WO2007083345A1

WO2007083345A1 - A competitive immunoassay for patulin detection

Info

Publication number: WO2007083345A1
Application number: PCT/IT2007/000045
Authority: WO
Inventors: Sabato D'auria; Mosè ROSSI; Paolo Bazzicalupo; Marcella De Champdore'
Original assignee: D Auria Sabato; Rossi Mose; Paolo Bazzicalupo; De Champdore Marcella
Priority date: 2006-01-23
Filing date: 2007-01-22
Publication date: 2007-07-26
Also published as: ITNA20060006A1

Abstract

A method to detect the content of patulin in foods is based on the use of polyclonal antibodies produced against the mycotoxin patulin by means of two synthetic derivatives (4) and (5) of the molecule conjugated to bovine serum albumin as carrier protein. The invention comprises also the synthesis of said patulin derivatives and the preparation of affinity columns used to develop an immunofluorescence competitive assay for the detection of patulin. (Formula 5 and 4).

Description

A competitive immunoassay for Patulin Detection

Description

The present invention relates to an immunochemical analytical method for patulin detection based on highly specific antigen-antibody interactions. More exactly the .invention relates to a method to detect the content of patulin in foods based on the use of antibodies produced against the mycotoxin patulin by means of two synthetic derivatives of the same molecule conjugated to bovine serum albumin as carrier protein.

The invention comprises also the synthesis of said patulin derivatives and the preparation of affinity columns used to develop an immunofluorescence competitive assay for the detection of patulin.

Patulin is a toxic secondary metabolite of a number of fungal species belonging to the genera Penicillum and Aspergillus. It has been mainly isolated from apples and apple products contaminated with the common storage-rot fungus of apples, Penicillum expaήsum, but has also been extracted from rotten fruits, mouldy feeds and stored cheese. (Buchanan, J. R. et al . J. Am. Soc.Hort. Sci . 1974,99,962 - Wilson, D.M. Advances in chemistry, Ser.149, p.90, 1976 Am.Chem. Soc. Washington DC- Bullerman,L.B. J. Food Sci 1976, 41,26)

Although several mycotoxins occur in nature, very few are regularly found in fruits, e.g. aflatoxins, ochratoxin A, patulin, Alternaria toxins. Mycotoxins may remain in fruits even when the fungal mycelium has been removed; furthermore the processing of fruits does not result in the complete removal of mycotoxins. Human exposure to mycotoxins occurs by ingestion of contaminated products and can lead to serious health problems, including immunosuppression and carcinogenesis. (CAST 2003, Council of Agricultural Science and Technology, Task force report n.139, CAST, Ames IA). In animal studies patulin has been shown to be acutely toxic, carcinogenic and teratogenic. One important aspect of patulin toxicity in vivo is an injury of the gastrointestinal tract including ulceration and inflammation of stomach and intestine. (Rychlich,M. et al. Food and Chem. Toxicol . 2004, 42,729-735.) Recently, patulin has been shown to be genotoxic by causing oxidative damage of DNA, (Liu, B-H. et al. Toxicol Appl.Pharm. 2003,252,255-263) and oxidative DNA base modifications have been considered to play a role in mutagenesis and cancer initiation (01inski,R. et al. Oxidative Acta Biochim.Po 1998, 45,561-572). Patulin is believed to exert its cytotoxic effects mainly by ^'forming covalent adducts with essential cellular thiols in proteins and aminoacids (Riley, R. T. et al. Toxicol . Appl . Pharmacol . 1991,205,108-126)

Indeed, from a chemical point of view patulin is a water soluble unsaturated lactone, readily reactive toward thiol groups and also amino groups. The electrophilic properties of patulin and its reactivity toward several model nucleophiles, such as N-acetylcysteine and glutathione, have been elucidated. (Fliege, R. et al. Chem. Res . Toxicol 2000, 13, 363-381) . Glutathione represents one of the most abundant cellular nucleophile and it has been shown to spontaneously form covalent adducts with patulin. The same reactivity is responsible for the ability of patulin to¹ induce in vitro intra- and intermolecular protein crosslinks involving cystein residues, lysine and histidine side chains

(Fliege, R. et al. Chem.Biol. Interact . 1999,

123(2) ,85-103) and for the capability to inhibit the activity of several enzymes .( Pfeiffer, E. et al MoI. Nutr. Food Res. 2005, 45(4) 329-336). Patulin is also reported to be endowed with selective DNA damaging activity. (Lee, KS. et al. Appl. Environ.

Microbiol. 1986, 252(5) , 1056-1054) .

The widespread presence of fungi in the environment, renders mycotoxins practically ubiquitous contaminants in food and feeds; therefore, one of the most effective measures to protect public health is to establish reasonable regulatory levels of these toxins on the basis of valid toxicological data. In order to ensure the safety and quality of foods a number of regulatory authorities have decreed maximum residues levels of several mycotoxins infoods. In agreement with the results of a long-term investigation in rats, the World Health Organisation (WHO) has set a tolerable weekly intake of 7 ppb body weight. The content of patulin in foods has been restricted to 50 ppb in many countries: the EU has set a limit of 10 ppb in children foods, but the objective is to reach 25 ppb of patulin in apple containing products. (European commission EC n.1425/2003, 2003) . A ready detection is the most prudent means to prevent the entry of patulin in commerce. Conventional analytical methods of detection involve chromatographic analyses, such as HPLC, GC and, more recently, techniques like LC/MS and GC/MS (Sforza,S. et al. Mass . Spectr.Rev. 2005). However, extensive protocols of sample cleanup are required before the analysis and expensive analytical instrumentation is necessary to accomplish it. Furthermore, the problem of precise quantitative determination of patulin and other mycotoxins by these methods is particularly significant due to the high variability of the real matrices and can be solved only by exploiting isotopically labelled internal standards. All these limitations make it difficult to extend the methods to detect patulin outside the laboratory.

