GR20210100441A - An innovative modified competitive -type immunoassay system for the quantitative detection of aflatoxin m1 in milk composition samples - Google Patents

Abstract

The invention relates to an innovative modified immunoassay of the competitive type, using polyclonal, monoclonal or recombinant antibodies, for the quantitative determination of Aflatoxin M1 in milk or liquid samples of milk composition. The invention provides procedures for the highest recovery quantitation of Aflatoxin M1 which utilize graded addition of Aflatoxin M1 antibody configuration and orienting factors during the binding process of the antibody to the solid support and standard solutions of powdered skim instant-dissolving milk containing non-zero fat acids. The immunochemical construction includes the indirect process of connecting the antibodies to the solid substrate, through a covalent or strong non-covalent bond with molecules of protein nature directly immobilized on the solid substrate and the graded addition of nonionic surfactants, polymers of low molecular weight and low degree of hydrolysis and disaccharides. This innovative modified immunoassay system quantifies Aflatoxin M1 with high recovery, in a range of 0-250 ng/L.

Description

AN INNOVATIVE MODIFIED COMPETITIVE-TYPE IMMUNOMETHOD SYSTEM FOR THE QUANTITATIVE DETECTION OF AFLATOXIN M1 IN MILK OR MILK COMPOSITION SAMPLES

DESCRIPTION

TECHNICAL FIELD

The invention relates to competitive immunoassay techniques, which use polyclonal, monoclonal or recombinant antibodies against Aflatoxin M1, for the quantitative determination of Aflatoxin M1 in milk or liquid samples of milk composition. The invention provides procedures for the highest recovery quantitation of Aflatoxin M1 in systems which employ the graded addition of conformational and orienting agents of the antibody to Aflatoxin M1 during the binding process of this antibody to a solid support and standard solutions of instant non-fatty skimmed milk powder. zero percentage of fatty acids. More specifically, the immunochemical construction involves the indirect process of linking the antibodies against Aflatoxin M1 to the solid substrate, through a covalent or strong non-covalent bond with different molecules of a protein nature directly immobilized on the solid substrate. The graded addition of conformational and orienting factors of the Aflatoxin M1 antibody in the process of its indirect binding to the solid support includes nonionic surfactants, low molecular weight and low degree of hydrolysis polymers, and disaccharides.

STATE OF THE PRIOR ART AND EVALUATION THEREOF

Aflatoxin M1 is the hydroxylated form of Aflatoxin B1 and is detected in milk, as well as milk products, obtained from animals that have consumed feed contaminated with Aflatoxin B1. The formation of Aflatoxin M1 from Aflatoxin B1 takes place in the liver of animals and then enters the bloodstream. Finally, through the blood circulation, it reaches the breast and is secreted in the milk. It is a fact that Aflatoxin M1 is detected in milk 12 - 24 hours after the intake of Aflatoxin B1 by the animal and after three days the highest levels of concentration (of Aflatoxin M1) are observed. When Aflatoxin B1-contaminated feed is stopped, Aflatoxin M1 levels in milk decrease but are detectable for at least 6 days.

Aflatoxin M1 is considered less carcinogenic than Aflatoxin B1. Nevertheless, it is considered particularly important for health because it exhibits similar hepatotoxicity and cytotoxicity and contributing significantly to the suppression of the immune system of infants. Exposure of children, including infants, to Aflatoxin M1 is of particular concern as they are considered more susceptible to its adverse effects. As a result, the International Agency for Research on Cancer has now classified Aflatoxin M1 in Group 1 (Category: Carcinogenic to humans). In Europe, the maximum concentration limit of Aflatoxin M1 in milk and milk products is 0.05 µg/kg or 50 ng/kg (or 50 ng/L). In infant formula and second infant formula, including infant milk and second infant formula as well as dietary food for special medical purposes intended specifically for infants, the maximum concentration limit of Aflatoxin M1 is 0.025 μg/kg or 25 ng/kg (or 25 ng/L). To date, the continued interest in the production of quality and safe food, which can ensure public health, has led to the development of many methods for the analysis of Aflatoxin M1. More specifically, for the detection of Aflatoxin M1 the methods that find frequent application are thin layer chromatography (TLC), liquid chromatography (LC), high performance liquid chromatography (HPLC) and immunomethods (for example the enzyme-linked immunosorbent method - ELISA). Immunomethods, among the applied methods, are widely used, low cost and show great ease of application. As an immunomethod, it requires small sample volumes and fewer sample preparation procedures compared to conventional methods (e.g. HPLC). The fact that an immunomethod (for example ELISA) does not require significant experimental procedure time and at the same time does not require specialized personnel, is the main reason that it can be used in laboratory milk control analyzes in the analysis of a large number of samples.

It is a method that, under specific conditions, can become very sensitive and highly reliable quantitatively. Although the sensitivity and specificity of immunoassays are mainly based on the nature of the antibodies used (which are mainly monoclonal or recombinant antibodies), the selection of the appropriate type of immunoassay and the way the antibodies are used are the factors that can make a method beyond the expected sensitivity and specialization. The accuracy of the method depends on the percentage of recovery of Aflatoxin M1 in the context of quantification. Even when the strategic design of the immunomethod and the type of antibody against Aflatoxin M1 give the immunochemical construction the advantage of high specificity and sensitivity, because the analyte is Aflatoxin M1 (mycotoxin), there is the matrix effect. The milk environment creates different physicochemical conditions for the antigen-antibody reaction, and at the same time various other compounds with a similar chemical structure can react with the antibodies. The matrix effect takes place in most immunomethods, thus contributing to overestimations or underestimations of Aflatoxin M1 concentrations in the sample.

Due to the fact that Aflatoxin M1 is a very small molecule, it has limited binding space and it is impossible for two antibodies to bind to its structure at the same time. Thus, the immunomethods for the detection and quantification of Aflatoxin M1 (like all hapten-mycotoxins), are of competitive type. That is, they use a single antibody against Aflatoxin M1 which is ideally monoclonal (ie produced in mice or rats), although recombinant or even polyclonal antibodies can be used. More specifically, for the detection and quantification of Aflatoxin M1, many variants of the competitive immunomethod type have been used, with the most common ones having:

(a) immobilized antigen (protein-conjugated Aflatoxin M1) on the solid support (for example polystyrene microplate in the case of ELISA), a soluble anti-Aflatoxin M1 antibody which binds the sample toxin or the immobilized toxin on the microplate (competition) and a second antibody (anti-Aflatoxin M1 antibody) conjugated to HRP peroxidase enzyme and (b) immobilized the anti-Aflatoxin M1 antibody on the solid support (for example polystyrene microplate in the case of ELISA), which binds its toxin sample or the toxin in conjugated form with the enzyme HRP peroxidase (competition).

Immunochemically, the second variant of competitive-type immunomethod is much simpler and usually gives better sensitivity, specificity and recovery results. For this reason, the immunoassays of commercially available kits usually use the second variant of the competitive immunoassay and monoclonal antibodies against Aflatoxin M1. The fixing of the monoclonal antibody to the solid substrate can be done directly and in more specific cases indirectly, through its binding by some other molecule of a protein nature. It is worth noting that most monoclonal antibodies against Aflatoxin M1 have been produced after immunization of mice or rats with BSA-Aflatoxin M1 immunogen. The fact that the carrier protein of the antigen is Bovine Serum Albumin (BSA), means that the antibody against Aflatoxin M1, in addition to the affinity it has for Aflatoxin M1, usually also has a low affinity for the BSA protein .

In the context of the process of direct or indirect immobilization of antibodies against Aflatoxin M1 on the solid substrate, an important role is played by the addition of the protein BSA (vacuity blocking step, for example when the immunomethod is ELISA), the presence of which contributes to the result of a final method which does not have the desired characteristics. This is due to the cross-reactions of antibodies against Aflatoxin M1 with the BSA protein, which as an unwanted antigenic determinant reduces the activity of the antibodies (by binding to the toxin). Also, that although the BSA protein is purified from other protein molecules (by the supplier), it is practically impossible for the purification fraction to be free of traces of bovine IgG, which can act as a source of cross-reactions with the Aflatoxin antibodies in the method. These non-specific interactions of the antibodies with the bovine BSA protein create background noise in the immunoassay (development of an unwanted signal) and low competition efficiency. Consequently, to balance the final signal and maintain the desired convexity of the standard curve, technical modifications are usually made to the immunomethod resulting in undesirable levels of sensitivity, specificity and, above all, recovery.

Also, the immunomethods of commercially available kits use standard solutions of milk composition which simulate the actual samples to be analyzed. This is because it is important to maintain the environment of the milk in the method so that the environment (matrix) of the standards does not present different physicochemical conditions and affect the antigen-antibody reaction. The composition of the standard solutions should be such as to enable the generation of a correct standard curve with a working quantification range and contribute to the result of a final method having the desired recovery levels. Commercially available kit immunoassays use skim milk standard solutions and this is because the presence of fat greatly affects the antigen-antibody reaction. For this reason, the commercially available immunomethods have as a basic requirement the defatting of the milk samples. In addition to the fact that no method is able to quantify the concentration of Aflatoxin M1 with satisfactory recovery when non-skimmed milk samples are used, these methods are unable to quantify the concentration of Aflatoxin M1 with satisfactory recovery when different types of skimmed milk are used of milk samples (raw milk, homogenized milk, milk powder).

Based on the data so far of the immunomethods that quantitatively determine the concentration of Aflatoxin M1, it appears that the combination of the procedure of immobilizing antibodies against Aflatoxin M1 on the solid support and the selection of standard solutions, in an interdependent manner, leads to a technical dead end the effort to create an immunomethod with the desired levels of recovery.

This report describes the development of a competitive immunoassay for the quantification of Aflatoxin M1 which can be highly sensitive and has a high degree of recovery, making it more suitable for the quantification of Aflatoxin M1 in milk.

SUMMARY OF THE INVENTION

In the present invention it was found that with the use of graded addition of factors for shaping and orienting the antibodies during the process of binding them to the solid substrate and using standard solutions of skim milk powder of instant dissolution of non-zero percentage of fatty acids, in immunomethod techniques of the competitive type, as for example the ELISA, the concentration of Aflatoxin M1 in milk can be determined with a high recovery rate in a range of 0-250 ng/L. (Figure 16).

In contrast to other immunochemical systems which use antibodies against Aflatoxin M1 and show non-zero non-specific interactions (a) with the BSA protein (cross-reactions, because they have been produced after immunization of mice or rats with the BSA-Aflatoxin M1 immunogen) and / or (b) with the bovine immunoglobulins (which are present in the BSA protein fraction from the manufacturer due to the undesirable purity and which BSA protein in turn is mainly used together with the antibodies in the direct or indirect immobilization process on the solid substrate -vacuity blocking stage), the use of a graded addition of conformation and orientation factors of the antibody against Aflatoxin M1 in the process of its indirect binding to the solid substrate (through covalent or strong non-covalent binding with different protein molecules directly immobilized on the solid substrate), contributes to the apot result of a competitive type final method with improved recovery levels. More specifically, graded addition of non-ionic surfactants (for example polysorbate 20), low molecular weight and low degree of hydrolysis polymers (for example polyvinyl alcohol (MW < 10000)) and disaccharides (for example mycose) contribute to improved recovery levels (Figure 1).

Antibodies against Aflatoxin M1 were found to have zero non-specific interactions with the nonionic surfactants, polymers and disaccharides used in the solid support antibody immobilization system. Also, these factors were found to contribute to the optimal conformation and orientation of the anti-Aflatoxin M1 antibody in the immunochemical construct imparting the desired activity.

