MXPA06000213A - Reagents, methods and kits for detecting feed enzymes - Google Patents

Abstract

This invention relates to the field of immunology and more specifically relates to immunoassay methods, kits, and reagents, for the detection of proteins and enzymes, in particular feed enzymes.

Description

Published: For rwo-letter codes and other abbreviations, refer to the "Guid- - wnhout intemational search repon and to be republished anee Notes on Codes and Abbrevialions" appearing at the beginning- upon receipt of the repon ning of the regular issue of the PCT Gazeite.

REAGENTS, METHODS AND KITS TO DETECT ENZYMES IN FOOD Field of the Invention This invention relates to the field of immunology and more specifically relates to immunoassay methods, including ELISA and assays, kits and immunotyping reagents, for the detection of proteins and enzymes, in particular food enzymes. Background of the Invention There is a significant need for a convenient, relatively easy test to detect the presence of enzymes in animal feed. To date, there is no standard immunological assay for such detection. There is also a tremendous need to determine if a plant has been genetically modified or if the grain or processed foods contain GMO traits. The need requires testing methods that can detect and quantify either the novel DNA or protein. The present invention meets this need by providing methods, reagents and immunological kits for detection and quantification of enzymes in foods. Summary of the Invention Methods, kits and reagents are provided to detect and measure an enzyme or enzymes in a sample. Preferably, the proteins to be detected include, Ref.:169216 but are not limited to one or more phytases, xylanases, cellulases, glucanases, amylases, glucoamylases and proteases. Proteases can be produced in various microorganisms, including but not limited to Escherichia coli, Schizosaccharamyces pombe, and Pichia pastoris or in plants, including but not limited to corn, wheat, cañola, and alfalfa, for example. In particular, the proteins are detected in food or in genetically modified plants that contain a gene that encodes the protein. Food is animal food. The animal feed can be for monogastric or ruminant. The food can be pureed food and / or granulated food. The reagents include purified protein and antibodies specific for the protein. In a particular embodiment, the protein is a phytase. The phytase protein can be isolated from inclusion bodies of E. coli and administered to animals to produce polyclonal or monoclonal antibodies. Alternatively, the protein can be isolated from a soluble cellular extract, such as a cellular extract of E. coli. The antibodies have high sensitivity and specificity for the protein and are useful in immunoassay methods for the detection of enzymatically active protein in animal or genetically modified organisms.

The methods are immunoassays that employ antibodies described herein and are capable of detecting low protein concentrations. The antibodies are purified and therefore react in a minimal way with other proteins that may be present in the sample. The antibodies and / or protein are assembled in a kit with conventional immunoassay reagents for detection of the protein. A method, kit and reagents are provided to detect and measure the protein in a sample. The proteins to be detected include one or more food enzymes, such as phytases, xylanases, cellulases, glucanases, glucoamylases and proteases. The proteins can be produced from various spices, including but not limited to JE ?, coli, S. pombe and P. pastoris. In particular, phytase proteins are detected in animal feed and in genetically modified plants that express a gene of food enzymes, such as phytase gene. The reagents may include antigenic peptides / proteins and antibodies. The antigenic peptides / proteins are immunoreactive with the antibodies. The peptides / antigenic proteins have common epitopes shared by the protein produced in different species. Peptides / proteins are isolated or synthesized and administered to animals to produce antibodies.

For phytase, the antibodies have high sensitivity and cross-reactivity for the phytase proteins produced in various species and are therefore useful in immunoassay methods for the detection of genetically modified organisms, particularly plants, which have been modified to express a gene of phytase. The methods are immunoassays employing antibodies described herein and are capable of detecting low concentrations of the phytase protein in animal feed and genetically enhanced culture samples. The antibodies are immunoreactive with common epitopes or epitopes in phytase expressed by phytase genes and react minimally with other proteins that may be present in the sample, thereby providing an accurate determination of the presence of a genetically modified organism in a sample, such as a sample of grain. The epitopes, antibodies, or both, are collectively assembled in a kit with conventional immunoassay reagents for detection of the protein. The kit can optionally contain both monoclonal and polyclonal antibodies and a standard for the determination of the presence of protein or enzyme in food in a sample. In view of the above, there is a real need for the development of technology that will allow the identification of specific proteins or enzymes in food samples. -; Other objects, features and advantages of the present invention will become apparent from the following detailed description. It should be understood, however, that the detailed description and specific examples, while indicating preferred embodiments of the invention, are given by the form of illustration only, since various changes and modifications within the spirit and scope of the invention will reach be apparent to those skilled in the art from this detailed description. Brief Description of the Figures Figure 1 is a graph which shows a standard curve for phytase activity. Figure 2 is a graph which shows the relative activity in percentage against the incubation time at 99 ° C of the enzyme phytase in both an ELISA assay and an enzyme activity, by comparison of activity data against ELISA. The detection of the enzyme phytase in ELISA is parallel to the amount of activity detected in the assay of enzymatic activity. Figure 3 is a scanned reproduction of immunotyping tests showing the detection of phytase (arrow) after incubation at 99 ° C for up to one hour.

