Enzyme-linkedImmunosorbentAssays(ELISAs)combinethespecificityofantibodieswiththesensitivityofsimpleenzymeassays,byusingantibodiesorantigenscoupledtoaneasily-assayedenzyme.ELISAscanprovideausefulmeasurementofantigenorantibodyconcentration.Therearetwomainvariationsonthismethod:TheELISAcanbeusedtodetectthepresenceofantigensthatarerecognizedbyanantibodyoritcanbeusedtotestforantibodiesthatrecognizeanantigen.AnELISAisafive-stepprocedure:

1. coatthemicrotiterplatewellswithantigen;
2. blockallunboundsitestopreventfalsepositiveresults;
3. addantibodytothewells;
4. addanti-mouseIgGconjugatedtoanenzyme;
5. reactionofasubstratewiththeenzymetoproduceacoloredproduct,thusindicatingapositivereaction.

1. ThesensitivityoftheSandwichELISAisdependentonfourfactors:
2. Thenumberofmoleculesofthefirstantibodythatareboundtothesolidphase.
3. TheAvidityofthefirstantibodyfortheantigen.
4. Theavidityofthesecondantibodyfortheantigen.
5. Thespecificactivityofthesecondantibody.

Theamountofthecaptureantibodythatisboundtothesolidphasecanbeadjustedeasilybydilutionorconcentrationoftheantibodysolution.Theavidityoftheantibodiesfortheantigencanonlybealteredbysubstitutionwithotherantibodies.Thespecificactivityofthesecondantibodyisdeterminedbythenumberandtypeoflabeledmoietiesitcontains.Antibodiescanbelabeledconvenientlywithiodine,enzymes,orbiotin.

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