ELISA(EnzymeLinkedImmunoSorbant

Assay)
ThepurposeofanELISAistodetermineifaparticularproteinispresentina
sampleandifso,howmuch.Therearetwomainvariationsonthismethod:you
candeterminehowmuchantibodyisinasample,oryoucandeterminehow
muchproteinisboundbyanantibody.Thedistinctioniswhetheryouaretrying
toquantifyanantibodyorsomeotherprotein.Inthisexample,wewillusean
ELISAtodeterminehowmuchofaparticularantibodyispresentinan
individualsblood.
ELISAsareperformedin96wellplateswhichpermitshighthroughputresults.
Thebottomofeachwelliscoatedwithaproteintowhichwillbindthe
antibodyyouwanttomeasure.Wholebloodisallowedtoclotandthecellsare
centrifugedouttoobtaintheclearserumwithantibodies(calledprimary
antibodies).Theserumisincubatedinawell,andeachwellcontainsadifferent
serum(seefigurebelow).Apositivecontrolserumandanegativecontrol
serumwouldbeincludedamongthe96samplesbeingtested.

Aftersometime,theserumisremovedandweaklyadherentantibodiesare
washedoffwithaseriesofbufferrinses.Todetecttheboundantibodies,a
secondaryantibodyisaddedtoeachwell.Thesecondaryantibodywouldbind
toallhumanantibodiesandistypicallyproducedinarodent.Attachedtothe
secondaryantibodyisanenzymesuchasperoxidaseoralkalinephosphatase.
Theseenzymescanmetabolizecolorlesssubstrates(sometimescalled

chromagens)intocoloredproducts.Afteranincubationperiod,thesecondary
antibodysolutionisremovedandlooselyadherentonesarewashedoffas
before.Thefinalstepistheadditiontheenzymesubstrateandtheproductionof
coloredproductinwellswithsecondaryantibodiesbound.
Whentheenzymereactioniscomplete,theentireplateisplacedintoaplate
readerandtheopticaldensity(i.e.theamountofcoloredproduct)isdetermined
foreachwell.Theamountofcolorproducedisproportionaltotheamountof
primaryantibodyboundtotheproteinsonthebottomofthewells.

Enzyme-Linked Immunosorbent Assay

(ELISA)
There are a number of ways to determine whether an antibody has bound to its
target antigen.
One simple method is an ELISA.
Generally, an ELISA has the following steps (each seperated by extensive
washes):

Antigen Binding:A solid support surface is initially coated with an

appropriate amountof the antigen of interest. The whole surface must be
blocked to ensurebiologically specific binding of the subsequent
components. Having theantigen bound to the solid surfaced makes it

simple to seperate boundfrom unbound molecules. Nonspecifically bound

materials are washed awayduring the process of the assay.
Blocking: All unbound sites on the solid support are blocked to prevent

nonspecific binding of the antibodies.

Primary Antibody: The primary antibody ia added and will be bound if

there is a recognized epitope within sample antigen.

Secondary Antibody:An enzyme-linked secondary antibody is added which
will bind to anyavailable primary antibody. Primary antibody will only be

available onthe surface if it has bound to the antigen.

Detection:After adding an enzyme substrate, a colored product will
develop ifthis binding has occurred. The color intensity reflects the
amount ofprimary antibody bound to the target antigen and can be
measured.

There are many different applications of ELISA and the assay may be modified in
a number of ways.
Direct versus Indirect ELISA
Indirect ELISA: Essentially the method described above.

Direct ELISA:This is similar to the process described above, except that the
primaryantibody is linked to the detection enzyme. Therefore, no
secondaryantibody is required. This method is very quick, but is typically
notas sensitive or flexible as the indirect method.

Sandwich ELISA
A less-common variant of this technique, called "sandwich" ELISA, is used

to detect sample antigen.

Asoild support is coated with a capture antibody. A solution containingthe
antigen is added followed by washing. Detection and quantitation ofbound
antigen is then accomplished by the direct or indirectmethodology as
above.

Competitive ELISA

The steps for this ELISA are somewhat different.

Theantigen is bound to the solid support. Unlabeled antibody is
incubatedin a solution containing the antigen. These bound
antibody/antigencomplexes are then added to an antigen coated well
followed byextensive washes to remove unbound complexes. (The
moreantigen in thesolution, the less antibody will be available to bind to

theantigen inthe well, hence "competition."). This is then processed

similar to theabove methods.
For competitive ELISA, the higher the original antigen concentration, the
weaker the eventual signal.

Microtiter plates were coated with the ANTIGEN of interest. Then filled
with the patients dilutions of serum. If antibodies against the antigen
are present, they will bind to the antigen fixed at the bottom of the
well. But only antigen-specific antibodies will bind to the well. The wells
are then washed out to remove the unbound antibodies. Next, a
solution of animal antibody against human antibodies is added. This
second antibody is covalently conjugated to an enzyme. The wells are
washed again this time to remove unbound enzyme conjugated
antibody. Finally, a solution of colorigenic enzyme substrate is added.
The interaction of the substrate with the enzyme on the second
antibody generate visible color. The devt of color in the wells, with the

specific antibody can be seen with the naked eye or quantified with the
reader.
Antigen antibody enzyme substrate color reaction