siti aziah

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%