Preparation of bioconjugates and antibodies for the immunodetection of anatoxin-a

Abstract

The present invention relates to bioconjugates and labeled derivatives of anatoxin-a, at different positions of the molecule, suitable for producing antibodies with high affinity and specificity for anatoxin-a. At the same time, the present invention also relates to the use of bioconjugates of anatoxin-a and labeled derivatives of anatoxin-a as assay antigens. Moreover, the present invention also relates to methods for analyzing, concentrating and extracting anatoxin-a using the antibodies obtained, sometimes together with assay antigens which are bioconjugates or labeled derivatives. This invention also provides a kit for analyzing anatoxin-a which comprises antibodies against this cyanotoxin sometimes together with assay antigens which are bioconjugates or labeled derivatives of anatoxin-a.

Claims

1. A bioconjugate of formula (Ib) ##STR00039## wherein P is bovine serum albumin and n is 14.5.

2. A method of producing a monoclonal antibody against anatoxin-a, the method comprising immunizing a mouse with the bioconjugate of claim 1.

Description

BRIEF DESCRIPTION OF THE FIGURES

(1) FIG. 1 structure of anatoxin-a.

(2) FIG. 2 diagram of the synthesis of the NHS-ANA-1 hapten.

(3) FIG. 3 diagram of the synthesis of the NHS-ANA-2 hapten.

(4) FIG. 4 diagram of the synthesis of the NHS-ANA-3 hapten.

(5) FIG. 5 diagram of the preparation of a bioconjugate of formula (I) from the corresponding functionalized derivative (hapten) of anatoxin-a.

(6) FIG. 6 standard curve for anatoxin-a with the monoclonal antibodies obtained in the indirect competitive ELISA format.

(7) FIG. 7 standard curve for anatoxin-a with the monoclonal antibodies obtained in the direct competitive ELISA format.

(8) FIG. 8 Enantiospecificity of the monoclonal antibodies against anatoxin-a.

EXAMPLES

(9) The invention is illustrated below by means of tests carried out by the inventors which reveals the efficacy of the bioconjugates of formula (I) for obtaining antibodies against anatoxin-a and developing an immunoassay with high sensitivity for the same. The numbers in bold make reference to the corresponding structure which is shown in the diagrams. These examples are presented as a demonstration, but in no way may they be assumed as limiting the invention.

1. Preparation of Bioconjugates of Formula (I)

Example 1

(10) Preparation of bioconjugates of formula (I) for T=R-I, L=CH.sub.2CH.sub.2CH.sub.2, Z(CO)NH and P=BSA (bovine serum albumin), OVA (ovalbumin) and HRP (horseradish peroxidase).

1.1. Preparation of the Ester of N-Hydroxysuccinimide of the ANA-1 Hapten (NHS-ANA-1)

(11) The starting material (1) was prepared from 1,2-epoxy-5-cyclooctene by adapting a previously published route [D. B. Kanne et al., J. Am. Chem. Soc. 1986, 108, 7864-7865].

(12) ##STR00011##

Preparation of tert-butyl 2-oxo-9-azabicyclo[4.2.1]nonane-9-carboxylate (2)

(13) A mixture of 300 mg (1.308 mmol) of bencylamine 1, 57 mg of 20% Pd(OH).sub.2 on carbon (50% moisture) and 1.12 equivalents (320 mg, 1.465 mmol) of di-tert-butyl carbonate in 3.2 mL of AcOEt was maintained with stirring under a pressure of 60 psi of H.sub.2 at room temperature for 12 hours. After this time, the mixture was filtered over celite and washed with AcOEt. The filtrate was washed with 20 mL of a NaHCO.sub.3 saturated solution and brine and dried over MgSO.sub.4 anhydrous. The resulting residue of the evaporation of the solvent was purified by means of silica gel chromatography, using AcOEt-hexane 9:1 as the eluent in order to obtain the protected N-Boc amine 2 as a colorless oil which solidifies at 20 C. (238 mg, 76%).

(14) IR v.sub.max/cm.sup.1 2967, 2931, 1709, 1687, 1392, 1343, 1168, 1102. .sup.1H NMR (300 MHz, CDCl.sub.3) (2 rotamers in a proportion of 2:1; the data of the majority are described) 4.58-4.18 (2H, m, H-1 and H-6), 2.59-2.47 (1H, m, H-3), 2.40-2.25 (2H, m, H-3 and H-7), 2.25-2.06 (2H, m, H-5 and H-8), 1.96-1.86 (1H, m, H-7), 1.81-1.62 (4H, m, H-4, H-5, H-8), 1.42 (9H, s, CMe.sub.3), .sup.13C NMR (75 MHz, CDCl.sub.3) 215.4 (C-2, CO), 153.1 (CO Boc) 80.4 (CMe.sub.3), 65.1 (C-6), 56.6 (C-1), 41.8 (C-3), 33.0 (C-5), 30.0 (C-7), 28.5 (CMe.sub.3), 26.9 (C-8), 19.4 (C-4); EMAR (ES) m/z calculated for C.sub.13H.sub.22NO.sub.3 [M+H].sup.+ 240.1594, found 240.1587.

(15) ##STR00012##

Preparation of tert-butyl 2-(((trifluoromethyl)sulfonyl)oxy)-9-azabicyclo[4.2.1]non-2-ene-9-carboxylate (3)

(16) In a flask, a solution was prepared with 123 mg (0.514 mmol) of the ketone 2 in 6.65 mL of THF anhydrous under inert atmosphere. The solution was cooled at 78 C. and 2.0 equivalents (2.05 mL, 1.027 mL) of 0.5 M bis(trimethylsilyl)potassium amide (KHMDS) in toluene were added dropwise; the reaction was left to progress for 1.5 hours and subsequently 2.5 equivalents (504 mg, 1.284 mmol) of a solution of N-(5-chloro-2-pyridinyl)bis(trifluoromethanesulfonamide) (Comins reagent) dissolved in 0.4 mL of THF anhydrous were added. This mixture was stirred for 1 additional hour, maintaining the reaction temperature at 78 C. Finally, 2 mL of an ammonium chloride saturated solution was added at 0 C., the reaction mixture was diluted with water and extracted with AcOEt (315 mL). The collected organic phases were washed with brine and were dried over MgSO.sub.4 anhydrous. The residue obtained after removing the solvent was purified by means of column chromatography, using hexane-AcOEt 9:1 as the eluent in order to obtain the enol triflate 3 as a colorless oil (174.0 mg, 91%). IR v.sub.max/cm.sup.1 2972, 1679, 1455, 1400, 1366, 1243, 1201, 1168, 1138, 1072, 884. .sup.1H NMR (300 MHz, CDCl.sub.3) (2 rotamers in a proportion of 3:1; the data of the majority are described) 5.79-5.72 (1H, m, H-3), 4.55 (1H, m, H-1), 4.44-4.23 (1H, m, H-6), 2.21 (2H, m, H-4), 2.20-1.64 (2H, m, H-5), 2.06 (2H, m, H-8), 2.04-1.67 (2H, m, H-7), 1.44 (9H, s, CMe.sub.3) .sup.13C NMR (75 MHz, CDCl.sub.3) 154.6 (C-2), 153.1 (CO Boc), 120.5 (C-3), 116.5 (CF.sub.3), 80.5 (CMe.sub.3), 58.9 (C-1), 54.9 (C-6), 32.4 (C-7), 31.0 (C-8), 30.5 (C-5), 28.3 (CMe.sub.3), 19.7 (C-4) .sup.19F-NMR (282 MHz, CDCl.sub.3) 74.4 (s), EMAR (ES) m/z calculated for C.sub.14H.sub.21F.sub.3NO.sub.5S [M+H].sup.+ 372.1009, found 372.1014.

