Consumers worldwide have long wanted the means to test, at home, the foods they purchase for themselves, their families and their pets to ensure that it has not been contaminated with Melamine. Prior to this invention, no one has conceived a way to provide consumers with this home-test kit which is dependent on an immunochromatographic assay for Melamine that can be read visually and that can be economical to commercially produce for home use. 
     It seems the principle reasons for this are three-fold: 1) Analytical techniques capable of detecting Melamine in foods or beverages are difficult to modify in a manner that would allow for visual readouts (and at levels low enough to be meaningful to food safety) without the use of sophisticated laboratory equipment such as the spectrophotometer. 2) The type of food and beverage products that have and can be contaminated with Melamine are so multifarious as to make it difficult to create a home-use system of detection that can be used in a variety of products and that does not require complex sample preparation. 3) To be meaningful to the consumer using it, a home test kit for Melamine detection must not only be reliable but also straightforward, easy to use and affordable. While immunochromatographic assay methods are frequently used in home test kits, the use of such a method is problematic when endeavoring to test specifically for Melamine in a home-test format because the variable conditions of samples and intended use require, not just a testing method, but a comprehensive system. 
     The present invention overcomes the above described and other problems and achieves its primary goal of providing consumers with a rapid, reliable and affordable home test kit for the analysis of foods and beverages that may be contaminated with Melamine. 
    
    
     
       BRIEF DESCRIPTION OF THE DRAWINGS 
       These and further objects of the present invention will be more fully understood from the following description of the invention reference to the illustrations appended hereto: 
         FIG. 1  is a view of the various components used in the home test kit of the present invention. 
         FIG. 2  is a cross-sectional view of a lateral flow test strip made in accordance with the present invention. 
         FIG. 3  is a perspective view of the test strip. 
         FIG. 3A  is an alternative embodiment of the test strip. 
         FIGS. 4-8  illustrate a proposed method of using the test kit of  FIG. 1 . 
         FIG. 4  shows a sample of prepared granules of a food product being inserted in a test vial containing a solution containing a melamine extract solution. 
     
    
    