A decrease in the detection of phytase is seen after approximately 20 minutes at 99 ° C. Figure 4 is a representation of an immunoassay test kit and the method for using same. Detailed Description of the invention Methods, kits and reagents for detecting proteins in a sample in the present invention are described. Preferably, proteins are enzymes in Foods, and most preferably enzymes in foods include, but are not limited to, phytases, xylanases, cellulases, glucanases, amylases, glucoamylases, and proteases. The methodology of the invention can be used to detect any enzyme in samples such as an animal feed. Many enzymes are known to those skilled in the art. For example, a number of phytases are known, the detection of which can be performed using the present invention. Known phytases include, but are not limited to, those described in the application WO 01/90333, entitled "Recombinant Bacterial Phytases and Uses Thereof", application WO 99/08539, entitled "Novel Phytase", Application of the United States of America. DO NOT. 10 / 334,672, entitled "Microbialy Expressed Thermotolerant Phytase for Animal Feed" and the application of the United States of North America. 10 / 334,671, entitled "Thermotolerant Phytase for Animal Feed", each of which is incorporated herein by reference in its entirety. It is important to detect when the immunoassays are made the protein in transgenic plants and the products produced from them (including food fractions), and that a test has the ability to detect the protein from several genes. In this way, cross-reactive antibodies are very important for the development of successful commercial products. The reagents are antigenic proteins or peptides that share common epitopes and antiprotein antibodies that are cross-reactive with the protein expressed from different genes. The method is an immunoassay for sensitive, protein-specific detection, specifically for the detection of protein in animal feed and in genetically modified plants, such as agricultural products. The kit contains the anti-protein antibodies described herein and other reagents, particularly those used in a strip test format, for use in the immunoassay described in more detail below. Antigenic Protein For the preparation of recombinant protein, such as phytase, after transformation of a suitable host strain and growth of the host strain to an appropriate cell density, eg, bacterial host, insect or yeast, a selected promoter can be induced by an appropriate means (e.g., temperature change or chemical induction) and cells cultured for an additional period to produce the recombinant enzyme. The cells are then harvested typically by centrifugation, altered by physical or chemical means, and the resultant extract without purifying retained for further purification. The microbial cells used in protein expression can be altered by any convenient method, including freeze-thaw cycle, sonication, mechanical alteration, or use of cell lysate agents, such methods are well known to those skilled in the art. The enzyme can be recovered and purified from recombinant cell cultures by methods that include ammonium sulfate or ethanol precipitation, acid extraction, anion or cation exchange chromatography, phosphocellulose chromatography, hydrophobic interaction chromatography, affinity chromatography, hydroxylapatite chromatography, and lectin chromatography. The reduplicated protein steps can be used, as necessary, to complement the mature protein configuration. Finally, high performance liquid chromatography (HPLC) can be used for the final purification steps. Antigenic peptides Antigenic peptides can be peptides of protein surfaces that share epitopes among the various species expressing the protein, preferably protein expressed from various microorganisms. The peptides are highly useful as diagnostic markers for the detection and quantification of the protein. Peptides are also useful for producing antibodies, tests and kits that have the superior sensitivity required of successful commercial products. In one embodiment, the peptides are either isolated from cell cultures in which the genes encoding the protein are expressed using conventional techniques known to those skilled in the art such as affinity column purification or the amino acid sequences of the Peptides are generated and the peptides synthesized according to methods known to those skilled in the art. Antigenic peptides having the characteristics indicated above are useful for the production of either monoclonal or polyclonal antibodies reactive with the phytase protein. Antibodies Antibodies useful in the invention can be made using a mammal, in particular, a rabbit, chicken, mouse or goat. The program for inoculation is not critical and can be used ordinarily for this purpose in the art. Such procedures are described, for example, in Antibodies A Laboratory Manual, Cold Spring Harbor Laboratory, 1988, pages 92-115. Preferred antibodies for the detection of phytase are rabbit antibodies, chicken antibodies, and goat antibodies that are purified by immuno affinity against the recombinant phytase produced in E. coli inclusion bodies. To detect and quantify the phytase, the antibodies are preferably labeled directly using labels that include enzymes, radioisotopes, and colorful particles such as latex beads or colloidal gold. In another embodiment, the antibodies are labeled directly, for example, by reaction with labeled substances that bind to the antibody such as secondary antibodies, protein A or protein G. Polyclonal antibodies In one embodiment, the antibodies are polyclonal antibodies. Methods for preparing polyclonal antibodies are known to the person skilled in the art. Polyclonal antibodies can be increased in an animal, for example, by one or more injections of an immunizing agent and, if desired, an adjuvant. Typically, the immunizing agent and / or adjuvant will be injected into the mammal by multiple subcutaneous or intraperitoneal injections. The immunizing agent includes the food enzyme or fusion protein thereof. For example, the agent is the phytase polypeptide or a fusion protein thereof. In another method, the immunizing agent is conjugated to a protein known to be immunogenic in the mammal that is immunized. Examples of such immunogenic proteins include, but are not limited to, limpet hemocyanin, serum albumin, bovine thyroglobulin, and soybean trypsin inhibitor. Examples of adjuvants include complete Freund's adjuvant and MPL-TDM adjuvant (monophosphoryl Lipid A, synthetic trehalose dicorinomiclate). The immunization protocol can be selected by one skilled in the art without prolonged experimentation. Preferred antibodies are highly sensitive for the detection of food enzymes such as, but not limited to, phytase proteins, for example transgenic phytase proteins at relevant concentrations in bulk samples of a commodity grain in the distribution channel. Preferably, the antibodies detect food enzymes, such as the phytase protein, at a high sensitivity of approximately 0.059 ng / ml. Antibodies with high sensitivity are useful for the detection of low concentrations of enzymes in foods, such as phytase proteins, in tissues of genetically modified crops, such as, but not limited to, leaves, stems, seeds, bark, roots and the like or products derived from such crops, such as food fractions. Monoclonal Antibodies Anti-food enzyme antibodies, such as anti-phytase antibodies are, alternatively, monoclonal antibodies. Monoclonal antibodies are prepared using hybridoma methods, such as those described by Kohier and Milstein, Nature 256: 495 (1975). In a hybridoma method, a mouse, hamster, or other appropriate host animal, is typically immunized with an immunizing agent to produce lymphocytes that produce or are capable of producing antibodies that will specifically bind to the immunizing agent. Alternatively, lymphocytes can be immunized in vitro. The immunizing agent typically includes the desired polypeptide or a fusion protein thereof. Generally, either peripheral blood lymphocytes ("PBL" for its acronym in English) are used if cells of human origin are desired, or vessel cells or lymph node cells are used if non-human mammalian sources are desired. The lymphocytes are then fused with an immortalized cell line using a suitable fusion agent, such as a polyethylene glycol, to form a hybridoma cell (Goding, Monoclonal Antibodies: Principles and Practice, Academic Press, (1986) pp. 59-103) . Immortalized cell lines are usually transformed mammalian cells, particularly rodent myeloma cells, of bovine and human origin. Usually, mouse myeloma cell lines are used. The hybridoma cells are cultured in a suitable culture medium preferably containing one or more substances that inhibit the growth or survival of unfused, immortalized cells. For example, if the parental cells lack hypoxanthine guanine phosphoribosyl transferase (HGPRT or PRET for its acronym in English), the culture medium for hybridomas typically includes hypoxanthine, aminopterin, and thymidine ("HAT medium"), whose substances prevent the growth of HGPRT deficient cells. Preferred immortalized cell lines are those that fuse efficiently, support high level stable expression of antibodies by the selected antibody producing cells, and are sensitive to a medium such as HAT medium. The most preferred immortalized cell lines are murine myeloma lines, which can be obtained, for example, from the Salk Institute Cell Distribution Center, San Diego, Calif., And the American type Culture Collection, Manassas, Va. Human myeloma cell lines and human mouse heteromyeloma have also been described for the production of human monoclonal antibodies (Kozbor, J. Immunol., 133: 3001 (1984), Brodeur et al., Monoclonal Antibody Production Techniques and Applications, Marcel Dekker, Inc., New York, 1987, pp. 51-63). The culture medium in which the hybridoma cells are cultured is then assayed for the presence of monoclonal antibodies directed against PRO. Preferably, the binding specificity of monoclonal antibodies produced by the hybridoma cells is determined by immune precipitation or by an in vitro binding assay, such as radio-immunoassay (RIA) or enzyme-linked immunosorbent assay ( ELISA). Such techniques and assays are known in the art. The binding affinity of the monoclonal antibody can, for example, be determined by the Scatchard analysis of Munson and Pollard, Anal. Biochem., 107: 220 (1980). After the hybridoma cells are identified, the clones are subcloned by limiting the dilution and growth procedures by standard methods (Goding, supra). The culture medium suitable for this purpose includes, for example, modified Eagle's medium of Dulbecco and half RPMI-1640. Alternatively, the hybridoma cells are grown in vivo as ascites in a mammal.

