Compounds and antibodies for the immunodetection of dihydroanatoxins

Abstract

The present invention relates to compounds, in particular to conjugates and labelled derivatives of dihydroanatoxin-a, suitable for the production of antibodies with a high affinity for dihydroanatoxins, particularly the cyanotoxins dihydroanatoxin-a and dihydrohomoanatoxin-a. The present invention also relates to the use of conjugates and labelled derivatives of dihydroanatoxin-a as test antigens. The present invention also relates to the use of the compounds for the analysis, concentration and extraction of dihydroanatoxin-a and dihydrohomoanatoxin-a using the obtained antibodies, sometimes together with test antigens that are conjugates or labelled derivatives of dihydroanatoxin-a. This invention also provides a kit for analysing dihydroanatoxins, particularly dihydroanatoxin-a and dihydrohomoanatoxin-a, comprising antibodies against these cyanotoxins, sometimes together with test antigens that are conjugates or labelled derivatives of dihydroanatoxin-a.

Description

[0001] DESCRIPTION

[0002] COMPOUNDS AND ANTIBODIES FOR IMMUNODETECTION OF

[0003] DIHYDROANATOXINS

[0004] FIELD OF INVENTION

[0005] The present invention relates to compounds, especially labeled conjugates and derivatives of dihydroanatoxin-a, suitable for the production of high-affinity antibodies against dihydroanatoxins, particularly the cyanotoxins dihydroanatoxin-a and dihydrohomoanatoxin-a. The present invention also relates to the use of labeled conjugates and derivatives of dihydroanatoxin-a as assay antigens. Furthermore, the present invention also relates to the use of the compounds for the analysis, concentration, and extraction of dihydroanatoxin-a and dihydrohomoanatoxin-a using the obtained antibodies, sometimes in conjunction with assay antigens that are labeled conjugates or derivatives of dihydroanatoxin-a.This invention also provides a kit for analyzing dihydroanatoxins, particularly dihydroanatoxin-a and dihydrohomoanatoxin-a, comprising antibodies against these cyanotoxins, sometimes together with assay antigens that are conjugates or labeled derivatives of dihydroanatoxin-a.

[0006] STATE OF THE ART

[0007] Biotoxins are a type of contaminant whose presence in food, water, and animal feed poses a significant problem for human health and animal welfare, causing substantial economic losses to the agri-food, agricultural, and fish farming sectors, and potentially severely impacting the safety of drinking water. Among the most relevant biotoxins due to their toxicity and prevalence are those produced by cyanobacteria, especially microcystins, nodularins, cylindrospermopsins, saxitoxins, and anatoxins. Based on their mode of action, cyanotoxins can be classified as hepatotoxic, neurotoxic, cytotoxic, dermatotoxic, and irritant toxins. Beyond these effects, alterations in odor and gastrointestinal problems have also been described as a result of contact with cyanotoxins or other cyanobacterial metabolites.The most relevant anatoxins, due to their toxicity and presence in environmental samples, are anatoxin-a, homoanatoxin-a, dihydroanatoxin-a, and dihydrohomoanatoxin-a. These toxins are produced by different species of cyanobacteria, primarily belonging to the genera Tychonema, Anabaena, Microcoleus, Aphanizomenon, Oscillatoria, Phormidium, Raphidiopsis, and Cylindrospermum. Under certain environmental conditions, these prokaryotic microorganisms are capable of proliferating excessively, leading to sudden blooms known in the scientific literature as massive algal blooms. These blooms are currently considered more frequent and intense than in the past due to eutrophication resulting from human activity and global warming.The main routes of exposure for humans and animals to dihydroanatoxin-a and / or dihydrohomoanatoxin-a are through contaminated water, either intentionally (drinking water from surface sources) or accidentally (through recreational and sporting uses), and through the consumption of dietary supplements derived from blue-green algae, which are not always subject to adequate controls and may therefore contain unwanted strains of toxin-producing cyanobacteria. A route of exposure that has become more relevant in recent years, and therefore constitutes an emerging risk, is through the consumption of fish and bivalve molluscs, from both traditional fishing and aquaculture, that have been raised in bodies of water affected by cyanobacterial blooms and have thus been exposed to these biotoxins.

[0008] As illustrated in the formulas below, dihydroanatoxin-a and dihydrohomoanatoxin-a—hereafter referred to collectively as “dihydroanatoxins”—are alkaloids with a common 9-azabicyclo[4.2.1]nonane skeleton, substituted at C-2 with an acetyl group (COCH3) in dihydroanatoxin-a and a propionyl group (COCH2CH3) in dihydrohomoanatoxin-a. Both cyanotoxins are structurally related to the C-2–C-3 dehydrogenated analogs, anatoxin-a and homoanatoxin-a. Due to the high basicity of the bridging nitrogen atom of the azabicyclic system, all of them are protonated at this atom at neutral pH in aqueous solution.

[0009] Dihydroanatoxins and their dehydrogenated analogues are potent neurotoxins that block nicotinic acetylcholine receptors on the postsynaptic membrane at the neuromuscular junction, causing muscle paralysis that leads to death by asphyxiation. The median lethal dose (LD50) of dihydroanatoxin-a by intraperitoneal injection is 0.73 mg / kg, indicating reduced toxicity compared to anatoxin-a (LD50). 50 = 0.23 mg / kg). However, when administered orally – both via feeding tube and through feeding – dihydroanatoxin-a is more toxic than anatoxin-a (LD50). 50 by feeding tube at 2.5 mg / kg for dihydroanatoxin-a and 10.6 mg / kg for anatoxin-a; LD 50The concentrations of dihydroanatoxin-a (8 mg / kg) and anatoxin-a (25 mg / kg) in feed are [Puddick et al. Chemosphere 2021, 263, 127937]. Dihydroanatoxin-a, in particular, has been shown to be highly toxic to dogs. Numerous dog deaths have been reported worldwide in France, the USA, and the Netherlands due to ingestion of water contaminated with dihydroanatoxin-a. The high acute oral toxicity of dihydroanatoxin-a and dihydrohomoanatoxin-a, and their presence in environmental samples, especially in spring [Fastner et al., Sci. Total Environ. 2023, 858, 159433], suggest that this is a significant contaminant requiring the utmost attention from agencies responsible for ensuring human health and animal welfare.

[0010] The analytical determination of dihydroanatoxin-a and dihydrohomoanatoxin-a can be carried out using gas or liquid chromatography with spectroscopic detectors, although in most cases derivatization of the toxin is required. Currently, the recommended analytical technique for the determination of these cyanotoxins in samples is high-resolution mass spectrometry [Testai et al., EFSA Supporting Publication, 2016, EN-998, 309]. This technique offers high sensitivity and reproducibility, and also allows for the simultaneous determination of several cyanotoxins; however, this approach is not without its drawbacks, like any other analytical technique. In fact, the necessary equipment is very expensive, not portable, and requires highly qualified personnel for its operation.As a complementary analytical strategy to instrumental methods, techniques based on antibody-analyte interaction (immunoassays, affinity chromatography, immunoreactive strips) are considered the best option when a large number of analyses need to be performed in a short time or when controls need to be carried out in situ and / or in technically limited environments. Currently, several immunodiagnostic companies market kit-type assays for the detection of the main cyanotoxins—including anatoxin-a and homoanatoxin-a—with the notable exception of the two dihydroanatoxins, as the necessary immunoreagents and technology are not yet available.

[0011] Immunoanalytical methods are based on the selective, reversible, and non-covalent binding between the substance to be detected (analyte) and an antibody that specifically recognizes it. Dihydroanatoxins, due to their low molecular weight, are not immunogenic and therefore cannot generate an immune response on their own when injected into an experimental animal. To generate antibodies against these toxins, it is necessary to covalently link the molecule to a protein, so that the resulting conjugate is immunogenic and allows for antibody production. Obtaining these conjugates often requires the de novo design and synthesis of a derivative, using strategies that allow the incorporation, at the optimal position on the molecule, of a hydrocarbon chain with a terminal functional group, while respecting its structure and characteristic chemical groups.This strategy makes it possible to present the molecule to the immune system in the most appropriate way to achieve antibodies with high affinity and specificity.

[0012] As mentioned previously, no specific immunoreagents for dihydroanatoxins have been described. Recently, the first conjugates and antibodies against anatoxin-a have been published [Quiñones-Reyes et al., Angew. Chim. 2019, 58, 9134-9139; Cevallos-Cedeño et al., Anal. Chem. 2022, 94, 10857-10864]. These are highly specific antibodies for anatoxin-a and homoanatoxin-a, but despite their relative structural similarity, they do not recognize either dihydroanatoxin-a or dihydrohomoanatoxin-a.

[0013] Therefore, there is a need to obtain antibodies with high affinity and specificity for dihydroanatoxins, suitable not only for the development of sensitive and selective immunoassays, but also for their implementation in new analytical platforms based on advanced technologies, such as different types of biosensors, multiplex assays, and fluorescence resonance energy transfer (FRET) methods. These antibodies, obtained from novel haptens, will form the basis for developing new immunoanalytical methods for the determination, detection, concentration, or extraction of dihydroanatoxins, preferably using a kit that can be used by the food, agricultural, clinical, and / or environmental industries.

[0014] DESCRIPTION OF THE INVENTION

[0015] The present invention provides an immunoanalytical procedure for the determination of the cyanotoxins dihydroanatoxin-a and dihydrohomoanatoxin-a by providing a method for the preparation of immunogenic and assay conjugates in which the hapten coupled to the protein maintains the complete structure of these molecules intact with all characteristic functional groups unaltered, and therefore available for recognition by the immune system during the immune response and, consequently, by the antibodies generated from it.The basis of the invention lies in the discovery of the ability of the conjugates of the molecules of dihydroanatoxin-a derivatized through the carbon atom of the methyl group of the acetyl group - incorporating at said position a hydrocarbon spacer arm of diverse length - to induce a very effective immune response that leads to the generation of antibodies of very high affinity and specificity towards dihydroanatoxin-a and dihydrohomoanatoxin-a, and therefore suitable for the development of immunodetection procedures of the two dihydroanatoxins.

[0016] In this document the term “dihydroanatoxins” refers to dihydroanatoxin-a and dihydrohomoanatoxin-a.

[0017] The present invention provides labeled conjugates and derivatives of dihydroanatoxin-a analogues and their use as immunogenic and assay antigens in immunoanalytical methods.

[0018] In this document, "dihydroanatoxin-a analogue" is understood to mean a hapten, defined as

[0019] TLY can be the functional group Y, one of the four functional groups listed below.

[0020] Y = -COOH, -CH2NH2, -CH2SH, -CH2N3.

[0021] T and L have the meanings indicated below for formula (I). Each of these Y groups is transformed after conjugation into the corresponding Z group of the compounds of formula (I), formula (II) or formula (III) defined below:

[0022] The structures of some of these TLY formula haptens, DA-1, DA-2 and DA-3 haptens, are illustrated below as examples. This does not, in any case, represent a limit to the variability in their structure and includes all the previously defined alternatives of the TLY formula:

[0023] The present invention relates to a compound having a general formula (I) where

[0024] T is Rl,

[0025] L is a linear hydrocarbon chain of 1 to 10 carbon atoms;

[0026] Z is a functional group selected from: m is a number with a value between 1 and 100;

[0027] X is selected from P and Q, such that

[0028] P is a peptide or polypeptide of non-enzymatic nature, natural or synthetic, with a molecular weight equal to or greater than 2000 Daltons, which may or may not be attached to a support, and

[0029] Q is a detectable non-isotopic marker.

