Enzyme By:

L
Sh. Nasirpour

inked
 Roya. Fathitil
L. Abedpour

I mmuno 

Instructor:
orbent


S Dr. Bazmi

ssays May2010



Basic Principles of ELISA

 Antigen  Antibody
Systems in ELISA

Co lo re d   Pro d u c t
S u b s t ra t e

Enz y m e ­ lin ke d  
a n t ib o d y
An t ib o d y
An t ig e n
STAGES IN ELISA
  Solid Phase
Coating
 50 mM carbonate, pH 9.6

COATING



Coating buffers


 20 mM Tris-HCl, pH 8.5


 10 mM phosphate-buffered saline
(PBS), pH 7.2
 WASHING
 Dipping Methods

 Multichannel Pipets

 Specialist Plate Washers

 ADDITION OF REAGENTS
Sing le ­channe l 
micropipe ts


Pipets
Multichanne l 
microtite r pipe ts
  Tips
ADDITION OF REAGENTS 
 INCUBATION
1 . Incubation of Re ag e nts while  
    Rotating  Plate s

2 . Stationary Incubation Conditions
Enzyme Conjugates

 Horseradish Peroxidase Plus

 Hydrogen Peroxide Substrate


 Alkaline Phosphatase Plus

 p-Nitrophenylphosphate


 β –Galactosidase Plus
 O-Nitrophenyl- β - d –Galactopyranoside


 Urease, pH Change, and

 Bromocresol Purple Indicator
 STOPPING REACTIONS


 Molar concentrations of strong acids

or strong bases


 Sodium azide is a inhibitor of HRP



 EDTA is a inhibitor AP
 READING


 Reading by Eye


 Spectrophotometric Reading
Main methods of Elisa
Direct Elisa

Antig e n Antibody Substrate  

Indirect Elisa

1 .  Antig e n 
adsorbe d 
to solid phase

4 .  Binding  to 
antibodie s 
2 .  Antibodie s are   attache d to antig e n
adde d
And incubate d

3 .  Antibodie s 
labe le d  5 .  Substrate  is 
with e nzyme adde d 
and color de ve lops
Sandwich Elisa

Conjug ate d 
Antibodie s  antibodie s
attache d  Are  adde d
to solid phase

Binding  of 
Antig e n  the  conjug ate
adde d During  
incubation

Antig e n is capture d  Substrate  is 

by coating   adde d
antibodie s and color 
during  incubation de ve lops
Competitive Direct Elisa

A. Pre ­titration of 
antig e n and 
conjug ate
in Dire ct ELISA

1 .  Antig e n 
1 .  Antig e n same   diffe re nt 
as  from that
that on solid  on solid phase
phase

2 .  Conjug ate   2 .  Conjug ate  binds 

binds  to 
to antig e n solid phase  antig e n
in liquid phase

3 . No conjugate   3 .  No re duction 

binds in 
that no color  color is 
de ve lops obse rve d
OCCURRENCE OF AFLATOXIN
M1 IN UHT MILK IN TURKEY

 Nurhan Unusan
Introduction
Materials and Methods

Preparation

Test procedure
Test procedure

Microtite r plate   Addition of AFM1   Addition of 

coate d with  standards anti­aflatoxin 
capture   Or the  pre pare d  M1  
antibodie s sample  solutions antibody

Addition of stop 
solution

Re ading  at 4 5 0  
nm

Addition of  Addition of 
e nzyme   substrate
conjug ate
FOOD AUTHENTICITY 
BY ELISA
Measurement of  Bovine IgG by
Indirect Competitive ELISA
as a Means of  Detecting Milk Adulteration
 Adultration

 Meat and meat-based

products
 Fish and fishery products
 Fruit juice
 Genetically modified products
 Irradiated food
 Feed stuffs
 Allergen ingredients
 Milk and dairy products



 te trame thyl be nzidine Wash
Bovine IgG

Monoclonal
anti-bovine IgG
antibody
Wash
Goat anti-mouse IgG
polyvalent HRP conjugate
me asuring abso rbanc e
at 450 nm
The enzymatic product concentration is 
inversely proportional to the analyte amount.
Detect cows milk in goat, Sheep
or buffalo milk with a detection
limit for all milks of 0.1%

Sensitive with pasteurized cows’ milk,
•

untreated milk and & frozen milk

Unable to detect bovine IgG in
UHT or reconstituted nonfat dried milk