IVD Technology Magazine | IVDT Article Index

Originally published July 1996

Artificial antibodies

Gary P. Henricksen and Mark T. Martin

Some researchers have tried to develop artificial molecular recognition systems to improve on the features of antibodies. These molecularly imprinted polymers show their potential for diagnostic assays.

Molecular imprinting is becoming increasingly recognized as a technique for the ready preparation of polymeric materials containing recognition sites of predetermined specificity. The technique of molecular imprinting allows the formation of specific recognition and catalytic sites in macromolecules by the use of templates. Molecularly imprinted polymers (MIPs) have been used in an increasing number of applications. Several useful reviews of the molecular imprinting field have recently been published.^1­3 Of special interest to the developers of diagnostic assays is the potential use of MIPs in sample preparation as antibody or receptor binding site mimics in recognition and assay systems, and as recognition elements in biosensors.

History of Molecular Imprinting

The concept of molecular imprinting has its roots in Linus Pauling's early theory of the formation of antibodies. Pauling suggested that antibodies were formed when serum proteins assembled around template antigen molecules. The assembled antibodies were thought to have specificity-endowing binding pockets complementary in shape to the antigens. Furthermore, strong antibody-antigen binding energy would result from multiple noncovalent binding interactions including hydrogen bonds, ionic bonds, and van der Waals forces. This theory led to the hypothesis by Pauling and Campbell that artificial antibodies could be assembled using these basic principles.⁴ Although Pauling's theory of antibody formation was later disproved, several groups subsequently tried to apply it to synthetic systems. Pauling's student, Frank Dickey, attempted to form specific absorbents using silica.⁵ In the 1970s Wulff, at the University of Düsseldorf (Germany), formed covalent bonds between a monomer and the template molecule, followed by polymerization and template cleavage to yield a specific binding site. This method is limited by the synthetic necessities of first preparing a monomer-template molecule conjugate, and later chemically cleaving the template molecule from the polymer.⁶

Pauling's original concept was finally applied to the synthesis of artificial antibodies with the development of molecular imprinting by Klaus Mosbach's group at the University of Lund (Sweden).⁷ Mosbach's approach of preassembling a noncovalently associated monomer-template complex in solution prior to polymer formation was the breakthrough that enabled molecular imprinting to be used in a variety of applications. Mosbach's group continues to lead in new developments including studies on various polymer systems, classes of template molecules, aqueous imprinting systems, and novel physical formats, and has extended the potential usefulness of molecular imprinting. Imprinting has now reached a high level of sophistication, and patent coverage in the field is extensive.

MIPs are achieving commercialization through a collaboration between Mosbach and IGEN, Inc. (Gaithersburg, MD). IGEN's Separations Business Group is researching new applications, developing molecular-imprinted products, and offering molecular imprinting services for use by diagnostic and drug manufacturers. IGEN holds the rights to numerous patents and patent applications in molecular imprinting.

Making an Imprint

The concept of molecular imprinting is shown in Figure 1. To make the imprinted polymer, the molecule to be imprinted is dissolved in an organic solvent, such as chloroform, with a functional monomer, a cross-linking monomer, and a polymerization initiator. The functional monomer is chosen to have a chemical functional group that will interact and preassociate with the imprint molecule (Figure 1A­B). Ionic, hydrogen bonds, ¼-¼, hydrophobic, metal coordination, and covalent bond interactions are typical. Functional monomers that have been used for noncovalent interactions are shown in Table I. Following preassociation, polymerization occurs by UV irradiation or mild heating (Figure 1C). Once the solid polymer has formed, it is ground in a mortar and pestle and sieved to obtain a desired size, and the print molecule is extracted by incubation in a solvent capable of disrupting the specific interactions between the imprint molecule and the polymer (Figure 1D). This extraction step often involves including an acid or base in the solvent. What remains are rigid stable polymer particles that have pockets complementary in shape and electron density to the imprint molecule (Figure 1E). Shape complementarity results in high specificity while multiple interactions between the polymer and individual imprint molecules yield high affinity. (Figures and tables not yet available on-line.)

