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Immunoassay

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u oassay

● 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 - kno n since the end of the !"th ct#-precipitation in gel- Agglutination

- turbidimetry assaysgradually developed until their potential has fully been appreciated since !"$% hen

higher sensitivity as achieved by labeling one of the components

Immunoassay

● simple● precise● sensitive (nano- and picomolar range)● high throughput measurementof more substances than any other analytical techni&ue

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ImmunoassayAll immunoassay require the same key reagents

● 'ne or more antibodies raised against epitopes believed to be specific tothe analyte in &uestion● A label (tracer) producing a measurable signal● alibrators in a fluid (matrix) similar to the sample

Antibodyhe antibody used in immunoassay is usually of the IgG class

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Immunoassay

Conjugated haptens● *maller molecules (called haptens ) are either eakly or not not at all immunogenic● o produce the antibody it is necessary to couple them to an immunogenic carrier(e+g+ *A, .H+++)he optimal molar ratio (excess may range from !% #! to /% #!) is important for

production of good antisera

Natural Antigen*ubstance that naturally elicit immune response0sually a larger molecule (over !% k1) ith severalepitopes (antigenic determinants)

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Immunoassay

Conjugated haptens● *maller molecules (called haptens ) are either eakly or not not at all immunogenic● o produce the antibody it is necessary to couple them to an immunogenic carrier(e+g+ *A, .H+++)he optimal molar ratio (excess may range from !% #! to /% #!) is important for

production of good antisera

Natural Antigen*ubstance that naturally elicit immune response0sually a larger molecule (over !% k1) ith severalepitopes (antigenic determinants)

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E I!A "En#yme$ inked Immuno!orbent Assay%& 'ypes andclassification

Analyte( antigen hapten "small molecule% one epitopemultivalent antigen) many epitopes

antibody

abelling( *irectIndirect

+esponse( Competitive assayNon competitive !andwich assay

Antigen immobili#edAntigen labelled

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E I!A "En#yme$ inked Immuno!orbent Assay%& 'ypes andclassification

*irect competitive E I!A

*23-H45

sulfonamida

anticuerpoanti-*23

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E I!A "En#yme$ inked Immuno!orbent Assay%& 'ypes andclassification

Indirect competitive E I!A

sulfonamida

anticuerpoanti-*23

*23- *A

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S P

S P

- I 6%

- .'1

- .inear range

- .'7

E I!A "En#yme$ inked Immuno!orbent Assay%& 'ypes andclassification

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E I!A "En#yme$ inked Immuno!orbent Assay%& 'ypes andclassification

Noncompetitive sandwich assay

Salmonella

anticuerpoanti-*almonella-H45

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E I!A "En#yme$ inked Immuno!orbent Assay%& 'ypes andclassification

Noncompetitive sandwich assay

Salmonella

anticuerposecundario-H45

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8lisa ompetitivo

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!ignal amplification

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