Description of the industrial invention entitled: "METHOD FOR DETECTING MYCOTOXINS IN MILK, DERIVATIVES AND DAIRY PRODUCTS"

FIELD OF THE INVENTION

The present invention relates to a method based on glucose oxidation for detecting mycotoxins in milk and derivatives or dairy products thereof, and suitable kits that make the performance of such method possible.

STATE OF THE ART

Mycotoxins are secondary metabolites, toxic substances, which are produced by some moulds. More than 300 of them have been identified. Toxicity for humans and animals is recognized for some of them, while for others the effects are not known yet. In any case, they are very stable substances that persist even after the disappearance of the moulds that have produced them.

Mycotoxins cause a reduction of performance in animals, an increase of incidence of diseases and eventually reproduction problems.

In humans, mycotoxins cause damage due to direct exposure to contaminants and indirect damage due to possible residues present in foodstuffs.

However, most of mycotoxins do not pass into foodstuffs as they are stopped or inactivated by the animal's metabolism (liver, rumen) . Sometimes metabolic conversion does not lead to inactive metabolites and their passing into food involves anyway inedibility of the latter, although the toxicity of metabolites can be lower than that of originary mycotoxins .

5 Mycotoxins present in feed administered to cows cause the presence of residues in milk, inedibility of the products, if they are present in an amount higher than the limits established by the law, and possible modifications of the dairy characteristics (non-positive effects on 10 microorganisms working in dairy transformations).

In the following table 1, mycotoxins of major interest in the specific field, the fungus producing such mycotoxins, the toxic effect and which contaminated food may contain them are reported.

15

Table 1

Mycotoxins Producing Toxic effect Contaminated fungus food

Aflatoxins - Bl, Flavus , Hepatitis , Peanuts and B2, Gl, G2, Ml, M2 Parasiticus , nephritis , other

other tumours leguminosae,

Aspergillus corn and spp. other metabolites cereals , oily seeds, walnuts and almonds . Milk and derivatives thereof

Zearalenones F. roseum, F . Hyperestrogenism, corn and

- zearalenone tricinctum, hypofertility other

- zearalenol F. cereals

moniliforme,

other

Fusarium spp. Ochratoxins Aspergillus nephritis , barley, corn

- ochratoxin A ocraceus , hepatitis and other

- ochratoxin B Penicillum cereals .

viridcans, Bread, pasta other and other

Aspergillus bakery spp. and products Penicillum

spp.

Trichothecenes F. rosum, F. Haemorrhage, corn, barley

- toxin T2 solani, F. leucopoenias , and other

- deoxymilvalenol tricintum, nervous cereals

and other disorders .

diacetoxyscirpenol Fusarium spp. Vomiting and food

- trivalenol refusal

Rubratoxin P. rubrum, P. Haemorrhage and corn and

- rubratoxin A purpurogenum, hepatic other

- rubratoxin B other degeneration cereals

Penicillum

spp.

Citrin Penicillum hepatitis, cereals

spp. nephritis

Table 1

Mycotoxicoses were identified for the first time forty years ago, but attention on mycotoxins has been increasing in these last years for a series of concomitant factors. 5 For example, there is better knowledge of their effects and of the fact that there are years with climatic conditions more favourable to their formation. Additionally, increased level of ingestion and the speeding up of feed passing at a ruminal level, caused by 10 the need of increased productivity, has lead to a reduction of degradation processes of mycotoxins at a ruminal level.

Increased productivity requested to zootechnic farming also involves the fact that feed rations with higher cereal contents are administered and consequently with higher risk of ingesting mycotoxin-contaminated feed.

Mycotoxins are classified according to the different effects they have on humans.

Class 1 mycotoxins are carcinogenic for humans, class 2A ones are probably carcinogenic, eventually class 2B ones are possibly carcinogenic.

Aflatoxins are produced mainly by Aspergillus flavus and Aspergillus parasiticus. Optimal temperatures for the development of these moulds are comprised between 27 °C and 37 °C. The animal body defends itself from the attack of mycotoxins through detoxification reactions leading to their excretion through body fluids such as milk, bile and urine. In cow milk almost exclusively aflatoxin Ml is found, while in buffalo milk also aflatoxin M2 and Bl are found. Aflatoxin Bl is certainly carcinogenic, hepatotoxic, it causes DNA damage and causes immunosuppression, then it belongs to said class 1, while Ml was classified as 2B, that is possibly carcinogenic.

The European legislation provided to set, for the safety of consumers' health, maximum limits for the presence of mycotoxin residues in food. In the case of milk, the content of aflatoxin Ml must not exceed 0.050 g/kg. For milk derivatives, the limit is set depending on the concentration factor of their components, this meaning, according to recent directions of the Ministry of Health, 0.150 g/kg for soft paste cheeses and for whey- derived products and 0.275 g/kg for hard paste cheeses.

Milk destined to infant formulas must display a maximum content of aflatoxin Ml lower than 0.025 g/kg.

Nevertheless, metabolites of other toxins can be found in milk, even if less toxic than aflatoxins.

For example, trichothecenes that can be found in milk are deoxynivalenol (DON) and its metabolite DOM-1, as well as DON-glucuronide conjugate, while ochratoxins that can be traced in cow milk are OTa and OTA. Eventually, zearalenones that can be found in milk are ZEA a, b metabolites and ZEA-glucuronide conjugate and zearalenol ZEL.

At present, known methods for detecting mycotoxins, and in particular aflatoxin Ml, are

- immunologic test and evaluation by reaction with monoclonal antibodies Ml

- evaluation of fluorescence

- HPLC chromatography.

Then, they all are methods that can be performed only in a laboratory, not directly in the field (at the farmhouse by a veterinary or a person in charge) .

Therefore there is the need of disposing of an easily employed analytical system able to determine the concentrations of mycotoxins - and in particular of aflatoxin Ml - that can be used non only in a highly specialized laboratory but directly in the place where cattle are, or in a milk room, or in a dairy. It would be extremely desirable to dispose of a detecting system able to measure a simple molecule, such as for example glucose, provided that it is released in equimolecular or anyway proportional amounts to the investigated toxin, and provided that the detector is able to measure the infinitesimal amounts possibly present.

In WO 2011/150186 a general methodology for the development of highly sensitive and selective sensors that can allow to employ glucometers usually employed by diabetics for the detection of some target molecules, among which also toxins, is disclosed.

In the Italian patent application NA 20009A000048 a methodology based on glucose detection for the detection of bacteria, virus, proteins, lipids, toxins, enzymes and/or nucleic acids in blood, sweat, urine, interstitial fluids or in water, food, is disclosed.

Both methods however imply the use of commercial glucometers for diabetics, which display a series of limitations. First of all, the fact that they allow the determination of glucose concentrations in a range between 1. OmM and about 12. OmM (compatible to measure hypo-, normo- , and hyper-glycaemias in humans), and then they are not suitable to evaluate the dosage of glucose in the scope of 0. OlmM-0. ImM, that is the requested range for the specific application in the field of mycotoxin detection in milk. Additionally, another problem that can be encountered using commercial glucometers for such type of application lays in the poor reproducibility of such detections. In fact, commercial glucometers are built so as to have precision oscillations as big as 20%. A similar instrumental accuracy is not suitable for example for the detection of mycotoxins, such as aflatoxin Ml, in milk.

Object of the present invention is then to identify a reproducible and readily performed analytical method, as well as related analytical devices having high sensitivity in detecting very low glucose concentrations (e.g.: 0.01- O.lmM) and then giving a more precise and reproducible measurement .

SUMMARY OF THE INVENTION

The Applicant has now found that it is possible to overcome the disadvantages of the state of the art through a method for detecting mycotoxins by immunoassay of the same with detection of glucose, by means of mycotoxin- monospecific primary antibodies, which are properly derivatized with an enzyme causing glucose formation starting from more complex sugars. This method preferably allows the detection of glucose ranging between O.OlmM and O.lmM.

According to a preferred embodiment, it comprises the following steps:

A. treating the milk to be analysed, to which methanol at the final concentration of 10% was added, with a recognition molecule comprising a mycotoxin-monospecific primary antibody bound to the enzyme invertase;

B. then adding said milk to a solid support on which the specific mycotoxin to be analysed is immobilized;

C. treating the solid support coming from step (B) with saccharose and a buffered aqueous solution;

D. detecting glucose formed by the enzymatic reaction by the invertase.

In particular, this method can be carried out through a direct methodology comprising the following steps:

a. adding a recognition molecule comprising an antibody specific for said mycotoxin, bound to the enzyme invertase, to properly prepared milk or a derivative thereof;

b. adding said milk or derivative thereof from step (a) to a solid support on which the mycotoxin is immobilized;

c. carrying out a washing of the solid support; d. treating the solid support with a buffered saccharose solution;

e. then detecting the formed glucose, in the presence of the invertase enzyme bound to the recognition molecule, with the glucose being lower the higher is the amount of mycotoxin contained in milk, by means of a nanoglucometer able to detect amounts of glucose at concentrations ranging between 0.0 ImM and O.lmM.