Several advantages make noncovalent imprinting the usual choice when a new method is being designed. For covalent imprinting, chemical modification of the print molecule with, for example, a vinyl group is necessary before forming the imprint. A wide range of monomers are available for noncovalent imprinting. In some cases the monomers can be combined to increase the strength of binding. In noncovalent interactions, association and dissociation of the imprint molecule to the imprinted polymer occur by the imprint molecule simply diffusing in and out of the complementary sites.

Recent developments in the formation of MIPs include imprinting of beaded polymers⁸ or silica resins³ and surface imprinting of membranes,⁹ which open additional methods of use, especially in diagnostic analysis.

Benefits and Current Limitations of Molecular Imprinting

Although molecular imprinting has some limitations, MIPs provide a combination of mechanical and chemical robustness with highly selective molecular recognition. They can be stored in the dry state at ambient temperatures for several years without loss of recognition capabilities, and are inexpensive, simple, and easily prepared. Generation of molecular imprints does not involve the use of laboratory animals or any material of biological origin other than the imprint molecule. MIPs are much more resistant to matrix effects than are biological antibodies. Highly lipophilic analytes can be assayed in organic extraction solvents using MIPs, which are formed in those solvents (Table II). Also, imprinted polymers can be made against print molecules that are too toxic for immunization in animals to raise antibodies. MIPs can also be made against molecules that are difficult to raise antibodies against--for example, short peptides. The preparation of antibodies against small organic recognition elements (haptens) requires hapten conjugation to a carrier protein before immunization. Preparing a molecular imprint avoids the need for derivatization of haptens.

Molecular imprints have been demonstrated against many classes of molecules. These include drugs,¹⁰ hormones,¹¹ pesticides,^2,12 proteins,¹³ amino acids,¹⁴ peptides,¹⁵ carbohydrates,¹⁶ coenzymes,¹⁷ nucleotides,¹⁸ nucleotide bases,¹⁹ steroids,²⁰ dyes,²¹ and metal ions.²² Some examples of interest for developers of in vitro diagnostics are shown in Table III.

Because noncovalent interactions are strongly dependent on the polarity of the solvent, the best imprints are made in organic solvents such as chloroform or toluene. Subsequent specific recognition of the imprint molecule by the imprint polymer is strongest under conditions that most closely resemble the cocktail used for polymer synthesis. If the MIP is transferred to aqueous solution, binding strength can be reduced significantly. Mosbach and others are currently working to improve aqueous imprints. As with polyclonal antibodies, the individual imprint cavities have varying degrees of selectivity. The distribution is usually around an average figure of high selectivity but with some sites of low selectivity. In chromatography, the low-selectivity sites do not influence the separation as long as the column is not overloaded with sample. In an assay format, specificity depends on the high- selectivity sites.

Examples of Molecular Imprints

MIPs have the potential for use in assay formats in a manner similar to the use of antibody-conjugated microspheres. For example, a radiolabeled ligand-binding assay, called the molecularly imprinted sorbent assay (MIA), was reported by Mosbach's group.¹⁰ Molecular imprints were made against two chemically unrelated drugs, theophylline and diazepam. Theophylline is a bronchodilating drug commonly used in the prevention and treatment of asthma. The assay accurately measured drug levels in human serum, with results comparable to those obtained using the established enzyme-multiplied immunoassay (EMIT). Specifically, the MIAs for theophylline and diazepam were linear over the ranges of 14­224 and 0.44­28 µm with detection limits of 3.6 and 0.2 µm, respectively, which are both satisfactory for therapeutic monitoring of the drugs. The specificity of the imprints was tested by determination of the cross-reactivity between major metabolites and of structurally related drugs. The imprints were shown to be highly selective, similar to those reported using commercial antibody­ based immunoassays (Table IV).

MIPs for leu-enkephalin and morphine have been made.¹¹ Because it is a closely related structure, codeine is a difficult cross-reactant for antimorphine antibodies. Cross-reactivity of the MIP with codeine is less than with most of the antimorphine antibodies (including monoclonal antibodies) reported to date. Although prepared in organic solvents, these MIPs were also efficient in aqueous solution with a performance sufficient for screening assays for drugs of abuse.

