CardiactroponinI(cTnI)wasseparatedandpurifiedfromhumanleftventriculartissuebyaffinitychromatographicmethodandusedtoimmunizeBalb/cmicebyintraperitonealinjectionandfourhybridomacelllines,whichsecretedmonoclonalantibody(mAb)againsthumancTnI,wereobtainedbycellfusion,identificationandcloningtwice.ThreemAbs(9F5,2F11,8C12)wereproducedfromtheascitesofBalb/cmiceinjectedintraperitoneallythehybridomacellsandcharacterizedbymeansofasurfaceplasmonresonance(SPR)biosensor.AnoptimalandspecificsensingmembranefortroponinIwaspreparedwithstaphylococcalproteinA(SPA)astheintermediatelayerandmAbagainsthumancTnIasthecaptureantibody.Onthebasisofthesensingmembrane,twomodesofoperationoftheSPRbiosensorweredeveloped,i.e.,adirectdetectionofantigen-antibodyaffinityandasandwichassay.Inthesandwichassaydetectionmode,themAbscompetitionwasmeasuredbymonitoringwhetherthesecondaryantibodyhadbeenattachedtothecTnIalreadycapturedbythefirstantibodyonthesensorsurface.TheSPRbiosensorwasshowntobeabletodirectlydetecttheantigen-antibodyaffinityandtheorderoftheaffinitywasfoundtobe9F5＞2F11＞8C12.Inthesandwichdetectionmode,itwasfoundthatthedifferentepitopesonthecTnImoleculeswererecognizedbythethreemAbsrespectively,buttheasymmetricalcompetitionwasshownbetween2F11and8C12andnocompetitionwasfoundbetween9F5and2F11or8c12.Basedontheseresults,adoublemonoclonalsandwichimmunoassayforcTnIwasdevelopedbyusingtheoptimalantibodypairof9F5and2F11andtheSPRbiosensorwithSPAsubstratemembrane,whichshowedanexcellentsensitivityof0.8μg/LforboththebufferandtheserumsamplescomparedwiththedirectdetectionofcTnIforthebufferwiththelowestdetectionlimitof4μg/LandconventionalELISAwiththesensitivityof1.9μg/L.
