1. OIE International Standard Reagents: IZSAM experience
Giovanni Savini Abu Dhabi February

2. Introduction OIE-approved International Standard Sera
The OIE Biological Standards Commission (Laboratories Commission) coordinates a programme for the preparation, validation and distribution of OIE-approved International Reference Standards for antibody assays for infectious diseases of animals.

3. Introduction OIE-approved International Standard Sera
The standards are prepared by an OIE Reference Laboratory in accordance with Guidelines drawn-up by the Laboratories Commission and in collaboration with other laboratories.
Such standard preparations are designated by the OIE as primary reference standards for use in conjunction with tests described in the OIE Manual of Diagnostic Tests and Vaccines for Terrestrial Aniamls (mammals, birds and bees).

4. Introduction OIE-approved International Standard Sera AIM
The aim of the programme is to harmonise diagnostic testing and encourage the mutual recognition of test results for international trade.

5. Introduction
Guidelines for the preparation, validation and distribution of antibodies as International Reference Standards for antibody assays for infectious diseases of animals

6. International Reference Standards (IRS)
Definition
International Reference Standards (IRS)
Reagents to which all assays are standardised

7. International Reference Standards
They are prepared by OIE Reference Laboratories (or other international reference labs) which use an internationally accepted Standard test Protocol

8. International Reference Standards
They are necessary to ensure that an antibody assay is capable of measuring antibody activity to a specified level of diagnostic sensitivity.

9. Reference Standards Secondary Standards Tertiary standards
are reagent calibrated by comparison with IRS
normally prepared by National Reference Laboratory
Commonly represent the National Standards
Tertiary standards
are reagents calibrated against the Secondary standards
usually prepared by normal diagnostic laboratories

10. Reference Standards
Secondary or Tertiary standards are used on a daily basis to standardize testing

11. International Reference Standards
How to prepare them?

12. Matrix Usually blood serum which should be free: from haemolysis
from excessive lipaemia
from infectious agents (safe)
it facilitates shipment between countries
from TSE

13. Matrix Treatments with binary ethyleneimine (BEI)
irradiation at kilogray and keeping at -78°C

14. International reference standards
Positive serum
It should origin from animals with a typical humoral immune-response to the selected pathogen (experimental infection, vaccination and exceptionally from naturally infected animals).
It should be free from cross-reactive antibodies
It could origin from a single animal or it could derive from a pool of animals

15. Our experience
The 10th of June 1993 IZSAM was appointed as OIE Reference Laboratory for CBPP
The mandate includes :
To function as a centre of expertise and standardization of techniques
To store and distribute biological reference products
To develop new procedures for diagnosis and control
To collect, process, analyze and disseminate epizootiological data
To place expert consultants at the disposal of the OIE

16. ISS for CBPP
Constitution: it would be better if matrix shows the desired level of reactivity (without further dilution)
Between October 1990-September 1993, 94 outbreaks of CBPP occurred in Italy
In May 1993: Outbreak of CBPP in Piedmont Region included 600 cattle
Donor animal:
fattening Charolais 18-months old beef serologically positive to CBPP:
CFT 1:320 (Campbell & Turner CFT- OIE Manual , par 2.1)

17. ISS for CBPP CBPP was diagnosed in the donor animal by:
by serological testing
post-mortem examination performed at slaughtering time
Confirmatory tests (IZSAM, NRC for CBPP):
isolation of MmmSC (OIE Manual Ch par 1.2)
immunohistochemistry (OIE Manual Ch par 1.3)
PCR (OIE Manual Ch par 1.4)
No clinical signs or pathological changes other than CBPP were observed

18. ISS for CBPP
Stability and storage: treatment to inactivate adventitious agents, freeze-dried
Blood was centrifuged, the serum separated and filtered (Sartorius capsule filters 0.8, 0.6, 0.45, 0.2µm)
No bacterial contaminants were detected in the serum
It was freeze-dried (1ml in 5 ml rubber capped bottles) and stored at -20°C

19. ISS CBPP
Presence of heterologous Abs and adventitious viruses on freeze-dried sera
Type of test
Reference
Result
S-VN BVD
IZSTE B2.1.4SOP012
Negative
S-VN Respiratory Syncytial Virus
IZSTE B2.1.4SOP026
1/8
S-VN BVH4
IZSTE B2.1.4SOP027
1/4
S-VN IBR
OIE Manual, Ch
S-VN Adenovirus
Internal standardised protocol
S-VN Parinfluenza 3
1/16
ELISA FMD
IZS Brescia
Adventitious Virus Isolation
Negative (after 3 pass. in BHK cell cultures)

20. ISS for CBPP Results of controls
There was no evidence that heterologous Abs detected in the serum interfere with the modified CFT
Vaccination against FMD had officially ceased in January 1992: no Abs to serotypes A, O, C, A22 Iraq, O Manisa and Asia were detected by ELISA
Prions were not present in the blood stream

21. ISS for CBPP
Stability and storage: treatment to inactivate adventitious agents, freeze-dried
After lyophilization, serum was divided in two lots of 400 bottles each:
Non-irradiated
Irradiated (25 kilogray)
e-beam or ɣ-irradiations could have detrimental effect on CBPP CFT titres:
Mediscan GmbH, Austria
Gammarad Italia

22. Irradiated positive reference standard
ISS for CBPP
Controls after treatment
CFT titres have been evaluated to confirm their activity:
Non irradiated positive reference standard
1:160
1:240
1:320
1:480
1:640
1:960
1:1280
Bottle 1
++++
+++ ++ +
Neg
Bottle 2
Irradiated positive reference standard
1:10
1:20
1:40
1:80
1:160
1:240
1:480
Bottle 1
++++
+++ ++ +
Neg
Bottle 2

23. ISS for CBPP Certification required
Data sheets: description of donor animal, nature of Abs response, details of organism used to elicit the immune response, details of experimental infection or immunization protocol, reference tests used, presence of heterologous Abs, safety testing, sterilization methods, batch number, date of production
The full technical and statistical data on the evaluation of the candidate reference standard, together with the full data sheet information, was submitted to OIE Standards Commission on 14 January 2003

24. ISS for CBPP
The OIE Biological Standards Commission has approved the OIE International Standard Serum for CBPP on 29 September 2003
The sera are available as two lots:
non-irradiated lot for CFT
irradiated lot for ELISA

25. ISS for CBPP
The OIE International Standard Serum for CBPP was added to the list of OIE-approved International Standard Sera

26. شكرا لاهتمامكم