In addition, unlike other immunochemical systems that use skim milk standard solutions and mimic real milk samples, the use of flash non-zero fat skim milk powder standard solutions makes it possible to generate a correct standard curve with a working range quantification and contributes to the result of a final method which has optimal levels of recovery. It was found that the standard solutions of skimmed milk powder instant dissolution of non-zero percentage of fatty acids 0.05 - 0.1%), maintain the desired environment (matrix) and in combination with an indirect system of immobilization of the antibodies, gives the optimal physicochemical conditions for the antigen-antibody reaction .

Accordingly, the invention provides in one aspect a competitive immunoassay for the detection of Aflatoxin M1 in samples of milk or milk composition, comprising:

(a) the indirect binding of shaped and oriented antibodies against Aflatoxin M1 to the solid substrate, through covalent or strong non-covalent bonding with different protein molecules which are directly immobilized on the solid substrate, where (i) in the immobilization step of the protein molecules, the remaining vacancies of the functional solid substrate are covered by nonionic surfactants and (ii) after the step of creating the complex of antibodies against Aflatoxin M1 with the immobilized protein molecules, a mixture of low molecular weight and low degree of hydrolysis polymers and disaccharides is added

(b) the binding of anti-Aflatoxin M1 antibodies to (i) the Aflatoxin M1 of the sample to be studied and (ii) the labeled form of the toxin or its conjugates with other molecules

(c) detecting whether the binding of anti-Aflatoxin M1 antibodies to the Aflatoxin M1 molecules to be studied in the sample is increased by the presence of the sample; and

(d) the quantification of the Aflatoxin M1 to be detected using non-zero fat flash skimmed milk powder standard solutions.

In the immunomethod of this invention, the rate of signal reduction is relative to the reaction solution in which the added Aflatoxin M1 has reacted with the anti-Aflatoxin M1 antibodies. Aflatoxin M1 concentration levels in milk can be determined using a calibration curve previously made from the rate of reduction of reactive standard solutions in which known concentrations of Aflatoxin M1 have occurred.

In conclusion, the invention provides a competitive immunoassay system that utilizes the graded addition of antibody conformers and orientators during their attachment to the solid support and standard solutions of non-zero fat instant skimmed milk powder, capable of quantifying the Aflatoxin M1 in high recovery milk samples.

ADVANTAGES OF THE INVENTION AND REMEDIES

The use of the method described, i.e., a competitive immunoassay system that uses graded addition of antibody conformers and orientators during their attachment to the solid support and standard solutions of non-zero fat instant skimmed milk powder, capable of determining quantitatively Aflatoxin M1 in milk samples, overcomes many disadvantages of other commercially available methods used.

It is known that chromatographic methods of analysis, such as thin layer chromatography (TLC), liquid chromatography (LC) and high performance liquid chromatography (HPLC), require significant experimental procedure time, specialized personnel, complex equipment. In addition, in these methods the cost per sample to be analyzed is very high, as a result of which they are not easily applicable in the context of daily milk analysis.

Focusing on immunoassays, commercially available solutions (kits) use antibodies, usually monoclonal (ie, produced in mice or rats) or recombinant, against Aflatoxin M1. These immunoassays are of the competitive type and use a single antibody against Aflatoxin M1. Based on the principles of the method of the most efficient immunomethods of the competitive type, this antibody is preferably indirectly immobilized on the solid support (for example a polystyrene microplate in the case of ELISA), which binds the sample toxin or the toxin in conjugated form with the enzyme HRP peroxidase (competition). However, most antibodies have been produced after immunization of rodents with the BSA-Aflatoxin M1 immunogen and the fact that the antigen carrier protein is the BSA protein means that there may be a low affinity of the antibody for this protein as well. (BSA). In the context of the process of indirect immobilization of antibodies against Aflatoxin M1 on the solid substrate, an important role is played by the addition of the BSA protein (vacancy blocking step, for example when the immunomethod is ELISA), the presence of which contributes to the result of a final method which does not have the desired characteristics. This happens:

(a) due to the cross-reactions of antibodies against Aflatoxin M1 with the protein BSA, which as an unwanted antigenic determinant reduces the activity of the antibodies (through its binding to the toxin) and

(b) due to the fact that although the BSA protein is purified from other protein molecules (by the supplier), it is practically impossible for the purification fraction to be free of traces of bovine IgG, which can act as a source of cross-reactions with the antibodies against Aflatoxin in the method.

These non-specific interactions of the antibodies with the bovine BSA protein create background noise in the immunoassay (development of an unwanted signal) and low competition efficiency. Consequently, to balance the final signal and maintain the desired convexity of the standard curve, technical modifications are usually made to the immunomethod resulting in undesirable levels of specificity and especially recovery. However, the existence of the BSA protein is not the only problem. The final immunochemical construct has the desired activity only with the optimal conformation and orientation of the anti-Aflatoxin M1 antibody in its indirect immobilization system on the solid support.

By using the competitive type immunomethod system which uses a graded addition of Aflatoxin M 1 antibody shaping and targeting factors such as polysorbate 20 non-protein molecules, polyvinyl alcohol (MW < 10000) (hydrolysis <85%) and mycosis, the spatial model of indirect binding of the antibody to a protein molecule already immobilized on the solid substrate (via covalent or strong non-covalent bonding), creates an immune system that exhibits zero non-specific interactions and improved recovery levels (%). The presence of non-ionic surfactants, low molecular weight and low degree of hydrolysis polymers and disaccharides, create the optimal stereochemistry so that the indirectly immobilized antibody against Aflatoxin M1 binds with excellent capacity the HRP-bound toxin and even greater capacity the free toxin sample (Figure 1). The optimal stereochemistry of the new immunochemical model gives the immunomethod the possibility to use a lower concentration of the antibody against Aflatoxin M1, a fact which is of key importance in competitive immunomethods. The low concentration of the antibodies contributes to the result of a final immunomethod which has a larger standard curve range and at the same time optimal levels of sensitivity.

At the same time, commercially available solutions (kits) use skim milk standard solutions that simulate the actual skim samples to be analyzed. This is because it is important to maintain the environment of the milk in the method so that the environment (matrix) of the standards does not present different physicochemical conditions and affect the antigen-antibody reaction. The formulation of standard solutions helps to create a correct standard curve with a working quantitation range and desired recovery levels. The presence of fat significantly affects the antigen-antibody reaction and the available immunomethods have as a basic condition the defatting of the milk samples. In addition to this fact, the methods fail to quantify the concentration of Aflatoxin M1 with satisfactory recovery when using different kinds of skimmed milk samples (raw milk, homogenized milk, milk powder), resulting in a final method that does not have the desired levels recovery.

By using the competitive type immunomethod system which uses non-zero fatty acid instant-dissolving skimmed milk powder, the method shows high retention of the desired environment (matrix) for the antigen-antibody reaction and a correct standard curve is created with a working range of quantitation and significant contribution to desired recovery levels. Standard non-zero fatty acid (0.05 - 0.1%) non-zero instant-dissolving skimmed milk powder solutions, combined with the indirect antibody immobilization system, provide the optimal physicochemical conditions for the antigen-antibody reaction. The immunochemical model is now capable of quantifying Aflatoxin M1 in milk samples with high recovery. Finally, the immunoassay, in a ready-to-use form, can determine the concentration of Aflatoxin M1 in milk or milk composition samples in a working range of 0-250 ng/L. (Figure 16).

BRIEF DESCRIPTION OF THE DRAWINGS

Figure 1: Schematic illustration of the indirect immunochemical model of antibody immobilization on the solid support. The model is performed by graded addition of conformation and orientation factors of the antibody. In the stage of direct immobilization of the protein molecules (antibodies), the remaining vacant positions of the functional solid substrate are covered by non-ionic surfactants and after the stage of creating the complex of antibodies against Aflatoxin M1 with the immobilized protein molecules (antibodies) a mixture of polymers of low molecular weight and low degree of hydrolysis and disaccharides.

Figure 2: Comparison of standard solutions of fresh skimmed cow's milk with standard solutions of reconstituted instant non-zero fatty acid skimmed cow's milk powder by competitive ELISA (with antigen immobilized on the solid support). Microplates were coated with Aflatoxin conjugated to BSA protein at a concentration of 0.07 µg/mL. Reagent dilutions were: standard solutions of Aflatoxin M1 with different concentrations, monoclonal antibody against Aflatoxin M1 (produced in rat), 1/1200, and detection polyclonal antibody against peroxidase (HRP)-conjugated rat IgG, 1/250. Data points are the mean ± standard deviation (SD), for n=8 samples.

Figure 3: Non-specific interactions of rat monoclonal antibody against Aflatoxin M1 with BSA protein or polyvinyl alcohol (PVA) by indirect ELISA. Microplates were coated with Aflatoxin conjugated to BSA protein at a concentration of 0.07 µg/mL. Reagent dilutions were: 2% BSA or 1% PVA blocking solution, 250 ng/L Aflatoxin M1 high concentration standard solution, Aflatoxin M1 monoclonal antibody (produced in rat), 1/1200, and polyclonal anti-rat IgG detection antibody peroxidase (HRP)-conjugated, 1/250. Data points are the mean ± standard deviation (SD), for n=8 samples.

Figure 4: Determination of the concentration of different coated polyclonal antibodies against rat IgG by direct ELISA. Microplates were coated with polyclonal antibodies against rat IgG, raised in goat, sheep or rabbit at different concentrations. Reagent dilutions were: peroxidase (HRP)-conjugated polyclonal anti-goat, sheep, or rabbit IgG detection antibody, 1/8000; Data points are the mean ± standard deviation (SD), for n=8 samples.

Figure 5: Determination of the concentration of rat monoclonal antibody against Aflatoxin M1 by indirect ELISA. Microplates were coated with goat polyclonal antibodies against rat IgG at a concentration of 10 pg/mL. Reagent dilutions were: monoclonal antibody against Aflatoxin M1 (rat produced) at different concentrations, peroxidase (HRP)-conjugated antigen (HRP-Aflatoxin), 1/2000. Data points are the mean ± standard deviation (SD), for n=8 samples.

Figure 6: Determination of the concentration of HRP-conjugated antigen (HRP-Aflatoxin) by indirect ELISA. Microplates were coated with rat monoclonal antibodies against Aflatoxin M1 at a concentration of 2 µg/mL, indirectly linked to goat polyclonal antibodies against rat IgG at a concentration of 10 µg/mL, which were directly linked to polystyrene. Reagent dilutions were: peroxidase (HRP)-conjugated antigen (HRP-Aflatoxin) at different concentrations; Data points are the mean ± standard deviation (SD), for n=8 samples.

Figure 7: Quantification of Aflatoxin M1 concentration by competitive ELISA, without the graded addition of Aflatoxin M1 antibody conformers and orienting factors. The microplates were coated with rat monoclonal antibodies against Aflatoxin M1 at a concentration of 2 µg/mL, indirectly linked to goat polyclonal antibodies against rat IgG at a concentration of 10 µg/mL, which were directly linked to polystyrene. Reagent dilutions were: standard solutions of Aflatoxin M1 from reconstituted instant non-zero fatty acid skimmed cow's milk powder, peroxidase conjugated antigen (HRP) (HRP-Aflatoxin), 0.125 x10<-3>μg/mL. Data points are the mean ± standard deviation (SD), for n=8 samples.