(17) ##STR00013##

Preparation of tert-butyl 2-((trimethylsilyl)-9-azabicyclo[4.2.1]non-2-ene-9 carboxylate (4)

(18) 4.0 equivalents (655 l, 4.64 mmol) of trimethylsilylacetylene were added to a mixture prepared from 430 mL (1.16 mmol) of the enol triflate 3, 81.4 mg (0.116 mmol, 10% mol) of PdCl.sub.2(PPh.sub.3).sub.2 and 11.0 mg (0.06 mmol, 5% mol) of Cul under inert atmosphere in 6.2 mL of DMF anhydrous. The resulting solution was degasified by sonication under a nitrogen current, 484 L (3.48 mmol, 3 equivalents) of Et.sub.3N were added and the mixture was stirred for 1 hour at room temperature. After checking the completion of the reaction by CCF (hexane: AcOEt, 8:2), the reaction mixture was diluted with 10 mL of water and was extracted with Et.sub.2O (315 mL), the collected organic phases were washed with a solution of 1.5% LiCl, brine and were dried over MgSO.sub.4 anhydrous. The residue obtained after the evaporation of the solvent was purified by means of silica column chromatography, using hexane-AcOEt 8:2 as the eluent in order to obtain the conjugated enone 4 as a colorless oil (348 mg, 94%). .sup.1H NMR (300 MHz, CDCl.sub.3) (2 rotamers in a proportion of 4:1; the data of the majority are described) 6.12 (1H, m, H-3), 4.58 (1H, m, H-1), 4.42-4.10 (1H, m, H-6), 2.26-2.14 (2H, m, H-4), 2.20-1.89 (2H, m, H-7), 2.15-1.63 (2H, m, H-8), 2.12-1.70 (2H, m, H-5), 1.47 (9H, s, CMe.sub.3), 0.16 (9H, s, SiMe.sub.3); .sup.13C NMR (75 MHz, CD.sub.3Cl) 153.4 (CO), 138.0 (C-3), 131.7 (C-2), 106.6 (C-1), 92.6 (C-2), 79.6 (CMe.sub.3), 59.8 (C-1), 55.6 (C-6), 31.4 (C-7), 31.3 (C-8), 28.8 (C-5), 28.6 (CMe.sub.3), 24.3 (C-4), 0.15 (SiMe.sub.3), EMAR (ES) m/z calculated for C.sub.18H.sub.30NO.sub.2Si [M+H].sup.+ 320.2040, found 320.2042.

(19) ##STR00014##

Preparation of tert-butyl 2-ethynyl-9-azabicyclo[4.2.1]non-2-ene-9-carboxylate (5)

(20) A mixture formed from 254 mg (0.795 mmol) of the trimethylsilylated enone 4 and 5.0 equivalents (550 mg, 3.978 mmol) of K.sub.2CO.sub.3 anhydrous in 10 mL of MeOH was stirred at room temperature under an N.sub.2 atmosphere, controlling its progression by CCF (hexane-AcOEt, 8:2 as the eluent). After 1 hour, the reaction mixture was diluted with 30 mL of water and extracted with CH.sub.2Cl.sub.2 (325 mL), the organic phases were washed with brine and were dried over MgSO.sub.4 anhydrous. After evaporating the solvent, the conjugated enone 5 was obtained as a clear yellow oil, almost pure as indicated by .sup.1H NMR, so further purification was not required (190 mg, 97%). IR v.sub.max/cm.sup.1 2973, 2928, 1685, 1404, 1362, 1248, 1168, 1106, 1007, 934, 852; .sup.1H NMR (300 MHz, CDCl.sub.3) (mixture of 2 rotamers in a proportion of 2:1; the data of the majority are described) 6.16 (1H, dd, J=6.1, 6.1 Hz, H-3), 4.59 (1H, m, H-1), 4.40-4.18 (1H, m, H-6), 2.89 (1H, s, H-2), 2.25 (2H, m, H-4), 2.20-2.12 (3H, m, H-5, H-7, H-8), 1.92-1.81 (1H, m, H-7), 1.71-1.60 (2H, m, H-5, H-8), 1.46 (9H, s, CMe.sub.3); .sup.13C NMR (75 MHz, CD.sub.3Cl) 155.6 (CO), 138.3 (C-3), 130.1 (C-2), 85.0 (C-1), 79.7 (CMe.sub.3), 76.0 (C-2), 60.0 (C-1), 55.6 (C-6), 31.8 (C-8), 31.2 (C-7), 29.2 (C-5), 28.6 (CMe.sub.3), 24.2 (C-4); EMAR (ES) m/z calculated for C.sub.15H.sub.22NO.sub.2 [M+H].sup.+ 248.1645, found 248.1645.

(21) ##STR00015##

Preparation of trifluoroacetate 2 ethynyl-9-azabicyclo[4.2.1]non-2-ene-9-io (6)

(22) A solution was prepared from 0.5 mL of recently distilled trifluoroacetic acid in 0.5 mL of dry CH.sub.2Cl.sub.2, which was added under inert atmosphere to 31.0 mg (0.125 mmol) of enone 5. The mixture was stirred at room temperature for 1 hour and subsequently the solvent and excess acid were removed under reduced pressure in order to obtain a brownish oily residue corresponding to the salt with the trifluoroacetic acid of the conjugated aza-bicyclo-enone 6 (23.5 mg, 89%) which was brought directly to the following reaction step without further purification. .sup.1H NMR (300 MHz, CDCl.sub.3) 6.43 (1H, m, H-3), 4.38 (2H, m, H-1), 4.28 (1H, m, H-6), 3.0 (1H, s, H-2), 2.45-1.77 (8H, m, H-5, H-8, H-7, H-4). .sup.19F NMR (282 MHz, CDCl.sub.3) 76.4.

(23) ##STR00016##

Preparation of tert butyl 4-(2-ethynyl-9-azabicyclo[4.2.1]non-2-ene-9-yl) butanoate (7)

(24) A solution was prepared from 81.0 mg (0.419 mmol) of the alkaline salt 6, 3.0 equivalents (1.26 mmol) of CsCO.sub.3 and 2.5 equivalents (1.04 mmol) of tert-butyl 4-bromobutanoate in 1.5 mL of anhydrous acetonitrile under an N.sub.2 atmosphere. The resulting reaction mixture was stirred at 50 C. for 3 hours and at the end of the reaction it was diluted with water and a saturated solution of NaHCO.sub.3, extracted with CH.sub.2Cl.sub.2 (320 mL) and the collected organic phases were washed with brine and were dried over MgSO.sub.4 anhydrous. The residue resulting after the evaporation of the solvent was purified by silica gel column chromatography using CHCl.sub.3-MeOH 9:1 as the eluent in order to obtain N-alkylated alkyne 7 as a slightly yellow color oil (61.0 mg, 51%). IR v.sub.max/cm.sup.1 3294.9, 2973.0, 2927.7, 1725.8, 1421.5, 1365.8, 1254.9, 1149.4, 951.4, 845.4; .sup.1H NMR (300 MHz, CDCl.sub.3) (mixture of 2 rotamers in a proportion of 6:1; the data of the majority are described) 6.25 (1H, ddd, J=8.2, 8.2, 0.6 Hz, H-3), 3.80 (1H, m, H-1), 3.47 (1H, m, H-6), 2.86 (1H, s, CCH), 2.60 (2H, m, H-4), 2.36 and 1.80 (2H, m, H-8 and H-8), 2.32-2.13 (2H, m, H-4), 2.26 (2H, m, H-2), 2.23-2.15 (2H, m, H-5), 2.06 and 1.64 (2H, m, H-7 and H-7), 1.80 (2H, m, H-3), 1.44 (9H, s, CMe.sub.3); .sup.13C NMR (75 MHz, CD.sub.3Cl) 173.3 (C-1), 138.9 (C-3), 129.1 (C-2), 86.8 (CCH), 80.1 (CMe.sub.3), 75.4 (CCH), 64.4 (C-1), 60.5 (C-6), 46.4 (C-4), 33.9 (C-2), 31.5 (C-8), 28.3 (C-7), 28.2 (CMe.sub.3) 25.24 (C-5), 24.54 (C-4), 23.84 (C-3); EMAR (ES) m/z calculated for C.sub.18H.sub.28NO.sub.2 [M+H].sup.+ 290.2115 found 290.2119.