As an alternative, immunochemical analytical methods, for instance enzyme-linked immunosorbent assay (ELISA) , offer several advantages compared to conventional analysis, i.e. low cost per sample, high selectivity, high sensitivity and high throughput. Extensive extraction and sample cleanup are not needed. Such methods have been shown to have a potential for the routine analysis of many mycotoxins. However no immunoassay has been developed yet for patulin detection. This is due in part to chemical reasons, since patulin is, like other non proteinaceous toxins, a low molecular weight compound (MW = 154) and, to develop antibodies, the use of a hapten-protein conjugates is necessary. In addition patulin, unlike other toxins, is highly reactive toward thio-containing compounds and highly unstable under basic conditions . (Harrison, M.A. J. Food Safety 1987,15,147-153). This instability may result in decomposition of patulin in the course of conjugation to the protein carrier. Furthermore, the exposed epitopes of the injected hapten-protein conjugate can be attacked by sulphydryl-containing compounds in the blood. For this reason few articles have appeared in the literature on the synthesis of patulin-protein conjugate and on the production of antibodies against this toxin, and the results are far from satisfactory in all cases. Recently, a review on the methods to detect the toxin in foods has been published. (Moake,M.M. et al. Comprehensive Reviews in Food Science and Food Safety 2005,2,8- 21) .

The aim of the present invention is to overcome the limits of the prior art, providing an immunochemical analytical method based on the use of antibodies obtained by conjugation of new derivatives of patulin to a carrier protein and apt to detect patulin with high selectivity, and high sensitivity.

Therefore, it is a first object of the invention to describe two new derivatives of patulin still maintaining the original skeleton of the natural toxin, but exhibiting a higher chemical stability.

It is another object of the invention the synthesis of said two patulin derivatives, as well as their conjugation to the bovine serum albumin carrier protein for the production of antibodies.

The invention comprises also the purification of the produced antibodies by affinity chromatography on columns obtained by covalently immobilizing said new patulin derivatives on solid support (Sepharose resin) .

The titre of the specific antibodies was determined by means of indirect ELISA assays. Finally, tetramethylrhodamine isothiocyanate labelled IgGs were prepared and used to develop a "hit and run" fluoroimmunoassay for patulin, based on the antibodies competition between the immobilized patulin derivative and free added patulin. This technigue was described for the detection of T-2 toxin (Warden B.A. et al. Anal.Biochem. 1987, 162,363-369) and more recently for the sensing of 2, 4, 6-trinitrotoluene in sea water (Green T.M. et al. Anal Biochem.2002, 310, 36-41) .

The present invention will be now described in more details with reference to a non limitative embodiment example, from which the features and the advantages thereof will be clear. The description that follows is given with reference to the drawings that are herein accompanied, in which:

Fig. 1 shows the chemical structure of patulin;

Fig.2 is a schematic representation of the conjiugates used for the production of antibodies against patulin; Fig.3 is a Western blot with specific antibodies against one of derivative;

Fig.4 is a chemical structure of tetramethylrhodamine isothiocyanate;

Fig.5 shows the fluorescence emission at λ= 575 nm of IgG-TMRI eluted from the column after incubation of patulin;

Fig.6 shows the synthesis of derivatives according to the invention, from L-arabinose;

Fig.7 shows the immunofluorescent assay for detection of patulin.

Synthesis of patulin derivatives 4 and 5.

The presence of a carrier protein conjugated to the toxin is required for the production of antibodies against patulin which is, like other non-proteinaceous toxins, too small to elicit any immunological response. However, the conjugation reaction is hampered by the reactivity of patulin functional groups in the reaction conditions required to functionalise the, OH group at the C-4 position (Fig. 1) . Indeed, from a chemical point of view patulin is a highly conjugated bicyclic lactone and, in general, very reactive at C7 position under basic conditions and toward nucleophiles, especially thiol and amino groups. Furthermore, even if the desired antigen could be obtained, the patulin epitopes would result sensitive, in vivo, to the SH- containing molecules in the blood, thus rendering the intact molecule unavailable and the recognition process disfavoured, with no or poor production of antibodies.

With the aim pf reducing the reactivity of patulin toward nucleophiles, two new derivatives, ' 4-[ (4-hydroxy~2-oxo-2_r 6, 7^', 7a-tetrahydro-4H-furo[3,2- c]pyran-7-yl) oxy] -4- oxobutanolc acid, having the chemical structure 4 (P-Sat-HS) of fig.6 and 4- [ (4-hydroxy-2-oxohexahydro-4H-furo[3,2-c]pyran-7- yl) oxy] -4-oxobutanoic acid having the chemical structure 5 (P- Ins-HS) of Fig.6, were synthesized starting from compound 3 (Fig.β), lacking the highly reactive C7-C7a double bond. The compound 3 in turn was obtained from derivative 2, prepared from commercial L-arabinose by a previously described synthetic scheme. (Bennett, M; Gill, G. B; Pattenden,G. ; Shuker, A: J. ; Stapleton, A. J. Chem.Soc.PerK:Trans.I 1991,929-937) .