The monoclonal antibodies secreted by the subclones are isolated or purified from the culture medium or ascites fluid by conventional immunoglobulin purification methods such as, for example, protein A-Sepharose, hydroxylapatite chromatography, gel electrophoresis, dialysis, or affinity chromatography. Monoclonal antibodies are also made by recombinant DNA methods, such as those described in US Pat. No. NO. 4,816,567. The 7DNA encoding the monoclonal antibodies of the invention is easily isolated and sequenced using conventional methods (for example, by using oligonucleotide probes that are capable of specifically binding to genes encoding the heavy and light chains of murine antibodies). The hybridoma cells of the invention serve as a preferred source of such DNA. Once isolated, DNA is placed in expression vectors, which are then transferred into host cells such as simian COS cells, Chinese hamster ovary cells (CHO), or myeloma cells that do not otherwise produce immunoglobulin protein, to obtain the synthesis of monoclonal antibodies in recombinant host cells. The DNA is also modified, for example, by substituting the coding sequence for human heavy and light chain constant domains in place of the homologous murine sequences (US Pat. No. 4,816,567; Morrison et al., Supra), or by covalently joining the immunoglobulin coding sequence to all or a portion of the coding sequence for a non-immunoglobulin polypeptide. Such a non-immunoglobulin polypeptide is replaced by the constant domains of an antibody of the invention, or is substituted by the variable domains of an antigen combining site of an antibody of the invention to create a chimeric bivalent antibody. In another embodiment, the antibodies are monovalent antibodies. Methods for preparing the monovalent antibodies are well known in the art. For example, one method involves recombinant expression of immunoglobulin light chain and modified heavy chain. The heavy chain is truncated generally at any point in the Fc region in order to prevent cross-linking of the heavy chain. Alternatively, the relevant cysteine residues are replaced with another amino acid residue or are deleted to prevent cross-linking. In vitro methods are also suitable for preparing monovalent antibodies. The digestion of antibodies to produce the fragments thereof, particularly the Fab fragments, can be performed using routine techniques known in the art. Other methods known in the art include the method of Kearney, et al., J. Immunol. 123: 1548-1558 (1979), which is incorporated for reference herein. Briefly, animals such as mice or rabbits are inoculated with the immunogen in adjuvant, and vessel cells are harvested and mixed with a myeloma cell line. The cells are induced to fuse by the addition of polyethylene glycol. Hybridomas are chemically selected by plating the cells in a selection medium which contains hypoxanthine, aminopterin and thymidine (HAT). Hybridomas are subsequently screened for the ability to produce monoclonal anti-phytase antibody. Hybridomas that produce antibodies are cloned, expanded and stored frozen for future production. In another embodiment, the antibody is directly labeled with a detectable label for identification and quantification of an enzyme in food, in particular, a phytase protein. Labels for use in immunoassays are generally known to those skilled in the art and include, but are not limited to, enzymes, radioisotopes and fluorescent, luminescent and chromogenic substances that include colorful particles such as colloidal gold and latex beads. Alternatively, the antibodies are indirectly labeled by the reaction with labeled substances having an affinity for immunoglobulin, such as .A or .G protein or second antibodies. The antibodies are conjugated to a second substance and detected with a third labeled substance having an affinity for the second substance conjugate to the antibody. For example, the antibody is conjugated to biotin and the antibody-biotin conjugate detected using avidin or labeled estrepavidin. In another embodiment, the antibody is conjugated to a hapten and the antibody-hapten conjugate is detected using labeled anti-hapten antibody. These and other methods of labeling antibodies and assay conjugates are well known to those skilled in the art. An anti-food enzyme, in particular anti-phytase, monoclonal and polyclonal antibodies having similar or higher sensitivity for food enzyme proteins are produced by immunization of an animal with the food enzyme, in particular the phytase protein described above, isolation of the antibody that it reacts with the protein, and the collection and purification of the antibodies from biological fluid such as blood according to methods well known to those skilled in the art.

Immunoassay Antibodies are assembled collectively in a kit with conventional immunoassay reagents for detection of the enzyme of the desired food or protein using the immunoassay described below. The .kit may optionally contain both monoclonal and polyclonal antibodies and a standard for determining the presence of the enzyme in a sample. The kit containing these reagents provides simple, rapid detection at the protein site. The antibodies described above are used as the basic reagents of a number of different immunoassays to determine the presence of the food enzyme in a sample. The antibodies are used in any type of immunoassay, either qualitative or quantitative. In a rapid quantitative interleaved test, there are three basic parts. For example, in such an assay as for a food enzyme, the phytase protein in a genetically modified plant extract or food extract, such as chicken feed, is captured on the solid phase using a primary antibody. In one embodiment, the primary antibody is an anti-rabbit food enzyme antibody.