Figure 8: Recovery of Aflatoxin M1 in fresh or homogenized cow's milk samples by competitive type ELISA, without the graded addition of conformation and orientation factors of rat monoclonal antibody against Aflatoxin M1. Data points are the mean ± standard deviation (SD), for n=8 samples.

Figure 9: Quantification of Aflatoxin M1 concentration by competitive ELISA, with graded addition of Aflatoxin M1 antibody conformation and targeting factors. Microplates were coated with rat monoclonal antibodies against Aflatoxin M1 at a concentration of 0.33 µg/mL, indirectly linked to goat polyclonal antibodies against rat IgG at a concentration of 10 µg/mL, which were directly linked to polystyrene. Reagent dilutions were: standard solutions of Aflatoxin M1 from reconstituted instant non-zero fatty acid skimmed cow's milk powder, peroxidase conjugated antigen (HRP) (HRP-Aflatoxin), 0.125 x 10<'3>μg/mL. Data points are the mean ± standard deviation (SD), for n=8 samples.

Figure 10: Recovery of Aflatoxin M1 in fresh or homogenized cow's milk samples by competitive type ELISA, with graded addition of conformation and orientation factors of the rat monoclonal antibody against Aflatoxin M1. Data points are the mean ± standard deviation (SD), for n=8 samples.

Figure 11: Quantification of Aflatoxin M1 concentration by competitive ELISA, with graded addition of Aflatoxin M1 antibody conformation and targeting factors and a single wash step. Microplates were coated with rat monoclonal antibodies against Aflatoxin M1 at a concentration of 0.33 pg/mL, indirectly linked to goat polyclonal antibodies against rat IgG at a concentration of 10 pg/mL, which were directly linked to polystyrene. Reagent dilutions were: standard solutions of Aflatoxin M1 from reconstituted instant non-zero fatty acid skimmed cow's milk powder, peroxidase conjugated antigen (HRP) (HRP-Aflatoxin), 0.125 x 10<'3>μg/mL. Data points are the mean ± standard deviation (SD), for n=8 samples.

Figure 12: Recovery of Aflatoxin M1 in fresh or homogenized cow's milk samples by competitive-type ELISA, with graded addition of conformation and orientation factors of rat monoclonal antibody against Aflatoxin M1 and a single washing step. Data points are the mean ± standard deviation (SD), for n=8 samples.

Figure 13: Recovery of Aflatoxin M1 in samples of skimmed or non-fresh cow's milk by competitive ELISA, with graded addition of conformation and orientation factors of rat monoclonal antibody against Aflatoxin M1 and a single washing step. Data points are the mean ± standard deviation (SD), for n=8 samples.

Figure 14: Quantification of Aflatoxin M1 concentration by competitive ELISA, with graded addition of Aflatoxin M1 antibody conformers and orientation factors and HRP-conjugated antigen of larger hydrodynamic radius. Microplates were coated with rat monoclonal antibodies against Aflatoxin M1 at a concentration of 0.25 µg/mL, indirectly linked to goat polyclonal antibodies against rat IgG at a concentration of 10 µg/mL, which were directly linked to polystyrene. Reagent dilutions were: standard solutions of Aflatoxin M1 from reconstituted instant non-zero fatty acid skimmed cow's milk powder, peroxidase (HRP)-conjugated antigen (HRP-Aflatoxin), 0.44x10<'3>µg/mL. Data points are the mean ± standard deviation (SD), for n=8 samples.

Figure 15: Recovery of Aflatoxin M1 in fresh or homogenized cow's milk samples by competitive-type ELISA, with graded addition of conformation and orientation factors of rat monoclonal antibody against Aflatoxin M1 and HRP-conjugated antigen of larger hydrodynamic radius. Data points are the mean ± standard deviation (SD), for n=8 samples.

Figure 16: Quantification of Aflatoxin M1 concentration by competitive ELISA, with graded addition of Aflatoxin M1 antibody conformation and targeting factors. Microplates were coated with mouse-produced anti-Aflatoxin M1 monoclonal antibodies at a concentration of 0.075 µg/mL, indirectly linked to goat-produced polyclonal anti-mouse IgG antibodies at a concentration of 10 µg/mL, which were directly linked to polystyrene. Reagent dilutions were: standard solutions of Aflatoxin M1 from reconstituted non-zero fatty acid instant skimmed cow's milk powder, peroxidase conjugated antigen (HRP) (HRP-Aflatoxin), 8.33 x 10<'3>μg/mL. Data points are the mean ± standard deviation (SD), for n=8 samples.

Figure 17: Recovery of Aflatoxin M1 in fresh or homogenized cow's milk samples by competitive type ELISA, with graded addition of conformational and targeting factors of mouse-produced Aflatoxin M1 monoclonal antibody. Data points are the mean ± standard deviation (SD), for n=8 samples.

Figure 18: Recovery of Aflatoxin M1 in samples of skimmed or raw cow's milk by competitive ELISA with graded addition of conformational and targeting factors of murine monoclonal antibody to Aflatoxin M1. Data points are the mean ± standard deviation (SD), for n=8 samples.

DISCLOSURE AND DETAILED DESCRIPTION OF THE INVENTION

Antibodies

In the present invention, the term ``antibody'' refers to an antibody which (a) may be polyclonal, monoclonal or recombinant, (b) recognizes a certain region or, more rarely, more than one region, in the case of a polyclonal antibody, because the antigen in this invention is a mycotoxin and (c) can be used in any immunomethod of this invention.

As noted above, a suitable antibody for use in any immunomethod of this invention will be capable of binding specifically to Aflatoxin M1. Desirably, the indirectly immobilized antibody shows zero cross-reactions with other toxins in the ELISA method, especially for those that could potentially be found in milk. It is worth noting that Aflatoxin M1 and Aflatoxin B1 share common epitopes due to their similar structure. By extension, but also based on the ISO 14675:2003 immunomethod for the detection of Aflatoxin M1, antibodies against Aflatoxin M1 could cross-react with Aflatoxin B1, as Aflatoxin B1 can be used in the HRP-conjugated form (instead of Aflatoxin B1).

In the competitive immunomethod of this invention, the antibody against Aflatoxin M1 is indirectly immobilized on the solid support. This can be accomplished by first immobilizing a proteinaceous molecule that has an affinity for the antibody against Aflatoxin M1 onto the solid substrate and then adding the antibody to form the indirect immobilization system. This proteinaceous molecule can be an antibody against the antibody against Aflatoxin M1 (Figure 1), an antibody against a molecule to which the antibody against Aflatoxin M1 has been conjugated, any molecule of proteinaceous nature which shows affinity to a another molecule to which the antibody against Aflatoxin M1 has been conjugated, proteins such as protein A or protein G, which bind most antibodies in the Fc region, avidin which has an affinity for biotin to which the antibody has been conjugated to of Aflatoxin M 1 or a combination thereof.

Also, it is important that the protein molecule directly immobilized on the solid substrate does not bind the HRP-conjugated toxin, but also that the indirectly immobilized antibody against Aflatoxin M1 binds Aflatoxin M1 at a different point of its structure than the one to which it has been linked with the HRP enzyme (so that the competition step can take place).

(a) Monoclonal antibodies against Aflatoxin M1

Monoclonal antibodies recognize only one and unique epitope in the structure of Aflatoxin M1 giving better results in immunomethods. Results in immunoassays depend on the affinity of the selected clone for both the sample toxin and the HRP-conjugated toxin. Monoclonal antibodies are antibodies made by cloning a single white blood cell, and all subsequent antibodies thus derived are localized to a single parent cell. The production of monoclonal antibodies is based on the production of hybridomas, which involves the identification of antigen-specific (Aflatoxin M1) plasma cells that produce antibodies specific for the antigen (Aflatoxin M1) and the fusion of these cells with myeloma cells. Hybridomas can be grown indefinitely in appropriate cell culture medium. It is worth noting that most monoclonal antibodies against Aflatoxin M1 are produced after immunization of mice or rats with BSA-Aflatoxin M1 immunogen. The fact that the antigen carrier protein can be the BSA protein means that the antibody against Aflatoxin M1, in addition to the affinity it has for Aflatoxin M1, usually also has a low affinity for the BSA protein.

Recombinant monoclonal antibodies can also be used through the process of cloning and phage display technology. Recombinant antibody engineering involves producing antibodies using viruses or yeast, rather than mice. These techniques rely on the rapid cloning of immunoglobulin gene segments to generate antibody libraries with slightly different amino acid sequences from which antibodies with desired specificities can be selected. Also, these techniques can be used to enhance the specificity with which antibodies recognize antigens, their stability in various environmental conditions, and their detectability in diagnostic applications.

In the case of immunomethods, if an antibody clone gives the desired results (affinity, specificity, etc.), the use of monoclonal antibodies gives the method excellent reproducibility in their continuous use, since the repeated immunizations of animals that are carried out in the case of production are avoided polyclonal antibodies.

Ideally, but not limitingly, the most suitable monoclonal antibodies against Aflatoxin M1 are those that can be produced using Aflatoxin M1 conjugated to a carrier protein (which is not the BSA protein) as the immunogen and the mouse as the carrier medium or the rat. These antibodies are an ideal case for the ELISA enzyme-linked immunosorbent assay method used for the quantification of Aflatoxin M1 in milk.

(a) Polyclonal antibodies against Aflatoxin M1

Polyclonal antibodies recognize more than one epitope in the structure of Aflatoxin M1 and can be used with satisfactory results in immunoassays. The fact that polyclonal antibodies recognize multiple epitopes means that those that recognize only the HRP-binding region of the toxin, while reacting with Aflatoxin M1, lack the ability to bind the HRP-conjugated toxin. This means that part of the antibodies immobilized indirectly on the solid substrate remains inactive in the competitive immunoassay.

Polyclonal antibodies are often used when the nature of the antigen in untested animal species is not known. This also creates the need to control the immunogen sequence for cross-reactivity. The animals in which the polyclonal antibodies are produced can be monkeys, rabbits, dogs, guinea pigs, mice, rats, sheep, goats and chickens.

(c) Antibody labeling against Aflatoxin M1

An antibody for use in the method of the invention can be directly or indirectly labeled with each cross-linking molecule, using cross-linking chemistry. A typical example is the cross-linking of the antibody against Aflatoxin M1 with biotin molecules.

Antigone

(a) Modification of antigens

An antigen, such as for example Aflatoxin M1, for use in the method of the invention can be directly or indirectly modified with each cross-linking molecule, using cross-linking chemistry. This is often necessary in order for the antigen to be labeled and used as a detection reagent.

(b) Antigen labeling

An antigen, such as for example Aflatoxin M1, for use in the method of the invention may be labeled directly or indirectly, e.g. in ways such as those of labeled detection molecules. A suitable labeling agent for this purpose is any conventional labeling agent used in immunomethods including radioactive, fluorescent and enzymatic such as horseradish peroxidase (hereafter referred to as "HRP") agents, as well as biotin.

