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Mohd Hairi Bin A.Hamid – Bsc.

Medical Laboratory Technology (Hons)

UniversitI Teknologi Mara, Malaysia – (January 2010)

Chemiluminescence immunoassay – Free thyroxine (T4)

1.0 Introduction

Chemiluminescence immunoassay (CLIA)

 Chemiluminescence immunoassay (CLIA) is the one of the detection method used in Clinical
Chemistry Laboratory.
 CLIA use chemiluminescent labels. Chemiluminescent molecules produce light when they
are excited by chemical energy.

 Isoluminol and acridinium esters are the mostly used as chemiluminescent labels.
 Oxidation of isoluminol by hydrogen peroxide in the presence of a catalyst produces a
relatively long lived light emission at 425 nm.
 Oxidation of acridium esters by alkaline hydrogen peroxide in the presence of a detergent
produces a flash of light at a 429 nm.

 Purpose: Determine the presence of thyroid disease in patient (Example: Hypothyroidism,

Hyperthyroidism, Grave’s Disease, Hashimoto thyroiditis).

 The advantages of Chemiluminescent immunoassay (CLIA) includes:

1. Not required long incubation time.
2. No addition of stopping reagent (colorimetric method is required).
3. More economical compared to conventional colorimetric method (Ex: Enzyme-
linked immunosorbent assays, ELISA)
4. More sensitive compare to the colorimetric method.

 In the Chemiluminescent immunoassay (CLIA) method involved:

1. Antibody Coated Microtiter Plate: Microplate with anti-T4 Antibody coated wells.
2. Enzyme Conjugate Reagent: Horseradish peroxidase (HRP) labelled T4.
3. PBS-T Powder: PBS-Tween (Washing solution)
4. Mixture of chemiluminescent substrate (Substrate A, B etc)
5. Pipettes (20-200μl, 100-1000μl)
6. Vortex Mixer or equivalent
7. Quality control specimens
8. Washer for Microplate
9. Absorbent paper
10. Distilled water
11. Luminometer
12. Incubator

 Chemiluminescent immunoassays (CLIA) are measured the substance’s level required (Ex:
Thyroxine, T4) based on the chemiluminescent reactions in the luminometer which Related
Light Unit, RLU is read and measured

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Mohd Hairi Bin A.Hamid – Bsc. Medical Laboratory Technology (Hons)
UniversitI Teknologi Mara, Malaysia – (January 2010)

Thyroid hormones

 Thyroid gland secrete 2 hormones including:

1. Thyroxine (T4) @ Tetraiodothyronine
The major hormone secreted by follicular cells of thyroid gland.
Less active form.
99.95% of secreted T4 being bound to Thyroxine Binding Protein (TBG),
thyroxine binding prealbumin (TBPA) and albumin.
Function: Control body’s metabolic process, energy and physical
development (growth).

2. Triiiodothyronine (T3)
More active form of thyroid hormone.
Only 15% of T3 is directly secreted by the follicular cells.
99.5% of the secreted T3 being bound to the same proteins as T4.

2.0 Principles

 In the Chemiluminescent immunoassays (CLIA) – Free Thyroxine (f-T4) test, amount of anti-
T4 antibody is coated on microtiter wells.
 Then, patient serum and T4 conjugated with horseradish peroxidase (HRP-T4) are added to
the microtiter wells.
 During incubation period, the f-T4 and f-T4 enzyme conjugate compete for the binding sites
on the anti-T4 antibody (in the wells)
 After 45 minutes incubation at 37℃, the wells are washed 5 times by washing solution to
remove unbound f-T4 enzyme conjugate.
 After that, mixture of chemiluminescent substrate is added and the relative light units (RLU)
were read.
 Relative light units (RLU) mean the intensity of the emitting light.
 Therefore, RLU is directly proportional to the amount of enzyme present and is inversely
proportional to the amount of unlabeled T4 in the patient’s sample.

Diagram shows the principles of the CLIA

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Mohd Hairi Bin A.Hamid – Bsc. Medical Laboratory Technology (Hons)
UniversitI Teknologi Mara, Malaysia – (January 2010)

3.0 Patient preparation

 Not validated for newborn testing

 Non-fasting specimen is acceptable – phlebotomist can collect the patient blood at random

4.0 Specimen collection

 Commonly serum is used as the sample.

 Plasma sample added with Ethylenediaminetetraaceticacid (EDTA), Oxalate and Heparin
should be avoided due to can interfere the procedures.
 Avoid also Lipemic, Hemolytic and turbid samples
 The sample should be allow to coagulate for 1 Hour
 The specimen should be capped and can stored up to 48 hours (2-8°C) @ -20°C (longer

5.0 Procedures

 Ensure that the microtiter wells are good enough to use in the test (already has anti T4 Ab)
 Dispense 50μl of standards, samples, and controls into appropriate wells.
 Dispense 100μl of Enzyme Conjugate Reagent (HRP-T4) into each well.
 Thoroughly mix for 30 seconds. It is important to have completed mixing in this step.
 Incubate at 37℃ for 45 minutes.
 Remove the incubation mixture into a waste container.
 Rinse the micro plate 5 times with wash solution (PBS-Tween)
 Tap the wells onto absorbent paper to remove residual water droplets.
 Dispense 50μl of Substrate A, then 50μl of Substrate B into each well. Gently mix for 10
 Put the microplate into the detecting chamber of Luminometer for 5 minutes, and then read
the RLU values of each well.

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Mohd Hairi Bin A.Hamid – Bsc. Medical Laboratory Technology (Hons)
UniversitI Teknologi Mara, Malaysia – (January 2010)

6.0 Reference range of Free Thyroxine (fT4)

 Normal: 0.8 – 1.5 ng/dL

 Hypothyroidism: < 0.8 ng/dL
 Hyperthyroidism: > 1.5 n/dL

7.0 Clinical significances

 Serum Free T4 values may increase or decrease due to:

Increased Decreased
 Hyperthyroidism  Hypothyroidism
 Pregnancy  Subclinical hyperthyroidism
 Administration of oral contraceptives  Protein-wasting disease
 Excessive therapy  Certain liver disease
 Administration of testosterone,
salicyclates or diphenylhydantoin.
 Euthyroid sick

8.0 References

 Carl A. Burtis, Edward R.Ashwood, Tietz Fundamentals of Clinical Chemistry (5 th

Edition) (2001), Saunders An Imprint of Elsevier.Pennsylvania

 Karen M.Escolas (1998), Saunders Manual of Clinical Laboratory Science (1 st

Edition): Endocrinology: Thyroid Function, W.B Saunders Company.

 MSDS of Free Thyroxine (f-T4) Chemiluminescence Immunoassay (Feb 2009),

Autobio; received on January 20, 2010

 Dr. Shireen Mansor, Hormone Assay from http://www.scribd.com/doc/6575314/1704

 T. G. Chasteen, The Chemiluminescence of Luminol and bis(2,4,6-

trichlorphenyl)oxalate (TCPO), retrieved on January 22, 2010 from

 Chemiluminescence (January 21, 2010) retrieved on January 22, 2010 from


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