[0030] L is preferably a linear hydrocarbon chain of 1 to 5 carbon atoms.

[0031] Rl salts can be of both inorganic and organic acids, for example:

[0032] - monoprotic inorganic acids, such as hydrochloric, hydrobromic, hydroiodic, hydrofluoric, nitric, perchloric, terafluoroboric, hypophosphorous acids,

[0033] - polyprotic inorganic acids, such as sulfuric, phosphoric, phosphorous, carbonic acids,

[0034] - aliphatic mono- and dicarboxylic acids, such as formic, acetic, chloroacetic, trichloroacetic, trifluoroacetic, propanoic, glycolic, lactic, oxalic, malonic, succinic, glutaric, citric, acrylic, aspartic, fumaric, and maleic acids,

[0035] - aromatic mono- and dicarboxylic acids, such as benzoic, phthalic, nitrobenzoic, dinitrobenzoic, chlorobenzoic, nicotinic, hydroxybenzoic, and mandelic acids,

[0036] - aliphatic or aromatic sulfonic acids, such as methanesulfonic, ethanesulfonic, benzenesulfonic, triflic, methylbenzenesulfonic, and dimethylbenzenesulfonic.

[0037] Examples of salts compatible with the object of the invention include acetate, trifluoroacetate, acrylate, ascorbate, aspartate, benzoate, bicarbonate / carbonate, bisulfate / sulfate, bromide, camphorsulfonate, citrate, chloride / hydrochloride, chlorobenzoate, dinitrobenzoate, phthalate, hydroxybenzoate, methoxybenzoate, methylbenzoate, o-acetoxybenzoate, naphthalene-2-benzoate, hydroiodide / iodide, formate, fumarate, glycolate, lactate, malate, maleate, malonate, mandelate, methyl sulfate, mesylate, nicotinate, nitrate, oxalate, pyrophosphate, propionate, phenylpropionate, succinate, sulfate, bisulfate, pyrosulfate, sulfite, bisulfite, sulfonate, benzenesulfonate, chlorobenzenesulfonate, ethanesulfonate, methanesulfonate, naphthalen-1-sulfonate, naphthalen-2-sulfonate, p-toluenesulfonate, succinate, tartrate, tosylate and phosphate / hydrogen osphate / dihydrogen phosphate salts.

[0038] In a preferred embodiment, Z is selected from the group consisting of ~(C=O)NH~ and

[0039] More preferably, Z is -(C=O)NH-.

[0040] There is no upper limit for the possible molecular weight of P, only a lower limit, as it depends on the immune system. For example, this lower limit could be 2000 Daltons.

[0041] The peptide or polypeptide P may or may not be attached to a support via covalent, electrostatic, or other interactions. This support may be a natural polymer, such as cellulose and its derivatives, or a synthetic one, such as nitrocellulose or polystyrene, or it may be composed of nanomaterials, such as colloidal gold, carbon, or latex nanoparticles. m is preferably a value between 1 and 50, more preferably between 1 and 25, and especially preferable between 1 and 10.

[0042] According to particular embodiments of the present invention, the compound is a conjugate of a dihydroanatoxin-a analogue of general formula (II), corresponding to formula (I) wherein X is P: where

[0043] T, L, Z, and P have the same meanings as given above. The value of m indicates the degree of conjugation, that is, the molar ratio between the fraction derived from the TLZ fragment and the peptide or polypeptide P, in the resulting conjugate of formula (II). m is a number with a value between 1 and 100, preferably between 1 and 50, and more preferably between 1 and 25.

[0044] According to a preferred embodiment of the present invention, the conjugate of formula (II) is characterized in that P is selected from the group consisting of albumin, thyroglobulin, and hemocyanin. More preferably, P is albumin, which may be egg albumin or serum albumin.

[0045] According to another preferred embodiment of the present invention, the conjugate of formula (II) is a conjugate of formula (lia) where

[0046] P and m are as defined above. Preferably, P is albumin and m is a value selected between 1 and 25.

[0047] According to another preferred embodiment of the present invention, the conjugate of formula (II) is a conjugate of formula (llb) where P and m are as previously defined for (lia).

[0048] According to another preferred embodiment of the present invention, the conjugate of formula (II) is a conjugate of formula (lie) where P and m are as previously defined for (lia).

[0049] The conjugate of formula (II) of the present invention can be obtained by a method comprising reacting the functionalized analogue of dihydroanatoxin-a of formula TLY (hapten), where T and L have been defined above and Y is a functional group selected from -COOH, --CH2NH2, --CH2SH, and -CH2N3, with P, a natural or synthetic polypeptide of non-enzymatic nature with a molecular weight greater than 2000 Dalton.

[0050] Depending on the Y group, the conjugation of the hapten TLY to the peptide P via this group may require its transformation into a more reactive functional group (activation), as in the case of Y = -COOH, or the transformation of peptide P itself, as in the case of Y = -CH2NH2. In other cases, the prior incorporation of functional groups suitable for reaction with the Y group into peptide P is required, such as Y = -CH2SH and -CH2N3. All these conjugation methods are widely known in the field (Greg T. Hermanson, Bioconjugate Techniques, Academic Press, London, UK, 2013).

[0051] For example, the carboxyl group of a hapten TLY, in which Y = -COOH, can be activated by transforming it into an active ester, for example, an N-hydroxysuccinimidyl ester [COON(COCH2)2], which reacts very efficiently with the free amino groups (NH2) of a peptide, as illustrated in Scheme 1:

[0052] Similarly, the amino groups of a hapten TLY, in which Y = -CH2NH2, can be reacted with the free carboxyl (COOH) groups of a previously activated peptide by transforming it into an active ester, for example, an A / -hydroxysuccinimidyl ester, as illustrated in Scheme 2: Scheme 2

[0053] In the case of a TLY hapten, in which Y = -CH2N3, the conjugation of the hapten to the peptide can be effected by first incorporating an alkyne group into it, sufficiently separated from the peptide structure with a spacer arm that facilitates the reaction of the alkyne group with the azide group (N3) of the hapten, as illustrated in scheme 3:

[0054] Scheme 3

[0055] Similarly, in the case of a hapten TLY, where Y = -CH2SH, the conjugation of the hapten to the peptide can be effected by first incorporating into it a group capable of reacting with the thiol group (SH), for example, a maleimide group [N(COCH)2], as illustrated in scheme 4:

[0056] Scheme 4

[0057] Functionalized analogues of formula TLY (haptens), as previously defined, can generally be obtained by chemical synthesis through a synthetic route involving as a key intermediate a compound such as A' or the oxidized analogue A, both compounds interconvertible through oxidation-reduction processes, as shown in scheme 5:

[0058] Scheme 5 In these compounds, L has the same meaning given above for (I) and (II) and G is a functional group of those commonly used for the protection of amino (NH) groups, such as, for example, tert-butoxycarbonyl (CO2CMe3, Boc), benzyl (CH2Ph, Bn), etc. (PGM Wuts and TW Greene, Greene's Protective Groups in Organic Synthesis. John Wiley & Sons, Inc., Hoboken, NJ, USA, pages 696-926, 2007).

[0059] Starting from compound A', the terminal hydroxymethylene group (-CH2OH) can be readily transformed by methods widely known in the synthetic technique into any of the functional groups previously defined as Y, a functional group selected from -COOH, -CH2NH2, -CH2SH, and -CH2N3. For example, the hydroxymethylene group (-CH SThe -OH group can be directly oxidized to the -COOH group with oxidizing agents such as oxygen in the presence of a catalyst such as platinum, or transformed into a -CH2N3 group through the prior transformation of the primary hydroxyl group into a sulfonate group (-CH2OSOaR') and subsequent substitution of the sulfonate group with an azide anion (Ns“) by conventional aliphatic nucleophilic substitution reactions. The -CH2NH2 group can also be prepared from the -CH2OH group, via the same sulfonate intermediate, by nucleophilic substitution of the sulfonate group with a nitrogenous nucleophile, such as NN3 or a synthetic equivalent thereof, or even from the -CH2N3 group itself by known reduction reactions of the azide group (N3) to amino (NH2), for example, via catalytic hydrogenation or by treatment with PhaP.Similarly, the --CH2SH group can be introduced through the same sulfonate by nucleophilic substitution of the sulfonate group by a sulfhydryl group (SH), either directly using NaSH as a nucleophile or indirectly using a synthetic equivalent thiolated nucleophile, for example, CH3COSK or (NH2)2C=S.

[0060] Alternatively, starting from compound A, the terminal formyl group (-CH=O) can be directly oxidized to the Y = -COOH group with reagents such as AgNO3, NaCIO2 or CrO3, or it can be reduced to the hydroxymethylene group (R - -CH2OH) by means of one of the reducing reagents commonly used to carry out this transformation, for example, NaBH4, LiAIH[OC(CH3)3]3 or Zn(BH4)2, to subsequently transform this group into any of the functional groups previously defined as Y, as mentioned above.

[0061] Examples of these functional group interconversions, illustrated in Scheme 6, are described, for example, in Delcanale et al., J. Org. Chem. 1986, 51, 567-569, for transforming -CH=O into -COOH; Wang et al., J. Am. Chem. Soc. 2021, 29, 10948-10962, for transforming -CH=O into -CH2OH; Shao et al., J. Agrie. Food Chem. 1993, 41, 1391-1396, for transforming -CH2OH into -COsH; Wang et al., J. Agrie. Food Chem. 2015, 63, 10013-10021, for transforming -CH2OH into -CH2N3; Minamoto et al., J. Chem. Soc., Perkin Trans 1, 1990, (11), 3027-3033 to transform -CH2OH through the sulfonate intermediate into -CH2NH2; Kerns et al., J. Am. Chem. Soc. 2000, 122, 12608-12609, to transform -CH2N3 into -CH2NH2; Wángler et al., J. Med. Chem. 2014, 57, 4368-4381 to transform -CH2OH into -CH2SH also through the same sulfonate intermediate:

[0062] Scheme 6

[0063] As illustrated in Scheme 7, the synthesis of intermediate A', with the hydrocarbon chain terminated in a hydroxymethylene group (R = CH2OH), can be carried out starting from bromo- or chloro-alcohols (i), where L has the same meaning given above, and 9-azabicyclo[6.2.0]dec-4-en-10-one (iv). Both the bromo- and chloro-alcohols i, for a length of L between 1 and 10 carbon atoms, and the p-lactam iv are known products that are commercially available. The synthesis begins with the appropriate derivatization of the hydroxymethylene group of the bromo- or chloro-alcohol i, either by transforming it into the corresponding alkoxide group by treatment with a strong base, for example, or by protecting it, for example, as a tetrahydropyranyl ether (G' = THP), a tert-butyldimethylsilyl ether (G' = SPBuMe^), or a benzyl ether (G != CH2Ph), to give an intermediate such as ii, followed by the transformation of this into the corresponding organomagnesian iii. On the other hand, the NH group of the p-lactam iv is also adequately protected with a G group, of the type indicated above, to obtain the p-lactam derivative v. The nucleophilic addition of the organomagnesian iii to the carbonyl group of the derivatized β-lactam v with concomitant opening of the p-lactam ring leads to the ketone vi, from which the construction of the characteristic azabicyclic system is completed by a regioselective oxidative aminocyclization leading to the unsaturated intermediate vil. The synthesis of intermediate A' is completed from the product formed in the previous reaction by hydrogenation of the double bond and deprotection of the terminal hydroxyl group of the hydrogenated derivative formed viii, according to scheme 7:

[0064] Scheme 7

[0065] As illustrated in Scheme 8, the synthesis of an intermediate such as A, with the hydrocarbon chain terminated in a formyl group (R = CH=O), can be carried out by an analogous synthetic strategy, but starting from a bromo- or chloro-acetal (ix), where L has the same meaning given above, and 9-azabicyclo[6.2.0]dec-4-en-10-one (iv). The bromo- and chloro-acetals ix, for a length of L between 1 and 10 carbon atoms, are also known products that are commercially available. The synthesis of intermediate A begins with the transformation of the bromo- or chloro-acetal ix into the corresponding organomagnesian x, which is reacted with the carbonyl group of the N-derivatized p-lactam v to provide the ketone xi, from which the construction of the azabicyclic system is completed by a regioselective oxidative aminocyclization leading to the unsaturated intermediate xü.The synthesis of intermediate A is completed from the product formed in the previous reaction by hydrogenation of the double bond to give xüi and hydrolysis of the acetal group of this to the formyl group:.

