EURO-DIAGNOSTICA B.V. The Netherlands For all information, please contact authorized European...

EURO-DIAGNOSTICA B.V. The NetherlandsFor all information, please contact

authorized European representative and distributor:

Diagnostics Center “EUROTEST” d.o.o.

Sarajevo, Bosnia and Herzegovina, EuropeTel/fax: +387(0)33-555-801, Cell Phone: +387(0)61-201-711

E-mail: info@eurotest.co.ba www.eurotest.co.ba

EURO-DIAGNOSTICA

EIA TECHNOLOGY FOR THE DETECTION OF THE ANTIBIOTIC

CHLORAMPHENICOL (CAP)IN SHRIMPS

EIA TECHNOLOGY FOR THE DETECTION OF THE ANTIBIOTIC

CHLORAMPHENICOL (CAP)IN SHRIMPS

O2N

OH H

O

CH2Cl2C

N

CH2

OH

EIA:ENZYME IMMUNOASSAY

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 is a rapid test used for detecting

and quantifying a large variety

of biological components, e.g.

viruses, bacteria, hormones,

antibiotics, allergens, etc.

EIA FORMATSEIA FORMATS

EIAs are divided into 3 main formats:EIAs are divided into 3 main formats:

• Direct EIAs

• Indirect EIAs

• Blocking or competitive EIAs

• Direct EIAs

• Indirect EIAs

• Blocking or competitive EIAs

The CAP-EIA is a competitive assay!The CAP-EIA is a competitive assay!

CAP-EIA KIT FROM EURO-DIAGNOSTICA CAP-EIA KIT FROM EURO-DIAGNOSTICA

O2N

OH H

O

CH2Cl2C

N

CH2

OH

REAGENTS IN THE CAP-EIAREAGENTS IN THE CAP-EIA

• Conjugate: labeled CAP (CAP-PO)• Conjugate: labeled CAP (CAP-PO)

• A 96-wells polystyrene microtitre plate

coated with sheep anti-rabbit antibodies

• A 96-wells polystyrene microtitre plate

coated with sheep anti-rabbit antibodies

• CAP-standards, negative controls• CAP-standards, negative controls

• Rabbit anti-CAP antibodies• Rabbit anti-CAP antibodies

• Sample material (not in the kit!)• Sample material (not in the kit!)

MICROTITRE-PLATE IN THE CAP-EIAMICROTITRE-PLATE IN THE CAP-EIA

The wells of the microtitre plate are coated

with sheep anti-rabbit (SAR) antibodies.

The wells of the microtitre plate are coated

with sheep anti-rabbit (SAR) antibodies.

The function of these antibodies is tocapture the rabbit anti-CAP antibodies.

The function of these antibodies is tocapture the rabbit anti-CAP antibodies.

IMMUNISATIIMMUNISATIONONOF RABBITOF RABBIT

IMMUNISATIIMMUNISATIONONOF RABBITOF RABBIT

ANTIBODIES AREANTIBODIES AREPURIFIED AND USEDPURIFIED AND USED

IN THE CAP-EIAIN THE CAP-EIA

ANTIBODIES AREANTIBODIES AREPURIFIED AND USEDPURIFIED AND USED

IN THE CAP-EIAIN THE CAP-EIA

ANTI-CAP ANTIBODIES IN THE EIAANTI-CAP ANTIBODIES IN THE EIAHAVE BEEN RAISED IN RABBITSHAVE BEEN RAISED IN RABBITS

ANTI-CAP ANTIBODIES IN THE EIAANTI-CAP ANTIBODIES IN THE EIAHAVE BEEN RAISED IN RABBITSHAVE BEEN RAISED IN RABBITS

CAPCAPCAPCAP COUPLING OF CAPCOUPLING OF CAPTO A CARRIER PROTEINTO A CARRIER PROTEIN

COUPLING OF CAPCOUPLING OF CAPTO A CARRIER PROTEINTO A CARRIER PROTEIN

RABBIT PRODUCESRABBIT PRODUCESANTI-CAP ANTIBODIES ANTI-CAP ANTIBODIES

RABBIT PRODUCESRABBIT PRODUCESANTI-CAP ANTIBODIES ANTI-CAP ANTIBODIES

CONJUGATE IN THE CAP-EIACONJUGATE IN THE CAP-EIA

THE CONJUGATE IN THE CAP-EIA CONSISTS OF

CAP THAT HAS BEEN LABELED WITH THE

ENZYME HORSE-RADISH PEROXIDASE (PO)

THE CONJUGATE IN THE CAP-EIA CONSISTS OF

CAP THAT HAS BEEN LABELED WITH THE

ENZYME HORSE-RADISH PEROXIDASE (PO)

+

POPOCAPCAP CAP-PO(CONJUGATE)

CAP-PO(CONJUGATE)