Next, a "sandwich" is formed between the primary antibody, the food enzyme protein, and the secondary antibody that has been added to the well. In one embodiment, the secondary antibody is an goat anti-food enzyme antibody. After a washing step, where the unbound secondary antibody has been removed, the bound secondary antibody is detected using a labeled antibody. In a particular embodiment, the detection antibody is an anti-carbonate antibody labeled with alkaline phosphatase. The substrate for the detection of enzyme, alkaline phosphatase, is added and the color development is measured by reading the absorbance of each well. The standard curve uses a four-parameter curve fixation to plot the concentrations against absorbance. ..:. In one embodiment of the invention, the immunoassay for the detection of enzyme in food comprises the steps of: a) preparing an extract of the sample; . . b) incubate a portion of the extract with. a primary anti-food enzyme antibody which binds to the food enzyme, the primary antibody that is bound to a solid carrier, and an anti-secondary food enzyme antibody which binds to the food enzyme to create the antibody complex- polymer-antibody, c) washing the antibody-polymer-antibody complex to remove unbound secondary antibody; d) adding a detection antibody that reacts immunologically with the secondary antibody where the detection antibody is tagged, and e) measuring the amount of labeled antibody bound or unbound to determine the concentration of the water treatment polymer in the fluid. In a particular embodiment, the food enzyme is a phytase, xylanase, cellulase, glucanase, amylase, glucoamylase, and / or a protease protein. In a more preferred embodiment, the phytase is a thermostable phytase. In another embodiment of the invention, the detectable label is an enzyme. In more preferred embodiments, the enzyme is alkaline phosphatase, peroxidase or β-galactosidase. In another embodiment, the enzyme produces an insoluble reaction product. The invention also provides a kit for detection and quantification by the immunoassay method which comprises: a) a means for extracting the enzyme from food from a sample; b) a solid support which comprises a primary anti-food enzyme antibody bound to the solid support; c) an anti-secondary food enzyme antibody; and d) a detection antibody capable of immunologically binding to the secondary antibody and wherein the detection antibody is labeled with a detection means. In a particular embodiment, the detection means is an enzyme. In more preferred modes, the detection enzyme is alkaline phosphatase, peroxidase, or β-galactosidase. In another embodiment, the enzyme produces a soluble or insoluble reaction product. In another embodiment, the kit further comprises a substrate for the enzyme. Such immunoassays are also referred to as enzyme-linked one-absorber assays (ELISA). The antibodies described above are also employed in a qualitative immunoassay for the detection of an enzyme in food, such as phytase. Such an assay is commonly referred to an immunotyre. Immunotirase is produced using membranes and filters through which a liquid sample is extracted by capillary action. The phytase in the sample reacts with the antibodies contained in the immunothirate as soon as it moves through the strip. To detect the protein .fitasa in chicken feed, the food is washed with a buffer, separated from the solid material, and added to the immunotyra. As soon as the liquid sample migrates to the opposite end of the immunotira, the phytase reacts with the specific antibodies and is captured in a line that becomes visible. The detection of the signal on the test line indicates that the phytase is in the sample. In one embodiment the invention provides an immunoassay for the detection of an enzyme in food in a sample comprising the steps of: a) preparing an extract of the sample in the presence of a primary antibody which immunologically recognizes the enzyme in food the extract in such a way that an enzyme complex is formed in food-primary antibody; b) preparing a solid phase format having a significant measurement in three dimensions to form a substantial volume with a plurality of interstitial spaces by binding to a desired secondary antibody capable of immunologically recognizing that enzyme in food and wherein the secondary antibody is conjugated to a detection means and wherein the secondary antibody also immunologically recognizes the enzyme in the food; c) combining the extract of step (a) with the format prepared from step (b) wherein the extract is extracted through the interstitial spaces of the solid phase format prepared by capturing the enzyme complex in food-primary antibody; d) detecting the food enzyme by the presence of the primaro-enzyme antibody complex of the captured food. In other embodiments, the food enzyme is a phytase, xylanase, cellulase, glucanase, amylase, glucoamylase, and / or protease protein. In a preferred embodiment, the phytase is a thermostable phytase. In other embodiments, the solid phase format is cellulose acetate, cellulose, nitrocellulose or nylon. In another embodiment, the solid phase format is comprised of multiple stacked and contiguous layers wherein each layer is capable of capturing a different food enzyme. In a preferred embodiment, the solid phase support further comprises a sample absorption pad of the solid phase format. In a more preferred embodiment, the immunoassay further comprises a strip comprising a labeled anti-food enzyme antibody. In a particular embodiment, the means of detection is colloidal gold. A highly sensitive immunoassay employing the antibodies described above is provided. The assay is useful for the detection of genetically modified organisms that have been modified to include a gene encoding a food enzyme or protein, such as a phytase gene. The immunoassay is capable of detecting low concentrations of the protein in samples, such as animal feed and in genetically enhanced culture samples. As described above, the antibodies used in the immunoassay are immunoreactive with epitopes or a common epitope in the food enzyme, in particular the phytase protein, expressed by various microorganisms and reacts minimally with other proteins that may be present in the sample. , thereby providing a more accurate determination of the presence of a genetically modified organism in a sample, such as a grain sample. The immunoassay is useful for detecting the presence or amount of the desired food enzyme protein, for example a phytase, in a variety of samples, including animal feed and agricultural samples such as plant material. The sample can be obtained from any source in which the desired protein is accessible to the antibody. For example, the sample may be any plant tissue or extract which includes root, stem, bark, leaf, or seed or products derived from such crops, such as food fractions. One or more of the antibodies described above are employed in any heterogeneous or homogeneous, sandwich or competitive immunoassay for the detection of a food enzyme, in particular protein phytase, for example. Either the antibody is labeled with a detectable label or coupled to a solid phase. Methods for coupling antibodies to solid phases are well known to those skilled in the art. According to the immunoassay method, the sample containing the food enzyme is reacted with the antibody for a sufficient amount of time under conditions to promote binding of the antibody to the phytase protein in the sample. It will be understood by those skilled in the art that the immunoassay and sample reagents can be reacted in different combinations and orders. A physical medium is used to separate reagents bound to the solid phase from unbound reagents such as particle filtration, decanting reaction solutions from coated tubes or wells, magnetic separation, capillary action, and other means known to those experts in the art. It will also be understood that a separate washing of the solid phase can be included in the method.