Competitive type methods

The method of the invention can be used in any form that lies within the framework of the sandwich competitive immunomethod, which uses the antibody against the analyte (toxin) indirectly immobilized on the solid support. The anti-Aflatoxin M1 antibody can be bound indirectly to a solid substrate, after first immobilizing a proteinaceous molecule which has an affinity for the anti-Aflatoxin M1 antibody and then adding the antibody to form the indirect immobilization system . This proteinaceous molecule can be an antibody against the antibody against Aflatoxin M1 (Figure 1), an antibody against a molecule to which the antibody against Aflatoxin M1 has been conjugated, any molecule of proteinaceous nature which shows affinity to a another molecule to which the antibody against Aflatoxin M 1 has been conjugated, proteins such as protein A or protein G, which bind most antibodies in the Fc region, avidin which has an affinity for the biotin to which the antibody has been conjugated against Aflatoxin M1 or a combination thereof. The solid substrate can be e.g. plastic cell surface (polystyrene) or solid pellets.

The method of the invention can thus e.g. to comply with the conventional competitive type of ELISA method using any indirect mode of immobilization of antibodies against Aflatoxin M1. Antibodies can be immobilized indirectly to the cells of a polystyrene microplate or solid beads. In each case, the antibody against Aflatoxin M1 competes for binding of milk toxin or label-conjugated toxin. The ELISA according to the invention is analyzed in detail immediately below and illustrated by typical examples.

Indirect immunochemical model of antibody immobilization on solid substrate

From a technical point of view, in immunoassays of the competitive type and especially in those that quantify the presence of a hapten, such as a mycotoxin, in a sample such as milk, the most important stage of the immunochemical construction is the stage of immobilization of the antibody. This step depends on how the antibody is immobilized (whether it is direct or indirect), how the antibody is immobilized, and the material covering voids on the solid support. The solid substrate can be e.g. plastic cell surface (polystyrene) or solid beads (especially when the immunomethod is ELISA). Practically the entire immunomethod depends on how the antibody chosen for the system will work. Representatively, in the present invention the solid substrate is polystyrene (Figure 1).

Based on the indirect immunochemical model of immobilization of the antibody on the solid substrate, a protein molecule that has an affinity for the antibody against Aflatoxin M1 is first immobilized on the solid substrate, and then the antibody against Aflatoxin M1 is added to form the complex (from hereafter referred to as "complex"). The direct binding protein molecule can be an antibody against the anti-Aflatoxin M1 antibody (Figure 1 ), an antibody against a molecule to which the anti-Aflatoxin M1 antibody has been conjugated, any molecule of a proteinaceous nature which shows affinity to another molecule to which the anti-Aflatoxin M1 antibody has been conjugated, proteins such as protein A or protein G, which bind most antibodies in the Fc region, avidin which has an affinity for the biotin to which the antibody has been conjugated against Aflatoxin M1 or a combination thereof. Representatively, in the present invention, the direct binding protein molecule is an anti-Aflatoxin M1 antibody. This antibody is usually a polyclonal against the Fc region of the Aflatoxin M1 antibody so that there is a correct orientation and the free Fab regions can bind Aflatoxin M1 (Figure 1).

Then, an important role is played by the addition of an agent inert to the system which, in addition to the role of the blocking agent of the vacancies of the solid substrate, must meet all the conditions of compatibility with the immunomethod to be developed. Under these conditions, an important role is played by the ability of this agent to adsorb onto the solid substrate (between the empty spaces not covered by the immobilized protein molecule) as well as the ability of this agent not to react with the already immobilized protein molecule on the solid substrate (the blocking step follows the immobilization step of the protein molecule). Also of importance is the ability of this agent not to react with any other reagent used in the immunoassay such as the antibody against Aflatoxin M1, the analyte antigen (Aflatoxin M1 in the case of the present invention) and the detection reagent (labeled toxin in the case of the present invention) when the immunomethod is of the competitive type. Ideally, the solid substrate vacancy blocking agent, in addition to being an adsorptive component for the solid substrate and an inert component for the immunochemical construct, is characterized by the ability to aid the structure and functionality of the substrate-anchored protein molecule, its bond with the solid substrate, its binding to the antibody against Aflatoxin M1 and the functionality of the antibody. It is worth noting that commercially available immunoassays usually use antibodies produced after immunization of rodents with the BSA-Aflatoxin M1 immunogen and the fact that the antigen carrier protein is the BSA protein means that there may be non-zero affinity of the antibody. and for this protein (BSA). However, the BSA protein is used in the step of blocking vacancies of the solid substrate and its existence contributes to the result of a final method which does not have the desired characteristics. This occurs: (a) due to the cross-reactions of antibodies against Aflatoxin M1 with the BSA protein, which as an unwanted antigenic determinant reduces the activity of the antibodies (via its binding to the toxin) and (b) due to the fact that although where the BSA protein is purified from other protein molecules (by the supplier), it is practically impossible for the purification fraction to not contain traces of bovine IgG, which can act as a source of cross-reactions with the anti-Aflatoxin antibodies in the method. These non-specific interactions of the antibodies with the bovine BSA protein create background noise in the immunoassay (development of an undesired signal) and low competition efficiency. Representatively, in the present invention, the site blocking agent of the solid substrate is polysorbate 20 which belongs to the nonionic surfactants. Due to its nature, it binds hydrophobically with great ease to polystyrene and due to its small molecular size it correctly orients the immobilized protein molecule to bind with the antibody against Aflatoxin M1 (Fig

1);

The addition of the antibody against Aflatoxin M 1 is carried out simultaneously with the addition of the non-ionic surfactant. That is, a mixture of the two reagents is added to the surface of the solid substrate to which the protein molecule is already fixed. Thus, it receives at the same time a double reaction in which there is no interaction. The nonionic surfactant binds hydrophobically to the voids of the solid substrate and the anti-Aflatoxin M1 antibody binds to the immobilized protein molecule. The reaction of the non-ionic surfactant is so fast that the undesired binding of the anti-Aflatoxin M1 antibody to the voids of the solid substrate is almost nil. At this stage of covalent binding of the anti-Aflatoxin M1 antibody to the immobilized protein molecule, the stoichiometry of the reaction is regulated. The stoichiometry of the reaction is the most important factor that will determine the shape of the final standard curve (the second most important factor is the composition and nature of the standard solutions). The less anti-Aflatoxin M1 antibodies bind to the (usually) proteinaceous solid substrate, the more functional the standard curve becomes, due to the greater degree of competition (Figure 1).

Finally, after the simultaneous addition of the nonionic surfactant and the antibody against Aflatoxin M1 (formation of the complex), of equal importance is the subsequent addition of a mixture of factors that will stabilize the complex, contribute to its correct formation and maintain the antibody activity against Aflatoxin M1 at maximum levels. The final orientation and the final configuration of the complex are two factors that characterize the final immunomethod. It is worth noting that the binding of the Fc region of the antibody (by the protein molecule) causes a remodeling of its Fab regions and, by extension, the way these regions will be able to bind (Aflatoxin M1 or the HRP-bound toxin). Conversely, binding of the Fab regions of the antibody (with Aflatoxin M1 or HRP-bound toxin) causes remodeling of its Fc region. Thus, in the present invention, it was hypothesized that the stabilization of bidirectional remodeling of the core structures of the complex can be accomplished by adding a suitable mixture of polymers and disaccharides. Polymers, such as low molecular weight and low degree of hydrolysis polyvinyl alcohol (PVA) (the polymer PVP can also be used), have been found to be able to protect the activity of soluble proteins such as peroxidase (Boyd and Yamazaki, 1994). With a corresponding mechanism, it has been found that disaccharides such as sucrose, mycose, etc., can equally contribute to the protection of the structural functions of proteins (Ansari et al., 1985, Leslie et al., 1995). In the present invention, using a mixture of polyvinyl alcohol (MW < 10000) with a low degree of hydrolysis (<85%) and mycose and in combination with polysorbate 20 already used (Figure 1), the spatial model of the complex binding stabilizes at optimal levels and the method exhibits zero non-specific interactions and improved recovery levels (%). The indirect immunochemical model creates the optimal stereochemistry so that the indirectly immobilized anti-Aflatoxin M1 antibody binds with excellent capacity the HRP-bound toxin and with even greater capacity the free toxin in the sample. The immunomethod has the potential to use a lower concentration of the antibody against Aflatoxin M1, which is of key importance in competitive-type immunomethods. The low concentration of the antibodies contributes to the result of a final immunomethod which has a larger standard curve range and at the same time optimal levels of sensitivity.

Standard solutions in immunoassays

In immunomethods which aim at the quantitative determination of the analyte and in particular in immunomethods which determine the concentration of a mycotoxin, standard solutions play a very important role.

A basic condition for the creation of the quantitative immunochemical construction is the correct preparation of the standard solutions. This can be done by using correct concentrations of the analyte (Aflatoxin M1 in the case of the present invention) and using the most compatible medium for diluting the analyte (milk in the case of the present invention). The analyte concentrations have no limitations and depend on the desired range of the standard curve.

Standard skimmed milk solutions can be used in immunoassays to simulate the actual skimmed samples to be analyzed. This is because it is important to maintain the environment of the milk in the method so that the environment (matrix) of the standards does not present different physicochemical conditions and affect the antigen-antibody reaction. The formulation of standard solutions helps to create a correct standard curve with a working quantification range and desired recovery levels. The presence of fat significantly affects the antigen-antibody reaction and the available immunomethods have as a basic condition the defatting of milk samples. In addition to this fact, the methods fail to quantify the concentration of Aflatoxin M1 with satisfactory recovery when using different kinds of skimmed milk samples (raw milk, homogenized milk, milk powder), resulting in a final method that does not have the desired levels recovery.

In the present invention, it has been hypothesized that the appropriate step by which standard solutions can be created which meet the requirements of the correct environment and preservation over time, is from non-zero fat instant skimmed milk powder. With the appropriate percentage of powder in the final solution (reconstituted milk) the method shows a high retention of the desired environment (matrix) for the antigen-antibody reaction and a correct standard curve is created with a functional quantification range and a significant contribution to the desired recovery levels. Standard non-zero fatty acid (0.05 - 0.1%) non-zero instant-dissolving skimmed milk powder standard solutions, combined with the indirect immunochemical model of antibody immobilization, provide the optimal physicochemical conditions for the antigen-antibody reaction. The immunochemical model is now capable of quantifying Aflatoxin M1 in milk samples with high recovery. Finally, the immunoassay, in a ready-to-use form, can determine the concentration of Aflatoxin M1 in milk or milk composition samples in a working range of 0-250 ng/L.

ELISA method

As far as the present invention is concerned, the specific binding of the selected antibody against Aflatoxin M1 leads to the easy detection of Aflatoxin M1 in milk and the creation of a method which can be in the form of a kit, as described below. A desirable kit may be based on the principles of the ELISA method.

In the case of the competitive ELISA, the kit includes standard solutions with specific concentrations of Aflatoxin M1 (from non-zero fat instant skimmed milk powder) which will be bound by the immobilized antibodies, a labeled toxin, a buffer solution and a chromophore detection solution.

The present invention relates to a method for the quantitative determination of Aflatoxin M1, which comprises the use of the selected antibody against Aflatoxin M1 and the detection means described above. This method of quantification has no particular limitations, since it uses the usual antigen-antibody reaction and there are many examples of such methods. Among these methods, ELISAs using β-galactosidase, glucosidase, alkaline phosphatase, peroxidase, malate dehydrogenase are particularly preferred in terms of sensitivity and utility.

The detection method of the standard ELISA includes the competitive type ELISA. For example, Aflatoxin M1 can be detected by a competitive ELISA using the appropriate anti-Aflatoxin antibody and the innovations of the present invention as described below.