(25) ##STR00017##

(26) Preparation of tert-butyl 4-(2-acetyl-9-azabicyclo[4.2.1]non-2-ene-9-yl) butanoate (8). 4.6 mL of a solution prepared from 2 mg of trichloroacetic acid and 50 L of BF.sub.3.Et.sub.2O in 10 mL of MeOH anhydrous were added under inert atmosphere over a solution of alkyne 7 (170.0 mg, 0.587 mmol) and 0.5 equivalents of HgO (63.7 mg, 0.293 mmol) in 2.4 mL of MeOH anhydrous. The resulting reaction mixture was stirred at room temperature, controlling the same by CCF (CHCl.sub.3-MeOH, 9:1), until its conclusion. After approximately 1 hour, the reaction mixture was poured over 15 mL of cold water and 5 mL of a saturated solution of NaHCO.sub.3, extracted with CH.sub.2Cl.sub.2 (315 mL) and the organic phases were washed with brine and were dried over MgSO.sub.4 anhydrous. After removing the solvent, a yellow oil was obtained which was subjected directly to acidic hydrolysis using 1.5 equivalents (167.2 mg, 0.88 mmol) of p-toluenesulfonic acid (PTSA H.sub.2O) in 3 mL of acetone; the reaction was stirred at room temperature for 20 minutes. Lastly, the reaction mixture was processed by dilution with 10 mL of H.sub.2O and 5 mL of a saturated solution of NaHCO.sub.3, extracted with CH.sub.2Cl.sub.2 (310 mL) and the collected organic phases were washed with brine and were dried over Na.sub.2SO.sub.4 anhydrous. The removal of the solvent at reduced pressure provides a slightly brown oily residue which was purified by column chromatography using CHCl.sub.3 with 0.5% of Et.sub.3N as the eluent in order to obtain the N-alkylated anatoxin-a 8 as a colorless oil (139 mg, 77%). .sup.1H NMR (300 MHz, CDCl.sub.3) 6.92 (1H, m, H-3), 4.52 (1H, apparent d, J=10.6 Hz, H-1), 3.49 (1H, m, H-6), 2.48-2.35 (2H, m, H-4), 2.43-2.35 (2H, m, H-4), 2.27 (3H, s, Me), 2.31 and 1.86 (2H, m, H-8 and H-8), 2.10 and 1.63 (2H, m, H-7 and H-7), 2.23-2.17 (2H, m, H-2), 1.85-1.72 (2H, m, H-5) 1.73-1.63 (2H, m, H-3), 1.41 (9H, s, CMe.sub.3); .sup.13C NMR (75 MHz, CDCl.sub.3) 199.2 (COMe), 173.2 (CO.sub.2), 148.6 (C-2), 143.2 (C-3), 80.0 (CMe.sub.3), 60.6 (C-6), 56.7 (C-1), 48.1 (C-4), 33.7 (C-2), 31.3 (C-8), 28.6 (C-7), 28.2 (CMe.sub.3), 25.6 (MeCO), 25.5 (C-5), 25.0 (C-4), 24.2 (C-3); EMAR (ES) m/z calculated for C.sub.18H.sub.30NO.sub.3 [M+H].sup.+ 308.2220 found 308.2234.

(27) ##STR00018##

Preparation of 2,2,2-trifluoroacetate of 4-2(2-acetyl-9-(3-carboxypropyl)-azabicyclo[4.2.1]non-2-ene-9-io (hapten ANA-1, 9)

(28) 30.5 mg (99.0 mol) of the tert-butylic ester 8 were treated with 1 mL of a 1:1 mixture of CF.sub.3CO.sub.2H:CH.sub.2Cl.sub.2. The resulting solution was stirred at room temperature for 1 hour. When the reaction concluded, the solvent was evaporated at reduced pressured until dryness, obtaining the hapten ANA-1 (9) in the form of the salt of trifluoroacetic acid as a colorless oil (36 mg, 99%). IR v.sub.max/cm.sup.1 2915.9, 2848.3, 1669.1, 1418.4, 1198.0, 1132.8, 754; .sup.1H NMR (300 MHz, CD.sub.3OD) 7.50 (1H, m, H-3), 5.19 (1H, apparent t, J=11.5 Hz, H-1), 4.22 (1H, m, H-6), 3.16 (2H, m, H-4), 2.65-2.55 (2H, m, H-4), 2.47 (2H, m, H-2), 2.35 (3H, s, Me), 2.23 and 2.03 (2H, m, H-8 and H-8), 2.19 and 1.85 (2H, m, H-7 and H-7), 2.03 (2H, m, H-3), 1.89-1.79 (2H, m, H-5); .sup.13C NMR (75 MHz, CDCl.sub.3) 196.0 (COMe), 176.0 (CO.sub.2), 161.4 (F.sub.3C CO.sub.2.sub.), 149.0 (C-2), 148.0 (C-3), 64.3 (C-6), 57.0 (C-1), 49.0 (C-4), 32.9 (C-2), 32.9 (C-8), 29.3 (C-7), 26.8 (C-5), 25.4 (Me), 23.5 (C-4), 20.7 (C-3); .sup.19F NMR (282 MHz, CD.sub.3OD) 77.73; EMAR (ES) m/z calculated for C.sub.14H.sub.22NO.sub.3 [M+H].sup.+ 252.1594, found 252.1596.

(29) ##STR00019##

Preparation of the hydrochloride of 4-(2-acetyl-9-azabicyclo[4.2.1]non-2-ene-9-yl) butanoic acid (hydrochloride of Hapten ANA-1, 10)

(30) 80 mg of amberlite resin IRA-400, previously conditioned, was added to a solution prepared from 36 mg (0.098 mmol) of the salt of the trifluoroacetic acid of the Hapten ANA-1 (9) in 1 mL of Milli-Q H.sub.2O and the suspension was left under gentle stirring overnight. The reaction mixture was filtered to separate the resin, and the aqueous filtrate was lyophilized in order to obtain a very hygroscopic foamy solid corresponding to the hydrochloride of the Hapten ANA-1 (10) (24.9 mg, 88%). .sup.1H NMR (300 MHz, CDCl.sub.3) 7.33 (1H, apparent t, J=5.9 Hz, H-3), 5.13 (1H, apparent d, J=7.9 Hz, H-1), 4.14 (1H, m, H-6), 3.01 (2H, m, H-4), 2.59 (2H, m, H-4), 2.42 (2H, m, H-2), 2.42 and 1.96 (2H, m, H-8 and H-8), 2.35 (3H, s, Me), 2.32 and 1.96 (2H, m, H-7 and H-7), 1.96 (2H, m, H-3), 1.81-1.72 (2H, m, H-5); .sup.13C NMR (75 MHz, CDCl.sub.3) 196.0 (COMe), 176.0 (CO.sub.2), 149.0 (C-2), 148.0 (C-3), 64.3 (C-6), 57.0 (C-1), 49.0 (C-4), 33.0 (C-2), 32.9 (C-8), 29.3 (C-7), 26.8 (C-5), 25.4 (Me), 23.5 (C-4), 20.7 (C-3); EMAR (ES) m/z calculated for C.sub.14H.sub.22NO.sub.3 [M+H].sup.+ 252.1594, found 252.1597.

(31) ##STR00020##

Preparation of the N-hydroxysuccinimidyl ester of the hapten ANA-1 (11)

(32) A solution was prepared from 13.2 mg (0.046 mmol) of the hydrochloride of the hapten ANA-1 (10), 1.5 equivalents (7.9 mg, 0.069 mmol) of N-hydroxysuccinimide and 1.5 equivalents (13.2 mg, 0.069 mmol) of EDC HCl in 900 mL of dry CH.sub.2Cl.sub.2 under N.sub.2 atmosphere. The reaction mixture was stirred at room temperature for 17 hours. After this time, the conclusion of the reaction was confirmed by CCF using acetone-chloroform 9:1 as the eluent, and the reaction mixture was concentrated until dryness under vacuum and the obtained residue was dissolved in DMF in order to obtain a solution of approximately 50 mM of the active ester NHS-ANA-1 (11) which was used directly for preparing the corresponding bioconjugates.

(33) ##STR00021##

(34) 1.2. Preparation of a bioconjugate of the hapten ANA-1 with BSA (BSA-ANA-1)

(35) 200 L of the solution of the active ester NHS-ANA-1 (11) in DMF obtained in the previous reaction (approximately 50 mM) was slowly added and with constant stirring over 1.8 mL of a BSA solution (15 mg/mL) in 50 mM carbonate buffer, pH 9.6. The conjugation reaction was incubated for 2 hours with gentle stirring at room temperature. After this time, the conjugates were purified by molecular exclusion using 100 mm sodium phosphate, pH 7.4, as the elution buffer. After purification, the collected fractions which contained the BSA bioconjugate were brought to a final concentration of 1 mg/mL with elution buffer and were stored at 20 C.

(36) In order to determine the haptenic charge (n) obtained in the conjugate, an aliquot of 100 L of the purified bioconjugate BSA-ANA-1 was dialyzed (dialysis against 5 l of deionized water with at least 2 to 3 changes of water per 24 hours at 4 C.; lastly, the dialyzed product was lyophilized and the number of hapten molecules conjugated per BSA molecule was determined by means of MALDI-TOF-MS (n=8, see Table 1, entry 2).

