Patent Application: US-62838196-A

Abstract:
the invention relates to artificial antibodies that are prepared by molecular imprinting , where methacrylic acid , ethylene glycol dimethacrylate and a print molecule are combined to form an artificial antibody having spatially positioned binding sites dictated by the corticosteroid print molecule , and the antibodies can be used in separation and analytical procedures .

Description:
in the present invention molecularly imprinted polymers were prepared against cortisol and corticosterone compounds . the polymers are prepared from the copolymerization of a monomer that is negatively charged , such as methacrylic acid ( maa ) ch 2 ═ c ( ch 3 ) cooh or itaconic acid with cross - linking ethylene glycol dimethacrylate monomer , with azo - bisisobutyronitrile as the initiator and were obtained from merck ( darmstadt , frg ). other negatively charged monomers include , but are not limited to acrylic acid , maleic acid , vinylbenzoic acid and 2 - trifluoromethyl acrylic acid . although the preferred cross - linking monomer is ethylene glycol dimethacrylate , other cross - linking monomers may be used , such as , divinylbenzene and trimethylolpropane trimethacrylate ( trim ). scintillation liquid , ecoscint o , was from national diagnostics ( manville , n . j ., usa ). all solvents were of either hplc or analytical grade . the cortisol and corticosterone compounds include , but are not limited to cortisol , deoxycortisol , 11 - deoxycortisol , 21 - deoxycortisol , corticosterone , 21 - deoxycortisone , 11 - dehydrocorticosterone , cortexolone , prednisolone , substituted prednisolone and cortisone . cortisol , corticosterone , 21 - deoxycortisone , cortexolone , prednisolone and cortisone were obtained from signa chemical co . ( st . louis , mo ., usa ). [ 1 , 2 , 6 , 7 - 3 h ] cortisol ( specific activity 2 . 22 tbq / mmol ) and ( 1 , 2 , 6 , 7 - 3 h ] corticosterone ( specific activity 3 . 03 tbq / mmol ) were from amersham international plc . ( little chalfont , uk ). in addition to the corticosteroids identified above , print molecules based on other steroid hormones , such as the androgens , estrogens , progestins , and gonadotropin releasing hormones identified in basic and clinical pharmacology , 4th ed ., 1989 , pg . 696 - 700 , incorporated herein by reference , may be used . ligand specificity was assayed using a radio - immunoassay technique and the binding characteristics of the cortisol and corticosterone imprinted polymers were estimated and equilibrium constants were determined . the self - assembly imprinting protocol used in the present invention , where only non - covalent interactions are utilized in the formation and maintenance of the complexes between the functionally - active monomers and the print species , relies , to a large extent , upon the solvent that is used . in the present invention , more polar solvents had to be used due to the low solubility of the print species in non - polar solvents that are conventionally used for increasing the selectivity of the artificial recognition sites . such conventional solvents include dichloromethane and toluene . the preferred polar solvents of the present invention include two different porogens , tetrahydrofuran and acetone . other solvents may be used so long as they solubilize the steroid of interest and have the requisite polarity and could include chloroform , ethylacetate , isopropanol and acetonitrile . the concentrations of the print species ( molecules or compounds ) in the protocols used was too high for reaching full solubility and were modified . addition of functional monomer , i . e ., methacrylic acid , to adjust the ratio to preferably about 10 : 1 ( functional monomer to print molecule ) provided clear solutions . the clear solutions indicated the establishment of strong interactions between the functional monomer and the print molecule ( species ). the molecularly imprinted polymers ( mips ) were prepared according to table 1 below . the print molecule was dissolved in dry porogen , either tetrahydrofuran or acetone , together with the functional monomer , methacrylic acid . the cross - linking monomer , ethylene glycol dimethacrylate , and the initiating agent , azo - bisisobutyronitrile , were added and the solutions were chilled on an ice - bath and purged thoroughly with nitrogen for ten minutes . the degassed solution was photolytically polymerized under nitrogen atmosphere at 4 ° c . overnight by use of a standard laboratory uv - source at 366 nm ( camag , bubendorf , ch ). the resulting polymer was crushed , ground in a mechanical mortar ( retsch , haan , frg ) and wet - sieved ( 25 : μm , retsch ) with water . fine particles were removed through repeated sedimentation in acetone . the print species were extracted by extensive washing with a methanol / acetic acid solution ( 9 : 1 , v / v ), followed spectrophotometrically at 242 nm until no more print molecule could be detected . as shown in fig1 the print molecule ( cortisol ) is initially dissolved in the porogen ( tetrahydrofuran or acetone ) and allowed to form non - covalent complexes to the functional monomer ( methacrylic acid ). following addition of cross - linker ( ethylene glycol dimethacrylate ) and initiator ( azo - bisisobutryonitrile ), these complexes are arrested by polymerization . finally , the print molecule is extracted by washing and the molecularly imprinted polymer is ready for association / dissociation studies . a procedure similar to that shown in fig1 is followed for the compounds of fig3 — cortisone ; fig4 — 21 - deoxycortisol ; fig5 — corticosterone ; fig6 — 11 - deoxycortisol ; and fig7 — prednisolone . the capacity of the methacrylic acid / ethylene diglycol dimethacrylate ( maa / egda ) polymers was measured by saturation studies . radiolabelled ligand to an activity of 500 bq was added to polymer particles ranging from a concentration of 0 . 