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Chemilluminescentassay

Basicprinciples,Instrumentation
andclinicalutility
By
Heratsoni
2ndYearResident
Biochemistry

Luminescence
X*---------->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)
AMPPD
Alkaline phosphatase

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

M-MOB- + hv(Light)

The suitable substrates for luminescent immunoassays


include (i) luminol (ii) acridine esters (iii)Isoluminol
(iv)Luciferin (v)AMPPD

Mechanism of pyruvate kinase


AMP+phosphoenolpyruvate
Pyruvate kinase

ATP+pyruvate
Luciferinluciferase
Reaction

Light

Instumentation

Chemilluminescent
Assay

ELISA

Radioimmunoassay

Nostopsolution

Stopsolutionrequired
(notinkineticassay)

Nostopsolution

Highersensitivity

Lesssensitive(stray
light)

Lesssensitive(?)

Nolightsource(Filter) Lightsourceneeded

Nolightsource

Clinical Utility
Thyroid Tumour
markers
AntiTG
AFP
AntiTPO
CA125II
FreeT3
CA199
FreeT4
CEA
TotalT3
PSA
TotalT4
Serum
TSH
HER2/neu

Anemia
Hormones
Ferritin
VitaminB12 AFP
FSH
hCG
Cardiac
LH
BNP
Progesterone
CKMB
Prolactin
DDimer
Testosterone

Therapeutic
Drug
Monitoring
Carbamazepi
ne
Digitoxin
Digoxin
Gentamicin
Phenytoin
Theophylline
Valproic
Acid
Vancomycin
Cyclosporine

Hepatitis
AntiHBe
AntiHBs
HAVIgM
HBcIgM
HBeAg
HBsAg
HBsAgConfirmatory
HCV

ToRCH
CMVIgG
CMVIgM
RubellaIgG
RubellaIgM
ToxoplasmaIgG
ToxoplasmaIgM

Diabetes
CPeptide
(Serum;Urine)
Insulin

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