(37) TABLE-US-00001 TABLE 1 Values of the haptenic charge of the protein conjugates as determined by MALDI-TOF-MS RM.sub.0 m/z (m/z) m/hapten n 1 BSA 66431.0 2 BSA-ANA-1 24 68316.2 1885.2 233.3 8.0 3 BSA-ANA-2 24 69810.5 3379.5 233.3 14.5 4 BSA-ANA-3 24 69952.8 3522.5 220.1 16.0 5 OVA 42749.0 6 OVA-ANA-1 8 43504.0 755.0 233.3 3.2 7 OVA-ANA-2 8 44715.0 1966.0 233.3 8.4 8 OVA-ANA-3 8 44500.0 1751.0 220.1 7.9 9 HRP 43973.5 10 HRP-ANA-1 10 n.d. n.d. n.d. n.d. 11 HRP-ANA-2 10 44937.4 963.5 233.3 4.1 12 HRP-ANA-3 10 44529.0 555.5 220.1 2.5 RM.sub.0: initial hapten/protein molar ratio used for the conjugation n: hapten/protein molar ratio obtained for each conjugate (m/z): (m/z conjugate) - (m/z reference protein) m/hapten: mass increase for each conjugated hapten molecule n.d.: not determined

(38) ##STR00022##

(39) 1.3 Preparation of a bioconjugate of the hapten ANA-1 with OVA (OVA-ANA-1)

(40) From a solution of 50 mm in DMF of the activated hapten NHS-ANA-1 (11), 100 L was taken and slowly added with constant stirring to a 1.9 mL OVA solution (15 mg/mL) in 50 mM carbonate buffer, pH 9.6. After 2 hours of reaction under gentle stirring at room temperature, the bioconjugate was purified as previously described for the BSA conjugate. The collected fractions were brought to a final concentration of 1 mg/mL in elution buffer with 0.01% (v/v) thimerosal and stored at 20 C. An aliquot of the conjugate OVA-ANA-1 recently obtained was dialyzed and lyophilized to calculate the efficacy of the conjugation in terms of the number of hapten molecules (11) coupled to the protein by means of MALDI-TOF-MS (n=3.2, see Table 1, entry 6).

(41) ##STR00023##

(42) 1.4 Preparation of a Bioconjugate of the Hapten ANA-1 with HRP (HRP-ANA-1)

(43) From a solution of 5 mm of the activated hapten NHS-ANA-1 (11) in DMF, 100 L was taken and slowly added with constant and gentle stirring over 0.9 mL of a HRP solution at a concentration of 2.5 mg/mL in 50 mM carbonate buffer, pH 7.4. The conjugation reaction was incubated for 2 hours at room temperature. Subsequently the bioconjugate was purified following the method previously described for the BSA and OVA bioconjugates and was brought to known concentrations of between 250-650 g/mL in PBS buffer with 1% BSA (w/v) and 0.02% (w/v) thimerosal and stored at 4 C.

Example 2

(44) Preparation of Bioconjugates of Formula (I) for T=R-II, L=CH.sub.2CH.sub.2CH.sub.2CH.sub.2, Z(CO)NH and P=BSA, OVA and HRP.

2.1 Preparation of the Ester of N-Hydroxysuccinimidyl of the Hapten ANA-2 (NHS-ANA-2)

(45) ##STR00024##

Preparation of methyl 2-(6-methoxy-6-oxohex-1-ene-1-yl)-9-azabicyclo[4.2.1]non-2-ene-9-carboxylate (12)

(46) 1.5 mL of DMF anhydrous and 4.0 equivalents (153 l, 1.167 mmol) of commercial methyl 5-hexenoate were added to a mixture prepared from 108 mg (0.291 mmol) of the enol triflate 3, 20.4 mg (10% mol, 0.029 mmol) of PdCl.sub.2(PPh.sub.3).sub.2 and 2.7 mg (5% mol, 0.014 mmol) of Cul under N.sub.2 atmosphere. The resulting solution was degasified by vacuum cycles under a nitrogen current and 3.0 equivalents (122 L, 0.875 mmol) of Et.sub.3N was added. After 1 hour of stirring at room temperature, it was checked by CCF that the starting triflate had been completely consumed. The reaction mixture was diluted with 10 mL of water and extracted with Et.sub.2O (315 mL); the organic phases were successively washed with a solution of 1.5% LiCl and brine and were dried over MgSO.sub.4 anhydrous. The residue obtained after the evaporation of the solvent was purified by silica gel column chromatography using hexane-AcOEt 9:1 as the eluent in order to firstly obtain the homocoupling product of the alkyne (10.9 mg, 11%), followed by the cross coupling product of the conjugated enone, 12, as a colorless oil (86.4 mg, 85%). IR v.sub.max/cm.sup.1 2953, 2400, 2350, 1733, 1690, 1402.5, 1365.1, 1164.7; .sup.1H NMR (300 MHz, CDCl.sub.3) (mixture of 2 rotamers in a proportion of 2:1; the data of the majority are described) 5.94 (1H, dd, J=11.6, 5.5 Hz, H-3), 4.51 (1H, m, H-1), 4.39-4.13 (1H, m, H-6), 3.66 (3H, s, CO.sub.2Me), 2.43 (2H, t, J=7.4 Hz, H-5), 2.31 (2H, t, J=6.9 Hz, H-3), 2.25-2.18 (2H, m, H-4), 2.16-2.04 (3H, m, H-8, H-7), 1.83 (2H, dt, J=6.8 Hz, H-4), 1.83-1.68 (3H, m, H-5, H-7), 1.44 (9H, s, CMe.sub.3 Boc); .sup.13C NMR (75 MHz, CDCl.sub.3) 173.1 (CO.sub.2CH.sub.3), 153.6 (CO Boc), 135.2 (C-3), 130.9 (C-2), 87.4 (C-2), 82.6 (C-1), 79.4 (CMe.sub.3 Boc), 60.5 (C-1), 55.1 (C-6), 51.7 (CO.sub.2CH.sub.3), 32.9 (C-5), 31.3 (C-7), 30.2 (C-8), 29.4 (C-5), 28.6 (CMe.sub.3 Boc), 24.1 (C-4), 23.6 (C-4), 18.9 (C-3), EMAR (ES) m/z calculated for C.sub.20H.sub.30NO.sub.4 [M+H].sup.+ 348.2169, found 348.2168.

(47) ##STR00025##

Preparation of methyl 2-(6-methoxy-6-oxohexanoyl)-9-azabicyclo[4.2.1]non-2-ene-9-carboxylate (13)

(48) 1.8 mL of a solution prepared from 2 mg of trichloroacetic acid and 50 L of BF.sub.3.Et.sub.2O in 10 mL of MeOH anhydrous was added dropwise under N.sub.2 atmosphere to a solution prepared from 80 mg of the alkyne 12 (0.230 mmol) and 0.5 equivalents of HgO (25 mg, 0.0115 mmol) in 900 L of MeOH anhydrous. The resulting reaction mixture was stirred at room temperature until the alkyne disappeared, monitoring the reaction by CCF using hexane-AcOEt 7:3 as the eluent. Approximately after 1 hour, the reaction mixture was poured over cold water, it was basified with a saturated solution of NaHCO.sub.3, extracted with CH.sub.2Cl.sub.2 (315 mL) and the collected organic phases were washed with brine and dried over Na.sub.2SO.sub.4 anhydrous. The evaporation of the solvent under vacuum provided a yellow oil which was purified by column chromatography, using hexane-AcOEt 8:2 as the eluent, the conjugated enone 13 being obtained as a slightly yellow oil (65 mg, 64%). .sup.1H NMR (300 MHz, CDCl.sub.3) (mixture of 2 rotamers in a proportion of 2:1; the data of the majority are described) 6.79 (1H, m, H-3), 5.13 (1H, m, H-1), 4.42-4.28 (1H, m, H-6), 3.65 (3H, s, CO.sub.2Me), 2.65 (2H, m, H-2), 2.42 (2H, m, H-5), 2.33 (3H, m, H-4, H-4), 2.17-2.07 (2H, m, H-8, H-4), 1.65 (7H, m, H-3, H-5, H-7, H-8), 1.39 (9H, s, CMe.sub.3Boc); .sup.13C NMR (75 MHz, CDCl.sub.3) 199.7 (C-1, CO), 173.0 (CO.sub.2Me), 153.2 (CO Boc), 150.0 (C-2), 141.1 (C-3), 79.4 (CMe.sub.3 Boc), 55.7 (C-6), 53.3 (C-1), 51.7 (CO.sub.2CH.sub.3), 36.9 (C-5), 35.5 (C-2), 31.7 (C-8), 30.5 (C-7), 28.9 (C-5), 28.2 (CMe.sub.3 Boc), 24.9 (C-3), 24.4 (C-4), 24.3 (C-4); EMAR (ES) m/z calculated for C.sub.20H.sub.32NO.sub.5 [M+H].sup.+ 366.2275, found 366.2274.