[0066] Scheme 8

[0067] The conjugate of formula (II) of the present invention can be used for the production of antibodies, or as an assay antigen in conjunction with a dihydroanatoxin-specific antibody to determine or detect these cyanotoxins in a sample using immunoassay technology. In another embodiment of the present invention, X is a non-isotopic marker Q, instead of the peptide P of formula (II), as the carrier material for the functionalized dihydroanatoxin-a analog TLY defined above. When the carrier material is a detectable non-isotopic marker, the dihydroanatoxin-a analog derivative is a compound of formula (III): where T, L, Z, and Q have the same meaning as defined above. The value of m in this case indicates the degree of titration, i.e., the molar ratio between the fraction derived from the TLZ fragment and a detectable non-isotopic marker Q, in the resulting conjugate of formula (III). m is a number with a value between 1 and 100, preferably between 1 and 50, more preferably between 1 and 25, and especially preferable between 1 and 10.

[0068] Q is a detectable non-isotopic marker.

[0069] "Detectable" means that the marker can be detected by an analytical technique or by the human eye, or both.

[0070] In the present invention, "marker" means any molecule or fragment that gives rise to a signal measurable by any type of analytical technique. In the present invention, Q identifies a fragment of a molecule, or a non-isotopic chemical detector, marker, or tracer molecule.

[0071] In a preferred embodiment, Q is selected from: an enzyme, biotin, a luminescent compound, a fluorophore, or a marker coupled to an indirect detection system. Preferably, Q is selected from the group consisting of biotin, fluorescein, a cyanine fluorophore, a rhodamine fluorophore, a coumarin fluorophore, a ruthenium bipyryl, luciferin or any of its derivatives, an acridinium ester, quantum dots, and colloidal gold, carbon, or latex nanoparticles.

[0072] In this document, an enzyme is understood to be a protein, peptide, or polypeptide that acts as a catalyst for biochemical reactions.

[0073] An indirect detection system is a system that does not directly measure the desired parameter, but rather obtains its value through the measurements of a different parameter.

[0074] According to a preferred embodiment, the derivative of formula (III) is a derivative of formula (Illa) where Q is selected from the group consisting of peroxidase, phosphatase, oxidase, biotin, fluorescein, or colloidal gold, carbon, or latex nanoparticles, and m is a value selected from 1 to 10.

[0075] According to another preferred embodiment, the derivative of formula (III) is a derivative of formula (lllb) where Q and m are as previously defined for (Illa).

[0076] According to another preferred embodiment, the derivative of formula (III) is a derivative of formula (lle) where Q and m are as previously defined for (Illa).

[0077] The labeled derivative of formula (III) of the present invention can be obtained by a process comprising reacting a functionalized analogue of dihydroanatoxin-a of formula TLY (hapten), where T, L and Y have been defined above, with Q, a non-isotopic marker, by methods analogous to those previously mentioned for the preparation of peptide conjugates and which are also widely known in the art (Greg T. Hermanson, Bioconjugate Techniques, Academic Press, London, UK, 2013).

[0078] This compound of formula (III) can be used together with a specific dihydroanatoxin antibody to determine or detect these cyanotoxins in a sample using immunoassay technology.

[0079] In this document the word “compound” includes all the alternatives of formula (I), formula (II) or formula (III) and is equivalent to the word “conjugated”.

[0080] To obtain antibodies with high affinity and specificity against dihydroanatoxins, specifically dihydroanatoxin-a and dihydrohomoanatoxin-a, and to develop immunoassays for analyzing these cyanotoxins, functionalized analogs of dihydroanatoxin-a with the formula TLY (haptens) have been prepared. These are structural analogs of this molecule that incorporate a functional group Y capable of being used for conjugation to a carrier P or marker Q. This functional group is separated from the azabicyclic system of the dihydroanatoxin molecule by a spacer L. The position at which the spacer L is attached to the dihydroanatoxin structure determines the position at which it binds to the carrier P or marker Q during conjugation via the functional group Y, thus influencing how the dihydroanatoxin structure is exposed to the immune system.Determining the optimal position on the dihydroanatoxin structure for incorporating the L spacer is not straightforward. The present invention demonstrates the viability of conjugates prepared by derivatization through the carbon atom of the methyl group of the acetyl group of dihydroanatoxin-a. Specifically, it demonstrates the viability of conjugates of formula (II) as inducers of the production of antibodies with suitable affinity and specificity against dihydroanatoxin-a and dihydrohomoanatoxin-a, and even the viability of conjugates of formula (II) or labeled derivatives of formula (III) as competitor molecules enabling the development of sensitive and specific immunoassays for these cyanotoxins.

[0081] In the context of this invention, the term “antibody” refers to the immunoglobulin that an animal generates or that a hybrid cell (such as a hybridoma) synthesizes specifically against the immunogen of the invention (conjugate of the invention).

[0082] Therefore, an additional aspect of the present invention relates to an antibody (hereinafter referred to as the antibody of the invention) generated in response to a conjugate of the invention, in particular the conjugate of formula (II). More preferably, the antibodies are generated in response to the conjugate of formula (Ha) or (llb).

[0083] Another additional aspect of the invention relates to the use of the previously described conjugate of formula (II) for obtaining antibodies, preferably these antibodies recognize dihydroanatoxin-a and / or dihydrohomoanatoxin-a.

[0084] A new aspect of the invention relates to an antibody that recognizes the conjugate of formula (II) or the conjugate of formula (III).

[0085] A new aspect of the invention relates to an antibody that recognizes Rl as part of the conjugate of formula (I). In a particular embodiment, the antibody may be polyclonal (or, equivalently, antiserum), monoclonal, recombinant, or an antibody fragment.

[0086] The process for obtaining the antibodies of the invention from conjugates of the invention can be carried out by methods widely known in the art, such as by immunizing an animal. The antibodies generated from a conjugate of the present invention can be polyclonal or monoclonal antibodies. The antibodies of the invention have high affinity and specificity for both dihydroanatoxins.

[0087] The terms “anti-dihydroanatoxin-a antibody” and “anti-dihydrohomoanatoxin-a” or “anti-dihydroanatoxin antibodies” refer to antibodies capable of binding to dihydroanatoxin-a and dihydrohomoanatoxin-a.

[0088] In a particular embodiment, the IC50 value towards dihydroanatoxin-a and / or dihydrohomoanatoxin-a of the antibodies of the invention is less than 100 nM, preferably less than 50 nM, more preferably less than 10 nM, even more preferably less than 5 nM, even more preferably less than 2 nM and even more preferably less than 1 nM.

[0089] In a particular embodiment, the antibody of the invention has the same or different IC50 value for dihydroanatoxin-a and dihydrohomoanatoxin-a, and may be lower for one of the two dihydroanatoxins.

[0090] Another aspect of the invention relates to the use of an antibody, obtained from conjugates of the invention, for the in vitro analysis of dihydroanatoxins. In particular, the use comprises contacting a sample containing dihydroanatoxins with said antibody; and more specifically comprises: a) contacting the sample with the antibody defined above; b) incubating the sample and the antibody from step (a) for a period of time suitable for an immunochemical reaction to occur; and c) determining the existence of an immunochemical reaction after the incubation of step (b).

[0091] In one particular embodiment, the sample containing dihydroanatoxins may comprise dihydroanatoxin-a or dihydrohomoanatoxin-a. A further aspect of the invention relates to the use of an antibody and a compound of formula (I), (II), or (HI) defined above for the in vitro analysis of dihydroanatoxins by competitive ELISA. In particular, this use comprises: a) contacting a sample with an anti-dihydroanatoxin antibody and with the compound of formula (II) or the labeled derivative of formula (HI) defined above; b) incubating the sample, the antibody, and the compound of step (a) for a period of time suitable for an immunochemical reaction to occur; and c) determining the existence of an immunochemical reaction after the incubation of step (b).

[0092] An additional aspect of the invention relates to the use of an antibody and a compound of formula (I), (II), or (III) defined above for the in vitro analysis of dihydroanatoxins by lateral flow chromatography. Preferably, this use comprises the following steps: a) contacting a sample with the antibody of the invention; b) incubating the sample and the antibody from step (a) for a period of time suitable for an immunochemical reaction to occur; and c) determining the existence of an immunochemical reaction after the incubation of step (b) by immunochromatographic separation and concentration.

[0093] Another aspect of the invention relates to the use of the antibody defined above for the purification and / or concentration of dihydroanatoxins from a sample. Specifically, this method involves immobilizing at least one antibody of the invention on any support and passing a sample through said support so that it retains the dihydroanatoxins present in the sample. Subsequent elution of the dihydroanatoxins retained on the support by methods widely known in the art (change in pH, modification of ionic strength, use of chaotropic agents) will allow their purification and / or concentration in a system known as immunoaffinity chromatography.In a preferred embodiment, this use comprises performing the following steps: a) immobilizing at least one antibody obtained from conjugates of formula (II) of the invention on a support; b) passing a sample through said support so that the antibody retains the dihydroanatoxins present in said sample; and c) eluting the dihydroanatoxins retained by the antibody on the support. The determination of the immunochemical reaction in step (c) can be carried out by a competitive assay, using as a competitor a compound of formula (II) or a labeled derivative of formula (III) as defined above.

[0094] An additional aspect of the present invention also relates to a dihydroanatoxin detection kit that uses at least one antibody of the invention or a compound of formula (II) or formula (III) as described in the present patent application.

[0095] The applications described in the present invention allow for the quantitative determination or qualitative analysis of the content of the cyanotoxins dihydroanatoxin-a and dihydrohomoanatoxin-a in a sample. Furthermore, the applications described in the present invention allow for the analysis of the dihydroanatoxin content in different types of samples, for example, environmental samples such as inland waters, and food samples such as dietary supplements prepared with algae. Preferably, the present invention provides a method for the in vitro analysis of dihydroanatoxin-a and dihydrohomoanatoxin-a in water.