SAMPLE PREPARATION CAP-EIASAMPLE PREPARATION CAP-EIA

SHRIMPS CONTAINING CAPSHRIMPS CONTAINING CAP

HOMOGENISATION OFHOMOGENISATION OFSHRIMPS IN BLENDERSHRIMPS IN BLENDER

HOMOGENISATION OFHOMOGENISATION OFSHRIMPS IN BLENDERSHRIMPS IN BLENDER

EXTRACTION OF CAP EXTRACTION OF CAP WITH ETHYL ACETATEWITH ETHYL ACETATE

EXTRACTION OF CAP EXTRACTION OF CAP WITH ETHYL ACETATEWITH ETHYL ACETATE

RECONSTITUTION RECONSTITUTION IN DILUTION BUFFERIN DILUTION BUFFER

RECONSTITUTION RECONSTITUTION IN DILUTION BUFFERIN DILUTION BUFFER

SAMPLE IN CAP-EIASAMPLE IN CAP-EIASAMPLE IN CAP-EIASAMPLE IN CAP-EIA

SHEEP ANTI-RABBIT ANTIBODIESSHEEP ANTI-RABBIT ANTIBODIES

REACTION WELLREACTION WELL

STEPS IN THE CAP-EIASTEPS IN THE CAP-EIA

STANDARDS, SAMPLES,NEG. CONTROLS

STANDARDS, SAMPLES,NEG. CONTROLSA

CONJUGATECONJUGATEB

RABBIT ANTI CAP ANTIBODIESRABBIT ANTI CAP ANTIBODIESC

1. THE SEVERAL REAGENTS ARE ADDED IN ORDER A, B, C1. THE SEVERAL REAGENTS ARE ADDED IN ORDER A, B, C

STEPS IN THE CAP-EIASTEPS IN THE CAP-EIA

2. THE PLATE IS INCUBATED FOR 60 MIN AT 4˚C 2. THE PLATE IS INCUBATED FOR 60 MIN AT 4˚C

3. THE WELLS ARE THEN WASHED WITH WASHING BUFFER3. 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

TWO SITUATIONS CAN NOW BE CONSIDERED:

THE SAMPLE CONTAINS A CERTAIN AMOUNT OF CAP

THE SAMPLE CONTAINS NO CAP

STEPS IN THE CAP-EIASTEPS IN THE CAP-EIA

SAMPLE WITH CAP:SAMPLE WITH CAP: SAMPLE WITHOUT CAP:SAMPLE WITHOUT CAP:

CONJUGATE AS WELL ASFREE CAP CAN BIND TO THE

ANTI-CAP ANTIBODIES

CONJUGATE AS WELL ASFREE CAP CAN BIND TO THE

ANTI-CAP ANTIBODIES

ONLY THE CONJUGATE ISAVAILABLE TO BIND TO THE

ANTI-CAP ANTIBODIES

ONLY THE CONJUGATE ISAVAILABLE TO BIND TO THE

ANTI-CAP ANTIBODIES

5. THE PLATE IS INCUBATED FOR 30 MIN AT 25˚C5. THE PLATE IS INCUBATED FOR 30 MIN AT 25˚C

STEPS IN THE CAP-EIASTEPS IN THE CAP-EIA

DURING INCUBATION THE COLORLESS CHROMOGEN ISCONVERTED BY THE ENZYME INTO A BLUE REACTION PRODUCT.

DURING INCUBATION THE COLORLESS CHROMOGEN ISCONVERTED BY THE ENZYME INTO A BLUE REACTION PRODUCT.

4. THE ENZYME SUBSTRATE/CHROMOGEN (H2O2/TMB) IS ADDED 4. THE ENZYME SUBSTRATE/CHROMOGEN (H2O2/TMB) IS ADDED

TMB/H2O2TMB/H2O2

STEPS IN THE CAP-EIASTEPS IN THE CAP-EIA

H2O2/TMBH2O2/TMB

THE BLUE COLOR IS INVERSELY PROPORTIONAL TO THEAMOUNT OF BOUND CAP. THE MORE CAP PRESENT IN THE

SAMPLE, THE LESS COLOR IS DEVELOPED.

THE BLUE COLOR IS INVERSELY PROPORTIONAL TO THEAMOUNT OF BOUND CAP. THE MORE CAP PRESENT IN THE

SAMPLE, THE LESS COLOR IS DEVELOPED.

STEPS IN THE CAP-EIASTEPS IN THE CAP-EIA

6. THE COLOR DEVELOPMENT IS STOPPED BY ADDING 0.5 M OF SULFURIC ACID (H2SO4)

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

IN THIS ACIDIC ENVIRONMENT THE BLUE COLOR CHANGES INTO YELLOW

READING THE RESULTSREADING THE RESULTS

Chloramphenicoly = -11,295Ln(x) + 44,093

R2 = 0,9717

0

10

2030

40

50

6070

80

90

0,01 0,1 1 10 100conc. (ng/ml)

% m

ax

.7. ABSORPTION OF EACH WELL IS MEASURED AT 450 nm7. ABSORPTION OF EACH WELL IS MEASURED AT 450 nm

8. AMOUNT OF CAP IN THE SAMPLES IS CALCULATED8. AMOUNT OF CAP IN THE SAMPLES IS CALCULATED

Thanks for your attention!

Thanks for your attention!

Greetings from HollandGreetings from Holland

CONTACT EURO-DIAGNOSTICACONTACT EURO-DIAGNOSTICA

EURO-DIAGNOSTICA B.V. The NetherlandsFor all information, please contact

authorized European representative and distributor:

Diagnostics Center “EUROTEST” d.o.o.

Sarajevo, Bosnia and Herzegovina, EuropeTel/fax: +387(0)33-555-801, Cell Phone: +387(0)61-201-711

E-mail: info@eurotest.co.ba www.eurotest.co.ba