(49) ##STR00026##

Preparation of 6-(9-(tert-butoxycarbonyl)-9-azabicyclo[4.2.1]non-2-ene-2-yl)-6-oxo-hexanoic acid (14)

(50) A solution of 97 mg (0.27 mmol) of methyl ester 13 was prepared in 2 mL of THF, it was cooled in an ice bath and a cold solution of 2 mL of an aqueous solution 2.7 m (5.4 mmol, 10 final equivalents) of LiOH was added dropwise with stirring. After agitating at room temperature for 30 mins, the reaction mixture was poured over ice water and it was acidified to pH 2-3 with a 1 M cold aqueous solution of KHSO.sub.4. The mixture was extracted with AcOEt (315 mL) and the combined organic phases were washed with brine and were dried over Na.sub.2SO.sub.4 anhydrous. The removal of the solvent under vacuum provided the carboxylic acid 14 as a colorless oil (93 mg, 98%) which appeared to be practically pure by means of NMR and therefore it was used in the following step without additional purification. IR v.sub.max/cm.sup.1 2973, 2931.6, 2356.8, 1662.4, 1409, 1365.1, 1246.5, 1216, 1166.9, 1115.8, 931.7, 750; .sup.1H NMR (300 MHz, CDCl.sub.3) (a mixture of 2 rotamers in a proportion of 2:1; the data of the majority are described), 6.79 (1H, m, H-3), 5.13 (1H, apparent d, J=8.9 Hz, H-1), 4.44-4.25 (1H, m, H-6), 2.65 (2H, m, H-5), 2.42 (5H, m, H-2, H-4, H-8), 2.10 (4H, m, H-3, H-8, H-7), 1.65 (5H, m, H-4, H-5, H-7), 1.39 (9H, s, CMe.sub.3 Boc); .sup.13C NMR (75 MHz, CDCl.sub.3) 199.6 (CO, C-6), 178.9 (CO.sub.2H), 157.6 (CO Boc), 149.9 (C-2), 141.2 (C-3), 79.6 (CMe.sub.3 Boc), 55.1 (C-6), 53.3 (C-1), 36.7 (C-5), 33.6 (C-2), 31.5 (C-8), 31.1 (C-7), 30.5 (C-4), 28.9 (C-5), 28.5 (CMe.sub.3 Boc), 24.5 (C-4), 24.3 (C-3); EMAR (ES) m/z calculated for C.sub.19H.sub.30NO.sub.5 [M+H].sup.+ 352.2118, found 352.2131.

(51) ##STR00027##

Preparation of tert-butyl 2-(6-((2,5-dioxopyrrolidine-1-yl)oxy)-6-oxohexanoyl)-9-azabicyclo[4.2.1]non-2-ene-9-carboxylate (15)

(52) A solution was prepared from 80 mg (0.228 mmol) of the acid 14, 1.5 equivalents (39.3 mg, 0.341 mmol) of N-hydroxysuccinimide and 1.5 equivalents (65.4 mg, 0.341 mmol) of EDC HCl in 3 mL of dry CH.sub.2Cl.sub.2 under N.sub.2 atmosphere. The reaction mixture was stirred at room temperature for 17 hours. After this time, the conclusion of the reaction was confirmed by CCF using acetone-chloroform 9:1 as the eluent. The reaction mixture was diluted with CH.sub.2Cl.sub.2, 5 mL of a saturated solution of NH.sub.4Cl was added and it was extracted with the same solvent (310 mL). The combined organic phases were washed with brine and were dried with Na.sub.2SO.sub.4 anhydrous. After evaporating the solvent, a colorless oil was obtained which was purified by column chromatography using CHCl.sub.3 as the eluent in order to obtain the N-hydroxysuccinimide ester 15 (77 mg, 75%). .sup.1H NMR (300 MHz, CDCl.sub.3) (a mixture of 2 rotamers in a proportion of 2:1; the data of the majority are described), 6.79 (1H, m, H-3), 5.13 (1H, m, H-1), 4.44-4.25 (1H, m, H-6), 2.83 (4H, s broad, COCH.sub.2CH.sub.2CO), 2.65 (2H, m, H-2), 2.42 (5H, m, H-5, H-4, H-8), 2.10 (4H, m, H-3, H-8, H-7), 1.65 (5H, m, H-4, H-5, H-7), 1.39 (9H, s, CMe.sub.3 Boc); .sup.13C NMR (75 MHz, CDCl.sub.3) 199.6 (CO, C-1), 168.9 (COCH.sub.2CH.sub.2CO), 165.5 (CO, C-6), 157.6 (CO Boc), 149.9 (C-2), 141.2 (C-3), 79.6 (CMe.sub.3 Boc), 55.1 (C-6), 53.3 (C-1), 36.7 (C-2), 33.6 (C-5), 31.5 (C-8), 31.1 (C-7), 30.5 (C-4), 28.9 (C-5), 28.5 (CMe.sub.3 Boc), 25.6 (COCH.sub.2CH.sub.2CO), 24.5 (C-4), 24.3 (C-3); EMAR (ES) m/z calculated for C.sub.23H.sub.33N.sub.2O.sub.7 [M+H].sup.+ 449.2282, found 449.2281.

(53) ##STR00028##

Preparation of 2,5-dioxopyrollidine-1-yl-6-(9-azabicyclo[4.2.1]non-2-ene-2-yl)-6-oxohexanoate as salt of trifluoroacetic acid (16)

(54) A solution of 25 mg (0.055 mmol) of the N-hydroxysuccinimidyl ester 15 in 1 mL of a 1:1 mixture of CF.sub.3CO.sub.2H:CH.sub.2Cl.sub.2 was stirred at room temperature under N.sub.2 atmosphere for 1 hour. The solvent and excess of CF.sub.3CO.sub.2H were evaporated at reduced pressure in order to provide the salt with the trifluoroacetic acid of the active ester NHS-ANA-2 (16) as a colorless oil (24.4 mg, 96%). .sup.1H NMR (300 MHz, THF-d.sub.8) 7.26 (1H, dd, J=8.0, 3.7 Hz, H-3), 5.13 (1H, apparent d, J=9.2 Hz, H-1), 4.30 (1H, m, H-6), 2.75 (4H, s broad, COCH.sub.2CH.sub.2CO), 2.75-2.70 (2H, m, H-5), 2.64-2.59 (4H, m, H-2, H-4), 2.51-2.40 (2H, m, H-8), 2.36-2.27 (1H, m, H-7), 2.10 (1H, m, H-5), 1.98 (1H, m, H-7), 1.95-1.82 (3H, m, H-4, H-5), 1.70 (2H, m, H-3), .sup.19F NMR (282 MHz, THF-d.sub.8) 76.86.

(55) ##STR00029##

2.2 Preparation of a Bioconjugate of the Hapten ANA-2 with BSA (BSA-ANA-2)

(56) The conjugate was prepared as previously described for the bioconjugate BSA-ANA-1 from 200 L of a 50 mM solution in DMF of the activated hapten NHS-ANA-2 (16) and 1.8 mL of a BSA solution (15 mg/mL) in 50 mM carbonate buffer, pH 9.6. After chromatographic purification, the collected fractions were brought to a final concentration of 1 mg/mL in elution buffer and were stored at 20 C. The number of molecules (16) conjugated to each BSA molecule as determined by MALDI-TOF-MS was n=14.5 (see Table 1, entry 3).

(57) ##STR00030##

2.3 Preparation of a Bioconjugate of the Hapten ANA-2 with OVA (OVA-ANA-2)

(58) The conjugate was prepared as previously described for the bioconjugate OVA-ANA-1 from 100 L of a solution of 50 mm in DMF of the activated hapten NHS-ANA-2 (16) and 1.9 mL of a OVA solution (15 mg/mL) in 50 mM carbonate buffer, pH 9.6. After the corresponding chromatographic purification, the collected fractions were brought to a final concentration of 1 mg/mL in elution buffer with 0.01% (v/v) thimerosal and were stored at 20 C. The number of molecules (16) conjugated to each OVA molecule as determined by MALDI-TOF-MS was n=8.4 (see Table 1, entry 7).

(59) ##STR00031##

2.4 Preparation of a Bioconjugate of the Hapten ANA-2 with HRP (HRP-ANA-2)

(60) Prepared from 100 L of a 5 mM solution of the activated hapten NHS-ANA-2 (16) in DMF and 0.9 mL of a HRP solution (2.5 mg/mL) in 50 mM carbonate buffer, pH 7.4. After the corresponding chromatographic purification, the fractions obtained containing the bioconjugate were brought to known concentrations of between 250-650 g/mL in PBS buffer with 1% (w/v) BSA and 0.02% (w/v) thimerosal and were stored at 4 C. The number of molecules (16) conjugated to each HRP molecule as determined by MALDI-TOF-MS was n=4.1 (see Table 1, entry 11).