Removal of the benzyl protecting group in 3 by treatment with SnCl₄ yielded compound 4; conversely, catalytic hydrogenation of 3 nicely afforded compound 5. In both cases the synthesis gave a mixture of diastereoisoπiers of 4 and 5, which were not separated and used as such in the successive protein conjugation reactions.

Conjugation of patulin derivatives to the Bovine Serum Albumin .

Bovine serum albumin (BSA) was chosen as the carrier protein, and haptens 4 and 5 were conjugated to it by the water soluble carbodiimide method, leading to antigens A and B (Figure 2) . The efficiency of conjugation reaction could not be determined because of the lack of UV chromophore in the case of derivative 5 and because of maximum absorption at λ= 278 nm of 4 and BSA overlapped.

Production of antibodies against patulin derivatives .

Both A and B were used to immunize rabbits for the production of polyclonal antibodies against patulin, following a standard protocol. At the end of the immunization period, the presence of antibodies against patulin derivatives in sera (SIl serum from rabbit 1 and SI2 from rabbit 2) was verified by Dot Blot assay. To exclude anti-BSA antibodies present in the sera, a different protein was conjugate to patulin derivatives P-Sat-HS and

P-Ins-HS. For this purpose, the glutamine-binding protein (GInBP) from E. coli was chosen as alternative protein and conjugates were prepared by the same procedures already used for BSA conjugates.

P-Sat-HS-GlnBP, P-Ins-HS-GlnBP, BSA as the positive control and GInBP as the negative one were spotted on four nitrocellulose membranes and each membrane was incubated with immune antisera (SIl and SI2, 1

: 250 dilution) and pre-immune sera from both rabbits (SPIl and SPI2 respectively, from rabbit 1 and rabbit 2, dilution 1:250) . After incubation of

HRP-conjugate secondary antibody and development with ECL specific reagent, pre- immune sera showed no response, while SIl and SI2 gave signals with both antigens P-Sat-HS-GlnBP and P-Ins-HS-GlnBP, with BSA but not with GInBP (not shown) .

Immunoglobulins purification from sera.

The immunoglobulin G (IgG) fraction of each serum

(IgGl from rabbit 1) and IgG2 from rabbit 2) was isolated by means of a protein A column kit by standard procedures. After elution, the IgG fraction was concentrated and dialyzed against PBS

20 inM, NaCl 50 mM at pH 7.0.

Preparation of affinity columns and purification of specific antibodies . Specific antibodies against compounds 4 and 5 were purified from the IgG fraction by means of affinity chromatography on two resins, previously loaded with 4 and 5, respectively. Elution of antibodies from the column was achieved firstly by varying the salt concentration of the phosphate buffer (0.5 M NaCl and then 1.0 M NaCl) and then the pH of the elution buffer (glycine 0.1 M, pH 2.7). Eluting the column as above, the fractions containing the antibodies, monitored by UV measurements at λ = 278 nm, were collected and each pool was tested against patulin derivatives by means of Western Blot experiments. Only antibodies eluted by changing the pH conditions were effective in the recognition of compounds 4 and 5, as shown in the Figure 3 (aPsHS and aPiHS, respectively) . These antibodies were used in the experiments of antibodies titration (aPsHSl and aPiHSl from rabbit 1; aPsHS2 and aPiHS2 from rabbit 2, respectively) . To the best of our knowledge, this is the first time that polyclonal antibodies produced against patulin have been purified by affinity chromatography, due to the chemical stability of derivatives 4 and 5, which allowed them to be conjugated to a NH-functionalised resin. The affinity columns were^* also used to develop the immunofluorescence competitive assay for the detection of patulin.

Specific Antibodies Titration. The titre of purified antibodies was determined by means of indirect ELISA, by coating on the microplates wells several different concentrations of antigens P-Sat-HS-GlnBP and P-Ins-HS-GlnBP, and by testing serially diluted aPsHSl and aPsHS2 against P-Sat-HS-GlnBP and P-Ins-HS-GlnBP, respectively. Each experiment was performed in triplicate and the results showed that the titre of antibodies, expressed as the reciprocal dilution giving 0.1 optical density units (OD) at λ = 450 ran, was 25000 for all the tested antibodies (data not shown) .

Patulin detection by affinity chromatography with IgG-TMRI . A "hit and run" fluoroimmunoassay (Warden B.A. et al. Anal. Biochem. 1987, 162, 363-369) was performed by labelling the IgG fraction with the fluorophore tetramethylrhodhamine isothiocyanate (TMRI, Fig. 4). The technique is based on the binding of fluorescently labelled IgG to the derivatives P-Sat- HS or P-Ins-HS, immobilized on a Sepharose 4B matrix as previously described, followed by displacement of the TMRI labelled antibodies by adding free patulin. Displaced antibodies are measured by the fluorescence in the eluate and represent a measure of the amount of free patulin added. (fig.6) . Labelling of IgG fraction was achieved by incubating it with TMRI, dissolved in a mixture of DMF and binding buffer, at pH 9.0. After purification by gel filtration, an aliquot of IgG-TMRI was applied to the column previously loaded with patulin derivatives 4 or 5, respectively. The column was washed and fluorescence emission was monitored at λ = 575 nm (excitation at λ=552 ran) . When the signal returned to the preloading level, samples of patulin (0, 10 and 100 μg-L ¹J in 2.0 mL of binding buffer PBS 0.1 M, NaCl 0.1 M at pH 7.4 were successively applied to the column and incubated for 10 min at room temperature. At the end of this period, 0.5 mL fractions were collected and their fluorescence emission signal was measured providing a measure of the IgG-TMRI complex released and thus of the free patulin added. Each experiment was performed in triplicate on each affinity column. Representative peaks of fluorescence are shown in Fig.5 for the experiments done with the column on which P-Ins-HS was immobilized. Similar results were obtained for the derivative P-Sat-HS. As seen, 10 μg-L of patulin are detectable by this technique. To test the specificity of the method, the antibiotic neomycin was also incubated with the matrix-IgG-TMRI complex under the same conditions used for patulin. In this case, as expected, no fluorescence emission at λ = 575 nm was detectable in the eluate fractions .

