Applications of Molecular Imprinting Technology to Immunoassays

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.
• 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
CH2-CH2-CH2-CH3
7
Molecular imprinting (Step 1)
Methacrylic acid Porogen
8
Molecular imprinting (Step 2)
9
Molecular imprinting (Step 3)
Cross-linking monomer Initiating reagent
10
Molecular imprinting (Step 4)
11
Comparison of MIPs and antibodies
Antibodies
MIPs

• In vivo preparation
• Limited stability
• Variable specificity
• General applicability

• In vitro preparation
• Unlimited stability
• Predictable specificity
• Limited applicability

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