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Applications of Molecular Imprinting Technology to ... - Bertholf

VIEWS: 6 PAGES: 13

									 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


                   • 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.
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.
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.
The birth of immunoassay


             • Rosalyn Yalow (1921-) and
               Solomon Berson described
               the first radioimmunoassay
               in 1957.
Fundamentals of antigen/antibody interaction



       CH2-CH2-CH2-CH3
                              NH 2


                         Cl
                     N
Molecular imprinting (Step 1)




                                        Methacrylic acid
             O
   H3C
                                          + Porogen
                   NH
         N                        O
                        H3C              NH
    O        N     N          N

             CH3         O        N      N


                                  CH3
Molecular imprinting (Step 2)




             O
   H3C             NH
         N                        O
                        H3C             NH
    O        N     N          N

             CH3         O        N     N


                                  CH3
Molecular imprinting (Step 3)

                          Cross-linking monomer
                          Initiating reagent



             O
   H3C             NH
         N                        O
                        H3C             NH
    O        N     N          N

             CH3         O        N     N


                                  CH3
Molecular imprinting (Step 4)
    Comparison of MIPs and antibodies


   Antibodies                    MIPs
• In vivo preparation     • In vitro preparation

• Limited stability       • Unlimited stability

• Variable specificity    • Predictable specificity

• General applicability   • Limited applicability
           Immunoassays using MIPs


• Therapeutic Drugs: Theophylline, Diazepam, Morphine,
  Propranolol, Yohimbine (2-adrenoceptor antagonist)

• Hormones: Cortisol, Corticosterone

• Neuropeptides: Leu5-enkephalin

• Other: Atrazine, Methyl--glucoside
                        Aptamers


1014-1015 random sequences             Target



                Oligonucleotide-Target complex

                                       Unbound oligonucleotides
+ Target
                      Aptamer candidates
                                 PCR

                   New oligonucleotide library

								
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