Novel haptens and monoclonal antibodies with subnanomolar affinity for a classical analytical target, ochratoxin A

Ochratoxin A is a potent toxic fungal metabolite whose undesirable presence in food commodities constitutes a problem of public health, so it is strictly regulated and controlled. For the first time, two derivatives of ochratoxin A (OTAb and OTAd) functionalized through positions other than the native carboxyl group of the mycotoxin, have been synthesized in order to better mimic, during the immunization process, the steric and conformational properties of the target analyte. Additionally, two conventional haptens making use of that native carboxyl group for protein coupling (OTAe and OTAf) were also prepared as controls for the purpose of comparison. The immunological performance in rabbits of protein conjugates based on OTAb and OTAd overcome that of conjugates employing OTAe and OTAf as haptens. After immunization of mice with OTAb and OTAd conjugates, a collection of high-affinity monoclonal antibodies to ochratoxin A was generated. In particular, one of those antibodies, the so-called OTAb#311, is very likely the best antibody produced so far in terms of selectivity and affinity to ochratoxin A.


Reagents, equipment, and general techniques used in the preparation of haptens and bioconjugates
Organic solvents were dried and distilled prior use using standard techniques. 1 Et 2 O and THF were distilled over Na and benzophenone under N 2 atmosphere just before being used. CH 2 Cl 2 and CH 3 CN were distilled from CaH 2 in the same way. MeOH was dried and stored on activated molecular sieves (3Å). DMF was distilled from CaH 2 at 7 mmHg and stored at -20 °C on 4Å molecular sieve. 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 N 2 or Ar, using syringes and/or cannulas, oven-dried (130 °C) glass material and freshly distilled and dried solvents. Reactions were monitored by thin-layer chromatography (TLC) 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 0.043-0.063 mm). Melting points were determined on a Büchi M-560 apparatus and are uncorrected. Proton and carbon nuclear magnetic resonance ( 1 H and 13 C NMR) spectra were recorded at room temperature (rt) on a Bruker Avance DPX300 spectrometer operating at 300.1 and 75.5 MHz, respectively, or on a Bruker Avance DRX500 spectrometer operating at 500.1 and 125.8 MHz, respectively. Chemical shifts (δ) are expressed in ppm recorded using the residual solvent as the internal reference in all cases [7.27/77.00 ppm and 2.50/39.51 for the 1 H/ 13 C spectra in CDCl 3 and DMSO, respectively (ACD/NMR Processor Academic Edition spectra processing program, version 12.0)]. Carbon substitution degrees were established by DEPT (Distortionless Enhancement by Polarisation Transfer) pulse sequences. A combination of COSY (COrrelation SpectroscopY) and HSQC (Heteronuclear Simple Quantum Coherence) experiments was used in most cases for the assignment of 1 H and 13 C chemical shifts. High resolution 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). Analysis of hapten bioconjugates was performed by mass spectrometry in a 5800 MALDI TOF-TOF (ABSciex) in positive linear mode (1500 shots for each position) in a mass range of 10000-120000 m/z.

Reagents and equipment used in antibody generation and immunoassays
Ochratoxin A was purchased from Sigma/Aldrich (Madrid, Spain) and ochratoxins B and C from Toronto Research Chemicals (Ontario, Canada). BSA fraction V was from Roche Applied Science (Mannheim, Germany). OVA, HRP, adult bovine serum, Freund's adjuvants, and ophenylenediamine were provided by Sigma/Aldrich (Madrid, Spain). Goat anti-rabbit immunoglobulins and goat anti-mouse immunoglobulins were obtained from Rockland Inc.
(Limerick, PA, USA) and Jackson ImmunoResearch Laboratories Inc. (West Grove, PA, USA), respectively. As secondary antibody, polyclonal rabbit anti-mouse immunoglobulin antibody peroxidase conjugate from Dako (Glostrup, Denmark) was used. Costar flat-bottom high-binding 96well polystyrene ELISA plates from Corning (Corning, NY, USA) were used. ELISA absorbances were read with a PowerWave HT from BioTek Instruments (Winooski, VT, USA). Microplate wells were washed with an ELx405 microplate washer also from BioTek Instruments. Figure S1. New synthetic route for the preparation of OTα Racemic OTα (5S) shown in Figure S1 was prepared through a new synthetic route starting from 4-chloro-3-methylphenol (p-chlorocresol, 1S), a very cheap industrial product widely used as an antiseptic and preservative. The synthesis began with the iodation of both positions ortho to the phenolic OH group to give the aryl diode 2S, which upon treatment with CO/MeOH under palladiumcatalyzed carbonylation reaction conditions afforded diester 3S. The construction of the S4 dihydroisocoumarin ring was accomplished based on the Kraus's method, 2 which involves the deprotonation of the methyl group with lithium diisopropylamide (LDA) and subsequent capture of the generated benzylic anion with excess acetaldehyde. The synthesis of OTα was readily completed by basic hydrolysis of the methoxycarbonyl group to the corresponding carboxylic acid group.
At the end of this time, the reaction mixture was treated with a 1:2 mixture of AcOH-Et 2 O and stirred for 10 minutes at 0 °C, diluted with EtOAc and washed with water and brine and dried over anhydrous

