Harnessing the Intrinsic Chemical Reactivity of the Mycotoxin Patulin for Immunosensing

Mycotoxins are globally pervasive contaminants that threaten food safety worldwide. Regulatory authorities have established maximum permissible levels for certain mycotoxins, and their presence is routinely monitored throughout the food chain to ensure the provision of healthy food and safe feed for humans and animals. While immunoanalytical methods are essential for mycotoxin screening, monoclonal antibodies for the detection of patulin are notably absent. Moreover, leading immunodiagnostic companies currently do not offer rapid tests for patulin in their product portfolios. This deficiency in mycotoxin testing is primarily due to the electrophilic reactivity of patulin. In this study, we exploit this reactivity to develop an innovative strategy that targets the stable adduct formed by the reaction of patulin with aryl-1,2-dithiolates, rather than analyzing the mycotoxin itself. Based on this previously unknown reaction, we present the first collection of monoclonal antibodies, enabling the long-sought goal of sensitive, simple, and user-friendly immunosensing of patulin.


Reagents, equipment, and general techniques in synthesis procedures
Tetrahydrofuran (THF) were distilled over Na and benzophenone under nitrogen atmosphere before use.CH 3 CN were distilled from CaH 2 in the same way. 1 Anhydrous N,N-dimethylformamide (DMF) and MeOH were purchased from Fisher Scientific (Madrid, Spain).Patulin was purchased from Fermentek (Jerusalem, Israel).The remaining solvents and commercial reagents were used without prior purification.The operations with air and/or moisture-sensitive reagents were carried out under an inert atmosphere of dry nitrogen, using syringes or cannulas, oven-dried (140 °C) glass material, and freshly distilled and dried solvents.
Reactions were monitored by thin-layer chromatography on precoated silica plates (0.25 mm layer thickness, Silica Gel 60 F 254 ) using UV light as the visualizing agent and ethanolic phosphomolybdic acid or aqueous ceric ammonium molybdate solutions and heat as developing agents.The synthesized compounds were purified by flash column chromatography using silica gel 60 (particle size 0.043-0.063mm).IR spectra were recorded using a Nicolet Avatar 320 FT-IR spectrophotometer equipped with ATR (IR band intensities: w = weak, m = medium, s = strong). 1H/ 13 C NMR spectra were recorded at 25 o C, in the solvent indicated, at 300/75 MHz (Bruker Avance DPX300 spectrometer), 400/101 MHz (Bruker AV400 spectrometer) or 500/125 MHz (Bruker Avance DRX500 spectrometer).The chemical shifts are expressed in ppm (δ scale) relative to the residual solvent as the internal reference in all cases [7.27/77.16ppm, 2.05/29.84ppm, 3.31/49.00ppm and 4.79 ppm for the 1 H/ 13 C spectra in CDCl 3 , acetone-d 6 , methanol-d 4 and D 2 O, respectively.Carbon substitution degrees were established by DEPT pulse sequences.Complete assignment of 1 H and 13 C chemical shifts of selected compound was made based on a combination of COSY, HSQC, HMBC and NOESY experiments.Highresolution mass spectra (HRMS) were obtained by electrospray ionization (ESI) mode in a premier Q-TOF mass spectrometer equipped with an electrospray source (Waters, Manchester, UK).The obtained data are expressed as mass/charge ratio (m/z).

Reaction of patulin with 4,5-dimethylbenzene-1,2-dithiol: Preparation of adduct III
i) Preparation of disodium 4,5-dimethylbenzene-1,2-thiolate.Sodium hydride 60 wt % dispersion in mineral oil (6.2 mg, 3.7 mg of NaH, 0.154 mmol, 2.2 equiv) was weighed into a round bottom flask fitted with a magnetic stir-bar and purged with nitrogen.The NaH-oil dispersion was then washed with several portions of dry pentane and back-flushed with N 2 , to give a dry white powder.The sodium hydride was then suspended in 150 μL of dry THF and cooled in an ice bath.A solution of 4,5-dimethylbenzene-1,2-dithiol 2,3 (12 mg, 0.071 mmol) in 300 μL of dry THF was then added dropwise (hydrogen evolution) and the mixture stirred for 1 h at 0 o C and for a further hour at rt.The resulting white suspension was transferred to a centrifuge tube and centrifuged at high speed.The supernatant was removed and the solid was washed with 500 μL of dry THF and centrifuged again.This procedure was repeated for two more times.The supernatant and washing were concentrated to dryness in the rotary evaporator and then placed under high vacuum to give disodium 4,5-dimethylbenzene-1,2-thiolate as a white powder (15 mg, 99%).

Preparation of bioconjugates of adduct I
For the preparation of the bioconjugate BSA-adduct I, 132 µL of a 50 mM solution in DMF of the NHS ester of adduct I (6.6 µmol, 30 equiv) were added slowly with stirring to 1.0 mL of a solution of BSA (15 mg/mL, 0.22 µmol) in PB, and the conjugation reaction mixture was stirred overnight at rt.For the I (6.3 µmol, 20 equiv) were added to 1.85 mL of a 15 mg/mL solution of OVA (0.63 µmol) in PB, and the conjugation reaction mixture was stirred overnight at room temperature.The bioconjugates were purified by size-exclusion chromatography and sterilized by filtration before being stored at -20 °C.

