EURO-DIAGNOSTICA
EURO-DIAGNOSTICA B.V. The Netherlands
For all information, please contact
authorized European representative and distributor:
Diagnostics Center “EUROTEST” d.o.o.
Sarajevo, Bosnia and Herzegovina, Europe
Tel/fax: +387(0)33-555-801, Cell Phone: +387(0)61-201-711
E-mail: info@eurotest.co.ba
www.eurotest.co.ba
EIA TECHNOLOGY FOR THE
DETECTION OF THE ANTIBIOTIC
CHLORAMPHENICOL (CAP)
IN SHRIMPS
OH
H
N
CH2
O2N
OH
C
O
CH2Cl2
EIA:
ENZYME IMMUNOASSAY
EIA is a rapid test used for detecting
and quantifying a large variety
of biological components, e.g.
viruses, bacteria, hormones,
antibiotics, allergens, etc.
EIA FORMATS
EIAs are divided into 3 main formats:
• Direct EIAs
• Indirect EIAs
• Blocking or competitive EIAs
The CAP-EIA is a competitive assay!
CAP-EIA KIT FROM EURO-DIAGNOSTICA
OH
H
N
CH2
O2N
OH
C
O
CH2Cl2
REAGENTS IN THE CAP-EIA
• A 96-wells polystyrene microtitre plate
coated with sheep anti-rabbit antibodies
• Rabbit anti-CAP antibodies
• Conjugate: labeled CAP (CAP-PO)
• CAP-standards, negative controls
• Sample material (not in the kit!)
MICROTITRE-PLATE IN THE CAP-EIA
The wells of the microtitre plate are coated
with sheep anti-rabbit (SAR) antibodies.
The function of these antibodies is to
capture the rabbit anti-CAP antibodies.
ANTI-CAP ANTIBODIES IN THE EIA
HAVE BEEN RAISED IN RABBITS
CAP
COUPLING OF CAP
TO A CARRIER PROTEIN
ANTIBODIES ARE
PURIFIED AND USED
IN THE CAP-EIA
IMMUNISATION
OF RABBIT
RABBIT PRODUCES
ANTI-CAP ANTIBODIES
CONJUGATE IN THE CAP-EIA
THE CONJUGATE IN THE CAP-EIA CONSISTS OF
CAP THAT HAS BEEN LABELED WITH THE
ENZYME HORSE-RADISH PEROXIDASE (PO)
+
CAP
PO
CAP-PO
(CONJUGATE)
SAMPLE PREPARATION CAP-EIA
SHRIMPS CONTAINING CAP
HOMOGENISATION OF
SHRIMPS IN BLENDER
EXTRACTION OF CAP
WITH ETHYL ACETATE
RECONSTITUTION
IN DILUTION BUFFER
SAMPLE IN CAP-EIA
STEPS IN THE CAP-EIA
1. THE SEVERAL REAGENTS ARE ADDED IN ORDER A, B, C
C
RABBIT ANTI CAP ANTIBODIES
B
CONJUGATE
A
STANDARDS, SAMPLES,
NEG. CONTROLS
SHEEP ANTI-RABBIT ANTIBODIES
REACTION WELL
STEPS IN THE CAP-EIA
2. THE PLATE IS INCUBATED FOR 60 MIN AT 4˚C
3. THE WELLS ARE THEN WASHED WITH WASHING BUFFER
TWO SITUATIONS CAN NOW BE CONSIDERED:
 THE SAMPLE CONTAINS A CERTAIN AMOUNT OF CAP
 THE SAMPLE CONTAINS NO CAP
STEPS IN THE CAP-EIA
SAMPLE WITH CAP:
SAMPLE WITHOUT CAP:
CONJUGATE AS WELL AS
FREE CAP CAN BIND TO THE
ANTI-CAP ANTIBODIES
ONLY THE CONJUGATE IS
AVAILABLE TO BIND TO THE
ANTI-CAP ANTIBODIES
STEPS IN THE CAP-EIA
4. THE ENZYME SUBSTRATE/CHROMOGEN (H2O2/TMB) IS ADDED
5. THE PLATE IS INCUBATED FOR 30 MIN AT 25˚C
DURING INCUBATION THE COLORLESS CHROMOGEN IS
CONVERTED BY THE ENZYME INTO A BLUE REACTION PRODUCT.
TMB/H2O2
STEPS IN THE CAP-EIA
THE BLUE COLOR IS INVERSELY PROPORTIONAL TO THE
AMOUNT OF BOUND CAP. THE MORE CAP PRESENT IN THE
SAMPLE, THE LESS COLOR IS DEVELOPED.
H2O2/TMB
STEPS IN THE CAP-EIA
6. THE COLOR DEVELOPMENT IS STOPPED BY ADDING
0.5 M OF SULFURIC ACID (H2SO4)
IN THIS ACIDIC ENVIRONMENT THE BLUE
COLOR CHANGES INTO YELLOW
READING THE RESULTS
7. ABSORPTION OF EACH WELL IS MEASURED AT 450 nm
8. AMOUNT OF CAP IN THE SAMPLES IS CALCULATED
% max.
Chloramphenicol
90
80
70
60
50
40
30
20
10
0
0,01
0,1
y = -11,295Ln(x) + 44,093
R2 = 0,9717
1 conc. (ng/ml) 10
100
Greetings from Holland
Thanks
for your attention!
CONTACT EURO-DIAGNOSTICA
EURO-DIAGNOSTICA B.V. The Netherlands
For all information, please contact
authorized European representative and distributor:
Diagnostics Center “EUROTEST” d.o.o.
Sarajevo, Bosnia and Herzegovina, Europe
Tel/fax: +387(0)33-555-801, Cell Phone: +387(0)61-201-711
E-mail: info@eurotest.co.ba www.eurotest.co.ba