OIE International Standard Reagents:
IZSAM experience
Giovanni Savini
Abu Dhabi
14-16 February
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.
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).
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.
Introduction
Guidelines for the preparation,
validation and distribution of
antibodies as International
Reference Standards for
antibody assays for infectious
diseases of animals
http://www.oie.int/fileadmin/Ho
me/eng/Our_scientific_expertise
/docs/pdf/GUIDELINE_3_REF_ST
ANDARDS_ANG.pdf
Definition
International Reference Standards (IRS)
Reagents to which all
assays are standardised
International Reference Standards
They are prepared by
OIE Reference
Laboratories (or other
international reference
labs) which use an
internationally accepted
Standard test Protocol
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.
Reference Standards
Secondary 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
Reference Standards
Secondary or Tertiary standards are used on a
daily basis to standardize testing
International Reference Standards
• How to prepare them?
Matrix
Usually blood serum which should
be free:
 from haemolysis
 from excessive lipaemia
 from infectious agents (safe)
 it facilitates shipment between
countries
 from TSE
Matrix
Treatments
 with binary ethyleneimine (BEI)
 irradiation at 25-30 kilogray and
keeping at -78°C
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
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
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 2.4.9, par 2.1)
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. 2.4.9 par
1.2)
 immunohistochemistry (OIE Manual Ch. 2.4.9
par 1.3)
 PCR (OIE Manual Ch. 2.4.9 par 1.4)
No clinical signs or pathological changes
other than CBPP were observed
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
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. 2.3.5
1/4
S-VN Adenovirus
Internal standardised protocol
1/8
S-VN Parinfluenza 3
Internal standardised protocol
1/16
ELISA FMD
IZS Brescia
Negative
Adventitious Virus
Isolation
Internal standardised protocol
Negative (after 3 pass. in
BHK cell cultures)
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
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
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
Neg
Bottle 2
++++
++++
+++
++
+
Neg
Neg
Irradiated positive reference standard
1:10
1:20
1:40
1:80
1:160
1:240
1:480
Bottle 1
++++
++++
++++
+++
++
+
Neg
Bottle 2
++++
++++
++++
+++
++
+
Neg
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
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:
1. non-irradiated lot for CFT
2. irradiated lot for ELISA
ISS for CBPP
The OIE International
Standard Serum for CBPP was
added to the list of OIEapproved International
Standard Sera
‫شكرا الهتمامكم‬