Chemiluminescence assay

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This document discusses the principles and clinical applications of chemiluminescent assays. Chemiluminescence involves the emission of light from a chemical reaction and can be enhanced through the use of enzymes like horseradish peroxidase. Chemiluminescent assays are commonly used for immunoassays and have higher sensitivity than ELISA or radioimmunoassays. The document outlines the mechanisms and substrates involved in chemiluminescence and lists many clinical applications for measuring markers of thyroid function, tumor progression, anemia, cardiac function, hormone levels, therapeutic drug monitoring, hepatitis, and diabetes.

Gesundheitswesen

Chemilluminescent assay
Basic principles , Instrumentation
and clinical utility
By
Herat soni
2nd Year Resident
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)

● 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
Luciferin-luciferase
Reaction
Light

Chemilluminescent
Assay
ELISA Radioimmunoassay
No stop solution
Stop solution required
(not in kinetic assay)
No stop solution
Higher sensitivity Less sensitive(stray
light)
Less sensitive(?)
No light source(Filter) Light source needed No light source

Clinical Utility
Thyroid
AntiTG
AntiTPO
Free
T3
Free
T4
Total
T3
Total
T4
TSH
Tumour
markers
AFP
CA
125II
CA
199
CEA
PSA
Serum
HER2/
neu
Anemia
Ferritin
Vitamin
B12
Cardiac
BNP
CKMB
DDimer
Hormones
AFP
FSH
hCG
LH
Progesterone
Prolactin
Testosterone
Therapeutic
Drug
Monitoring
Carbamazepi
ne
Digitoxin
Digoxin
Gentamicin
Phenytoin
Theophylline
Valproic
Acid
Vancomycin
Cyclosporine

Hepatitis
AntiHBe
AntiHBs
HAV IgM
HBc IgM
HBeAg
HBsAg
HBsAg Confirmatory
HCV
ToRCH
CMV IgG
CMV IgM
Rubella IgG
Rubella IgM
Toxoplasma IgG
Toxoplasma IgM
Diabetes
CPeptide
(Serum;Urine)
Insulin

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