(1) Molecules of a proteinaceous nature which have an affinity for the anti-Aflatoxin M1 antibody (or a conjugated molecule to the anti-Aflatoxin M1 antibody) are immobilized on a support substrate. The support substrate used is preferably a 96, 48 or 192 cell polystyrene microplate. According to the present invention, the protein molecule is a polyclonal antibody against the Fc region of the anti-Aflatoxin M1 antibody, so that there is a correct orientation and the free Fab regions can bind Aflatoxin M1. The polyclonal antibody can be immobilized on the solid support substrate by introducing a buffer containing the protein molecule to be immobilized and incubating for a certain period of time e.g. at 37 °C for 2 - 4 hours. Then follows washing with a washing liquid which has no limitations and can be the same buffer solution of the incubation of the polyclonal antibody to be fixed. The concentration of the polyclonal antibody in the buffer is usually 1 - 10 µg/mL. The buffer can be a known solution, depending on the detection means. (2) In order to avoid non-specific adsorption of proteins on the surface of the solid support substrate, the surface of the solid phase to which the polyclonal antibody was not adsorbed is blocked with an agent unrelated to the polyclonal antibody. Based on the present invention, non-ionic surfactants such as polysorbate 20 (triton X-100 etc. can also be used) are used as the blocking agent instead of BSA protein. The blocking step is carried out after adding the blocking agent or agents to the support substrate with the simultaneous addition of the antibody against Aflatoxin M1 and incubating them e.g. at 37 °C for 2 - 4 hours. The percentage of nonionic surfactant and the concentration of anti-Aflatoxin M1 antibody in the buffer are usually 0.01 - 0.1% and 0.01 - 1 µg/mL respectively.

(3) In order to stabilize the two-way remodeling of the basic structures of the formed complex, a mixture of agents is added to ensure the optimal stereochemistry of the indirect immunochemical model. Based on the present invention, the mixture consists of polymers such as polyvinyl alcohol (PVA) of low molecular weight (MW < 10000) and low degree of hydrolysis (<85%) (PVP polymer can also be used) in a final concentration of 0.1 - 2 % and disaccharides such as mycose (sucrose etc. can also be used) in a final concentration of 0.5 - 10%. The stabilization step is carried out after adding the mixture and incubating it e.g. at 37 °C for 1 - 2 hours.

(4) Standard solutions with specific concentrations of Aflatoxin M1 are prepared. These concentrations also have no limitations and depend on the desired range of the standard curve. Based on the present invention, solutions of reconstituted skimmed milk powder of instant dissolution of non-zero percentage of fatty acids (0.05 - 0.1%), in the presence of sodium hydrazote, are used as standard solutions.

(5) The mixture of step (4) is reacted with the antibody of the solid phase substrate complex obtained in step (3) above. The antigen-antibody reaction between Aflatoxin M1 and indirectly immobilized anti-Aflatoxin M1 antibodies results in binding of Aflatoxin M1 indirectly to the solid phase substrate. The reaction is carried out for example at 25 °C for about 45 minutes. (6) HRP-conjugated toxin molecules are diluted for example 30000 times and create a detection solution.

(7) The indirectly immobilized anti-Aflatoxin M1 antibodies and Aflatoxin M1 of step (5) are washed with washing liquid, and then the detection solution (6) is added and reacted with the remaining antibodies that have not bound Aflatoxin M1 for example at 25 °C for 15 min. After the reaction is terminated, the substrate is washed with washing liquid and the enzyme-linked toxin molecules that were not bound to the solid-phase bound antibodies against Aflatoxin M1 are removed. By detecting the amount of antibody-HRP-conjugated toxin complex bound to the solid phase, the amount of Aflatoxin M1 in the sample is determined from a calibration curve which depends only on the standard solutions used.

(8) A chromogenic substrate solution for the reaction of the bound enzyme-labeled complex is added to detect the absorbance of the reaction solution, whereby the amount of Aflatoxin M1 can be calculated from the calibration curve. When peroxidase is used as the labeling enzyme, a chromogenic substrate solution containing for example hydrogen peroxide and 3',3',5',5'-tetramethylbenzidine (TMB) or o-phenylenediamine (OPD) can be used (typically, the TMB). The mixture is reacted for about 15 minutes at room temperature and finally phosphoric acid is added to terminate the enzyme reaction. When TMB is used, the absorbance of the reaction solution is measured at 450 nm. When phenylenediamine is used, the absorbance of the reaction solution is measured at 492 nm. To compensate for the background value, the absorbance at 630nm is measured at the same time.

When, for example, alkaline phosphatase is used as a labeled enzyme, p-nitrophenylphosphoric acid is used as a chromogenic substrate, sodium caustic is used as a termination solution for the enzyme reaction, and the absorbance of the reaction solution is measured at 415 nm. The rate of decrease in absorbance is relative to the Aflatoxin M1 reacted. The Aflatoxin M1 concentration of milk can be determined using a calibration curve from the rate of reduction of reaction solutions of known concentrations of Aflatoxin M1 added.

Example 1

The concentration of all antibodies used, before and after their use, was quantified by the “coomassie dye binding” method (Bradford, 1976), using standard goat IgG (Sigma) as a standard and adjusted to a final protein concentration of 1 mg/m .

Example 2

Preparation of concentrated polymer solution

Polyvinyl alcohol (PVA) of low molecular weight (MW < 10000) and low degree of hydrolysis (<85%) was dissolved in deionized distilled water at a concentration of 20%. Dissolution was carried out under boiling and magnetic stirring conditions for 3 hours. The final solution was filtered with a 0.22 µm filter and stored at room temperature until use.

Example 3

Preparation of concentrated disaccharide solution

Mycose was dissolved in deionized distilled water at a concentration of 20%. Dissolution was carried out at room temperature and magnetic stirring for 3 hours. The final solution was filtered with a 0.22 µm filter and stored at room temperature until use.

Example 4

Preparation of HRP-Aflatoxin Detection Antigen

The HRP-Aflatoxin detection antigen used was from commercial ELISA kits for the detection of Aflatoxin M1 in milk, either in dilute or concentrated form. In each case the reagent was filtered with a 0.22 µm filter, diluted once (1:1) with glycerol and stored at -20 °C until use. Alternatively, a corresponding detection reagent from a commercial source (Immunechem) was used.

Example 5

Preparation of HRP-BSA-Aflatoxin M1 Longer Hydrodynamic Radius Detection Antigen

To generate the HRP-BSA-Aflatoxin M 1 larger hydrodynamic radius detection antigen, conjugation between the HRP enzyme (SIGMA) and the BSA-Aflatoxin M 1 molecule was performed using metaperiodic sodium, according to a published literature reference (Farzamfar et at. , 2006).

Example 6

Preparation of standard solutions of skimmed fresh cow's milk

Raw milk, whose Aflatoxin M1 concentration had previously been measured < 5 ng/L by a reference method (HPLC), was used to create standard solutions of skimmed fresh cow's milk. The milk was centrifuged for 10 minutes at 3000 rpm and the fat layer was removed. Then sodium hydrazide was added at a rate of 0.05% or a mixture of 2-methyl-4-isothiazolin-3-one (0.6 - 1 %) and 5-chloro-2-methyl-4-isothiazolin-3-one (2.1 - 2.8%) at a rate of 0.05%. After establishing a high maternal concentration of Aflatoxin M1 (SIGMA) in this milk, the different concentrations were performed using a yoke. In any case, due to the high microbial load (unpasteurized milk), it was not possible to use these standards in ELISA kits. An ELISA kit should contain reagents that are stable for at least 9 - 12 months. The specific standards were used for comparison purposes in the studies of the present invention.

Example 7

Preparation of standard solutions from reconstituted non-zero fatty acid (0.05 - 0.1 %) flash-dissolved skimmed cow's milk powder

To create standard solutions from reconstituted non-zero fat instant skimmed milk powder, commercial instant skimmed cow's milk, which had a fat content of 0.08%, was used. The Aflatoxin M1 concentration of the reconstituted powder was measured < 5 ng/L previously by reference method (HPLC). The amount of powder dissolved in deionized distilled water was according to the manufacturer's instructions. Then sodium hydrazide was added at a rate of 0.05% or a mixture of 2-methyl-4-isothiazolin-3-one (0.6 - 1%) and 5-chloro-2-methyl-4-isothiazolin-3-one (2.1 - 2.8%) at a rate of 0.05%. After establishing a high maternal concentration of Aflatoxin M1 (SIGMA) in this milk, the different concentrations were performed using a yoke. Unlike standard solutions of skimmed fresh cow's milk, presenting the required stability for 12 months. The specific standards were used in the studies of the present invention.

Example 8

Comparison of standard solutions of fresh skimmed cow's milk with standard solutions of reconstituted instant non-zero fatty acid skimmed cow's milk powder by competitive ELISA (with antigen immobilized on the solid support)

(1) BSA protein-conjugated aflatoxin M1 (SIGMA) was added in a volume of 100 μL/cell to each cell of a 96-cell microplate at a concentration of 0.07 μg/mL in PBS pH 7.5 and then immobilized on the solid substrate at 4 °C for 12 hours. Fluid from each cell was removed by aspiration, PBS pH 7.5 containing 2% BSA was added to a volume of 300 μL/cell, and each cell was blocked at 37 °C for 1.5 h, followed by removal of the blocking solution also by aspiration.

(2) 1:1 mixtures of rat monoclonal antibody against Aflatoxin M1 (diluted 1200 times with PBS-Tween) and standard solutions with concentrations of 0, 6.25, 25, 50, 100, 200 and 400 ng/ml_ were prepared. The standard solutions were solutions of fresh skimmed cow's milk (Example 6) or solutions of reconstituted non-zero fat flash skimmed cow's milk powder (Example 7).

(3) Plated cells (1) were washed three times with PBS-Tween pH 7.5 and then each mixture (standard solution-antibody) (2) was added in a volume of 100 µL/cell to the plate and reacted at 25 °C for 45 min .

(4) HRP-labeled anti-rat IgG antibody (Sigma) was diluted 250-fold with PBS-Tween pH 7.5 to prepare the detection antibody solution.

(5) The cells that underwent the reaction (3) were washed three times with PBS-Tween pH 7.5, and then the detection antibody solution (4) was added in a volume of 100 µL/cell to the plate and reacted at 25 °C for 45 min .

(6) Each cell after the reaction (5) as above, was washed three times with PBS-Tween pH 7.5, and 100μL of the TMB substrate solution was added to each cell, followed by incubation at 25 °C for 15 minutes. Then 100 µL/cell of 15% phosphoric acid was added to each cell to terminate the chromogenic reaction and absorbance at 450nm was measured with a dedicated microplate photometer.

The results of the comparison of standard solutions of fresh skimmed cow's milk with standard solutions of reconstituted instant non-zero fatty acid skimmed cow's milk powder (with the antigen immobilized on the solid support) are shown in Figure 2. In each case, the standard inhibition curve was same. This fact means that the reconstituted instant non-zero fat skim milk powder (Example 7) can closely mimic fresh skim milk (Example 6) and be used to produce standard solutions of an ELISA kit, providing the maximum sustainability. Thus, the standard solutions of Experiment 7 were then used.

Example 9

Study of Non-Specific Interactions of Anti-Aflatoxin M1 Monoclonal Antibody (Rat Produced) with BSA Protein or Polyvinyl Alcohol by Indirect ELISA

(1) The antigen used in Example 8 was added in a volume of 100 μL/cell to each cell of a 96-cell microplate at a concentration of 0.07 μg/mL in PBS pH 7.5 and then fixed to the solid substrate at 4 °C for 12 hours . Fluid from each cell was removed by aspiration, PBS solution pH 7.5 containing 2% BSA or 1% PVA (Example 2) was added to a volume of 300 µL/cell and each cell was blocked at 37 °C for 1.5 h, followed by removal of the blocking solution also by aspiration.