[0096] The term “immunoassay” refers to an analytical assay in which an immunochemical reaction occurs for the detection or quantification of an analyte. Competitive immunoassays are those in which the analyte competes with another molecule for binding to the antibody.

[0097] The terms “immunogen” and “immunogenic” as used in the present invention refer to a substance that is recognized as foreign to a living organism and is therefore capable of producing or generating an immune response in a host. In the present invention, the immunogen is a conjugate of formula (II). The term “antigen” in this patent application refers to a molecule capable of specifically interacting with an antibody. The immunochemical interaction or reaction consists of the specific, non-covalent binding between an antibody and an antigen, which may be the analyte or an assay antigen.

[0098] In this document, the term “assay antigen”, “enzyme antigen” or “tracer” refers to a conjugate of formula (II) or a labeled derivative of formula (III) used in the competitive assay.

[0099] Throughout the description and claims, the word "comprises" and its variants are not intended to exclude other technical features, additives, components, or steps. For those skilled in the art, other objects, advantages, and features of the invention will become apparent partly from the description and partly from the practice of the invention. The terms "the" or "the" may refer to one or a plurality of the modifying elements (for example, "the dihydroanatoxins" may mean dihydroanatoxin-a and / or dihydrohomoanatoxin-a).

[0100] The following examples illustrate how functionalized analogues of dihydroanatoxin-a of formula TLY (haptens) and the corresponding conjugates of formula (II) and (III) can be prepared. These examples are not intended to be limiting to the present invention and serve to show not only how they can be prepared but also the importance of the structural nature of the conjugate of formula (II) and (III) for the production of antibodies with adequate affinity and specificity towards dihydroanatoxin-a and dihydrohomoanatoxin-a, suitable for the development of an effective immunoanalytical method.

[0101] BRIEF DESCRIPTION OF THE FIGURES

[0102] Fig. 1. Standard curves for dihydroanatoxin-a (solid line) and dihydrohomoanatoxin-a (dashed line) in the direct competitive ELISA format using the monoclonal antibody lla#17, produced from the conjugate (Ha) where P is BSA (BSA_DA-1 conjugate), with the enzyme tracer (lllb) where Q is HRP (HRP__DA-2 conjugate).

[0103] EXAMPLES

[0104] The invention will now be illustrated by means of tests carried out by the inventors, which demonstrate the effectiveness of the conjugates of formulas (II) and (III) for obtaining antibodies against dihydroanatoxins and the development of an immunoassay with high sensitivity and selectivity for them. The numbers in bold refer to the corresponding structure shown in the diagrams. These examples are presented by way of demonstration, but in no way constitute a limitation of the invention.

[0105] 1. General Techniques

[0106] 1.1. Reagents, Equipment, and General Techniques Used in the Preparation of Haptens and Conjugates. All anhydrous solvents were dried using standard methods. Air- and moisture-sensitive reactions were carried out under a nitrogen atmosphere using material previously oven-dried at 135 °C overnight. The progress of the reactions was monitored by thin-layer chromatography on 0.25 mm silica gel plates. The plates were visualized under ultraviolet light at 366 and 254 nm, using an aqueous solution of ammonium ceric molybdate or an ethanolic solution of phosphomolybdic acid and heat as developing agents. Chromatography refers to flash column chromatography, which was performed on silica gel 60 (particle size 40–63 pm) with the solvents indicated in each case. The NMR spectra of 1 H and 13The C values ​​were recorded at 25 °C, in the indicated solvent, at 300 / 75 MHz (Bruker Avance 300 spectrometer) or 500 / 126 MHz (Bruker Avance DRX500). The chemical shifts of 1 H and !3 C (scale 5) values ​​are expressed in parts per million (ppm) relative to tetramethylsilane and refer to the residual proton or carbon in the NMR solvent and 77.16 ppm, respectively). A combination of COSY and edited HSQC experiments was used to assign the chemical shifts of 1 H and 13 C. The multiplicities of the spectra of !H values ​​are given using the following abbreviations: singlet (s), doublet (d), triplet (t), multiplet (m), double doublet (dd), double triplet (dt), double quintuplet (dq), double double doublet (ddd), double triplet doublet (dtd), and double doublet triplet (ddt). High-resolution mass spectra (HRMS) were obtained using a TripleTOF TM 5600 LC / MC / MC System from AB Sciex (Nieuwerkerk aan den IJssel, The Netherlands) equipped with an electrospray source. The data obtained are expressed as mass-to-charge ratios (m / z). The analysis of the conjugates was performed with a matrix-assisted laser desorption / ionization time-of-flight (MALDI-TOF) mass spectrometer, model 5800 (AB Sciex MALDI-TOF / TOF), in positive linear mode (1500 shots for each position) in a mass range of 12,000-100,000 m / z.

[0107] 1.2. Reagents, equipment and general techniques used in antibody generation and immunoassay development

[0108] The reference natural enantiomers (1R,2S,6R)-dihydroanatoxin-a and (1R,2S,6R)-dihydrohomoanatoxin-a were synthesized from [ ] en-10-one (Forro et al., Tetrahedron: Asymmetry, 2001, 15, 2875-2880), according to the procedure previously used for the preparation of racemic analogues (Mann et al., Toxicon 2012, 60, 1404-1414). BSA (bovine serum albumin fraction V) was from Roche Applied Science (Mannheim, Germany). Sigma / Aldrich (Madrid, Spain) provided OVA (egg albumin), HRP (horseradish peroxidase), bovine serum, and Freund's adjuvants. The 3,3',5,5'-tetramethylbenzidine (TMB) used was from Acros Organics. For the purification of the conjugates of formula (II) and (III), prepacked Sephadex G-25 HiTrap™ desalination columns from GE Healthcare (Uppsala, Sweden) were used with PB (100 mM sodium phosphate buffer, pH 7.4) as the elution buffer. A polyclonal anti-mouse immunoglobulin (GAM) antibody from Jackson Immunoresearch Europe (Ely, UK) was used as the capture antibody.Corning (Corning, NY, USA) 96-well, high-adsorption, flat-bottom polystyrene Costar ELISA plates were used. ELISA absorbances were read using a BioTek Instruments PowerWave HT microplate reader (Winooski, VT, USA). Microplate wells were washed using a BioTek Instruments ELx405 microplate washer.

[0109] 2. Preparation of protein conjugates of formula (II)

[0110] To a solution of p-lactam (1S,8RZ)-9-azabicicium[6.2.0]dec-4-en-10-one (Forro et al., Tetrahedron: Asymmetry, 2001, 15, 2875-2880) [(— )— 1.239 mg, 1.58 mmol] and DMAP (192.8 mg, 1.58 mmol) in anhydrous CH2Cl2 (3.6 mL) at 0 °C under nitrogen, BOC2O (730 pL, 3.16 mmol) and EtsN (220 pL, 1.58 mmol) were added. The resulting solution was allowed to reach room temperature and stirred for 5 hours. After this time, the reaction mixture was decanted onto a 1 M HCl solution (10 mL) and stirred carefully. Once gas emissions ceased, the organic phase was separated and the aqueous phase was extracted with CHCl3. The combined organic phases were washed with brine, dried over anhydrous Na2SC4, filtered, and concentrated under vacuum. The resulting crude was purified by chromatography (silica gel, CHCl3 / MeOH 99:1) to obtain the p-lactam

[0111] A crystal of l was added to a suspension of Mg shavings (126.4 mg, 5.2 mmol) in dry THF (2.5 mL). 2) followed by bromoacetal 3 (350 pL, 2.58 mmol) and the mixture was refluxed for 5 hours under a nitrogen atmosphere. The resulting grayish solution was allowed to reach room temperature before use and was titrated with benzoic acid using 4-(phenylazo)diphenylamine (PDA) as an indicator, yielding a concentration of 0.16 M of organomagnesian 4.

[0112] A solution of organomagnesian 4 (3.5 mL, 0.560 mmol) was added dropwise to a stirred solution of p-lactam 2 (109.5 mg, 0.435 mmol) in dry THF (0.5 mL) at -30 °C under nitrogen, and the resulting mixture was stirred for 1 hour at the same temperature. After this time, the reaction mixture was diluted with a saturated solution of NH4Cl and water and extracted with EtAc. The pooled organic phases were washed with brine, dried over anhydrous MgSCU, filtered, and concentrated under vacuum. The resulting crude reaction was purified by chromatography (silica gel, hexane / acetate 8:2) to obtain ketone 5 (135 mg, 85%) as a colorless oil. NMR reveals that it is a mixture of carbamate rotamers, and for simplicity, only the signals of the major rotamer are shown. NMR of 1H (300 MHz, CDCh) 0 5.74-5.56 (m, 2H, H-4 and H-5), 4.86 (s width, 1 H, NH Boc), 4.81 (t, J ~ 4.3 Hz, 1 H, H-5'), one 4.13 (da, H8, H-3 (1 y), width m, each 2H, OCH2CH2O), 2.83 (d width, J = 8.8 Hz, 1 H, H-8), 2.60-2.42 (m, 2H, H2-2'), 2.42-2.28 (m, 2H, H-3 and H-6, H-2, H-3, 2,40, m H'-6 and H-7), 1 ,80-1 .58 (m, 6H, H'-2, H'-7, H2-3' and H2-4'), 1 ,39 (s, 9H, CMe3Boc); NMR of 13 C (101 MHz, CDCh) 6 212.3 (CT) 155.2 (CO2Boc), 130.2 and 129.5 (C-4 and C-5), 104.4 (C-5'), 79.4 (CMe3Boc), 64.91 and 60.24.925 (O (C-1), 41 ,1 (C-2'), 33.3 (C-4'), 33.1 (C-2), 28.4 (C / Vte3Boc), 26.0 (C-7), 25.5 (C-6), 23.9 (C-3), 18.2 (C-3'); EMAR (TOP, ESI+) calculated for C20H34NO5 [M+H] + 368.2431 , found 368.2427.

[0113] 2.1.1.3. Preparation of (1S,5S,6R)-5~(4~(1 ,3-d¡o>:oiar-2-¡')butane¡')-9-azab¡c¡clo[4.2. 1] non-2-en-9-terebutyl carboxyate (6).

