chemist-Specialist in clinical biochemistry

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.

7. • proteins produced by the immune
system which help defend against
antigens .

8. 2-Antigen

10. ELISA
Why
known as……………??
Enzyme-Linked ImmunoSorbent Assay

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 .

18. Competitive ELISA

21. • 2- The reagent strip contains pre-
dispensed reagents:
• Conjugate: alkaline phosphatase-labelled
mouse monoclonal anti-human
immunoglobulin .
• Fluorescent substrate: 4-MUP (4-Methyl-
umbelliferyl phosphate) & DEA
(diethanolamine).
• Washing buffers .

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 .

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

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.

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