Immunoassay

Immunoassay
Highly specific in vitro tests that use antigen-antibody reaction to detect extremely
low concentrations of a broad range of analytes in different types of samples
(clinical, environmental, food, etc)
Antigen-antibody reaction - known since the end of the 19th ct:
-precipitation in gel
-Agglutination
- turbidimetry assays
gradually developed until their potential has fully been appreciated since 1960 when
higher sensitivity was achieved by labeling one of the components
simple
precise
sensitive (nano- and picomolar range)
high throughput measurement
of more substances than any other analytical
technique

Immunoassay
All immunoassay require the same key reagents
One or more antibodies raised against epitopes believed to be specific to
the analyte in question
A label (tracer) producing a measurable signal
Calibrators in a fluid (matrix) similar to the sample

Antibody
The antibody used in immunoassay is usually of the IgG class

Immunoassay
Natural Antigen
Substance that naturally elicit immune response
Usually a larger molecule (over 10 kD) with several
epitopes (antigenic determinants)

Conjugated haptens
Smaller molecules (called haptens) are either weakly or not not at all immunogenic
To produce the antibody it is necessary to couple them to an immunogenic carrier
(e.g. BSA, KLH...)
The optimal molar ratio (excess may range from 10 :1 to 80 :1) is important for
production of good antisera

Immunoassay
Natural Antigen
Substance that naturally elicit immune response
Usually a larger molecule (over 10 kD) with several
epitopes (antigenic determinants)

Conjugated haptens
Smaller molecules (called haptens) are either weakly or not not at all immunogenic
To produce the antibody it is necessary to couple them to an immunogenic carrier
(e.g. BSA, KLH...)
The optimal molar ratio (excess may range from 10 :1 to 80 :1) is important for
production of good antisera

ELISA (Enzyme-Linked ImmunoSorbent Assay). Types and

classification
Analyte:

antigen hapten (small molecule) one epitope

multivalent antigen, many epitopes
antibody

Labelling:

Direct
Indirect

Response:

Competitive assay
Non competitive Sandwich assay

Antigen immobilized
Antigen labelled

ELISA (Enzyme-Linked ImmunoSorbent Assay). Types and

classification
Direct competitive ELISA

sulfonamida
SFM-HRP

anticuerpo
anti-SFM

ELISA (Enzyme-Linked ImmunoSorbent Assay). Types and

classification
Indirect competitive ELISA

anticuerpo
anti-SFM
sulfonamida

SFM-BSA

ELISA (Enzyme-Linked ImmunoSorbent Assay). Types and

classification
Direct competitive ELISA
(with antigen immobilized)
anticuerpo
anti-SFMHRP

sulfonamida

SFM-BSA

ELISA (Enzyme-Linked ImmunoSorbent Assay). Types and

classification

- LOD
- LOQ
- IC50
- Linear range

ELISA (Enzyme-Linked ImmunoSorbent Assay). Types and

classification
Noncompetitive sandwich assay

Salmonella
anticuerpo
anti-Salmonella-HRP

ELISA (Enzyme-Linked ImmunoSorbent Assay). Types and

classification
Noncompetitive sandwich assay

Salmonella
anticuerpo
secundario-HRP

Elisa Competitivo

Signal amplification