[0114] A suspension of ketone 5 (135 mg, 0.367 mmol), Pd(OAc)2 (23.6 mg, 0.096 mmol), and Cu(OAc)2 (150.9 mg, 0.83 mmol) in DMF (1.2 mL) was stirred at 70 °C for 8 hours under a nitrogen atmosphere. Afterward, the reaction mixture was diluted with EtAc and filtered through silica gel. The filtrate was washed with water, followed by small portions of 1.5% LiCl and brine. After drying over anhydrous MgSCU and concentrating under reduced pressure, the aza-bicyclic alkene 6 (133 mg, 99%) was obtained as a yellowish oil, which was used in the next step without further purification. NMR reveals that it is a mixture of carbamate rotamers, and for simplicity, only the signals of the major rotamer are shown. NMR of 1H (300 MHz, CDCh) 5 5.63 (m, 1 H, H-3), 5.56 (m, 1 H, H-2), 4.81 (t, J = 4.4 Hz, 1 H, H-5'), 4.67 (dd width, J = 7.2 y, J 1 d 1 4 H , H -4 = 8.6 and 2.9 Hz, 1 H, H-6), 3.92 and 3.80 (each m, each 2H, OCH2CH2O), 2.68–2.56 (m, 1 H, H–5), 2.56–2.46 (m, 2.4H, H2–2m–2.2), H-7), 2.15-2.04 (m, 1 H, H'-4), 1 ,92-1 .77 (m, 2H, H'-7 and H-8), 1 ,74-1 .58 (m, 5H, H'-8, H2-3' and H2-4'), CMe (BocH38); NMR of 13 C (126 MHz, CDCh) 5 209.1 (CT), 153.4 (CO2Boc), 132.5 (C-2), 125.6 (C-3), 104.3 (C-5'), 80.1 (CMe3Boc), 64.9 (OCH2CH2O), -62,-62 54.8 (C-6), 40.1 (C-2′), 34.2 (C-7), 33.1 (C-4′), 29.6 (C-8), 28.3 (CMe3Boc), 24.4 (C-4), 18.4 (C-3′); EMAR (TOP, ESI+) calculated for C20H32NO5 [M+H] + 366.2275, found 366.2281. 2.1.1.4. Preparation of (1R,2S,6R)-2-(4-(1,3-dioxolan-2-yl)butanoyl)-9-azabicithium[4.2. 1] nonan-9-tert-butthium carboxyate (7).

[0115] A solution of azabicyclic aikene 6 (79.6 mg, 0.217 mmol) and Wilkinson's catalyst (28.5 mg, 0.03 mmol) in anhydrous THF (3 mL) was introduced into a Büchi Tinyclave reactor equipped with magnetic stirring and pressurized to 3 atm with H₂ at room temperature. Vigorous stirring was maintained for 16 hours. The reactor was then depressurized, and the reaction mixture was dried. The resulting residue was purified by chromatography (silica gel, hexane / acetate 7:3) to yield compound 7 (73.4 mg, 92%) as a yellowish oil. NMR spectroscopy revealed it to be a mixture of carbamate rotamers; for simplicity, only the signals are reported.

[0116] 2.1.1.5. Preparation of the acid (1'R : 2'S,6'R)-5-(9-(tert-butoxycarbonii)-9- azabicicio[4.2. 1]nonan-2-ii)-5~oxopenianoic acid (8). To a stirred solution of acetal 7 (33.8 mg, 0.092 mmol) in acetone (2 mL) cooled to 0 °C, Jones reagent was added dropwise using a Pasteur pipette (34 drops, 1 drop / mg of 7), and the resulting mixture was allowed to reach room temperature, at which point a greenish precipitate appeared. Once the reaction was complete (monitored by TLC, CH3C / Methanol 9:1), the reaction mixture was diluted with water and extracted with CH3C. The collected organic phases were washed with brine and dried over anhydrous Na2SO4. After removing the solvent under reduced pressure, the residue was purified by chromatography (silica gel, CH3C / MeOH 9:1) to obtain acid 8 (27.7 mg, 88%) as a colorless oil. NMR shows that it is a mixture of carbamate group rotamers and, for simplicity, only the

[0117] 2.1.1.6. Preparation of ( 1R.2S, 6R)-2-(5-((2, 5-dioxopyrrolidine- 1-ii)o:<i)-5-c>:aperitsrioi)-9- azabicyclo [4.2. 1]nonan-9-carboxyate of tere-butyl (9). at 0 °C under nitrogen. The reaction mixture was allowed to reach room temperature with stirring for 3 hours, diluted with CH2Cl2, washed with water and brine, and dried over anhydrous MgSO4. After filtration and removal of the solvent under reduced pressure, the β-hydroxysuccinimidil ester 9 (8.5 mg, 75%) was obtained as a colorless oil, which was used in the next step without further purification. NMR spectroscopy shows that it is a mixture of carbamate rotamers, and for simplicity, only the signals of the major rotamer are given. NMR of 1 H (500 MHz, CDCh) 5 4.42 (d width, J 2.1.1.7. Preparation of the N-hydroxysuccinimidyl ester of hapten DA 1 as a salt of trifluoroacetic acid (10).

[0118] The A / -hydroxysuccinimidyl ester 9 (8.0 mg, 0.018 mmol) was dissolved in a 3:1 mixture of anhydrous CH2Cl2 and CF3CO2H (300 pL), and the mixture was stirred at room temperature for 15 minutes. The reaction mixture was then dried using a rotary evaporator, and the excess CF3CO2H was removed by cycles of dilution and concentration with anhydrous benzene (3 times). The resulting residue was maintained under high vacuum overnight to provide the A / -hydroxysuccinimidyl ester of the hapten DA-1 as

[0119] 2.1.2. Preparation of DA-1 hapten conjugates with proteins 2.1.2.1. Preparation of the DA-1 hapten conjugate with BSA [BSA_DA-1 conjugate].

[0120] An approximately 50 mM solution in DMF of the active ester of the DA-1 hapten (10, 136 pL) obtained in the previous reaction was slowly added with stirring to 1.21 mL of a BSA solution (15 mg / mL) in 100 mM phosphate buffer, pH 7.4 (PB). The conjugation reaction was incubated for 20 hours with stirring at room temperature. After this time, the BSA bioconjugate formed was purified by size-exclusion chromatography on three coupled 5 mL Sephadex G-25 HiTrap™ desalination columns, using PB as the elution buffer. Following the purification process, the collected fractions containing the BSA bioconjugate were pooled, sterilized by filtration through a 0.45 pm nylon filter, brought to a final concentration of 3 mg / mL with filtered elution buffer, and stored at -20 °C.

[0121] To determine the hapten loading (m) obtained in the conjugate, a 33.3 μL aliquot of the purified BSA_DA-1 conjugate solution was diluted to 100 pL with deionized water and dialyzed (dialysis against 5 L of deionized water with at least 3 to 4 water changes for 24 hours at 4 °C); finally, the dialyzed product was used to calculate the conjugation efficiency in terms of the average number of DA-1 hapten molecules coupled per BSA molecule by MALDI-TOF-MS (m = 21, see Table 2, entry 1).

[0122] 2.1.2.2. Preparation of the DA-1 hapten conjugate with OVA [OVA_DA-1 conjugate].

[0123] 136 pL of a 50 mM solution in DMF of the A / -hydroxysuccinimidyl ester of the DA-1 hapten (10) was slowly added with stirring to a volume of 2.13 mL of an OVA solution (15 mg / mL) in PB. After 20 hours of reaction with slow stirring at room temperature, the conjugate formed was purified as described above 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. A 100 pL aliquot of the freshly purified OVA_DA-1 conjugate was dialyzed, and the average number of DA-1 hapten molecules per OVA molecule was determined by MALDI-TOF-MS (m = 5.3, see Table 2, entry 3). A suspension of magnesium shavings (536.4 mg, 21.95 mmol) in dry THF (10 mL) cooled to 0 °C under nitrogen was mixed with an iodine crystal and 6-chlorohexanol (11.980 pL, 1.003 mg, 7.3 mmol). The mixture was stirred under the same conditions, and a 3 M solution of methyl chlorite phosphate (MC12) in anhydrous THF (2.8 mL, 7.3 mmol) was added dropwise. Once gas emission ceased, the mixture was refluxed at the solvent temperature for 2 hours. The resulting solution was allowed to cool to room temperature before use and was titrated with benzoic acid using (phenylazo)diphenylamine as an indicator, yielding a 0.25 M concentration of the organomagnesian compound.

[0124] The freshly prepared solution of organomagnesian 12 (5 mL, 1.25 mmol) was added dropwise to a solution of racemic p-lactam 2 (236 mg, 1.020 mmol)—prepared as described in Parsons et al., Tetrahedron 1999, 56, 309–315—in anhydrous THF (1 mL) at −30 °C under a nitrogen atmosphere. The reaction mixture was stirred for 1 hour under the same conditions, treated with a saturated solution of NH₄Cl and water, and extracted with EtAc. The pooled organic phases were washed with brine, dried over anhydrous MgSCU, filtered, and concentrated under vacuum. The resulting crude reaction product was purified by chromatography (silica gel, hexane / acetate 8:2) to obtain ketone 13 (181 mg, 57%) as a colorless oil. NMR analysis revealed it to be a mixture of carbamate rotamers, and for simplicity, only the NMR signals of the major rotamer are shown. 1 H (300 MHz

[0125] 2.2.1.2. Preparation of rac-(1S,5S,6R)-5-(7-((hydroxyheptanoyl)-9-azabicyclo[4.2.1]non- 2-en-9-carboxyate of tert-butylium (14).

[0126] A suspension of alkene 13 (49.7 mg, 0.14 mmol), Pd(OAc)2 (10.6 mg, 0.028 mmol), and Cu(OAc)2 (57.3 mg, 0.294 mmol) in DMF (460 pL) was stirred at 70 °C under nitrogen for 8 hours. After this time, the reaction mixture was cooled to room temperature, diluted with Et2O, and filtered through silica gel, using Et2O for washing. The filtrate and washings were combined and successively washed with water, a 1.5% aqueous LiCl solution, and brine. After drying over anhydrous MgSO4 and concentrating under reduced pressure, alkene 14 (48 mg, 74%) was obtained as a yellowish oil, sufficiently pure by NMR 1H to be used in the next stage without further purification. NMR shows that it is a mixture of carbamate rotamers and, for simplicity, only the signals of the major rotamer are given.

[0127] 2.2.1.3. Preparation of rac-(1R,2S,6R)-2-(7-hydroxyheptanoyl)-9-azabicyclo[4.2. 1]nonan- 9-butyl carboxyate (15).

[0128] A suspension of alkene 14 (43 mg, 0.12 mmol) and 10% Pd / C (13.5 mg, 0.012 mmol Pd) in 2 mL of EtAc was hydrogenated at room temperature for 5 hours under a hydrogen pressure of 3 bar in a Büchi Tinyclave pressurized reactor equipped with magnetic stirring. The reactor was then depressurized, and the mixture was filtered through Celite and washed with EtAc. After evaporating the solvent under vacuum, the saturated azabicyclic compound 15 (40 mg, 92%) was obtained as a colorless oil, which was used in the next step without further purification. NMR spectroscopy revealed it to be a mixture of carbamate rotamers, and for simplicity, only the

[0129] To a solution of alcohol 15 (25.2 mg, 0.0713 mmol) in CH2Cl2 (2.8 mL), Dess-Martin periodinane (36.2 mg, 0.085 mmol) was added, and the mixture was stirred at room temperature for 40 minutes. After this time, the reaction mixture was treated with a saturated solution of Na2SO3 (3 mL) and stirred for 30 minutes. The mixture was then diluted with CH2Cl2 and washed successively with a 5% NaHCO3 solution, water, and brine. After drying the organic phase over anhydrous MgSO4, filtering, and concentrating under vacuum, aldehyde 16 (22.3 mg) was obtained as a colorless oil, which was immediately oxidized to the corresponding carboxylic acid, as described below.