The concentration of food enzyme protein such as phytase in the sample is determined either by comparing the intensity of the color produced by the sample with a color card or by using a reflectometer. The resulting reaction mixture is prepared, or combination of antibody and sample, in a solution that optimizes the kinetics of food-antibody enzyme binding. An appropriate solution is an aqueous solution or buffer. The solution is preferably provided under conditions that will promote specific binding, minimize non-specific binding, solubilize the food enzyme, stabilize and preserve the reactivity of the reagent, and may contain buffers, detergents, solvents, salts, chelants, proteins, polymers, carbohydrates, sugars, and other substances known to those skilled in the art. The reaction mixture solution is reacted for a sufficient amount of time to allow the antibody to react and bind to the feed enzyme to form an antibody-food enzyme complex. The shortest amount of reaction time resulting in the link is desired to minimize the time required to complete the assay. A suitable reaction time period for an immunotyping test is less than or equal to 10 minutes or between about one minute and 10 minutes.

A reaction time of less than five minutes is preferred. More preferably, the reaction time is less than three minutes. By optimizing the reagents, the link can be substantially completed as soon as the reagents are combined. The reaction is performed at any temperature at which the reagents do not degrade or become inactive. A temperature between about 18 ° C and 30 ° C is preferred, and most preferred the reaction temperature is ambient or room temperature (about 22 ° C). A solid phase format such as an immunotyra is ideally suited for this immunoassay. The test strips are comprised of multiple porous components, membranes and filters, through which the liquid sample is extracted by capillary action. The food enzyme in the sample reacts with the test reagents contained within the test tape as it traverses the length of the strip. To detect the protein in the grain or seed, the grain is ground to a powder and the protein is extracted from the powder with a liquid that is then separated from the solid material and tested using the test. The liquid is applied to the immunotyra, and the food enzyme migrates to the distal end of the tape. As soon as it migrates towards the strip, the food enzyme reacts with reagents applied to or immobilized on the strip causing a detectable signal product. The detection of the signal indicates the presence of the food enzyme in the sample. In one embodiment the solid phase format is cellulose acetate, cellulose, nitrocellulose or nylon. In a preferred embodiment, the solid phase format is nitrocellulose. In another embodiment, the solid phase format comprises a sample absorption pad, a nitrocellulose tape and a bottom pad which comprises a labeled anti-food enzyme antibody. In yet another embodiment, the solid phase format is comprised of multiple stacked and contiguous layers wherein each layer is capable of capturing a different food enzyme. Immunoassay Kit An immunoassay kit for the detection of food enzyme protein in a sample contains one or more of the antibodies described above. The kit may additionally contain kit for obtaining the sample, a vessel for containing the reagents, a means of synchronization, a buffer for diluting the sample, and a colorimeter, reflectometer, or a standard against which a color change may be measured. The kit can include the reagents in the form of an immunotyre as described above. In a preferred embodiment, the reagents, including the antibody, are dry. The addition of the aqueous sample to the vial or strip results in the solubilization of the dry reagent, causing it to react. The reagents, immunoassay methods, and kits described above will also be understood with reference to the following non-limiting examples. The following examples show typical experimental protocols and reagents that can be used in the detection of food enzymes, in particular, phytase, in samples such as food or other plant materials. Such examples are provided by the form of illustration and not by the form of limitation. All of the numerous references cited above are incorporated herein in their entirety. EXAMPLES These methods and materials describe the general procedure for preparing the corn samples and the production of the monoclonal antibodies used in the examples described below. Materials and methods Corn sample: Corn extract is derived from either Hi II seed or A188 seed (non-GM) or genetically modified phytase seed. Five grains are pulverized using a Kleco tissue grind. The resulting corn flour is suspended in 5 ml of distilled water to solubilize the proteins. The supernatant is tested in either ELISA or immunostains. Production of polyclonal antibodies For immunization: After the initial injection, the animal (rabbit or goat) is reinforced after 28 days. Each subsequent reinforcement after this is every 21 days. The animals are bled 10 days after each reinforcement. For chickens, the first reinforcement is 7 days after the initial injection, followed by reinforcements each 28 days. Chickens are bled 10 days after each booster, and if an antibody titer is detected, eggs laid after boosters are collected. The immunizing agent is the purified total phytase protein of an E. coli expression system. With the first injection in the animal, the protein is emulsified in complete Freund's adjuvant. The reinforcements are in incomplete Freund's adjuvant. The animals used here to produce the polyclonal antibodies are rabbit, chicken and goat. Purification of phytase (Nov9X) Phytase (Nov9X) formulated with 10% sorbitol, 10% NaCl and pH 4.2 is dialysed overnight against 25 mM Tris-HCl, pH 8.0 at 4 ° C using a SankeSkin 10K dialysis tube MWCO (Pierce, Rockford, IL). The following dialysis solid (NH4) 2S04 is added to the phytase mixture, initially at 25% saturation, then at 50 and finally 75% saturation at 0 ° C. Upon addition of (NH4) 2S04 for 25% saturation, the mixture is stirred for 30 minutes at 0 ° C, then centrifuged at 20,000 rpm for 20 minutes. To the decanted supernatant, (NH4) 2S04 is added at 50% saturation while the pellet is resuspended in 25 mM Tris-HCl, pH 9.0. This procedure is performed 3 times which produces Nov9X (NH4) 2S04 granules of saturation 0-25%, 25-50% and 50-75%. The SDS-PAGE analysis demonstrates the presence of Nov9X in the 50-75% fraction. This fraction is dialyzed against 25 mM Tris-HCl, pH 9.0 and prepared for purification of column chromatography. -. . Nov9X Unpurified TAM from 50-75% (NH4) 2S04 fractionation is loaded onto a HiTrapQ anion exchange column (Amersham Biosciences, Piscataway, NJ) using a flow rate of 5.0 ml / min. A linear gradient of 0-0.4 M NaCl in 25 mM Tris-HCl, pH 9.0 developed over 30 minutes is used to elute Nov9X. The absorbance measurements at 280 nm are used to follow the progress of the chromatography run. After analysis of SDS-PAGE, the purest fractions containing Nov9X are grouped with a Centricon Plus-20 centrifuge concentrator (Millipore, Bedford, MA), and loaded onto a SlOO 26/60 size exclusion column Sephacryl (Amersham Biosciences , Piscataway, NJ) run at 1 ml / min. The eluant buffer is 25 mM Tris-HCl, pH 9.0. Fractions containing pure Nov9X are pooled, concentrated and dialysed against 25 mM Tris-HCl, pH 8.0, and used for the studies described below. EXAMPLE 1: Phytase ELISA This example describes the detection and quantitative measurement of the phytase enzyme in a corn sample using the ELISA immunological technique. Procedure: Multipoint plates (Nunc, Maxisorp) are coated at 4 ° C overnight with the rabbit anti-phytase antibody at a concentration of 2 μg / ml, diluted in borate-buffered saline pH 8.5 (sodium borate / 50 mM boric acid, 75 mM NaCl). The plates are washed five times with a Tris base buffer pH 8.0 (10 mM Tris containing 0.05% Tween-20 and 0.03% sodium azide) (wash buffer). Note: the same washing step is performed after each incubation period to remove the unbound / sample antibodies. Plates are then blocked for 45 minutes at room temperature with PBS / Tween-20 / BSA buffer pH 7.4 (1% bovine serum albumin, 0.05% Tween-20, 0.03% sodium azide, 150 mM NaCl in phosphate 100 mM sodium, pH 7.4 (diluent) Fifty microliters of each sample is added to the plate and incubated for 1.5 hours at room temperature, then the goat anti-phytase antibody (diluted to 2 μg / ml in diluent) is added to plates and incubated for 1 hour at 37 ° C. The detection antibody (alkaline phosphatase-anticuepro anticabra de chango labeled with alkaline phosphatase is diluted to 1 μg / ml in diluent) is added to the plates and incubated for 1 hour at 37 ° C. The substrate is added paranitrophenyl phosphate (pNPP) and allowed to develop for 30 minutes at room temperature The absorbance is measured at 405 nm with 492 nm as reference Test characteristics The standard curve of phytase is a curve fixation of 4 pa raters (see Figure 1). The curve is plotted linearly against log with a range of 0.04 to 16 ng / ml. - To plot the standard curve of 4 parameters on a log X axis, the standard 0 ng / ml should be introduced in the analysis program at 0.01 ng / ml instead of 0 ng / ml. The analysis program used is WinSelect ™ software for the Tecan Sunrise ™ microplate reader, although any four parameter curve setting program will work. The minimum detectable dose (MDD) is the lowest level of the phytase protein that is statistically distinguished from the zero standard. The minimum detectable dose is determined by analysis of 24 replicates of corn seed extract of negative control in 1 mg / ml of total protein. Two standard deviations of the O.D. standard mean zero (95% confidence limits) are added to the average, and the dose of this value O.D. Total is determined using a standard curve. The minimum detectable dose is 0.044 ng / ml. The accuracy between the runs is determined by testing 4 different control samples in 21 different tests. The samples are purified phytase taken in ELISA diluent. The results are indicated in Table 1. The accuracy is good, less than 15%, for the concentrations of samples that are measured in the ar portion of the standard curve. Table 1. Accuracy test between runs The precision within the run is determined by testing 20-24 replicates of the following samples. Samples are phytase taken in ELISA diluent. • The results are indicated in Table 2 below. All samples result in very good accuracy, indication of good reproducibility within a test run. Table 2. Accuracy test within the run Four extracts of corn seeds are diluted with ELISA thinner in order to test the linearity of the assay. The corn extract is derived from either the Hi II seed or the A188 (non-GM) seed or genetically modified phytase seed. Five grains are pulverized using a Kleco tissue mill. The resulting corn flour is suspended in 5 ml of distilled water to solubilize the proteins. The supernatant is tested in either ELISA or with the strips. The percent recovery of the phytase from the diluted samples is acceptable.