In this case, further object of the present invention is a kit (1) that allows performing such direct method, comprising :

i. a recognition molecule comprising a mycotoxin- monospecific antibody: said antibody is bound to the enzyme invertase ;

ii. a solid support on which the specific mycotoxin to be detected is immobilized;

iii. a buffered aqueous saccharose solution;

iv. a set of data, graphs/diagrams in the form of paper or electronic material, for example CD, DVD, etc. that allows to correlate the presence or absence or the increase/reduction of glucose, and the amount of mycotoxin present in the analysed milk;

v. a nanoglucometer able to detect glucose concentrations ranging between O.OlmM and O.lmM. Alternatively, the method of the invention can be carried out through an indirect methodology comprising the following steps:

a' ) adding a recognition molecule including a monospecific antibody directed against the mycotoxin to milk;

b' ) adding the milk coming from step a' ) to a solid support on which the target molecule is immobilized;

c' ) carrying out a washing of the solid support coming from the previous step;

d' ) submerging the solid support from the previous washing in an aqueous solution containing an invertase enzyme-bound secondary antibody, specific for the primary antibody directed against the mycotoxin;

e' ) carrying out a washing of the support coming from the previous step;

f' ) adding a buffered aqueous saccharose solution; g' ) then detecting the formed glucose, in the presence of the invertase enzyme-bound secondary antibody, with the glucose being lower the higher is the amount of mycotoxin contained in milk.

A further object of the present invention is a kit (2) to perform the method object of the present invention according to the indirect methodology and comprising the following components: i' ) a recognition molecule comprising a mycotoxin- specific antibody;

ii' ) a solid support on which the specific mycotoxin to be detected is immobilized;

iii' ) a buffered aqueous solution containing at least one invertase enzyme-bound secondary antibody, directed against the primary antibody specific against the mycotoxin (recognition molecule) ;

iv' ) a buffered aqueous saccharose solution;

v' ) a set of data, graphs/diagrams in the form of paper or electronic material, for example CD, DVD, etc. that allows to correlate the presence or absence or the increase/reduction of glucose, and the amount of mycotoxin present in the analysed milk;

vi' ) a nanoglucometer able to detect glucose concentrations ranging between O.OlmM and O.lmM.

Further object of the present invention is the nanoglucometer comprised in the kits according to the present invention able to detect concentrations ranging within said range, which was properly modified and is able to detect power variations in the order of nano-Ampere and then at least 1000-fold lower than those used for traditional nanoglucometers.

DESCRIPTION OF THE FIGURES Figure 1 represents a picture of a TLC plate eluted with dichloromethane : methanol to verify the completion of the reaction described in example C.

Figure 2 is a picture of the plate following the Western Blot experiment described in example C, on which the conjugate dimer Gln-BP-AFMi and Gln-BP previously dissolved in Laemmli buffer were allowed to run in a 12% SDS-PAGE experiment together with specific molecular weight markers in kDa designated as WM.

Figure 3 represents the diagram of immunoreactivity of rabbit IgGs against GLNBP-AFMi, as described in the ELISA assay of example D, where on the ordinate the optical density at 450 nm is reported and on the abscissa the reciprocal of the dilution (1/dilution) is reported and wherein NO-Coating stands for no coating, cntr+ stands for positive control, while contr- stands for negative control .

Figure 4 represents a picture of the gel, on which the following IgG fractions were charged:

· IgG not bound to the resin EAH - AFMi, designated with the acronym FT in the picture;

• IgG washed away during washings, designated with Wl- 5 in the picture,

• IgG eluted following drastic pH change, designated with E1-E7 in the picture, ^• specific molecular weight markers in kDa designated with WM,

which were allowed to run in 12% SDS-PAGE as reported in assay E.

Figure 5 reports, in the form of a block diagram, the ELISA assay of reactivity of IgGs measured as a function of light absorption measured at the wavelength of 450 nm in the case of non-coated IgGs (no coating) and against GlnBP-AFMi, GlnBP and BSA at a dilution 1:8000 as reported in example E.

Figure 6 shows results of competitive indirect ELISA assay of reactivity of monospecific IgGs, expressed as % inhibition of binding of anti-AFMi IgGs, against aflatoxin Ml, in solution as a function of aflatoxin concentration, as described in example G.

Figure 7 reports the picture of a tube containing a dialysis membrane clamped between 2 pincers on which the enzymatic assay and reactions involved are carried out.

Figure 8 represents the reactions involved in the method according to the present invention.

DETAILED DESCRIPTION OF THE INVENTION

For the objects of the present invention, in the definition of milk all the types of mammal milk are comprised, such as for example cow's milk, buffalo's milk, ovine milk, such as for example goat's, sheep's milk, pig milk such as sow's, boar's milk, horse milk such as for example donkey's or horse's milk. By milk derivatives, for example, cream and fermentation products too, such as yoghurt, kefir, etc. are meant.

By dairy products, in addition to cheeses be they fresh (stretched curd and not) or seasoned, soft, hard or semihard, also dairy products such as butter, melted cheeses, whey, ricotta and ricotta cheese are meant.

For the objects of the present invention, dairy products before being subjected to this method are treated with a sodium citrate solution to dissolve casein.

For the objects of the present invention, by recognition molecule a molecule comprising a primary antibody directed against the mycotoxin possibly present in milk and the immobilized mycotoxin on the solid support, bound to invertase, is meant.

By target molecule the immobilized mycotoxin on the solid support (ii) and able to compete with the same mycotoxin possibly contained in milk is meant.

For the objects of the present invention, by nanoglucometer (iv) an instrument able to detect glucose concentrations ranging between O.OlmM and 0.1M is meant.

This instrument is a properly modified traditional glucometer able to detect power variations in the order of nano-Ampere and then at least 1000-fold lower than those used for traditional glucometers.

For the objects of the present invention, by primary antibody a monospecific antibody against the aflatoxin to be analysed in milk is meant.

Hereinafter some preferred embodiments of some fundamental components to carry out the method object of the present invention and contained respectively in kits (1) and (2) are reported more in detail.

RECOGNITION MOLECULE

The recognition molecule (i) object of the present invention employed in the method for the detection of mycotoxins possibly present in milk, derivatives or dairy products thereof, and in the kit to perform such method includes in turn a monospecific primary antibody of said mycotoxin bound to invertase.

This recognition molecule, when it comes into contact with milk, a derivative or a dairy product thereof contaminated by a mycotoxin, for example AFMi, binds the present mycotoxin that competes with the one immobilized on the solid support. Such recognition molecule, once it comes into contact with the saccharose solution, produces a lower amount of glucose than the amount of glucose produced in the absence of such mycotoxin. Based on the amount of glucose produced, it is then possible to go back to the amount of mycotoxin.

Preferably the enzyme invertase contained in it is directly bound to said monospecific antibody by a conjugation ^' agent that, according to a preferred embodiment, is glutaraldehyde . In particular, the antibody present in the recognition molecule is monospecific against a mycotoxin present in milk and in particular belonging for example to aflatoxins, zearalenones , trichothecenes and ochratoxins.

Preferred recognition molecules are those containing monospecific antibodies against aflatoxins, since aflatoxins are the most dangerous and most frequently found mycotoxins in milk and derivatives thereof. Particularly preferred recognition molecules are those containing monospecific antibodies, against aflatoxins Ml, Bl and M2 and even more preferable are those containing monospecific antibodies against aflatoxin Ml.

SOLID SUPPORT

The solid support (ii), used in the method for detecting mycotoxins and contained in the kit according to the present invention, generally consists of a solid material of a known type and characterized by properties such as for example: - an activation surface, which, following stimulation, is able to form with the target molecule according to the present invention,

- chemical inertia, so that those areas on the support not occupied by the recognition molecule able to bind to the target molecule, do not form aspecific bonds or, if they do, the latter are readily removable without removing the recognition molecule.

The solid support can have characteristics promoting binding between recognition molecule and target molecule immobilized on the support. The solid support has additionally an immobilized enzyme able to oxidize glucose, produced by the invertase, contained in the recognition molecule. This type of enzyme is preferably glucose oxidase that allows the detection of glucose by the nanoglucometer .

The surface of a solid support can be activated through chemical processes able to form stable bonds with the solid support. However, any other method can be used for the aims object of the present invention, such as for example ionic interactions, hydrophobic, covalent interactions, and the like.

In a preferred embodiment, the solid support is in the form of a sub-micronic or micronic bead. These beads can consist of metal, metal derivatives, such as for example metal oxides, derivatives, non-metal, magnetic particles.

In another preferred embodiment, solid supports are porous, preferably in the form of a material such as paper, membrane, water-insoluble polymer. For example, the solid support can comprise a membrane that allows some materials to pass and that selectively traps others. Preferably this material comprises nitrocellulose. In some preferred embodiments, solid supports such as nitrocellulose are in the form of sheets, strips, such as those in use in a lateral flow device.

