Chemilluminescent assay Basic principles , Instrumentation 

and clinical utility

By

Herat soni

2nd Year Resident

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+pyruvate

Pyruvate kinase

Luciferin- luciferase

Reaction

Light

Instumentation

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 UtilityThyroid          

­Anti­TG­Anti­TPO­Free T3­Free T4­Total T3­Total T4­TSH

Tumour markers­AFP­CA 125II­CA 19­9­CEA­PSA­Serum HER­2/neu

Anemia­Ferritin­Vitamin B12

Cardiac­BNP­CKMB­D­Dimer

Hormones

­AFP­FSH­hCG­LH­Progesterone­Prolactin­Testosterone

Therapeutic Drug Monitoring­Carbamazepine­Digitoxin­Digoxin­Gentamicin­Phenytoin­Theophylline­Valproic Acid­Vancomycin­Cyclosporine

HepatitisAnti­HBeAnti­HBsHAV IgMHBc IgMHBeAgHBsAgHBsAg ConfirmatoryHCV

ToRCHCMV IgGCMV IgMRubella IgGRubella IgMToxoplasma IgGToxoplasma IgM

DiabetesC­Peptide (Serum;Urine)Insulin