1. Applications of Molecular Imprinting Technology to Immunoassays Roger L. Bertholf, Ph.D. Associate Professor of Pathology University of Florida Health Science Center/Jacksonville Roger L. Bertholf, Ph.D. Associate Professor of Pathology University of Florida Health Science Center/Jacksonville

2. Early theories of antibody formation Paul Ehrlich (1854-1915) proposed that antigen combined with pre-existing side-chains on cell surfaces. Ehrlichs theory was the basis for the genetic theory of antibody specificity.

3. The Template theory of antibody formation Karl Landsteiner (1868-1943) was most famous for his discovery of the A/B/O blood groups and the Rh factor. Established that antigenic specificity was based on recognition of specific molecular structures; he called these haptens; formed the basis for the template theory of antibody formation.

4. History of molecular imprinting Linus Pauling (1901-1994) first suggested the possibility of artificial antibodies in 1940 Imparted antigen specificity on native globulin by denaturation and incubation with antigen.

5. The birth of immunoassay Rosalyn Yalow (1921-) and Solomon Berson described the first radioimmunoassay in 1957.

6. Fundamentals of antigen/antibody interaction O O-O- O O-O- NH 3 + CH 2 -CH 2 -CH 2 -CH 3 OH N NH 2 Cl

7. Molecular imprinting (Step 1) N NO N NH O H 3 C CH 3 N NO N NH O H 3 C Methacrylic acid + Porogen

8. Molecular imprinting (Step 2) N NO N NH O H 3 C CH 3 N NO N NH O H 3 C

9. Molecular imprinting (Step 3) N NO N NH O H 3 C CH 3 N NO N NH O H 3 C Cross-linking monomer Initiating reagent

10. Molecular imprinting (Step 4)

11. Comparison of MIPs and antibodies In vivo preparation Limited stability Variable specificity General applicability In vivo preparation Limited stability Variable specificity General applicability In vitro preparation Unlimited stability Predictable specificity Limited applicability AntibodiesMIPs

12. Immunoassays using MIPs Therapeutic Drugs: Theophylline, Diazepam, Morphine, Propranolol, Yohimbine ( 2 -adrenoceptor antagonist) Hormones: Cortisol, Corticosterone Neuropeptides: Leu 5 -enkephalin Other: Atrazine, Methyl- -glucoside Therapeutic Drugs: Theophylline, Diazepam, Morphine, Propranolol, Yohimbine ( 2 -adrenoceptor antagonist) Hormones: Cortisol, Corticosterone Neuropeptides: Leu 5 -enkephalin Other: Atrazine, Methyl- -glucoside

13. Aptamers 10 14 -10 15 random sequences Target Oligonucleotide-Target complex Unbound oligonucleotides Aptamer candidates PCR New oligonucleotide library + Target