In another preferred embodiment, the solid support consists of an organic polymer such as for example polypropylene, polyethylene, polybutylene, polyisobutylene, polybutadiene , polyisoprene , polyvinylpyrrolidone, polyvinilidenefluoride, polytetrafluoroethylene, etc.

The solid support for example, can be the in form of a coating or laminate coated on an inert material such as paper, glass, plastic film or fabrics.

The solid support can have any form such as micro- titre plates, ELISA plates, test probes, immersion rods, lateral flow devices.

According to a particularly preferred solution, the solid support is preferably in the form of a paper strip, more preferably of nitrocellulose, on which the target molecule able to bind the recognition molecule is immobilized (see figure 8).

SACCHAROSE AQUEOUS SOLUTION

The saccharose concentration employed in the method according to the present invention is not a critical parameter for the methodology object of the invention, even if preferably saccharose aqueous solutions with concentrations between 5 and 20% weight/volume, even more preferably between 8 and 15% w/v, are used.

GLUCOSE DETECTOR (NANOGLUCOMETER)

The nanoglucometric detector used in the method according to the present invention, in the 2 kits further object of the present invention, preferably includes a software that allows to perform a high number of glucose determinations in a short time, for example 10 determinations per minute, and to provide the mean value of such determinations.

Additionally, it can comprise LEDs able to report the glucose value and/or coloured LEDs that are lit up depending on the presence or absence of aflatoxins and/or their concentration.

The nanoglucometer can be wireless connected by GPS, GSM and Wi-Fi, to an operation centre where results can be recorded and/or processed. DIRECT METHODOLOGY/INDIRECT METHODOLOGY

Using the direct method, a measurement is obtained faster, on the other hand, employing the indirect method measurements can be performed on more types of mycotoxins using the same an invertase-bound secondary antibody and different specific primary antibodies targeted against different mycotoxins. With the method object of the present invention that allows to detect glucose concentrations ranging between 0. ΙμΜ and ΙΟΟμΜ, it is possible to evaluate in a reliable manner the amount of mycotoxins in milk, derivatives and dairy products thereof, without need of complex laboratory equipment.

For illustrative purpose the method of the invention is reported in detail.

Selection of most suitable glucometers for the aim of the invention

Glucometers that appeared to be suitable for this strategy use, as a detector enzyme on the reactive strips, glucose oxidase. Glucometers that use other enzymatic systems give non-reliable results due to the presence of lactose recognized as a substrate (example A)

Obtaining anti-aflatoxin Ml antibodies

To obtain a high titre of antibodies targeted against AFMi, two rabbits were hyper-immunized with a (marketed) preparation of BSA conjugated with AFMi . Once the reactivity of sera against AFMi was confirmed (challenging them against AFMi conjugated with GlnBP) , the two rabbits were sacrificed for the final draw of sera, of about 50 ml per rabbit. From these sera IgGs were purified using an affinity resin with protein A (example B) .

Obtaining conjugates of aflatoxin Ml with GlnBP protein (Glutamine Binding Protein) and assay

To obtain an antigen that could be used for antibodies specificity tests, aflatoxin was conjugated with a different protein from that used as a carrier for immunization (GlnBP-AFMi) (example C) .

Validation

IgGs were challenged in a Western Blot assay and they identified a band of about 50 kDa, corresponding to a dimer of GlnBP formed during conjugation as the main product conjugated with AFMi. Such experiment allowed to confirm the specificity of IgG recognition of the GlnBP-AFMi conjugate, which was then used to carry out all the ELISA assays .

ELISA Assays of purified IgGs with reference antigen

GlnBP-AFMi

Such experiments allowed to confirm a high titre of reactive IgGs in both immunized rabbits (# 068 and # 069) detectable up to a dilution 1:16,000 with a 12.5 ng coating of GlnBP-AFMi per well (example D) . Monospecific IgGs anti-aflatoxin Ml

Monospecific IgGs for aflatoxin Ml (IgGMS) were obtained purifying them from total IgGs by affinity chromatography .

a. Preparing the affinity resin

b. Purification of the monospecific antibodies (example E)

IgGMS were challenged in ELISA assays which allowed to demonstrate, as compared to total IgGs, a significant reduction of the reactivity for BSA used as a carrier during immunization and to confirm a high titre of reactive IgGs against GlnBP-AFMi at a 1:8000 dilution, and no reactivity against non-conjugated GlnBP.

Competitive assay

Indirect competitive ELISA assays allowed to confirm high affinity of IgGMS for aflatoxin Ml in solution, what allowed to set up a highly sensitive assay (example F) .

Antibodies derivatization (total IgGs) with the enzyme invertase

The strategy of the project involves detecting binding of the antigen (AFMi) with antibodies through the invertase activity. A protocol was set up, which uses glutaraldehyde as a homofunctional and bifunctional binding agent, which allowed to obtain a conjugate of total IgGs with invertase (Saccharomyces cerevisiae, FLUKA) . The conjugate was affinity immobilized on a resin able to bind IgGs (example G) .

Assays to verify that conjugation with antibodies has not destroyed the enzymatic activity of invertase

Subsequently, the invertase activity of the conjugate was assayed. The enzymatic assay allowed to confirm the invertase activity of the conjugates with total IgGs at 15 min with a glucose production at the concentration of 2 mg/dl, also taking into account the time needed for the diffusion of substrates and products and the dilution volume (example H) .

Purification of the conjugate

The conjugate was purified with an affinity resin able to bind the constant portion of the antibodies (example I) .

Assay to verify the antibody activity of the conjugate

Purified conjugates were challenged in a Dot blot assay, after an incubation time of 5 min. Small squares of 1 cm ² on which BSA had been adsorbed, gave a positive result for invertase activity, while those on which GlnBP had been adsorbed gave a negative result (example J) .

In the whole, these assays demonstrate that the conjugation procedure preserves both antibody and enzymatic reactivity. Purification of conjugated monospecific antibodies anti-aflatoxin Ml (Example K) .

Immunoenzymatic assays of IgGMS-Ml-I V

For these assays a protein that is commercially available conjugated with aflatoxins (HRP-AFL) was used, the assay allowed to detect the protein derivatized with aflatoxins (HRP-AFL) , and not the other proteins incubated with the conjugates IgGMS-Ml-INV (example L) .

Assay to verify interference of the binding conditions in the presence of milk and methanol

The experiment allowed to demonstrate that there was no interference of milk during incubation of the IgGMS-Ml- INV conjugate with nitrocellulose strips, and it was possible to detect invertase activity after 1 h of incubation at room temperature. Additionally, it was assessed that there was no interference in binding of IgGMS-Ml-INV against HRP-AFL, by adding 10% methanol to the solution. The solvent in solution will be necessary to add free aflatoxin Ml in the next competitive assays or to allow solubilisation of aflatoxin Ml in possibly contaminated milk (example M) .

Competitive assay in the presence of milk

After incubation in the absence of AFMi, about 1.5 μg of glucose was produced, and it was possible to detect 80% and 50% reduction of produced glucose, after incubation of free AFMi at 54 ppt and 27 ppt, respectively (example N) .

EXAMPLE A

• Saccharase activity assay

Materials :

° 1.0 ml of 0.1M acetate buffer pH 4.6.

° 0.5 ml of 10% w/v saccharose solution (freshly prepared) .

° 0.1 ml of 7 U/ml invertase (Saccharomyces cerevisiae, FLUKA) or water (Sample Blank (blank) ) .

° 0.4 ml of 0.3M Tris Base Buffer (Stop Solution). Procedure :

° Add 0.1 ml of enzyme to the solution to assay the invertase activity: 1 ml of 0.1M acetate buffer pH 4.6 and 0.5 ml of a 10.0% w/v saccharose solution and incubate for 5 min at room temperature.

Add Stop Solution, shake well and place in ice.

Assay of produced glucose (D-GLUCOSE GOPOD-FORMAT

Megazyme)

Materials :

o 0.1 ml of assay invertase.

°0.1 ml of 100 mg/dl Standard Glucose (1 pg/μΐ) .

°1.0 ml of assay mix.

Procedure : ° Incubate at 45°C for 20 min and read absorption on a spectrophotometer at 510 nm.

° Reading vs. Sample Blank (blank) and determination of the concentration of produced glucose.

• Glucose assay with Breeze 2 (Bayer) and with ACCU CHEK AVIVA (Roche) .

° 2 μΐ of assay invertase.

° 2 μΐ of Sample Blank.

° Assay conditions 0.7 U invertase at RT for 5 min. GLUCOMETERS

Conditions required to carry out the enzymatic assays of invertase activity were optimized, measuring produced glucose, both with commercial glucometers and, in parallel, with a laboratory kit for glucose detection in food as a control.

