Upload
wilfrid-cannon
View
215
Download
0
Tags:
Embed Size (px)
Citation preview
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: [email protected] 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: [email protected] www.eurotest.co.ba