Description:

TECO®αGSTisanELISAforthequantitativedeterminationofalpha GlutathioneS-Transferase(αGST)inhumanserum,plasmaor urine.

αGST isametabolicenzymeinvolvedwith cellulardetoxificationreactions.Itis approximately51kDaanditcomprises ~5%ofsolublecytosolicproteininhepatocytes.Uponcellulardamage,αGSTisrapidlyreleasedfromcells.Anincreasedconcentrationinbloodisasensitiveindicatorofacutehepaticinjury.StudieshaveshownthatincreasedserumorplasmaαGSTcan beindicativeofhepatotoxicity,allograftrejection,orviralhepatitis.

Inthekidney, αGSTcomprises~2%ofthecytosolicsolubleproteincontent intheepithelialcellsoftheproximaltubule.Likewithhepatocytes,itisrapidlyreleasedupondamagetothe cells.Increasedurineconcentrationsareindicativeofacutekidneyinjury.

Advantages:

• UnlikewithothertoxicitybioMarkers,αGSThasnotbeenshowntoincreasewithmuscledamage,hemolysisorgeneralinflammation(i.e.Rheumatoidarthritis).
• αGSTisrapidlyreleaseduponcellulardamageandthususefulasanearlyindicatorofacuteliver(orkidney)damageinresearchsettings.
• SuitabletousewiththeM30Apoptosense®ELISA(apoptosisdetection),M65®ELISA(necrosisdetection),andTECO®HyaluronicAcidELISA(chronic,fibrosisdetection)asapanelofresearchassaysusefulforassessingliverdamage.
• Applicationsforcellcultureandculturespheroids.
• SandwichELISAwith a96-wellplateinaconvenient,ready-to-useformat.
• Easytoperformassaywithminimalpipettingsteps.

KitComposition:

• AntibodyCoatedMicroassayPlate: 96well(12×8break-apartwellstripscoatedwithIgGdirectedagainstαGST)
• Calibrator,0.2mL(2mg/L): PurifiedαGSTinstABIlizingdiluentcontainingProClin950andBronidoxLaspreservatives.25xConcentrate.
• SampleDiluent,30mL: ProteincontainingsolutionwithaddedstabilizersandProClin950andBronidoxLaspreservatives.
• WashBuffer,45mL: Tris-bufferedsaline/Tween-20(TBST)containingProClin950aspreservative.25xConcentrate.
• PositiveControl,1.5mL: PurifiedαGSTinstabilizingdiluentcontainingProClin950andBronidoxLaspreservatives.
• EnzymeConjugate,11mL: Antibodysolutioncontaininganti-αGSTIgGlabelledwithhorserADIshperoxidaseandProClin950andBronidoxLaspreservatives.
• Substrate,11mL: StabilizedliquidTMBsolution.
• StopSolution,11mL: 0.5MSulphuricAcid
• UrineStabilisingBuffer(USB),10mL: ProteincontainingsolutionwithaddedstabilizersandProClin950andBronidoxLaspreservatives.

AssayPrinciple:

Theα-GSTassayisastandardsandwich ELISA.The96-wellmicroplateiscoatedwithanti-αGSTIgG.Sampleaddedtothewellsforanincubationbindsto the microwell-coatedantibody.  Additionofantibody-enzymeconjugatebindsinasandwichfashiontosamplescontainingalpha-GSTandthenTMBsubstrate allowsforcolorimetricdeterminationof αGSTconcentration.Thecolorintensityisdirectlyproprotionaltotheamountof αGSTinasampleandtheassayrangeis 2.5–80μg/L.

TypicalcalibrationcurveobtainedusingtheAlphaGSTEIA.4-parameterplotofA450/630nmversus[alphaGST]μg/L.Assayrangeis2.5–80μg/LalphaGST.

Background:

Inliver, αGST islocatedinthehepatocyteswhereaspiGSTisfoundin theintrahepaticbileductcells.ThisheterogeneousGSTsubclassdistributionsuggeststhattheisoenzymeshaveuniqueinvivofunctionsindifferenthepaticregionsandthatthedetectionofGSTsubclasslevelsinBIOLOGicalfluidscouldbeofsignificantuseinmonitoringtheintegrityofspecifichepaticregions.Currently,liverinjuryisstudiedbythemeasurementofliverenzymessuchasalanineaminotransferase(ALT)andaspartateaminotransferase(AST).Adisadvantageofthesemarkersisthattheyarenotdistributeduniformlythroughouttheliverandthattheirperiportalconcentrations are greaterthantheircentrilobularconcentrations.Incontrast,αGSThasbeenfoundtobeequallydistributedinboththecentrilobularandperiportalregionsoftheliver.SincethecentrilobularhepatocytesareverysusceptIBLetodamageinavarietyofconditionsincludingallograftrejection,viralhepatitis,andhepatotoxicity,αGSTisamoresensitiveindicatorofhepaticinjury.The AlphaGSTEIAisaspecific,preciseimmunoassay and,beingaquantitativetest, itisnotaffected bymodulatorsofenzymeactivitysuchas bilesaltsandbilirubin.

Inkidney,αGSTisfoundintheproximaltubuleregion. Inhealthy individuals,lowlevelsofαGSTarereleasedintotheurineasconfirmedbyimmunoassayandWesternblotanalyses.Eventsthatleadto proximaltubulardamageoften causeincreasedreleaseofαGSTintourine.Elevated urinaryαGSTlevelshavebeenshowntoindicate proximaltubuledamagedueto nephrotoxicity,environmentaltoxicity,surgery,acuterenalfailureandtransplantation.

αGSTisoftendescribedasa“leakage”biomarkersinceitisrapidlyreleasedfromdamagedcells.ThoughintendedforresearchuseonlyintheUSandCanada,theAlphaGSTEIAcanbeausefulindicatorofacuteliver(orkidney)damageandisthusausefulresearchassayforassessingtoxicity.TheTECOAlphaGSTEIAcanalsobeusedwithhumanlivercellculturesamples,providinganapplicationfor invitrodrugscreening.