Subsequently, commercial glucometers adopting reactive strips that used enzymes recognizing glucose only, and not other simple or complex sugars as a substrate, were identified (Breeze 2, Bayer) . Glucometers that appeared suitable for this purpose used glucose oxidase as a detector enzyme on the reactive strips.

EXAMPLE B

• 63-Day hyper-immunization protocol

Materials :

° BSA-AFMi Conjugate 400 μq . ° Rabbits 068 and 069 (Covalab) .

Procedure :

° Injection on days 14, 28 and 42.

° Blood draws for test on days 0, 39, 53.

° Final draw on day 63.

• Total IgGs purification

Materials :

° 0.5 ml of nProtein A Sepharose™ 4 Fast Flow resin (GE Healthcare) .

° 50mM Tris buffer Binding Buffer pH 7.4.

° Affinity chromatography 10 ml columns (Biorad) . ° 2 ml of sera of rabbits 068 and 069 filtered and diluted 1:1 in Binding Buffer.

Procedure :

° Wash the resin with water and Binding Buffer and add diluted sera, incubate ON at 4°C under slow stirring.

° Wash at RT with 5 ml of Binding Buffer.

° Elute at RT with 5 1-ml fractions in 0.1M glycine buffer pH 3, and buffer at pH 7 with Tris Buffer pH 8.8.

° Quantify on a spectrophotometer and on SDS-PAGE fractions containing IgGs, then pool them in a single sample .

OBTAINING ANTIBODIES ANTI AFLATOXIN Ml

For this purpose a BSA-AFMi conjugate was purchased; 0.4 mg of the conjugate were sent to the Covalab company to immunize two rabbits, in order to obtain antibodies targeted against aflatoxin Mi.

Two rabbits, defined 068 and 069 respectively, underwent a hyper-immunization protocol lasting 63 days. 3 injections (boost) of BSA-AFMi conjugate were performed on days 14, 28 and 42; and serum samples drawn on days 0, 39 and 53 of the immunization protocol before the final draw on the 63 ^rd day.

During the various phases of the immunization protocol in cooperation with Covalab, titre and reactivity of IgGs present in sera drawn at various time points of the immunization protocol were monitored in our laboratory.

To do so, IgGs were purified from serum of the two rabbits, by affinity chromatography thanks to the use of a protein A-conjugated Sepharose resin, which is able to bind with high affinity the constant portion of the rabbit IgGs, for their subsequent high efficiency purification.

For this purpose 2 ml of rabbit sera were purified with 0.5 ml of nProtein A Sepharose™ 4 Fast Flow resin (GE Healthcare) . To test the reactivity of IgGs against aflatoxin Mi alone and not against BSA (Bovine Serum Albumin) , the carrier protein used for immunization, ELISA assays were carried out (Enzyme-linked immunosorbent assay) challenging IgGs against aflatoxin Mi conjugated with another protein (Glutamine Binding Protein) . Subsequently, once reactivity against another protein conjugated to AFMi was confirmed, the two rabbits were sacrificed for the final draw of sera, of about 50 ml per rabbit .

EXAMPLE C

• Derivatization of AFMi with a carboxylic arm

Materials :

° 1 mg of aflatoxin Ml.

° 3 mg of carboxylic arm (0- (Carboxymethyl ) - hydroxylamine, hemi-hydrochloride, Sigma-Aldrich) .

Procedure :

° Incubate at reflux for 2 h in H ₂ 0:Py:MeOH (1:1:4). ° Take to dryness.

° Redissolve the raw material in H2O and alkalinize with NaOH (pH 8 ) ..

° Wash with CH ₂ C1 ₂ 3 times.

⁰ Acidify the aqueous phase with HCl pH 3.

0 Extract in CH2CI2 3 times.

• Conjugation with GlnBP

Materials:

° 3.3 mg of EDC-HC1 (N- ( 3-Dimethylaminopropyl) -N ' - ethylcarbodiimide hydrochloride) (Sigma-Aldrich) .

° 160 μΐ of 0.7 mg/ml GlnBP (Glutamine Binding Protein) .

Procedure : ° Take to dryness.

° Resuspend in H ₂ 0, 20% EtOH.

° Add 1.1 mg of EDC-HC1, incubate for 5 min at RT.

° Add GlnBP and incubate for 48 h at RT under slow stirring.

° Add 1.1 mg of EDC-HC1 again at 16 and 32 h.

CONJUGATION OF AFLATOXIN Mi WITH GLNBP AND VALIDATION

For this purpose the protein GlnBP (Glutamine Binding Protein) was purified through ion exchange chromatography involving the use of DEAE-Sepharose .

Commercial aflatoxin Mi was chemically modified introducing an arm containing a carboxylic group that, properly activated, allowed to conjugate, by a peptide bond, aflatoxin Mi to the lateral chains of the glutamine residues present in GlnBP.

For this purpose 1 mg of aflatoxin Mi and 3 mg of the 0- (carboxymethyl) -hydroxylamine hemi-hydrochloride arm ( Sigma-Aldrich) were allowed to react at reflux for 2 hours in 1.2 ml of a solution of pyridine, deionised water and methanol having the following ratio 1:1:4.

At the end of the reaction a thin layer chromatography is carried out (TLC) to verify the formation of the derivative, using an eluting solution of dichloromethane and methanol, in a 95:5 ratio. Once the formation of the aflatoxin Mi derivative is assessed, the reagent mixture is put to dry.

To purify the aflatoxin Mi derivative with the carboxylic arm a liquid-liquid extraction was carried out to remove excess reagents. The dry reagents mixture was resuspended in a solution of deionised water taken to pH 8, and three 1-ml washings were performed with dichloromethane .

After the washings, the aqueous solution was acidified at pH 3, and three 1-ml extractions were performed in dichloromethane. Afterwards, a thin layer chromatography (TLC) of the derivative recovered from the three extractions was carried out, using the same running conditions as previously described (see figure 1).

All the derivative obtained was taken to dryness and resuspended in 20% ethanol solution and incubated for 5 min with 1.1 mg of EDC-HC1 (N- ( 3-dimethylaminopropyl ) -N ' - ethylcarbodiimide hydrochloride) (Sigma-Aldrich) to activate the carboxylic group, and was allowed to react with 260 g of GlnBP so as to have a molar ratio of 1:250 with respect to the aflatoxin Mi derivative.

The reaction occurred at room temperature for 48 hours during which 1.1 mg of EDC was added twice again.

The conjugate thus obtained was tested in a Western Blot experiment. In the experiment 0.1 g of the GlnBP- AFMi conjugate and 1 pg of GlnBP were dissolved in Laemmli buffer and run on 12% SDS-PAGE. Proteins were transferred on a PVDF membrane (Millipore) , previously activated in methanol, using the trans Blot Semy-dry System (Biorad Laboratories) , and assayed with the IgGs of rabbit 068, as a primary antibody at a dilution of 1:500.

The IgGs identified a band of about 50 kDa, corresponding to a dimer of GlnBP formed during conjugation as a main product conjugated to AFMi; they do not recognize GlnBP alone that has a molecular weight of about 27 kDa. Such experiment allowed to confirm the specificity of IgG recognition of the GlnBP-AFMi conjugate, which was used to carry out all the ELISA assays.

Plate after Western Blot (FIG. 2) :

The arrow shows the GlnBP-AFMi conjugate recognized by antibodies from rabbits immunized with BSA-AFMi.

The band of the molecular weight marker at 66 kD is BSA. GlnBP alone has a molecular weight of 27 kD.

EXAMPLE D

· Indirect ELISA assays

Materials :

° MaxiSorp™ 96-well plates (Nunc) .

° Coating Buffer 25mM carbonate buffer pH 9.6

° Wash Buffer 0.05% TBST. ° Blocking Solution 0.5% ovalbumine (OVA) in TBS Buffer (20mM Tris, 130mM NaCl) .

° Diluting solution 0.25% ovalbumine in 0.05% TBST Buffer.

° Development solution T B ( 3 , 3 ' , 5 , 5 ' -Tetrarnethyl- benzidine, Sigma) .

° Stop Solution 2.5M HC1.

Procedure :

° To adsorb the GlnBP-AFMi conjugate in the plate wells, Coating Buffer was used and placed ON at 4°C.

° Incubate 50 μΐ of various dilutions of the GlnBP- AFMi conjugate in Coating Buffer, starting from a solution 2 ng/μΐ concentrated (total 100 ng) ON (overnight) at 4°C.

° Perform 3 50-μ1 washings for 10 min with Wash Buffer.

° Assay serial 1:2 dilutions of the GlnBP-AFMi conjugate with serial dilutions of IgGTot purified from both 068 and 069 rabbits, use a starting dilution of 1:4000 and serial 1:2 dilutions until 1:32000, and perform each point in duplicate.

° Add 50 μΐ of Blocking Solution per well and incubate at 37°C for 1 h.

° Perform 3 50-μ1 washings for 10 min with Wash Buffer.

