Post on 16-Nov-2015
description
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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