EXAMPLES

All reagents used in the following examples were of the highest commercially available quality and were used as received. L-arabinose was purchased from Sigma. NMR spectra were recorded on Bruker WM- 400, Varian Gemini 300, Varian Gemini 200 and Varian Inova 500 spectrometers. All chemical shifts are expressed in ppm with respect to the residual solvent signal. For the electrospray ionization spectroscopy mass spectrometry (ESI MS) analyses, a Waters Micromass ZQ instrument, equipped with an electrospray source, was used in the positive and/or negative mode. 1- (3-dimethylamino-propyl) -3- ethylcarbodiimide (EDC), bovine serum albumin (BSA; fraction V) , ovalbumin (OVA; gradeV) were purchased from Sigma. PURElA Protein A Antibody Purification kit was purchased from Sigma, Goat polyclonal to rabbit IgG-HRP conjugate (secondary antibody) was from Abeam. Affinity resin EAH Sepharose 4B was purchased from Amersham Biosciences. Nitrocellulose Transfer Membrane PROTRAN from Schleicher & Schuell and ECL detection reagents from Amersham Biosciences were used in Dot Blot and Western Blot experiments. 96-wells microplates Lock Well Maxisorp from Nunc, 3, 3', 5,5'- tetramethyl-benzidine (TMB) enzyme substrate from Sigma and a microplates reader Multiskan EX from Thermo were used for ELISA experiments. UV measurements (detection at λ= 278 nm) were carried out on a Varian Cary 50 Bio spectrophotometer. Tetramethyl rhodhamine isothiocyanate (TMRI) was purchased from Sigma. Fluorescence experiments were carried out on a ISS K2 fluorometer (ISS Champaign US) .

Synthesis and characterisation of patulin derivatives .

Synthesis of 4- [4- (benzyloxy) -2-oxo-2,6,7, 7a- tetrahydro-4H-furo [3,2-c]pyran-7-yl]oxy}-4-oxo- butanoic acid (3) .

4- (benzyloxy) -7-hydroxy-7, 7a-dihydro-4H-furo [3, 2- c]pyran-2 (6H) -one (2, 350 ing^", 1.37 mmol) was dissolved in 9 mL of dry THF and treated with 16.7 mg of DMAP (0.137 mmol) and 1.37 g of succinic anhydride (13.7 mmol), under stirring at r.t. After 8 h, a second aliquot of succinic anhydride (685 mg, 6.85 mmol) was added and the reaction left for additional 15 h. The reaction mixture was concentrated under reduced pressure and then redissolved in 25 mL of ethyl acetate, washed three times with water and purified by silica gel chromatography (eluent system: acetone in CHGl₃, from 5 to 10 %) , which resulted in 330 mg (0.91 mmol, 68%) of 3 as a pure compound.

¹HNMR (400 MHz, CDCl₃): δ_H 7.38 - 7.33 (5H, complex signals, aromatic protons); 5.98 (IH, d, H-3) ; 5.70 (IH, s, H-4); 5.48 (IH, d, H-7a) ; 5.23 (IH, m, H-7); 4.81 (IH, dd, J = 11.7 Hz, CHHPh); 4.65 (IH, dd, J =11.7 Hz, CHHPh); 4.05 (IH, d, J = 12.6 Hz, H-6); 3.90 (IH, dd, J = 12.6 Hz, H-6'); 2.62 (4H, m, H-IO and H-Il) ; ¹³C NMR (100 MHz, CDCl₃): δ_c 176.9 (C-9) ; 171.7 (C- 2); 171.0 (C-12); 158.9 (C-3a) ; 136.1, 128.7, 128.4 and 128.1 (aromatic carbons); 115.0 (C-3) ; 93.3 (C- 4); 77.6 (C-7a); 70.3 (C-7); 69.7 (CH₂Ph); 59.5 (C- 6); 28.8 and 28.6 (C-IO and C-Il); ESI-MS calculated for C₁₈H₁₈O₈: 362.04 Found (positive ions): 385.28 (M + Na⁺); 401.25 (M + K⁺).

Synthesis of 4-[ (4-hydroxy-2-oxo-2, 6,7,7a- tetrahydro-4H-furo [3,2-c]pyran-7-yl) oxy] -4- oxobutanoic acid (4, P-Sat-HS) .

To a solution of 3 (150 mg, 0.41 mmol) in dry DCM (10 mL) SnCl4 was slowly added (150 μl) at r.t. After 24 h additional 75 μL of SnCl4 were added and the reaction allowed to proceed for 24 h. The reaction mixture was poured into cold H₂O (20 mL) and diluted with CHCl₃ (5 mL) . The organic layer was separated and the aqueous phase was concentrated under reduced pressure, purified by silica gel chromatography (eluent system: MeOH in CHCl₃ from 20 % to 30 %) and passed through a DOWEX H⁺ ion exchange resin to obtain 75 mg (0.28 mmol, 67 %) of pure 4.