° Dilute primary and secondary antibodies in Diluting Solution, add 50 μΐ per each well and incubate for 1 h at 37°C. ° Perform 3 50-μ1 washings for 10 min with Wash Buffer. ° Use secondary anti-rabbit antibody at 1:6000 dilution .

° Perform 3 50-μ1 washings for 10 min with Wash Buffer. ° Add 100 μΐ of TMB for development, 10 min in the dark, and stop reaction adding 100 μΐ of Stop Solution.

° Perform reading on the plate analyzer measuring absorption at 450 nm.

ELISA ASSAYS OF ANTIBODIES (IgG) WITH REFERENCE ANTIGEN GLNBP-AFMl

Purified immunoglobulins were challenged in indirect ELISA assays, which involve the use of a secondary antibody conjugated with peroxidase, able to recognize IgGs of rabbit .

For this purpose for the ELISA assays MaxiSorp™ 96- well plates (Nunc) were used.

To adsorb the GlnBP-AFMi conjugate on the plate wells (coating) a 25mM carbonate Coating buffer pH 9.6 was used and placed ON at 4°C.

In each well 50 μΐ of various dilutions of the GlnBP-

AFMi conjugate (coating) were incubated starting from a solution 2 ng/μΐ concentrated (total 100 ng) . Serial 1:2 dilutions of the conjugate were assayed with serial dilutions of total IgGs purified from both 068 and 069 rabbits. IgGs were used at a starting dilution of 1:4000 and serial 1:2 dilutions until 1:32000, each point was performed in duplicate.

After coating, 3 washings were performed for 10 min with 0.05% TBST Buffer (20mM Tris, 130m NaCl, 0.05% Tween- 20) .

Subsequently a 0.5% OVA solution (ovalbumine) was used in TBS buffer (20mM Tris, 130mM NaCl) as a Blocking solution, 50 μΐ per each well and the plate was placed in an incubator at 37 °C for 1 h.

3 washings were performed for 10 min with 0.05% TBST

Buffer (20mM Tris, 130mM NaCl, 0.05% Tween-20).

Antibodies were diluted in 0.25% OVA solution (ovalbumine), 0.05% TBST (20mM Tris, 130mM NaCl, 0.05% Tween-20), 50 μΐ were used per each well and incubated for 1 h at 37°C.

3 washings were performed for 10 min at RT with 0.05% TBST Buffer.

The secondary anti-rabbit antibody was used at 1:6000 dilution .

Once washings were finished after incubation of the secondary antibody, for the development of the peroxidase reaction, the wells were incubated in the dark for 10 min with 0.1 ml of TMB (3, 3' , 5, 5' -Tetramethyl-benzidine, Sigma). To stop the colorimetric reaction 0.1 ml of a 2.5M HC1 solution were added. The absorbance reading was carried out on a plate analyzer at the wavelength of 450 nra.

Such experiments allowed to confirm a high titre of reactive IgGs in both immunized rabbits 068 and 069 at 1:16,000 dilution, with a coating of 12.5 ng of GlnBP-AFMi per well. In the ELISA assay, as a positive control 25 ng BSA, and as a negative control 25 ng of GlnBP (see Fig.3) were used.

EXAMPLE E

· Preparing affinity resin

Materials :

° 1.2 ml of EAH Sepharose™ 4B (GE Healthcare).

° 1.0 mg of carboxylic derivative used in example B ° 10 ml columns for affinity chromatography (Biorad) . ° deionised water at pH 4.5.

° EDC-HC1 to be used at 0.1M final concentration.

Procedure :

° Dissolve the derivative in 2 ml of EtOH.

° Add EDC-HC1 at 0.1M final concentration and incubate for 5 min at RT .

° Add EAH Sepharose™ 4B resin, take to 4 ml volume and incubate at 4°C under slow stirring ON.

• Purification of monospecific antibodies

Materials : ° 8 mg total IgGs (2 mg/ml) 1:1 diluted in Binding Buffer.

° Binding Buffer 50mM Tris buffer pH 7.4.

° 1.2 ml of EAH-AFMi resin (GE Healthcare) .

° Washing buffer 50mM Tris pH 7.4.

° 10 ml columns for affinity chromatography (Biorad) . Procedure :

° Wash the resin with water and Binding Buffer and add diluted IgGs, incubate overnight at 4°C under slow stirring.

° Wash at room temperature with 12 ml of Binding Buffer.

° Elute at room temperature with 10 1-ml fractions in 0.1M glycine buffer pH 3 and buffer at pH 7 with Tris Buffer pH 8.8.

° Quantify on a spectrophotometer and on SDS-PAGE the fractions containing IgGMS, then pool them in a single sample .

° ELISA Test to assess reactivity against BSA and GlnBP-AFMi.

ISOLATION OF MONOSPECIFIC IgGs ANTI-AFLATOXIN Mi a) Preparing affinity resin

For biosensor setup, once verified the high titre of IgGs, monospecific IgGs have to be isolated. For this purpose a resin conjugated with the carboxylic derivative of aflatoxin Mi was generated. Such conjugation occurred through the formation of a peptide bond with a primary amine group exposed through a 10-atom arm bound to the EAH Sepharose™ 4B resin (GE Healthcare) , which allows the formation of the antigen antibody complex required for subsequent affinity purification of monospecific IgGs.

For this purpose 1.2 ml of resin were allowed to react ON at 4°C under continuous shaking with 1 mg of a new preparation of the carboxylic derivative of AFMi previously dissolved in 2 ml of 100% ethanol, and diluted in a final volume of 4 ml in deionised water acidified at pH 4.5, in the presence of the activator of the carboxylic group EDC at 0.1M final concentration.

b) Purification of monospecific antibodies

The thus generated resin allowed to purify, by affinity chromatography, 1.8 mg of monospecific IgGs targeted against aflatoxin Mi alone, with a high yield.

Purification was carried out challenging the resin with 8 mg of total IgGs in 50mM Tris buffer pH 7.4 (Binding Buffer) , subsequently washings followed with a volume 10- fold that of the settled resin (12 ml final). Elution was carried out with 0.1M glycine buffer pH 3.0. Fractions were immediately buffered at pH 7 with 1.0M Tris pH 8.8. Fractions were checked with 12% SDS-PAGE and protein concentration was determined on a spectrophotometer measuring absorption at 280 nm (figure 4) .

Affinity purification on the thus generated resin allowed to purify monospecific IgGs against aflatoxin Mi with high yield. The first 4 2-ml fractions containing monospecific IgGs anti AFMi were pooled in 8 ml at a concentration of 0.22 μς/μΐ (IgGMS-Mi).

IgGs were challenged in ELISA assays, which allowed to demonstrate a significant reduction of reactivity against BSA, used as a carrier during immunization and to confirm a high titre of reactive IgGs against GlnBP-AFMi _. at a dilution of 1:8000, and no reactivity against GlnBP alone ( figure 5 ) .

EXAMPLE F

Indirect competitive ELISA assays

Materials :

° MaxiSorp™ 96-well plates (Nunc) .

° Coating Buffer 25mM carbonate buffer pH 9.6.

° Wash Buffer 0.05% TBST.

° Blocking Solution 0.5% ovalbumine (OVA) in TBS Buffer

(20mM Tris, 130mM NaCl) .

° Diluting solution 0.25% ovalbumine in 0.05% TBST Buffer .

° Development solution TMB (3, 3' , 5, 5' -tetramethyl- benzidine, Sigma) . ° Stopping Solution 2.5M HC1.

Procedure :

° To adsorb the GlnBP-AFMi conjugate in the plate wells, Coating Buffer was used and placed ON at 4°C.

° Perform 3 50-μ1 washings for 10 min with Wash Buffer.

° Incubate 50 μΐ of the GlnBP-AFMi conjugate 0.5 ng/μΐ in Coating Buffer (25 ng total) ON at 4°C.

° Add 50 μΐ of Blocking Solution per well and incubate at 37°C for 1 h.

° Perform 3 50-μ1 washings for 10 min with Wash Buffer.

° Dilute primary and secondary antibodies in Diluting Solution, add 50 μΐ per each well, perform every point in duplicate, and incubate for 1 h at 37°C.

° Assay Coating with purified IgGMS-Mi as primary antibodies, use them at a 1:6000 dilution.

° Before assaying coating with IgGMS-Mi, pre-incubate them for 30 min at 37 °C with different final concentrations of free AFMi from 0.12 ppt until 8 ppt, add a 10-fold more concentrated solution in a methanol volume 10% of the final volume.

° Perform 3 50-μ1 washings for 10 min with Washing Buffer .

° Use the secondary anti-rabbit antibody at a 1:6000 dilution . ° Perform 3 50-μ1 washings for 10 min with Washing Buffer .

° Add 100 μΐ of TMB for development, 10 min in the dark, and stop the reaction by adding 100 μΐ of Stopping Solution.

° Reading is performed on a plate analyzer measuring absorption at 450 nm.

