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Applications of Molecular Imprinting Technology to Immunoassays

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

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Applications of Molecular Imprinting Technology to Immunoassays

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  1. Applications of Molecular Imprinting Technology to Immunoassays 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. • Ehrlich’s 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. OH NH3+ N H 2 C l N O O O- O- Fundamentals of antigen/antibody interaction CH2-CH2-CH2-CH3

  7. O O H H C C N N H H 3 3 N N N N O O N N CH3 CH3 Molecular imprinting (Step 1) Methacrylic acid + Porogen

  8. O O H H C C N N H H 3 3 N N N N O O N N CH3 CH3 Molecular imprinting (Step 2)

  9. O O H H C C N N H H 3 3 N N N N O O N N CH3 CH3 Molecular imprinting (Step 3) Cross-linking monomer Initiating reagent

  10. Molecular imprinting (Step 4)

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

  12. Immunoassays using MIPs • Therapeutic Drugs: Theophylline, Diazepam, Morphine, Propranolol, Yohimbine (2-adrenoceptor antagonist) • Hormones: Cortisol, Corticosterone • Neuropeptides: Leu5-enkephalin • Other: Atrazine, Methyl--glucoside

  13. Aptamers Target 1014-1015 random sequences Oligonucleotide-Target complex Unbound oligonucleotides + Target Aptamer candidates PCR New oligonucleotide library

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