[0130] A solution of aldehyde 16 (22.3 mg, 0.0634 mmol) in a 5:1 mixture of !To BuOH- H2O (1 mL) cooled to 0 °C, 2-methyl-2-butene (65 pL, 1.3 mmol), NaH2PO4 (26.8 mg, 0.19 mmol), and NaClO2 (17.9 mg, 0.19 mmol) were sequentially added. Once the solids were dissolved, the reaction mixture was stirred for 10–15 minutes. After this time, the reaction mixture was acidified with a 10% KHSO4 solution and extracted with CHCh. The collected organic phases were washed with brine and dried over anhydrous MgSO4. After filtration and removal of the solvent under reduced pressure, acid 17 (22.4 mg, 85% from alcohol 15) was obtained as a colorless oil, which was used in the next step without further purification. NMR shows that it is a mixture of carbamate group rotamers and, for simplicity, only the signals are given

[0131] A solution of acid 17 (12.5 mg, 0.034 mmol) and A / , / V'-disuccinimidyl carbonate (13 mg, 0.050 mmol) in anhydrous CH2Cl2 (430 pL) cooled to 0 °C under nitrogen was treated with Et3N (17 pL, 0.012 mmol), and the resulting mixture was allowed to reach room temperature slowly for 3 hours. The reaction mixture was diluted with CH2Cl2 and water and extracted with CH2Cl2. The collected organic phases were washed with brine and dried over anhydrous MgSCh. After filtration and solvent removal under vacuum, the A / , / V'-hydroxysuccinimidil ester 18 (10.7 mg, 67%) was obtained as a colorless oil. NMR analysis revealed it to be a mixture of carbamate rotamers.

[0132] 2.2.1.6. Preparation of the N-hydroxysuccinimidiium ester of the DA-2 hapten as a salt of trifluoroacetic acid (19).

[0133] Ester 18 (10.7 mg, 0.023 mmol) was dissolved in a 3:1 mixture of anhydrous CH2Cl2 and CF3CO2H (370 pL), and the resulting solution was stirred at room temperature for 15 minutes. The reaction mixture was then dried using a rotary evaporator, and the excess CF3CO2H was removed by dilution and concentration cycles with benzene (3 times) to obtain the A / -hydroxysuccinimidyl ester of the DA-2 hapten as a salt of

[0134] 2.2.2. Preparation of DA-2 hapten conjugates with proteins

[0135] 2.2.2.1 . Preparation of the DA-2 hapten conjugate with BSA [BSA... DA-2 conjugate], active ester hapten DA-2 Conjugate BSAJ3A-2; m - 12,9

[0136] Prepared following the same procedure described above for the BSA-DA-1 conjugate (section 2.1.2.1) from 205 pL of a 50 mM solution in DMF of the A / -hydroxysuccinimidyl ester of the DA-2 hapten (19) and 1.5 mL of a BSA solution (15 mg / mL) in PB. After the purification process, the collected fractions containing the BSA conjugate were pooled, sterilized by filtration through a 0.45 µm nylon filter, brought to a final concentration of 2 mg / mL with filtered elution buffer, and stored at -20 °C. The mean number of DA-2 hapten molecules conjugated per BSA molecule, determined by MALDI-TOF-MS, was m = 12.9 (see Table 2, entry 2).

[0137] 2.2.2.2. Preparation of the DA-2 hapten conjugate with OVA [OVA__DA~ 2 conjugate]. Active ester of the DA-2 hapten conjugate OVA„DA-2; m = 3.3. Prepared as described above for the OVA_DA-1 bioconjugate (section 2.1.2.2) from 122 µL of a 50 mM solution in DMF of the N-hydroxysuccinimidyl ester of the DA-2 hapten (19) and 1.8 mL of an OVA solution (15 mg / mL) in PB. After appropriate chromatographic purification, the collected fractions were brought to a final concentration of 1 mg / mL in elution buffer containing 0.01% (v / v) thimerosal and stored at -20 °C. The mean number of conjugated DA-2 hapten molecules per OVA molecule, determined by MALDI-TOF-MS, was m = 3.3 (see Table 2, entry 4).

[0138] To a solution of alcohol 15 (14.4 mg, 0.0407 mmol) in anhydrous CH2Cl2 (600 pL), Et3N (15 pL, 0.104 mmol) was added under nitrogen. The resulting mixture was cooled to 0 °C, and mesyl chloride (4.7 pL, 0.061 mmol) was added. Stirring was maintained at the same temperature for 15 minutes. After this time, the reaction mixture was diluted with CH2Cl2 and successively washed in cold water, a 0.5 M HCl solution, a saturated NaHCO3 solution, and brine. The organic phase was dried over anhydrous MgSCl, filtered, and concentrated under vacuum to give mesylate 20 (13.4 mg, 76%) as a colorless oil, which was used immediately without purification in the next step.

[0139] A solution of freshly prepared mesylate 20 (11 mg, 0.031 mmol) and NaNs (8.9 mg, 0.13 mmol) in anhydrous DMF (500 pL) was stirred for 15 hours at room temperature under nitrogen. The reaction mixture was then diluted with water and extracted with CHCh. The pooled organic phases were washed with water, then with a 1.5% aqueous LiCl solution, and finally with brine. After drying over anhydrous MgSCh, filtration, and vacuum concentration, the resulting crude reaction mixture was purified by chromatography (silica gel, CHCh) to obtain azide 21 (7 mg, 70%) as a colorless oil. NMR analysis revealed it to be a mixture of rotamers from the group

[0140] 2.3.1.2. Preparation of the hapten DA-3 as a trifluoroacetic acid (22).

[0141] The α / -hydroxysuccinimidyl ester of 5-oxo-5-(prop-2-yn-1-iamino)pentanoic acid (i) was dissolved in DMF and added dropwise to a solution of each protein (BSA or OVA) in PB. The alkyne-to-protein molar ratios (RMo) used in the reaction mixture were 25 and 10 for the derivatization of BSA and OVA, respectively (see Table 1, entries 1 and 2). The conjugation reaction was carried out with a maximum DMF content of 10% (v / v). After overnight incubation at room temperature, the alkynylated proteins were purified by size-exclusion chromatography using a 5 mL Sephadex G-25 HiTrap© desalination column and PB as the eluent, at a flow rate of 5 mL / min, collecting 1 mL fractions, and stored at 4 °C until use. The mean number of incorporated alkyne residues per protein / enzyme molecule (hapten:protein molar ratio, MR) was calculated by MALDI-TOF-MS.The RMs obtained for BSA-alkyne and OVA-alkyne were approximately 20 and 5, respectively (see Table 2, entries 7 and 8). 2.3.2.2. Preparation of the DA-3 hapten conjugate with BSA [BSA__DA-3 conjugate].

[0142] A solution of the hapten DA-3 (22, 6.0 mg, 15.3 pmol, 3.8 hapten equivalents per alkyne residue) in DMSO (790 pL) was added dropwise to a solution of the BSA-alkyne conjugate described in section 2.3.2.1 (RM = 20, 15 mg, 4 pmol of alkyne groups) in PB (31 mL), with stirring at room temperature. To the resulting solution, 107 pL of a solution previously prepared from solutions of ir / s(3-hydroxypropyltriazolylmethyl)amine (THPTA, 120 mM, 150 pL, 18.0 pmol) and CuS₄ (40 mM, 90 pL, 3.6 pmol) in Milli-Q water (2 equivalents of THPTA and 0.4 equivalents of CuS₄ per alkyne residue) were added dropwise. The resulting mixture was degassed by vacuum / nitrogen cycling, and 317 pL of a 250 mM sodium ascorbate solution in Milli-Q water (20 equivalents per alkyne group), also previously degassed by vacuum / nitrogen cycling, were added.The resulting solution was stirred for 20 hours at room temperature, and the BSA_DA-3 conjugate formed was separated from the remaining reagents using Amicon® Ultra-4 10K filters. Three 5-minute cycles were performed at 4800 rpm using PB, yielding a final volume of 2 mL. The BSA_DA-3 conjugate was purified by size-exclusion chromatography on three coupled 5 mL Sephadex G-25 HiTrap™ desalination columns, using PB as the elution buffer. After purification, the collected fractions containing the BSA conjugate were pooled, sterilized by filtration through a 0.45 µm nylon filter, diluted to a final concentration of 2 mg / mL with filtered elution buffer, and stored at -20 °C. The average number of conjugated DA-3 hapten molecules per BSA-alkyne molecule, determined by MALDI-TOF-MS, was m = 18.1 (see Table 2, entry 10). 2.3.2.3.Preparation of the DA-3 hapten conjugate with OVA [OVA_DA-3 conjugate].

[0143] The OVA__DA-3 conjugate was prepared as described in section 2.3.2.2 for the BSA analogue conjugate, using a solution of the DA-3 hapten (22, 2.0 mg,

[0144] 5.1 pmol, 3.1 hapten equivalents per alkyne residue) in DIVISO (265 pL), a solution of the OVA-alkyne conjugate described in section 2.3.2.1 (RM = 5.15 mg, 1.66 pmol of alkyne groups) in PB (18.8 mL), 35 pL of the THPTA and CuS₄ solution, and 106 pL of the sodium ascorbate solution in Milli-Q water. The conjugate obtained after purification was brought to a final concentration of 1 mg / mL in PB and stored at -20 °C. The average number of DA-3 hapten molecules conjugated per OVA-alkyne molecule, determined by MALDI-TOF-MS, was m = 4.2 (see Table 2, entry 11).

[0145] Table 1. Conditions for the preparation of proteins / enzymes modified with alkyne groups. 3. Preparation of enzyme conjugates of formula (III)

[0146] Example 3.1. Preparation of conjugates of formula (III) for T = Rl, L = -CH2CH2- Z = ~(C=O)NH~ and Q = HRP.

[0147] 3.1.1. Preparation of the DA-1 hapten conjugate with HRP [HRPJDA-1 conjugate], active ester hapten DA-1 Conjugate HRP_DA-1; m = 1,2

[0148] The HRP_DA-1 conjugate was prepared as described above for the BSA_DA-1 conjugate (section 2.1.2.1) from 140 pL of a 5 mM solution of the active ester of the DA-1 hapten (10) in DMF and 1 mL of an HRP solution (3 mg / mL) in PB. After chromatographic purification, the resulting tracer-containing fractions were diluted to 228 pg / mL with the stabilizer Biostab and stored at 4 °C. The mean number of conjugated DA-1 hapten molecules per HRP molecule, determined by MALDI-TOF-MS, was m - 1.2 (see Table 2, entry 5).

[0149] 3.1.2. Preparation of the DA-2 hapten conjugate with HRP [HRP_DA-2 conjugate],

[0150] The HRP_DA-2 conjugate was prepared as described above for the BSA_DA-1 conjugate (section 2.1.2.1) from 100 pL of a 5 mM solution of the active ester of the DA-2 hapten (19) in DMF and 900 pL of an HRP solution (3 mg / mL) in PB. After chromatographic purification, the resulting tracer-containing fractions were diluted to 180 pg / mL with the stabilizer Biostab and stored at 4 °C. The average number of conjugated DA-2 hapten molecules per HRP molecule, determined by MALDI-TOF-MS, was m = 1.5 (see Table 2, entry 6).