SET UP OF COMPETITIVE ASSAY

Monospecific IgGs thus obtained were challenged in indirect competitive ELISA assays. In these assays 25 ng of GlnBP-AFMi were used per reaction well.

Competitive assays were carried out pre-incubating suitable dilutions of these antibodies with increasing amounts of aflatoxin Mi in solution, before performing a traditional indirect ELISA assay. Before performing an indirect competitive ELISA assay we determined linearity conditions of the signal in response to different concentrations of antibodies and different amounts of Coating (adsorptions) used. 50 μΐ of 1:2 serial dilutions of GlnBP-AFMi were used, starting from the concentration of 2 ng/μΐ (100 ng in 50 μΐ) . Such coatings were challenged with increasing dilutions of antibodies starting from 1:4000 to 1:16000 dilutions. Thus, conditions to perform competitive indirect ELISA assay were optimized using coating of 25 ng of GlnBP-AFMi (0.5 ng/μΐ) per well and 1:6000 dilution of IgGMS-Mi. We proceeded to previously incubate IgGMS-Mi with increasing concentrations of free AFMi starting from 0.12 ppt until 8 ppt for 30 min, and subsequently an ELISA assay was performed. In this range of concentrations, inhibition of binding of IgGMS-Mi to GlnBP-AFMi adsorbed to the reaction well increasing from 40% up to 80% was observed.

Indirect competitive ELISA assays allowed to confirm high affinity of IgGs against aflatoxin Mi in solution, which allowed to set up a highly sensitive assay, that allowed to detect high affinity of IgGs for free AFMi antigen in solution as shown in the graph of Fig. 6 where, in particular, increasing the free concentration, binding of monospecific IgGs to the antigen attached to the well decreases.

EXAMPLE G

Protocol for antibody derivatization (total IgGs) with the enzyme invertase from Saccharomyces cerevlslae

Materials :

° 6.5 mg of invertase from yeast (Saccharomyces cerevisiae, FLUKA) at a concentration of 6.5 mg/ml in 0.1M sodium phosphate buffer pH 6.8.

° 25% glutaraldehyde (SIGMA) .

° 0.2 ml of IgGTot at a concentration of 8 mg/ml (1.6 mg) . ° 0.5M sodium carbonate buffer pH 9.6.

° 0.2M ethanolamine pH 7 (SIGMA).

Procedure :

° Dialyse 10 mg of invertase at the concentration of 10 mg/ml in sodium phosphate buffer pH 6.8.

° Take 1 ml of this 6.5 mg/ml preparation and add glutaraldehyde at the final concentration of 1.25%.

° Incubate under gentle stirring at room temperature for 12 hours.

° Remove the excess glutaraldehyde dialysing 3 times for 2 h at 4°C against 250 ml of 0.1M sodium phosphate buffer pH 6.8, 0.15M NaCl.

° Add 0.8 ml of 0.5M sodium carbonate buffer pH 9.6 to 0.2 ml of IgGTot 8 mg/ml, lOmM sodium phosphate buffer pH 7.4.

° Add 1 ml of invertase 6.5 mg/ml activated with glutaraldehyde and incubate ON at 4°C under gentle stirring .

° Add 0.1 ml of 0.2M ethanolamine solution pH 7 and incubate the solution under gentle stirring for 2 h at 4°C.

° The conjugate was centrifuged at 12000 g for 10 min at 4°C, and the supernatant was dialysed again 5 times with 200 ml of 50mM Tris Buffer pH 7.4. SET UP OF A PROTOCOL FOR ANTIBODY DERIVATIZATION (TOTAL IgGS) WITH INVERTASE

Subsequently, an experimental protocol for conjugation of invertase from yeast (Saccharomyces cerevisiae, FLUKA) with purified IgGs was set up. Since IgGs of the two rabbits did not show significant differences in titre and binding affinity for GlnBP-AFMi, for conjugations purified IgGs from pooled sera of both rabbits were used, and secondly affinity purifications of the . generated conjugates were carried out. For the set up of an IgG- invertase conjugation protocol, different conjugation strategies were adopted using glutaraldehyde as a homobifunctional crosslinking agent.

We proceeded activating invertase with glutaraldehyde and subsequently adding IgGs in the appropriate buffer that allows conjugation.

For this purpose 10 mg of invertase from yeast were dialysed 4 times for 2 h at room temperature against 250 ml of 0.1M sodium phosphate buffer pH 6.8. Afterwards glutaraldehyde was added at the final concentration of 1.25% to 1 ml of invertase (6.5 mg) and the solution was placed under gentle stirring ON at room temperature.

Subsequently, the excess glutaraldehyde was removed by dialysing 3 times for 2 h at 4°C against 250 ml of 0.1M sodium phosphate buffer pH 6.8, 0.15M NaCl. The molar ratio between invertase and IgGs in this reaction was 5:1. For this purpose 0.2 ml of a new preparation of IgGs at the concentration of 8 mg/ml (1.6 mg) , obtained from sera of both rabbits, were dissolved in 0.8 ml of 0.5M sodium carbonate buffer pH 9.6 and were placed in a 2 ml Eppendorf.

Subsequently, 1 ml containing 6.5 mg of invertase activated with glutaraldehyde was added, the whole was placed at 4°C under gentle stirring ON.

Subsequently, 0.1 ml of 0.2M ethanolamine solution pH

7 were added and the solution was placed under stirring for 2 h at 4°C. The conjugate was centrifuged at 12000 g for 10 min at 4°C, and dialysed 5 times against 200 ml of 50mM Tris Buffer pH 7.4.

EXAMPLE H

Purification of conjugated IgGs from still free invertase

Materials :

° 0.3 ml of nProtein A Sepharose™ 4 Fast Flow resin (GE Healthcare) .

° Binding Buffer 50mM Tris buffer pH 7.4.

° 10 ml columns for affinity chromatography (Biorad) .

° Conjugate IgG Tot-INV was taken to 4 ml in 50mM buffer pH 7.

Procedure : ° Wash resin with water and binding buffer and add diluted conjugate and incubate overnight at 4°C under slow stirring .

° Wash at RT with Binding Buffer until invertase activity is no longer detectable.

° Draw 100 μΐ from each washing and assay invertase activity, as described in example A.

° Draw the resin and place it in a dialysis membrane KO 30 kD and seal it.

· Enzymatic assay of conjugated invertase

° Place the membrane in a 50 ml tube containing 30 ml of 0.1M acetate buffer pH 4.6 and 15 ml of 10% w/v freshly prepared saccharose solution.

° Place in an incubator at 37 °C under continuous slow stirring.

° Draw 0.1 ml of the external solution of the dialysis at various time points.

° Determine the amount of glucose present with D- GLUCOSE kit (Megazyme) .

TESTS TO VERIFY QUALITY AND EFFICIENCY OF CONJUGATION

Purification of conjugated IgGs from still free invertase

To verify that there were conjugated IgGs and to verify that invertase activity was still present, the conjugation reaction mixture was taken to 4 ml in 50mM Tris Buffer pH 7.4 and placed to incubate with 0.3 ml of Protein A SepharoseTM 4 Fast Flow resin (GE Healthcare) ON at 4°C under gentle stirring. Such resin is able to bind IgGs with high affinity and remove the part of unreacted invertase. For this purpose, the resin was washed with 50mM Tris Buffer pH 7.4 until, in the performed washings, there was no detectable invertase activity. For this purpose, 0.1 ml of each washing were assayed for residual invertase activity, by adding 1 ml of 0.1M acetate buffer pH 4.6 and 0.5 ml of 10% w/v freshly prepared saccharose solution, incubating for 15 min at 37°C. The reaction was stopped by adding 0.4 ml of 0.3M Tris base buffer and immediately placed in ice. When the reaction terminated, 33 μΐ of it were assayed to determine the amount of produced glucose through a commercial kit for the determination of glucose in food (D-GLUCOSE GOPOD-FORMAT Megazyme) that uses glucose oxidase as a revealing enzyme. Thus, the phase of resin washing was terminated after no amount of glucose was detectable any longer with this kit.

Enzymatic assay of conjugated invertase

Subsequently, the resin was placed inside a dialysis membrane, such membrane closed at the ends was submerged in a 50 ml tube containing 30 ml of 0.1M acetate buffer pH 4.6 and 15 ml of 10% w/v freshly prepared saccharose solution, the whole was placed in an incubator at 37 °C under continuous slow stirring. Subsequently 0.1 ml of external solution of dialysis were drawn at various time points, and the amount of glucose present was determined with the D-GLUCOSE kit (Megazyme) .

The enzymatic assay schematically illustrated in figure 7 allowed to confirm the invertase activity of the conjugates with total IgGs already at 15 min with a concentration of produced glucose of 2 mg/dl, also taking into account the time necessary for diffusion of the substrates and products and the dilution volume.

EXAMPLE I

a

Purification of conjugated antibodies

Procedure :

° Recover resin from the dialysis tube and place it in a column for affinity chromatography.