(2) A 1:1 mixture of standard solution of concentration 250 ng/mL and the monoclonal antibody used in Example 8, diluted 1200 times with PBS-Tween was prepared.

(3) The plated cells (1) were washed three times with PBS-Tween pH 7.5, and then the standard solution-antibody mixture (2) was added in a volume of 100 μL/cell to the plate and reacted at 25 °C for 45 min.

(4) HRP-labeled anti-rat IgG antibody (used in Example 8) was diluted 250-fold with PBS-Tween pH 7.5 to prepare the detection antibody solution.

(5) The cells that underwent the reaction (3) were washed three times with PBS-Tween pH 7.5, and then the detection antibody solution (4) was added in a volume of 100 µL/cell to the plate and reacted at 25 °C for 45 min .

(6) Each cell after the reaction (5) as above, was washed three times with PBS-Tween pH 7.5, and 100μL of the TMB substrate solution was added to each cell, followed by incubation at 25 °C for 15 minutes. Then 100 µL/cell of 15% phosphoric acid was added to each cell to terminate the chromogenic reaction and absorbance at 450nm was measured with a dedicated microplate photometer.

The results of the study of the non-specific interactions of the monoclonal antibody against Aflatoxin M1 (produced in rat) with BSA protein or polyvinyl alcohol are shown in Figure 3. By using the standard solution of high concentration of Aflatoxin M1 (so that there is a low optical absorbance due to competition and the existence of non-specific interactions can be studied) it was shown that, in contrast to the BSA protein, the addition of a polymer such as polyvinyl alcohol worked exceptionally well. There was a 37.8% reduction in method noise, thus avoiding the use of BSA protein.

Example 10

Determination of the concentration of different coated polyclonal antibodies against rat IgG by direct ELISA

(1) Different anti-rat IgG polyclonal antibodies, produced in goat, sheep, or rabbit, were diluted to different concentrations of 0, 0.15625, 0.3125, 0.625, 1.25, 2.5, 5, and 10 μg/mL in PBS pH 7.5. The solutions of different concentrations were added in a volume of 100 μL/cell to each cell of a 96-cell microplate and then fixed to the solid substrate at 4 °C for 16 h. Fluid from each cell was removed by aspiration, PBS solution pH 7.5 containing 1% PVA (Example 2) was added in a volume of 300 µL/cell and each cell was blocked at 37 °C for 1.5 h, followed by removal of the blocking solution as well with suction.

(2) Different HRP-labeled antibodies against goat, sheep, or rabbit IgG (Sigma) were diluted 8000-fold with PBS-Tween pH 7.5 to prepare each solution of the respective detection antibody. (3) The plated cells (1) were washed three times with PBS-Tween pH 7.5, and then each respective solution of detection antibody (2) was added in a volume of 100 μL/cell to the plate and reacted at 25 °C for 30 min.

(4) Each cell after the reaction (3) as above, was washed three times with PBS-Tween pH 7.5, and 100μL of the TMB substrate solution was added to each cell, followed by incubation at 25 °C for 10 minutes. Then 100 µL/cell of 15% phosphoric acid was added to each cell to terminate the chromogenic reaction and absorbance at 450nm was measured with a dedicated microplate photometer.

The results of determining the concentration of the different anti-rat IgG polyclonal antibodies coated in this example are shown in Figure 4. Three typical saturation curves were observed for the various concentrations of each anti-bovine IgG polyclonal antibody immobilized on the microplate, with the saturation of the largest of percent binding sites to be achieved at 10 µg/mL. The goat polyclonal antibody against rat IgG showed the strongest signal at each concentration used and was therefore chosen for each indirect immunosystem used.

Example 11

Determination of the concentration of rat-produced monoclonal antibody against Aflatoxin M1 by indirect ELISA -Indirect binding to the solid substrate through the immobilized polyclonal antibodies

(1) The goat anti-rat IgG polyclonal antibody used in Example 10 was added in a volume of 100 µL/cell to each cell of a 96-cell microplate at a concentration of 10 µg/mL in PBS pH 7.5 and then fixed on the solid substrate at 4 °C for 16 h. Fluid from each cell was removed by aspiration, PBS solution pH 7.5 containing 1% PVA (Example 2) was added to a volume of 300 µL/cell and each cell was blocked at 37 °C for 1.5 h, followed by removal of the solution blocking also with suction.

(2) The rat monoclonal antibody against Aflatoxin M1 used in Example 8 was diluted to different concentrations of 0, 0.5, 1, 2, 4 and 8 µg/mL with PBS-Tween pH 7.5. The solutions of the different concentrations of the monoclonal antibody were added in a volume of 100 µL/cell into the microplate in which the polyclonal anti-rat IgG antibody had been coated on the solid support, and then the microplate was incubated at 4 °C for 12 h. This was followed by removal of the monoclonal antibody solutions by aspiration.

(3) HRP-labeled antigen, HRP-Aflatoxin (Example 4) was diluted to a concentration of 0.5x1 0<'3>µg/mL with PBS-Tween pH 7.5 to prepare the detection antigen solution.

(4) The plated cells (2) were washed three times with PBS-Tween pH 7.5, and then the detection antigen solution (3) was added in a volume of 100 µL/cell to the plate and reacted at 25 °C for 15 min.

(5) Each cell after the reaction (4) as above, was washed three times with PBS-Tween pH 7.5, and 100μ of the TMB substrate solution was added to each cell, followed by incubation at 25 °C for 10 minutes. Then 100 pL/cell of 15% phosphoric acid was added to each cell to terminate the chromogenic reaction and the absorbance at 450nm was measured with a dedicated microplate photometer.

The results of determining the concentration of the rat monoclonal antibody against Aflatoxin M1 in this example are shown in Figure 5. For the indirect binding of the monoclonal antibody to the solid support via the immobilized polyclonal antibodies, the concentration of 2 μg/mL was chosen, so that the immunomethod uses the linear part of the curve.

Example 12

Determination of the concentration of HRP-conjugated antigen (HRP-Aflatoxin) by indirect ELISA

(1) The goat anti-rat IgG polyclonal antibody used in Example 10 was added in a volume of 100 µL/cell to each cell of a 96-cell microplate at a concentration of 10 µg/mL in PBS pH 7.5 and then fixed on the solid substrate at 4 °C for 16 h. Fluid from each cell was removed by aspiration, PBS solution pH 7.5 containing 1% PVA (Example 2) was added in a volume of 300 µL/cell and each cell was blocked at 37 °C for 1.5 h, followed by removal of the blocking solution as well with suction.

(2) The rat-produced anti-Aflatoxin M1 monoclonal antibody used in Example 8 was added in a volume of 100 µL/cell to each microplate on which the anti-rat IgG polyclonal antibody had been coated at a concentration of 2 µg/mL in PBS- Tween pH 7.5 and then indirectly fixed to the solid substrate at 4 °C for 12 h. This was followed by removal of the monoclonal antibody solution by aspiration.

(3) HRP-labeled antigen, HRP-Aflatoxin (Example 4) was diluted to different concentrations of 0, 0.03125, 0.04167, 0.0625, 0.0833, 0125, 0.25 and 0.5 (x10<-3>) μg/mL with PBS-Tween pH 7.5 for the preparation of detection antigen solutions.

(4) Plated cells (2) were washed three times with PBS-Tween pH 7.5, and then detection antigen solutions (3) were added in a volume of 100 µL/cell to the plate and reacted at 25 °C for 15 min.

(5) Each cell after the reaction (4) as above, was washed three times with PBS-Tween pH 7.5, and 100μL of the TMB substrate solution was added to each cell, followed by incubation at 25 °C for 10 minutes. Then 100 µl/cell of 15% phosphoric acid was added to each cell to terminate the chromogenic reaction and the absorbance at 450nm was measured with a special microplate photometer.

The results of the concentration of the HRP-conjugated antigen (HRP-Aflatoxin) by indirect ELISA in this example are shown in Figure 6. To create the competitive type ELISA, the concentration of 0.125 x 10<'3>μg/mL was chosen so that it could to predominate the binding of the sample antigen to the monoclonal antibody (versus the binding of the HRP-conjugated antigen to the monoclonal antibody) and to favor competition without, however, disrupting the immunochemical construct.

Example 13

Quantification of Aflatoxin M1 Concentration by Competitive Type ELISA Without Graded Addition of Modulating and Orienting Factors of Aflatoxin M1 Rat Monoclonal Antibody

(1) The goat anti-rat IgG polyclonal antibody used in Example 10 was added in a volume of 100 µL/cell to each cell of a 96-cell microplate at a concentration of 10 µg/mL in PBS pH 7.5 and then fixed on the solid substrate at 4 °C for 16 h. Fluid from each cell was removed by aspiration, PBS solution pH 7.5 containing 1% PVA (Example 2) was added in a volume of 300 µL/cell and each cell was blocked at 37 °C for 1.5 h, followed by removal of the blocking solution as well with suction.

(2) The rat monoclonal antibody against Aflatoxin M1 used in Example 8 was added in a volume of 100 pL/cell to each microplate on which the polyclonal antibody against rat IgG had been coated at a concentration of 2 µg/mL in PBS- Tween pH 7.5 and then indirectly fixed to the solid substrate at 4 °C for 12 h. This was followed by removal of the monoclonal antibody solution by aspiration.

(3) Aflatoxin M1 standard solutions (0, 5, 10, 25, 50, 100, and 250 ng/L) from reconstituted non-zero fat instant skimmed cow's milk powder were prepared and added in a volume of 100 μL/cell into the microplate in which the rat monoclonal antibody against Aflatoxin M1 had been indirectly coated on the solid support (2) and then the microplate was incubated at 25 °C for 60 min.

(4) HRP-labeled antigen, HRP-Aflatoxin (Example 4) was diluted to a concentration of 0.125x10<'3>µg/mL with PBS-Tween pH 7.5 to prepare the detection antigen solution.

(5) The plated cells (3) were washed three times with PBS-Tween pH 7.5, and then the detection antigen solution (4) was added in a volume of 100 µL/cell to the plate and reacted at 25 °C for 15 min.

(6) Each cell after the reaction (5) as above, was washed three times with PBS-Tween pH 7.5, and 100μL of the TMB substrate solution was added to each cell, followed by incubation at 25 °C for 15 minutes. Then 100 µL/cell of 15% phosphoric acid was added to each cell to terminate the chromogenic reaction and absorbance at 450nm was measured with a dedicated microplate photometer.

The results of quantification of Aflatoxin M1 concentration by competitive ELISA, without the graded addition of Aflatoxin M1 antibody modulators and orientations in this example are shown in Figure 7. A standard competitive ELISA method curve was observed.

Example 14

Recovery of Aflatoxin M1 in fresh or homogenized cow's milk samples by competitive-type ELISA without the graded addition of conformational and orienting factors of rat monoclonal antibody to Aflatoxin M1

Samples of fresh or homogenized cow's milk whose Aflatoxin M1 concentration had previously been measured < 5 ng/L by a reference method (HPLC) were collected to study the recovery of Aflatoxin M1 in fresh or homogenized cow's milk samples by a competitive ELISA. In this study, exogenous Aflatoxin M1 (SIGMA) was added to the samples to generate different Aflatoxin M1 concentrations of 0, 10, 20, 40 and 80 ng/L and the samples were kept at 4 °C for one day. The milk samples were then centrifuged for 10 minutes at 3000 rpm, the fat layer removed and used in the method of Example 13.

