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Hormone Assay Detection: ELISA, ECL, and Chemiluminescence Compared
1.
2. • Introduction
• Hormone measurement is necessary
for the diagnosis of a wide range of
clinical conditions and is essential for
monitoring the effectiveness of
treatment.
• As the number of hormone requests
in the clinical field rises exponentially,
it has become importance to create
hormone assays accessible to
researchers with a different range of
devices.
3. • Before the introduction of
radioimmunoassay most hormones were
measured by bioassay and/or chemical
methods.
• The sensitivity of these methods was
low, and large amounts of samples were
needed .
• Radioimmunoassay first applied In
1959, to measurement of insulin, after
which many protein hormones were
determined by this method.
4. • Because radioactivity poses a
potential health threat, so Research
had to be go on for finding a safer
alternative .
• The first paper on the ELISA
procedure published in 1971 as a
replacement for radioimmunoassays
by Sweden and Netherlands
Scientists independently .
5. • Recently, non-isotopic immunoassay
methods utilizing
chemiluminescence, fluorescence
and enzymes as labels are widely
used.
• These methods have resulted in
sensitivities adequate to replace
radioimmunoassays.
6.
7. • proteins produced by the immune
system which help defend against
antigens .
11. • 1. Antigen/antibody of interest is
absorbed on to plastic surface
(‘sorbent’).
• 2. Antigen is recognised by specific
antibody (‘immuno’).
• 3. This antibody is recognised by second
antibody (‘immuno’) which has enzyme
attached (‘enzyme-linked’).
• 4. Substrate reacts with enzyme to
produce product, usually coloured.
12. •ELISA
• Is used to the detection of
1)Antibodies .
2)Proteins .
3)Peptides .
4)Biomolecules .
22. 1- Measuring Principle
The reaction occurs within
the interior of the SPR
whereby antibodies and
conjugate form a
sandwich.
(4-MUP) is cycled into SPR
and conjugate enzyme
catalyses the hydrolysis of
the substrate into 4-
Methyl-umbelliferone
which is measured at
450nm.
23. The intesity of the Fluorescence is
inversely proportional to the
concentration of antigen present in
the sample .
24.
25.
26.
27. “Electro” refers to electrical stimulation.
+
“Chem” indicates a chemical reaction.
+
“Luminescence”means “produces light.”
=
Electrochemiluminescence (ECL)
28. Principle
Electrochemiluminescence
Chemiluminescence is light produced by a
electrochemical reaction from a substance as
it returns from an electronically excited state
to ground state.
The chemiluminescent substance is excited
by the oxidation and catalysis forming
intermediates. When the excited
intermediates return back to their stable
ground state, a photon is released, which is
detected by the luminescent signal
instrument .
29. An enzyme converts a substrate to a
reaction product that emits photons
of light instead of developing a visible
color.
ECL is a unique and highly sensitive
luminescence (light) detection system
that amplifies the signal (you want) to
detect ultra-low concentrations of
analyte. and reduces any signals you
don’t want to deliver .
30. • Source kicks an electron of an atom out of its lowest
energy “ground” state into a higher energy
“excited” state .
• Electron returns the energy in the form of light so it
can fall back to its “ground” state .
• [A] + [B] → [◊] → [Products] + light
• [A], [B]: reactants
• [◊]: excited intermediate .
31. For example, if [A] is luminol and [B] is
hydrogen peroxide in the presence of a
suitable catalyst we have:
• luminol + H2O2 →3-APA[◊] →3-APA +
light
• Where:
• 3-APA is 3-aminophthalate
• 3-APA[◊] is the excited state producing
light as it return to a lower energy level.
32. • What happens in Chemiluminescence
immunoassays
• Monoclonal antibody coated well
↓
Test specimen (serum)
↓
HRP labelled antibody conjugate
↓
Test antigen: sandwich between solid
phase Ab and enzyme labelled Ab
33. Incubate at 37° C
↓
Remove unbound enzyme labeled Ab
↓
Chemiluminescence reagent added
↓
Read relative light unit with luminometer
34.
35.
36. luminescence is the most sensitive
detection method currently in use due to
the ability of signal multiplication and
amplification.
Luminescent reactions are measured in
relative light units (RLU) that are typically
proportionate to the amount of analyte
present in a sample.
37. • Properties
Ultra-high sensitivity (10, 000 times higher
than and the light absorption method and
1000 times higher than the fluorescence
detection method) .
Show a linear relationship between luminous
intensity and the concentration of measured
substance .
No preincubation step .
Substrate can be added several minutes prior
to detection.
38.
39.
40. • Diagnoses acute toxoplasmosis during the first
trimester of pregnancy.
• Sensitivity and specificity are calculated for
each technique:
• ELISA technique showed a specificity of 83%
and a sensitivity of 95%.
• ELFA technique showed a specificity of 90%
and a sensitivity of 97.5%.
• ECL technique showed a specificity of 100%
and a sensitivity of 100%.
41. Comparison Between
Chemilluminescent
Assay
• No stop solution
• Higher sensitivity
• No light
source(Filter)
ELISA
• Stop solution
required (not in
kinetic assay)
• Less sensitive
• Light source needed