Example 3

(61) Preparation of bioconjugates of formula (I) for T=R-III, L=NOCH.sub.2, Z(CO)NH and P=BSA, OVA and HRP.

3.1 Preparation of the Ester of n-Hydroxysuccinimidyl of the Hapten ANA-3 (NHS-ANA-3)

(62) ##STR00032##

Preparation of tert-butyl 2-acetyl-9-azabicyclo[4.2.1]non-2-ene-9-carboxylate (17)

(63) A solution prepared from 1 mg of trichloroacetic acid and 22 L of BF.sub.3.Et.sub.2O in 1.9 mL of methanol, was added dropwise over a mixture prepared from 140 mg (0.569 mmol) of the alkyne 5 (0.283 mmol) and 0.5 equivalents of HgO (62 mg, 0.284 mmol) in 3.6 mL of MeOH anhydrous under inert atmosphere. The resulting reaction mixture was stirred for 1 hour at room temperature. After this time, 300 mL of distilled water was added to the reaction mixture and the stirring was continued for 15 additional minutes. Thereafter, the reaction mixture was poured over 10 mL of cold water and 5 mL of a saturated aqueous solution of NaHCO.sub.3, extracted with CH.sub.2Cl.sub.2 (315 mL) and the collected organic phases were washed with brine, dried over Na.sub.2SO.sub.4 and concentrated at reduced pressure, providing a yellow oil. The purification by column chromatography using hexane-AcOEt 9:1 as the eluent provided N-Boc anatoxin-a (17) as a colorless oil (134 mg, 90%). IR v.sub.max/cm.sup.1 2978.5, 2925.8, 2853.6, 1690.6, 1662.9, 1404.7, 1390, 1363.2, 1337.5, 1231, 1168, 1108.1, 991.3; .sup.1H NMR (300 MHz, CDCl.sub.3) (mixture of 2 rotamers in a proportion of 3:1; the data of the majority are described) 6.81 (1H, m, H-3), 5.12 (1H, m, H-1), 4.44-4.23 (1H, m, H-6), 2.52-2.43 (2H, m, H-4), 2.29 (3H, s, Me), 2.22-2.0 (3H, m, H-8, H-7), 1.69-160 (3H, m, H-5, H-7), 1.37 (9H, s, CMe.sub.3); .sup.13C NMR (75 MHz, CDCl.sub.3) 197.9 (COMe), 150.5 (CO Boc), 142.2 (C-2), 141.4 (C-3), 79.5 (CMe.sub.3 Boc), 55.8 (C-6), 53.2 (C-1), 31.6 (C-8), 30.5 (C-7), 29.1 (C-5), 28.9 and 28.6 (CMe.sub.3 Boc). 25.5 (Me), 24.3 (C-4); EMAR (ES) m/z calculated for C.sub.15H.sub.24NO.sub.3 [M+H].sup.+ 266.1751, found 266.1747.

(64) ##STR00033##

Preparation of 2-((((E)-1-(-9-(tert-butoxycarbonyl)-9-azabicyclo[4.2.1]non-2-ene-2-yl)ethylidene)amine)oxy)acetic acid (18)

(65) 46 L of pyridine was added to a solution of 50 mg (0.178 mmol) of N-Boc anatoxin-a (17) and 3.0 equivalents (0.534 mmol) of aminooxyacetic acid hydrochloride in 3.2 mL of dry ethanol under argon atmosphere. The mixture was stirred at room temperature until the consumption of all the starting material was confirmed (2 hours) by CCF (using CHCl.sub.3CH.sub.3CO.sub.2H-MeOH, 92:4:4 as the eluent). The reaction mixture was poured over a mixture of 15 mL of ice water and 10 mL of 1 M HCl, extracted with CHCl.sub.3 (410 mL) and the organic phases were washed with brine and were dried over Na.sub.2SO.sub.4 anhydrous. The evaporation of the solvent at reduced pressure provided a slightly yellow oil corresponding to the oxime 18 (59 mg, 98%) which appeared to be nearly pure by NMR analysis, so further purification was not required. IR v.sub.max/cm.sup.1 2972.6, 2929.2, 2539.5, 1739.9, 1674.0, 1593.5, 1476.5, 1408.4, 1364, 1336.8, 1257.2, 1168.8, 1,109.8, 1066.4; .sup.1H NMR (300 MHz, CDCl.sub.3) (mixture of 2 rotamers in a proportion of 3:1; the data of the majority are described) 6.07 (1H, m, H-3), 5.15 (1H, m, H-1), 4.66 (2H, m, H-2), 4.36 (1H, m, H-6), 2.39-2.25 (2H, m, H-4), 2.22-2.12 (3H, m, H-7, H-8), 2.01 (3H, s, Me-CN), 1.78-1.51 (3H, m, H-5, H-7), 1.38 (9H, s, CMe.sub.3 Boc) .sup.13C NMR (75 MHz, CDCl.sub.3) 172.9 (C-1), 157.7 (CNO), 153.7 (CO.sub.2 Boc), 144.9 (C-2), 132.1 (C-3), 79.1 (CMe.sub.3 Boc), 68.6 (C-2), 55.5 (C-6), 55.2 (C-1), 34.3 (C-8), 31.5 (C-7), 28.6 (CMe.sub.3 Boc), 24.2 (C-4), 11.3 (Me-CN); EMAR (ES) m/z calculated for C.sub.17H.sub.27N.sub.2O.sub.5 [M+H].sup.+ 339.1914, found 339.1912.

(66) ##STR00034##

Preparation of tert-butyl 2-((E)-1-((2-((-1-yl)oxy-2-oxoethoxy)imine)ethyl-9-azabicyclo[4.2.1]non-2-ene-9-carboxylate (19)

(67) A solution of 86.6 mg (0.256 mmol) of the acid 18, 1.5 equivalents (44.2 mg, 0.384 mmol) of N-hydroxysuccinimide and 1.5 equivalents (73.6 mg, 0.384 mmol) of EDC HCl in 6.0 mL of dry CH.sub.2Cl.sub.2 was stirred at room temperature under N.sub.2 atmosphere and the progress of the reaction was controlled by CCF, using acetone-chloroform 9:1 as the eluent. After 17 hours, the complete disappearance of the starting material was confirmed, and the reaction mixture was diluted with CH.sub.2Cl.sub.2. Then, 2 mL of a saturated solution of NH.sub.4Cl was added and extracted with the same solvent (310 mL). The collected organic phases were washed with brine and were dried over Na.sub.2SO.sub.4 anhydrous. After evaporating the solvent in vacuum, a colorless oil was obtained which was purified by column chromatography using CHCl.sub.3 as the eluent, obtaining the N-hydroxysuccinimidyl ester 19 (59 mg, 53%). IR v.sub.max/cm.sup.1 2973, 2928.6, 1826.7, 1787.8, 1737.8, 1682.6, 1407.4, 1363.4, 1199.6, 1169.3, 1109.8, 1071.4, 861, 750.7; .sup.1H NMR (300 MHz, CDCl.sub.3) (a mixture of 2 rotamers in a proportion of 2:1; the data of the majority are described) 6.05 (1H, ddd, J=6.1, 6.1, 0.5 Hz, H-3), 5.15 (1H, m, H-1), 4.97 (2H, m, H-2), 4.31 (1H, m, H-6), 2.84 (4H, s broad, COCH.sub.2CH.sub.2CO), 2.37-2.20 (3H, m, H.sub.2-4 and H-8), 2.20-2.03 (2H, m, H-5 and H-7), 2.00 (3H, s, Me), 1.80-1.55 (3H, m, H-8, H-7 and H-5), 1.37 (9H, s, Me.sub.3C-Boc); .sup.13C NMR (75 MHz, CDCl.sub.3) 168.8 (COCH.sub.2CH.sub.2CO), 165.5 (C-1), 157.7 (CNO), 153.7 (CO.sub.2-Boc), 144.9 (C-2), 132.1 (C-3), 79.1 (Me.sub.3C-Boc), 68.6 (C-2), 55.5 (C-6), 55.2 (C-1), 31.9 (C-5), 31.5 (C-8), 29.8 (C-7), 28.6 (Me.sub.3C-Boc), 25.7 (COCH.sub.2CH.sub.2CO), 24.2 (C-4), 11.3 (Me); EMAR (ES) m/z calculated for C.sub.21H.sub.30N.sub.3O.sub.7 [M+H].sup.+ 436.2078 found 436.2083.

