1. OPTIMISATION OF
OF HAZELNUTS
FOR DETECTION
AN ENZYME-LINKED
(ELISA)
by
SITI AZIAH
ARIPIN
IMMUNOSORBENT ASSAY

2. • ELISA is a plate-based assay technique
designed for detecting and quantifying
substances such as peptides, proteins and
hormones.
• Types: Competitive
Non-competitive
INTRODUCTION

3. • Hazelnut is an edible tree nut that
mainly grown in Turkey, Italy, Spain, Portugal,
France, Greece and US.
• Hazelnut is beneficial for human health –
HDL LDL total cholesterol
• Hazelnut consumption may trigger IgE-
hypersensitivity reactions in certain
individuals.

4. To compare the
effectiveness of different
hazelnuts extract for use
as standard materials in
the ELISA
i. Crude, de-fatted
extract
ii. Ammonium sulfate
precipitates of de-
fatted extract
OBJECTIVE

5. De-shelled
hazelnuts De-fatting
Ammonium sulphate
precipitationELISA
METHODS
PBS extraction
(1:10, w:v)

6. AMMONIUM SULPHATE
PRECIPITATION
300 ml of clear hazelnuts extract
Increase the saturation from 0 to 80% by adding (NH4)2SO4 while
stirring at 4 °C
Continue mix the solution for 2 hours
Centrifuge at 15,000g for 10 min
Collect pellet
30 ml Supernatant
Remaining
supernatant
De-salting and freeze
dry *Saturation was increased
by 2% each time.

7. • At low concentrations, salt stabilises various charged groups on a
protein molecule  enhance protein solubility (salting in)
• As more salt was added, the salt used the available water to keep
itself soluble  protein starts to precipitate (salting out)

8. • At low concentrations, salt stabilises various charged groups on a
protein molecule  enhance protein solubility (salting in)
• As more salt was added, the salt used the available water to keep
itself soluble  protein starts to precipitate (salting out)
Advantages:
• Pure (NH4)2SO4
widely available and
inexpensive
• Stabilise the protein
• Prevent proteolysis
and bacterial action
• Can concentrate the
protein

9. METHODTITRE CURVE OF ANTISERA
(direct noncompetitive ELISA)
0.0
1.0
2.0
3.0
4.0
5.0
6.0
10 100 1000 10000 100000 1000000 10000000
Absorbance(450nm)
1/Dilution factor
Pre B1 B2 B3 T

10. METHODTITRE CURVE OF ANTISERA
(direct noncompetitive ELISA)
0.0
1.0
2.0
3.0
4.0
5.0
6.0
10 100 1000 10000 100000 1000000 10000000
Absorbance(450nm)
1/Dilution factor
Pre B1 B2 B3 T

11. 0
1
2
3
4
5
6
7
1 10 100 1,000 10,000 100,000 1,000,000 10,000,000
Absorbance(450nm)
[standard] (ng/ml)
Crude extract 80% pellet 84% pellet 86% supernatant
METHOD
STANDARD CURVE OF CRUDE AND (NH4)2SO4
PRECIPITATE HAZELNUT EXTRACTS
(indirect cELISA)

12. CONCLUSION
• The use of hazelnuts protein
precipitated using 80% ammonium
sulphate as a standard did improve the
sensitivity of ELISA in detecting the
presence of the hazelnuts.

13. FURTHER WORKS
• Develop a standard curve using 80% pellet as
the standard and use it detect or quantify
hazelnut in real food sample.
• Carried out SDS-Page
• Check cross-reactivity with other proteins
• Improve assay by blocking
• Determine LOD and LOQ of the assay

15. HAZELNUTS EXTRACTION
USING PBS
De-fatted nut flour
Mixing with PBS in 1:10 (w:v)
at room temperature
Filter the mixture
using cheese cloth
Centrifuge filtrate at
29,100 g for 30 min
Collect the clear extract
and store at -20 °C
1 2 3 4 5

16. ELISA
Coat plate with
coating buffer
containing
1mg/L hazelnuts
Incubate for
overnight at 4 °C
Wash plate with
PBST five time
and blot dry
Dispense
100uL/well of
standard
Dispense
100uL/well
primary antibody
Incubate for 2
hrs at 37 °C
Wash plate five
times with PBST
Add 2nd antibody
200uL/well
Incubate for 2
hrs at 37 °C
Wash plate five
times with PBST
Add TMB
substrate
200uL/well
Incubate at RT
for 30 mins
Add 2M H2SO4
(50uL/well)
Measure
absorbance at
450nm
Construct the
standard curve

17. Supernatant 20 to 90% Pellet 20 to 90%