elisa online version

7/23/2019 Elisa Online Version

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ELISA:

Enzyme-Linked Immunosorbent Assay

Advances in Drug Delivery

23rd of May 2011

Slides by Mathias Bader and Simon Loew


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ELISA: Enzyme-Linked Immunosorbent Assay

2

ELISA detects substances with antigenicproperties (mainly proteins

Based on en!ymatic color"reaction



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Basic principle of ELISA

#

En!yme is used to detect the binding o$ Antibody " Antigen

En!yme converts colorless substrate into colored product% indicating

the presence o$ Antibody " Antigen comple&

ELISA can be used to detect either presence o$ Antigens or Antibodies



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Direct and Indirect ELISA

'

Direct ELISA Indirect ELISA



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Direct ELISA



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)

Antibody is adsorbed

onto microtiter plate

Add serum to test $or

antigen

Speci$ic antigen binds i$

contained



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*

+emove test $luid and

unbound antigen

Binding o$ antigen is

strong enough to

withstand rinsing



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,

+emove test $luid and

unbound antigen

Binding o$ antigen is

strong enough to

withstand rinsing



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-Slides by Mathias Bader and Simon Loew


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./

E

E

E

E

E

E

Add en!yme labeled

antibody

E E

Labeled antibody bindsto antigens



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..

EE

EE

E

E

0ash sample to remove

unbound antibodies

E

E



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.2

EE

E


unbound antibodies

E

E

E

E

E



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.#

EE


unbound antibodies

E



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.'

Add substrate

EEE

Measure color change

1ositive egativ



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Direct and Indirect ELISA

.

Direct ELISA Indirect ELISA



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.)

Indirect ELISA

Antigen is adsorbed onto

microtiter plate


antibody



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.*


microtiter plate


antibody



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.,


microtiter plate


antibody

0ash sample



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.-


microtiter plate


antibody

0ash sample



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2/Slides by Mathias Bader and Simon Loew


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2.

E

E

E

E

E

E

E

E

Add secondary antibody

1rimary antibody binds

to secondary anitbody



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22

EEE

E

E

E

E

E


0ash unbound antibodies



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2#

EEE

E

E

E

E

E





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2'

EEE





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2

EEE

1ositive egativ

Add substrate

Measure color change



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Direct vs. Indirect ELISA

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3uic4 methodology 5 only one AB

Advantages o$ Indirect ELISA6

Immunoreactivity o$ primary

antibody is not a$$ected by labeling

7ross"reactivity o$ second AB

eliminated

0ide variety o$ secondary

antibodies available on the mar4et

Signal ampli$ication (several epitopes

EEE

Advantages o$ Direct ELISA6

EEE



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Immobilized Antien vs. Sand!ic"

2*

Mainly used $or antien

detection

Mainly used $or antibody

detection

Immobili!ed

Antigen6

Sandwich

ELISA6

EEE

EEE



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#ompetitive ELISA

2,

Step .6

Add sample

Step 26

Add antibodys

E E

E

Step #6

0ashing

EE

E

Step '6

Add substrate

E

3uic4 methodology 5 8ust one washing step



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Applications

2-

Medical diagnostic to detect presence o$ antibodies in patient

Detection o$ potential allergens in $ood

9I: ;est

But6 high material costs

Drug tests

0est ile :irus

;est 4its $or eggs and peanuts



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Thankyou

#/Slides by Mathias Bader and Simon Loew

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