03 to 20 mg / ml in a total volume of 1 . 0 ml solvent in polypropylene micro - centrifuge tubes ( brand , wertheim , frg ). the binding was allowed to reach equilibrium at ambient temperature on a rocking table overnight . subsequently , the polymer particles were removed from the samples by centrifugation at 10 , 000 g for five minutes and 500 μl of the supernatant was added to 10 ml of scintillation cocktail in 20 ml scintillation vials ( national diagnostics , atlanta , ga ., usa ) and the radioactivity was measured using a model 2119 rackbeta β - radiation counter ( lkb wallac , solentuna , sweden ). the competition assays were performed in a similar way . non - radiolabelled ( cold ) ligand ranging from 0 . 01 to 250 μg was mixed with 1 . 0 mg of polymer particles in polypropylene microcentrifuge tubes . radiolabelled ( hot ) ligand to an activity of 500 bq was added and the volume was made up to 1 . 0 ml with solvent . the samples were allowed to reach equilibrium overnight at ambient temperature on a rocking table . the amount of bound ligand was estimated after centrifugation at 10 , 000 g for five minutes and measuring the radioactivity of 500 μl supernatant by addition of the latter to 10 ml of scintillation liquid and measuring the radioactivity using a β - radiation counter . the concentration of ligand capable of displacing 50 % of bound ligand ( ic 50 ) was calculated using the computer software package ebda / ligand ( elsevier - biosoft , amsterdam , nl ). the capacities of the molecularly imprinted polymers for the print species were investigated by saturation of the polymer with increasing amount of ligand . the assays were performed in several different solvents , but optimal binding performance were achieved with mixtures of tetrahydrofuran and n - heptane . in order to achieve a higher solubility of the ligands for further studies , a small amount of acetic acid was added to the solvent . the resulting imprinting performance , as measured by the saturation studies , revealed no difference between the imprinting porogens , tetrahydrofuran or acetone , but tetrahydrofuran was chosen as the best porogen because of a closer resemblance with the solvent used in the analysis system . the amount of polymer capable of binding 50 % of added radiolabelled print species was similar in the imprinted polymers , − 1 . 4 mg in the anti - cortisol polymers ( mip1 and mip2 ) and 2 . 0 mg in the anti - corticosterone polymer ( mip3 ). the corresponding values for the reference polymer ( ref1 ) were 6 . 3 mg and 7 . 0 mg , respectively . using a polymer concentration of 1 mg / ml , the bindings by the blank polymers were 10 - 16 % of the binding by the imprinted polymers and this concentration level was chosen for further experiments . the binding characteristics of the polymers are heterogeneous in nature , as reflected by the non - linear scatchard plots shown in fig8 and 9 . this “ polyclonal ” behavior is an unavoidable effect from the imprinting procedure , in which the weak , non - covalent , interactions between the template molecules and the functional monomers lead to the formation of gradually differing sites in the finished polymer . thus , sites are formed of which the binding strength ranges from reasonably high affinity , exerted by a small amount of binding sites , down to larger number of sites with low affinity . of the polymers studied , a two - site model can be readily employed to describe the binding of the templates to the polymers . the resulting figures of the dissociation constants and the corresponding binding site densities are displayed in table 2 below . these values are of the same order for the polymers , where the two - site model is somewhat more pronounced in the anti - cortisol polymer as reflected by a smaller number of sites with higher affinity and a larger density of sites with a lower affinity than the anti - corticosterone polymer . in comparison to natural antibodies these binding constants are lower , possibly resulting from the experimental conditions used . if a more sensitive assay method could be used , it would be feasible to measure the stronger binding affinities of the best sites . the selectivities of the artificial antibodies were estimated from the measures of 50 % displacement of radiolabelled template species from the polymers by non - radiolabelled displacing ligand ( ic 50 ). the experimental design is analogous to standard competitive immunoassays used , where unlabelled ligands compete with the radio - labelled ligand for admission and binding to the sites . dose - response curves obtained from