Download - SITI AZIAH
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OPTIMISATION OF
OF HAZELNUTSFOR DETECTION
AN ENZYME-LINKED(ELISA)
by SITI AZIAH
ARIPIN
IMMUNOSORBENT ASSAY
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• ELISA is a plate-based assay technique designed for detecting and quantifying substances such as peptides, proteins and hormones.
• Types: Competitive Non-competitive
INTRODUCTION
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• 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.
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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
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De-shelled hazelnuts De-fatting
Ammonium sulphate precipitationELISA
METHODS
PBS extraction (1:10, w:v)
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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.
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• 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)
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• 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
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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
)
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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
)
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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)
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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.
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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
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THANK YOU
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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
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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
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Supernatant 20 to 90% Pellet 20 to 90%