The results of the recovery of Aflatoxin M1 in fresh or homogenized cow's milk samples by a competitive ELISA, without the graded addition of Aflatoxin M1 antibody modulators and orienting factors, in this example are shown in Figure 8. In each case, the mean recovery value of the different concentrations of Aflatoxin M1. For the case of fresh cow's milk the recovery was 101.26%. For the case of homogenized cow's milk the recovery was 75.26% (undesired levels).

Example 15

Quantification of Aflatoxin M1 concentration by competitive ELISA with graded addition of conformation and orientation factors of rat monoclonal antibody to Aflatoxin M1

(1) The goat anti-rat IgG polyclonal antibody used in Example 10 was added in a volume of 100 µL/cell to each cell of a 96-cell microplate at a concentration of 10 µg/mL in PBS pH 7.5 and then fixed on the solid substrate at 4 °C for 16 h. The fluid from each cell was removed by aspiration and a solution containing Tween-20 (vacancy blocking) and a concentration of 0.33 pg/mL of the rat-produced anti-Aflatoxin M1 monoclonal antibody used in Example 8 was added in a volume of 100 µL/cell. and each cell was incubated at 4 °C for 12 h. The fluid from each cell was removed by aspiration, a solution containing PVA and mycose (Example 2 & 3) (ensuring optimal stereochemistry of the indirect immunochemical model) was added in a volume of 300 µL/cell and each cell was incubated at 37 °C for 3 h, followed by removal of the solution also by suction.

(2) Aflatoxin M1 standard solutions (0, 5, 10, 25, 50, 100, and 250 ng/L) from reconstituted non-zero fat instant skimmed cow's milk powder were prepared and added in a volume of 100 μL/cell into the microplate in which the rat monoclonal antibody against Aflatoxin M1 had been indirectly coated on the solid support (1 ) and then the microplate was incubated at 25 °C for 60 min.

(3) HRP-labeled antigen, HRP-Aflatoxin (Example 4) was diluted to a concentration of 0.125x10<'3>μg/mL with PBS-Tween pH 7.5 to prepare the detection antigen solution.

(4) The plated cells (2) were washed three times with PBS-Tween pH 7.5, and then the detection antigen solution (3) was added in a volume of 100 pL/cell to the plate and reacted at 25 °C for 15 min.

(5) Each cell after the reaction (4) as above, was washed three times with PBS-Tween pH 7.5, and 100μL of the TMB substrate solution was added to each cell, followed by incubation at 25 °C for 15 minutes. Then 100 pL/cell of 15% phosphoric acid was added to each cell to terminate the chromogenic reaction and the absorbance at 450nm was measured with a dedicated microplate photometer.

The results of quantification of the concentration of Aflatoxin M1 by competitive type ELISA, with graded addition of conformation and orientation factors of the rat monoclonal antibody against Aflatoxin M1 in this example are shown in Figure 9. An optimized competitive type ELISA method curve was observed. The graded addition of conformation and orientation factors of the antibody against Aflatoxin M1, gave the antibody greater activity and consequently a lower concentration of it was achieved (6 times lower 0.33 pg/mL instead of 2 pg/mL). In this way, competition was favored without disturbing the immunochemical construct, by predominating the binding of the sample antigen to the monoclonal antibody (versus the binding of the HRP-conjugated antigen to the monoclonal antibody).

Example 16

Recovery of Aflatoxin M1 in fresh or homogenized cow's milk samples by a competitive-type ELISA with graded addition of conformational and targeting factors of rat monoclonal antibody to Aflatoxin M1

Corresponding samples of fresh or homogenized cow's milk to those of Example 14 were used in the method of Example 15.

The results of the recovery of Aflatoxin M1 in fresh or homogenized cow's milk samples by a competitive ELISA, with graded addition of Aflatoxin M1 antibody modulators and orienting agents, in this example are shown in Figure 10. In each case, the mean value was calculated of the recovery of the different concentrations of Aflatoxin M1. For the case of fresh cow's milk the recovery was 100.01%. For the case of homogenized cow's milk the recovery was 102.13% (acceptable levels). The optimal stereochemistry of the new immunochemical model, using nonionic surfactants, low molecular weight and low degree of hydrolysis polymers and disaccharides, created the conditions for the desired levels of recovery in homogenized cow's milk.

Example 17

Quantification of Aflatoxin M1 Concentration by Competitive Type ELISA with Graded Addition of Modulating and Orienting Factors of Aflatoxin M1 Rat Monoclonal Antibody and a Single Washing Step (1) The anti-rat IgG polyclonal antibody, produced in goat, which used in Example 10, was added in a volume of 100 µL/cell to each cell of a 96-cell microplate at a concentration of 10 µg/mL in PBS pH 7.5 and then fixed to the solid substrate at 4 °C for 16 h. The fluid from each cell was removed by aspiration and a solution containing Tween-20 (vacancy blocking) and a concentration of 0.33 µg/mL of the rat monoclonal antibody against Aflatoxin M1 used in Example 8 was added in a volume of 100 µL/cell. and each cell was incubated at 4 °C for 12 h. The fluid from each cell was removed by aspiration, a solution containing PVA and mycose (Example 2 & 3) (ensuring optimal stereochemistry of the indirect immunochemical model) was added in a volume of 300 µL/cell and each cell was incubated at 37 °C for 3 h, followed by removal of the solution also by suction.

(2) Aflatoxin M1 standard solutions (0, 5, 10, 25, 50, 100, and 250 ng/L) from reconstituted non-zero fat instant skimmed cow's milk powder were prepared and added in a volume of 100 μL/cell into the microplate in which the rat monoclonal antibody against Aflatoxin M1 had been indirectly coated on the solid support (1) and then the microplate was incubated at 25 °C for 60 min.

(3) HRP-labeled antigen, HRP-Aflatoxin (Example 4) was diluted to a concentration of 0.125x10<'3>μg/mL with PBS-Tween pH 7.5 to prepare the detection antigen solution and added in a volume of 100 μL/cell to the plate and reacted at 25 °C for 15 min.

(4) Each cell after reaction (3), was washed three times with PBS-Tween pH 7.5, and 100μL of the TMB substrate solution was added to each cell, followed by incubation at 25 °C for 15 minutes. Then 100 µL/cell of 15% phosphoric acid was added to each cell to terminate the chromogenic reaction and absorbance at 450nm was measured with a dedicated microplate photometer.

The results of quantification of the concentration of Aflatoxin M1 by competitive ELISA, with graded addition of conformation and orientation factors of rat monoclonal antibody against Aflatoxin M1 and a single step of washings in this example are shown in Figure 11. An optimized method curve was observed. Competitive type ELISA. This was because each standard solution acted as a simultaneous competition antigen.

Example 18

Recovery of Aflatoxin M1 in fresh or homogenized cow's milk samples by a competitive ELISA, with graded addition of rat monoclonal anti-Aflatoxin M1 conformation and orientation factors and a single washing step

Corresponding samples of fresh or homogenized cow's milk to those of Example 14 were used in the method of Example 17.

The results of the recovery of Aflatoxin M1 in fresh or homogenized cow's milk samples by a competitive ELISA, with graded addition of Aflatoxin M1 antibody conformers and orienting agents and a single wash step in this example are shown in Figure 12. In each case , the average value of the recovery of the different concentrations of Aflatoxin M1 was calculated. For the case of fresh cow's milk the recovery was 94.58%. For the case of homogenized cow's milk the recovery was 97.33% (acceptable levels). It is apparent that the new indirect immunochemical model, using the graded addition of Aflatoxin M1 antibody modulators and targeting agents and Aflatoxin M1 standard solutions from reconstituted non-zero fat instant skimmed cow's milk powder, has no limitations regarding the existence mixture of the standard solutions and the detection antigen (no washing step). The new immunochemical construction shows great resistance to the environment phenomenon (matrix effect).

Example 19

Recovery of Aflatoxin M1 in samples of skimmed or non-fresh cow's milk by competitive ELISA, with graded addition of rat monoclonal anti-Aflatoxin M1 conformation and orientation factors and a single washing step

Corresponding samples of skimmed or raw cow's milk to those of Example 14 were used in the method of Example 17.

The results of the recovery of Aflatoxin M1 in samples of skimmed or non-fresh cow's milk by competitive type ELISA, with graded addition of conformation and orientation factors of the rat monoclonal antibody against Aflatoxin M1 and a single washing step in this example are shown in Figure 13. In each case, the average value of the recovery of the different concentrations of Aflatoxin M1 was calculated. For the case of skimmed fresh cow's milk the recovery was 100.0%. For the case of non-skimmed cow's milk the recovery was 95.07% (acceptable levels). It is apparent that the new indirect immunochemical model, using the graded addition of Aflatoxin M1 antibody modulators and targeting agents and Aflatoxin M1 standard solutions from reconstituted non-zero fat instant skimmed cow's milk powder, has no limitations regarding the existence or no fat. The new immunochemical construction shows great resistance to the environment phenomenon (matrix effect).

Example 20

Quantification of Aflatoxin M1 Concentration by Graded Addition of Modulating and Orienting Factors of Aflatoxin M1 Rat Monoclonal Antibody and HRP-conjugated Larger Hydrodynamic Radius ELISA by Competitive Type ELISA

(1) The goat anti-rat IgG polyclonal antibody used in Example 10 was added in a volume of 100 µL/cell to each cell of a 96-cell microplate at a concentration of 10 µg/mL in PBS pH 7.5 and then fixed on the solid substrate at 4 °C for 16 h. The fluid from each cell was removed by aspiration and a solution containing Tween-20 (vacancy blocking) and a concentration of 0.25 µg/mL of the rat-produced anti-Aflatoxin M1 monoclonal antibody used in Example 8 was added to a volume of 100 µL/cell. and each cell was incubated at 4 °C for 12 h. The fluid from each cell was removed by aspiration, a solution containing PVA and mycose (Example 2 & 3) (ensuring optimal stereochemistry of the indirect immunochemical model) was added in a volume of 300 µL/cell and each cell was incubated at 37 °C for 3 h, followed by removal of the solution also by suction.

(2) Aflatoxin M1 standard solutions (0, 5, 10, 25, 50, 100, and 250 ng/L) from reconstituted non-zero fat instant skimmed cow's milk powder were prepared and added in a volume of 100 μL/cell into the microplate in which the rat monoclonal antibody against Aflatoxin M1 had been indirectly coated on the solid support (1) and then the microplate was incubated at 25 °C for 60 min.

(3) HRP-labeled longer hydrodynamic radius antigen, HRP-BSA-Aflatoxin M1 (Example 5) was diluted to a concentration of 0.44x10<-3>μg/mL with PBS-Tween pH 7.5 to prepare the longer hydrodynamic radius detection antigen solution.

(4) The plated cells (2) were washed three times with PBS-Tween pH 7.5, and then the longer hydrodynamic radius detection antigen solution (3) was added in a volume of 100 μL/cell to the plate and reacted at 25 °C for 15 min.

(5) Each cell after the reaction (4) as above, was washed three times with PBS-Tween pH 7.5, and 100μL of the TMB substrate solution was added to each cell, followed by incubation at 25 °C for 15 minutes. Then 100 µL/cell of 15% phosphoric acid was added to each cell to terminate the chromogenic reaction and absorbance at 450nm was measured with a dedicated microplate photometer.

