Post on 15-Jan-2017
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