MALDI mass spectrometry analysis of bioconjugates
For sample preparation, 100 μL of bioconjugates (0.5-1 mg/mL) were dialyzed against milli-Q water and lyophilized.The samples were dissolved in Milli-Q water to a theoretical final concentration of 1 mg/mL, and 0.8 μL was spotted onto the MALDI plate.After the droplet was air-dried at rt, 0.8 μL of matrix (10 mg/mL sinapinic acid in 70% MeCN, 0.1% trifluoroacetic acid) was added and allowed to air-dry at rt.Then, samples were analyzed in a 5800 MALDI TOF/TOF (ABSciex) apparatus in positive linear mode (1500 shots every position) in a mass range of 12000-100000 m/z.Previously, the plate was calibrated with 1 μL of the TOF/TOF calibration mixture (ABSciex), in 13 positions.Every sample was calibrated by 'close external calibration' method with a BSA, OVA or HRP spectrum acquired in a close position.The analysis of the results was performed using the mMass program (http://www.mmass.org/).Thereafter, the solutions were added to microtiter wells precoated with OVA-Ia or OVA-Ib at 1 µg/mL followed by the corresponding antiserum diluted 1/3000 in PBS-T.Error bars represent the standard deviation (n=3).

Monoclonal antibody generation, selection and purification
Six two-month old female Balb/c mice were inoculated by intraperitoneal injection with 100 μg of BSA-Ia conjugate in a 1:1 (v/v) emulsion (200 µL) between PB and Freund's adjuvant (complete for the first injection and incomplete for the second and third injections).After a resting period of at least three weeks from the third boost, a fourth injection in sterile phosphate buffer was administered four days before cell fusions.
For hybridoma generation, a modified procedure from the original protocol developed by Köhler and Milstein was followed which entails the cellular fusion of myeloma cell line P3-X63-Ag8.635with B lymphocytes isolated from the spleen of 2 equally-inoculated mice. 6Once animal sacrifice had taken place, blood samples were obtained by intracardiac puncture, followed by spleen extirpation.Physical force using the piston of a sterile syringe was applied in order to extract cells from the spleen.Erythrocytes were lysed by osmotic shock employing 1 mL of lysis buffer at 4 °C for 1 min, and after washing with complete medium at 4 °C, the suspension was filtered to separate possible blood clots.Cellular fusion was carried out by mixing cultured myeloma cells with the isolated B lymphocytes at a 4:1 lymphocyte/myeloma ratio.The resulting mixture was washed 3 times with incomplete medium and then collected by centrifugation, and 1 mL of PEG 1500 at 37 °C was added for 1 min.Afterwards, the cell mixture was gradually diluted with 50 mL of complete medium and incubated for 30 min.After centrifugation, fused cells were distributed in 96-well culture plates at a density of 1.5 × 10 5 lymphocytes per well in 100 µL of DMEM containing 15% (v/v) FBS and incubated at 37 °C (5% CO 2 , 95% RH).Twenty-four hours after plating, 100 µL of selection medium (DMEM supplemented with HAT containing 20% (v/v) FBS and 1% (v/v) HFCS) was added to each well and the plates were incubated under the same conditions for 12 days.
Antibody-producing cells were identified by a double screening procedure.Twelve days after cell fusions, hybridoma culture supernatants were first screened by differential antigen-coated competitive ELISA on microtiter plates coated with 0.1 μg/mL (100 μL per well) of the OVA-Ia bioconjugate.Fifty microliters of each supernatant was added to two adjacent wells of an ELISA plate, one containing 50 μL of PBS (blank) and the other one containing 50 μL of 200 nM adduct I in PBS.The ratio between the signals of both wells was used as the criterion for selecting the antibodies with the highest affinity.Fresh culture medium was added to the selected wells, and the next day they were revaluated by checkerboard competitive ELISA.This second screening assay was carried out with two coating concentrations of the OVA-Ia conjugate (0.01 and 0.1 μg/mL), four supernatant dilutions (1/10, 1/50, 1/250, and 1/1250), and three analyte levels (0, 10, and 100 nM).Selected hybridomas were cloned by limiting dilution in cloning medium (DMEM containing 20% (v/v) FBS and supplemented with HT and 1% (v/v) HFCS), and stable antibody-producing clones were expanded and cryopreserved in liquid nitrogen.
Figure S3.Global minimum energy conformation of adduct I methyl ester.Calculations were performed using Molecular Mechanics (MM3) as implemented in the SCIGRESS program (version 3.0.0).A systematic conformational search was performed (all rotatable bonds were rotated by 15 degree steps) and the geometry of the generated conformers was refined by performing an optimize geometry calculation in MOPAC using PM3 parameters and including solvation effects of water simulated by COSMO [MO-G-PM3_H2O].Dotted arrows denote relevant NOESY correlations.

Figure S7 .
Figure S7.Recognition of patulin by rabbit polyclonal antibodies elicited by immunization with the bioconjugates BSA-Ia (green curve) and BSA-Ib (red curve).For the competitive step of the immunoassay, the patulin standards were prepared in PBS and 2% (v/v) of a 1 mg/mL of 7a in water was added to each calibrator.The standards were incubated at rt for 30 min to quantitatively convert patulin to adduct I.

Position assignment Chemical shift (ppm) a 1 H multiplicity b 1 H-13 C connectivity at 2 bonds c 1 H-13 C connectivity at 3 or more bonds c Observed NOE effects d 1 H 13 C
Chemical shift assigned from the experiments of 1 H, 13 C, HSQC and HMBC in CDCl 3 as solvent.