ECL Technology

Mehul Rana
TRAINING TOPICS

• ECL Technology

• Test Principles

• Reagent Concept
ELECTROCHEMILUMINESCENCE

• Luminescence generated during electrochemical reaction in solutions.

• The development of ECL immunoassays is based on the use of

ruthenium(II)-tris(bipyridyl) Ru(bpy)32+ and tripropylamine (TPA)
attached to Streptavidin coated microbeads.

• Reactions are triggered electrically rather than chemically.

ECL - Technology

Ru

Ruthenium Conjugated
Antibody

Antigen

Biotinylated antibody

Streptavidin

Microparticle
Advantages of streptavidin-biotin concept

• High stability of coating (also at 37°C)

• High binding capacity towards the solid phase

• High immunoreactivity of the bound protein

• Uniform and defined quality of 1 universal solid phase for all parameters

• Improvement of within and between-batch consistency

• Long term stability of microparticle reagent

(>18 months at 4°C)
Ruthenium – A good immunoassay label

• High activity at low concentration

(free label has 1 pmol detection limit)

• Stable and easily conjugated

• Small size to minimize steric interference

• Available in high purity

• Easily measurable by available techniques over multiple decades (free label

linear over 6 decades)

• Not present in biological fluids

Small size compared to Enzyme labels

• ß-galactosidase M.W. 540.000

• Alkaline phosphatase M.W. 100.000

• Horseradish peroxidase M.W. 40.000

• Ru(bpy)3 M.W. 1.000

• Acridiniumester M.W. 500

 Steps involved in ECL (Electrochemiluminescence) Measurement

Fig 1- Addition of R1+R2+Serum Fig 2- Addition of M, specific binding

Fig 3- Measuring cell Step 4- Addition of TPA (Procell),

Step 5- Emission of light

Step 6- Flushing by Clean cell
Magnetic microparticles Photomultiplier Counter electrode
with bound antigen-
antibody complex Unbound label

Magnet
Working electrode Flow channel

Reaction within Measuring Cell

ECL Signal Generation

ECL intensity (counts) applied voltage [mV]

1500
350,000
1200
300,000

250,000 900

200,000
600
150,000
300
100,000

50,000 0
0
0.00 0.20 0.40 0.60 0.80 1.00 1.20 Time [sec.]
Advantages of ECL Technology

• Extremely stable non-isotopic labels

• Increased Sensitivity

• Short Incubation Time

• Wide Measuring Range

• Less Sample Volume

Test Principles
 SANDWICH PRINCIPLE

FIRST IMMUNOLOGICAL REACTION

SERUM CONSTITUENTS

SECOND REACTION

LIGHT REACTION
SIGNAL (LIGHT)

TPA  ECL

CONCENTRATION
MAGNETIC FORCE &
ELECTRICAL POTENTIAL

RUTHENIUM LABELLED
ANTIGEN
ANTIBODY TPA TRIPROPYLAMINE
BIOTINYLATED STREPTAVIDIN-COATED
ANTIBODY MICROPARTICLE
 COMPETITIVE PRINCIPLE

FIRST REACTION

SECOND REACTION

LIGHT REACTION
SIGNAL (LIGHT)

TPA  ECL

CONCENTRATION
MAGNETIC FORCE &
ELECTRICAL POTENTIAL

RUTHENIUM LABELLED
ANTIGEN
ANTIBODY TPA TRIPROPYLAMINE

BIOTINYLATED STREPTAVIDIN-COATED
ANTIGEN MICROPARTICLE
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