Table 3 Linearity of the test test EXAMPLE 2: Phytase immunotoxins This example describes the use of immunotyping assays to test the presence of phytase in a sample. Procedure Pureed chicken feed extracts are prepared by adding feed to a 50 ml centrifuge tube to the 15 ml designation. The amount of the feed is added to one side of the mesh insert inside the extraction bag. Add the extraction buffer (25 ml borate 0.1 M, pH 7.5 which contains 0.5% Tween-20) and gently press the buffer on the food to ensure all the food is moist. The extract is incubated at room temperature for at least 10 minutes before applying 3-5 drops to the test immunocyte. Immunotira Briefly, the lateral flow immunotyping comprises a nitrocellulose detection membrane (2.5 x 18 cm), supported on a plastic reinforcement (plastic cassettes brand Arista ™, Bethlehem, PA), in which a line of 1 mm is sprayed of rabbit-specific antifungal polyclonal antibody line (chicken antibodies can also be used). A reagent control line of monkey anti-carver antibody is sprayed in parallel above the first line of the antibody. The final bottom portion of the nitrocellulose ribbon is overlaminated with a treated polyester web part. The polyester strip is first treated with a solution B (0.5% BSA, 0.5% polyvinylalcohol and 0.1% Triton X-100, 50 mM phosphate buffer pH 7.4) and the goat anti-phytase antibody conjugated to colloidal gold. The polyester tape is allowed to dry. The polyester tape is then overlaminated with a sample application pad. The sample application pad is also pretreated with a solution C (Triton X-10 0.1 and borate buffer 0.1 M pH 8.5) and allowed to dry. Flanking the other end or top end of the nitrocellulose strip is another cotton pad to absorb the solution from the sample after it passes over the test antibody and areas of control antibody in the nitrocellulose. This complete card is then cut into 4 mm test strips to be attached to a plastic cassette with an oval sample application well placed on top of the sample pad and a rectangular detection window positioned above the detection area of the nitrocellulose membrane. The test is performed by adding 150 μl (3-5 drops) of extract to the sample well. After waiting approximately 5-10 minutes, the results appear in the result window. If the phytase is present in the sample, a double red line appears in the result window. The lower line indicates the presence of phytase while the upper line is the control line which demonstrates an appropriate working device. If the phytase is absent, a single red control line appears in the result window. See Figures 3 for the sample immunotopes. Figure 3 shows the detection of the presence of phytase. The detection of phytase decreases after 20 minutes as indicated by the arrow, because that's when the phytase is starting to lose activity. Detailed preparation of the immunotirase 2nd generation strands of the phytase - membrane coating Materials 1. Cards, 2.25 inches (5.75 cm) x 180 mm with AE100 membrane 2. chicken antipysate IAP at 1.0 mg / ml in PBS 3. Anticabra antibody of Chango Jackson at 0.15 mg / ml in PBS 4. Pierce's RBS detergent. Procedure: Coat the test line: 1. Set the volume of the Camag ™ sprinkler to 18 (1 μl / cm) by pressing the volume, 18, press, enter. 2. Set the track by pressing the track, 1, press, enter. 3. Place the card on the platform. The portion of the card with the two pieces of paper is placed near the front of the instrument. Secure the card with the magnets. 4. Fill the syringe with 1.0 mg / ml IAP anti-phytase chicken. 5. Fix the spray head 30 mm. 6. Activate the gas supply and start the spraying by pressing the gas, calculation, run. See the spray pattern closely for consistency and accuracy. 7. Repeat stages 3-6 for each additional card. 8. Remove the syringe and wash 5 times with RBS Pierce detergent (concentrate 20 μl / ml dH20), then wash 10 times with dH20. Cover the control line: 9. Set the Camag volume to 18 (1 μl / cm) by pressing vol, 18, press, enter. 10. Set the track by pressing the track, 1, press, enter. 11. Fill the syringe with 0.15 mg / ml of monkey anticabra. 12. Fix the spray head 36 mm. 13. Activate the gas supply and start spraying by pressure gas, calculation, run. Wash the spray pattern closely for consistency and accuracy. 14. Repeat steps 11-13 for each additional card.