° Perform 2 0.5-ml washings in 50mM Tris buffer pH

7.4.

° Elute at RT with 5 1-ml fractions in 0.1M glycine buffer pH 3 and buffer at pH 7 with Tris buffer pH 8.8.

° Monitor absorption of fractions containing IgGTOT-

INV on a spectrophotometer at 280 nm, then pool them in a single sample.

PURIFICATION OF CONJUGATED ANTIBODIES

Subsequently, the resin with bound conjugates was recovered from the dialysis tube and was placed in a column for subsequent purification of the conjugates. 2 0.5-ml washings were performed in 50mM tris buffer pH 7.4, and subsequently 3 l-ml elutions were performed in 0. IM glycine pH 3, immediately buffered with 50 μΐ of IM Tris buffer pH 8.8. Absorption of fractions at 280 nm was monitored on a spectrophotometer, and the first two l-ml fractions were pooled and used for subsequent assays.

EXAMPLE J

Verification of the activity of antibodies and enzyme of the conjugates

Materials :

° 0.50 g of BSA in TBS deposed on 1 cm ² as a positive control .

° 0.50 g of GlnBP in TBS deposed on 1 cm ² as a negative control.

° Purified IgGTOT-INV conjugate.

° 25 cm ² Nitrocellulose sheets.

° Blocking Solution 5% OVA TBS.

° Diluting Solution 1% OVA, 0.05% TBST.

Procedure :

° Adsorb 2 μΐ of BSA and GlnBP 0.250 g/μΐ protein solutions so as to get various 1 cm ² spots.

° Block the reacting sites of nitrocellulose with Blocking Solution. 1 h at room temperature under gentle stirring. ° Perform 3 washings of 10 min in 0.05% TBST.

° Dilute all the purified conjugate in Diluting Solution and incubate with nitrocellulose sheet ON at 4°C under gentle stirring.

° Perform 3 washings of 10 min in 0.05% TBST.

° Cut the sheet in as many small strips as deposed proteins are.

° Incubate them for 5 min with 50 μΐ of the solutions, to assay saccharase activity according to example A.

° Draw 33 μΐ and assay them with D-GLUCOSE GOPOD-FORMAT

(Megazyme) to determine the produced glucose concentration .

VERIFICATION OF THE ACTIVITY OF ANTIBODIES AND ENZYME OF THE CONJUGATES

To verify that in conjugates purified from the resin antibodies retained their structure and are still able to recognize an antigen, we proceeded to perform a Dot Blot assay using the carrier of immunization, that is BSA (Bovine Serum Albumin) as a control antigen.

0.5 pg of BSA were adsorbed as a positive control and

0.5 g of GlnBP as a negative control on a nitrocellulose sheet so that each spot had the maximum size of 1 cm ² . Each spot was highlighted since it will be cut and tested individually for invertase activity. Subsequently, nitrocellulose sheet with the spots of adsorbed proteins was submerged in a Blocking solution (saturation) of 5% OVA in TBS Buffer (20m Tris, 130mM NaCl) for 1 h at room temperature under gentle stirring. 3 washings were performed in TBS Buffer of 10 min at room temperature. The conjugate total IgGs-invertase was taken to a volume of 10 ml in 1% OVA solution in TBS Buffer and was incubated with the nitrocellulose sheet under gentle stirring at 4°C overnight.

3 washings in TBS for 10 min at room temperature were performed, and subsequently the sheet was cut so as to obtain a square of 1 cm ² for each spot of deposed protein.

Each small square was placed in a 1.5 ml Eppendorf and was assayed for invertase activity given by the conjugate total IgGs-invertase bound to it. For this purpose, a solution to assay invertase activity with 1 ml of 0.1M acetate buffer pH 4.6 and 0.5 ml of saccharose 10% w/v solution was prepared.

On each small square of nitrocellulose inserted in an Eppendorf, 50 μΐ of this solution were added, the Eppendorf was closed and placed to incubate at room temperature.

At various time points, 33 μΐ were drawn from each Eppendorf and the amount of produced glucose was determined through the D-GLUCOSE kit GOPOD-FORMAT (Megazyme) . After an incubation time of 5 min the small squares on which BSA had been adsorbed gave a positive result for invertase activity, while those on which GlnBP had been adsorbed gave a negative result. Then the procedure retains both antibody and enzymatic reactivity.

EXAMPLE H

Purification of monospecific antibodies anti- conjugates of aflatoxin Ml with invertase

Materials :

° IgGTOT-INV conjugate prepared as in example G.

° 250 μΐ of EAH-AFMi resin prepared as described in example E.

° Wash buffer 50mM Tris pH 7.4.

Procedure :

° Incubate conjugate with 250 μΐ of EAH-AFMi resin in a 2 ml Eppendorf overnight at 4°C under slow stirring.

° Perform 2 washings in Eppendorf and one last washing for transfer in a 0.8 ml column in 50mM Tris buffer pH 7.4.

° Perform 10 0.1-ml elutions in 0.1M glycine buffer pH

3 immediately buffered at pH 7 with 1M Tris pH 8.8.

° Monitor absorption at 280 nm on a spectrophotometer and pool fractions containing the conjugate with monospecific IgGs anti-aflatoxin Ml ( IgGMS-Ml-INV) . ° Add isodiazide as a preservative at the final concentration of 5μΜ.

Purification of monospecific antibodies anti- conjugates of aflatoxin Mi with invertase

Once invertase activity and ability to recognize antigens of the conjugates then generated were confirmed, we proceeded to produce another IgG-invertase conjugate to be purified on the resin (EAH Sepharose™ 4B) conjugated with the carboxylic derivative of aflatoxin Mi EAH-AFMi) . a. Purification of antibodies and production of invertase conjugates prepared as in example G

b. Purification of monospecific antibodies conjugated with invertase

Conjugate was incubated with 250 μΐ of EAH-AFMi resin in a 2 ml Eppendorf ON at 4°C under slow stirring. 2 washings in Eppendorf and one last washing for transfer in a 0.8 ml column in 50mM Tris buffer pH 7.4 were performed. Afterwards, 10 0.1-ml elutions were performed in 0.1M glycine buffer pH 3 immediately buffered at pH 7 with 1M Tris pH 8.8. Fractions were monitored on a spectrophotometer measuring absorption at 280 nm and pooled in a new Eppendorf, to which isodiazide was added as a preservative at the final concentration of 5μΜ.

EXAMPLE L

• Immunoenzymatic assays of monospecific conjugates Materials :

° 1 cm x 3 cm nitrocellulose strips.

° IgGMS-Ml-INV conjugate diluted 1:100 and 1:250. ° Blocking Solution 0.5% OVA, 0.05% TBST.

° Diluting solution 0.05% TBST buffer, 0.25% OVA.

° Saccharase activity buffer (2/3 of 0.1M acetate buffer pH 4.6 and 1/3 of 10.0% w/v saccharose solution).

° Peroxidase-aflatoxins (HRP-AFL ORSell)

° 0.1% OVA.

° 0.7 mg/ml GlnBP.

Procedure :

° Incubate 150 g of OVA and GlnBP and 50 μΐ of HRP- AFL (6.6 μς/μΐ) in a 1.5 ml Eppendorf in 1 ml TBS for 1 h at room temperature.

° Perform 3 1-ml washings in 0.05% TBST for 15 min at

4°C under slow stirring.

° Incubate strips with 1 ml of Blocking Solution for 1 h at room temperature under slow stirring.

° Perform 3 1-ml washings in 0.05% TBST for 15 min at 4°C under slow stirring.

° Incubate strips with two dilutions of IgGMS-Inv conjugate overnight at 4°C.

° Perform 3 1-ml washings in 0.05% TBST for 15 min at 4°C under slow stirring. ° Incubate strips with 500 μΐ of Buffer to assay saccharase activity.

° Draw 33 μΐ at various time points and measure concentration of produced glucose, with the D-GLUCOSE kit (Megazyme) ₌

IMMUNOENZYMATIC ASSAYS OF MONOSPECIFIC CONJUGATES

The purified conjugate on resin was challenged on nitrocellulose strips previously incubated with different protein solutions. The strips, of a size of 1 cm x 3 cm, were placed in an Eppendorf and incubated with 1 ml of TBS Buffer, wherein different proteins had been diluted. For this purpose 150 μg of BSA and GlnBP were used as negative controls after affinity purification of the conjugates on a EAH-AFMi resin. In this experiment the ability of the conjugates to monitor a commercial protein conjugated with various aflatoxins used in ELISA competitive quantitative assays was verified. For this purpose 50 μΐ of a solution of peroxidase-aflatoxins conjugate (HRP-AFL ORSell) was diluted in 1 ml of TBS before incubating it with the nitrocellulose strip for 1 h at RT under slow stirring. Subsequently, the nitrocellulose strips were incubated with 1 ml of 0.5% OVA, 0.05% TBST, for 1 h at room temperature under slow stirring. 3 1-ml washings in 0.05% TBST were performed, and the strips were incubated ON at 4°C with 2 dilutions of monospecific conjugates IgG anti- AFMi-invertase ( IgGMS-Ml-Inv) . The IgGMS-Ml-Inv conjugates were used at dilutions of 1:250 and 1:500 in 0.05% TBST Buffer, 0.5% OVA (20mM Tris, 130mM NaCl, 0.05% Tween-20).

