siti aziah
TRANSCRIPT
OPTIMISATION OF
OF HAZELNUTSFOR DETECTION
AN ENZYME-LINKED(ELISA)
by SITI AZIAH
ARIPIN
IMMUNOSORBENT ASSAY
• ELISA is a plate-based assay technique designed for detecting and quantifying substances such as peptides, proteins and hormones.
• Types: Competitive Non-competitive
INTRODUCTION
• 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.
To compare the effectiveness of different hazelnuts extract for use as standard materials in the ELISAi. Crude, de-fatted
extractii. Ammonium sulfate
precipitates of de-fatted extract
OBJECTIVE
De-shelled hazelnuts De-fatting
Ammonium sulphate precipitationELISA
METHODS
PBS extraction (1:10, w:v)
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 pellet30 ml Supernatant
Remaining supernatant
De-salting and freeze dry *Saturation was increased
by 2% each time.
• 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)
• 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
METHODTITRE CURVE OF ANTISERA (direct noncompetitive ELISA)
10 100 1000 10000 100000 10000000.0
1.0
2.0
3.0
4.0
5.0
6.0
Pre B1 B2 B3 T
1/Dilution factor
Abs
orba
nce
(450
nm
)
METHODTITRE CURVE OF ANTISERA (direct noncompetitive ELISA)
10 100 1000 10000 100000 10000000.0
1.0
2.0
3.0
4.0
5.0
6.0
Pre B1 B2 B3 T
1/Dilution factor
Abs
orba
nce
(450
nm
)
1,000 10,000 100,000 1,000,000 10,000,000 100,000,0000
1
2
3
4
5
6
7
Crude extract 80% pellet 84% pellet 86% supernatant
[standard] (ng/ml)
Abs
orba
nce
(450
nm
)METHOD STANDARD CURVE OF CRUDE AND (NH4)2SO4
PRECIPITATE HAZELNUT EXTRACTS (indirect cELISA)
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.
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
THANK YOU
HAZELNUTS EXTRACTIONUSING 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
ELISACoat 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
Supernatant 20 to 90% Pellet 20 to 90%