. Remove the syringe and wash 5 times with Pierce's RBS detergent (20 μl concentrate / dH20), then wash 10 times with dH20. 16. Dry the cards at 33 ° C overnight, then transfer to room temperature. 17. It is stored dried at room temperature. Phytase strips - coating the conjugate in the polyester Materials 1. Anti-N0V9X goat conjugated to gold, OD = 50 2. Polyester sheets, grade 2033, treated with a solution B 3. Sucrose 4. Trehalose 5. RBS detergent Pierce Procedure 18. Dilute the gold conjugate to OD = 50 using gold diluent. 19. Add 20% sucrose and 5% trehalose - to the gold conjugate to stabilize (0.2 g of sucrose and 50 mg of trehalose per 1 ml of gold conjugate) -. Mix until dissolved completely. 20. Set the volume of Carnag ™ to 27 (1.5 μl / cm) by pressing volume 27, press, enter. 21. Set the track by pressing track 1, press, enter. 22. Place the polyester sheet on the platform and secure with the magnets. 23. Fill the syringe with goat anti-N0V9X conjugated to gold (OD = 50). 24. Fix the spray head 15 mm. 25. Activate the gas supply and start the spraying by pressing gas, cale, corrida. See the spray pattern closely for consistency and accuracy. 26. Move the spray head 9 mm (the fixing will be 24 mm). 27. Activate the gas supply and start the spray by pressing the gas, cale, corrida. 28. Continue the sprinkling of the conjugate, move 9 mm for each run, until the total polyester sheet is filled. Eight lines of conjugate will be filled in one sheet. 29. Dry the leaf at 37 ° C for 1 hour. 30. Cut into strips of U "(0.56 cm) so that the line of the gold conjugate runs along the top of each tape 31. Store dried at room temperature Cleaning the instrument Remove the syringe and wash 5 times with RBS Pierce detergent (20 μl of concentrate / ml of dH20), then washed 10 times with dH20 Cleaning the instrument platform with dH or phytase strips - assembly Materials 6. Cards coated with IAP anti-phytase chicken antibody at 1.0 mg / ml and 1 μg / cm 7. 5/8"(1.58 cm) x 180 mm tapes # 40 Absorbent paper (upper pad) 8.%" strips (1.9 cm) x 180 mm # 903 paper treated with solution C, pH 8.6 (lower pad) 9. Vi tapes (0.63 cm) x 180 mm gold-sprayed conjugate (goat anti-NOV9X, PD = 50 in 1.5 μg / cm). 10. gloves. Procedure: Note: Assemble the strips under conditions of less than 40% humidity. Carry the gloves to apply all the components .. 1. Remove the two linings from the strips of gluing on the bottom of the card. 2. Place the gold strip with the gold conjugate line along the top and overlap the membrane by 1-1.5 mm. 3. Place the bottom cushion along the bottom edge of the card, being careful to leave the gold strip exposed. 4. Remove the lining from the gluing strips along the top of the card. Place the pad on the top along the top of the card that overlaps the membrane by 1-1.5 mm. 5. Store finished cards dried at room temperature until easily cut into strips. 6. Cut the strips in 4 mm lengths. One card will produce approximately 40 strips. Store the dried strips at room temperature. EXAMPLE 3: Detection of Enzymatically Active Phytase Procedure: The purified phytase produced by Pichia is inactivated by heating at 99 ° C for up to 60 minutes.

The phytase is then tested for enzymatic activity and compared for reactivity in phytase ELISA (Figure 2) and reactivity with phytase immunothiras (Figure 3). ELISA comparison: Figure 2 shows a graph of the residual activity of Nov9X after incubation at 99 ° C 04-28-03, FPLC purified TAM lot # PHY-PP9XR-PB200L comparison of activity against ELISA data. This shows that the ELISA assay and the inmuntiras seem to detect the active phytase only. The phytase inactivated by heating is not detected in any test. EXAMPLE 4: Phytase immunoassay kit This diagnostic test (see Figure 4) is designed for the rapid detection (10 min) of phytase in food. The kit contains all the reagents and kit necessary to perform the test. The kit can be stored at ambient temperatures that do not exceed 100 ° F (38 ° C). The tests are packaged in a moisture proof aluminum bag sealed with a silica gel desiccant capable of absorbing some moisture. Keep the test in its packaging until before use. Avoid placing the test in a damp place. Test Procedure 1. Fill the large tube with feed to the 15 mark. Add this amount of feed to one side of the mesh insert inside the extraction bag. 2. Remove a plastic extraction buffer cup (25 ml) from the kit and place in the extraction bag. 3. Close the bag and gently move the buffer over the food to ensure that all the food is moist. Wait for at least 10 minutes. 4. Remove a field test from the aluminum bag and place on a flat dry surface. Check the desiccant. It must be blue. If it's pink, the tests are not valid and must be downloaded. 5. Using the transfer pipette, transfer 3-5 drops of the food extract to fill the sample well of the field test. 6. Wait approximately 5 minutes for the results that appear in the window above the sample well. Results If the phytase is present in the sample, a double red line appears in the result window of the field test. The lower line indicates the presence of phytase, while the upper line is the control line that indicates an appropriate working device. The test line should not be as strong as the control line. Any reaction on the test line is considered positive. If the phytase is not present, only a simple red control line appears in the result window. EXAMPLE 5 Detection of phytase in granulated food This example demonstrates the use of immunotyping assays to detect phytase in granulated animal feed. The methods and reagents are described as above in Example 4, with the exception that the granulated animal feed is crushed to a grainy or powdery consistency with any mechanized mechanism, and that the extraction buffer is 5% methanol, with Tween-20 0.5% in water instead of the borate absorber. Also, the anti-phytase antibody is chicken instead of rabbit. The results are indicated below in Table 4. Table 4 shows that Quantum® phytase is detectable in both pureed diets (before granulation) and granulated diets both in ELISA and immunotyping assays. Table 5 shows that the Quantum phytase is detectable in the starting diets (before granulating) and the shredded diets (granulated diets) with both the immunotyra and ELISA. The activity is also confirmed with the enzyme assay.

