Lab.7 2nd stage

Hawler Medical UniversityCollege of Health Sciences

Clinical Biochemistry Dept.Ass. Lec. Amer Ali Khaleel

(M.Sc. Medical Immunology)

Electrochemiluminescent (ECL)Technology

Practical Immunology and Serology

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Introduction:• The last years have seen the development and refinement of many

new immunoassay measurement principles and systems.

• The major trend has been away from liquid phase assays with radio isotopic labels, and toward fast solid-phase assays based on monoclonal antibodies.

• This development is moving further towards precise and reliable non-isotopic, automated or semi automated assays with detection limits low molecular weight.

• This new technology has many distinct advantages over other detection systems.

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Electro-chemiluminescence (ECL) Technology

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“ Electro” refers to electrical stimulation.“Chem” indicates a chemical reaction.

“Luminescence”means “produces light.”=Electrochemiluminescence (ECL)

ECL Assay Principles:

• ECL processes are known to occur with numerous molecules including compounds of ruthenium, osmium, rhenium or other elements.

• The development of ECL immunoassays is based on the use of a ruthenium chelate as the complex for the development of light.

• The chemiluminescent reactions that lead to the emission of light from the ruthenium complex are initiated electrically rather than chemically.

• This is achieved by applying a voltage to the immunological complexes (including the ruthenium complex) that are attached to Streptavidin – coated micro particles, Streptavidin, isolated from Streptomyces avidinii is preferred to avidin

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ECL Assay Principles:• Three test principles are used for the estimation of analytes and

antibodies in the samples:

1-Competitive principle for extremely small analytes.(such as FT3, FT4, Cortisol, Testosterone, Estradiol and others).

2-Sandwich principle for larger analytes and (such as, Thyroid stimulating hormone (TSH), Follicle stimulating

hormone (FSH), Luteinizing hormone (LH) and others).

3-Bridging principle to detect antibodies in the sample.

(such as detect antibodies, (e.g. IgG, IgM and IgA).

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Principle of ECL:• The sequences of the reactions involved are given below with FT3 (Hormone) as an example.

• In the first step, sample and a specific anti – T3 antibody labeled with a ruthenium complex are combined in the assay cup.

• After the first incubation, biotinylated T3 and Streptavidin- - coated paramagnetic micro particles are added. The still free binding sites of the labelled antibody become occupied with the formation of an antigen – hapten complex. The entire complex is bound to the micro particle via interaction of biotin and streptavidin. 05/02/23 6

• After the second incubation, the reaction mixture containing the immune complexes is transported into the measuring cell. The immune complexes are magnetically entrapped on the working electrode, but unbound reagent and sample are washed away by a system buffer.

• In the ECL reaction, the conjugate is a ruthenium based derivative and the chemiluminescent reaction is electrically stimulated to produce light. The amount of light produced is indirectly proportionately to the amount of antigen in the patient sample.

• Evaluation and calculation of the concentration of the antigen (FT3) are carried out by means of a calibration curve that was established using standards of known antigen concentration.

• All These Steps Done Fully Automated Not Manually OR semi-manually.

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Instruments

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Cobas e 411

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LIAISON® XL

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ARCHITECT c16000

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Immulite 1000

Advia Centaur XP

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UniCel DxC 600 Synchron Clinical Systems

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VITROS 3600 Immunodiagnostic System

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Advantages of ECL:1. Excellent sensitivity, comparable to EIA and RIA.2. No radioisotopes are used.3. Measurement is simple and rapid.4. The reagents are stable and relatively nontoxic.5. Easy to use: Immunoassay method similar to conventional ELISA, but quicker.6. ECL is a highly successful detection system that achieves clinical quality data in a

variety of sample types, including cell supernatant, serum, plasma, and whole blood.

7. ECLs are simple and relatively inexpensive.8. This technology has begun to be more widely applied to immunologic testing and

has great potential for the future.9. The advantage of electrically initiating the chemiluminescent reaction is that the

entire reaction can be precisely controlled. 10. Applicable for the detection of all analytes providing a solid platform for menu

expansion

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Limitations:

•False results may be some biological materials such as urine or plasma cause quenching of the light emission.

•Not good for Viral marker determination.

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Applications of ECL (Assay menu):In vitro diagnostics testing:

•Thyroid Function•Hormones•Anemia•Tumor Markers•Cardiac Markers•Bone Markers•Infectious Disease•Others

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Any

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