Tuesday, August 25, 2009
3-4. Give the advantages of CLIA over the conventional colorimetric
5. What is the labeled antibody used?
6. Why is there a need of washing the solution?
7-9. What is the relationship of emitted light to the amount of enzyme present and to the amount of unlabelled T4 in the sample?
10. What is the possible result if you fail to wash the solution?
In your own words, how will you explain the principle of CLIA?
Thursday, August 13, 2009
Immunoassay is a convenient and said to be the most reliable screening test to determine any presence of thyroid disorders in patient.
Recent study said that chemiluminescent immunoassay (CLIA) has been shown to be more sensitive than of the conventional colorimetric method(s). One advantage of chemiluminescent is that there’s no need for long incubation or addition of stopping reagents, as in case of some colorimetric assays.nad in terms of methological advantages, Chemiluminescent immunoassays will play an important part in the diagnostic and research areas that ELISA can not do.
Chemiluminescent immunoassay involve the use of horseradish peroxidase (HRP) labeled antibody or antigen and a mixture of chemiluminescent substrate, hydrogen peroxide, and enhancers. CLIA kits are intended to identify glow based chemiluminescent reactions.
Principle of the test
Amount of anti-T4 antibody, a measured amount of patient serum, and a constant amount of T4 conjugated with horseradish peroxidase are all added to the microtiter wells. In the incubation period, the anti-T4 antibody is bound to the second antibody, while the T4 and conjugated T4 compete for the left binding sites on the anti-T4 antibody.
After the incubation ( 1 Hour) at room temperature, the microtiter wells are washed five times by a washing solution. The purpose of washing is to remove the unbound T4 conjugate. And the washing procedure is said to be critical, why? Because insufficient washing will result in poor precision and falsely elevated absorbance reading. Then a certain solution of chemiluminescent substrate is being added and read the relative light units (RLU).
The result means that the intensity of the emitting light is directly proportional to the amount of enzyme present and is inversely proportional to the amount of unlabeled T4 in the patient’s sample.