     DETAILED DESCRIPTION OF THE INVENTION 
     The invention is for a method, device  10  and test kit system  100  that can be used by a person unskilled in the art of analytical chemistry to test a food or beverage product for the presence or absence of Melamine (1,3,5-triazine-2,4,6-triamine). 
     Method: The preferred method relates to a direct double-antibody immunochromatographic assay using a monoclonal melamine antibody and a melamine horseradish peroxide (HRP) enzyme label with a color tracer reagent such as latex, gold, a colored liposome or colored polymeric bead, tetramethylbenzidine (TMB) or any triarylmethane chromogen that is capable of undergoing a detectable color change upon oxidation with HRP. 
     Device  10 : A lateral flow membrane-based test strip  10  modified to be compatible with the intended use of the test kit  100  and specifically pre-treated for optimal performance of the preferred testing method and to allow for a visual reading indicating the presence or absence of Melamine  2 . 
     Test Kit: Home Test Kit system comprising materials preferred for optimal performance and reliability of the testing method, easy use and straightforward readability of the test results. 
     The preferred system is a boxed kit  100  comprising a plastic measuring scoop  20  with fill line  22  indicated, sample extract container  30  with a closure or lid  31 , preferably the lid or closure  31  is colored blue to match a melamine color indicator, the container cylinder  32  containing Melamine extract solution  40  (such as an acetronitrile/water or methanol/water solution); pipette  50 ; test tube  60 ; lateral flow device  10 ; the lateral flow device being the test strip  10 ; and brochure insert  70  containing directions for use and other information pertinent to the use of the kit  100 . 
     Depending on the consistency of the food or beverage sample  4  (dry, semi-moist or liquid) the user prepares the sample  4  by means of grinding, blending, crushing or simple pouring. Sample is then measured to the fill line indicated on the Measuring Scoop  20 . The prepared and measured sample  4  is then added to the Sample Extract Container  30  in which a volume of extraction solution  40  is present as shown in  FIG. 4 . 
     Sample  4  with extraction solution  40  is then shaken/mixed to produce an aqueous sample extract solution  42  as shown in  FIG. 5 . Using the Pipette  50 , user then transfers the entire contents  42  of the Sample Extract Container  30  to the Test Tube  60  component of the kit  100  as shown in  FIGS. 6 and 7 . The Lateral Flow test strip component  10  of the kit  100  is then inserted into the Test Tube  60  as shown in  FIG. 8 . Alternately, the test strip  10  can be inserted directly into the mixed sample/extract solution container  30 . The user then waits the instructed time of preferably 15 to 60 minutes, more preferably 20 to 30 minutes then removes the test strip  10  and observes for any color changes. 
     Method and Device: An immunochromatographic assay with a direct (double antibody sandwich) reaction scheme performed within a lateral flow test strip device  10  is preferred. In this scheme, a lateral flow test strip device  10  has a nitrocellulose (NC) membrane  12 , sample pad “dipstick”  11 , conjugate pad  13 , absorbent pad and plastic adhesive backing card  15  is used. Antibody specific to Melamine such as mouse monoclonal antibodies to Triazines is sprayed and immobilized in a test line  10 A on the surface of the NC membrane. As a Detection Conjugate  16 , a second antibody reagent such as horseradish peroxide (HRP) enzyme, also recognizing Melamine and labeled with a color tracer reagent is contained within the conjugate pad  13  on the membrane  12 . 
     Alternate Version of the strip  10  may use an Antibody specific to Melamine sprayed and immobilized in a test line  10 A on the surface of the NC membrane  12 . As a Detection Conjugate  16 , a second antibody reagent, also recognizing Melamine and labeled with a color tracer reagent is contained within the conjugate pad  13  on the membrane  12 . 
     When applied to the Sample Pad  14 , liquid from the sample extract  42  moves by capillary action into the conjugate pad  13  and re-hydrates the detection conjugate  16 . There, the detection conjugate  16  specifically binds to Melamine and the complex of sample extract  42  and detection conjugate  16  moves into and up the membrane  12 . The sample passes through the test line  10 A where the immobilized Melamine Antibody captures the Melamine-antibody-substrate complex forming an antibody sandwich. If the sample  42  is positive for the presence of Melamine  2 , the test line  10 A develops a color and if no melamine 2 is present in the sample  42 , no antibody-melamine sandwich is formed and the test line  10 A does not turn a color. Immobilized reagents at the control line capture excess color reagent that has passed through the test line. 
     This causes the control line  10 B to develop a color which indicates that the test was performed correctly. The user can then visually read the results of the test. A single control line  10 B on the NC membrane  12  indicates the test was performed correctly and that the sample  42  tested negative. Two lines on the NC Membrane  12  indicate that the test was performed correctly and that the sample  42  tested positive meaning melamine was detected. 
     Foodstuffs can sometimes be contaminated with remarkably high doses of melamine. For instance, an October 2008 report revealed that one Chinese company sold infant formula spiked with Melamine at a level of 6,196 parts per million. This is an incredible 2,500 times higher than the FDA safe limit. 
     This can present a problem with detection because very high doses of Melamine  2  in a sample  4  can create a condition in which unbound Melamine can block the test line  10 A and interfere with the labeled-antibody-bound antigen, resulting in a “hook effect” or false negative. 
     This problem is solved by adding a “hook line”  10 C to the test strip  10  as shown in  FIG. 3A . A third capture line  10 C can be immobilized on the surface of nitrocellulose membrane  12  using a binding partner for the labeled antibody which will bind labeled antibody that did not form a complex with the sample  42  and facilitate its capture on that line  10 C. 
     This allows the user to determine if a negative or weak result is due to low levels of Melamine  2  in the sample  4  or to excessively high levels. When the user reads the test, if the results are weak or negative and the Hook Line is present, that means the results are weak or negative. If the results are weak or negative and the Hook Line is NOT present, this indicates a “hook effect” or very high levels of Melamine  2  in the sample  4 . 
     If a sample tests positive or if a high dose effect is indicated, the user should clearly not use the food of beverage for consumption. Such a test result could be used to alert FDA or any responsible authorities to retest the product for a possible recall. The advantage of the test kit is consumers can assist regulatory agencies in monitoring human and pet food supplies while using the test results themselves to establish a “first defense” determination of safety for immediate home use of a suspect food or beverage. 
     Alternatively, it is feasible to create new antibodies that specifically bind to Melamine and that can serve to detect Melamine in the present invention. 
     Variations in the present invention are possible in light of the description of it provided herein. While certain representative embodiments and details have been shown for the purpose of illustrating the subject invention, it will be apparent to those skilled in this art that various changes and modifications can be made therein without departing from the scope of the subject invention. It is, therefore, to be understood that changes can be made in the particular embodiments described which will be within the full intended scope of the invention as defined by the following appended claims.