[0151] -CH2CH2CH2C

[0152] 3.3.1. Functionalization of the free amino groups of the HRP enzyme with alkynyl groups. [Preparation of the HRP-alkyne conjugate]

[0153] The free amino groups of the HRP enzyme were derivatized with alkynyl groups following the same protocol previously described for the analogous derivatization of the amino groups of the BSA and OVA proteins, as detailed in section

[0154] 2.1.2, using in this case a molar ratio of the active alkynylated ester (i) to protein (MRo) of 10 (see Table 1, entry 3). After overnight incubation at room temperature, the alkynylated enzyme was purified by size-exclusion chromatography using a 5 mL Sephadex G-25 HiTrap© desalination column and PB as the eluent, at a flow rate of 5 mL / min, collecting 1 mL fractions (final concentration obtained of 1 mg / mL), and stored at 4 °C until use. The average number of incorporated alkyne residues per enzyme molecule (hapten:enzyme molar ratio, MR) was calculated by MALDI-TOF-MS. The MR obtained for the HRP-alkyne conjugate was 1.8 (see Table 2, entry 9).

[0155] 3.3.2. Preparation of the DA-3 hapten conjugate with HRP [HRP_DA-3 conjugate],

[0156] HRP The HRP_DA-3 conjugate was prepared as described in section 2.3.2.2 for the BSA analogue conjugate, using a solution of the DA-3 hapten (22, 110 pg, 0.28 pmol, 2.3 hapten equivalents per alkyne residue) in DIVISO (14.1 pL), a solution of the HRP~alkyne conjugate described in section 3.3.1 (RM = 1,8, 3 mL, 3 mg, 0.12 pmol of alkyne groups) in PB (33.8 mL), 63 pL of the THPTA and CuSO4 solution, and 191 pL of the Milli-Q sodium ascorbate solution in water. The HRP__DA-3 conjugate was purified by size-exclusion chromatography on two coupled 5 mL Sephadex G-25 HIT rap™ desalination columns, using HEPES as the elution buffer. The purified conjugate was diluted to 236 pg / mL with Biostab stabilizer and stored at 4 °C. The mean number of DA-3 hapten molecules conjugated per HRP molecule, determined by MALDI-TOF-MS, was m = 1.6 (see Table 2, entry 12).

[0157] TABLE 2. Hapten load values ​​of protein and enzyme conjugates of dihydroanatoxin haptens determined by MALDI-TOF-MS 4. ELISA Procedure

[0158] Polystyrene plates with 96 wells were used. Each antibody was evaluated by competitive ELISA in the immobilized antibody format using direct detection capture antibody (direct format), using in all cases as an enzymatic tracer a labeled derivative of dihydroanatoxin-a of formula (lllb) or (lile), in which Q is horseradish peroxidase (HRP), both for the evaluation of the antibodies generated from the conjugates of formula (Ha) as (llb), in which P is bovine serum albumin (BSA).On the other hand, hybridoma selection and antibody specificity were determined by competitive ELISA in the immobilized conjugate format with indirect detection (indirect format) using homologous conjugates, i.e., a labeled derivative of dihydroanatoxin-a of formula (Ha) or (llb), wherein P is ovalbumin (OVA), for the evaluation of antibodies generated from conjugates of formula (Ha) or (llb), respectively, wherein P is bovine serum albumin (BSA).

[0159] After each incubation stage, the plates were washed four times with a washing solution, using an ELx405 96-channel washer (Biotek Instruments, Winooski, USA).

[0160] For direct assays, plates were coated with 100 pL per well of a 1 pg / mL capture antibody (GAM) solution in 50 mM carbonate buffer, pH 9.6, by incubation overnight at room temperature. The next day, the plates were washed, and 100 pL per well of a monoclonal antibody solution at different concentrations was added in PBST (10 mM phosphate buffer, pH 7.4, with 140 mM NaCl and 0.05% Tween 20), and incubated for 1 hour at room temperature. After washing the plates, 50 pL per well of a standard analyte solution in PBS (10 mM phosphate buffer, pH 7.4, with 140 mM NaCl) was dispensed into each column, followed by 50 pL per well of an enzyme tracer in PBST, which is a derivative of formula (III) where Q is HRP, at various concentrations. The immunochemical reaction was carried out for 1 hour at room temperature, and then the plates were washed.Next, the retained peroxidase activity was revealed with 100 pL per well of a 1 mM TMB solution in 205 mM citrate buffer, pH 4.0, containing 0.012% (v / v) H2O2. This reveal was carried out for 10 minutes at room temperature and stopped using 100 pL per well of 1 M sulfuric acid.

[0161] For indirect assays, plates were coated with 100 pL per well of an assay antigen solution, which is a conjugate of formula (II), where P is OVA, at various concentrations in 50 mM carbonate buffer, pH 9.6, by overnight incubation at room temperature. After washing the plates, 50 pL per well of PBS or analytical standard in PBS was dispensed into each column, followed by 50 pL per well of culture supernatant or a selected antibody diluted in PBST (0.05% Tween 20). The immunochemical reaction was carried out for 1 hour at room temperature, after which the plates were washed. Subsequently, each well received 100 pL of a 1 / 2000 dilution of GAM-HRP (peroxidase-labeled goat anti-mouse immunoglobulin antibody) in PBST. This reaction was left at room temperature for 1 hour.After washing the plates, the retained peroxidase activity was revealed as described for direct format assays.

[0162] At the end of the assays, the absorbance of each well was read at 450 nm using a reference wavelength of 650 nm on a PowerWave HT microplate reader (Biotek Instruments, Winooski, USA). The sigmoidal standard curves obtained by plotting absorbance against the logarithm of the analyte concentration were fitted to a four-parameter logistic equation using the SigmaPlot software package of SPSS (Chicago, USA).

[0163] The antibody affinity (IC50) was estimated as the concentration of free analyte capable of halving the maximum signal (A max ).

[0164] The antibody specificity was estimated as the percentage of the ratio between the IC50 value of dihydrohomoanatoxin-a and that of the analyte under study.

[0165] 5. Production of monoclonal antibodies

[0166] 5.1. Immunization of mice

[0167] For immunization, the conjugates of formula (Ha) and (llb), in which P is BSA (immunizing conjugates BSA_DA-1 and BSA__DA-2), were used, obtained as described in the previous examples. BALB / c female mice were used, with an age at the start of the process between 6 and 8 weeks.

[0168] In each dose, 100 pg of conjugate was administered intraperitoneally to each mouse, with a total administered volume of 200 pL. For the first immunization, the conjugate was administered in an emulsion prepared with complete Freund's adjuvant (1:1, v / v). At 3-week intervals, the mice received two additional immunizations, in these cases emulsifying the conjugates with incomplete Freund's adjuvant. Four days before each cell fusion, the selected mice received a final dose of 100 pg of the corresponding conjugate diluted in PBS. 5.2. Cell fusions for obtaining hybridomas

[0169] The fusions with the immunized mice were carried out following previously described and well-established methodologies in the state of the art. Immediately after mouse sacrifice, the spleen was removed and homogenized using the plunger of a sterile syringe. After osmotic shock of the red blood cells with 1 mL of lysis buffer for 1 minute in a cold environment, the lymphocytes were washed twice with cold complete medium (containing serum) and filtered to remove any clots.

[0170] The P3-X63-Ag8.653 myeloma cell line was cultured in the days prior to fusion in DMEM (Dulbecco's modified Eagle medium) supplemented [2 mM L-alanine-L-glutamine, 1 mM non-essential amino acids, 25 pg / mL gentamicin, fetal bovine serum (FBS) 10% (v / v)], keeping the cells in the exponential growth phase, so that a sufficient number of them would be available on the day of fusion.

[0171] After two washes with serum-free medium, both cell populations were pooled at a lymphocyte:myeloma ratio of 4:1. The cells were then centrifuged, and cell fusion was immediately performed. For this, the chemical fusion agent PEG1500 (1 mL per spleen, 1 minute) was used, which partially dissolves the membranes, allowing the cells to fuse. Once both populations were fused, the cells were resuspended in supplemented DMEM medium [15% (v / v) SBF] and seeded in 96-well culture plates (100 pL per well) at a cell density of 150 x 10⁶ 3 lymphocytes per well, and were incubated at 37 °C in an atmosphere with 5% CO2 and 95% humidity. 24 hours after fusion, 100 pL per well of HAT hybridoma selection medium [DMEM supplemented with 100 pM hypoxanthine, 0.4 pM aminopterin, 16 pM thymidine, and 20% (v / v) SBF] containing 1% (v / v) HFCS (High Fusion and Cloning Supplement) was added.

[0172] 5.3. Selection, cloning and conservation of hybridomas

[0173] Approximately 10–12 days after cell fusion, the supernatants from the seeded wells were evaluated to identify those containing hybridomas secreting antibodies capable of recognizing dihydroencotatoxins in both their free and conjugated forms. Prior to this, fusion efficiency was determined by visual inspection, defined as the percentage of wells exhibiting at least one clone clearly visible under a microscope. To identify competing clones, the culture supernatants were analyzed using an indirect differential ELISA technique. This technique involves parallel analysis of each supernatant in adjacent wells, both in the absence of analyte and in the presence of a predetermined analyte concentration, typically 100 nM.For this purpose, the plates were coated with the homologous conjugate, which is a conjugate of formula (Ha) or (llb) in which P is OVA (assay conjugates OVA_DA-1 and OVA_DA-2), at a concentration of 0.1 pg / mL, and the assay was carried out by adding 50 pL of the culture supernatant. The conditions for the indirect ELISA format are detailed in section 4.

[0174] Next, wells containing antibody-producing hybridomas capable of providing an absorbance signal of 0.5 or greater in the assay in the absence of dihydroanatoxin-a and signal inhibition of 80% or greater in the assay in the presence of dihydroanatoxin-a were selected. Additionally, for all positive wells, a second, more thorough screening was performed in competitive two-dimensional mode to more reliably select the best hybridomas. For this purpose, the supernatant from each hybridoma was assayed at four dilutions (1 / 8, 1 / 32, 1 / 128, and 1 / 512) on plates coated with the homologous conjugate at 0.01 and 0.1 pg / mL, using dihydroanatoxin-a as a competitor at 10 and 100 nM (in assay). Thus, 200 pL of the culture supernatant were diluted in 600 pL of PBST and subsequent dilutions were made serially from this first one.The assay was performed by adding 50 pL per well of the corresponding supernatant dilution and 50 pL of the dihydroanatoxin-a solution in PBS at the concentration of 0, 10 and 100 nM.

[0175] The cells from the finally selected wells were cloned by the limit dilution method, seeding from each well a new plate of 96 wells at 2 cells per well in HT medium, of the same composition as HAT but without aminopterin, and containing HFCS at 1% (v / v).

[0176] Generally, 7–10 days after the first cloning, wells containing a single clone were identified by visual inspection, and the culture supernatant was re-evaluated in the same way as previously described for fusion supernatants. This process was repeated as many times as necessary (at least twice) to ensure the monoclonality and stability of the selected hybridomas. Finally, the selected cell lines were expanded by progressively culturing the hybridoma in larger volume containers. Once the clone had grown, the cells were frozen in liquid nitrogen at a concentration of 10 7 cells per vial (2-4 vials for each hybridoma) in an SBF solution with 10% (v / v) DMSO as a cryoprotectant. The vials were kept at -80 °C inside a polystyrene box for 24 hours before being transferred to the liquid nitrogen container.

