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
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ELISA detects substances with antigenicproperties (mainly proteins
Based on en!ymatic color"reaction
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Basic principle of ELISA
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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
0ash sample to remove
unbound antibodies
E
E
E
E
E
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.#
EE
0ash sample to remove
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
Slides by Mathias Bader and Simon Loew
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.)
Indirect ELISA
Antigen is adsorbed onto
microtiter plate
Add serum to test $or
antibody
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.*
Antigen is adsorbed onto
microtiter plate
Add serum to test $or
antibody
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.,
Antigen is adsorbed onto
microtiter plate
Add serum to test $or
antibody
0ash sample
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.-
Antigen is adsorbed onto
microtiter plate
Add serum to test $or
antibody
0ash sample
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2/Slides by Mathias Bader and Simon Loew
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2.
E
E
E
E
E
E
E
E
Add secondary antibody
1rimary antibody binds
to secondary anitbody
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22
EEE
E
E
E
E
E
Add secondary antibody
0ash unbound antibodies
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2#
EEE
E
E
E
E
E
Add secondary antibody
0ash unbound antibodies
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2'
EEE
Add secondary antibody
0ash unbound antibodies
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2
EEE
1ositive egativ
Add substrate
Measure color change
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Direct vs. Indirect ELISA
2)
3uic4 methodology 5 only one AB
Advantages o$ 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 $or antibody
detection
Immobili!ed
Antigen6
Sandwich
ELISA6
EEE
EEE
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#ompetitive ELISA
2,
Step .6
Add sample
Step 26
Add antibodys
E E
E
Step #6
0ashing
EE
E
Step '6
Add substrate
E
3uic4 methodology 5 8ust one washing step
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Applications
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Medical diagnostic to detect presence o$ 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