A molecularly imprinted membrane was recently developed that specifically bound theophylline over other purine bases.⁹ Such artificial affinity membranes should have applications for rapid analytical method development or for sample preparation methods. An additional benefit could be the combination of sample preparation and detection on the membrane.

MIPs that specifically bind particular metal ions (such as calcium and magnesium) have been produced using polymerizable metal-binding complexes. These have been used to make ion- selective electrodes.³ Imprints against pesticides and industrial pollutants have been reported by several groups.^2,12,16 Since many of these compounds are only soluble in organic solution, a solvent extraction step is necessary before the assay. Because MIPs can act as artificial antibodies in organic solution, they provide the possibility for developing formats similar to immunoassays, simplifying the current procedures.

Assays using molecular imprints with dissociation constants in the order of 10^­7­10^­8 M for cortisol and corticosterone were recently demonstrated.¹³ When the imprints were used in water systems, the interactions were disrupted because of the strong hydrogen bonding capacity of the water molecules, and the selectivity was severely reduced. Although many antibody-based steroid analytical methods have been developed for immediate use in aqueous environments such as plasma or urinary samples, several techniques use an extraction step for the steroids with organic solvents to avoid interferences with native binding proteins. In this perspective, anticorticosteroid MIPs may be an alternative for analytical applications, either by direct analysis or as a sample preparation step.

In some studies by Mosbach's group, target molecules have been chosen against which it is difficult or expensive to prepare natural antibodies. One example involves imprints against various macrolide antibiotics, such as erythromycin. Another example is imprints against immunosuppressants, such as cyclosporin.

Besides the many studies on resolution of the enantiomers of amino acids and sugars, molecular imprints against ß-blockers²⁴ and antiinflammatories²⁵ have also been reported.

Future Directions

The development of sample preparation methods and assays for small organic molecules using molecular imprints is expected to be completed in the near future. The preparation of MIPs for the isolation and detection of biomolecules is under development. Preparing an artificial antibody by molecular imprinting may be far more efficient in time and effort than is raising and producing a new antibody when none exists. Still needed are improvement of the binding strength of molecular imprints in aqueous solution and further development of surface imprinting and beaded imprints for higher-molecular-weight biopolymers such as proteins.

Molecular imprints have been included in several experimental biosensor designs.^1,3 Special advantages of MIPs in this application is their long-term stability at ambient conditions and in harsh environments. However, one obstacle to such biosensors is the current lack of a suitable interface between the MIP and the sensor element. Further developments in molecular-imprinted membranes may help in this area.

The creation of artificial antibodies through the combination of polymer chemistry and biochemistry is an ambitious goal, and one that promises considerable benefits. After many years of experimental development, MIPs are examples of progress toward this goal.

References

1. Mosbach K, and Ramstrom O, "The Emerging Technique of Molecular Imprinting and Its Future Impact on Biotechnology," Bio/Technol, 14:163­170, 1996.

2. Muldoon M, and Stanker L, "Plastic Antibodies: Molecularly-Imprinted Polymers," Chem Indust, pp 204­207, 1996.

3. Wulff G, "Molecular Imprinting in Cross-Linking Materials with the Aid of Molecular Templates--A Way Towards Artificial Antibodies," Angew. Chem Indust Ed Engl, 19:9­14, 1995.

4. Pauling L, and Campbell D, "The Manufacture of Antibodies in Vitro," J Exper Med, 76:211­220, 1942.

5. Dickey FH, "Specific Adsorption," J Phys Chem, 59:695­707, 1955.

6. Wulff G, Sarhan A, and Zabrocki K, "Enzyme-Analogue Built Polymers and Their Use for the Resolution of Racemates," Tetrahedron Lett, 44:4329­4332, 1973.

7. Andersson L, Sellergren G, and Mosbach K, "Imprinting of Amino Acid Derivatives in Macroporous Polymers," Tetrahedron Lett, 25:5211­5214, 1984.

8. Mayes A, and Mosbach K, "Molecularly Imprinted Beads: Suspension Polymerization Using a Liquid Perfluorocarbon as the Dispersing Phase," J Molec Recogn, in press.