The signal interpretation in NMR spectrum relates to diastereoisomer most present. ¹HNMR (500 MHz, D20) : δ_H 6.25 (IH, s, H-3) ; 6.15 (IH, s, H-4); 5.62 (2H, overlapped signals, H-7 and H-7a); 4.36 (IH, d, J = 13.5 Hz, H-6, ) ; 3.96 (IH, dd, J = 13.5 Hz and J = 13.0 Hz, H-6'); 2.67 - 2.57 (4H, m, 4 x H of butanoic acid); ¹³C NMR (125 MHz, D₂O): δ_c 178.0 (C-4 of butanoic acid); 173.8 (C-I of butanoic acid); 165.1 (C-2); 161.7 (C-3a) ; 114.5 (C-3) ; 89.0 (C-4); 77.1 (C-7a) ; 71.6 (C-7); 70.1 (C-6) ; 29.6 (C-2 and C-3 of butanoic acid) ; ESI-MS: calculated for C₁₁H₁₂O₈: 272.05 Found (positive ions): 255.15 (M - H20 + H⁺), 295.14 (M + Na⁺) , 313.10 (M + K⁺) .

Synthesis of 4-[ (4-hydroxy-2-oxohexahydro-4H- furo [3,2-c]pyran-7-yl) oxy] -4-oxobutanoic acid (5, P- Ins-HS) .

To a solution of 3 (150 mg, 0.41 mmol) in 5 mL of MeOH a suspension of 150 mg of 10 % Pd/C in MeOH

(3 mL) was added under a flux of argon and the mixture was hydrogenated at atmospheric pressure for 16 h. The reaction mixture was filtered through a celite-pad and concentrated under reduced pressure. The residue was taken up in 40 mL of H₂O and washed twice with DCM. The aqueous layer was concentrated to yield 90 mg (0.32 mmol, 80%) of pure

5.

The signal interpretation in NMR spectrum relates to diastereoisomer most present.

¹HNMR (400 MHz, D₂O): δ_H 5.32 (IH, m, H-7); 5.21 (IH, d, J = 3.2 Hz, H-4); 5.10 (IH, m, H-Ia) ; 4.16 (IH, dd, J = 12.8 Hz and J ^ 2.4 Hz, H-6) ; 3.75 (IH, dd, J = 13.2 Hz and J = 1.6 Hz, H-6' ); 2.80 - 2.60 (7H, overlapped signals, H-3/ H-3a; 2CH₂ of butanoic acid) ; ¹³C NMR (100 MHz, D2O) : δ_c 183.9 (C-I of butanoic acid); 183.1 (C-4 of butanoic acid); 177.2 (C-2); 95.9 (C-4); 77.9 (C-7a) ; 70.1 [C-I); 62.1 (C-3a) ; 42.7 (C-3); 34.7 (C-2 of butanoic acid); 33.1 (C-3 of butanoic acid) ; ESI-MS: calculated for C₁₁H₁₄O₈: 274.07. Found (positive ions): 297.15 (M + Na⁺), 313.12 (M + K⁺).

Synthesis of BSA conjugates (antigens A and B) .

Synthesis of P-Ins-HS-BSA conjugate (antigen A) .

To a solution of P-Ins-HS (2 mg, 0.0073 mmol) in 0.5 mL of MES buffer 0.1 M, pH 5, 0.2 mL of a solution of BSA (4 mg mL ) in the same buffer and 0.1 mL of an EDC solution in H₂O (10 mg mL ) were added. The reaction mixture was incubated for 2 h at r.t. and then dialysed against 0.5 L of PBS 0.01 M NaCl 0.01 M, pH 7.4 (0.5 L, for three days with daily buffer changes). The concentration of the conjugate, spectrophotometrically determined at λ = 278 nm, was 2.8 mg-mL^""1.

Synthesis of P-Sat-HS-BSA conj ugate (antigen B) .

To a solution of P-Sat-HS (1 . 5 mg, 0 . 0054 mmol) in Tris pH 8 /dioxane, 1 : 1 (v/v, 0 . 4 mL) 20 μL ( 1 . 0 mg, 0 . 0054 mmol) of an EDC solution in H₂O (50 mg mL^~ ) and 0.5 inL of a BSA solution (8 mg itiL ¹J in PBS 0.1 M at pH 7.4 were added. After 2 h at r.t., the reaction mixture was dialysed against PBS 0.01 M, 0.01 M NaCl, pH 7.4 (0.5 L, for 3 days with daily buffer changes) . Conjugate concentration determined spectrophotometrically at λ «= 278 nm was 4.2 mg-mL .

Antibodies production and purification.

Antibodies production.

Two rabbits were immunized following a standard protocol by intradermal inoculation of a mixture of antigens A and B (0.5 mg each per rabbit). After the immunization period, rabbits were sacrificed and blood was centrifuged to separate blood cells from serum (SIl from rabbit 1 and SI2 from rabbit 2) . IgG purification.

2.0 mL of antiserum (SIl and SI2) were applied to a protein-A column of PURElA Protein A Antibody Purification kit, Sigma, and the IgG fraction was purified according to the manufacturer's instructions. Elution of proteins was monitored by absorbance at λ = 280 nm. The IgG fraction was eluted with glycine 0.1 M at pH 2.8 and immediately buffered in Tris 1 M at pH 8.0. Concentration on an Amicon XM50 membrane and dialysis against PBS 20 mM, pH 7.0, NaCl 50 mM resulted in 4.0 mL of IgGl and 4.0 mL of IgG2, respectively.