[0177] 5.4 Production and purification of monoclonal antibodies

[0178] In the final phase of hybridoma cell expansion, the cells were progressively divided in culture plates until a final volume of 100–200 mL of supernatant was reached. The cells were allowed to grow until confluence, and once the nutrients in the culture medium were exhausted, the contents of the plates were collected. The collected volume was centrifuged to remove cell debris, and the supernatant was precipitated by adding a volume of a saturated ammonium sulfate solution. It was then maintained at 4 °C until purification.

[0179] Antibody purification was performed by affinity chromatography using protein G columns, following the manufacturer's instructions. For this purpose, the precipitated antibody was centrifuged for 20 minutes at 5000 rpm (4000 x g), and the supernatant was discarded. The antibody-containing precipitate was redissolved in 20 mM sodium phosphate buffer, pH 7.4, and filtered through nitrocellulose membranes (0.45 µm pore diameter) to remove suspended particles. Antibody elution from the column was performed using 100 mM sodium citrate buffer, pH 2.5. Antibody-containing fractions were identified by UV spectrophotometry and collected. The solution was neutralized by adding 1 M Tris-HCl, pH 9.5. Finally, the concentration of the purified antibody [A2so (1 mg / mL IgG) = 1.4] was determined by UV spectrophotometry and a working solution was prepared 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.

[0180] 6. Results

[0181] 6.1. Generation of hybridomas producing monoclonal antibodies against dihydroanatoxins

[0182] To demonstrate the suitability of the conjugate of formula (II) for obtaining high-affinity monoclonal antibodies against dihydroanatoxins, mice were immunized with the conjugates of formula (Ha) and (llb) in which P is BSA (immunizing conjugates BSA_DA-1 and BSA_DA-2), and cell fusions were carried out to generate hybridomas. Six cell lines (hybridomas) producing high-affinity monoclonal antibodies against dihydroanatoxins were obtained from the conjugate of formula (lia) and another three cell lines from the conjugate of formula (llb).The monoclonal antibodies produced from these hybridomas have been designated, for the purposes of the present invention and the examples included herein, as mAb lla#17, Ha#22, lla#23, Ha#122, lla#129 and lla#145 [monoclonal antibodies obtained from the conjugate of formula (Ha), the BSA_DA-1 conjugate], and mAb Hb#116, llb#130 and Hb#132 [monoclonal antibodies obtained from the conjugate of formula (llb), the BSA_DA-2 conjugate].

[0183] 6.2. Determination of the affinity of monoclonal antibodies

[0184] Once the nine monoclonal antibodies obtained were purified by immunoaffinity chromatography, their affinity for dihydroanatoxin-a and dihydrohomoanatoxin-a was determined by direct competitive ELISA, using as a competitor the enzyme tracer of a labeled derivative of formula (lllb) or (lile), in which Q is HRP (HRP_DA~2 or HRP_DA~3 conjugates). Each monoclonal antibody was tested at two concentrations (100 and 1000 ng / mL) against different tracer concentrations (10, 30 and 100 ng / mL). The IC50 values ​​for dihydroanatoxin-a and dihydrohomoanatoxin-a of each monoclonal antibody are shown in Table 3 and correspond to the optimal combination, i.e., the concentration of immunoreagents that generated the calibration curves with a lower IC50 value for dihydroanatoxin-a and dihydrohomoanatoxin-a, and therefore a higher affinity.In this assay format, the best antibodies showed IC50 values ​​for dihydroanatoxin-a and dihydrohomoanatoxin-a below 1 nM, with the highest affinity antibodies produced towards both dihydroanatoxins being the monoclonal antibodies Ha#17 (IC50 = 0.8 nM for dihydroanatoxin-a and IC50 = 0.2 nM for dihydrohomoanatoxin-a) and Ha#145 (IC50 = 0.7 nM for dihydroanatoxin-a and IC50 = 0.4 nM for dihydrohomoanatoxin-a).

[0185] TABLE 3. Results of the assays in direct competitive ELISA format using as competitor the enzyme tracer of a labeled derivative of formula (lllb) or formula (lile) (n = 3).

[0186] These results again demonstrate that the conjugates of formula (II) of this invention are suitable for obtaining monoclonal antibodies with very high affinity against dihydroanatoxin-a and dihydrohomoanatoxin-a. Furthermore, these results demonstrate that the conjugates of formula (III) of this invention are highly suitable for implementation in various immunoanalytical platforms that allow the determination or quantification of dihydroanatoxins at the most demanding sensitivity levels.

[0187] 6.3. Determination of the specificity of monoclonal antibodies Most antibodies are highly specific for dihydroanatoxin-a and dihydrohomoanatoxin-a. When comparing the IC50 values ​​obtained for dihydroanatoxin-a and dihydrohomoanatoxin-a, using competitive ELISA in indirect format with homologous conjugate - that is, the labeled derivative of formula (lia) or (llb), in which P is OVA (conjugates OVA_DA-1 and OVA_DA-2), for the evaluation of the antibodies generated from the conjugates of formula (Ha) or (llb), in which P is BSA (conjugates BSA_DA-1 and BSA_DA-2) respectively - high cross-reactivity values ​​were obtained, even close to 100% in some cases, such as with antibody lla#23. In contrast, cross-reactivity with anatoxin-a and homoanatoxin-a was generally less than 10%, except with antibodies lla#22 and llb#116.This result again demonstrates that the conjugates of formula (II) that are the subject of this invention are suitable for obtaining monoclonal antibodies specific to dihydroanatoxins.

[0188] Table 4. Cross-reactivity of monoclonal antibodies by indirect competitive ELISA with homologous conjugate. (a)

Claims

CLAIMS A compound of formula (I): where T is Rl, or its salts L is a linear hydrocarbon chain of 1 to 10 carbon atoms; Z is a functional group selected from: m is a number with a value between 1 and 100; X is selected from P and Q, such that P is a peptide or polypeptide of non-enzymatic nature, natural or synthetic, with a molecular weight greater than 2000 Daltons and Q is a detectable non-isotopic marker.

2. A compound according to claim 1, wherein L is a linear hydrocarbon chain of 1 to 5 carbon atoms.

3. A compound according to claim 1 or 2, wherein Z is selected from the group consisting of -~(C=O)NH-~, -CH2SCH-, and, more preferably, Z is -(C=O)NH-, 4. A compound according to any one of the preceding claims, wherein the peptide or polypeptide P is attached to a support.

5. A compound according to any one of the preceding claims, wherein m has a value between 1 and 25.

6. A compound according to any one of the preceding claims, which is a conjugate of a dihydroanatoxin-a analogue of general formula (II), where T, L, Z, P and m have the same meanings given above in one of claims 1 to 5.

7. A compound according to claim 6, wherein P is selected from the group consisting of albumin, thyroglobulin and hemocyanin, preferably P is albumin, more preferably egg albumin or serum albumin.

8. A compound according to any one of the preceding claims 6 or 7, which is a conjugate of formula (Ha) where P and m are as defined in claim 6 or 7.

9. A compound according to any one of the preceding claims 6 or 7, which is a conjugate of formula (llb) where P and m are as defined in claim 6 or 7.

10. A compound according to any one of the preceding claims 6 or 7, which is a conjugate of formula (lie) where P and m are as defined in claim 6 or 7 11. A compound according to any one of the preceding claims 1 to 5, which is a marked derivative of formula (HI) [TLZ] m -Q (III) wherein T, L, Z and m have the same meaning as defined above in one of claims 1 to 5 and Q is a detectable non-isotopic marker.

12. A compound according to the preceding claim, wherein Q is selected from a non-isotopic detector, marker or tracer chemical molecule, preferably selected from: an enzyme, biotin, a luminescent compound, a fluorophore, preferably a marker coupled to an indirect detection system, more preferably Q is selected from the group consisting of biotin, fluorescein, a cyanine fluorophore, a rhodamine fluorophore, a coumarin fluorophore, a ruthenium bipyryl, luciferin, an acridinium ester, quantum nanoparticles, and colloidal gold, carbon or latex nanoparticles.

13. A compound according to claim 11 or 12, of formula (Illa) where Q is selected from the group consisting of peroxidase, phosphatase, oxidase, biotin, fluorescein, and colloidal gold, carbon, or latex nanoparticles, and m is a value selected from 1 to 10.

14. A compound according to claim 11 or 12, of formula (lllb) where Q and m are as defined in claim 13.

15. A compound according to claim 11 or 12, of formula (lile) where Q and m are as defined in claim 13.

16. An antibody obtained by using a compound defined in any one of claims 1 to 15 that recognizes dihydroanatoxin-a and / or dihydrohomoanatoxin-a.

17. An antibody obtained by using a compound defined in any one of claims 1 to 15 that recognizes Rl as part of a compound of formula (I).

18. The antibody according to claim 16 or 17, wherein the antibody is selected from monoclonal, polyclonal and recombinant.

19. Use of a compound defined in any one of claims 1 to 15 for obtaining an antibody.

20. Use of an antibody defined in any one of claims 16 to 18, comprising contacting a sample containing dihydroanatoxin-a and / or dihydrohomoanatoxin-a with said antibody for in vitro analysis of dihydroanatoxin-a and / or dihydrohomoanatoxin-a.

21. Use according to claim 20 comprising: a) contacting a sample with the antibody defined in any one of claims 16 to 18; b) incubating the sample and the antibody of step (a) for a period of time suitable for an immunochemical reaction to take place; and c) determining the existence of an immunochemical reaction after incubation of step (b).

22. Use of a compound of formula (I), (II) or (III) defined in any one of claims 1 to 15 for the in vitro analysis of dihydroanatoxin-a and / or dihydrohomoanatoxin-a.

23. Use according to claim 22 comprising: a) contacting a sample with an anti-dihydroanatoxin-a and / or anti-dihydrohomoanatoxin-a antibody with the compound defined in any one of claims 1 to 15; b) incubating the sample, the antibody and the compound of step (a) for a period of time suitable for an immunochemical reaction to take place; and c) determining the existence of an immunochemical reaction after incubation in step (b), 24. Use according to claim 23, wherein the determination of the immunochemical reaction in step (c) is performed by a competitive assay, using as a competitor a labeled compound or derivative defined in any one of claims 1 to 15.

25. Use of the antibody defined in any one of claims 16 to 18 for the purification and / or concentration of dihydroanatoxin-a and / or dihydrohomoanatoxin-a from a sample.

26. Use according to claim 25 wherein the purification and / or concentration is carried out by affinity chromatography comprising the following steps: a) immobilizing at least one antibody described in any of claims 16 to 18 on a support; b) passing the sample through said support so that it retains the dihydroanatoxin-a and / or dihydrohomoanatoxin-a present in said sample; and c) eluting the dihydroanatoxin-a and / or dihydrohomoanatoxin-a retained on the support.

27. A kit for the detection and / or determination of dihydroanatoxin-a and / or dihydrohomoanatoxin-a comprising at least one antibody as described in any of claims 16 to 18.

28. The Kit according to the preceding claim comprising a compound described in any of claims 1 to 15.

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