9. Kobayashi T, Wang HY, and Fujii N, "Molecular Imprinting of Theophylline in Acrylonitrile-Acrylic Acid Copolymer Membrane," Chem Lett, 10:927­ 928, 1995.

10. Vlatakis G, Andersson L, Müller R, et al., "Drug Assay Using Antibody Mimics Made by Molecular Imprinting," Nature, 361:645­647, 1993.

11. Andersson L, Müller R, Vlatakis G, et al., "Mimics of the Binding Sites of Opioid Receptors Obtained by Molecular Imprinting of Enkephalin and Morphine," Proc Natl Acad Sci, 92:4788­4792, 1995.

12. Siemann M, Andersson L, and Mosbach K, "Selective Recognition of the Herbicide Atrazine by Noncovalent Molecularly Imprinted Polymers," J Ag Food Chem, 44: 141­145, 1996.

13. Kempe M, Glad M, and Mosbach K, "An Approach towards Surface Imprinting Using the Enzyme Ribonuclease A," J Molec Recogn, 8:35­39, 1995.

14. Kriz D, Ramström O, Svensson A, et al., "Introducing Biomimetic Sensors Based on Molecularly Imprinted Polymers as Recognition Elements," Anal Chem, 67:2142­2144, 1995.

15. Ramström O, Nicholls IA, and Mosbach K, "Synthetic Peptide Receptor Mimics: Highly Stereoselective Recognition in Nonconvalent Molecularly Imprinted Polymers, Tetrahedron: Assymetry, 5(4):649­656, 1994.

16. Mayes AG, Andersson LI, and Mosbach K, "Sugar Binding Polymers Showing High Anomeric and Epimeric Discrimination by Noncovalent Molecular Imprinting," Anal Biochem, 222:483­488, 1994.

17. Andersson LI, and Mosbach K, "Molecular Imprinting of the Coenzymer Substrate Analogue N-Pyridoxyl-L-Phenylalaninanalide," Makromolec Chem, Rapid Commun, 10:491­495, 1989.

18. Norrlöw O, Mänsson MO, and Mosbach K, "Improved Chromatography: Prearranged Distances between Boronate Groups by the Molecular Imprinting Approach," J Chromatog, 396:374­377, 1987.

19. Shea KJ, Spivak DA, and Sellergren B, "Polymer Complements to Nucleotide Bases: Selective Binding of Adenine Derivatives to Imprinted Polymers," J Am Chem Soc, 115:3368­3369, 1993.

20. Ramström O, Ye L, and Mosbach K, "Artificial Antibodies to Corticosteroids Prepared by Molecular Imprinting," Chem Biol, in press.

21. Norrlöw O, Glad M, and Mosbach K, "Acrylic Polymer Preparations Containing Recognition Sites Obtained by Imprinting with Substrates," J Chromatog, 299:29­ 41, 1984.

22. Rosatzin T, Andersson LI, and Mosbach K, "Preparation of Ca²⁺ Selective Sorbents by Molecular Imprinting Using Polymerisable Ionophores," J Chem Soc, Perkin Trans, 2:1261­1265, 1990.

23. Andersson L, Nicholls I, and Mosbach K, "Antibody Mimics Obtained by Noncovalent Molecular Imprinting," in Immunoanalysis of Agrochemicals, Nelson J, Karu A, and Wong R (eds), Washington, DC, American Chemical Society, pp 89­96, 1995.

24. Fisher L, Müller R, Ekberg B, et al., "Direct Enantioseparation of ß-Adrenergic Blockers Using a Chiral Stationary Phase Prepared by Molecular Imprinting," J Am Chem Soc, 113:9358­9360, 1991.

25. Kempe M, and Mosbach K, "Direct Resolution of Naproxen on a Non-Covalently Molecularly Imprinted Chiral Stationary Phase," J Chromatog A, 664:276­279, 1994.

Gary P. Henricksen is vice president, Separations Business Group, and Mark T. Martin, PhD, is director, Discovery Research Group, IGEN, Inc. (Gaithersburg, MD).