Affinity columns preparation. Two affinity columns were obtained by conjugating derivatives P-Ins-HS and P-Sat-HS, respectively, to EAH Sepharose 4B as follows. 1.O mL of resin was washed with H₂O at pH 4.5 (20 iriL) , with NaCl 0.5 M (20 mL) , again with H₂O at pH 4.5 (20 mL) and finally suspended in 2.0 mL of H₂O. The Sepharose resin was added to a solution of 4 or 5, respectively (5 mg in 0.5 mL of H20 at pH 4.5) and the resulting suspensions gently shaken. The slurry was cooled to 0 ⁰C and EDC was added in two steps to a final concentration of 0.1 M (52 mg) . After 12 h at 4 ⁰C, the reaction mixture was taken to r.t. and after an additional 4 h the resin was extensively washed with H20 at pH 4.5 and then treated with 1 mL of AcOH (0.1 M) and 38 mg of EDC, for 1 h at r.t. The suspension was washed with H20 at pH 4.5 (20 mL) , acetate buffer 0.1 M containing NaCl 0.5 M (20 mL) , pH 4.0, PBS 0.1 M containing NaCl 0.3 M, pH 7.4 (20 mL) and finally packed into a polystyrene column (2 mL, BIORAD) .

Antibodies purification by affinity chromatography.

For the affinity purification, a 2.0 mL aliquot of IgG (4 mL, see above) was dropwise applied to the affinity column prepared as described above. The resin was washed with PBS 0.01 M containing NaCl 0.1 M, pH 7.0 (20 mL) , with PBS 0.01 M containing NaCl 0.5 M, pH 7.0 (20 mL) , then with PBS 0.01 M containing NaCl 1 M, pH 7.0 (20 mL) and finally with glycine 0.1 M, pH 2.7 (2.5 mL) . The eluate was collected in 0.5 mL fractions, monitored by absorbance measurements at λ = 278 nm. The fractions containing the antibodies were collected, concentrated by means of a Centricon YM-3 membrane to a volume of 1.0 mL and dialyzed against PBS 0.1 M, NaCl 0.1 M, pH 7.4. The concentration of the antibodies was spectrophotometrically determined by absorbance measurements at λ = 278 nm.

Synthesis of GlnBP-conjugates (P-Sat-HS-GlnBP, P- Ins-HS-GlnBP) .

In order to avoid interference by the carrier protein in the polyclonal antibodies detection process P-Ins-HS e P-Sat-HS were conjugated to GInBP, a bacterial protein different from the protein used to carry out the immunization. The following procedure was used: 2.5 mg (0.0092 iranol) of 4 or 5 were dissolved in 0.25 mL of MES buffer 0.1 M, pH 5. The solution was incubated at r.t. with 0.25 mL of a GInBP solution (5 mg itiL^" ) in the same buffer and 0.1 mL of an aqueous solution of EDC (10 mg mL ) . After 5 h, the reaction mixture was dialyzed against PBS 0.01 M containing NaCl 0.1 M, at pH 7.4 (0.5 L, 3 days with daily buffer changes). Dot Blot experiments .

P-Sat-HS-GlnBP, P-Ins-HS-GlnBP and GInBP were spotted on four nitrocellulose membranes (10 μg and 1 μg of each antigen on each filter) and the spots allowed to dry. The filters were washed twice with PBS 0.1 M, NaCl 0.4 M at pH 7.4 (PBS) and then incubated with the blocking buffer (PBS containing 5 % skim milk, 0.2 % Tween 20 and 0.05 % Tryton, pH 7.4) for 1 h at r.t. After two washing with PBS 0.1 M, NaCl 0.4 M, Tween 0.2 %, Tryton 0.05 % at pH 7.4 (PBS-TT) and one with PBS • (10 min per washing) , the filters were incubated with SIl, SI2, SPIl and SPI2 (each diluted 1 : 250 in blocking buffer), respectively, for 1 h at r.t. Filters were washed (twice with PBS-TT and one time with PBS, 10 min per washing) before being incubated with the secondary antibody (goat anti-rabbit HRP-conjugate, 1:3000 in blocking buffer), for 1 h at r.t. The filters were washed three times as described above and developed with the detection reagent ECL.

Western Blot experiments .

Proteins (BSA, P-Ins-HS-GlnBP or P-Sat-HS-GlnBP and GInBP, 10 μg each) were loaded and separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (12 % SDS-PAGE) and then transferred overnight at 4 ⁰C onto a nitrocellulose membrane. Membranes were blocked for 1 h at r.t. by rocking in 50 mL of the blocking buffer (PBS containing 5 % skim milk, 0.2 % Tween 20 and 0.05 % Tryton ). After two washing with PBS-TT and one with PBS (10 min per washing) , the filters were incubated with a-PiHS and a-PsHS, respectively (1 : 500 in the blocking buffer), for 1 h at r.t. After two washing with PBS-TT and one with PBS (10 min per washing), the filters were incubated with secondary antibody (goat anti-rabbit HRP-conjugate, 1:3000 in the blocking buffer), for 1 h at r.t. The filters were washed three times as described above and then developed with the detection reagent ECL. Antibodies titration .