The results of quantification of Aflatoxin M1 concentration by competitive ELISA, with graded addition of conformation and orientation factors of rat monoclonal antibody against Aflatoxin M1 and HRP-conjugated antigen of larger hydrodynamic radius in this example are shown in Figure 14. A optimized competition-type ELISA method curve relative to that of Example 15. Due to steric hindrance, using the HRP-conjugated antigen with a larger hydrodynamic radius, the competition step was relatively favored.

Example 21

Recovery of Aflatoxin M1 in fresh or homogenized cow's milk samples by competitive-type ELISA with graded addition of conformation and orientation factors of rat monoclonal antibody to Aflatoxin M1 and HRP-conjugated antigen of larger hydrodynamic radius

Corresponding samples of fresh or homogenized cow's milk to those of Example 14 were used in the method of Example 20.

The results of the recovery of Aflatoxin M1 in samples of fresh or homogenized cow's milk menu by competitive ELISA, with graded addition of Aflatoxin M1 antibody conformation and orientation factors and HRP-conjugated antigen of larger hydrodynamic radius in this example are shown in Figure 15. In each case, the mean value of the recovery of the different concentrations of Aflatoxin M1 was calculated. For the case of fresh cow's milk the recovery was 100.51%. For the case of homogenized cow's milk the recovery was 102.63% (acceptable levels). It is apparent that the new indirect immunochemical model, using the graded addition of Aflatoxin M1 antibody modulators and targeting agents and Aflatoxin M1 standard solutions from reconstituted non-zero fat instant skimmed cow's milk powder, has no limitations regarding the existence of different radius of HRP-conjugated antigen. The new immunochemical construction shows great flexibility in the molecular weight of the molecules that make it up.

Example 22

Quantification of Aflatoxin M1 concentration by competitive ELISA with graded addition of conformational and targeting factors of murine monoclonal antibody to Aflatoxin M1

(1) Anti-mouse IgG polyclonal antibody, produced in goat, was added in a volume of 100 μL/cell to each cell of a 96-cell microplate at a concentration of 10 μg/mL in PBS pH 7.5 and then fixed to the solid substrate at 4 °C for 16 hours. The fluid from each cell was removed by aspiration and a solution containing Tween-20 (vacancy blocking) and a concentration of 0.075 μg/mL mouse monoclonal antibody against Aflatoxin M1 was added in a volume of 100 μL/cell and each cell was incubated at 4 °C for 12 hours. The fluid from each cell was removed by aspiration, a solution containing PVA and mycose (Example 2 & 3) (ensuring optimal stereochemistry of the indirect immunochemical model) was added in a volume of 300 µL/cell and each cell was incubated at 37 °C for 3 h, followed by removal of the solution also by suction.

(2) Aflatoxin M1 standard solutions (0, 5, 10, 25, 50, 100, and 250 ng/L) from reconstituted non-zero fat instant skimmed cow's milk powder were prepared and added in a volume of 100 μL/cell into the microplate in which the mouse-produced monoclonal antibody against Aflatoxin M1 had been indirectly coated on the solid support (1) and then the microplate was incubated at 25 °C for 45 min.

(3) HRP-labeled antigen, HRP-Aflatoxin (Example 4) was diluted to a concentration of 8.33x1 0<'3>μg/mL with PBS-Tween pH 7.5 to prepare the detection antigen solution.

(4) The plated cells (2) were washed three times with PBS-Tween pH 7.5, and then the detection antigen solution (3) was added in a volume of 100 µL/cell to the plate and reacted at 25 °C for 15 min.

(5) Each cell after the reaction (4) as above, was washed three times with PBS-Tween pH 7.5, and 100μL of the TMB substrate solution was added to each cell, followed by incubation at 25 °C for 15 minutes. Then 100 µL/cell of 15% phosphoric acid was added to each cell to terminate the chromogenic reaction and absorbance at 450nm was measured with a dedicated microplate photometer.

The results of quantification of the concentration of Aflatoxin M1 by competitive type ELISA, with graded addition of modulating and targeting factors of the mouse produced monoclonal antibody against Aflatoxin M1 in this example are shown in Figure 16. A standard curve of competitive type ELISA method (optimized due to the higher affinity of the antibody).

Example 23

Recovery of Aflatoxin M1 in samples of fresh or homogenized cow's milk by competitive ELISA with graded addition of conformational and targeting factors of murine monoclonal antibody to Aflatoxin M1

Corresponding samples of fresh or homogenized cow's milk to those of Example 14 were used in the method of Example 22.

The results of the recovery of Aflatoxin M1 in samples of fresh or homogenized cow's milk by a competitive ELISA, with graded addition of Aflatoxin M1 antibody modulators and orienting factors, in this example are shown in Figure 17. In each case, the mean value was calculated of the recovery of the different concentrations of Aflatoxin M1. In the case of fresh cow's milk the recovery was 100.0%. For the case of homogenized cow's milk the recovery was 100.1% (acceptable levels). Clearly, the new indirect immunochemical model, using the graded addition of anti-Aflatoxin M1 antibody structuring and targeting agents and standard solutions of Aflatoxin M1 from reconstituted non-zero fat instant skimmed cow's milk powder, has no species limitations of the monoclonal antibody. The new immunochemical construction shows great flexibility in the type of molecules that compose it.

Example 24

Recovery of Aflatoxin M1 in Skimmed or Raw Cow's Milk Samples by Competitive Type ELISA with Graded Addition of Modulating and Orienting Factors of Rat Monoclonal Antibody to Aflatoxin M1 Corresponding skim or raw cow's milk samples to those of Example 14 were used in the method of Example 22.

The results of the recovery of Aflatoxin M1 in samples of skimmed or raw cow's milk by a competitive ELISA, with graded addition of conformational factors and orientation of the murine monoclonal antibody against Aflatoxin M1 in this example are shown in Figure 18. In each case , the average value of the recovery of the different concentrations of Aflatoxin M1 was calculated. For the case of skimmed fresh cow's milk the recovery was 100.0%. For the case of non-skimmed cow's milk the recovery was 99.33% (acceptable levels). Clearly, the new indirect immunochemical model, using the graded addition of anti-Aflatoxin M1 antibody structuring and targeting agents and standard solutions of Aflatoxin M1 from reconstituted non-zero fat instant skimmed cow's milk powder, has no species limitations of the monoclonal antibody and the presence or absence of fat. The new immunochemical construction shows great resistance to the environment phenomenon (matrix effect) and to the type of molecules that compose it.

BIBLIOGRAPHY REFERRING TO THE INVENTION

Ansari AA, Hattikudur NS, Joshy SR. and Medeira M.A. ELISA Solid Phase: Stability and Binding Characteristics. J. Immunol. Methods 1985, 84: 117-124.

Boyd S. and Yamazaki H. Use of polyvinyl alcohol as a stabilizer of peroxidase antibody conjugate for enzyme immunoassay. Biotechnol Tech 1994, 8: 123-128.

Bradford M. A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal Biochem 1976, 72: 248-254.

Farzamfar B., Bayanolhagh S., Mahboudi F. and Zahrai M, The effect of Different Stabilizers on Stability of Horseradish Peroxidase-Bovine Serum Albumin-Aflatoxin B1, a Conjugated Tracer for Detection of Aflatoxin B1 in Immunoassay-Based Methods. Iran J Pharm Res 2007, 6(3): 179-184.

Leslie SB, Israeli E, Lighthart B, Crowe JH. and Crowe L.M. Trehalose and sucrose protect both membranes and proteins in intact bacteria during drying. Appl Environ Microbiol 1995, 61(10): 3592-3597.

Claims (10)

1. An innovative modified competitive type immunomethod system for the quantitative detection of Aflatoxin M1 in milk or milk composition samples, comprising: (a) the indirect binding of shaped and oriented antibodies against Aflatoxin M1 to the solid substrate, through covalent or strong non-covalent bonding with different protein molecules which are directly immobilized on the solid substrate, where (i) in the immobilization step of the protein molecules, the remaining vacancies of the functional solid substrate are covered by non-ionic surfactants and (ii) after the step of creating the complex of antibodies against Aflatoxin M1 with the immobilized protein molecules, a mixture of low molecular weight and low degree of hydrolysis polymers and disaccharides is added (b) the binding of anti-Aflatoxin M1 antibodies to (i) the Aflatoxin M1 of the sample to be studied and (ii) the labeled form of the toxin or its conjugates with other molecules (c) detecting whether the binding of anti-Aflatoxin M1 antibodies to the Aflatoxin M1 molecules to be studied in the sample is increased by the presence of the sample; and (d) the quantification of the Aflatoxin M1 to be detected using non-zero fat flash skimmed milk powder standard solutions. 2. The immunomethod according to Claim 1, in which the non-ionic surfactants used are polysorbate 20 or Triton X-100 or a combination thereof at a rate of 0.01-0.2% and the polymers used are polyvinyl alcohol (PVA) or polyvinyl pyrrolidone (PVP) or a combination of these in a final concentration of 0.1 - 2% and the disaccharides used are mycose or sucrose or a combination of these in a final concentration of 0.5-10%. 3. The immunomethod according to Claim 2, in which the percentage of fatty acids of the instant skimmed milk powder is 0.05 - 0.1% and the percentage of said powder in the final solution of the standard solutions is 5 - 10%. 4. The immunomethod according to Claim 3, wherein the antibodies against Aflatoxin M1 are monoclonal or recombinant or polyclonal or a combination thereof and the directly immobilized protein molecules are antibodies against antibodies against Aflatoxin M1 or antibodies against molecules with which they have conjugate the anti-Aflatoxin M1 antibodies or proteins which show affinity to the anti-Aflatoxin M1 antibodies or proteins which show affinity to the molecules to which the anti-Aflatoxin M1 antibodies have been conjugated or a combination thereof. 5. The immunomethod according to Claim 4, in which the antibodies against Aflatoxin M1 are at a concentration of 0.01 - 1 µg/mL. 6. The immunomethod according to Claim 5, in which the directly immobilized protein molecules are monoclonal antibodies or recombinant antibodies or polyclonal antibodies or protein A or protein G or avidin or a combination thereof at a concentration of 1-10 µg/mL. 7. The immunomethod according to Claim 6, in which the antibodies against Aflatoxin M1 have not been conjugated or if they have been conjugated to biotin molecules. 8. The method according to Claim 7, in which the labeling of the toxin of the same or similar structure or the conjugates of the toxin of the same or similar structure has been done with peroxidase (HRP) or biotin or a fluorescent substance or a combination thereof. 9. The immunomethod according to Claim 8, wherein the solid support is an ELISA polystyrene plate (cells) or solid beads and is used in the ELISA. 10. An ELISA kit which uses the immunomethod of Claim 9, quantitatively detects Aflatoxin M1 in samples of milk or milk composition and has the following technical characteristics: 1) Polystyrene microplate to which the indirect binding of antibodies against Aflatoxin M1 has already been done. 2) Standard solutions of Aflatoxin M1 from reconstituted non-zero fat instant skimmed cow's milk powder, with a quantification range of at least 0-80 ng/L (e.g. 0, 5, 10, 25, 50, 100 and 250 ng/L L). 3) HRP-conjugated antigen solution. 4) Wash solution (eg PBS-Tween). 5) Substrate solution (eg TMB). 6) Termination solution (eg 15% phosphoric acid).