1. 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 EURO-DIAGNOSTICA

2. EIA TECHNOLOGY FOR THE DETECTION OF THE ANTIBIOTIC CHLORAMPHENICOL (CAP) IN SHRIMPS EIA TECHNOLOGY FOR THE DETECTION OF THE ANTIBIOTIC CHLORAMPHENICOL (CAP) IN SHRIMPS

3. 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.

4. EIA 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!

5. CAP-EIA KIT FROM EURO-DIAGNOSTICA

6. REAGENTS IN THE CAP-EIA 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 Rabbit anti-CAP antibodies Sample material (not in the kit!)

7. MICROTITRE-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 to capture the rabbit anti-CAP antibodies. The function of these antibodies is to capture the rabbit anti-CAP antibodies.

8. IMMUNISATION OF RABBIT IMMUNISATION OF RABBIT ANTIBODIES ARE PURIFIED AND USED IN THE CAP-EIA ANTIBODIES ARE PURIFIED AND USED IN THE CAP-EIA ANTI-CAP ANTIBODIES IN THE EIA HAVE BEEN RAISED IN RABBITS ANTI-CAP ANTIBODIES IN THE EIA HAVE BEEN RAISED IN RABBITS CAPCAP COUPLING OF CAP TO A CARRIER PROTEIN RABBIT PRODUCES ANTI-CAP ANTIBODIES RABBIT PRODUCES ANTI-CAP ANTIBODIES

9. 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) THE CONJUGATE IN THE CAP-EIA CONSISTS OF CAP THAT HAS BEEN LABELED WITH THE ENZYME HORSE-RADISH PEROXIDASE (PO) + PO CAP CAP-PO (CONJUGATE) CAP-PO (CONJUGATE)

10. SAMPLE PREPARATION CAP-EIA SHRIMPS CONTAINING CAP HOMOGENISATION OF SHRIMPS IN BLENDER HOMOGENISATION OF SHRIMPS IN BLENDER EXTRACTION OF CAP WITH ETHYL ACETATE RECONSTITUTION IN DILUTION BUFFER SAMPLE IN CAP-EIA

11. SHEEP ANTI-RABBIT ANTIBODIES REACTION WELL STEPS IN THE CAP-EIA STANDARDS, SAMPLES, NEG. CONTROLS STANDARDS, SAMPLES, NEG. CONTROLS A CONJUGATE B RABBIT ANTI CAP ANTIBODIES C 1. THE SEVERAL REAGENTS ARE ADDED IN ORDER A, B, C

12. 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 TWO SITUATIONS CAN NOW BE CONSIDERED:  THE SAMPLE CONTAINS A CERTAIN AMOUNT OF CAP  THE SAMPLE CONTAINS NO CAP

13. STEPS IN THE CAP-EIA SAMPLE WITH CAP: SAMPLE WITHOUT CAP: CONJUGATE AS WELL AS FREE CAP CAN BIND TO THE ANTI-CAP ANTIBODIES 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 ONLY THE CONJUGATE IS AVAILABLE TO BIND TO THE ANTI-CAP ANTIBODIES

14. 5. THE PLATE IS INCUBATED FOR 30 MIN AT 25˚C STEPS IN THE CAP-EIA DURING INCUBATION THE COLORLESS CHROMOGEN IS CONVERTED BY THE ENZYME INTO A BLUE REACTION PRODUCT. DURING INCUBATION THE COLORLESS CHROMOGEN IS CONVERTED BY THE ENZYME INTO A BLUE REACTION PRODUCT. 4. THE ENZYME SUBSTRATE/CHROMOGEN (H 2 O 2 /TMB) IS ADDED TMB/H 2 O 2

15. STEPS IN THE CAP-EIA H 2 O 2 /TMB THE BLUE COLOR IS INVERSELY PROPORTIONAL TO THE AMOUNT OF BOUND CAP. THE MORE CAP PRESENT IN THE SAMPLE, THE LESS COLOR IS DEVELOPED. THE BLUE COLOR IS INVERSELY PROPORTIONAL TO THE AMOUNT OF BOUND CAP. THE MORE CAP PRESENT IN THE SAMPLE, THE LESS COLOR IS DEVELOPED.

16. STEPS IN THE CAP-EIA 6. THE COLOR DEVELOPMENT IS STOPPED BY ADDING 0.5 M OF SULFURIC ACID (H 2 SO 4 ) IN THIS ACIDIC ENVIRONMENT THE BLUE COLOR CHANGES INTO YELLOW IN THIS ACIDIC ENVIRONMENT THE BLUE COLOR CHANGES INTO YELLOW

17. READING THE RESULTS 7. ABSORPTION OF EACH WELL IS MEASURED AT 450 nm 8. AMOUNT OF CAP IN THE SAMPLES IS CALCULATED

18. Thanks for your attention! Thanks for your attention! Greetings from Holland

19. 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