ChemilluminescentassayBasicprinciples,Instrumentation

andclinicalutility

By

Heratsoni

2ndYearResident

Biochemistry

LuminescenceX*---------->X+hv(light)

Chemiluminescence Luminol + H2O2 ---> oxidised luminol* + H2O

-----> oxidised Luminol + hv (light)

Bioluminescence

seen in firefly

Enhanced chemiluminescence-In presence of HRP

Iodophenol(R-OH) + H2O2 +luminol

RO*(phenolate radical) +H2O +luminol

Hydroxyquinone +oxidised luminol*

Oxidised luminol + hv(Light)

Electrochemiluminescence

Electrochemiluminescence video

Principle of CLIA

The widely used enzymes for luminescent immunoassays are AP(Alkaline phosphatase) and HRP(Horseradish peroxidase) and pyruvate kinase(in bioluminescence)

The suitable substrates for luminescent immunoassays include (i) luminol (ii) acridine esters (iii)Isoluminol (iv)Luciferin (v)AMPPD

AMPPD

m-AMPD

(m-MOB-)*=m-oxybenzoate anion

M-MOB- + hv(Light)

Alkaline phosphatase

Mechanism of pyruvate kinase

AMP+phosphoenolpyruvate

ATP+pyruvatePyruvate kinase

Luciferin- luciferase

Reaction

Light

Instumentation

Chemilluminescent Assay

ELISA Radioimmunoassay

Nostopsolution

Stopsolutionrequired(notinkineticassay)

Nostopsolution

Highersensitivity Lesssensitive(straylight)

Lesssensitive(?)

Nolightsource(Filter) Lightsourceneeded Nolightsource

Clinical UtilityThyroid

AntiTGAntiTPOFreeT3FreeT4TotalT3TotalT4TSH

TumourmarkersAFPCA125IICA199CEAPSASerumHER2/neu

AnemiaFerritinVitaminB12

CardiacBNPCKMBDDimer

Hormones

AFPFSHhCGLHProgesteroneProlactinTestosterone

TherapeuticDrugMonitoringCarbamazepineDigitoxinDigoxinGentamicinPhenytoinTheophyllineValproicAcidVancomycinCyclosporine

HepatitisAntiHBeAntiHBsHAVIgMHBcIgMHBeAgHBsAgHBsAgConfirmatoryHCV

ToRCHCMVIgGCMVIgMRubellaIgGRubellaIgMToxoplasmaIgGToxoplasmaIgM

DiabetesCPeptide(Serum;Urine)Insulin

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