applications of molecular imprinting technology to immunoassays roger l. bertholf, ph.d. associate...
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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 formationEarly 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.
• 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 formationThe “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.
• 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 imprintingHistory 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.
• 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 immunoassayThe birth of immunoassay
• Rosalyn Yalow (1921-) and Solomon Berson described the first radioimmunoassay in 1957.
• Rosalyn Yalow (1921-) and Solomon Berson described the first radioimmunoassay in 1957.
Fundamentals of antigen/antibody interactionFundamentals of antigen/antibody interaction
O
O-
O
O-
NH 3
+
CH2-CH2-CH2-CH3
OH
N
NH2
Cl
Molecular imprinting (Step 1)Molecular imprinting (Step 1)
N
NO N
NH
O
H3C
CH3
N
NO N
NH
O
H3C
CH3
Methacrylic acid+ Porogen
Molecular imprinting (Step 2)Molecular imprinting (Step 2)
N
NO N
NH
O
H3C
CH3
N
NO N
NH
O
H3C
CH3
Molecular imprinting (Step 3)Molecular imprinting (Step 3)
N
NO N
NH
O
H3C
CH3
N
NO N
NH
O
H3C
CH3
Cross-linking monomerInitiating reagent
Molecular imprinting (Step 4)Molecular imprinting (Step 4)
Comparison of MIPs and antibodiesComparison 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
• In vitro preparation
• Unlimited stability
• Predictable specificity
• Limited applicability
Antibodies MIPs
Immunoassays using MIPsImmunoassays using MIPs
• Therapeutic Drugs: Theophylline, Diazepam, Morphine, Propranolol, Yohimbine (2-adrenoceptor antagonist)
• Hormones: Cortisol, Corticosterone
• Neuropeptides: Leu5-enkephalin
• Other: Atrazine, Methyl--glucoside
• Therapeutic Drugs: Theophylline, Diazepam, Morphine, Propranolol, Yohimbine (2-adrenoceptor antagonist)
• Hormones: Cortisol, Corticosterone
• Neuropeptides: Leu5-enkephalin
• Other: Atrazine, Methyl--glucoside
AptamersAptamers
1014-1015 random sequences Target
Oligonucleotide-Target complex
Unbound oligonucleotides
Aptamer candidates
PCR
New oligonucleotide library
+ Target