VariousTypesofELISAAssays(Biosolutions) Thestepsofthegeneral,"indirect,"ELISAfordeterminingserumantibody concentrationsare: 1. Applyasampleofknownantigenofknownconcentrationtoasurface,oftenthe wellofamicrotiterplate.Theantigenisfixedtothesurfacetorenderitimmobile. Simpleadsorptionoftheproteintotheplasticsurfaceisusuallysufficient.These samplesofknownantigenconcentrationswillconstituteastandardcurveusedto calculateantigenconcentrationsofunknownsamples.Notethattheantigenitself maybeanantibody. 2.

Aconcentratedsolutionofnoninteractingprotein,suchasbovineserumalbumin (BSA) or casein, is added to all plate wells. This step is known as blocking, becausetheserumproteinsblocknonspecificadsorptionofotherproteinstothe plate. 3. Theplatewellsorothersurfacearethencoatedwithserumsamplesofunknown antigenconcentration,dilutedintothesamebufferusedfortheantigenstandards. Since antigen immobilization in this step is due to nonspecific adsorption, it is important for the total protein concentration to be similar to that of the antigen standards. 4. Theplateiswashed,andadetectionantibodyspecifictotheantigenofinterestis appliedtoallplatewells.Thisantibodywillonlybindtoimmobilizedantigenon thewellsurface,nottootherserumproteinsortheblockingproteins. 5. Secondaryantibodies,whichwillbindtoanyremainingdetectionantibodies,are added to the wells. These secondary antibodies are conjugated to the substrate specificenzyme.Thisstepmaybeskippedifthedetectionantibodyisconjugated toanenzyme. 6. Washtheplate,sothatexcessunboundenzymeantibodyconjugatesareremoved. 7. Apply a substrate which is converted by the enzyme to elicit a chromogenic or fluorogenicorelectrochemicalsignal. 8. View/quantifytheresultusingaspectrophotometer,spectrofluorometer,orother optical/electrochemicaldevice. SandwichELISA Alesscommonvariantofthistechnique,called"sandwich"ELISA,isusedtodetect sampleantigen.Thestepsareasfollows: 1. Prepareasurfacetowhichaknownquantityofcaptureantibodyisbound. 2. Blockanynonspecificbindingsitesonthesurface.

3. 4. 5. 6.

Applytheantigencontainingsampletotheplate. Washtheplate,sothatunboundantigenisremoved. Applyprimaryantibodiesthatbindspecificallytotheantigen. Apply enzymelinked secondary antibodies which are specific to the primary antibodies. 7. Washtheplate,sothattheunboundantibodyenzymeconjugatesareremoved. 8. Applyachemicalwhichisconvertedbytheenzymeintoacolororfluorescentor electrochemicalsignal. 9. Measure the absorbance or fluorescence or electrochemical signal (e.g., current) oftheplatewellstodeterminethepresenceandquantityof antigen. CompetitiveELISA AthirduseofELISAisthroughcompetitivebinding.ThestepsforthisELISAare somewhatdifferentthanthefirsttwoexamples: 1. Unlabeledantibodyisincubatedinthepresenceofitsantigen. 2. These bound antibody/antigen complexes are then added to an antigen coated well. 3. Theplateiswashed,sothatunboundantibodyisremoved.(Themoreantigenin thesample,thelessantibodywillbeabletobindtotheantigeninthewell,hence "competition.") 4. The secondary antibody, specific tothe primary antibody is added. This second antibodyiscoupledtotheenzyme. 5. Asubstrateisadded,andremainingenzymeselicitachromogenicorfluorescent signal. For competitive ELISA, the higher the original antigen concentration, the weaker the eventualsignal. (Note that some competitive ELISA kits include enzymelinked antigen rather than enzymelinked antibody. The labeled antigen competes for primary antibody binding sites with your sample antigen (unlabeled). The more antigen in the sample, the less labeledantigenisretainedinthewellandtheweakerthesignal). ELISAReversemethod&device(ELISARm&d) Anewertechniqueusesasolidphasemadeupofanimmunosorbentpolystyrenerodwith 412 protruding ogives (curved shape/feature). The entire device is immersed in a test tube containing the collected sample and the following steps (washing, incubation in

conjugate and incubation in chromogenous) are carried out by dipping the ogives in microwellsofstandardmicroplatesprefilledwithreagents. Advantages: Theogivescaneachbesensitizedtoadifferentreagent,allowingthesimultaneous detectionofdifferentantibodiesanddifferentantigensformultitargetassays Thesamplevolumecanbeincreasedtoimprovethetestsensitivityinclinical(saliva, urine),food(bulkmilk,pooledeggs)andenvironmental(water)samples Oneogiveisleftunsensitizedtomeasurethenonspecificreactionsofthesample Theuseoflaboratorysuppliesfordispensingsamplealiquots,washingsolutionand reagentsinmicrowellsisnotrequired,facilitatingreadytouselabkitsandonsitekits.