Antibodies titre was determined by an indirect ELISA assay, by the following general procedure. The antigens (P-Ins-HS-GlnBP or P-Sat-HS-GlnBP) , in PBS 0.1 M, pH 7.4, were used to coat 96-well microplates, varying the concentration by a factor

3, from 11 μg mL —1 to 1.7 10—3 μg mL—1 (one column for every antigen concentration, 100 μL per well) , overnight at 4° C. Control wells were incubated for the same period with BSA in the same buffer. The wells were rinsed three times with PBS 0.1 M containing 0.05 % Tween (PBS-T) pH 7.4, and blocked by incubation for 2 h at r.t. with PBS-T containing BSA 1 % (100 μL each well) . After three washing with PBS-T, serially diluted antibodies, aPiHSl, aPsHSl, aPiHS2 or aPsHS2 respectively, were added to the wells and incubated at r.t. for 1 h, then rinsed three times with PBS-T. Horsedish peroxidase- conjugated anti-rabbit IgG antibodies, diluted 1:4000 in PBS-T containing BSA 1 %, were added to the wells (100 μl) and incubated for 1 h at r.t. After three washeing with PBS-T, the enzyme substrate TMB was added (100 μL per well) and the colour reaction was quenched after 5 min by addition of 1 M H₂SO₄ (100 μL per well) . The absorbance was measured at λ = 450 nm. Antibodies titre was graphically determined by plotting the reciprocal of antibodies dilution vs absorbance for each dilution of antibodies. The titre was taken as the maximum antibodies dilution able reading 0.1 absorbance units. The following values were found: 1/25000 for aPsHSl and aPsHS2, and 1/25000 for aPiHSl and aPiHS2.

Synthesis of IgG-TMRI conjugate.

IgG fractions from each rabbit serum were labelled with TMRI as follows. 0.7 mL of IgG in buffer NaHCO₃ 10 mM at pH 9.0 (2.5 mg-mlT¹) were incubated with a solution of TMRI (0.15 mg-mL ) in DMF (50 μL) /NaHCO₃ 10 mM pH 9.0 (450 μL) , for 2 h at r.t. The labelled antibodies were purified on a Sephadex G-25 column. The fractions containing labelled IgG were pooled and concentrated to a volume of 2.0 mL on a Centricon microconcentrator. The degree of labeling (DOL) , as calculated from the absorbance values at λ = 280 and 552 nm, by applying a correction factor for label absorption at λ = 280 nm, was found to be 3.5.

Binding of IgG-TMRI to patulin derivatives on affinity columns and detection of free patulin.

The previously prepared affinity column was washed and equilibrated with the binding buffer PBS 0.1 M, NaCl 0.1 M, pH 7.4. 1.5 itiL of diluted IgG- TMRI (dilution 1 : 3 in binding buffer) were applied dropwise applied onto the resin. The column was washed with the binding buffer (20 mL) , then with PBS 0.1 M, NaCl 0.5 M pH 7.4 (20 mL) and finally with PBS 0.1 M, NaCl 1 M pH 7.4, until the fluorescence signal was returned to the preloading level (λ = 552 nm excitation; λ = 575 nm emission) . Samples of patulin (0, 1, 10, 100 ppb) in 2.0 mL of binding buffer were applied to the column at r.t. and incubated for 10 min. At the end of the incubation period, the patulin-IgG-TMRI complex was eluted from the column and collected in 0.5 mL fractions. The fluorescence emission signal was measured at λ = 575 nm. The column was washed with 5 mL of the binding buffer and could be re-used for the analysis of the successive patulin sample.

CONCLUSIONS

The polyclonal antibodies produced against the mycotoxin patulin, by means of two synthetic derivatives of the molecule conjugated to bovine serum albumin as carrier protein, after affinity purification, showed a high titre for the recognition of the haptene. The immunoglobulins were successfully used to develop a competitive immunofluorescent assay for the detection of small quantities of patulin. The method was based on the binding of fluorescently labelled antibodies to the synthetic patulin derivative, covalently immobilized on a Sepharose matrix. The displacement of the antibodies by addition of free patulin was detected as fluorescence signal at the emission wavelength of the fluorophore. The assay allowed the detection of 10 μg/L of patulin, and thus represents an alternative methodology compared to the analytical chromatographic techniques in use.

Claims

1) Patulin derivative of formula:

4-[ (4-hydroxy-2-oxo-2, 6, 7, 7a-tetrahydro-4H-furo [3,2- c]pyran-7-yl) oxy] -4- oxobutanoic acid, (P-Sat-HS) , having the following chemical structure (4):

(4) 2) Patulin derivative of formula:

4-[ (4-hydroxy-2-oxohexahydro~4H-furo [3,2-c]pyran-7- yl) oxy]-4-oxobutanoic acid,, (P-Ins-HS) , having the following chemical structure (5) :

(5) 3 Method for producing the patulin derivative as defined in claim 1 comprising the steps of: a) preparing a derivative (2) having the following chemical structure:

(2) from commercial L-arabinose by a known synthetic scheme; b) treating said derivative (2) with succinic anhydride and DMAP in dry THF for 15 h at r.t. to obtain an acid derivative (3) having the following chemical structure:

(3) c) treating said acid derivative (3) with SnCL₄ to remove the benzyl protecting group and obtain a mixture of diastereoisomers wherein said acid derivative (4) is present in higher amount; d) treating said mixture in order to obtain a pure compound (4) .

4) Method for producing the patulin derivative as defined in claim 2 comprising the steps of: a) preparing a derivative (2) having the following structure :

(2) from commercial L-a^'rabinose by a known synthetic scheme;

b) treating said derivative (2) with succinic anhydride and DMAP in dry THF for 15 h at r.t. to obtain an acid derivative (3) having the following structure:

c) adding to a solution of said acid derivative (3) in MeOH, a suspension of Pd/C in MeOH under flux of argon and idrogenating the obtained mixture at atmospheric pressure for 16 hours, in order to obtain a reaction mixture of diastereoisomers wherein said acid derivative (5) is present in higher amount; d) treating said reaction mixture in order to yield an amount of pure compound (5) .