Table 4 Detection of phytase in granulated food Table 5 Phytase activity and quantification of phytase ELISA in starting and shredded diets Modifications of the reagents, methods and kits present to detect enzyme proteins in foods, in particular phytase, will be obvious to those skilled in the art from of the description mentioned above. While the present invention has been described with reference to specific embodiments thereof, it will be appreciated that numerous variations, modifications and additional modalities are possible, and consequently, all such variations, modifications and modalities are to be seen as being within the scope of the invention. scope of the present invention. Numerous patents, applications and references are discussed and cited within this specification, and all are incorporated for reference in their entirety. It is noted that in relation to this date, the best method known to the applicant to carry out the aforementioned invention, is that which is clear from the present description of the invention.

Claims (25)

1. CLAIMS Having described the invention as above, the content of the following claims is claimed as property: 1. An immunoassay for the detection of an enzyme in food in a sample characterized in that it comprises the steps of: a) preparing an extract of the sample in the presence of a primary antibody which immunologically recognizes the enzyme in food in the extract in such a way that a complex of primary antibody-food enzyme is formed; b) preparing a solid phase format having a significant measurement in three dimensions to form a substantial volume with a plurality of interstitial spaces by binding to a desirable secondary antibody capable of immunologically recognizing the food enzyme and wherein the secondary antibody is conjugated to a detection means and wherein the secondary antibody also immunologically recognizes the food enzyme; c) combining the extract from step (a) with the format prepared from step (b) wherein the extract is extracted through the interstitial spaces of the solid phase format that captures the primary antibody-food enzyme complex; d) detecting the food enzyme by the presence of the primary antibody-enzyme complex of captured food.
2. 2. The immunoassay according to claim 1, characterized in that the food enzyme is a phytase, xylanase, cellulase, glucanase, amylase, glucoamylase, and / or protease protein.
3. 3. The immunoassay according to claim 2, characterized in that the phytase is a thermostable phytase.
4. 4. The immunoassay according to claim 1, characterized in that the solid phase format is cellulose acetate, cellulose, nitrocellulose or nylon.
5. 5. The immunoassay according to claim 4, characterized in that the solid phase format is composed of multiple stacked and contiguous layers wherein each layer is capable of capturing a different food enzyme.
6. 6. The immunoassay according to claim 4, characterized in that it also comprises a sample absorption pad of the solid phase format.
7. 7. The immunoassay according to claim 6, characterized in that it also comprises a strip which comprises a labeled anti-food enzyme antibody.
8. 8. The immunoassay according to claim 1, characterized in that the detection means is colloidal gold.
9. 9. A kit for detection by the immunoassay according to claim 1, characterized in that it comprises: a) means for extracting the food enzyme from a sample; and b) a solid phase format which comprises a primary anti-food enzyme antibody and which has a significant measurement in three dimensions to form a substantial volume with a plurality of interstitial spaces by binding to a desired secondary antibody capable of immunologically recognizing the enzyme of food and wherein the secondary antibody is conjugated to a means for detection and wherein the secondary antibody also immunologically recognizes the food enzyme.
10. 10. The kit according to claim 9, characterized in that it also comprises a container that contains a shock absorber.
11. 11. The kit according to claim 10, characterized in that it also comprises a medium for dispersing the sample in the solid phase format.
12. 12. An immunoassay for the detection and quantification of a food enzyme characterized in that it comprises the steps of: a) preparing an extract of the sample; b) incubating a portion of the extract with a primary anti-food enzyme antibody which binds to the food enzyme, the primary antibody that binds to a solid carrier, and a secondary anti-food enzyme antibody which binds to an enzyme of food to create an antibody-polymer-antibody complex, c) wash the antibody-polymer-antibody complex to remove unbound secondary antibody; d) adding a detection antibody that reacts immunologically with the secondary antibody wherein the detection antibody is labeled; and e) measuring the amount of labeled antibody bound or unbound to determine the concentration of the water treatment polymer in the fluid. The immunoassay according to claim 12, characterized in that the food enzyme is a phytase, xylanase, cellulase, glucanase, amylase, glucoamylase, and / or protease protein. The immunoassay according to claim 12, characterized in that the phytase is thermostable phytase. 15. The immunoassay according to claim 13, characterized in that the detectable label is an enzyme. 16. The immunoassay according to claim 15, characterized in that the enzyme is alkaline phosphatase, peroxidase or β-galactosidase. 17. The immunoassay according to claim 16, characterized in that the enzyme produces an insoluble reaction product. 18. A kit for detection and quantification for the immunoassay according to claim 12, characterized in that it comprises: a) a means for extracting the enzyme from food from a sample; b) a solid support which comprises a primary anti-food enzyme antibody bound to a solid support; c) a secondary anti-food enzyme antibody; and d) a detection antibody capable of immunologically binding to the secondary antibody and wherein the detection antibody is labeled with a detection means. 19. The kit according to claim 18, characterized in that the detection means is an enzyme. 20. The kit according to claim 19, characterized in that the detection enzyme is alkaline phosphatase, peroxidase, or β-galactosidase. 21. The kit in accordance with the claim 20, characterized in that the enzyme produces a soluble or insoluble reaction product. 22. The kit in accordance with the claim 21, characterized in that it also comprises a substrate for the enzyme. 23. An antibody characterized in that it immunologically recognizes phytase. 24. The antibody according to claim 23, characterized in that the antibody is a polyclonal antibody. 25. The antibody according to claim 23, characterized in that the antibody is a monoclonal antibody.