experiments when unlabelled print species , cortisol or corticosterone , were used as competing ligands against mip1 and mip3 are displayed in fig1 and 11 . the resulting values from the competition assays are presented in table 3 together with the calculated cross - reactivities . from the structures of the various corticosteriods analyzed ( fig2 - 7 ), the structural implications of the recognition can be deduced . for the anti - cortisol polymer ( mip1 ) the removal of one hydroxyl functionality of the imprinted structure leads to a decrease in binding as reflected by the increased ic 50 - values . removal of the 11 -, 17 -, or 21 - oh groups , respectively , all reduce the binding by a factor of 11 - 25 , where the 21 - oh seems to be of higher importance for the recognition than the other hydroxyl groups . this can be viewed as the loss in hydrogen bonding between the hydroxyl groups of the corticosteroids and the carboxyl groups of the surrounding polymer will inevitably reduce the binding . by introduction of an additional unsaturation in the structure in the δ - 1 position , prednisolone , the binding is not as severely affected . the cross - reactivity is as high as 36 % compared to cortisol . this can be understood as a minor locking of the flexibility of the a - ring will not drastically affect the binding to the sites . a slight reduction in binding can be perceived since the prednisolone structure is unable to adapt to all of the configurations possible for cortisol . on the other hand , changing the 11 - oh group for a keto functionality as in cortisone , the binding is unprecendently reduced . one reason for this effect can be changes in hydrogen bonding capabilities of the cortisone as compared to the cortisol molecule . in the cortisol structure the hydroxyl group is able to act as both hydrogen donor and hydrogen acceptor , thereby stabilizing an interaction with the carboxyl functionality of the methacrylic acid residues of the polymer . the keto functionality of cortisone is unable to act as a hydrogen donor , leading to weaker interactions . another explanation of this effect may be the sterical constraints of the cortisone structure in comparison to cortisol . the planar keto functionality , as opposed to the hydroxyl pointing in the β - position , may lead to changes in ring structure . for the anti - corticosterone polymer ( mip3 ), introduction of an additional hydroxyl group in a 17 - α - position ( cortisol ) reduces the binding to the polymer by a factor of 10 . this is most probably due to stearic constraints of the cortisol molecule to fit into the more tightly formed site of corticosterone . although further changes from the basic corticosterone structure of the other ligands studied , which differ from corticosterone in two positions , lead to higher selectivity of the anti - corticosterone polymer in comparison to the anti - cortisol polymer , the tendencies of the structural implications for the recognition can be seen . similarly , as with the case of the anti - cortisol polymer , removal of one hydroxyl functionality from the template species , reduces the binding to the sites by a factor of 6 - 19 . also very similar to the anti - cortisol polymer is the reduction in binding resulting from the introduction of a double bond in the a - ring ( prednisolone ), where the binding is reduced by 17 %. the exchange of the 11 - β - oh group for the planar keto group reduces binding by an additional 39 %, much alike the binding by the anti - cortisol polymer . in comparison to commercially used antibodies and antibodies reported in literature ( table 4 ), the artificial antibodies prepared by molecular imprinting exhibit strong similarities . the cross - reactivities of the anti - cortisol antibodies are roughly in the same order as those obtained by the anti - cortisol polymer . normally , the naturally - raised antibodies are either subjected to some sort of screening process , or monoclonal , thus leading to an optimized performance . in the case of molecularly imprinted polymers , a range of binding sites are obtained , i . e ., the polymers can be perceived as “ polyclonal ” in appearance . this fact will inevitably reduce the binding selectivities . the sensitivities of the assays using the artificial antibodies of the present invention , lie in the order of 10 − 7 - 10 − 6 m for cortisol and corticosterone using the anti - cortisol and anti - corticosterone polymers of the present invention , respectively , as indicated in the dose response curves displayed in fig1 and 11 . this limitation is in part a consequence of the detection method used and may be further forced by finding a more sensitive analysis . although the foregoing invention has been described in some detail by way of illustration and example for purposes of clarity of understanding , it will be obvious to those skilled in the art that certain changes and modifications may be practiced without departing from the spirit and scope thereof as described in the specification and as defined in the appended claims . h . dugas , bioorganic chemistry . a chemical approach to enzyme action , 1989 . d . r . smith , chem . ind ., supramolecular chemistry , 1994 , pp . 14 - 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