CHEMILUMINESCENT ASSAY“CLIA”

Dr. Hafsa Bashir

ObjectivesImmuno assay: Definition Types:

Competitive Non Competitive

Homogenous Non Homogenous

Types Depending on reagent Used: ELISA RIA

CLIA

IMMUNOASSAYDefinition:

Immunoassay is an assay based on the reaction of an antigen with an antibody

specific for the antigen.

“Immuno” refers to an immune response that causes the body to generate antibodies.

“Assay” refers to a test.

Examples:Detect the presence of macro molecules such as proteins,

hormones etc.

Used for measurement of drugs, hormones, specific proteins, tumor markers, and markers of cardiac injury. 

Detect antigens on Cryptococcus  and Streptococcus  organisms in the cerebrospinal fluid (CSF) of meningitis

patients.

Detect infectious and autoimmune diseases

TYPES

Competitive

Non Competitive

Heterogeneous

Homogenous

Competitive:Unlabeled antigen in the test sample is measured

by its ability to compete with labeled antigen in the immunoassay.

The unlabeled antigen blocks the ability of the labeled antigen to bind because that binding site on the antibody is already occupied.

Conc. Is inversely proportional to result.

Non Competitive:Add patient sample, e.g. looking for antibody to

known reagent antigen.Reaction occurs & the conc. Is directly related

to the amount of antibody in patient sample.

This format is referred to as sandwich assay because analyte is bound (sandwiched) b/w two highly specific antibody reagents.

Heterogeneous Assay & Homogenous AssayHeterogeneous assays called separation assays.Immuno assay methods that require separation

of bound Ab-Ag complex are referred to as “Heterogeneous immunoassay”.

Those that do not require separation of unbound complexes from the bound complexes referred to as

“Homogenous immunoassays”.

Types depending of the reagent used:

•These immunoassays make use of certain enzymes.ELISA•Radioactive isotopesRIA•Fluorescent materialsFluor

immunoassay

CLIAChemiluminescent

material

Chemiluminescence Immunoassay“CLIA”

Definition:

An immunoassay technique in which the antigen or 

antibody is labeled with a molecule capable 

of emitting light during a chemical reaction; this light is used to measure the formation of the antigen-antibody complex.

Principle:Chemiluminescence is the light emission produced during

chemical reaction.

In CLIA, a chemiluminescent molecule is used as an indicator label to detect or quantify immunological reactions.

Large number of molecules capable of chemiluminescenceLuminolAcridium estersRuthenium derivativesNitro phenyl oxalates

Sodium hydroxide can be use as a catalyst and widely used enzyme are Alkaline phosphates & Horseradish peroxidase.

Luminol + H2O2 ---> oxidised luminol* + H2O -----> oxidised Luminol + hv (light)

Oxidation of luminol by hydrogen peroxide in the presence of catalyst (enzyme) Intermediates are produced that are of a higher energy state.

These intermediates spontaneously return to their

original state, giving off energy in the form of light.

Light emission ranges from quick burst or flash to light which remains for a longer time.

Principle of CLIA

Have an excellent sensitivity .

Reagents are stable and relatively non toxic. Because very little reagent is used, they are inexpensive to

perform.

Detection systems basically consist of photomultiplier tubes which are simple and relatively inexpensive .

Measurements possible in colored samples, even at Extreme pH.

Advantages:

Disadvantages:Provides Limited Structural Information

False results may be obtained if there is lack of precision in injection of the hydrogen peroxide

If some biological materials such as urine or plasma cause diminishing of the light emission.

The Most Frequently Used Type of Chemiluminescence Set-up

Applications:Can be used for Heterogeneous or

Homogeneous assays.

Can attach label to Antigen or Antibody.

Heterogeneous assays use Competitive and Sandwich assay.

Competitive assays used to measure Smaller analytes.

Sandwich assays used to measure Larger analytes.

Clinical UtilityThyr

oid

• AntiTG

• AntiTPO

• Free T3

• Free T4

• Total T3

• Total T4

• TSH

Tumor Markers

• AFP

• CA125II

• CA 19-9

• CEA

• PSA

Hormon

es

•FSH•hCG•LH•Progesterone•Prolactin•Testosterone

Clinical UtilityTherapeuti

c Dru

g mo

nitorin

g

• Carbamazepine

• Digitoxin

• Digoxin

• Gentamicin

• Phenytoin

• Valproic Acid

• Vancomycin

• Cyclosporine

Hep

atitis

• Anti-HBs

• HAV IgM

• HBc IgM

• HBeAg

• HBsAg

• HBsAg confirmatory

• HCV

Anemia

•Ferritin•Vitamin B12

No stop solution Stop solution required

Higher sensitivity Less sensitive

No Light source (filter) Light source needed

CLIA ELISA

References:http://web.indstate.edu/thcme/PSP/labtests/

precip.htmWild, David (Ed.). (2005).The Immunoassay Handbook.

Kidlington, Oxford: Elsevier. http://www.medschool.lsuhsc.edu/microbiology/

DMIP/dmex03.htm