(68) ##STR00035##

Preparation of 2,5-dioxopyrrolidin-1-yl 2-((((E)-1-(-9-azabicyclo[4.2.1]non-2-ene-2-yl)ethylidene)amino)oxy)acetate as the salt of trifluoroacetic acid (NHS-ANA-3, 20)

(69) A solution of 28.0 mg (0.064 mmol) of the N-hydroxysuccinimidyl ester 19 in 1 mL of a 1:1 mixture of CF.sub.3CO.sub.2H:CH.sub.2Cl.sub.2 was stirred for 1 hour at room temperature under N.sub.2 atmosphere. The course of the reaction was monitored by CFF using acetone-chloroform 8:2 as the eluent in order to check the absence of the starting material. When the reaction concluded, the solvent and the excess of CF.sub.3CO.sub.2H were evaporated at reduced pressure and a colorless oil corresponding to the activated ester NHS-ANA-3 as the salt of trifluoroacetic acid (20) was obtained (27.7 mg, 96%). IR v.sub.max/cm.sup.1 2948.1, 2359.6, 1783.8, 1737.4 f, 1705.2, 1668.8, 1429.5, 1191.0, 1132.7, 1074.1, 720.1; .sup.1H NMR (300 MHz, CDCl.sub.3) 6.45 (1H, ddd, J=6.0, 6.0, 0.5 Hz, H-3), 5.43 (1H, m, H-1), 4.92 (2H, apparent s broad, H-2), 4.37 (1H, m, H-6), 2.82 (4H, s broad, COCH.sub.2CH.sub.2CO), 2.56 (3H, m, H.sub.2-4 and H-8), 2.34 (1H, m, H-5), 2-06-1.98 (3H, m, H-8, H-5 and H-7), 2.01 (3H, s, Me), 1.88 (1H, m, H-7); .sup.13C NMR (75 MHz, CDCl.sub.3) 169.4 (COCH.sub.2CH.sub.2CO), 165.3 (C-1), 156.6 (CNO), 140.3 (C-2), 136.3 (C-3), 69.4 (C-2), 59.6 (C-6), 54.4 (C-1), 30.1 (C-8), 28.8 (C-7), 27.9 (C-5), 25.1 (COCH.sub.2CH.sub.2CO), 23.3 (C-4), 10.8 (Me); .sup.19F NMR (282 MHz, CDCl.sub.3) 76.34 (s); EMAR (ES) m/z calculated for C.sub.16H.sub.22N.sub.3O.sub.5 [M+H].sup.+ 336.1554, found 336.1565.

(70) ##STR00036##

3.2 Preparation of a Bioconjugate of the Hapten ANA-3 with BSA (BSA-ANA-3)

(71) Prepared as has been previously described for the bioconjugate BSA-ANA-1 from 200 l of a solution of 50 mm in DMF of the activated hapten NHS-ANA-3 (20) and 1.8 ml of a BSA solution (15 mg/ml) in carbonate buffer 50 mm, pH 9.6. After the corresponding chromatographic purification, the collected fractions were brought to a final concentration of 1 mg/mL in elution buffer and were stored at 20 C. The number of molecules of 20 conjugated to each BSA molecule as determined by MALDI-TOF-MS was n=16 (see Table 1, entry 4).

(72) ##STR00037##

3.3 Preparation of a Bioconjugate of the Hapten ANA-3 with OVA (OVA-ANA-3)

(73) The conjugate was prepared as previously described for the bioconjugate OVA-ANA-1 from 100 L of a 50 mM solution in DMF of the activated hapten NHS-ANA-3 (20) and 1.9 mL of a OVA solution (15 mg/mL) in 50 mM carbonate buffer, pH 9.6. After chromatographic purification, the collected fractions were brought to a final concentration of 1 mg/mL in elution buffer with 0.01% (v/v) of thimerosal and were stored at 20 C. The number of molecules of 20 conjugated to each OVA molecule as determined by MALDI-TOF-MS was n=7.9 (see Table 1, entry 8).

(74) ##STR00038##

3.4 Preparation of a Bioconjugate of the Hapten ANA-3 with HRP (HRP-ANA-3)

(75) Prepared from 100 L of a 5 mM solution of the activated hapten NHS-ANA-3 (20) in DMF and 0.9 mL of a HRP solution (2.5 mg/mL) in 50 mM carbonate buffer, pH 7.4. After chromatographic purification, the fractions obtained containing the bioconjugate were brought to known concentrations of between 250-650 g/mL in PBS buffer with 1% (w/v) BSA and 0.02% (w/v) thimerosal and were stored at 4 C. The number of molecules of 20 conjugated to each HRP molecule as determined by MALDI-TOF-MS was n=2.5 (see Table 1, entry 12).

(76) 2. ELISA Method

(77) 96-well polystyrene plates were used. Each antibody was evaluated in the two classic competitive ELISA formats (the immobilized antigen or conjugate with indirect detection format and the immobilized antibody with direct detection format) using homologous assay antigens, that is to say, an assay antigen from the same bioconjugate of formula (I) as the one used for obtaining the immunogen, but in which P=OVA or HRP. After each incubation step, the plates were washed four times with a washing solution, using a ELx405 96-channel washer (Biotek Instruments, Winooski, USA). The signal produced by the peroxidase used as a label was revealed with 100 L per well of a 2 mg/mL solution of o-phenylenediamine in 25 mM citrate, 62 mM phosphate, pH 5.4, containing 0.012% (v/v) of H.sub.2O.sub.2. The enzymatic reaction was performed for 10 minutes at room temperature and was stopped using 100 L per well of 2.5 M sulfuric acid. Upon concluding the assays, the absorbance of each well was read at 492 nm using a reference wavelength at 650 nm in a PowerWave HT microplate reader (Biotek Instruments, Winooski, USA). The sigmoid standard curves obtained when representing the absorbance versus the analyte concentration were adjusted to a four-parameter logistic equation using the SigmaPlot computer package from SPSS (Chicago, USA).

(78) The affinity of the antibody (IC.sub.50) was estimated as the concentration of free analyte capable of reducing the maximum signal (A.sub.max) to half.

(79) 2.1 Competitive ELISA Tests in the Immobilized Antigen or Conjugate Format with Indirect Detection (Indirect Assay)

(80) The plates were coated with 100 Lper well of an assay antigen solution which is a bioconjugate of formula (I) where P is OVA, at 0.01 or at 0.1 g/mL in 50 mM carbonate buffer, pH 9.6, by overnight incubation at room temperature. After washing the plates, 50 Lper well of a complete standard curve of the analyte in PBS was dispensed in each column, followed by 50 L per well of antibody diluted in PBST (0.05% Tween 20). The immunochemical reaction was carried out for 1 hour at room temperature and then the plates were washed. Subsequently, each well received 100 L of a 1/2000 RAM-HRP dilution (rabbit anti-mouse immunoglobulins labeled with peroxidase) in PBST. This reaction was left at room temperature for 1 hour. After washing the plates, the retained peroxidase activity was revealed and the absorbance was read at 492 nm as previously described.

(81) 2.2. Competitive ELISA Tests in the Immobilized Antibody Format with Direct Detection (Direct Assay)

(82) The plates were coated with 100 L per well of an antibody solution in 50 mM carbonate buffer, pH 9.6 by overnight incubation at room temperature. After washing the plates, 50 Lper well of a complete standard curve of the analyte in PBS was dispensed in each column, followed by 50 Lper well of a specific dilution in PBST of the enzymatic bioconjugate which is a bioconjugate of formula (I) where P is HRP.

(83) The same reagent distribution was repeated for each plate with a different antibody. The immunochemical reaction was carried out for 1 hour at room temperature and the plates were then washed. Lastly, the retained peroxidase activity was revealed and the absorbance was read at 492 nm as previously described.

(84) 3. Production of Mouse Monoclonal Antibodies

(85) 3.1 Mouse Immunization

(86) For the immunization, the bioconjugates of formula (I) in which P is BSA (immunizing conjugates) obtained in the previous examples were used. BALB/c female mice, aged between 6 and 8 weeks at the start of the process, were employed.

(87) In each dose, 100 g of bioconjugate per mouse was administered via the intraperitoneal route, the total volume injected being 200 L. In the first immunization, the bioconjugate was supplied as an emulsion prepared with Freund's Complete Adjuvant (1:1, v/v). At intervals of 3 weeks, the mice received two additional immunizations, in these cases, emulsifying the bioconjugates with Freund's Incomplete Adjuvant. Four days before each cellular fusion, the mouse selected received a final dose of 100 g of the corresponding bioconjugate diluted in PBS.

(88) 3.2 Cellular Fusions for Hybridoma Generation

(89) The fusions with immunized mice were carried out basically following previously described methodologies that are well established in the state of the art.

