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