Preparation of 3-(5-(benzyloxy)pentyl)-5-chloro-8-hydroxy-1-oxoisochromane-7-carboxylic
acid (10). A solution of LiOH•H 2 O (135.0 mg, 3.21 mmol, 10 equiv) in water (1.40 mL) was added to a solution of dihydroisocoumarin 9 (139.0 mg, 0.32 mmol) in anhydrous THF (1.30 mL) and the mixture was heated at reflux for 3.5 h. The reaction mixture was then cooled in an ice bath and treated with a 1M aqueous solution of HCl (5.80 mL, 5.80 mmol, 18 equiv), stirred at 0 °C for 2 h, diluted with water and extracted with EtOAc. The combined organic phases were washed with brine, dried over anhydrous MgSO 4 and concentrated to dryness under reduced pressure to obtain acid 10 (134.5 mg, 100%) as a yellowish semi-solid, whose 1 H NMR showed to have a sufficiently high purity to be used in the next stage without further purification. 1 H NMR (MeOD, 300 MHz) δ (ppm) 1.42-

Antibody generation
Two 2-kg female New Zealand white rabbits were immunized with 21-day intervals by subcutaneous injection of 0.3 mg of BSA-hapten conjugate in 1 mL of a 1:1 emulsion between sterile 100 mM phosphate, pH 7.4, and Freund's adjuvant (complete for the first dose and incomplete for subsequent boosts). Ten days after the fourth injection, rabbits were exsanguinated by intracardiac puncture. Blood samples were allowed to coagulate overnight at 4 °C, and sera were separated by centrifugation (3000×g, 20 min). Finally, antibodies were precipitated with 1 volume of saturated ammonium sulfate solution. Salting out was performed twice, and precipitates were stored at 4 °C.
Four two-month old female Balb/c mice were immunized by intraperitoneal injection of 0.1 mg of BSA-OTAb or BSA-OTAd conjugate in 200 µL of a water-in-oil emulsion that was prepared using Freund's adjuvants as described above for rabbit immunization. Three doses were applied with 21-day intervals, and a fourth injection in sterile PBS with the same amount of protein conjugate was given at least 21 days after the third injection and 4 days before the animal was sacrificed.
Hybridomas were prepared by fusion of mouse myeloma cells with lymphocytes b from two equallyimmunized mice.
Antibody-producing cells were screened by a double sequential procedure. Twelve days after cell fusion, hybridoma culture supernatants were first screened by differential competitive ELISA on microtiter plates coated with 0.1 µg/mL (100 µL per well) of the homologous OVA-hapten conjugate. Fifty microliter of each supernatant was added to two adjacent wells of an ELISA plate, one containing 50 µL of PBS (blank) and the other containing 50 µL of 200 nM OTA 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 those culture plate wells giving signals higher than 3.0 in the absence of mycotoxin. Next day, they were reevaluated by checkerboard competitive ELISA. This second screening assay was carried out with two coating concentrations of the homologous OVA-hapten conjugate (0.01 and 0.1 µg/mL), four supernatant dilutions (1/10, 1/50, 1/250, and 1/1250), and three OTA levels (0, 10, and 100 nM). Hybridomas selected from the first and the second screening assays were cloned twice by limiting dilution and cryopreserved in liquid nitrogen. Monoclonal antibodies were purified from late stationary phase hybridoma cell cultures by affinity chromatography using 5-mL protein G columns from GE Healthcare (Uppsala, Sweden) and stored at 4 °C as ammonium sulfate precipitates.