(90) Immediately after slaughtering the mice, the spleen was removed and homogenized with the plunger of a sterile syringe. After erythrocytes were lysed by osmotic shock with 1 mL of cold lysis buffer for one minute, the lymphocytes were washed twice with cold complete medium (with serum) and filtered to remove blood clot.

(91) The myeloma line P3-X63-Ag8.653 was cultivated the days prior to the fusion in DMEM-supplemented medium [2 mM L-alanine-L-glutamine, 1 mM non-essential amino acids, 25 g/mL gentamicin, 10% (v/v) fetal bovine serum (FBS)], maintaining the cells in the exponential growth phase such that the day of the fusion there was a sufficient number of the same.

(92) After two washes with medium without serum, both cellular populations were combined at a lymphocyte:myeloma ratio of 4:1. Then, the cells were centrifuged in order to carry out the cellular fusion immediately thereafter. To this end, the chemical fusing agent PEG 1500 (1 mL per spleen, 1 minute) was used which, upon partially dissolving the membranes, allows for the fusion of the cells. Once both populations had been fused, the cells were resuspended in DMEM-supplemented medium [15% (v/v) FBS] and were seeded into 96-well culture plates (100 L per well) at a cellular density of 15010.sup.3 lymphocytes per well and were incubated at 37 C. in an atmosphere with 5% of CO.sub.2 and 95% humidity. 24 hours after the fusion, 100 L per well of HAT medium was added for hybridoma selection [DMEM-supplemented with 100 M hypoxanthine, 0.4 M aminopterin, 16 M thymidine and 20% (v/v) FBS which contained 1% (v/v) HFCS (high fusion and closing supplement)].

(93) 3.3. Selection, Cloning and Preservation of Hybridomas

(94) Approximately 10-12 days after the cellular fusion, supernatant screening was carried out with the aim of identifying which one contained antibody-secreting hybridomas capable of recognizing anatoxin-a both in its conjugated and free form (competitor clones). Beforehand, the efficacy of the fusion was determined by visual inspection, defined as the percentage of wells which presented at least one clone that was clearly visible to the microscope.

(95) In order to carry out the identification of competitor clones, supernatants were assayed by means of the differential ELISA technique consisting of analyzing in adjacent wells, in a parallel manner, each supernatant in the absence of analyte and in the presence of a prefixed analyte concentration, usually 100 nM. To this purpose, the plates were coated with the homologous conjugate which is a bioconjugate of formula (I) in which P is OVA at a concentration of 0.1 g/mL and the test was carried out by adding 50 L of the culture supernatant. The conditions for the indirect ELISA format, as detailed in section 2.1, were followed

(96) Thereafter, the wells containing antibody-producing hybridomas capable of providing an absorbance equal to or greater than 0.5 in the test in the absence of anatoxin-a and inhibiting the signal equal to or greater than 80% in the test in the presence of anatoxin-a were selected for further work. Additionally, for all the positive wells, a second more exhaustive screening was carried out in a bidimensional competitive manner with the aim of selecting the best hybridomas with greater certainty. To this end, the supernatant of each hybridoma was tested at 4 dilutions (, 1/32, 1/128 and 1/512) in plates coated with the homologous bioconjugate at 0.01 and 0.1 g/mL and using anatoxin-a as competitor at 5 and 50 nM. 200 L of the culture supernatant was thus diluted in 600 L of PBST and the following dilutions were serially made from this first dilution. The test was performed by adding 50 Lper well of the corresponding dilution of supernatant and 50 L of the solution of anatoxin-a in PBS at the concentration of 100, 10 and 0 nM.

(97) The cells into the wells ultimately selected were cloned by limiting dilution, seeding from each original well a new 96-well plate at 2 cells per well in HT medium (HAT medium without aminopterin and containing 1% (v/v) HFCS).

(98) Usually 7-10 days after the first cell cloning, the wells which contained a unique cell clone were identified by means of visual inspection, and the culture supernatants were assayed as previously described for the screening of supernatants after cell fusion. This process was carried out as many times as it was necessary (at least twice) in order to ensure the monoclonality of the selected hybridomas as well as their stability. Lastly, the expansion of the selected cellular lines was carried out, progressively cultivating the hybridoma in containers of higher size. Once the clone had grown, the cells were frozen in liquid nitrogen at a concentration of 10.sup.7 cells per vial (2-4 vials for each hybridoma) in a FBS solution with 10% (v/v) DMSO as the cryoprotective agent. The vials were maintained at 80 C. in the interior of a polystyrene box for 24 hours before passing them to the liquid nitrogen container.

(99) 3.4 Production and Purification of Monoclonal Antibodies

(100) In the last phase of the cellular expansion of the hybridomas, they were progressively divided into culture plates until a final volume of between 100 and 200 mL of supernatant was reached. The cells were left to grow until the confluence was reached and once the culture medium nutrients were depleted the content of the plates was collected. The collected volume was centrifuged in order to eliminate the cellular residues and the supernatant was precipitated by adding a volume of an ammonium sulfate saturated solution, being maintained at 4 C. until it was purified.

(101) The purification of the antibodies was carried out by affinity chromatography with protein G columns following the manufacturer's instructions. To this end, the precipitated antibody was centrifuged for 20 minutes at 5000 rpm (4000g) and the supernatant was discarded. The pellet which contained the antibodies was solubilized with 20 mM sodium phosphate buffer, pH 7.4 and filtered through nitrocellulose membranes (pore diameter 0.45 m) to eliminate suspended solids. The elution of the antibody from the column was carried out with 100 mM sodium citrate buffer, pH 2.5. The fractions which contained the antibody were identified by means of UV spectrophotometry and were collected. The solution was neutralized by adding 1 M Tris-HCl, pH 9.5. Finally, the concentration of the purified antibody was determined by UV spectrophotometry [A.sub.280 (1 mg/mL IgG)=1.4] and a working solution was prepared at a concentration of 500 g/mL in PBS with 1% (w/v) BSA and 0.01% (w/v) thimerosal which was stored at 4 C. The remaining solution was precipitated with saturated ammonium sulfate [1:1, (v/v)] which guarantees its stability at 4 C. for years.

(102) 4. Results

(103) 4.1 Generation of Monoclonal Antibody-Producing Hybridomas Against Anatoxin-a

(104) Based on mice immunized with bioconjugates of formula (I), in which P is BSA, cellular fusions were carried out aimed at generating hybridomas. The bioconjugates of formula (I) in which T is R-I or R-III were not capable of inducing the generation of monoclonal antibody-secreting hybridomas with a high affinity towards anatoxin-a. On the other hand, based on the bioconjugate of formula (I) in which T is R-II, it was possible to obtain 5 monoclonal antibody-producing cellular lines with high affinity towards anatoxin-a. The monoclonal antibodies obtained from said hybridomas have been named for the purpose of the present invention and the examples included here: mAb #38, mAb #44, mAb #325, mAb #417 and mAb #39. This result reveals that the bioconjugate of formula (I) most suitable for obtaining antibodies to anatoxin-a is not a clear and obvious question. The results also demonstrate the suitability of a bioconjugate of formula (I) in which T is R-II for inducing the production of antibodies capable of recognizing anatoxin-a.

(105) 4.2 Determination of the Affinity of the Antibodies

(106) Once the 5 monoclonal antibodies obtained (see 4.1, antibodies mAb #38, mAb #44, mAb #325, mAb #417 and mAb #39) were purified, their affinity towards anatoxin-a was determined by means of homologous competitive ELISA. In the indirect format (FIG. 6), the antibodies exhibited IC.sub.50 values for anatoxin-a ranging from 3.15 nM to 11.00 nM. In the direct format (FIG. 7), the antibodies exhibited IC.sub.50 values for anatoxin-a ranging from 2.18 nM to 9.49 nm.

(107) 4.3. Determination of the Specificity of the Antibodies

(108) Anatoxin-a exists chemically in 2 enantiomeric forms. The natural cyanotoxin is exclusively dextrorotatory (+), while the levorotatory form () can be obtained by organic synthesis. In order to test the extent to which the obtained monoclonal antibodies were actually specific, they were tested in the homologous indirect ELISA format against the two enantiomers of anatoxin-a. As it may be observed in FIG. 8, all the antibodies recognized (+)-anatoxin-a with an affinity at least 100 times greater compared to the affinity they exhibited towards their enantiomer, ()-anatoxin-a. This test convincingly demonstrates the significant specificity and discrimination ability of the monoclonal antibodies described in this invention and therefore their high capacity for determining the natural enantiomer of the anatoxin-a in samples, even in the presence of other potential contaminants.