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.

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

2. ● Luminescence
X*---------->X+hv(light)
● Chemiluminescence
Luminol + H2O2 ---> oxidised luminol* + H2O
-----> oxidised Luminol + hv (light)
● Bioluminescence
seen in firefly

3. ● Enhanced chemiluminescence
-In presence of HRP
Iodophenol(R-OH) + H2O2 +luminol
RO*(phenolate radical) +H2O +luminol
Hydroxyquinone +oxidised luminol*
Oxidised luminol + hv(Light)

4. ● Electrochemiluminescence

5. Electrochemiluminescence video

6. Principle of CLIA

7. ● 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

8. Mechanism of pyruvate kinase
AMP+phosphoenolpyruvate
Pyruvate kinase
ATP+pyruvate
Luciferin-luciferase
Reaction
Light

9. Instumentation

11. 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

12. 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

13. 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