5) Method to detect the content of patulin in foods characterized by the fact that it provides for the use of polyclonal and/or monoclonal antibodies produced against the micotoxin patulin by means of the two following patulin derivatives:

4-[ (4-hydroxy-2-oxo-2, 6, 7^', 7a-tetrahydro-4H- furo[3,2-c]pyran-7-yl) oxy]-4- oxobutanoic acid (4), and

4-[ (4-hydroxy-2-oxohexahydro-4H-furo [3,2-c]pyran- 7-yl)oxy] -4-oxobutanoic acid (5) which are conjugated to a protein and/or to other molecules used as carrier to produce polyclonal and/or monoclonal antibodies in any organism.

6) Method according to claim 5 characterized by the fact that the derivatives (4) and (5) are conjugated to bovine serum albumin (BSA) as carrier protein.

7) Method according to claim 5 characterized by the fact that it further provides the purification of the produced antibodies by affinity chromatography on columns obtained by covalently immobilizing said two patulin derivatives on a solid support

8) Method according to claim 5 wherein the solid support is a Sepharose matrix.

9) An immunoassay for detection of small amounts of patulin down to 10 μg/L in foods characterized by the fact that it comprises the step of binding between patulin to antipatulin derivatives antibodies.

10) A competitive immunoassay for detection of patulin down to 10 μg/L in foods characterized by the fact that there is a competition between patulin and derivatives thereof and antipatulin derivatives antibodies.

11) A competitive immunofluorescent assay for the detection of small quantities of patulin down to 10 μg/L in foods characterized by the fact that it comprises the step of labelling IgGs with the fluorophore tetramethylrhodhamine isothiocyanate (TMRI) , binding the labelled IgGs to derivatives of formula 4 (P-Sat-HS) or 5 (P-Ins-HS) , immobilized on solid support, and detecting the displacement of TMRI labelled antibodies (fluorescence signal detection) upon binding of patulin.

12) Use of derivatives of formula 4 (P-Sat-HS) and formula 5 (P-Ins-HS) according to claims 1 and 2 for the preparation of solid supports for affinity chromatography.

13) Use of solid supports for affinity chromatography according to claim 8 for the purification of specific antibodies and other patulin-binding proteins to be used in patulin detection.

14) Use of derivatives of formula 4 (P-Sat-HS) and 5 (P-Ins-HS) according to claims 1 and 2 for the production of polyclonal and/or monoclonal antibodies antiP-Sat-HS and anti-P-Ins-HS in any organism, which recognise also patulin. 15) Use of antibodies produced against the derivatives of formula 4 (P-Sat-HS) and/or 5 (P-Ins-HS) according to claims 1 and 2, for patulin detection trough immunoenzymatic assay. 16) Use of antibodies produced against the derivatives of formula 4 (P-Sat-HS) and/or 5 (P-Ins-HS) according to claims 1 and 2, modified with fluorescent probes and/or radioactive probes and /or magnetic probes . 17) Use of antibodies produced against the derivatives of formula 4 (P-Sat-HS) and/or 5 (P-Ins-HS) according to claims 1 and 2, for patulin detection through optical method.

18) Use of antibodies produced against the derivatives of formula 4 (P-Sat-HS) and/or 5 (P-Ins-HS) according to claims 1 and 2, for patulin detection in any kind of foods and vegetables.

19) Use of antibodies produced against the derivatives of formula 4 (P-Sat-HS) and/or 5 (P-Ins-HS) according to claims 1 and 2, for patulin detection through cromatographic method.

20) Use of antibodies produced against the derivatives of formula 4 (P-Sat-HS) and/or 5 (P-Ins-HS) according to claims 1 and 2, for patulin detection through mass spectrometry method.

21) Use of antibodies produced against the derivatives of formula 4 (P-Sat-HS) and/or 5 (P-Ins-HS) according to claims 1 and 2, for patulin detection through micro and nanotechnologies . 22) Use of derivatives of formula 4 (P-Sat-HS) and/or 5 (P-Ins-HS) according to claims 1 and 2, for patulin detection trough immunoenzymatic assay.

23) Use of derivatives of formula 4 (P-Sat-HS) and/or 5 (P-Ins-HS) according to claims 1 and 2, modified with fluorescent probes and/or radioactive probes and /or magnetic probes.

24) Use of derivatives of formula 4 (P-Sat-HS) and/or 5 (P-Ins-HS) according to claims 1 and 2, for patulin detection through optical methods. 25) Use of derivatives of formula 4 (P-Sat-HS) and/or 5 (P-Ins-HS) according to claims 1 and 2, for patulin detection in any kind of foods and vegetables.

26) Use of derivatives of formula 4 (P-Sat-HS) and/or 5 (P-Ins-HS) according to claims 1 and 2, for patulin detection through cromatographic methods.

27) Use of derivatives of formula 4 (P-Sat-HS) and/or 5 (P-Ins-HS) according to claims 1 and 2, for patulin detection through mass spectrometry methods.

28) Use of derivatives of formula 4 (P-Sat-HS) and/or 5 (P-Ins-HS) according to claims 1 and 2, for patulin detection through micro and nanotechnologies .

29) Use of derivatives of formula 4 (P-Sat-HS) and/or 5 (P-Ins-HS) according to claims 1 and 2, for immobilization on a solid support and inclusion in micellar structures.

30) Use of solid supports for affinity chromatography according to claims 7 and 8 for the purification of patulin-binding proteins from natural sources or from transgenic organism to be used in patulin detection.