3 washings were performed for 10 min with 0.05% TBST Buffer (20mM Tris, 130mM NaCl, 0.05% Tween-20).

The strips were incubated at room temperature with 0.5 ml of a buffer solution optimized to assess invertase activity, containing an excess of substrate (2/3 of 0.1M acetate buffer pH 4.6 and 1/3 of 10% w/v saccharose solution) .

After various incubation times, drawings of 33 μΐ and one final draw of 100 μΐ were performed, which were assayed with the D-GLUCOSE kit (Megazyme) to dose glucose, produced by the activity of the IgGMS-Ml-I V conjugate.

The assay allowed to detect the protein derivatized with aflatoxins (HRP-AFL) , and not other proteins incubated with the IgGMS-Ml-INV conjugate.

EXAMPLE M

• Assays of monospecific conjugates in the presence of milk and methanol

Materials :

° nitrocellulose strips 1 cm x 3 cm.

° IgGMS-Ml-INV conjugate diluted 1:100.

° Blocking Solution 0.5% OVA, 0.05% TBST. ° Diluting Solution 33% Granarolo whole milk in 0.05% TBST or 0.05% TBST Buffer, 0.25% OVA, plus 10% MeOH.

° Saccharase activity buffer (2/3 of 0.1M acetate buffer pH 4.6 and 1/3 of 10.0% w/v saccharose solution).

° Peroxidase-a latoxins (HRP-AFL ORSell) .

° 10 g/ ΐ OVA in TBS.

Procedure :

° Incubate 100 μΐ of OVA (10 μς/μΐ) and 100 μΐ of HRP- AFL (6.6 g/μΐ) in a 1.5 ml Eppendorf in 1 ml TBS ON at 4°C.

° Perform 3 1-ml washings in 0.05% TBST for 15 min at 4°C under slow stirring.

° Incubate strips with 1 ml of Blocking Solution for 7 h at 4°C under slow stirring.

° Perform 3 1-ml washings in 0.05% TBST for 15 min at

4°C under slow stirring.

° Incubate strips with the 1:100 dilution of IgGMS- l- INV conjugate ON at 4°C.

° Perform 3 1-ml washings in 0.05% TBST for 15 min at 4°C under slow stirring.

° Incubate strips with 0.5 ml of buffer to assay saccharase activity.

° Draw 100 μΐ at two hours and measure concentration of produced glucose with the D-GLUCOSE kit (Megazyme) . ASSAYS OF MONOSPECIFIC CONJUGATES IN THE PRESENCE OF MILK AND METHANOL

Subsequently, we verified that such assay worked in the presence of milk and methanol.

The assay was performed diluting the IgGMS-Ml-INV conjugate in 33% Granarolo whole milk solution in 0.05% TBST (20mM Tris, 130mM NaCl, 0.05% Tween-20) or in 0.125% OVA solution in 0.05% TBST adding 10% methanol in each sample.

In this experiment nitrocellulose strips were incubated with 100 μΐ of HRP-AFL conjugate (6.6 μς/μΐ) diluted in TBS and placed to incubate ON at 4°C under slow stirring. 3 1-ml washings were performed in 0.05% TBST for 15 min at 4°C under slow stirring. Afterwards, strips were incubated with 1 ml of 0.5% OVA solution for 7 h at 4°C under slow stirring to saturate all the binding sites present on the strips. 3 1-ml washings were performed in 0.05% TBST for 15 min at 4°C under slow stirring. Subsequently, conjugates diluted 1:100 were added.

3 1-ml washings in 0.05% TBST were performed for 15 min at 4°C under slow stirring. Nitrocellulose strips were incubated at room temperature with 0.5 ml of a solution 2/3 of 0.1M acetate buffer pH 4.6 and 1/3 of 10% w/v saccharose solution. At various time points, various drawings of 33 μΐ and one final of 100 μΐ were performed for the dosing of released glucose with the D-GLUCOSE kit (Megazyme) .

The experiment allowed to demonstrate that there was no interference of milk during incubation of the IgGMS-Ml- INV conjugate with the nitrocellulose strips, and it was possible to detect the invertase activity after 1 h of incubation at room temperature. Additionally, we verified that there was no interference in binding by IgGMS-Ml-INV against HRP-AFL, adding 10% methanol to the solution. The solvent in the solution will be necessary to add free aflatoxin Ml in the next competitive assays or to allow solubilisation of aflatoxin Ml in possibly contaminated milk.

EXAMPLE N

Competitive assays of monospecific conjugates in the presence of milk

Materials :

° IgGMS-Ml-INV conjugate diluted 1:100.

° Blocking Solution 0.5% OVA, 0.05% TBST.

° Diluting Solution 33% Granarolo in 0.05% TBST or 0.05% TBST Buffer, 0.25% OVA, plus 10% MeOH.

° Saccharase activity buffer (2/3 of 0.1M acetate buffer pH 4.6 and 1/3 of 10.0% w/v saccharose solution).

° Peroxidase-aflatoxins 6.6 g/μΐ (HRP-AFL ORSell) .

° OVA in TBS 10 ug/μΐ. Procedure :

° Place 100 μΐ of OVA, OVA 10 g/μΐ, and 100 μΐ of HRP-AFL (6.6 μς/μΐ) in a 1.5 ml Eppendorf in 1 ml TBS ON at 4°C.

° Perform 3 1-ml washings in 0.05% TBST for 30 min at

4°C under slow stirring.

° Place strip in a new 1.5 ml Eppendorf.

° Incubate strips with 1 ml of Blocking Solution ON at 4°C under slow stirring.

° Perform 3 1-ml washings in 0.05% TBST for 30 min at

4°C under slow stirring.

° Incubate for 1 h at room temperature IgGMS-Ml-INV conjugates diluted 1:100 in diluting solution with 54 ppt and 27 ppt of AFMi dissolved in MeOH.

° Incubate strips with IgGMS-Ml-INV conjugate at 1:100 dilution already preincubated with aflatoxin Mi solution in MeOH, ON at 4°C.

° Perform 3 1-ml washings in 0.05% TBST for 30 min at 4°C under slow stirring.

° Incubate strips with 1.0 ml of buffer to assay the saccharase activity.

° Draw 100 μΐ at two hours and measure concentration of produced glucose with the D-GLUCOSE kit (Megazyme) .

COMPETITIVE ASSAYS OF MONOSPECIFIC CONJUGATES IN THE PRESENCE OF MILK To carry out competitive assays, the assay was properly optimized to allow a careful comparison of results with suitable controls and verify reduction of the signal after addition of free aflatoxin Mi.

For this purpose it was necessary to: i) increase incubation time of washings to 30 min at 4°C to reduce possibility of having false positives; ii) place strips in a new 1.5 ml Eppendorf after incubation with the protein conjugated with aflatoxins, so as to verify the activity obtained only from HRP-AFL bound to nitrocellulose strips; iv) increase incubation time of Blocking solution ON to be unfavourable to aspecific bonds of the conjugate with the plastic of the Eppendorf; v) eventually, increase the volume of assay of the invertase activity to 1 ml to allow for an optimal incubation of solution on the strips in the 1.5 ml Eppendorf, this latter carried out at room temperature without shaking, aware that this will prolong incubation times to obtain a detectable signal.

For competitive assays IgGMS-Ml-INV conjugates, diluted in 33% Granarolo whole milk solution in 0.05% TBST, were first incubated for 1 h at room temperature under slow stirring with various concentrations of aflatoxin Mi, 54 ppm and 27 ppm respectively, adding a methanol volume equal to 10% of the total volume of the solution, wherein a suitable dilution of free aflatoxin Mi was dissolved. Afterwards, 1 ml of this solution was incubated with each strip ON at 4°C under slow stirring. 3 1-ml washings were performed in 0.05% TBST for 30 min at 4°C under slow stirring.

After adding 1 ml of solution to assay invertase activity, 100 μΐ were drawn after two hours, assayed with the D-GLUCOSE kit (Megazyme) . After incubation in an Eppendorf in the absence of AFMi about 1.5 μg of glucose was produced, and it was possible to detect 80% and 50% reduction of the glucose produced with an incubation of free AFMi 54 ppt and 27 ppt, respectively.

Competitive assay of conjugates in the presence of milk

The table shows that, in the presence of 54 ppt of free aflatoxin Mi dissolved in milk, binding of conjugated IgGs to the invertase and consequently the amount of produced glucose is dramatically reduced. In the presence of another protein, ovalbumine (OVA) , bound to the strips, no glucose production is observed.